Optimizing the Effectiveness of Induced Resistance in Tomato for Bacterial Disease Management. Cheryl Trueman

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1 Optimizing the Effectiveness of Induced Resistnce in Tomto for Bcteril Disese Mngement by Cheryl Truemn A Thesis presented to The University of Guelph In prtil fulfilment of requirements for the degree of Doctor of Philosophy in Environmentl Biology Guelph, Ontrio, Cnd Cheryl Truemn, December, i

2 ABSTRACT OPTIMIZING THE EFFECTIVENESS OF INDUCED RESISTANCE IN TOMATO FOR BACTERIAL DISEASE MANAGEMENT Cheryl Lynn Truemn University of Guelph, 2017 Advisor: Professor P. H. Goodwin As lterntives for mnging bcteril speck (Pseudomons syringe pv. tomto (Pst)) nd bcteril spot (Xnthomons grdneri (Xg)) of tomto (Solnum lycopersicum), the synthetic chemicl defense ctivtor, cibenzolr-s-methyl (ASM) combined with the synthetic plnt growth regultor (PGR) uniconzole (UNI), the nturl chemicl defense ctivtor, pr-minobenzoic cid (PABA), nd the puttive biologicl defense ctivtor, B. mycoides/weihenstephnensis R17, were exmined. No consistent benefits for bcteril spot nd speck control or tomto growth were observed in field experiments from with the combintion of ASM nd UNI, whether pplied to seedlings (<6 weeks old) or post-trnsplnting. However, greenhouse pplied ASM or ASM+UNI reduced lte seson disese severity 18 to 24% compred to nontreted nd CuOH-treted controls for cv. TSH4 in 2012, indicting tht ASM pplied to tomto seedlings cn hve long-term benefits under certin conditions. Fitness costs in terms of reduced growth re concern with ASM, so UNI ws dded to ASM to determine if tht could be meliorted, but this pproch ws ineffective. Greenhouse ASM pplictions to cv. H9909 in 2012 reduced totl yield by 20% compred to the nontreted control, indicting fitness cost, nd ASM+UNI treted plnts showed similr loss. Fitness costs my hve occurred in 2012 due to stress from dry conditions fter trnsplnting. For PABA, effectiveness ws ffected by ppliction method, concentrtion nd host genotype. Despite optimizing PABA efficcy under controlled conditions, PABA ws ineffective in the field. A bcteril endophyte, R17, ws isolted from Solnum rcnum nd its bility to puttively induce resistnce ginst Pst under controlled conditions ws ffected by its concentrtion, ppliction method, nd host genotype. PABA nd R17 reduced bcteril speck lesion incidence up to 43 nd 51%, respectively, but the lesions tht developed were lrger in treted thn nontreted plnts resulting in no reduction in Pst popultion or totl symptomtic lef re. This suggests tht certin defense ctivtors cn reduce the bility of Pst to infect but then llow for greter Pst popultion growth post-infection. While defense ctivtors hve potentil, they need to be more effective nd consistent before they re integrted into bcteril disese mngement strtegies of tomto. 2 ii

3 DEDICATION For Justin, you believed in me, nd Alexis, you helped me believe in myself. Also for Dd, who told me to find summer job in Ontrio, nd Mom for ll those kilometres you drove with me nd for me. iii

4 3 ACKNOWLEDGEMENTS Thnk you to my dvisor, Dr. Pul Goodwin, nd the members of my dvisory committee, Dr. Kri Dunfield, Dr. Tom Hsing, Dr. Annette Nssuth, nd Dr. Istvn Rjcn for their guidnce nd dvice during this journey. Your time nd ptience is truly pprecited. This reserch ws completed t the Ridgetown Cmpus, University of Guelph, nd there re mny people who contributed time nd effort towrd its completion. These people include the Field Vegetble Pest Mngement Technicin, Phyllis My, former co-op students Sherri Tedford nd Tin Simonton, nd mny summer students nd reserch ssistnts. Thnk you for your dediction to the progrm. I would lso like to thnk the mny collegues t the Ridgetown Cmpus who provided dvice nd support during this journey including Jnice LeBoeuf, Steve Loewen, Ken McEwn, Eline Roddy, Dr. Drren Robinson, Dr. Art Schfsm, nd Dr. Lur Vn Eerd. I would lso like to thnk Dr. Dine Cuppels, with whom I hd vluble converstion bout bcteril disese in tomto fter her retirement from Agriculture nd Agri-Food Cnd in London. To my prtner in life, Justin Kritikos, thnk you for being my soft plce to fll. Your ptience nd encourgement did not go unnoticed. Thnk you for embrcing wht it mens to be n equl prtner in life. Funding for this reserch ws provided in prt by the Ontrio Tomto Reserch Institute, Syngent Cnd, Vlent Cnd, Ridgetown Cmpus University of Guelph, nd the Ontrio Ministry nd Food nd Rurl Affirs. iv

5 4 TABLE OF CONTENTS ABSTRACT... ii DEDICATION... iii ACKNOWLEDGEMENTS... iv TABLE OF CONTENTS... v LIST OF TABLES... x LIST OF FIGURES... xv LIST OF ABBREVIATIONS... xxi Chpter 1: Literture Review Introduction Processing tomtoes in Ontrio Tomto genetics Bcteril endophytes of tomto Bcteril speck pthogen of tomto Bcteril spot pthogen of tomto Current bcteril speck nd spot mngement prctices in tomto SIR nd its use in bcteril disese mngement SAR Mechnisms of SAR Biologicl ctivtors of SAR PAMP ctivtors of SAR Plnt ctivtors of SAR Synthetic ctivtors of SAR Plnt fitness costs ssocited with SAR Vrition in SAR response mong cultivrs SAR in disese mngement SAR in mngement of bcteril speck nd spot of tomto ISR Mechnisms of ISR Biologicl ctivtors of ISR PAMP ctivtors of ISR v

6 Synthetic ctivtors of ISR Plnt growth effects ssocited with ISR Fctors ffecting ISR ISR s disese mngement tool ISR s disese mngement tool in tomto Interctions nd reltionships between SAR nd ISR pthwys GA-relted PGRs Hypothesis Objectives Chpter 2: Effects of pr-minobenzoic cid on the incidence of bcteril speck disese, P. syringe pv. tomto growth, nd plnt growth in processing tomto Introduction Mterils & Methods Direct ntimicrobil effects of PABA on Pst Growth room evlution of folir pplictions of PABA ginst bcteril speck Optiml concentrtion Host genotype effect, folir PABA ppliction number, folir PABA response durtion nd soil PABA ppliction Pst popultions in tomto leves Pst lesion sizes in tomto leves Field evlution of folir pplictions of PABA Sttisticl nlysis Results PABA direct ntimicrobil effect PABA soil drench ppliction PABA folir ppliction Effect of plnt genotype on PABA response PABA pplictions PABA response durtion PABA effect on Pst popultions nd lesions Field evlution Discussion Direct ntimicrobil effects of PABA vi

7 2.4.2 PABA concentrtions nd ppliction methods Host genotype effect Prmeters ffecting protection nd the durtion of protection by PABA Effect of PABA on bcteril speck lesion incidence versus Pst popultion Effects of PABA on bcteril speck incidence in the field Effects of PABA on plnt growth nd development Conclusions Chpter 3: Effects of the plnt growth regultor uniconzole with the plnt defense ctivtor cibenzolr- S-methyl on incidence nd severity of bcteril speck (P. syringe pv. tomto) nd bcteril spot (X. grdneri) in tomto Introduction Mterils & Methods Wether conditions Greenhouse tretment with UNI followed by field tretment with ASM Experimentl design nd tretments Pthogen inocultions Disese nd plnt growth ssessments Sttisticl nlysis Greenhouse tretment with UNI followed by greenhouse tretment with ASM or CuOH Experimentl design nd tretments Pthogen inocultions Disese nd plnt growth ssessments Sttisticl nlysis Results Wether Conditions Greenhouse tretment with UNI followed by field tretment with ASM Effect of greenhouse UNI followed by field ASM on bcteril speck nd bcteril spot Effect of greenhouse UNI followed by field ASM on tomto growth, yield, nd qulity Greenhouse tretment with UNI followed by greenhouse tretment with ASM Effect of greenhouse UNI followed by greenhouse ASM on bcteril speck nd bcteril spot vii

8 Effect of greenhouse UNI followed by greenhouse ASM on tomto growth, yield nd qulity Discussion Greenhouse UNI with field ASM ppliction Greenhouse UNI nd greenhouse ASM ppliction Greenhouse UNI with greenhouse CuOH ppliction Improvement of ASM effectiveness Environmentl impcts on disese nd control mesures Durtion of ASM effectiveness Comprison of disese ssessment methods Conclusions Chpter 4: Use of bcteril endophytes to control the incidence of bcteril speck disese (Pseudomons syringe pv. tomto) nd lter plnt growth in processing tomto seedlings Introduction Mterils & Methods Isoltion of endophytes from tomto nd wild tomto species Screening of endophytes for induced resistnce nd plnt growth effects Stndrd curve of CFU versus A Direct ntimicrobil effects of R17 nd R Optimiztion of bcteril endophyte tretment Effect of R17 inocultion on popultions in plnt Effect of R17 inocultion on Pst popultions nd lesion size in plnt Identifiction of endophytes R17 nd R Sttisticl nlysis Results Bcteril endophytes isolted from tomto nd wild tomto species Screening of bcteril endophytes for induced resistnce nd plnt growth effects Direct ntimicrobil effects of R17 nd R Optimiztion of bcteril endophyte tretment Effect of R17 inocultion on Pst popultions nd lesion size in plnt Effect of R17 inocultion on popultions in plnt Identifiction of R17 nd R Discussion viii

9 4.4.1 Isoltion of endophytes from domestic nd wild tomto Screening for ctivity of endophytes for induced resistnce nd plnt growth promotion Identifiction of strins R17 nd R Optimiztion of bcteril endophyte tretment Direct ntimicrobil effects of promising endophytes Coloniztion of R17 inside tomto tissues Effect of R17 on bcteril speck lesion incidence versus Pst popultion Conclusions Chpter 5: Generl Discussion References Appendix ix

10 LIST OF TABLES Tble 2.1 Effect of PABA concentrtion on growth of P. syringe pv. tomto (Pst) in filter disc ssy. Filter discs soked in 0, 1, 9, 18, nd 27 mm pr-minobenzoic cid (PABA) dissolved in wter, nd 0, 18, nd 72 mm PABA dissolved in 70% ethnol were plced on tryptic soy gr covered thoroughly with Pst, incubted t room temperture, nd ssessed for zones fter three dys Tble 2.2 Folir, root, nd totl dry weight of six tomto cultivrs treted with pr-minobenzoic cid (PABA). Plnts were coted with 18 mm PABA 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto 20 DAS. Dry weight of plnts five dys post inocultion is shown Tble 2.3 Folir, root, nd totl dry weight of tomto cv. H5108 nd cv. TSH33 treted with zero, one, two, or three pplictions of pr-minobenzoic cid (PABA). One, two, or three folir PABA tretments (18mM) were pplied by coting fine mist on plnts 10, 12, nd/or 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Dry weight of plnts five dys post inocultion is shown Tble 2.4 Folir, root, nd totl dry weight of tomto cv. H5108 inoculted with P. syringe pv. tomto (Pst) five, seven, or 10 dys fter pr-minobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 dys fter seeding (DAS)). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five, seven, or 10 dys fter the second PABA ppliction. Dry weight of plnts five dys post inocultion is shown Tble 2.5 Incidence of bcteril speck symptoms, popultion of P. syringe pv. tomto (Pst) per cm 2, nd per lesion on the third youngest terminl leflet of tomto cv. H5108 fter pr-minobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 dys fter seeding (DAS)). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five dys fter the second PABA ppliction Tble 2.6 Are under the disese progress curve (AUDPC) for erly nd lte disese in tomto cv. H5108. Erly seson disese ws mesured by clculting the percentge of leves with disese symptoms nd lte seson disese ws mesured by estimting defolition in plnts treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, in Tble 2.7 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. H5108, hrvested from plots treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted Tble 2.8 Totl, red, green, nd rotten fruit yield in 2m section of tomto cv. H5108 treted with CuOH nd pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, in Tble 3.1 Tretment descriptions nd ppliction timings for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Further detils on pplictions methods re described in the text Tble 3.2 Tretment descriptions nd ppliction timings for tomto cv. TSH4 nd H9909 treted with uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Further detils on pplictions methods re described in the text x

11 Tble 3.3 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted Tble 3.4 Totl, red, green, nd rotten fruit yield in 2m section of tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Tble 3.5 Soluble solids, Agtron colour redings, nd juice ph of ripe tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws processed Tble 3.6 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted Tble 3.7 Stem dimeter nd folir nd root dry weight of tomto seedlings, cvs. TSH4 nd H9909, treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse, Ridgetown, ON, Tble 3.8 Soluble solids, Agtron colour redings, nd juice ph of ripe tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws processed Tble 4.1 Source nd colony description of endophytes isolted from roots of S. lycopersici, S. rcnum, S. chmielewskii, S. cheesmnie, nd N. benthmin fter growth on tryptic soy gr t room temperture (~22 C) for two dys. Endophytes were isolted from mcerted roots of plnts growing outdoors in the field t Ridgetown Cmpus, in medi contining 40 to 50% psteurized field soil mixed with snd under controlled conditions (Ridgetown R isoltes), or in 1:1 mixture of psteurized soil from the Guelph Turfgrss Institute (Guelph, ON) nd potting mix (Guelph G isoltes) Tble 4.2 Disese incidence of bcteril speck on non-fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes listed in Tble 4.1, Serende Mx (B. subtilis QST713) or Promix PGX (B. subtilis MBI600). Endophytes were inoculted following the Vlenzuel-Soto et l. (2010) method using solution of ech endophyte with A600=1.000 then diluted 1:10 in 10 mm MgCl 2, except for MBI600 which ws included in the potting mix (1 x 10 7 CFU/mL) nd QST713 which ws pplied t 1 x 10 6 CFU /ml. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto t 20 dys fter seeding, nd disese incidence ssessed t seven dys post inocultion. Disese ws ssessed on ll leflets of ech plnt Tble 4.3 Growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of nonfertilized tomto cv. TSH4 following inocultion with the endophytes listed in Tble 4.1, Serende Mx (B. subtilis QST713) or Promix PGX (B. subtilis MBI600). Endophytes were inoculted following the Vlenzuel-Soto et l. (2010) method using solution of ech endophyte with A600=1.000 tht ws diluted 1:10 in10 mm MgCl 2, except for MBI600 which ws included in the potting mix (1 x 10 7 CFU/mL) nd QST713 which ws pplied t 1 x 10 6 CFU/mL. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were ssessed t 27 dys fter seeding on the sme dy s ssessment of disese incidence Tble 4.4 Disese incidence of bcteril speck on non-fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes R9, R17, R19, R20 nd R21. Endophytes were inoculted using the seed xi

12 sok + seed drench + seedling drench inocultion method (A600=1.000 then diluted 1:10 in10 mm MgCl 2 ). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with Pst t 20 dys fter seeding, nd disese incidence ssessed t seven dys post inocultion on ll leflets of ech plnt Tble 4.5 Growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of nonfertilized tomto cv. TSH4 following inocultion with the endophytes R9, R17, R19, R20 nd R21. Endophytes were inoculted using the seed sok + seed drench + seedling drench inocultion method (A600=1.000 diluted by fctor of 10 in10 mm MgCl 2 ). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were ssessed t 27 dys fter seeding on the sme dy s ssessment of disese incidence Tble 4.6 In vitro inhibition of P. syringe pv. tomto (Pst) strin DC06T2-4 by co-incubtion with endophytes R17 nd R21 using cross streking ssy. Ech endophyte ws streked in single line cross the centre of plte of tryptic soy gr nd incubted for two dys. Pst ws then streked in stright line from the edge of the plte to the endophyte strek t 90 ngle. Pltes were ssessed two dys fter streking Pst Tble 4.7 In vitro inhibition of P. syringe pv. tomto (Pst) strin DC06T2-4 by co-incubtion with endophytes R17 nd R21 using n gr overly ssy. The overly ws tryptic soy gr (TSA) (1.50% gr) (50 C tht ws spiked with 0.1 ml of 2 x 10 7 CFU/mL Pst. This ws poured over 2-cm dimeter circle of endophyte tht hd grown for 48 hours on hrd TSA gr bse (1.50% gr), incubted for 48 hours, nd ssessed for the presence of zone of inhibition Tble 4.8 Disese incidence of bcteril speck nd growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes R17 nd R21. Plnts were fertilized 10, 15 nd 21 dys fter seeding (DAS) with 20 (three experiments) or 80 ml (one experiment) of 1.26 g/l micronutrients fertilizer solution. Endophytes were inoculted using the seed sok + seed drench + seedling drench inocultion method t concentrtions of 1 x 10 7 CFU/ml (R17) or 1 x 10 8 CFU/ml (R21). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS. The incidence of bcteril speck lesions on ll leflets on the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst Tble 4.9 The effect of seed sok, seed drench nd/or seedling drench ppliction methods of R17 nd R21 on disese incidence of bcteril speck nd growth prmeters of fertilized tomto cv. TSH4. Plnts were inoculted with the seed sok, seed drench, seedling drench, seed sok + seed drench or seed sok + seed drench + seedling drench methods using 1 x 10 7 CFU/ml (R17) or 1 x 10 8 CFU/ml (R21) in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15 nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS. The incidence of bcteril speck lesions on ll leflets of the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst Tble 4.10 The effect of rw nd pelleted seed with seed sok nd/or seed drench ppliction methods of R17 tretment on disese incidence of bcteril speck nd growth prmeters of fertilized tomto cv. TSH4. Plnts were inoculted using the seed sok, seed drench, or seed sok + seed drench method using 1 x 10 7 CFU/ml in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. Plnts were inoculted with P. syringe pv. tomto (Pst) t 20 DAS. The incidence of bcteril speck lesions on ll leflets of the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst xii

13 Tble 4.11 Effect of different doses of R17 s seed drench tretment on the incidence of bcteril speck disese incidence nd growth prmeters (reltive chlorophyll, plnt height, folir dry weight, nd root dry weight) of fertilized tomto cv. TSH4. R17 ws pplied s seed drench t zero dys fter seeding (DAS) t 0, 1x10 5, 1x10 6, 1x10 7, 1x10 8 or 1x10 9 cfu/ml in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15 nd 21 DAS with 80 ml of micronutrients fertilizer solution mixed t concentrtion of 1.26 g/l. Plnts were inoculted with P. syringe pv. tomto t 20 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves Tble 4.12 The effect of R17 seed drench tretment on the incidence of bcteril speck lesions, P. syringe pv. tomto (Pst) popultion (LOG CFU) per cm 2 nd Pst popultion (LOG CFU) per lesion on terminl leflets of the third youngest leves of fertilized tomto cv. TSH4. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of micronutrients fertilizer solution mixed t concentrtion of 1.26 g/l. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Plnts were inoculted with Pst 20 DAS, nd disese incidence ssessed t five dys post inocultion. Plnts receiving 10 mm MgCl 2 were used s control Tble 4.13 Comprison of R17 seed drench tretments with wt nd rifmpicin-resistnt strins on disese incidence of bcteril speck nd growth prmeters of fertilzed tomto cv. TSH4. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench t 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS, nd disese incidence ssessed t five dys post inocultion (DPI) on ll leflets of the second nd third youngest leves. The reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were lso determined t five DPI Tble 4.14 Popultions of rifmpicin resistnt R17 mutnt R17-RfpC in leves, roots nd rhizosphere of tomto cv. TSH4. Plnts were fertilized t 10, 15 nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17-RfpC ws pplied s seed drench t 0 DAS t 1 x 107 CFU/mL in10 mm MgCl2. Plnts receiving 10 mm MgCl2 were used s control. R17-RfpC popultions in rhizosphere growing medi, roots nd cotyledon leves t 10 DAS or the rhizosphere growing medi, roots nd terminl leflet on the second nd third youngest leves t 25 DAS. Smples were from 10 (10 DAS) or five (25 DAS) plnts per replicte Tble A.1 Effect of different pr-minobenzoic cid (PABA) concentrtions nd ppliction volumes on the reltive chlorophyll content in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). PABA ws pplied to the root zone with pipette 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Reltive chlorophyll ws mesured t five dys post inocultion Tble A.2 Effect of different PABA concentrtions on the reltive chlorophyll content in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). Plnts were coted with 0, 0.01, 0.1, 0.5, 1, 4, 9, or 18 mm pr-minobenzoic cid (PABA) 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Reltive chlorophyll ws mesured t five dys post inocultion Tble A.3 The incidence of bcteril speck nd bcteril spot on green tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 green fruit hrvested in 2m section of ech plot ws evluted Tble A.4 Incidence nd severity of nthrcnose symptoms on red tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 xiii

14 red fruit ws collected from ll red fruit hrvested in 2m section of ech plot, stored for three dys t room temperture, nd then ssessed for nthrcnose symptoms Tble A.5 Number of dys fter trnsplnting to begin inflorescence, fruit set, nd ripening for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Five plnts per plot were monitored t seven to 12 dy intervls fter trnsplnting Tble A.6 The incidence of bcteril speck nd bcteril spot on green tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 green fruit hrvested in 2m section of ech plot ws evluted Tble A.7 Incidence nd severity of nthrcnose symptoms on red tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit ws collected from ll fruit hrvested in 2m section of ech plot, stored for three dys t room temperture, nd then ssessed for nthrcnose symptoms Tble A.8 Number of dys fter trnsplnting to begin inflorescence, fruit set, nd ripening for tomto cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Five plnts per plot were monitored t seven to 12 dy intervls fter trnsplnting Tble A.9 Response of commercil processing tomto cvs. H2401 (ll vribles) nd H9553 (reltive chlorophyll) for the effect of endophyte R17 tretment on the incidence of bcteril speck lesions, reltive chlorophyll, plnt height, dry root weight, nd folir dry weight. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of micronutrients fertilizer solution mixed t concentrtion of 1.26 g/l. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto t 20 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves xiv

15 6 LIST OF FIGURES Figure 1.1. Symptoms of bcteril speck on ) tomto folige, b) ripe fruit, nd c) peeled processing tomto, nd bcteril spot on d) tomto folige, b) ripe fruit, nd c) peeled processing tomto Figure 2.1 Effect of different pr-minobenzoic cid (PABA) concentrtions nd ppliction volumes on the systemic cquired resistnce response in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). A pipette ws used to pply 10 ( ), 20 ( ), or 40 ( ) ml PABA to the root zone 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Disese incidence five dys post inocultion is shown. Dt points with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction Figure 2.2 Phytotoxicity symptoms on tomto ) true lef, nd b) nd c) cotyledon lefs coted to runoff with 18 mm PABA t 10 nd 15 dys fter seeding Figure 2.3 Effect of different pr-minobenzoic cid (PABA) concentrtions on the systemic cquired resistnce response in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). Plnts were coted with 0, 0.01, 0.1, 0.5, 1, 4, 9, or 18 mm PABA (LOG + 1 = 1, , , , , , 1.000, mm PABA) 10 nd 15 dys fter seeding (DAS), nd then plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Disese incidence five dys post inocultion is shown. Errors brs represent stndrd error of the men. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction Figure 2.4 Incidence of bcteril speck symptoms on the second nd third youngest leves of six tomto cultivrs treted with pr-minobenzoic cid (PABA). Plnts were coted with 18 mm PABA 10 nd 15 DAS. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto (Pst) 20 dys fter seeding (DAS). Disese incidence five dys post inocultion is shown for the nontreted control ( ) nd PABA ( ). Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for cvs. H2401, H9553, TSH33, nd TSH4 with five replictions of ech tretment, nd five independent trils for cv. H5108 nd cv. H9909 with five or four replictions of ech tretment, ws pooled together becuse ANOVA showed no tretment x tril interction. One outlier ws removed ech dt set for cv. H9909 nd cv. H Figure 2.5 Incidence of bcteril speck symptoms on the second nd third youngest leves of tomto cultivrs ) cv. H5108, nd b) cv. TSH33 treted with zero, one (15 dys fter seeding (DAS)), two (10 nd 15 DAS), or three (10, 12, nd 15 DAS) pplictions of pr-minobenzoic cid (PABA). Folir PABA tretments (18mM) were pplied by coting fine mist on plnts. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Disese incidence five dys post inocultion is shown. Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Dt for cv. H5108 ws log trnsformed to meet ssumptions of ANOVA Figure 2.6 Incidence of bcteril speck symptoms on the second nd third youngest leves of tomto cv. H5108 inoculted with P. syringe pv. tomto (Pst) five, seven, or 10 dys fter pr-minobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five, seven, or 10 dys fter the second PABA ppliction. Disese incidence five dys post inocultion is shown for the nontreted control ( ) nd PABA ( ). Errors brs represent stndrd error xv

16 of the men. Dt points with the sme letter for the sme inocultion timing re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. One outlier ws removed from the nlysis for the dt from seven dys fter the lst PABA ppliction Figure 2.7 The effect folir pplictions of pr-minobenzoic cid (PABA) on ) the incidence of bcteril speck symptoms, b) men lesion size, c) men lesion circumference, nd d) percent lef re with lesions on the third youngest terminl leflet of tomto cv. H5108. Folir PABA tretments (18mM) were pplied 10 nd 15 dys fter seeding (DAS) by coting fine mist on plnts. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Disese incidence nd men lesion size of 10 to 15 lesions is shown for the nontreted control ( ) nd PABA ( ). Error brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with 10 (tril 1) nd seven (tril 2) replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction Figure 2.8 Erly seson disese progress of bcteril speck symptoms in tomto cv. H5108 treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON. The percentge of leves with disese symptoms in the nontreted control ( ), eight pplictions of CuOH t 7-dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5-dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Tble 2.6. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 2.9 Lte seson disese progress of bcteril speck symptoms in tomto cv. H5108 treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON. Defolition in the nontreted control ( ), eight pplictions of CuOH t 7-dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5-dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in whole plots is shown. The corresponding re under the disese progress curve is shown in Tble 2.6. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 2.10 Reltive chlorophyll mesured 24, 30, nd 36 dys fter trnsplnting in tomto cv. H5108 treted with CuOH nd pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto in Ridgetown, ON, SPAD redings in the nontreted control ( ), eight pplictions of CuOH t 7- dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5- dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in whole plots is shown. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.1 Dily mximum ( ) nd minimum ( ) tempertures, nd totl dily rinfll ( ) for ) 2011, b) 2012, c) 2013, nd d) 10-yer verge ( ) t Ridgetown Cmpus, University of Guelph Figure 3.2 Men monthly ) mximum temperture, b) minimum temperture, nd c) monthly rinfll in 2011 ( ), 2012 ( ), 2013 ( ) nd 10-yer verge ( ) ( ) t the Ridgetown Cmpus, University of Guelph Figure 3.3 Erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. xvi

17 syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011, b) 2012, c) The number of leves with disese symptoms in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.2. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.4 Are under the disese progress curve (AUDPC) for erly seson disese. Erly seson disese ws mesured by the number of leves with disese symptoms (shown in fig. 2) for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (22 June-27 July), b) 2012 (7 June-4 July), c) 2013 (11 June-29 June). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD Figure 3.5 Lte seson progress of bcteril spot nd speck on tomto cv. TSH4 treted with uniconzole (UNI_ in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011, b) 2012, c) Defolition in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.4. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.6 Are under the disese progress curve (AUDPC) for lte seson disese. Lte seson disese ws mesured by the percent defolition (shown in fig. 4) for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (27 July-23 Aug), b) 2012 (4 July-27 Aug), c) 2013 (29 June-19 Aug). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD Figure 3.7 SPAD chlorophyll redings mesured 18, 36, nd 56 dys fter trnsplnting in tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments ) 2011, b) 2012, c) Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.8 Erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011-TSH4, b) TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. The number of leves with disese symptoms in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.9. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. An * indictes differences mong tretments which re discussed in the text Figure 3.9 Are under the disese progress curve (AUDPC) for erly seson disese. Erly seson disese ws mesured by the number of leves with disese symptoms (Figure 3.8) for tomto cv. TSH4 (blck brs) nd cv. H9909 (grey brs) treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (22 June-27 July), b) 2012 (8 June-5 July), c) 2013 (22 June-8 July). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD Figure 3.10 Lte seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd xvii

18 inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011-TSH4, b) TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Defolition in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments in whole plots is shown. The corresponding re under the disese progress curve is shown in Figure Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.11 Are under the disese progress curve (AUDPC) for lte seson disese. Lte seson disese ws mesured by the percent defolition (shown in fig. 8) for tomto cv. TSH4 (blck brs) nd cv. H9909 (grey brs) treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (27 July-29 Aug), b) 2012 (5 July-8 Aug), c) 2013 (1 July-23 Aug). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD Figure 3.12 Height of tomto seedlings cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse, Ridgetown, ON, in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.13 SPAD chlorophyll redings mesured 18, 37, nd 56 dys fter trnsplnting in tomto cv. TSH4 nd cv. H9909 treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) H9909. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure 3.14 Totl ( ), red ( ), green ( ), nd rotten ( ) fruit yield in 2m section of tomto cv. TSH4 nd cv. H9909 treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD Figure 4.1 Comprison of four commercil processing tomto cultivrs for the effect of R17 seed drench tretment on the ) incidence of bcteril speck lesions, b) reltive chlorophyll, c) plnt height, d) dry root weight, nd e) folir dry weight. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench t 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with P. syringe pv. tomto t 20 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves. Dt for ech vrible is shown for the nontreted control ( ) nd R17 ( ). Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with six replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Results for cv. H2401 (most vribles) nd cv. H9553 (reltive chlorophyll) were inconsistent nd re presented in Tble A Figure 4.2 The durtion of the plnt response to R17 ( ) or10 mm MgCl 2 ( ) seed drench tretment s mesured by the ) incidence of bcteril speck lesions, b) reltive chlorophyll, c) plnt height, d) dry root weight, nd e) folir dry weight in fertilized tomto cv. TSH4. Plnts were fertilized 10, 15, 21 nd 26 DAS (plnts inoculted with P. syringe pv. tomto t 25 dys fter seeding (DAS) only) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 xviii

19 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with Pst 15, 20 or 25 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves. Dt points with the sme letter t the sme time point re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with six replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Error brs represent stndrd error of the men Figure 4.3 The effect of R17 seed drench tretment on ) the incidence of bcteril speck symptoms, b) men lesion size, c) men lesion circumference, nd d) percent lef re with lesions on the terminl leflet of the third youngest lef of fertilized tomto cv. TSH4. Disese incidence nd men lesion size of 10 to 15 lesions is shown for the nontreted control ( ) nd R17 ( ). Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with P. syringe pv. tomto 20 DAS, nd disese incidence ssessed t five dys post inocultion. Errors brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with seven (experiment 1) nd six (experiment 2) replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction Figure 4.4 Neighbour joining tree of prtil 16S sequences (1160 bp) from representtive members of the E. cloce complex, other Enterobcter sp., closely relted Enterobctere, nd R Figure 4.5 Neighbour joining tree of prtil rpob sequences (934 bp) from representtive members of the E. cloce complex, other Enterobcter sp., closely relted Enterobctere, nd R Figure 4.6 Neighbour joining tree of prtil 16S sequences (1234 bp) from representtive members of the B. cereus group, other Bcillus sp., nd R Figure 4.7 Neighbour joining tree of prtil gyrb sequences (696 bp) from representtive members of the B. cereus group, other Bcillus sp., nd R Figure A.1. Lesion size ws determined by ) tking photo (12.2 MB, 4288 x 2824 pixels) of ech leflet ws tken using Nikon D300s cmer with ruler for reference, b) uploding, enlrging nd printing ech photo in colour, c) trcing lesion circumference using fine point mrker on cette, nd d) scnning lesion circumference imges nd nlyzing using Imge J Figure A.2 Lef counts nd erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in The ) totl number of symptomless nd symptomtic leves, nd b) % of leves with symptoms in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) is shown for 1.24 m 2 re. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure A.3 Lef counts nd erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 nd H9909 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in Figures represent ) the totl number of symptomless nd symptomtic leves in cv. TSH4, b) the % of leves with symptoms in cv. TSH4, c) the totl number of symptomless nd symptomtic leves in cv. H9909, nd d) the % of leves with symptoms in cv. H9909, in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments is shown for 1.24 m 2 re. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference Figure A.4 Stndrd dilution curves of ) R17 nd b) R21 endophytes of A600 versus CFU/ml. A xix

20 stndrd growth curve ws developed for R17 nd R21 by plting dilutions of the bcteri grown overnight in tryptic soy broth nd then diluted in sterile distilled wter to 0.2, 0.4, 0.6, 0.8, nd 1.0 bsorbnce (OD = 600). Seril dilutions t ech bsorbnce level were plted onto tryptic soy gr nd counted t 48 hours fter incubtion t pproximtely 22 C. From this, formul ws developed for R17: popultion (CFU/mL) = [(9x10 7 ) (bsorbnce)] - 1x10 7 (R 2 = 0.93) nd for R21: popultion (CFU/mL) = [(2x10 9 ) (bsorbnce)] - 2x10 8 (R 2 = 0.89). Vlues t 1.0 bsorbnce for R21 were omitted becuse using the vlues from to provided curve with higher R 2 vlue. Second vlues for R21 t 0.4 bsorbnce re missing due to missing plots becuse of plte contmintion xx

21 LIST OF ABBREVIATIONS 4-AA ABA ACC AHO AM AON AUDPC ASM BABA BIT BSX CFU CMPA CMV DAPG DAS DAT DPI DPT EBDC ECM efl18 EIN3-like ET 4-minobenzoic cid bscisic cid 1-minocyclopropne-1-crboxylte 3-cetyl-3-hydroxyindole Vesiculr rbusculr mycorrhiz Autoregultion of nodultion Are under the disese progress curve Acibenzolr-s-methyl β-minobutyric cid 1, 2-benzisothizol-3 (2H)-one 1,1-dioxide Bcteril spot cusing Xnthomonds Colony forming units 3-chloro-1-methyl-1Hpyrzole-5-crboxylic cid Cucumber Mosic Virus 2,4-dicetyphloroglucinol Dys fter seeding Dys fter trnsplnting Dys post inocultion Dys post tretment ethylenebis(dithiocrbmte) Ectomycorrhiz fungi Trnsltionl elongtion fctor-tu ET-insensitive 3-like Ethylene xxi

22 ETI ETS flg22 GA HR hrc hrp IAA ICS INA IPM ISR JA JA-Ile JAZ LPS MAMP MeS MTI NPR1 PABA PAL PAMP PGPB PGPF Effector-triggered immunity Effector triggered susceptibility Bcteril flgellin Gibberellins Hypersensitive response HR nd conserved genes HR nd pthogenicity genes Indole cetic cid Isochorismte synthse 2,6-dichloro isonicotinic cid Integrted pest mngement Induced systemic resistnce Jsmonic cid JA-isoleucine jsmonte ZIM-domin Lipopolyscchrides Microbe ssocited moleculr pttern Methyl slicylte Microbe ssocited moleculr pttern triggered immunity Nonexpressor of PR genes1 Pr-minobenzoic cid Phenyllnine mmoni lyse Pthogen ssocited moleculr pttern Plnt growth promoting rhizobcteri Plnt growth promoting fungi xxii

23 PGR PTI PR PRR PR1 PR1b Pst ROS SA SAR SGT1 SIR T3SS TMV TSA UNI VBTr wt Xcv Xe Xv Xg Xp Plnt growth regultor PAMP-triggered immunity Pthogenesis-relted Pttern recognition receptor Acidic PR1 Bsic PR1 Pseudomons syringe pv. tomto Rective oxygen species Slicylic cid Systemic cquired resistnce Suppressor of G 2 llele of skp1 Systemic induced resistnce Type III secretion system Tobcco mosic virus tryptic soy gr Uniconzole Vogel Bonner-trtrte medi Wild type Xnthomons cmpestris pv. vesictori Xnthomons euvesictori Xnthomons vesictori Xnthomons grdneri Xnthomons perforns xxiii

24 1 Chpter 1: Literture Review 1.1 Introduction The use of biologicl nd chemicl plnt defense ctivtors offer novel pproches for disese mngement, but their use hs been limited becuse of inconsistent results nd yield reductions ssocited with plnt growth fitness costs due to the relloction of plnt metbolites to defense compound production from norml growth nd development (Byrne et l., 2005, Heil, 2001, Heil et l., 2000, Wlters & Heil, 2007, Louws et l., 2001). Further testing nd optimiztion of novel defense ctivtors s well s better understnding of the fctors tht my improve plnt response to existing ctivtors could led to strtegies involving defense ctivtors tht provide consistent results with cceptble levels of resistnce induction nd limited fitness costs. Fctors include selection of highly responsive genotypes, ctivtor ppliction method nd concentrtion, the durtion of host response to ctivtors, nd synergies mong ctivtors nd PGRs ssocited with stress tolernce. This pproch could be pplied to reduce losses in processing tomto (Solnum lycopersicum L. (Lycopersicon esculentum)) from plnt diseses such s bcteril speck cused by Pst (Okbe) Young, Dye, & Wilkie nd bcteril spot cused by one of four species of bcteril spot-cusing Xnthomonds (BSX). 1.2 Processing tomtoes in Ontrio Tomtoes re the second most populr vegetble in the world, nd the most populr vegetble for home grdeners (Foold & Pnthee, 2012). Processing tomtoes re used to produce vriety of processed goods including crushed, diced, nd whole pck tomtoes, pstes used in ketchup nd other suces, nd tomto juice. Globlly, verge nnul production of processing tomtoes verged over 39 million metric tonnes per yer from 2014 to 2016, 1.0 % of which ws grown in Ontrio (WPTC, 2017). The frm gte vlue of the Ontrio processing tomto production from 2014 to 2016 ws pproximtely $55 million (OPVG, 2017b). The industry is primrily locted in Essex nd Kent counties in southwestern Ontrio, nd is economiclly importnt in those res. Processing tomto plnts, unlike greenhouse nd some fresh mrket cultivrs, hve determinnt growth hbit. In Ontrio, processing tomtoes re grown from pproximtely 6-week old trnsplnts tht re plnted in twin-row system. Rinfll is often supplemented with overhed or drip irrigtion (OMAFRA, 2010). It is lso common for growers to pply Ethrel (ethepon) to trigger fruit ripening of mture green fruit ner hrvest nd mximize the number of red fruit hrvested (OMAFRA, 2010). Importnt fetures of processing tomto fruit qulity include colour, low ph, nd high soluble solids. Low fruit ph is necessry to prevent spoilge, nd rnge of 4.1 to 4.3 is recommended (Gould, 1992). 1

25 Higher levels of soluble solids in the fruit re desirble for production of tomto pste (S. Loewen, University of Guelph, pers. comm.). Growers delivering tomtoes with poor colour rting, s defined by n Agtron colour rting greter thn 37 units, receive lower price for their tomtoes nd risk hving lods rejected by processors. Abiotic stresses cn gretly dmge tomtoes. Tempertures less thn 10 C cn hve detrimentl effects on tomto growth nd development becuse of shrp reduction in cellulr ctivities (Breidenbch & Wring, 1977, Lyons, 1973). Tomtoes re sometimes subject to these tempertures in Ontrio in My during the weeks immeditely fter trnsplnting. Tomto seedlings exposed to chilling t 5 C for three dys exhibited loss of cell turgor, vcuoliztion, reduction in cytoplsm nd vcuolr protein bodies, disorgniztion of plstids, loss of cytoplsmic structure, nd cell deth (Ilker et l., 1976). Other biotic stresses often found for field tomtoes in Ontrio re het stress tht cn result in blossom drop when tempertures exceed 30 C dytime or 21 C nighttime, puffiness, nd sunscld, drought tht cn result in poor growth nd development nd excerbte blossom end rot, nd excess wter tht cuses wter wilt s lck of oxygen limits respirtion nd lters phytohormone concentrtions (Scott, 2014). A tomto crop cn often be exposed to severl of these in the sme growing seson. Growth nd development outcomes for tomto exposed to multiple biotic stresses cn be positive, negtive or neutrl, but prolonged exposure to biotic stresses most often results in greter susceptibility to biotic stresses, like plnt diseses (Suzuki et l., 2014). Diseses re mjor source of biotic stresses for Ontrio tomtoes. The mjor field tomto bcteril diseses in Ontrio re bcteril spot nd bcteril speck, which re sometimes found together nd re difficult to distinguish bsed on symptoms lone (Pitbldo & Trtier, 1994, Trtier & Pitbldo, 1994, Miller & Jones, 2014, Jones, 1991b). Bcteril cnker is lso present but is now rre in its dmging systemic form of infection (Jones et l., 1991, OMAFRA, 2010, LeBoeuf et l., 2009). Most of the other diseses in field tomtoes in Ontrio cusing economic losses re cused by fungi, including Verticillium wilt (Verticillium dhlie; Verticillium lbo-trum), erly blight (Alternri solni), Septori lef spot (Septori lycopersici) nd nthrcnose (Colletotrichum coccodes) (Jones et l., 1991, OMAFRA, 2010). The fungl diseses gry mold (Botrytis cinere) nd white mold (Sclerotini minor, Sclerotini sclerotiorum) re occsionlly seen in Ontrio, but re not usully economiclly importnt (Jones et l., 1991, OMAFRA, 2010). Diseses of significnce cused by Oomycetes in Ontrio include lte blight (Phytophthor infestns) nd Phytopthor root rot (Phytophthor cpsici). For virl diseses, Cucumber Mosic Virus (CMV), tomto mosic virus nd tomto spotted wilt virus re ll known in Ontrio, lthough sightings re spordic nd rrely result in economic losses (OMAFRA, 2010). 2

26 1.3 Tomto genetics Tomto (S. lycopersicum) is member of the Solncee, which lso includes griculturlly significnt crops such s potto (S. tuberosum), pepper (Cpsicum nnuum), eggplnt (S. melongen), tobcco (Nicotin tbcum), petuni (Petuni hybrid), nd number of weed species including estern blck nightshde (S. ptycnthum) (Knpp et l., 2004). The cultivted tomto originted from Centrl nd South Americ but the time of domestiction is unknown (Bi & Lindhout, 2007). Wild tomtoes, such s S. chilense nd S. peruvinum hve greter genetic diversity thn S. lycopersium, but only S. lycopersium hs been domesticted (Bi & Lindhout, 2007). Open-pollinted cultivrs once dominted the processing tomto mrket, but incresingly hybrid cultivr seed, which is produced using hnd pollintion, is used to produce processing tomtoes becuse of perceived benefits with tomto qulity nd yield (Bi & Lindhout, 2007). Tomto hs set of 12 chromosomes nd is diploid, which is similr to the mjority of other Solncee species (Olmsted et l., 1999). An interntionl consortium of reserchers strted sequencing the entire tomto genome in 2004 (Mueller et l., 2009) nd it ws publiclly relesed in 2012 (Consortium, 2012). The tomto genome is predicted to contin more thn 30,000 genes (Consortium, 2012). 1.4 Bcteril endophytes of tomto Bcteril endophytes re culturble or non- culturble orgnisms tht re detected in plnt tissues following surfce steriliztion nd hve no dverse effects on the host (Hllmnn et l., 1997, Bulgrelli et l., 2013, Giero et l., 2013). The soil environment is considered the most importnt source of plnt endophytes, but other sources include seeds, propgtive mteril, nd the phylloplne (Hllmnn et l., 1997, Hrdoim et l., 2008, Rosenblueth & Mrtinez-Romero, 2006). Bcteril endophytes re diverse with members of Actinobcteri, Bcteroidetes, Firmicutes nd lph, bet nd gmm Proteobcteri. However, bcteril endophyte communities re typiclly less diverse thn the rhizosphere communities (Berg et l., 2005, Bulgrelli et l., 2013, Gottel et l., 2011, Mrquez-Sntcruz et l., 2010). Higher diversity of endophytes in roots, shoots, nd seeds were found in corn, tomto, melon nd pepper grown in orgniclly cultivted soil compred to conventionlly mnged soil (Xi et l., 2015), possibly becuse of differences in the soil environment, such s higher levels of soil crbon nd nitrogen, nd different C:N, which cn ffect bcteril diversity nd bundnce (Trivedi et l., 2016). For tomto, n exmintion of the diversity of culturble endophytes found 32 endophyte species in five phyl mong 336 bcteril isoltes from roots, shoots nd seeds of n unspecified tomto cultivr (Xi et l., 2015), nd 27 endophyte species mong 34 bcteril isoltes from roots of cvs. Ark Abh nd Ark Viks (Upreti & Thoms, 2015). Xi et l. (2015) found tht the predominnt species were members 3

27 of the Firmicutes, while Upreti & Thoms (2015) found gmm-proteobcteri were the most bundnt. Using non-culturing dependent methods of PCR-restriction frgment length polymorphism nd 16S rdna sequencing, 41 isoltes in seven genetic groups were identified from tomto cv. Xihong-1 roots nd stems nd 38 isoltes in four genetic groups were identified in cv. Boshi-5 roots nd stems (Feng et l., 2013). The sequences mtched those of Sphingomons ynoikuy, Pseudomons pseudolcligenes, Serrti mrcescens, Bcillus megterium, Penibcillus polymyx, B. pumilus, B. cereus, Pseudomons fluorescens nd Arthrobcter globiformis in Xinhong-1 nd S. ynoikuye, P. fluorescens, Arthrobcter globiformis nd P. polymyx in Boshi-5. Furthermore, 80 opertionl txonomic units from tomto cv. Pltense leves were found using mss sequencing of 16S-ribosoml RNAs (Romero et l., 2014). Most were members of the Proteobcteri (>94%) with most of the rest belonging to the Actinobcteri, Plnctomycetes, Verrucomicrobi nd Acidobcteri. In contrst, more thn 1700 opertionl txonomic units from the tomto cv. Pltense rhizosphere soil were identified with members of twelve different txonomic groups being most common, mong which the Acidobcteri, Verrucomicrobi, Proteobcteri nd Gemmtimondetes comprised 22, 24, 17 nd 16% of the popultion, respectively. Romero et l. (2014) concluded tht the diversity of lef endophytes in tomto is less thn the rhizosphere nd dominted by Proteobcteri. Thus, similr to other crops, ll tissues of tomto pper to contin both culturble nd non-culturble bcteril endophytes, which hve considerble diversity but less thn tht found in the environment round tomto plnts. 1.5 Bcteril speck pthogen of tomto Bcteril speck is cused by the hemibiotrophic bcterium Pst (Preston, 2000, Young et l., 1978). Pst ws formerly clssified s Pseudomons tomto (Okbe) prior to being renmed Pst (Hirno & Upper, 1990, Young et l., 1978). Pst is grm-negtive, rod-shped bcterium clssified in phylum Proteobcteri, clss Gmmproteobcteri, nd fmily Pseudomondcee (Kdo, 2010). Pst is only reported to be of economic importnce in tomto, lthough it hs been isolted from numerous other crop nd weed species s n epiphyte (Preston, 2000). Pst strin DC3000 is commonly used in moleculr reserch studies s it lso infects the model plnt Arbidopsis thlin (Cuppels & Ainsworth, 1995, Preston, 2000), nd ws originlly isolted from the Chnnel Islnds, Guernsey, United Kingdom, in 1960 (Cuppels & Elmhirst, 1999). Its complete genome sequence ws published in 2003 (Buell et l., 2003). Pst cn spred in the environment through the movement of wter, especilly during rin or overhed irrigtion events tht llow for splsh dispersl onto leves within the crop cnopy, nd infections cn be excerbted by hevy winds tht my force the bcteri through nturl openings like stomt, or crete wounds tht enble pthogen entry (Jones, 1991, Pitbldo & Trtier, 1994). There is potentil tht insects might lso ct s vector of the pthogen; however, there re no reports of nonpersistent, 4

28 semipersistent, or persistent trnsmission of Pst by insects in the literture. Once rriving on lef, Pst is similr to other P. syringe pthovrs, in tht it hs two relted phses. The first phse is epiphytic, on the surfce of the lef, nd the second stge is endophytic nd occurs in the lef poplst (Melotto et l., 2008). Bcteri such s Pst cn survive on the lef surfce under rnge of high UV, moisture, nd temperture conditions (Hirno & Upper, 2000). The lef surfce contins number of peks, vlleys, nd cvities tht enble survivl, such s the re in nd round the trichomes nd epistomtl cvities (Hirno & Upper, 2000, Schneider & Grogn, 1977b). Furthermore, Pst possesses chrcteristics tht enble epiphytic coloniztion. For exmple, DC3000 mutnts deficient in type IV pili hd lower popultions on the lef surfce thn the wild-type DC3000 under field conditions, possibly becuse type IV pili re responsible for ggregrtion of bcteri tht my enhnce UV tolernce, (Roine et l., 1998). The reltive importnce of the epiphytic phse of Pst on bcteril speck epidemics under field conditions is not entirely cler. In study in Ontrio greenhouses nd fields in the erly 1990s, Pst popultions on helthy trnsplnts did not correlte with disese severity in the field, even though epiphytic popultions of Pst were detected on trnsplnts from ll three greenhouses included in the study (Cuppels & Elmhirst, 1999). The difference in severity mong fields ws lrgely ttributed to environmentl vribles such s the number of intense rins tht occurred during the field seson, which would hve creted wounding sites tht would esily fcilitte pthogen entry (Cuppels & Elmhirst, 1999). Pst shifts from n epiphytic to n endophytic phse when it enters the poplst through stomt nd other nturl openings or wounds (Preston, 2000). Environmentl conditions tht fvour invsion nd infection of Pst include cooler tempertures (18 to 24 C) (Jones, 1991, Smitley & McCrter, 1982) nd extended periods of lef wetness (Preston, 2000). Lef morphology is thought to ply limited role in the bility of Pst to successfully invde plnt tissues. In study of the number of stomt, trichomes, nd other nturl openings on 15 tomto cultivrs with rnge of susceptibility to Pst, positive correltion ws observed between n incresed number of openings nd in increse in disese susceptibility; however it ws felt tht even in very tolernt cultivrs, the number of nturl openings would still llow for infection to occur (Bshn et l., 1985). Previously, it ws believed tht bcteril pthogens such s Pst entered plnts through purely pssive mens; however, recent evidence indictes tht Pst my be ble to ctively induce the opening of stomt. Stomtl closure within one hour of exposure of leves to bcteri is due to recognition of pthogen ssocited moleculr ptterns (PAMPs), lso known s microbe ssocited moleculr ptterns in nonpthogenic bcteri (Melotto et l., 2008). These hve been observed s resistnce mechnism ginst plnt pthogenic bcteri in A. thlin nd tomto. Melotto et l. (2006) reported tht exposure 5

29 to live Pst or the purified PAMPs lipopolyscchrides (LPS) nd flgellin triggered stomtl closure in A. thlin within one hour, but the phytotoxin corontine nd the type III secretion system (T3SS) effector vrrpt2 suppressed stomtl closure nd resulted in re-opening of stomt. Inocultion with wild type (wt) DC3000, corontine mutnt DC3000 mutnt, nd the non-pthogenic humn pthogen Escherichi coli O157:H7 lso resulted in stomtl closure, but in the cse of the wt DC3000, the stomt reopened lter. This effect ws lso explored in tomto, where similr to A. thlin, LPS-induced stomtl closure ws overcome by Pst DC3000 (Melotto et l., 2006). Thus, corontine ppers to be n importnt molecule tht helps suppress the PAMP-triggered immunity (PTI) responsible for stomtl closure in tomto. Corontine production by Pst field strins is common, s ll 244 Pst isoltes collected in survey of Ontrio tomto fields in 1989 were ble to produce corontine (Cuppels et l., 1990). An dditionl 6- yer survey completed from 1997 to 2002 found tht 68 of 70 isoltes collected rected positively with the COR1/2 primer, which ws derived from the bcteril speck probe TPRI. The TPRI probe is derived from gene cluster in Pst DC3000 tht is responsible for the production of corontine, indicting tht the vst mjority of bcteril speck symptoms occurring in Ontrio re ssocited with corontine producing strins of Pst (Cuppels et l., 2006). Once inside the lef, vrious PAMPs of Pst re rpidly detected by membrne-bound receptor kinses known s pttern recognition receptors (PRRs) (Abrmovitch et l., 2006). This form of resistnce is known s PTI nd results in mitogen-ctivted protein kinse signling cscde tht induces the expression of genes responsible for the ctivtion. This triggers number of integrted defenses including deposition of cllose for cell wll fortifiction, production of ntimicrobil secondry metbolites such s the lkloid tomtine (Friedmn, 2002) nd phytolexin rishitin (Le Floch et l., 2005), pthogenesis-relted (PR) proteins such s PR3 nd PR4 (chitinse), PR2 (glucnse) nd PR5 (osmotin or thumtin-like proteins) (Ji & Mrtin, 1999, Ren et l., 2011, Vn Loon & Vn Strien, 1999), nd production of ntimicrobil defensins nd defensin-like peptides (Stotz et l., 2009, Pieterse et l., 2009). In ddition, expression of number of other plnt hormones, such s the uxin indole cetic cid (IAA) nd gibberellins (GA) cn lso be induced or repressed s result of PTI (Pieterse et l., 2009). A key step in the ctivtion of PTI is slicylic cid (SA) production, which is relted to rective oxygen species (ROS) production nd controlled by n increse in cytoplsmic clcium levels (Nicise et l., 2009). Furthermore, stomtl closure in A. thlin ginst Pst ws ccelerted for 48 h following exposure to rhizobcterium B. subtilis FB17, indicting tht stomtl resistnce cn be triggered systemiclly (Kumr et l., 2012). Interestingly, recent evidence in A. thlin indictes tht PTI ginst Pst vries ccording to the plnt circdin clock, with A. thlin hving greter immunity to Pst chllenge in the morning thn the evening (Bhrdwj et l., 2011). The implictions for this discovery on 6

30 bcteril speck mngement in the field re not yet cler. Pst cn overcome PTI defenses by relesing effectors through its T3SS tht interfere with tomto PRRs or the downstrem signling of resistnce (Kdo, 2010b). This is defined s effector triggered susceptibility (ETS) since the relese of these effector proteins interct with vriety of trgets inside plnt cells undergoing PTI resulting in the suppression of PTI nd coloniztion of the plnt by Pst nd leding to the development of bcteril speck symptoms. The pthogenecity of Pst is relint on the T3SS, which is encoded by the hrp ((HR nd pthogenicity))/hrc (HR nd conserved) genes (Kdo, 2010b). hrc genes re locted in highly conserved region in mny bcteri, wheres hrp genes re common only in group I T3SS pthogenic bcteri (Preston, 2000). For Pst DC3000, there re seven wekly expressed effectors nd 28 fully ctive effector proteins introduced into host cells using this system. Two effectors, AvrPto nd AvrPtoB, re implicted in interfering with PRRs such s FLS2 nd BAK1 tht re prt of the PTI response in tomto (Munkvold & Mrtin, 2009, Segonzc & Zipfel, 2011, Xing et l., 2011). In the cse of FLS2, AvrPto nd AvrPtoB prevent it from binding with the PAMP bcteril flgellin (flg22) peptide (Abrmovitch et l., 2006). This interference results in suppression of PTI nd enbles infection nd coloniztion of the tomto host. AvrPtoB cn lso degrde some PRRs, suppress kinse ctivtion, nd suppress PAMP-induced gene expression nd microrna production (Munkvold & Mrtin, 2009). Similr effects re lso reported for AvrPto (Munkvold & Mrtin, 2009). In ddition to interfering or suppressing the ctions of PRRs nd PAMPs-medited response, effectors relesed by Pst lso ply other roles in overcoming PTI. For AvrPto, these host processes include inducing ethylene (ET) production, nd for AvrPtoB, inducing both bscisic cid (ABA) nd ET production. These hormones re thought to ntgonize SA-dependent defense responses in the host (Munkvold & Mrtin, 2009). Furthermore, the effectors HopQ1-2 nd Hopl1 re implicted in directly reducing plnt levels of SA (Cunnc et l., 2009). Tomtoes my successfully recognize these effector proteins resulting in effector-triggered immunity (ETI) nd hypersensitive response (HR) (Preston, 2000). Tomto cultivrs with the Pto gene re ble to recognize the effectors AvrPto nd AvrPtoB in Pst rce 0. Both AvrPto nd AvrPtoB physiclly interct with Pto to ctivte plnt immunity (Kim et l., 2002, Pedley & Mrtin, 2003). A second gene, Prf, is lso required for Pto-medited resistnce to Pst in tomto (Pedley & Mrtin, 2003). Previously, it ws thought tht Pto does not recognize Pst rce 1 becuse it lcks the AvrPto effector (Pedley & Mrtin, 2003). However, in n nlysis of Pst rce 1 strins collected over 13 yers in Cliforni, functionl vrpto with key mino cid polymorphism tht prevents its interction with Pto ws identified in four isoltes (Kunkew et l., 2010). vrptob ws still expressed in rce 1 strins, but AvrPtoB protein expression ws mintined t low levels using n unknown mechnism, nd this enbles rce 1 strins to overcome Pto-medited resistnce. Pto-medited resistnce for bcteril speck mngement hs been used commercilly for pproximtely 20 yers, nd over 90 percent of processing 7

31 tomto cultivrs grown in Cliforni crry the Pto nd Prf genes. Therefore, it is possible tht Pst rce 1 hve evolved to overcome this resistnce (Kunkew et l., 2010). In ddition to its role in overcoming PTI, corontine is lso importnt s phytotoxin in the lter stges of pthogenesis. Corontine production in DC3000 mutnts deficient in expression of hrp/hrc genes nd their hrpfgctv operon still occurred, but typicl speck lesions did not develop nd the mutted bcteri were unble to reproduce (Penloz-Vzquez et l., 2000). However, some hrp muttions resulted in overexpression of corontine, indicting the existence of cross-tlk between hrp nd cor gene clusters (Penloz-Vzquez et l., 2000). Corontine lso mimics the ction of the phytohormone jsmonic cid (JA), nd this plys key role in the virulence of Pst. Corontine induces the expression of JA-dependent wound-relted genes ssocited with herbivory leding to suppression of SA relted genes (Zho et l., 2003). JA-insensitive mutnts re resistnt to DC3000, nd the expression of JA-dependent wound-relted genes is reduced while PR gene expression increses. In contrst, in wild-type plnts, both corontine nd the T3SS repress expression of PR genes (Zho et l., 2003). Corontine is lso implicted in suppression SA-dependent defense responses in tomto (Upplpti et l., 2007). If Pst overcomes PTI-relted defenses nd is not detected by plnt R proteins, it successfully infects tomto tissues nd continues coloniztion in biotrophic fshion. Tomto poplstic fluid is complete nutrient medium for Pst, nd Pst uses both constitutive nd plnt-induced ssimiltion pthwys to use nutrients vilble in the poplst (Rico & Preston, 2008). The influence of environmentl conditions on the nutrient profile in the poplst nd subsequent impct on Pst growth hs not been explored, but might be n interesting venue for future reserch (Rico & Preston, 2008). The shift from biotrophy to necrotrophy nd subsequent development of symptoms is not clerly understood, but number of fctors hve been identified tht contribute to symptom development. The chlorotic hlo surrounding lesions is ttributed to the phytotoxin corontine, nd is dependent on SGT1 (suppressor of G 2 llele of skp1), gene tht is lso required in progrmmed cell deth ssocited with HR (Upplpti et l., 2011). Bender et l. (1987) observed lck of chlorosis nd reduction in lesion size in Pst mutnts deficient in corontine production. Furthermore, effectors lso ply role in necrosis. Both AvrPto nd AvrPtoB re implicted in incresing plnt ET levels. High levels of ET re linked to plnt stress response nd cn result in cell deth which might improve Pst ccess to nutrients, but results in plnt tissue necrosis (Abrmovitch et l., 2006). Lesion formtion by Pst ws lso reduced when the effector HopAA1-1 nd the chlorosispromoting fctor PSPTO4723 were bsent (Munkvold et l., 2009). The effector vre lso contributes to lesion formtion more thn bcteril growth (Bdel et l., 2006). Undoubtedly, dditionl effectors probbly ply roles in necrosis nd lesion development which re yet to be determined. 8

32 Bcteril speck symptoms re typiclly visible five dys following inocultion under controlled conditions, nd seven to 10 dys fter inocultion under field conditions. It is possible tht tomto susceptibility to Pst is dependent on tomto developmentl stge, since this phenomenon is reported in tomto nd other plnt species for other pthogens, nd for Pst in A. thlin (Develey-Rivière & Glin, 2007, Kus et l., 2002, Shh et l., 2015, Pnter et l., 2002, Shrbni et l., 2013). Bcteril speck symptoms my pper on ll folir plnt prts, including tomto leves, fruit, stems, nd flowers. Smll, round, drk brown to blck lesions develop on leves tht often hve chlorotic yellow hlo (Jones, 1991) (Figure 1.1). Eventully, lesions colesce cusing lrge res of tissue to die. This my led to defolition, erly fruit ripening, nd sunscld, which in the cse of processing tomtoes, negtively ffects hrvesting schedules nd the quntity of fruit meeting cceptble stndrds. Smll (<1mm) lesions develop on green fruit less thn 3 cm in dimeter, but do not develop on lrger green fruit or on red fruit (Getz et l., 1983, Jones, 1991, Preston, 2000). These specks reduce the qulity of fresh mrket tomtoes, nd cn negtively ffect the peeling process for processing tomtoes (Figure 1.1b & c). Incresing bcteril speck severity is normlly positively correlted with Pst popultion in lef tissues (Bysl et l., 2007, Vlld et l., 2003, Scrponi et l., 2001). Following infection, Pst my survive s sprophyte on crop residue for up to 30 weeks nd in soil for less thn 30 dys (Jones, 1991, Preston, 2000). Pst cn lso survive on infested seed for long periods (Bshn et l., 1982, Jones, 1991, McCrter et l., 1983). In one instnce, Pst ws isolted from 20 yer old tomto seeds in Isrel (Bshn et l., 1982). Potentil mechnisms of seed infection include internl coloniztion vi the host xylem, coloniztion through the pistil, nd externl contmintion fter contct with symptomtic fruit tissues (Mude, 1996). Other potentil sources of inoculum include volunteer tomto plnts, infected weed hosts, crop residue, nd contminted equipment such s frm mchinery, greenhouse structures, insects, niml nd tools (Bshn, 1986, McCrter et l., 1983, Schneider & Grogn, 1977). 1.6 Bcteril spot pthogen of tomto Bcteril spot is lso common bcteril disese in Ontrio (Cuppels et l., 2006, LeBoeuf et l., 2009). The bcterium cusing this disese ws previously clssified s Xnthomons cmpestris pv. vesictori (Doidge) Dye (Xcv) (Young et l., 1978). Xcv ws first divided into Group A nd Group B strins bsed on strch hydrolysis nd pectolytic ctivity, nd it ws suggested tht these groups should be reclssified s two seprte species, where Group A is X. xonopodis pv. vesictori nd Group B is X. vesictori (Vuterin et l., 1995). A second X. xonopodis pv. vesictori strin ws lter chrcterized 9

33 ) b) c) d) e) f) Figure 1.1. Symptoms of bcteril speck on ) tomto folige, b) ripe fruit, nd c) peeled processing tomto, nd bcteril spot on d) tomto folige, b) ripe fruit, nd c) peeled processing tomto. 10

34 s Group C, long with X. grdneri in Group D (Potnis et l., 2015). Four groups known s A, B, C, nd D re now distinguished bsed on DNA reltedness (Jones et l., 2004). DNA:DNA hybridiztion reveled tht Group A nd Group C hve less thn 70 percent DNA reltedness with ech other, nd Group D hs less thn 70 percent ny of the other three groups (Jones et l., 2004). Groups A, B, C nd D re referred collectively s BSX, nd it hs been proposed tht these groups be renmed s seprte species: Group A s X. euvesictori Jones, sp. nov. (Xe), Group B s X. vesictori (Doidge) Vuterin, sp. nov., nom. rev. (Xv), Group C s X. perforns Jones, sp. nov. (Xp), nd Group D s X. grdneri (ex Sutic) Jones, nom rev., comb. nov. (Xg) (Jones et l., 2004). A fifth species, Xnthomons rboricol Vuterin, sp. nov., is lso reported to cuse bcteril spot of tomto but so fr this report is limited to Tnzni (Mbeg et l., 2012). The pthogens re lso clssified into rces bsed on different interctions with stndrd group of tomto genotypes, which is bsed on the production of different T3SS effector proteins. The rces tht hve been found thus fr re T1 in Xe, T2 in Xv, Xp nd Xg, T3 in Xp, nd T4 in Xp (Jones et l., 2005). Xe nd Xv hve worldwide distribution, wheres Xp is limited to Mexico, Cnd, USA, Thilnd nd the Seychelles, Comoros nd Muritius Islnds in the southwestern Indin Ocen (Hmz et l., 2010, Jones et l., 2000). Xg is confirmed in Ontrio, Pennsylvni, Michign, Ohio, Brzil, the former Yugoslvi, Cost Ric, nd the Reunion Islnd in the southwestern Indin Ocen (Cuppels et l. 2006). Xg, which is considered more ggressive thn other strins, is the predominnt strin in Southwestern Ontrio (Cuppels et l., 2006, Abbsi et l., 2015). Previously Xv ws the secondry species of importnce in Ontrio (Cuppels et l. 2006), but more recently Xp is more common (Abbsi et l., 2015). Cuppels et l. (2008) reported tht Xv, Xp, Xg were detected on crop residue fter one winter, but not fter two winters, nd detection of Xp nd Xg on volunteer tomtoes, whet, nd weeds ner tomto fields in Ontrio ws rndom. In ddition to these potentil inoculum sources, infested seed nd equipment cn lso be sources of ll four BSX (Bshn et l., 1982, Bshn, 1986, Koike et l., 2007, Trtier & Pitbldo, 1994). Similr to Pst, BSX hve hemibiotrophic lifestyle. Initil interctions with the host plnt begin on the lef surfce; however, the epiphytic lifestyle my not be s gret for BSX s Pst. Zhng et l. (2009b) reported congregtions of Xe cells round lef depressions, gurd cells nd stomtl openings 48 nd 72 h fter inocultion but found little extensive growth on the remining tomto lef surfce. Insted, bcteril cells were found in sub-stomtl chmbers nd mesophyll tissues. The growth nd development of the BSX is fvoured by wrm tempertures rnging from 24 C to 30 C, nd similr to Pst, they re lso fvoured by wet conditions (Koike et l., 2007). These conditions encourge entry into the host plnt through nturl lef openings or wounds. Expression of hrc nd hrp genes, which re responsible for the induction of the T3SS, re required 11

35 for BSX virulence. There is evidence tht induction of these genes occurs erly in the infection process, prior to estblishment within lef tissues. Fluorescence ws observed on the surfce of tomto leves 24 h fter inocultion with Xe cells crrying hrpg- nd hrpx-gfp reporter constructs, which ws prior to detection of bcteri within lef tissues (Zhng et l., 2009b). HrgG begins regultory cscde tht induces expression of hrc nd hrp. Thus, these results my indicte tht the T3SS is ctivted prior to pthogen entry. Becuse the HrpG/HrpX regulon includes other genes tht re unrelted to the T3SS, ctivtion on the lef surfce might lso be relted to enhnced survivl on the lef surfce or priming for ETS, s opposed to immedite suppression of PTI (Zhng et l., 2009b). There is lso evidence tht X. cmpestris pv. cmpestris is cpble of interfering with stomtl closure on A. thlin induced by the bcteri on the lef surfce (Gudesblt et l., 2009, Gudesblt et l., 2009b). This mechnism might lso be importnt in the infection process of BSX. Estblishment of BSX in tomto tissues is lso dependent on T3SS effector proteins. Seventeen different effector groups hve been identified for BSX, most of which belong to the Xop nd Avr protein fmilies (Ky & Bons, 2009). Ten of these effectors re fully or prtilly homologous to known effectors in P. syringe (Ky & Bons, 2009). The effector XopD re present in Xnthomons spp., Acidovorx spp., nd Pseudomons spp. nd re known to suppress PTI (Kim et l., 2011). Effectors in the AvrBs3 fmily re unique in tht they hve only been detected in Xnthomons spp. nd Rlstoni solncerum (Ky & Bons, 2009). These effectors, which re known s trnscription ctivtor-like (TAL), induce plnt gene expression by cting s trnscription fctors in plnt, nd hve lso been demonstrted to be importnt in dissemintion in the field (Ky & Bons, 2009). Although the homology of effectors mong the BSX is not clerly understood, it ppers t lest some T3SS effectors re homologous mong the Xnthomons spp. The effector protein XopE2 lso hs role in virulence nd suppression of the HR for Xe nd Xv (Lin et l., 2011), nd vrbs3 genes re common in BSX, X. cmpestris pv. cmpestris, nd Xnthomons oryze pv. oryze (Oh et l., 2011). However, recently vrhh1 ws identified s novel member of the vrbs3 gene fmily in Xg (Schornck et l., 2008). Once estblished within the host, BSX strts obtining nutrients from the host. The enzymtic ctivity of Xg in A. thlin reveled tht the production of the cell wll degrding enzymes cellulse nd -rbinofurnosidse, but not pectinse, invertse nd xylnse were involved in nutrient cquisition (Cndido et l., 2008). Furthermore, BSX my lso interfere with nutrient export from lef tissues to plnt sinks (Kocl et l., 2008). Bcteril spot symptoms of chlorosis nd necrosis re relted to the ction of T3SS effectors (Slomon et l., 2011). The production nd relese of T3SS effectors by BSX in yest led to n ttenution of cell growth nd toxicity, nd these chrcteristics were ssocited with chlorosis nd 12

36 necrosis in plnts (Slomon et l., 2011). Both virulent nd virulent BSX bcteri induce ET- nd SAdependent defense responses in tomtoes, which re ssocited with reduction in symptom development from secondry chllenges with these pthogens (Block et l., 2005). Folir symptoms of bcteril spot on tomto re difficult to distinguish from bcteril speck (Figure 1.1d). They re lesions tht re drk brown, circulr to irregulr nd usully remin less thn 5 mm in dimeter. As the symptoms dvnce, lesions colesce nd premture defolition cn occur (Koike et l., 2007). Lesions on fruit begin s smll brown blisters tht eventully enlrge to 5 to 8 mm in dimeter, which re distinct from those of bcteril speck becuse they re lrger in size (Koike et l., 2007) (Figure 1.1e & f). Occsionlly the initil lesions on green fruit re surrounded by white hlo, which cn be confused with symptoms of bcteril cnker cused by Clvibcter michignensis subsp. michignensis (Trtier & Pitbldo, 1994). 1.7 Current bcteril speck nd spot mngement prctices in tomto The current strtegy for bcteril disese mngement in Ontrio relies on severl tenets of integrted pest mngement, including culturl, biologicl, nd chemicl controls. The use of clen seed nd proper greenhouse snittion for trnsplnt production re criticl to prevent erly infections (Jones, 1991, Jones, 1991b, Koike et l., 2007, Pitbldo & Trtier, 1994, Trtier & Pitbldo, 1994, Truemn, 2016, Truemn & LeBoeuf, 2015). Genetic resistnce is used to some extent to help limit the severity of bcteril speck in Ontrio, nd processing tomto cultivrs crrying the Pto, Prf genes, or both re vilble tht confer resistnce to Pst rce 0, but not rce 1 (Chng et l., 2000). Approximtely 25 % of Pst isoltes collected in Ontrio during the erly 1980s were identified s rce 1, but extensive survey work hs not been done since tht time (Lwton & Mcneill, 1986). In New York, Pst phenotype tht reches n intermedite popultion size in Pto-expressing tomto leves ws recently reported, which the uthors hypothesize mens Pst is recognized by Pto but then suppressed by nother fctor (Krus et l., 2017). There re no commercilly vilble tomto cultivrs with full resistnce to the BSX (Pei et l., 2012). Some biologicl control gents, such s Bcillus subtilis QST 713 (Serende Mx) nd Streptomyces lydicus WYEC 108 (Actinovte) re lso reported to directly ntgonize or outcompete bcteril pthogens (Roberts et l., 2008). The mechnisms of ntgonisms by B. subtilis QST 713 re not fully explored (Fousi et l., 2016). The ntibiotic compound streptomycin is used in the USA for bcteril disese mngement, but bcteril popultions re known to develop resistnce to this compound (Ritchie & Dittpongpitch, 1991). The use of bcteriophges hs lso been explored, nd these re used to some extent in the southestern US, in combintion with other control methods (Obrdovic et l., 2009, Obrdovic et l., 2004). Use of streptomycin nd bcteriophges in Cnd re hindered by regultory obstcles.ksugmycin (Ksumin 13

37 2L) is registered in Cnd for suppression of BSX on tomto (PMRA, 2016), but dt on its efficcy in field trils is limited (Truemn, 2015). Ksugmycin hs only been shown to be effective when mixed with other products like copper (Griffin et l., 2017). Thus, mngement of bcteril disese in the field is hevily relint on copper bctericides. Copper bctericides (copper hydroxide) re historiclly the most common tools used for mngement of bcteril spot nd bcteril speck diseses of tomto (OMAFRA, 2010). Copper is trce element tht hs importnt function in living orgnisms, including within the superoxide dismutses nd within enzymes involved in the respirtory chin tht produce energy to pump protons cross cytoplsmic membrnes. However, t high levels, the intrcellulr concentrtion of copper ions cn no longer be controlled nd toxic compounds re formed (Nies, 1999). These bctericides offer some benefit in suppressing levels of bcteril speck nd bcteril spot, but do not lwys provide the level of control required to prevent yield or qulity losses ssocited with these diseses (Lnge & Smrt, 2005, Lewis Ivey et l., 2004, Griffin et l., 2017). High levels of copper cn lso ffect cellulr processes in tomtoes nd led to toxicity symptoms (Iseri et l., 2011). Mixtures of copper with mncozeb or other ethylenebis(dithiocrbmte) (EBDC) fungicides my improve the ctivity of copper bctericides under field conditions, lthough in some reports no dditionl benefit over copper lone or unspryed control tretments is reported (Dmicone & Trent, 2003, Grves & Alexnder, 2002, Lewis Ivey et l., 2004, Louws et l., 2001, Obrdovic et l., 2004, Roberts et l., 2008). One of the mjor limittions of the repeted use of copper bctericides is the development of resistnce in popultions of bcteri (Griffin et l., 2017, Abbsi et l., 2015). All Pst isoltes tested since the lte 1970s in Ontrio hve some level of tolernce to copper (Cuppels & Elmhirst, 1999). Bender nd Cooksey (1986) demonstrted tht the Pst copper sensitive strins PT12.2 nd PT17.2 becme copper insensitive fter cquiring the plsmid ppt23c, which confers copper resistnce by n undetermined mechnism, vi horizontl gene trnsfer. However, nother ntive plsmid in Pst, pt23d, lso confers copper resistnce, nd contins four genes, copa, copb, copc, nd copd tht re periplsmic nd outer membrne proteins tht bind nd sequester copper outside the cytoplsm s copper-resistnce mechnism (Ch & Cooksey, 1991, Cooksey & Azd, 1992, Cooksey et l., 1990). The copper resistnt sprophytic species Pseudomons putid, nd yellow Pseudomons sp., s well s the pthogenic BSX nd Pseudomons cichorii, ll contined plsmid DNA tht ws homologous with DNA encoded by ppt23d, suggesting tht copper resistnce genes cn be trnsferred between bcteril species (Cooksey et l., 1990). In n effort to improve mngement of bcteril disese, plnt ctivtors tht induce or re presumed to induce systemic cquired resistnce (SAR) or induced systemic resistnce (ISR) hve been registered in Cnd. These include the SAR compound ASM (Actigrd) (PMRA, 2011) nd the ISR ctivtor B. 14

38 subtilis QST 713 (Serende Mx, Serende Opti, Serende Soil, Cese) (Byer, 2017, Byer, 2014, Bioworks, 2016, Fousi et l., 2016), which is vilble in Cnd for suppression of BSX on field tomtoes nd vrious other crop disese combintions (Byer, 2017, Byer, 2014b, Byer, 2014, Bioworks, 2016). However, field performnce of these ctivtors hs been inconsistent for bcteril spot nd speck control in field tomtoes (Roberts et l., 2008, Truemn, 2015). B. mycoides isolte J (LifeGrd) (Certis, 2017b) nd n extrct from Reynoutri schlinensis (gint knotweed) (Regli Mxx) (PMRA, 2016d) re lso registered for suppression or prtil suppression of bcteril speck, bcteril spot or both on field tomtoes in Cnd (PMRA, 2016d, OMAFRA, 2010, Syngent, 2012). Applictions of B. mycoides isolte J increse defense enzyme production in sugrbeet, but the defense signling pthwy in tomto hs not been identified (Brgbus et l., 2002). Another potentil SIR ctivtor is B. myloliquefciens strin D747 (Double Nickle 55) (Certis, 2017). It lcks evidence for SIR, lthough t lest one other B. myloliquefciens strin induced resistnce ginst Pst in tomto (Lnn-Filho et l., 2017). The possible modes of ction of ASM, B. subtilis QST 713, nd extrct from R. schlinensis is discussed lter in this review. 1.8 SIR nd its use in bcteril disese mngement The bility of plnts to suppress or resist plnt pthogens depends on intruder recognition through PTI nd ETI (Pieterse et l., 2009). The ctivtion of PTI or ETI lso ctivtes nother plnt immune response, known s systemic induced resistnce (SIR). SIR is further subdivided into SAR nd ISR (Pieterse et l., 2009). Both SAR nd ISR result in phenotypiclly similr reductions in disese severity, but the modes of ction of the two systems re different. Evidence in A. thlin suggests tht SAR generlly ctivtes defense mechnisms tht re effective ginst biotrophic nd hemibiotrophic pthogens, wheres ISR generlly ctivtes defense mechnisms effective ginst necrotrophic nd hemibiotrophic pthogens, s well s insects (Ton et l., 2006). In other words, pests controlled through the SA-dependent defenses re generlly suppressed by SAR, nd pests controlled through JA- nd ET-dependent defenses re generlly suppressed through ISR (Pieterse et l., 2009) SAR Mechnisms of SAR SAR is systemic defense response to preventtively suppress disese symptoms. A chrcteristic of SAR is tht the defense system is ctivted systemiclly in distl plnt prts s result of locl ctivtion, thus preventing the spred of disese. SAR ws first reported in 1961 (Ross, 1961) where it ws found tht locl inocultion of tobcco plnts with tobcco mosic virus (TMV) resulted in brod- 15

39 spectrum systemic resistnce to TMV nd other pthogens in distl leves. SAR is dependent on the genertion of ROS in cell, which triggers n ccumultion of SA tht chnges the oxidtion stte of the regultory protein nonexpressor of PR1 gene (NPR1) (Durrnt & Dong, 2004, Nicise et l., 2009). Inctive NPR1 oligomers re reduced to ctive monomers tht re trnslocted from the cytosol to the nucleus (Pieterse et l., 2009, Spoel & Dong, 2012). Nucler import is dependent on phosphoryltion of NPR1 by the SNF1-RELATED PROTEIN KINASE2.8 (Birkenbihl et l., 2017) s well s thioredoxins TRX-h5 nd TRX-h3 (Crls et l., 2015). In A. thlin, the NPR1 prlogues, NPR3 nd NPR4, re SA receptors (Fu et l., 2012). NPR3 nd NPR4 help modulte levels of NPR1 nd SA in the nucleus by binding with SA nd degrding NPR1 (Crls et l., 2015). NPR3 hs low binding ffinity with SA, nd SA promotes NPR1-NPR3 interction, wheres NPR4 hs high binding ffinity with SA, but SA disrupts NPR4-NPR1 interction (Withers & Dong, 2017). Intermedite levels of SA led to interction between NPR3 nd NPR1, which results in ccumultion of NPR1 nd ctivtion of SA-dependent defenses (Crls et l., 2015). NPR1 intercts with trnscription fctor proteins in the nucleus, which results in the expression of PR genes nd production of PR proteins ssocited with disese suppression (Spoel & Dong, 2012, Pieterse et l., 2009) s well s WRKY trnscription fctor genes. Nucler interctions with NPR1 lso include sumoyltion by SUMO3. This shifts NPR1 ssocition from WRKY70 trnscription fctor to the trnscription ctivtor TGA3, which promotes PR gene expression nd estblishes SAR (Withers & Dong, 2017, Birkenbihl et l., 2017). For exmple, the expression of cidic PR1 (PR1) is induced during SAR in tomto nd this gene hs been used s mrker for SAR gene expression in this crop (Block et l., 2005, Hermn et l., 2007, Tornero et l., 1997). Although NPR1-dependent SAR is well documented, there is lso some evidence tht SA cn ccumulte nd induce defense response in n NPR1-independent mnner tht still results in the ccumultion of PR proteins (An & Mou, 2011), nd this is dependent on ccumultion of ROS nd nitric oxide (Go et l., 2015). While SAR is dependent on the SA signling pthwy, locl ccumultion of SA is not necessrily required for the systemic nture of SAR to occur (Pieterse et l., 2009, Spoel & Dong, 2012). The mechnism by which the SAR signl trvels from the site of locl infection to helthy plnt prts is not entirely understood; however methyl slicylte (MeSA), JA, glycerol-lipid fctors, nd vrious peptides re ll implicted in the signling process (Liu et l., 2011b, Vlot et l., 2008). Environmentl fctors such s the durtion of light fter pthogen inocultion cn lso influence the reltive importnce of MeSA in SAR ctivtion (Liu et l., 2011). It hs been proposed tht immune signls MeSA, lipidderived glycerol-3-phosphte, nd zelic cid, might work coopertively to induce SAR, nd the prticulr signling molecules involved might be pthogen-dependent (Spoel & Dong, 2012). Regrdless of the signling mechnism, the SAR signl trvels through the phloem to helthy plnt tissues (Spoel & 16

40 Dong, 2012, Vlot et l., 2008). In the cse of MeSA, it must be hydrolised to SA in the distl plnt cell prior to ctivting the SAR response (Vlot et l., 2008). SA my ccumulte through mechnisms other thn MeSA, but these re unknown (Spoel & Dong, 2012) Biologicl ctivtors of SAR SAR is nturl defense response to loclized infection by pthogen; therefore, nturl ctivtors of SAR include virulent plnt pthogens nd non-pthogens. Activtion of SAR in tobcco occurs ginst vriety of pthogens including P. syringe pv. tbci, Phytophthor prsitic, nd TMV fter inocultion with P. syringe nd TMV (Edrev, 2004). In nother exmple, inocultion of tomto plnts with virulent BSX resulted in ccumultion of SA nd ET in plnt tissues, incresed expression of PR mrker genes for SAR, nd decresed tissue dmge for secondry chllenges with BSX even though there ws not decrese in the BSX popultion (Block et l., 2005). Primry inocultion with Pst elicited decresed tissue dmge due to secondry chllenge by Pst nd BSX, but primry inocultion by BSX result in reduction in tissue dmge from secondry chllenge from BSX but not Pst (Block et l., 2005). Inocultion of tomto plnts with n virulent strin of BSX nd virulent strin of BSX ws lso shown to reduce symptoms of bcteril spot fter subsequent chllenge with virulent BSX by reducing plnt cell deth, s indicted by mesurement of cellulr ion lekge (Block et l., 2005). Thus, initil loclized infections helped suppress further spred of the sme or sometimes different pthogens, but clerly do not reduce disese severity of bcteril spot nd bcteril speck to economiclly cceptble levels (Jones, 1991, Jones, 1991b, Koike et l., 2007, Pitbldo & Trtier, 1994, Trtier & Pitbldo, 1994). Avirulent mutnts of plnt pthogens, prticulrly those with hrp muttions, hve lso been reported to reduce disese severity in the greenhouse nd field. The Pst DC3000 hrps, hrph, nd hrpa mutnts reduced severity of disese in tomtoes inoculted with Pst wild-type DC3000 in the greenhouse. However, only hrps provided significnt reduction under field conditions (Wilson et l., 2002). Similrly, hrpg, hrpx, hrpf, nd hrpei mutnts of Xe reduced the severity of bcteril spot in tomtoes grown under greenhouse conditions while only hrpg nd hrpf mutnts significntly reduced disese severity in the field (Moss et l., 2007). Although these mutnts re useful for studying the mechnisms of SAR, their prcticl ppliction is limited by regultory bureucrcy nd other obstcles ssocited with their commerciliztion (Wilson et l., 2002). Non-pthogenic microorgnisms re lso ssocited with SAR, lthough this ssocition is less frequent thn with ISR, which is discussed lter in this review. Living ctivtors ssocited with SAR include isoltes of B. pumilus nd B. myloliquefciens tht induced higher PAL ctivty fter Pst inocultion in tomto (Lnn-Filho et l., 2017). An increse in PR1, PDF1.2 nd PAL gene expression 17

41 in A. thlin fter inocultion with Pepper mild mottle virus suggests tht B. myloquefciens induces both SA nd JA-pthwys (Ahn et l., 2002), nd n enhnced expression of SA, JA nd ET-dependent genes in A. thlin chllenged with Pst suggests B. cereus AR156 primes host defenses for SAR nd ISR (Niu et l., 2011). Streptomyces sp. from helthy whet tissue re lso reported to prime for SAR nd ISR in A. thlin ginst Fusrium oxysporum or E. crotovor subsp. crotovor (Conn et l., 2008) PAMP ctivtors of SAR Hrpin proteins promote the trnsloction of T3SS effectors nd re ssocited with number of effects on plnts including induction of SAR (Chen et l., 2008, Dong et l., 1999). There re four hrpin genes in the Pst DC3000 genome (Cunnc et l., 2009, Kvitko et l., 2007, Preston, 2000). The hrpin protein produced by the hrpn gene of Erwini mylovor (Wei et l., 1992) hs been commercilized nd is mrketed in the United Sttes under the brnd nme Messenger by the Eden Bioscience Corportion (Wlters & Fountine, 2009). Additionl PAMP ctivtors of induced resistnce include chitosn (such s Elex mrketed by SfeScience), sulfted lminrin, which induces SA ccumultion nd PR1 expression, the flg22, nd trnsltionl elongtion fctor-tu (elf18) proteins, which re ssocited with ROS production nd cllose ccumultion, nd LPS, which re specific to grm-negtive bcteri (Schreiber & Desveux, 2008, Wlters, 2009). Smll proteins secreted by Pythium nd Phytophthor spp, clled elicitins, nd the Phytophthor protein cellulose-binding elicitor lectin re lso ssocited with induced resistnce, lthough their dependence on SA-signling is not cler (Schreiber & Desveux, 2008) Plnt ctivtors of SAR A number of compounds extrcted from vriety of plnts cn ctivte SAR. For exmple, SA is phenolic compound nd plnt secondry metbolite tht is produced in plnts vi the isochorismte synthse (ICS)-medited isochorismte pthwy nd the phenyllnine mmoni lyse (PAL)-medited phenyllnine pthwy (An & Mou, 2011). Its ccumultion in plnt cells is usully necessry for the development of SAR (Durrnt & Dong, 2004, Ysud et l., 2003). Exogenous pplictions of SA to plnts elicits SAR (Sticher et l., 1997). Presumbly, these pplictions result in SA ccumultion in plnt cells tht leds to the ccumultion of PR proteins ssocited with SAR. However, SA does not trnslocte efficiently in plnts, nd cn be phytotoxic to plnts if the concentrtion pplied is too high. Members of the B vitmin group, including thimine (vitmin B 1 ), riboflvin (vitmin B 2 ), nd PABA re lso ssocited with SAR (Song et l., 2013, Yng et l., 2011b, Azmi-Srdooei et l., 2010, Boubkri et l., 2013, Dong & Beer, 2000, Liu et l., 2010, Nie & Xu, 2016, Theri & Trighi, 2010, Zhng et l., 2009, Tzhoor, 2014, Ahn et l., 2005, Ahn et l., 2007, Boubkri et l., 2013b). PABA, lso known s 4-minobenzoic cid (4-AA), vitmin H 1, B x nd B 10, is benzoic cid derivtive 18

42 synthesized by plnts, fungi, bcteri nd protistns s folic cid precursor (Bsset et l., 2004). Endogenous levels of PABA occur in tomto leves nd fruit t the nm level (Bsset et l., 2004, Quinlivn et l., 2003). The bility of PABA to reduce symptoms is reported for diseses cused by Puccini striiformis in whet (Kelmn & Cook, 1977), Xnthomons xonopodis pv. vesictori (Xe or Xp) for soil drench pplictions in pepper for up to 77 dys (Song et l., 2013), Pectobcterium crotovorum subsp. crotovum for growth medi pplictions in tobcco seedlings (Yng et l., 2011b), nd Pst in some tomto breeding lines using folir pplictions (Tzhoor, 2014). The efficcy of suppression of Xe/Xp using PABA ppliction to growth medi ws dependent on concentrtion (Song et l., 2013). PABA ws ssocited with the induction of the SAR mrker gene PR1 in tomto (Tzhoor, 2014). Likewise, n increse in SA-relted gene expression but not JA or ET-dependent gene expression in PABA-treted pepper ws observed fter pthogen chllenge (Song et l., 2013), suggesting PABA primes plnts for SAR. An ethnolic extrct of gint knotweed (R. schlinensis) (Milsn / Regli mrketed by Mrrone Bio Innovtions) is lso implicted in SAR (Wlters & Fountine, 2009). The knotweed extrct is implicted in induced resistnce through induction of phytolexin nd phenolic compound production (Dyf et l., 1997), production of defense-relted proteins (Schneider & Ullrich, 1994), ccumultion of ROS (Vechet et l., 2005) nd incresed lignifictions nd ppill formtion in cell wlls in vriety of host-pthogen systems (Fofn et l., 2005, Wurms et l., 1999). However, it is not cler if extrct from R. schlinensis induces SAR, ISR or nother plnt defense pthwy. Extrct from fruit of Azdircht indic lso ppers to induce resistnce in cucumber ginst Podospher xnthii, s ppliction resulted in n increse in PAL nd tyrosine mmoni lyse ctivity nd phytolexin ccumultion (Aboellil, 2007). Bet-minobutyric cid (BABA) hs lso been implicted in SAR (Kunz et l., 1997), but is rrely found nturlly in plnts nd there re conflicting reports on its bility to induce resistnce through SAdependent mnner consistent with other SAR ctivtors (Jkb et l., 2001). Other plnt derived ctivtors include zelic cid (Jung et l., 2009), humic cids (Abdel-Monim et l., 2011), extrcts of Strobilnthes cusi (Li et l., 2008) nd β-1,4 cellodextrins (Aziz et l., 2007) Synthetic ctivtors of SAR ASM is functionl nlog of SA nd hs been commercilized s plnt ctivtor by Syngent Crop Protection (formerly Novrtis Crop Protection) for number of griculturl crops, nd is mrketed s Actigrd in North Americ nd Bion in Europe (Vlld & Goodmn, 2004). ASM is derivtive of benzol [1,2,3] thidizol-7-crbothioic cid-s-methyl ester (Kunz et l., 1997). ASM ws initilly found to hve the bility to induce plnt resistnce ginst wide rnge of pthogens, including Colletotrichum lgenrium in cucumber, Pseudomons lchrymns in cucumber, Erysiphe grminis in whet, P. 19

43 infestns nd Pst in tomto, Pyriculri oryze in rice nd Peronospor tbcin in tobcco (Kunz et l., 1997). ASM ctivtes SAR by inhibiting the enzymes ctlse nd scorbte peroxidse, which results in the genertion of ROS within the cell (Wendehenne et l., 1998). Effects of ASM on induced resistnce defense enzyme production or gene expression hve been observed up to 10 to 22 dys fter ppliction in tomto (Goodwin et l., 2017b, Cvlcnti et l., 2006), up to 20 dys fter ppliction in tobcco (Friedrich et l., 1996) nd 21 dys fter ppliction in cnol (Potlkyl et l., 2007). Bcteril speck lesion dimeter decreses with incresing concentrtions of ASM (Scrponi et l., 2001). 2,6-dichloro isonicotinic cid (INA) nd its derivtives lso function s nlogs of SA in the SAR signling pthwy (Oostendorp et l., 2001). Commerciliztion of INA nd SA hs not occurred becuse of problems with phytotoxicity (Oostendorp et l., 2001). However, the widely used systemic neonicotinoid insecticide imidcloprid breks down into n nlog of INA in plnt nd induces SAR (Ford et l., 2010). The chemicl 3-llyloxy-1,2-benziothizole-1,1-oxide (probenzole) nd its ctive metbolite 1, 2- benzisothizol-3 (2H)-one 1,1-dioxide (BIT; scchrin) re lso synthetic ctivtors of SAR (Schreiber & Desveux, 2008, Yoshiok et l., 2001). Bsed on SA- nd NPR1-dependent induced resistnce, SAR ws demonstrted s the mode of ction of probenzole in A. thlin using plnts deficient in SA ccumultion, insensitive to JA nd ET, nd n npr1 mutnt (Yoshiok et l., 2001). Probenzole is thought to ct upstrem of SA ccumultion becuse it filed to stimulte SAR or PR gene expression in NhG plnts tht cnnot ccumulte SA (Yoshiok et l., 2001), nd similr pthwy ws lter confirmed in tobcco (Nkshit et l., 2002). Probenzole-dependent SA ccumultion in rice my be dependent on plnt growth stge, s probenzole incresed SA nd PR protein ccumultion in rice t the 8-lef stge but not t the 4-lef stge (Iwi et l., 2007). 3,4-dichloro-N-(2-cynophenyl)-1,2-thizole-5-crboxmide (tidinil), the tidinil metbolite SV-03, 3-chloro-1-methyl-1Hpyrzole-5-crboxylic cid (CMPA), cthechin, nd 3-cetyl-3-hydroxyindole (AHO) re lso ssocited with SAR (Schreiber & Desveux, 2008). Other chemicls reported to induce resistnce include phosphte compounds (Reuveni et l., 1997, Schreiber & Desveux, 2008), micronutrient sprys (Reuveni et l., 1997), metl phthlocynines (Vol pin et l., 2000), dipic cid derivtives, sulfmethoxzole, oxltes, trehlose, cholic cid, ergosterol, nd syringolin re lso reported to induce resistnce. However, the mechnisms of induced resistnce of these compounds hve not been thoroughly exmined; therefore it is not cler if SAR or nother induced resistnce mechnism is implicted in the host response Plnt fitness costs ssocited with SAR The defense response induced by SAR depends on the production of PR proteins tht require plnt nutrients nd energy tht would otherwise be llocted to plnt growth nd development, thus SAR is 20

44 ssocited with some plnt fitness costs, prticulrly in pthogen-free environment (Durrnt & Dong, 2004, Wlters & Heil, 2007). Reductions in plnt growth nd development resulting from ASM pplictions hve been reported in sunflower, tobcco, culiflower, strwberry, melons, pepper nd tomto (Wlters & Heil, 2007, Lnn-Filho et l., 2017). A single ppliction of the endophytes, B. pumilus nd B. myloliquefciens, to tomto seeds lso resulted in reductions in plnt height nd totl dry weight five weeks fter seeding, lthough the negtive effects were not s severe s those of ASM (Lnn-Filho et l., 2017). However, in mny other cses no negtive effects of ctivtors were found nd reported yield ws equivlent to the pesticide stndrd (Wlters & Heil, 2007, Vlld & Goodmn, 2004). PABA is reported to hve vrious effects on plnt growth including stimultion of seed germintion in winter whet nd winter brley (Bekusrov et l., 2013), reduction in root length in A. thlin, nd chnges in the number of lterl roots of A. thlin (Crisn et l., 2014). The effects on root structure pper to be concentrtion dependent, since no effects re reported in nother study (Hong et l., 2007). PABA effects on plnt growth my be relted to its uxin-like root growth regulting ctivity (Crisn et l., 2014), but plnt growth evlutions using concentrtions reported for induced resistnce ctivity hve not been completed. The presence of sufficient or excess resources for plnt growth nd development likely contributes to reduction in fitness costs ssocited with SAR, s this improves the likelihood tht plnts cn obtin the energy nd nutrients required for defense responses (Wlters & Heil, 2007). The production of the defense relted enzymes chitinse, chitosnse nd peroxidse in ASM-treted A. thlin ws lower in plnts grown under limited nitrogen (Dietrich et l., 2004). ASM-induced A. thlin initilly hd reduced rtes of growth under the low, medium, nd highest nitrogen fertiliztion regimes, but not the high regime (Dietrich et l., 2005). Lter in the study period, plnts compensted for this growth reduction by incresing growth rtes in the highest nitrogen regime, but not the low nd medium regimes. In ddition, induced plnts produced more seed thn nontreted controls during shortened growing period initited by seizing wtering one week fter ASM tretment, but not during full growing period tht lsted 4.5 weeks fter ASM tretment. Greter reductions in biomss, shoot, nd er production in ASM-treted whet were lso observed in plnts growing in low nitrogen conditions, s compred to nontreted control plnts growing in the sme conditions (Heil et l., 2000). In A. thlin, competition between intrspecific neighbours ws not found to excerbte fitness costs ssocited with SAR (Cipollini, 2002). It my lso be possible tht some plnt species re ble to compenste for the increse in inputs required for defense by incresing their rte of photosynthesis (Wlters & Fountine, 2009). However, ASM-dependent ctivtion of the SAR regultory protein NPR1 by creting monomeric NPR1 is demonstrted to downregulte expression of genes responsible for bsic cellulr processes such s photosynthesis in rice (Sugno et l., 2010). 21

45 Reports of yield losses ssocited with ASM in tomto under field conditions re limited to few field trils in fresh mrket cultivrs (Dmicone & Trent, 2003, Lnge & Smrt, 2005, Louws et l., 2001) In those three studies of yield losses, ppliction rtes rnged from 26.3 to 35.0 g ASM/H, which is more thn twice the Cndin lbel rte of 12.5 g/h (PMRA, 2011). In other studies where no sttisticlly significnt yield reductions were observed, the rtes rnged from 17 to 26.5 ASM/H, 10.5 g ASM/H, 11.6 to 26.3 g ASM/H, 38 g ASM/H, 12 g ASM/H, nd 9.5 g ASM/H (Alexnder & Wldenmier, 2003, Grves & Alexnder, 2002, Lnge et l., 2007, Lewis Ivey et l., 2004, Miller et l., 2002, Roberts et l., 2008, Truemn, 2015). A yield reduction in grfted tomto plnts growing in R. solncerum infested soil nd treted with folir ASM is lso reported (Kunwr et l., 2017), nd in processing tomtoes tht received more thn eight pplictions in seson (Pontes et l., 2016). Nevertheless, the observtion tht ASM-treted plnts produced lower yields hs probbly resulted in negtive imge for the use of plnt defense ctivtors s disese mngement tools Vrition in SAR response mong cultivrs Plnt response to SAR ctivtors results in chnges in gene expression, nd therefore vrition in response to ctivtors mong cultivrs is not surprising. The severity of white mold symptoms cused by S. sclerotiorum ws reduced in four soyben cultivrs treted with INA or ASM, nd the gretest reductions in disese severity were observed in the two highly susceptible cultivrs, s compred to the two cultivrs, which hd greter tolernce to white mold (Dnn et l., 1998). In contrst, the concentrtion of INA required to control powdery mildew in cucumber using INA ws lower in prtilly resistnt cultivr thn two susceptible cultivrs (Hijwegen & Verhr, 1994). In pepper, five cultivrs with no or prtil resistnce to bcteril spot (Xe) ll responded to ASM with equivlent reductions in disese, lthough yield reductions ssocited with ASM ppliction vried mong cultivrs (Romero et l., 2001). The levels of induced resistnce by vlidmycin A nd PABA to F. oxysporum f. sp. lycopersici nd Pst, respectively, lso vried mong tomto genotypes (Ishikw et l., 2007, Tzhoor, 2014). The conclusion from this is tht response to SAR ctivtors cn vry by plnt cultivr, but tht my or my not be relted to the resistnce level of cultivr to prticulr pthogen. Attempts hve been mde to relte the degree of response to SAR ctivtors with the level of plnt defense gene expression. The expression of tomto PR1, mrker gene for induction of SAR, incresed in cv. Supersonic one dy fter tretment but returned to bseline level three dys fter tretment, while in cv. Rutgers, there ws similr but reltively greter response, nd for cv. Rio Grnde, expression lso rose fter one dy, declined more slowly thn in the other two cultivrs nd fell below the nontreted control fter seven dys (Hermn et l., 2007). In ll three cultivrs, PR1 gene expression ws higher fter second ppliction of ASM, with cv. Supersonic nd cv. Rio Grnde hving 22

46 similr PR1 response to the first ASM ppliction, but cv. Rutgers hving pek PR1 expression two dys fter the second ASM ppliction nd then flling shrply the next dy. ASM lso ctivted PR1b gene expression, which is ssocited with ET-dependent ISR, nd followed similr expression ptterns s PR1 but t lower levels (Hermn et l., 2007). This study did not include chllenge of the plnts with Pst or BSX fter induction by ASM, thus vrition in the field effectiveness of ASM mong cultivrs ws not reported. Furthermore, Cipollini (2002) observed vrition in peroxidse ctivity mong five A. thlin lines treted with exogenous pplictions of SA under pthogen-free conditions. Tzhoor (2014) lso reported differentil response in tomto breeding lines to pplictions of PABA, nd this ws relted to fster nd stronger increse in SlPR1 expression fter Pst inocultion in the responsive breeding line. These results indicte tht lthough SAR is common response in plnts, genotype differences within plnt species my dictte the speed nd intensity of SAR response. The genetic vrition in response to defense ctivtors cn be mde more complicted by n epigenetic effect, defined s the control of gene expression bsed on chromtin orgniztion insted of primry DNA sequence informtion (Bender & Scholz-Schroeder, 2004). Chromtion structure cn be influenced by the ctivities of different enzymes tht cn modify DNA or disrupt its interctions with histones which occur s response to stress (Alvrez et l., 2010). The induction of SAR might hve effects on the plnt immunity in subsequent plnt genertions. Progeny of A. thlin plnts ctivted for SAR by Pst DC3000 hd lower levels of coloniztion by Hyloperonospor rbidopsidis nd Pst thn those tht hd mock-inoculted prents (Lun et l., 2012). This effect ws lso observed for second genertion progeny inoculted with H. rbidopsidis. Gene expression nlysis reveled tht PR1 gene expression in the DC3000-inoculted progeny ws fster nd stronger thn in control plnts nd ws NPR1-dependent. Similr results re lso reported by Slughter et l. (2012) who successfully primed A. thlin progeny ginst ttck by H. rbidopsidis nd Pst by pplying BABA nd n virulent strin of Pst to prentl line. Furthermore, priming tretment on the first genertion progeny resulted in n even stronger priming effect in second-genertion progeny chllenged with H. rbidopsidis SAR in disese mngement The potentil for SAR s disese mngement tool hs been explored on vriety of economiclly importnt plnts. For exmple, ASM reduced the severity of severl diseses including downy mildew (Peronoscleropor sorghii) nd powdery mildew (Blumeri grminis f. sp. tritici) in whet, bcteril wildfire (Pseudomons syringe pv. tbci) nd blue mold (Perenospor hyoscymi f. sp. tbcin) in tobcco, bcteril spot (Xcv) in pepper, white mold in soyben, bcteril blight (Xnthomons cmpestris pv. mlvcerum) in cotton, white rust (Albugo occidentlis) in spinch, fire blight (E. mylovor) in 23

47 pple, nd rust (Gymnosporngium siticum) nd scb (Venturi nshicol) in per (Vlld & Goodmn, 2004). ASM lso reduced post-hrvest rots in rockmelon cused by Fusrium spp., Alternri spp., nd Rhizopus spp. (Bokshi et l., 2006), nd the severity of rust (Uromyces vicie-fbe), scochyt blight (Ascochyt fbe), nd broomrpe (Orobnche crent) in fb ben (Sillero et l., 2012). However, it did not reduce levels of lte lef spot (Cercosporidium persontum) in penut (Vlld & Goodmn, 2004), nd its effectiveness t reducing citrus cnker on grpefruit trees ws dependent on the ppliction rte, intervl nd timing (Grhm & Myers, 2011). The combintion of ASM, BABA, nd cisjsmone induced resistnce in brley ginst Rhynchosporium seclis, but there ws no effect on PR1 expression, defense-relted enzyme production, nd the bility of the host to prevent new infection when the elicitor combintion ws pplied to plnts with R. seclis infections on lower leves (Wlters et l., 2011b). Probenzole, which is mrketed s Oryzemte, is plnt ctivtor used in Asi ginst rice blst nd bcteril lef blight cused by Mgnporthe grise nd X. oryze pv. oryze (Wlters & Fountine, 2009, Yoshiok et l., 2001). Probenzole suppressed symptoms of southern corn lef blight (Cochliobolus heterostrophus) in corn with no dverse effects on plnt growth or yield (Yng et l., 2011). Probenzole lso reduced symptoms of te gry blight (Pestlotiopsis longiset) nd nthrcnose (Colletotrichum thee-sinensis) in field grown te (Yoshid et l., 2010). Fewer field studies re vilble in the literture regrding the field efficcy of INA, imidcloprid nd exogenous SA. INA reduced levels of rust (Uromyces ppendicultus) in ben, white mold in soyben, lternri lef spot, nd Verticillium wilt in cotton, but not lte lef spot in penut (Vlld & Goodmn, 2004). Exogenous SA reduced levels of rust but not scochyt blight nd broomrpe in fb ben (Sillero et l., 2012). In ddition, one or two soil pplictions of imidcloprid were effective t reducing the severity of citrus cnker in grpefruit trees, except during conditions of high disese pressure (Grhm & Myers, 2011). In tomto, ASM reduced the severity of bcteril wilt in greenhouse experiments under conditions of low disese pressure (Anith et l., 2004), but did not reduce symptoms in lter unrelted field study (Hong et l., 2011). In Florid, drip pplictions of ASM reduced bcteril wilt incidence in non-grfted plnts in two trils, but drip ASM pplictions to grfted plnts with tolernt rootstock provided no dditionl benefits to grfting lone (Kunwr et l., 2017). ASM reduced the severity of lef mold (Fulvi fulv), erly blight, nthrcnose, nd trget spot (Corynespor cssiicol) (Abbsi et l., 2002, Vlld, 2010, Vlld & Goodmn, 2004), but resulted in only slight reduction in Fusrium crown nd root rot (Fusrium oxysporum f. sp. rdicis-lycopersici) (Myresiotis et l., 2012) SAR in mngement of bcteril speck nd spot of tomto 24

48 The effectiveness of SAR ctivtors to control bcteril spot nd speck on tomto hs yielded mixed results. Bcteril speck lesion dimeter decreses with incresing concentrtions of ASM (Scrponi et l., 2001). In series of efficcy trils cross North Americ in fresh nd processing tomto cultivrs, ASM reduced the severity of bcteril spot on tomto folige below tht observed in control plots nd the stndrd copper + EBDC tretment in 13 of 14 nd 4 of 14 experiments (Louws et l., 2001). Furthermore, ASM reduced the severity of bcteril speck on folige in six of six nd two of six experiments compred to nontreted control plots nd the stndrd bctericide tretment. However, the degree of disese reduction rnged from 17 to 85% for bcteril spot with men of 51%, nd 23 to 100% for bcteril speck with men of 56%. The ppliction rtes nd ppliction intervls of ASM vried mong experiments nd rnged from 10.5 to 70.0 g/h, nd every seven dys to every 14 dys depending on the loction. Another report showed tht ASM reduces bcteril spot nd speck severity; however disese severity ws not lwys sttisticlly lower thn the control nd rrely better thn stndrd copper + EBDC tretments (Obrdovic et l., 2004, Roberts et l., 2008, Wilson et l., 2002, Truemn, 2015). Appliction rtes nd intervls of ASM my be importnt in determining its efficcy. In Florid, weekly pplictions of ASM using rtes rnging from 75 to 200 µm effectively reduced disese severity, nd incresing rtes were inversely relted to disese severity ginst Xp (Hung et l., 2012). Bi-weekly pplictions of ASM using the sme rtes were not s effective, nd some phytotoxicity symptoms were observed t the high rte of 200 µm. Similrly, in study in processing tomto nd Xg nd Xp in Brzil, the idel ppliction intervl for ASM ws identified to be between eight nd 10 dys (Pontes et l., 2016). The current Cndin lbel dicttes tht Actigrd be pplied up to eight times t 7-dy intervls t rte of 12.5 g ASM/H, in 280 to 655 L wter/h, which is equivlent to 91 to 212 µm (PMRA, 2011). The Actigrd lbel for the United Sttes differs from the Cndin lbel in tht it dicttes tht ASM be pplied t 7-dy intervls t rte equivlent to 95 to 158 µm strting to two weeks fter trnsplnting, then 103 to 120 µm t three to four weeks fter trnsplnting, nd finlly t 108 to 154 µm t five to eight weeks fter trnsplnting (CDMS, 2016). Although Hung et l. (2012) found significnt inverse reltionship between re under the disese progress curve (AUDPC) nd ASM concentrtion in tomto ginst Xp, this only ccounted for 37 nd 35 percent of the vrition observed, suggesting tht other fctors lso influence the effectiveness of ASM nd other defense ctivtors in the field. Plnts re subject to vriety of biotic nd biotic stresses, which could influence the degree tht SAR cn be induced, resulting in priming for stronger defence response or negtively ffecting the defence response due to excessive stress or cross-tlk with other signling pthwys such s ISR (Wlters, 2009). For exmple, the efficcy of ASM for reducing B. cinere infection in tomto ws lso reduced in tomtoes exposed to wounding, wter, nd N deficiency stress 25

49 (Mymoune et l., 2015). Excessive stress is lso indicted by reports of phytotoxicity with ASM. For exmple, tobcco treted with incresed concentrtions of ASM hd greter SAR ginst tomto spotted wilt virus, but the highest rte of ASM lso cused temporry folir spotting nd stunting of the plnts (Mndl et l., 2008). Chlorotic phytotoxicity symptoms were lso observed in tobcco treted with ASM, lthough this problem ws corrected with top dressing clcium nitrte fertilizer (Cole, 1999). Therefore, the dditionl stress due to n SAR ctivtor coupled with biotic nd biotic stresses could reduce the field effectiveness of SAR ISR Mechnisms of ISR ISR is systemic defense response to suppress pthogen infections fter coloniztion by nonpthogenic root-ssocited microbes, such s mycorrhizl fungi, plnt growth promoting fungi (PGPFs), plnt growth promoting rhizobcteri (PGPRs) nd bcteril endophytes. ISR is phenotypiclly similr to SAR (Pieterse et l., 2009). ISR ws first reported by Alstrom (1991) who found reduction in hlo blight on bens fter seed tretment with the PGPR, P. fluorescens S97; however, lter studies tht used root inocultions tht sptilly seprted the beneficil microorgnism from folir pthogen re more conclusive, s they eliminted the potentil for direct ntgonism between the orgnisms nd confirmed the systemic nture of the response (vn Loon et l., 1998). A hllmrk chrcteristic of ISR is tht the defense system is primed systemiclly in distl plnt prts s result of root coloniztion, thus preventing the spred of disese in roots or leves. Priming is defined s the sensitiztion of plnt tissues to express bsl defense mechnisms more rpidly nd more strongly following pthogen chllenge, s opposed to induction soon fter ppliction (Conrth et l., 2006). Priming hs been studied in SAR nd BABA-induced resistnce nd is most likely due to the ccumultion of inctive protein kinses following tretment with n ctivtor (Beckers et l., 2009). The ccumultion of inctive unphosphorylted MPK3 nd MPK6 resulted fter ASM ppliction in A. thlin, however, fter pthogen chllenge MPK3 nd MPK6 were phosphorylted. This ctivtion enbled their interction with downstrem trgets, thus inititing plnt defense gene expression. Furthermore, low levels of SA-dependent trnscription fctors were induced by BABA, resulting in priming but not expression of SA-inducible defense genes. Priming by riboflvin in A. thlin is ssocited with ROS- nd C2+-signling dependent pthwys involving MPK3 nd MPK6 ctivtion (Nie & Xu, 2016). Unlike in SAR, SA does not ccumulte in plnts cells during ISR, nd NPR1 remins in its oligomer stte in the cytoplsm (Vn Wees et l., 2008) becuse of the presence of intermoleculr disulfide bonds (Crls et l., 2015). NPR1 is still required for ISR to occur but its exct role is not 26

50 currently understood (Pieterse et l., 2009). In the bsence of JA, JA-responsive genes re repressed within the cell by the formtion of complex between the E3 ubiquitin ligse SCFCOI1 complex, jsmonte ZIM-domin (JAZ) proteins, the dpter protein NINJA, or HDA6 (Crls et l., 2015, Pieterse et l., 2009b). This represses trnscription fctors like MYC2, EIN3, nd EIL1 (Crls et l., 2015). When JA ccumultes in the cell during induction of ISR, JA-isoleucine (JA-Ile) binds to the F-box protein COI1 in the SCFCOI1 complex, which eventully results in degrdtion of the JAZ proteins, nd ctivtion of JA-responsive genes. There re two JA-dependent signling pthwys. The first pthwy is regulted by MYC2, which ctivtes mrker genes VSP2 nd LOX2, nd is lso dependent on ABA. The second pthwy is regulted by the trnscription fctors ET-insensitive 3-like (EIN3), EIL1, nd ERF, which ctivtes the mrker gene PDF1.2, nd is lso regulted by ET(Crls et l., 2015). The MED25 enhncer-bound trnscription fctor in the Meditor complex regultes JA signling for both pthwys (Birkenbihl et l., 2017). Mrker genes for ISR include PR1b for ET-dependent gene expression in tomto (Hermn et l., 2007, Block et l., 2005), the plnt defensin genes, PDF1.2 (PR12), PR3 nd PR4, for ET- nd JAdependent gene expression in A. thlin (Pieterse & vn Loon, 1999, Ahn et l., 2007), nd the thionin gene, Thi2.1 (PR13), for JA-dependent gene expression in A. thlin (Pieterse & vn Loon, 1999, vn Loon et l., 2006). Lox1, Pl1, ETR1, nd CTR1 hve lso been used s mrkers of JA nd ET-signling in cucumber (Shoresh et l., 2005). The dependence of ET, JA nd SA on different PR protein induction cn vry by plnt species (vn Loon et l., 2006). Gene expression is monitored to determine if the priming phenomenon ssocited with ISR cn be identified. In other words, ET- nd JA-dependent mrker genes re often used to look t the difference in expression before ppliction of the ISR ctivtor, before pthogen infection, nd fter pthogen infection in ctivted, inoculted nd control plnts. The dependence of ISR on JA nd ET signling ws demonstrted in the A. thlin - P. putid LSW17S system ginst Pst DC3000 (Ahn et l., 2007). Monitoring the expression of defense-relted genes before nd fter inocultion with DC3000 confirmed tht LSW17S primed the system to respond fster to pthogen ttck, s demonstrted by the difference in gene expression in LSW17S- nd DC3000- only inoculted plnts, nd mock-inoculted plnts. Furthermore, disese suppression remined intct in NhG plnt deficient in SA ccumultion, suggesting tht induced resistnce ws not dependent on the SA-signling pthwy (Ahn et l., 2007). Disese suppression ws compromised in the mutnt npr1 which does not ccumulte PR1, the etr1 mutnt with ltered perception of ET, nd the jr1 mutnt with reduced sensitivity to JA, indicting dependence on the JA- nd ET-signling pthwys (Ahn et l., 2007). A similr priming response by LSW17S ws lso observed for Pst in tomto, but not for R. solncerum (Ahn et l., 2011). While most ISR signling occurs through JA- nd ET-dependent pthwys, there re few reports of ctivtion through other signling pthwys by PGPRs (Conn et l., 27

51 2008, Niu et l., 2011, Pvlo et l., 2011, Ryu et l., 2003). The MYB72 trnscription fctor gene in A. thlin is lso responsive to root coloniztion by P. fluorescens WCS417r nd is required in erly signling for ISR (Vn der Ent et l., 2008). Trnsgenic A. thlin lines with constitutive expression of MYB72 do not express ISR, potentilly becuse t lest one other component is required upstrem or in prllel with MYB72 trnscription fctor (Vn der Ent et l., 2008). The trnscription fctor ethylene-insensitive 3, which is linked to the ET-signling pthwy, is one possible cndidte, s it ws found to interct in vitro with MYB72 (Vn der Ent et l., 2008). MYB72 is lso required for ISR ctivtion by the beneficil fungi Trichoderm sperellum T34, indicting tht MYB72 my be n essentil trnscription fctor for ISR for wide vriety of microorgnisms (Segrr et l., 2009, Vn Wees et l., 2008). The signling components required to communicte the MYB72 signl to distl plnt prts signl re currently unknown Biologicl ctivtors of ISR Induced resistnce is often ssocited with PGPRs including Bcillus spp., Pseudomons spp., nd members of Actinobcteri, such s Streptomyces, Scchropolyspor nd Micromonospor spp. (Bent, 2006, Hllmnn & Berg, 2006). Bcillus spp. re grm-positive, erobic, nd endospore forming bcteri in the phylum Firmicutes nd re ubiquitous in rhizospheric soils (Govindsmy et l., 2010, Kloepper et l., 2004). Common PGPR species tht ctivte ISR include B. subtilis, Bcillus pumilus, Bcillus myloliquefciens, nd Bcillus cereus (Bent, 2006). Direct ntimicrobil effects ginst pthogens nd insects re lso reported for certin strins of Bcillus spp. (Govindsmy et l., 2010, Kloepper et l., 2004). Pseudomons spp. nd Pseudomons-like gener (Burkholderi, Rlstoni, Acidovorx, Commons) re lso ubiquitous in the rhizosphere (Hs & Defgo, 2005, Weller, 2007). Pseudomons spp. re members of clss Gmmproteobcteri nd re grm-negtive nd erobic (Hs & Defgo, 2005). Common PGPR species tht induce ISR re Pseudomons ureofciens, Pseudomons chlororphis, P. fluorescens, P. putid, P. brssiccerum, P. thevervlensis, nd nonpthogenic strins of P. syringe (Bent, 2006, McSpdden-Grdener, 2007). Similr to Bcillus spp., some Pseudomons strins lso directly ntgonize plnt pthogens nd insects (Hs & Defgo, 2005, Sveeth et l., 2010, Sevim et l., 2012, Weller, 2007). The Actinobcteri PGPRs tht induce ISR re Streptomyces, Scchropolyspor, nd Micromonospor spp. tht re filmentous, grm-positive bcteri tht re ubiquitous in soils nd cn lso cuse direct ntgonism ginst plnt pthogens nd insects (Conn et l., 2008, El-Trbily et l., 2010, Pn et l., 2011, Qin et l., 2011, Verm et l., 2011). The plnt defense ctivtion by Bcillus spp. is most commonly ssocited with ISR nd is independent of SA nd dependent on JA, ET, nd NPR1 (Govindsmy et l., 2010, Kloepper et l., 2004). However, specific ctivtion mechnisms differ between strins, host plnt nd pthogen. In some 28

52 cses, induced resistnce from Bcillus spp. is ssocited with the SA pthwy (Govindsmy et l., 2010, Kloepper et l., 2004, Lnn-Filho et l., 2017). These bcteri my lso be ntgonistic towrd microorgnisms nd certin insects vi the production of ntibiotics (Govindsmy et l., 2010). Mny, but not ll, PGPRs cn invde roots to become endophytes, nd these bcteri cn lso ctivte ISR. Rhizobium spp., which belong to clss Alphproteobcteri nd form nodules with plnts fixing tmospheric nitrogen (Kdo, 2010), re the best studied bcteril endophytes of plnt roots (Zmioudis & Pieterse, 2012). At lest one species, Rhizobium etli, is cpble of inducing ISR in tomto (Mrtinuz et l., 2012). In n incomptible Rhizobium-legume interction, host resistnce (R) proteins recognize Rhizobium effectors nd ctivte ETI nd nodultion fils to occur. However, in susceptible legume roots, Rhizobium secrete microbe ssocited moleculr ptterns (MAMPs) which re detected by PRRs (leucine-rich repet receptor-like-kinses) triggering PTI (lso known s microbe-ssocited moleculr pttern triggered immunity; MTI). However, surfce polyscchrides nd protein effectors relesed by Rhizobium spp. counterct PTI, perhps resulting in response nlogous to ETS. The recognition of Nod fctors secreted by the bcteri results in symbiotic plnt gene expression reprogrmming, which further prevents PTI. Rhizobium peptides produced t infection sites re trnsported to the shoot nd perceived by leucine-rich repet receptor-like kinses resulting in the production of plnt molecules tht re trnsported bck to the roots vi the phloem tht restrict nodule formtion. This process is known s utoregultion of nodultion (AON) (Ok-Kir & Kwguchi, 2006, Stehelin et l., 2011). AON shres similrities with ISR nd SAR s ll three rections re triggered by locl infection of n orgnism nd result in systemic response tht limits coloniztion in distl plnt prts (Zmioudis & Pieterse, 2012). Studies of Rhizobium spp. indictes tht the interctions mong endophytes nd PGPRs tht elicit ISR re similr to the wy plnts interct with pthogens, nd involve the recognition of PAMPs nd the elicittion of PTI, ETS nd ETI (Zmioudis & Pieterse, 2012, Vn Wees et l., 2008). Interctions between PGPFs nd plnts pper to shre similrities with endophytes nd PGPRs (Zmioudis & Pieterse, 2012). In tomto, reports of ISR by bcteril endophytes include Acinetobcter johnsonii, S. mrcescens, Sinorhizobium sp. nd B. megterium tht induced resistnce ginst erly blight (A. solni) nd bcteril speck pplied to cut stems (Brretti et l., 2009), nd Bcillus pumilus SE34 tht induced resistnce ginst Fusrium oxysporum f. sp. rdicis-lycopersici (Benhmou et l., 1998). For the lter, the endophyte induced ultrstructurl chnges in tomto, such s the production of root wll ppositions nd electron-dense substnces tht re ssocited with resistnce. Inocultion of tomto with endophytes from other plnt species cn lso hve positive effects on reducing disese. For exmple, bcteril endophytes from oilseed rpe, grpe, wtermelon, nd ppy reduced wilt symptoms cused by F. oxysporum f. sp. lycopersici (Nejd & Johnson, 2000) or R. solncerum (Thoms & Upreti, 2014). In most cses, ISR 29

53 by bcteril endophytes is ssumed; however, in some cses, SAR is suggested bsed on higher PAL ctivity (Lnn-Filho et l., 2017), s discussed erlier in this review. In ddition to bcteri, some fungi cn lso ctivte ISR. Trichoderm spp. colonize the root surfce nd re lso endophytes of roots (Druzhinin et l., 2011, Shoresh et l., 2010). Induced resistnce in T. sperellum cucumber system implicted JA- nd ET-dependent signlling in the defence response, which is consistent with ISR (Shoresh et l., 2010). However, gene expression nlysis of tomto inoculted with T. hrzinum 382 reveled upregultion of 36 genes including the SAR mrker, PR5, but not the ISR mrkers, Lox1, ETR1, or CTR1 (Alfno et l. 2007). Another group of ISR-relted fungi re the Sebcinles fungi, such s Piriformospor indic (Shoresh et l., 2010, Zmioudis & Pieterse, 2012). P. indic is root colonizing filmentous Bsidiomycete tht colonizes wide rnge of plnts, including A. thlin (Zuccro et l., 2011). Like R. etli, P. indic my lso initilly ctivte plnt defenses during root coloniztion but then suppress PTI nd ETI through s of yet not well understood mechnisms (Zmioudis & Pieterse, 2012). A third group of ISR-relted fungi re the vesiculr rbusculr mycorrhiz (AM) nd ectomycorrhiz fungi (ECM) (Brundett, 2006). Reports of ISR ssocited with AM nd ECM include reduction in the number of penetrtion sites by root-knot nemtode (Meloidogyne incognit) in tomto in plnts treted with the AM fungus, Glomus mossee (Vos et l., 2012), induction of PR genes in potto growing in the presence of AM fungus, Glomus irregulr, nd inoculted with Fusrium smbucinum (Ismil & Hijri, 2012), nd reduction in the severity of poplr cnker in poplr trees growing in the presence of G. mossee nd the ECM Boletus luridus (Zhn et l., 2010) PAMP ctivtors of ISR In ddition to living cells, bcteril PAMPs cn lso elicit ISR. The voltile orgnic compound (2R,3R)-butnediol, relesed by B. subtilis GB03 nd B. myloliquefciens IN937 cts s PAMP to trigger ISR in the A. thlin - Erwini crotovor subsp. crotovor system (Ryu et l., 2003). Seedlings exposed to trnsgenic B. subtilis tht emitted low levels of (2R,3R)-butnediol hd higher disese levels thn non-trnsgenic lines, nd hd no disese suppressing effect in ET insensitive line but not n JA insensitive line nd n NhG line, indicting tht (2R,3R)-butnediol elicits ISR in A. thlin vi n ET-dependent pthwy (Ryu et l., 2004). Exogenous pplictions of (2R,3R)-butnediol, which is produced by some Bcillus spp., induced ET-dependent ISR in A. thlin ginst E. crotovor subsp. crotovor, in Nicotin benthmin ginst Colletotrichum orbiculre, nd in Agrostis stolonifer ginst Microdochium nivle, Rhizoctoni solni, nd Sclerotini homoecrp (Ryu et l., 2004, Cortes- Brco et l., 2010). The voltile orgnic compound cetoin (3-hydroxy-2-butnone), lso relesed by B. subtilis, is implicted in ISR ginst Pst in A. thlin, nd when relesed by B. myloliquefciens nd B. subtilis induced ISR ginst E. crotovor subsp. crotovor in A. thlin (Rudrpp et l., 2010, Ryu et 30

54 l., 2004). PAMPs re lso importnt chrcteristics of Pseudomons spp. with the bility to induce resistnce in plnts, nd include flgellins, the min protein of flgell, LPS, iron-regulted metbolites (siderophores), N-lkylted benzylmine derivtive, nd the phenolic compound 2,4- dicetyphloroglucinol (DAPG) (Bkker et l., 2007, Schreiber & Desveux, 2008). However, mny of these mechnisms re not universl triggers mong Pseudomons spp, nd the exct mode of ction of Pseudomons spp. my depend on the specific strin, host nd pthogen. For exmple, the flgellins of P. putid WCS358 triggered ISR ginst Pst in A. thlin but not in ben or tomto, nd mutnt WCS358 lcking flgellin lso induced resistnce in A. thlin (Gl et l., 2003). Siderophores, which re lowmoleculr weight Fe 3+ cheltors, lso directly compete with plnt pthogens for iron cting in nother mode for disese suppression (Duffy & Defgo, 1999, Weller, 2007). In ddition, DAPG is produced by some fluorescent Pseudomons spp. nd is n ntibiotic tht is lso ssocited with induction of ISR, but lso cts directly s n ntgonist of plnt pthogens (Cronin et l., 1997, Notz et l., 2001). PAMPs produced by fungi tht ctivte ISR re not s well understood s those of PGPRs nd bcteril endophytes, but re thought to include chitin, cellulose, cutinse, glucns, xylnse, nd the hydrophobin-like elicitor Sm1, peptibols, swollenin nd certo-pltnins (Druzhinin et l., 2011, Schreiber & Desveux, 2008). Extrcts from other orgnisms my lso elicit ISR, s demonstrted by the tretment of A. thlin roots with extrcts from the brown microlg, Ascophyllum nodosum, which induced resistnce ginst Pst nd S. sclerotiorum in JA-dependent mnner (Subrmnin et l., 2011). The plnt compounds, glctinol, ET precursor 1-minocyclopropne-1-crboxylte (ACC), nd methyl jsmonte (MeJA) re ll linked to ISR (Kim et l., 2008, Pozo et l., 2004, vn Wees et l., 1999). Exogenous pplictions of JA cn induce ISR, such s tretment of tomto seeds with JA resulting in incresed resistnce to B. cinere, which ws JA-, ET- nd bsicisic cid dependent (Worrll et l., 2012) Synthetic ctivtors of ISR The isoprffin mixture PC1, which is mrketed in North Americ s Civits, is mixture of foodgrde synthetic isoprffins nd food-grde emulsifiers nd does not hve direct ntimicrobil effects (Cortes-Brco et l., 2010). Gene expression studies hve reveled tht reductions in disese severity in N. benthmin ginst C. orbiculre, nd in A. stolonifer ginst M. nivle, R. solni, nd S. homoecrp, re consistent with ISR, lthough the mechnisms of priming nd gene expression differed from (2R,3R)-butnediol (Cortes-Brco et l., 2010, Cortes-Brco et l., 2010b). Thus fr, the mode of ction of Civits is unknown Plnt growth effects ssocited with ISR The priming tht results from ISR ctivtion hs dvntges over induction of gene expression nd 31

55 production of PR nd other proteins tht occurs with SAR (Pieterse et l., 2009, Vn Wees et l., 2008). vn Hulten et l. (2006) reports tht fitness costs ssocited with ISR re not s high s those observed through induction of SAR in A. thlin, becuse the system is primed nd thus no energy is diverted to the production of plnt defenses in the bsence of pthogen. Priming my be dvntgeous in both nturl nd griculturl systems by reducing the net plnt fitness cost ssocited with defense. In A. thlin, exogenous pplictions of 10 mg/l nd 25 mg/l of BABA, which ctivtes type of resistnce is not controlled by the sme signlling pthwy s ISR, only primed PR1 gene expression, while ASM or higher concentrtion of BABA (60 mg/l) ctivted PR1 gene expression immeditely. At the lower levels of BABA, the plnts produced the sme number of seeds s the wter control under disese-free conditions, nd the 25 mg/l tretment produced more seeds thn the wter control when chllenged with P. syringe or Hyloperonospor prsitic (vn Hulten et l., 2006). In contrst, direct induction of PR1 with ASM or 60 mg L -1 BABA resulted in lower seed production thn the wter control in disese-free conditions, nd no increse in seed production under pthogen chllenge. It is tempting to speculte tht similr dvntges of priming would be extended to ISR. Repeted pplictions of T. hrzinum T39 under disese-free conditions resulted in no negtive effects in plnt growth or chlorophyll content in greenhouse-grown grpevines, which is in contrst to repeted pplictions of ASM tht negtively ffected these prmeters (Perzzolli et l., 2011). T. hrzinum T39 ppers to prime the expression of some but not ll PR genes. In grpevine treted with T39, systemic expression of PR2 nd PR10 incresed bove levels of the nontreted control fter inocultion with Plsmopor viticol, but systemic expression of PR4 nd LOX9 incresed slightly bove levels in control plnts prior to pthogen chllenge. T39 lso upregulted the JA- nd ET-dependent signling pthwys, which is lso consistent with priming (Perzzolli et l., 2011). However, it is possible tht fitness costs could occur with ISR. The effectiveness of mycorrhiz-induced resistnce ginst B. cinere in tomto by the rbusculr mycorrhizl fungus, Rhizophgus irregulris, which is similr to ISR, ws reduced under low nitrogen (Snchez-Bel et l., 2016) indicting tht host nitrogen sttus my ffect the response to coloniztion by PGPR nd bcteril endophytes. For bcteril endophytes tht induce ISR, there re no reports of fitness costs, nd reports of plnt growth promotion re frequent (Hrdoim et l., 2015), including tomto (Pilly & Nowk, 1997, Brretti et l., 2009, Amresn et l., 2012, Nv-Diz, 2006). Plnt growth promotion by endophytes is ssocited with increses in the vilbility of nitrogen nd phosphorous nd modulting levels of phytohormones suhc s IAA, cytokinins, nd ET (Hrdoim et l., 2015). Plnt growth promotion my lso be due to competition with plnt pthogens, such s the production of ntimicrobil compounds, nutrient competition, nd siderophore production (Hllmnn et l. 1997; Lugtenberg nd Kmilov 2009; Rosenblueth nd Mrtinez-Romero 2006). For exmple, Bcillus nd Arthrobcter species from tomto 32

56 cused plnt growth promotion nd this ws ssocited with IAA nd siderophore production, nd solubilized inorgnic phosphte (Amresn et l., 2012) Fctors ffecting ISR Induction of ISR by competent microorgnisms occurs in diverse plnt species, but specific plntmicrobe interctions re dependent on gene-for-gene interctions mong the microorgnism nd its potentil host. The importnce of host genotype differences ws demonstrted by Ton et l. (1999) who observed tht P. fluorescens WCS417r filed to trigger ISR in two of 10 A. thlin ecotypes chllenged with Pst. Reciprocl crosses from the ISR-responsive Col-type nd the ISR-nonresponsive RLD reveled tht the F 2 segregtion pttern for susceptibility to Pst in plnts growing in P. fluorescens WCS417r mended soil ws 3:1. Further nlysis reveled tht inducibility of ISR nd reltively high bsl resistnce ginst the pthogen were geneticlly linked t the sme locus, ISR1, nd the 3:1 segregtion is likely the result of the sme dominnt gene. This gene ws lter found to cosegregte with trit for reduced sensitivity to ET but not JA, nd to be involved in WCS417r medited resistnce ginst Xnthomons cmpestris pv. morcie nd Phytophthor prsitic (Ton et l., 2001, Ton et l., 2002). Cultivr-dependent expression of trnscripts encoding defense-relted enzymes nd PR10 is lso reported in whet (Mketon et l., 2012). The men expression rtios of eight trnscripts were greter in cv. Buchnn thn cv. Tr within the first 24 hours fter inocultion with the P. fluorescens Q8r1-96. Although the study period ws reltively short nd the gene expression of the whet cultivrs ws not monitored fter chllenge with pthogen, the results still indicte tht host rection to rhizosphere coloniztion is dependent on the combintion of the host genotype nd the bcteril strin (Mketon et l., 2012). The influence of plnt genotype on ISR ginst B. cinere ws lso evluted fter T. hrzinum T22 nd T. troviride P1 seed tretment using two inbred processing tomto lines M82 nd TA209, the lndrce Corbrino, the dvnced breeding line SM26, nd the Peruvin ccession of the wild tomto species Solnum hbrochites LA1777 (Tucci et l., 2011). There ws significnt interction between the tomto lines nd the Trichoderm spp. with regrds to disese severity, nd further nlysis of gene expression reveled tht upregultion of mrker genes for both SA- nd JA-pthwys differed mong lines. Root coloniztion by PGPRs nd tissue coloniztion by endophytes cn be ffected by genotype. Pseudomons sp. PsJN colonized the tomto root surfce more for cultivr Celebrity thn for cultivrs Blzer, Scoti nd Mountin Delight (Pilly & Nowk, 1997). Root surfce popultions incresed linerly with incresing endophyte inocultion concentrtion from 4.6 x 10 7 to 8.8 x 10 8 CFU/mL, when seedlings were inoculted by suspending seedlings with trimmed root tops in the endophyte solution for 15 minutes. 33

57 Induced resistnce ws not exmined in this study, but there ws no reltionship between root surfce coloniztion nd plnt growth promotion. Endophytic coloniztion of inner root tissues ws lso highest t similr inoculum density (4 x 10 1 CFU/mL) but did not vry with tomto genotype. Inoculum levels (3 x 10 8 to 7 x 10 8 CFU/mL) tht promoted endophytic coloniztion were lso the best for promoting tomto seedling growth. The bility of the rhizobcteri nd endophyte B. pumilus SE34 nd rhizobcteri P. fluorescens 89B61 to induce tomto seedling growth nd resistnce ginst P. infestns ws linerly relted to incresing inoculum (rnging from 10 3 to 10 9 CFU/g soil mix) (Yn et l., 2000). The diversity of endophytes colonizing the bcteril wilt resistnt tomto cv. Ark Abh ws greter thn the susceptible cv. Ark Viks (Upreti & Thoms, 2015), nd coloniztion of the spermosphere by over 20 seed inoculted B. cereus strins differed mong inbred tomto lines (Simon et l., 2001). The reltionship between microbil popultions nd the induction of resistnce hve been contrdictory. Pilly nd Nowk (1997) proposed tht there is biostimultion threshold within tissues of inoculted tomto plnts, which could lso pply to PGPRs or bcteril endophytes for ISR induction. Endophyte concentrtions rnging from seedling soil drench t 1 x 10 9 CFU/ml for Azospirillium sp. 510 (Fujit et l., 2017), seed tretment t 1 x 10 9 CFU/ml for B. pumilus SE34 nd B. myloliquifciens IN937 (Ji et l., 2006), nd soil drench t 1 x 10 7 CFU/ml for B. pumilus SE34 nd B. myloliquifciens IN937 (Ji et l., 2006) induced resistnce ginst Pst in tomto. However, it is not cler to wht extent host coloniztion is required to chieve induced resistnce (Hrdoim et l., 2015). Coloniztion by the PGPR S. mrcescens nd P. putid 89B-27 resulted in greter reduction in C. orbiculre disese symptoms in older leves even though root coloniztion by these bcteri dropped over time. Endophyte coloniztion my lso be influenced by the timing nd method of inocultion. In tomto, seed sok or seed drench (Lnn-Filho et l., 2017, Ji et l., 2006), seedling drench (Fujit et l., 2017), nd combintions of seed tretment nd soil drenches (Ji et l., 2006) hve been used successfully. Plnt developmentl stge lso influences coloniztion of plnts by microorgnisms, lthough the reltionship of this with ISR hs not been explored. For exmple in vetiver, dizotrophic bcteril popultions were lower 1 month fter trnsplnting compred to 3, 6, or 12 months (Monteiro et l. 2011). In soyben, the vegettive stge of soyben hd higher popultions of phosphte solubilizing bcteri compred to the flowering nd senescence stges (Kuklinsky-Sobrl et l., 2004). Pseudomons spp. nd Actinobteri popultions were lower t the senescence stge in potto compred to flowering nd vegettive stges (vn Overbeek nd vn Elss 2008), nd popultions of B. subtilis peked before heding wheres popultions of other endophytes in the sme whet plnts remined constnt throughout ll developmentl stges (Comby et l., 2016). Thus, endophyte community composition nd diversity is dynmic nd ppers to be dependent on the host developmentl stge. This my be due to chnges in host defense (Andreote et l., 2010), the level of soluble crbohydrtes, clcium nd phenolic compounds 34

58 (Hunter et l., 2010), or strch content (Inceoglu et l., 2010) ISR s disese mngement tool There re numerous reports from greenhouse nd field-level reserch tht implicte ISR in economiclly vible levels of disese suppression. Mny Bcillus spp. reduce the severity of rnge of diseses nd rnge of plnts species (Govindsmy et l., 2010, Kloepper et l., 2004). Specific exmples from greenhouse nd field studies using either folir pplictions, soil drenches, or seed tretments include B. pumilus nd B. mycoides nd cercospor lef spot (Cercospor beticol) on sugrbeet, Bcillus spp. nd CMV, B. pumilus nd ngulr lef spot (P. syringe pv. lchrymns) on cucumber, nd B. pumilus nd lte blight on tomto (Kloepper et l., 2004). The level of disese reduction is vrible within individul Bcillus species, with some strins ppering more effective thn others (Kloepper et l., 2004). Other exmples of ISR-relted disese suppression by PGPRs in the field include sheth blight (R. solni) of rice by two strins of P. fluorescens, bcteril wilt (Erwini trcheiphill) of cucumber by Serrti mrcecens, ngulr lef spot of cucumber by S. mrcescens, P. putid, Flvomons oryzhbitns nd Curtobcterium flccumfciens, nthrcnose (C. orbiculre) of cucumber by Burkholderi gldioli nd C. flccumfciens, blue mold (P. tbcin) of tobcco by P. fluorescens nd S. mrcesens, botrytis (B. cinere) of bens by P. eruginoz, nd lef spot (X. cmpestris pv. rmorcie) of rdish by Pnteo gglomerns (Bent, 2006, Vlld & Goodmn, 2004). For endophytic bcteri, there re lso mny reports of disese suppression. For exmples, screening of 150 isoltes reveled tht two endophytes closely relted to Pseudomons rhodesie nd Pntoe nntis promoted plnt growth nd induced resistnce to Xe in one pepper cultivr, lthough the mechnism of resistnce ws not explored further (Kng et l., 2007). Similrly, four of 190 bcteril endophytes isolted from wtermelon roots in Vietnm were ssocited with reducing gummy stem blight becuse of increses in hydrogen peroxide nd peroxidse levels (Ng et l., 2010). The bility of endophytes to induce resistnce in hrvested fruit is lso documented in P. putid MGY2 nd ppy ginst Colletotrichum gloesporides becuse of decreses in fruit symptosm nd increses in phenyllnine mmoni-lyse, ctlse, peroxidse, nd phenolics, nd expression of PAL1, CAT1, nd POD genes (Shi et l., 2011). Some PGPRs nd endophytes hve been developed s commercilly vilble products. For endophytes, there is YieldShield (B. pumilus strin INR7) (Kloepper et l., 2004, Govindsmy et l., 2010, Kloepper & Ryu, 2006). For PGPRs, there is Ecogurd (Bcillus licheniformis), Kodik (B. subtilis GB03), Subtilex (B. subtilis MBI600), HiStick (B. subtilis MBI600 nd Rhizobium sp.) (Govindsmy et l., 2010), Bio-Sve (P. syringe ESC-10), (PMRA, 2010) nd BlightBn A506 (P. fluorescens A506) (PMRA, 2009). However, it is possible tht severl of these PGPRs could lso be endophytes. BioYield is 35

59 combintion product contining endophyte B. myloliquefciens IN937 nd PGPR B. subtilis GB03 (Kloepper et l., 2004, Govindsmy et l., 2010). Although Pseudomons spp. possess number of positive chrcteristics such s rpid growth in vitro nd good bility to compete well with other microorgnisms, they do not produce endospores like Bcillus spp. This hs led to chllenges in developing formultions tht hve long shelf lives (Weller, 2007). For exmple, Biosve cn be stored up to one yer t 4 C nd BlightBn cn be stored up to one yer t -27 C (PMRA, 2010, PMRA, 2009). Commercilly vilble PGPFs re T. sperellum T34, T. hrzinum ATCC 20476, T. hrzinum Rifi T-22, T. hrzinum T-39, nd Trichoderm polysporum ATCC (EPA, 2012), nd T. hrzinum Rifi strin KRL-AG2, which is mrketed in Cnd s Rootshield (PMRA, 2010b). Trichoderm spp. hve issues relted to shelf life. For exmple, Rootshield must be refrigerted nd used within twelve months of mnufcturing ISR s disese mngement tool in tomto For tomto fungl disese control, twelve PGPF Trichoderm spp. isoltes reduced the lef re ffected by erly blight (A. solni) when dded to the growing medi seven dys before pthogen inocultion (Fontenelle et l., 2011). Inocultion of tomto growing medi with the bcteril endophytes Acinetobcter johnsonii, S. mrcescens, Sinorhizobium sp., nd Bcillus megterium lso reduced the severity of disese symptoms cused by A. solni (Fontenelle et l., 2011). For tomto virl diseses, the endophyte B. pumilus SE34 nd B. myloliquefciens IN937 nd PGPR B. subtilus IN937b reduced severity of CMV nd tomto mottle virus (Vlld & Goodmn, 2004). For tomto nemtode control, soil drench of four endophytic bcteri, P. gglomerns MK-29, Cedec dvise MK-30, Enterobcter spp. MK-42 nd P. putid MT-19, reduced juvenile penetrtion nd the number of root glls by M. incognit (Vlenzuel-Soto et l., 2010, Munif et l., 2001). It thus ppers tht PGPFs, PGPRs, nd endophytes in tomtoes cn suppress vriety of diseses nd pests, lthough ISR hs not been clerly demonstrted in ll cses. For tomto bcteril disese control, greenhouse experiments demonstrted tht seed tretment followed by drenching trnsplnt medi with the rhizobcteri P. putid 89B61 reduced the severity of bcteril wilt in two experiments. The effectiveness of formultion contining the rhizobcteri B. subtilis GB03 nd endophyte B. myloliquefciens IN973, nd nother formultion contining the endophyte B. pumilus SE34 ws inconsistent (Anith et l., 2004). For Pst control, the PGPRs, P. syringe TLP2, P. syringe Cit7, nd P. fluorescens A506, were pplied to folige in greenhouse screening nd field trils in North Americ (Wilson et l., 2002). P. syringe Cit7 ws the most consistent biologicl control gent evluted nd reduced disese symptoms by n verge of 28% in field trils, possibly becuse of preemptive competitive exclusion on lef surfce (Wilson et l., 2002). However, due to the 36

60 numerous reports in the literture reporting the induction of resistnce by vrious Pseudomons spp., it my be possible tht these gents lso ct through ISR mechnisms. In ddition, seed tretment nd soil drenching with the PGPRs, P. putid 89B61, B. psteurii M38, B. cereus, 83-6, Burkholderi gldioli IN26, Stenotrophonoms mltophili IN287, B. cereus M-22, nd endophytes B. pumilus SE34, B. myloliquifciens IN937, nd B. megterium reduced the incidence of Pst infection in greenhouse screening ssys (Brretti et l., 2009, Ji et l., 2006). Combining 89B61 with P. syringe Cit7 resulted in better disese control in one greenhouse experiment, but not second, compred to 89B61 lone (Ji et l., 2006). In field experiments using the sme ppliction methods, Cit7 reduced Pst severity in three of three trils, 89B61 ws effective in two of three trils, nd SE34 ws not consistently effective (Ji et l., 2006). The effectiveness of seed tretment following by soil drench with these microorgnisms t reducing the severity of BSX ws lso reported, where SE34, 89B61, nd Cit7 ll reduced disese severity in two trils. For both Pst nd BSX control evlutions, the bcteri rrely provided disese control tht ws equivlent or better thn copper + mncozeb stndrd (Ji et l., 2006). The disese severity nd Pst popultion in plnt ws lso reduced in tomto following seedling drench inocultion of the endophyte Azospirillum sp. B510 from rice (Fujit et l., 2017). In series of field experiments cross North Americ, the efficcy of weekly folir pplictions of P. syringe Cit7 nd P. putid B56 were lso evluted for BSX control (Byrne et l., 2005). The severity of BSX symptoms on folige nd fruit ws reduced in Cit7 treted tomtoes in five of nine experiments nd one of five experiments (Byrne et l., 2005). Disese suppression rnged from 28 to 44% for folige nd 77% for fruit. Likewise, weekly folir pplictions of B56 reduced the severity of BSX on folige nd fruit in seven of nine experiments nd one of five experiments (Byrne et l., 2005). B56 reduced the severity of BSX from 16 to 34% on folige, nd 67% on fruit. Applictions of B. subtilis QST 713 reduced the severity of BSX below tht of the notreted control in two of four field trils completed in Florid (Roberts et l., 2008). In both cses where it reduced the severity of BSX, it ws lso equivlent to copper + mncozeb stndrd. In ddition to PGPRs, ppliction of PGPF Trichoderm spp. to the growing medi tomtoes growing under greenhouse conditions successfully reduced the severity of BSX reducing disese from 24 to 95% for the twelve isoltes tested (Fontenelle et l., 2011). Amendments with one isolte successfully reduced disese severity when pplied three, seven, 14, or 21 dys prior to inocultion with Xe. There re currently no commercilly vilble biocontrol gents mrketed s bcteril endophytes for tomto disese mngement. However, further reserch in this re my yield benefits s endophytes re well dpted to colonize plnts nd there my be secondry benefits of endophytes such s plnt growth promotion (Mercdo-Blnco & Lugtenberg, 2014). 37

61 1.8.3 Interctions nd reltionships between SAR nd ISR pthwys A significnt level of cross-tlk exists between SA/JA, JA/ET, nd ET/SA signling pthwys my further complicte the interctions between phytohormone levels nd ctivtion of SAR nd ISR (Pieterse et l., 2009). SA is ntgonistic towrd JA-responsive genes in A. thlin (Pieterse et l., 2009, Gimenez-Ibnez & Solno, 2013). Suppression of JA/ET defense signling is linked to n increse in disese symptoms by the necrotrophic fungi Alternri brssiccol nd Rmulri collo-cygni in A. thlin fter prior inocultion with Pst nd in brley fter prior ppliction with ASM, BABA, nd cisjsmone, respectively (Spoel et l., 2007, Wlters et l., 2011). In ddition, SA nd JA re considered positive nd negtive regultors of stomtl defense, respectively (Melotto et l., 2017). On the other hnd, ET-SA signling nd ET-JA signling re often synergistic, nd in the cse of ET nd JA there is often n integrted response with the sme trnscription fctors mediting response (Pieterse et l., 2009). There is lso evidence linking ABA, IAA, GA nd cytokinins to the SA, JA nd ET defense signling pthwys, nd these dditionl phytohormones cn lso be modulted by some PGPRs nd bcteril endophytes (Hrdoim et l., 2008, Pieterse et l., 2009, Zmioudis & Pieterse, 2012). For exmple, ABA is reported to weken JA/ET-dependent gene expression nd hve role in JA biosynthesis during the response of A. thlin to ttck by Pythium irregulr, but ABA incresed susceptibility of A. thlin to Pst by suppressing the induction of SAR (Adie et l., 2007, Mohr & Chill, 2007). ABA is lso considered positive regultor of stomtl defense (Melotto et l., 2017). GA my interct with the SA-JA-ET network by modulting levels of proteins responsible for promoting susceptibility to biotrophs nd resistnce to necrotrophs (Nvrro et l., 2008). Combintions of SAR nd ISR ctivtors my lso led to synergistic or ntgonistic response. Koornneef et l. (2008) demonstrted tht SA is cpble of suppressing JA-responsive genes t certin concentrtions nd timings, suggesting possible ntgonisms between the SAR nd ISR signling pthwys. Conversely, induction of SAR nd ISR in A. thlin resulted in lower disese index for Pst thn SAR or ISR plnts lone (vn Wees et l., 2000) nd ppliction of the PGPR P. putid 89B61 to mrnthus limited fitness cost effects by ASM (Nir et l., 2007). The complex nd not yet fully understood mechnisms of cross-tlk between SAR nd ISR pthwys no doubt contribute to the mixed results of combining SAR nd ISR ctivtors. Combintions of SAR nd presumed ISR ctivtors hve lso been tested under field conditions in tomto. Combintions of ASM + P. fluorescens A506 resulted in reduction in the incidence of Pst infections on tomto leves tht ws greter thn ASM or P. fluorescencs lone, but the severity of Pst on folige ws not lower thn ASM lone (Wilson et l., 2002). In seprte experiment, ASM combined 38

62 with P. syringe Cit7 resulted in lower disese severity thn ASM lone on one ssessment dte, but combintions of ASM + P. syringe TLP2 nd ASM + P. fluorescens A506 provided the sme level of disese control s ASM lone (Wilson et l., 2002). Combintions of ASM nd mixture of the PGPR B. subtilis GB03 nd endophyte B. myloliquefciens IN937 for Pst control in tomto resulted in synergies in only one of three experiments (Hermn et l., 2008). 1.9 GA-relted PGRs PGRs re chemicl compounds tht increse or decrese levels of phytohormones in plnts upon ppliction resulting in chnges in plnt growth. Severl PGRs hve been commercilized nd re used in the production of food nd ornmentl crops. These include the trizole group growth regultors, UNI nd pclobutrzol, which re mrketed s Sumgic nd Bonzi, respectively, to inhibit GA formtion, nd there re lso GA products, mrketed s Activol nd Flgro. The trizole-type PGRs re involved in inhibiting the formtion of GA in plnts (Rdemcher, 2000). GAs re implicted in longitudinl shoot growth, germintion, bolting, nd fruit set nd development, thus ppliction of GA inhibitors re pplied to food nd ornmentl crops to prevent stem elongtion (Rdemcher, 2000). For exmple, pclobutrzol nd UNI prevent stem elongtion in greenhouse tomto trnsplnts, which prevents the production of over-sized plnts tht re difficult to ship nd trnsplnt into the field (Zndstr et l., 2006, Agehr & Leskovr, 2017). UNI nd pclobutrzol re structurlly similr, nd both inhibit GA biosynthesis by blocking the cytochrome P-450-dependent monooxygenses, which prevents the oxidtion of ent-kurene into ent-kurenoic cid in the GA biosynthesis pthwy (Rdemcher, 2000). UNI cuses mny plnt physiologicl responses, including those relted to reductions in plnt stress. UNI reduced symptoms of chilling injury in tomto, slt stress in Dtur spp., nd drought stress in soyben nd whet (Al-Rumih & Al-Rumih, 2007, Dun et l., 2008, Senrtn et l., 1988, Zhng et l., 2007). These stress tolernces re linked to n increse in ntioxidnt levels, protein content, photosynthetic rte, nd chlorophyll content, s well s chnges in other phytohormone levels. These effects my be relted to the fct tht some enzymes involved in GA biosynthesis lso cuse inhibition of ABA, inhibition of ET, nd chnges in sterol production which serve multiple functions in plnt physiology (Fletcher et l., 2000, Rdemcher, 2000, Todoroki et l., 2008). The effects of UNI cn be long term, s tretement t the seedling stge incresed dys to flower in Argyrnthemum nd scevol nd reduced flower number in clibrocho, scevol, nd verbn t high concentrtions (Blnchrd & Runkle, 2007). Thus, PGRs such s UNI cn hve multiple beneficil effects on plnt growth nd development. Conversely, GAs re pplied to some griculturl crops to dely ripening, increse shoot 39

63 development, reduce tuber size, or void long vernliztion periods. GA increse fruit size, weight nd firmness in cherry, which cn enble lter hrvest without the loss of fruit qulity (Cnli & Orhn, 2009, Cline & Trought, 2007). Appliction of GA to pottoes results in smller tuber size, s tuber initition is delyed in fvour of shoot growth (Lovell & Booth, 1967). Furthermore, pplictions of GA to rhubrb crown cn result in erlier emergence nd higher yield in some cultivrs (Mynrd, 1990). There is only one report on the effect of GA-PGRs on the effectiveness of induced resistnce ginst plnt disese. Applictions of pclobutrzol with the plnt ctivtor ASM reduced symptoms of Pst on tomto leves under greenhouse conditions to greter extent thn ASM lone, lthough the mechnism of this synergy ws not exmined (Mhesniy, 2002). This is not surprising given the known effects of PGRs on phytohormones, nd the ssocition of phytohormones either directly or indirectly with the plnt defense system. In summry, plnt genotype, GA-PGRs, ctivtor ppliction method, ctivtor concentrtion nd novel chemicl nd biologicl ctivtors ll hve the potentil to increse the effectiveness of induced resistnce of tomto ginst pthogens, such s Pst nd BSX. These fctors cn be explored using controlled conditions nd field studies to better understnd induced resistnce t the whole plnt level Hypothesis Induced resistnce of tomto ginst Pst nd BSX cn be chieved by using lterntive ctivtors, such s PABA nd endophytes from tomto, nd the level of effectiveness of n ctivtor requires optimiztion for fctors such s ppliction method, concentrtion nd host genotype. In ddition, induced resistnce of tomto ginst Pst nd BSX by the well-estblished ctivtor ASM cn be improved by combining it with the PGR, UNI, to reduce possible stresses creted by ASM Objectives 1. Evlute the effects of PABA on inducing resistnce ginst Pst nd optimize for its effectiveness by evluting the impct of ppliction method, timing, number of pplictions, concentrtion nd plnt genotype. 2. Exmine combintoril effects of UNI nd ASM on reducing the severity of bcteril folir diseses of tomto under field conditions, nd exmine chnges in plnt growth nd development ssocited with the combintion. 3. Screen tomto root endophytes for their bility to induce resistnce ginst Pst nd optimize their effectiveness by exmining inocultion timing, inocultion method, endophyte concentrtion nd host genotype. 40

64 2 Chpter 2: Effects of pr-minobenzoic cid on the incidence of bcteril speck disese, P. syringe pv. tomto growth, nd plnt growth in processing tomto 2.1 Introduction The folic cid precursor PABA, lso known s 4-AA, is benzoic cid derivtive synthesized by plnts, fungi, bcteri nd protistns (Bsset et l., 2004). PABA is lso known s vitmin H 1, B x nd B 10 s it is member of the B vitmin group which lso includes thimine (vitmin B 1 ) nd riboflvin (vitmin B 2 ). PABA is vitmin becuse it is folte precursor nd foltes re essentil nutrients tht cnnot be synthesized by nimls (Biley & Gregory, 1999). Synthesis of PABA by plnts occurs in chloroplsts, nd it is then exported to the cytosol where it is reversibly esterified nd then tken up by the mitochondri for folte synthesis (Quinlivn et l., 2003, Bsset et l., 2004). Foltes re essentil cofctors for one-crbon rections in plnts, but endogenous levels of PABA re low, such s t the nm level in tomto leves nd fruit (Quinlivn et l., 2003, Bsset et l., 2004). PABA, s well s the relted compounds thimine nd riboflvin, re lso ssocited with SIR in plnts, which is brodly defined s enhnced resistnce of plnt to pthogen fter being stimulted by biotic or biotic gent (Vn Loon, 1997). Induced resistnce is n ttrctive lterntive to conventionl pesticides for plnt disese mngement becuse mny conventionl pesticides hve negtive environmentl nd humn helth impcts nd there re chllenges with effectiveness due to the evolution of pesticide resistnce by pthogens (Wlters & Fountine, 2009, Osborn, 2012). The two mjor types of induced resistnce in plnts re SAR nd ISR. They re distinguished by the phytohormone signling pthwys, which SA for SAR versus ET nd JA for ISR (Pieterse et l., 2009). The two types lso differ s biotrophic pthogens tend to be suppressed by SAR wheres necrotophic pthogens suppressed by ISR (Pieterse et l., 2009, Ton et l., 2006). Hemibiotrophic pthogens such s those tht cuse bcteril lef spots my be suppressed by both SAR nd ISR (Ton et l., 2006). The first report of pplying PABA for mngement of plnt diseses ws for whet stripe rust (Puccini striiformis) in Chin (Kelmn & Cook, 1977); however, detils on its use pttern nd mode of ction were not described nd so it is not cler whether the reserchers considered the control to be due to induced resistnce. More recently, soil drench pplictions of PABA to pepper seedlings reduced the severity of the hemibiotroph Xe/Xp in leves in greenhouse experiments nd one field experiment (Song et l., 2013). Induced resistnce by PABA ws inferred from n increse in expression of defense relted genes of pepper by PABA ppliction fter inocultion compred to control plnts. The form of induced resistnce ws considered to be SAR rther thn ISR since the expression of the SA signlling mrker defense genes CPR4 nd CPR9 were primed by PABA, but not the expression of Pin2, which is relted to JA signlling, or Tin1, which is relted to ET signlling. Yng et l. (2011) showed tht root pplied 41

65 PABA reduced the severity of the necrotroph, Pectobcterium crotovorum subsp. crotovotrum, in tobcco seedlings. PABA lso reduced the incidence of the hemibiotrophic bcteril speck pthogen, Pst in tomto, which ws ssocited with induction of the SAR mrker gene SlPR1 fter pthogen inocultion but not the induction or priming of ISR mrker genes, SlPin2 nd SlPR2b (Tzhoor, 2014). Induction of SAR is sometimes ssocited with plnt fitness cost (i.e., reduced growth nd yield) since the defense response requires nutrients nd energy tht would otherwise be used for plnt growth nd development (Durrnt nd Dong 2004; Wlters nd Heil 2007). It is unknown if this occurs with PABA. Soking seeds in PABA solution stimulted seed germintion in winter whet nd winter brley (Bekusrov et l., 2013). A. thlin seeds sown on PABA-mended gr medi hd reduced root length with incresing PABA concentrtion, n increse in lterl roots t low concentrtions, nd decrese in lterl root growth t higher concentrtions possibly due to its uxin-like root growth regulting ctivity t 0.02 to 2 mm (Crisn et l., 2014). However in nother study, A. thlin seeds grown on 0.1 mm PABA mended gr medi hd no effect on root length (Hong et l., 2007). Thus, PABA my hve effects on the growth nd development of plnts but these effects pper to be dependent on concentrtion nd my be more relted to its plnt growth hormone ctivity. Also, such studies hve not tested PABA t the often higher concentrtions used for SAR, such s 0.1 to 10 mm in pepper, 1 nd 18 mm in tobcco, nd 18 mm in tomto (Song et l., 2013, Yng et l., 2011b, Tzhoor, 2014). Although there hve been three studies on the mode of ction of PABA nd its use under controlled conditions, only one study (Song et l., 2013) tested it under field conditions showing tht single soil drench ppliction of PABA reduced disese severity cused by the hemibiotrophic pthogen Xe/Xp nd the biotrophic pthogen CMV resulting in incresed yield. However, this ws done only for one field seson. If PABA is to be used in the field, number of fctors should be ssessed. The objective of this study ws to evlute these fctors including; PABA concentrtion, plnt cultivr, ppliction method, number of pplictions nd pthogen inocultion timing on disese incidence of bcteril speck (Pst) on tomto, s well s exmine plnt growth of PABA-treted plnts under growth chmber nd field conditions. 2.2 Mterils & Methods Direct ntimicrobil effects of PABA on Pst For the filter pper ntimicrobil ssy, 0, 1.0, 9.0, 18.0, nd 27.0 mm PABA, nd 0, 18.0, nd 72.0 mm PABA were prepred by dissolving in distilled wter or 70% ethnol, respectively. Filter discs (1.2 or 2.5 cm qulittive P5, Fisher Scientific, Pittsburg, PA) were then soked in ech PABA solution, blotted to prevent dripping, nd plced on TSA plte covered with 100 µl of Pst from n overnight TS 42

66 broth culture. Pltes were incubted for three dys nd ssessed for the presence of zone of inhibition. The zone of inhibition t three points ws mesured for ech disc nd the men clculted. The experiment ws repeted three times, with 10 pltes per experiment, ech contining one disc of ech PABA concentrtion Growth room evlution of folir pplictions of PABA ginst bcteril speck Optiml concentrtion Tomto cv. H9909 ws sown in 3.5-inch squre pots filled with Ffrd germintion mix (Ffrd et Frères, St. Bonventure, PQ), covered with vermiculite, nd plced in growth chmber t 24 C, 16 h photoperiod, nd with light intensity of pproximtely 95 µmol/m 2 /s. The pots were rrnged in rndomized complete block with five replictions per tretment nd one plnt per pot per replicte. PABA (MP Biomedicls, Solon, OH) t 0.01, 0.1, 0.5, 1, 4, 9 or 18 mm ws pplied to the upper side of the leves until just before runoff t 10 nd 15 dys fter seeding (DAS) using hnd-held mist pplictor. Tomtoes were t the cotyledon stge t 10 DAS nd the two-true lef stge t 15 DAS. PABA ws mixed in distilled wter using mgnetic stir br for pproximtely 10 minutes prior to trnsfer to the pplictor. Concentrtions greter thn 18 mm were not tested becuse they could not be dissolved. Ech pot ws fertilized with 80 ml of Plnt-Prod Ultimte fertilizer (Plnt Products Co Ltd, Brntford, ON) t 10, 15, nd 21 DAS using 1.26 g/l. Plnts were inoculted with pproximtely 2 x 10 7 CFU/mL of Pst 06T2-4 with 0.01% Sylgrd 309 (Dow Corning Cnd Inc., Georgetown, ON) t 20 DAS (five dys fter the second PABA tretment) when the third nd fourth compound leves were developing. Pst 06T2 ws originlly isolted by Dr. Dine Cuppels from processing tomtoes in southwestern Ontrio in 2006 (Cuppels, pers. comm.). Bcteril speck symptoms include folir lesions, defolition, erly fruit ripening nd sunscld (Preston 2000; Young et l. 1978). The upper nd lower lef surfces of ech plnt were covered with inoculum until just before runoff using hnd-held mist pplictor nd covered with trnslucent plstic continer for 24 hours. The number of bcteril speck lesions on the second nd third youngest leves of ech plnt ws recorded five dys post inocultion (DPI). Lef re ws determined by detching compound leves, trcing the circumference of ech leflet on trnsprency, scnning the imge, nd determining the number of pixels using Imge J v.1.47 ( Pixel number ws converted to re (cm 2 ) using the following eqution: re = ((0.0171*no. of pixels) ) * The eqution ws developed by mesuring the numbers of pixels in circles with known res nd creting stndrd curve using the number of pixels. The number of lesions per cm 2 on the whole plnt, nd the second nd third youngest leves on ech plnt ws clculted. The second youngest lef ws identified using the definition of the youngest lef s the first developed lef from the picl meristem with terminl leflet 43

67 midrib length of 1.5 cm or longer. Reltive chlorophyll mesurements were tken from the terminl leflet of the youngest lef using Minolt SPAD-502 Chlorophyll Meter (Konic Minolt, Osk, Jpn) Host genotype effect, folir PABA ppliction number, folir PABA response durtion nd soil PABA ppliction To evlute host genotype effects, tomto cvs. H2401, H5108, H9553 nd H9909 (H.J. Heinz, Lemington, ON), nd cvs. TSH33 nd TSH4 (Tomto Solutions, Chthm, ON) were grown, treted with 18 mm PABA nd disese evluted s described previously, except folir nd root dry weights were recorded fter fresh smples were plced in greenhouse for 1 to 2 weeks until dry. Three experiments with five replictions per tretment were completed for cv. H2401, cv. H9553, cv. TSH33, nd cv. TSH4, nd five experiments with five replictions per tretment for three of the experiments or four replictions per tretment for two of the experiments were completed for cv. H5108 nd cv. H9909. The effect of the number pplictions of PABA on bcteril speck suppression ws determined with tomto cvs. TSH33 nd H5108. Plnts were grown, 18 mm PABA ws pplied zero, once (15 DAS), twice (10 nd 15 DAS) or three times (10, 12, nd 15 DAS) nd disese ws evluted s described previously. Three experiments were completed with five replictions per tretment. The durtion of tomto response to PABA ws determined with tomto cv. H5108. Plnts were grown, treted with 18 mm PABA t 10 nd 15 DAS, nd inoculted with Pst 06T2-4 t five, seven, or 10 dys fter the second PABA tretment s described previously. Three experiments were completed with five replictions per tretment. To evlute the effect of soil drench pplictions of PABA, tomto cv. H9909 ws grown s described previously, nd 10, 20 or 40 ml of 0.0, 1.0, 4.5 nd 9.0 mm PABA ws pplied to the growing medi t 10 nd 15 DAS. Two experiments were completed with five plnts per tretment Pst popultions in tomto leves Tomto cv. H5108 plnts were grown nd treted with 18 mm PABA t 10 nd 15 DAS nd then inoculted t 20 DAS s previously described, except on the dy of disese ssessment (five DPI), the third terminl leflet of control nd PABA treted plnts ws collected to determine Pst popultion. Lef re for ech terminl leflet ws determined s previously described. Smples were wrpped in pper towel nd stored in plstic bgs until processing, which occurred on the sme dy s smpling. Ech smple ws weighed, then surfce sterilized in 3.1% sodium hypochlorite solution for 90 seconds, 70% lcohol for 60 seconds, nd then rinsed in sterile distilled wter for 30 seconds. The leflet smple ws then blot dried on filter pper, nd homogenized in 2 ml of sterile distilled wter using mortr nd pestle. Seril dilutions were then performed. The diluted homogenized lef solution (100 µl) from dilutions 10-2 through 10-6 were plted on Vogel Bonner-trtrte medi (VBTr), semi-selective medi 44

68 for Pst (Cuppels & Elmhirst, 1999), with two pltes per dilution. The rinse wter from ech smple ws lso plted on tryptic soy gr (TSA) to confirm the efficcy of the surfce steriliztion method. Pltes were incubted t room temperture, nd the number of colonies on ech plte recorded fter four dys. The number of colony forming units (CFU) per cm 2 of lef tissue nd per lesion ws then clculted. The number of CFU per lesion ws clculted by dividing the popultion per cm 2 by the number of lesions per cm 2. The tril ws repeted three times with five plnts per tretment in ech tril. To compre Pst popultions between symptomtic nd symptomtic regions of tomto leflets, 0.78 cm 2 lef tissue discs were excised from leflets with bcteril speck lesions nd from pprently helthy tissue t lest 5 mm wy from ny visible lesions in plnts not treted with PABA. There were five replictions per tretment nd five lef discs per repliction Pst lesion sizes in tomto leves Tomto cv. H5108 ws grown, treted with PABA t 10 nd 15 DAS nd inoculted with Pst s previously described, except on the dy of disese ssessment (five DPI), the third terminl leflet of control nd PABA treted plnts ws collected to determine men lesion size (Figure A.1). A photo (12.2 MB, 4288 x 2824 pixels) of ech leflet ws tken using Nikon D300s cmer (Nikon Cnd Inc., Mississug, ON) set to JPEG fine qulity. A ruler ws lso included within ech imge. Photos were uploded to PowerPoint 2010 (Microsoft, Redmond, Wshington, U.S.A.), incresed in size to llow for trcing of the lesion circumference, printed in colour, nd the circumference of 12 to 15 lesions per leflet trced using fine point permnent mrker. Lesions were selected by trcing ll lesions within rndomly plced 225 mm 2 qudrnt, nd then 12 to 15 lesions were rndomly selected. The lesion circumferences were then scnned nd the number of pixels determined s previously described for lef re, except the size of ech imge ws stndrdized by clibrting ech imge. This ws completed in Imge J by using the line tool to drw stright line representing distnce of 2 cm on the ruler included in the originl imge, clicking nlyze nd set scle, setting the distnce in pixels to 40 nd the known distnce to 2 cm. The experiment ws repeted twice with 10 (experiment 1) nd seven (experiment 2) plnts per tretment Field evlution of folir pplictions of PABA Tomto cv. H5108 seedlings produced using norml grower prctices were cquired from locl commercil trnsplnt grower nd trnsplnted into twin-rows in the field using mechnicl trnsplnter on 26 My Plnts were spced 33 cm within rows, nd twin-rows were spced 75 cm prt on 1.5 m centers. Ech plot consisted of one 7 m twin-row. Tretments were rrnged in rndomized complete block design with four replictions per tretment. Tretments were nontreted control, Kocide 45

69 2000 (E.I. du Pont Cnd Co, Missisug, ON; 53.8% CuOH) with eight folir pplictions of 69 mm CuOH for first four pplictions using 200 L per H of wter nd 46 mm for finl four pplictions using 200 L per H on 7-dy intervl beginning 3 dys fter trnsplnting (DAT), 1 mm PABA with one soil drench ppliction pplied 0 DAT, 18 mm PABA with two folir pplictions on 5-dy intervl beginning 3 DAT, 18 mm PABA with eight folir pplictions on 7-dy intervl beginning 3 DAT nd 18 mm PABA with nine folir pplictions on 5-dy intervl beginning 3 DAT, nd. For PABA tretments of two folir pplictions, 200 L wter per H ws pplied, wheres for PABA tretments of eight nd nine folir pplictions, 200 L wter per H for the first four pplictions nd 300 L/H for remining pplictions. Folir CuOH nd PABA tretments were pplied using hnd-held CO2 spryer (40 psi) with ULD (Pentir Ltd., New Brighton, MN, USA) nozzles for the first four pplictions nd ULD (Pentir Ltd., New Brighton, MN, USA) nozzles for the remining pplictions. For soil drench PABA ppliction, ppliction wter volume ws 3 L in trnsplnt bottom trys nd then plug cells were incubted in the solution of PABA or wter for 60 minutes so tht roots, but not stems or folige, were exposed to the PABA solution. Men bsorption per cell ws 3.1 ml. Folir pplictions were mde on 29 My, 5, 12, 19, 26 June, 3, 10, nd 17 July for eight pplictions t 7-dy intervls, on 29 My nd 3 June for the two ppliction t 5-dy intervls, on 29 My, 3, 8, 12, 18, 25, 30 June, 4 nd 9 July for the nine ppliction t 5-dy intervls. Soil drench ppliction ws on 29 My Sttisticl nlysis Sttisticl nlysis ws completed using SAS v9.4 (SAS Institute, Cry, NC, USA). Dt were tested for normlity using the Shpiro-Wilk sttistic. Outliers were identified using Lund s test of stndrdized residuls (Lund 1975). Anlysis of vrince on plnt growth nd disese incidence dt ws completed using Proc Mixed with tretment s fixed effect nd repliction s rndom effect. Dt from repeted experiments ws pooled together when sttisticl nlysis showed no tretment x experiment interction (P 0.05), except where noted in some tbles nd figures. Experiment ws treted s fixed effect. Mens comprisons were performed when P 0.05, nd mens were seprted using Tukey s HSD. Regression nlysis for PABA concentrtion ws completed using Proc Reg to determine model significnce nd Proc GLM to obtin prmeter estimtes. The LOG of ech PABA concentrtion tested + 1 ws used for regression nlysis to obtin liner curve nd llow the clcultion of LOG vlues for ll concentrtions tested from 0 to 18 mm. 2.3 Results PABA direct ntimicrobil effect 46

70 A filter disc ssy with distilled wter control nd 1.0, 9.0, 18.0 or 27.0 mm PABA dissolved in wter did not result in ny mesurble zone of inhibition of Pst (Tble 2.1). However, higher PABA concentrtions were not ble to dissolve completely in distilled wter but could be dissolved in ethnol (dt not shown). A zone of inhibition ws observed with the ethnol control in the filter disc ssy, indicting tht the ethnol inhibited the growth of Pst. Concentrtions of 18.0 nd 72.0 mm PABA in ethnol resulted in significntly higher zones of inhibition thn the ethnol control t 15 nd 50 %, respectively. The presence of zone of inhibition by 18.0 mm PABA greter thn the ethnol control but not the wter control implies tht PABA my hve been ble to be tken up more redily by Pst when ssocited with ethnol thn only distilled wter. The lrger zone of inhibition for 72.0 mm PABA compred to 18.0 mm PABA in ethnol shows tht the ntimicrobil effect of PABA ws dose dependent. These results indicte tht PABA would hve no direct ntimicrobil effect on Pst t 18.0 mm or less in wter, nd thus PABA t concentrtions 18.0 mm or less in wter ws used in ll subsequent experiments PABA soil drench ppliction Soil drench ppliction of 18 mm PABA to cv. H9909 seedlings in growth chmbers showed sufficient toxicity to the plnts tht disese ssessment ws not possible becuse the plnts were stunted or died (dt not shown). Therefore, 1.0, 4.5, nd 9.0 mm PABA with ech concentrtion pplied t 10, 20, or 40 ml per pot, ws used s soil drench. There ws no difference mong tretments for ny PABA concentrtion (Figure 2.1). There ws no effect of soil pplictions of PABA on reltive chlorophyll (Tble A.1) compred to the control PABA folir ppliction Folir ppliction to cv. H9909 seedlings in growth chmbers with 9.0 nd 18.0 mm PABA resulted in symptoms of phytoxicity pprox. three dys post tretment (DPT) on true leves nd cotyledon leves (Figure 2.2 -c). Folir ppliction of 0.01, 0.1, 0.5, nd 1.0 did not result in phytotoxicity, nd symptoms were less severe t 4.0 mm. The number of bcteril speck lesions per cm 2 of lef tissue on the second nd third youngest leves on ech plnt ws evluted, which developed fter the PABA pplictions, nd thus hd not come in direct contct with PABA nd hd no phytotoxicity symptoms. Results of n ANOVA nlysis indicted significnt effects of two PABA pplictions t 10 nd 15 DAS on disese incidence t concentrtions of 9 or 18 mm. Two pplictions of PABA to the sme leves ws chosen bsed on preliminry results. Regression nlysis of bcteril speck lesion number versus PABA dose indicted dose response tht could be explined by the eqution disese incidence =

71 0.32(LOG10 (PABA concentrtion)) (R 2 = 0.53, p < ) (Figure 2.3). Bsed on these results, 18 mm PABA ws selected for dditionl experiments since this provided the gretest reduction in disese. There ws no effect of PABA concentrtion on reltive chlorophyll redings (Tble A.2) Effect of plnt genotype on PABA response Among the six commercil processing tomto cultivrs evluted, PABA reduced bcteril speck lesions per cm 2 of lef tissue by 28, 27, nd 24% in cvs. H5108, H9553 nd H9909, but there ws no reduction in bcteril speck in cvs. H2401, TSH33 nd TSH4 (Figure 2.4). Phytotoxicity to PABA similr to tht observed in Figure 2.2-c ws observed on the PABA spryed leves for ll the cultivrs. Folir nd dry root weight ws not ffected by PABA ppliction in cv. H2401, H5108, H9553 or TSH4, but cv. TSH33 hd lower folir dry weight nd cvs. H9909 nd TSH33 hd lower root dry weights thn control plnts (Tble 2.2). Bsed on these results, cv. H5108 ws used in subsequent experiments becuse PABA ppliction reduced disese incidence without ffecting plnt growth, nd thus ws considered to show incresed resistnce due to PABA without negtive growth effects. Also, cv. TSH33 ws used in subsequent experiments becuse PABA ppliction did not reduce disese incidence but did reduce folir nd root dry weight, nd thus ws considered to show no chnge in resistnce due to PABA but with growth negtively ffected PABA pplictions To determine the effect of the number of folir PABA pplictions on bcteril speck suppression, PABA ws pplied once (15 DAS), twice (10 nd 15 DAS) or three times (10, 12 nd 15 DAS) before pthogen inocultion. One, two, or three pplictions of PABA to tomto cv. H5108 ll resulted in significnt reductions in bcteril speck compred to the control (Figure 2.5). In contrst, one, two, or three pplictions of PABA to tomto cv. TSH33 did not reduce the incidence of bcteril speck (Figure 2.5b). Thus, the number of pplictions did not lter whether tomto cultivr showed incresed disese resistnce with PABA tretment. Detrimentl growth effects of PABA were observed for cv. H5108 (Tble 2.3). One, two or three PABA pplictions resulted in reduction in root dry weight compred to the control, nd one or three PABA pplictions resulted in lower folir dry weight thn the control (Tble 2.3). This ws unlike the results of the previous experiment with two folir pplictions of PABA for root dry weight with this cultivr (Tble 2.2). In contrst, no detrimentl growth effects of PABA were observed for cv. TSH33 with ny number of PABA pplictions (Tble 2.3). Once gin, this ws inconsistent with previous results tht showed lowered folir nd dry root weights with two PABA pplictions (Tble 2.2). It 48

72 ppers tht the effect of PABA tretment on plnt growth is vrible PABA response durtion For the 10 nd 15 DAS PABA ppliction method, inocultion of tomto cv. H5108 t five, seven, or 10 dys fter the second PABA ppliction (15 DAS) showed tht bcteril speck lesions per cm 2 of lef tissue ws reduced when inocultion occurred t five nd seven but not 10 dys fter the second PABA ppliction (Figure 2.6). The effect t five dys fter the 15 DAS PABA ppliction ws consistent with tht observed in previous experiments (Figure 2.4 nd Figure 2.5). This indictes tht the protection provided by PABA hd been lost by 10 dys fter tretment. However, bcteril speck incidence in control plnts vried over time incresing between five nd seven dys nd then dropping between seven nd 10 dys (Figure 2.6). The incresed growth of the plnts between seven nd 10 dys versus between five nd seven dys for both PABA-treted nd control plnts cn be seen in the chnges in folir nd root dry weights (Tble 2.4). There were no negtive effects of PABA on folir or root dry weight, which is consistent with the results for cv. H5108 from the host genotype evlution (Tble 2.2), but different from the results of the evlutions tht ssessed the effects of the number of PABA pplictions on host response (Tble 2.3) PABA effect on Pst popultions nd lesions Even though bcteril speck lesions per cm 2 of lef tissue in PABA treted plnts ws significntly lower thn the non-treted control, the Pst popultion per cm 2 of lef tissue in PABA treted plnts ws equivlent to the non-treted control (Tble 2.5). Thus, the fewer number of lesions ws not reflected in the presence of fewer cells of the pthogen when Pst popultion in whole leflets ws ssessed. When the popultion ws exmined on per lesion bsis, the Pst popultion per lesion ws 38% higher in PABA treted plnts compred to the non-treted control. This indictes tht the verge lesion in PABA treted leves contined more bcteri thn the verge lesion in the control leves. One possibility is tht Pst popultions with lesions re similr between PABA nd no PABA treted leves, but Pst popultions outside of the lesions (i.e., symptomtic tissue) could be different. A comprison of Pst popultions in no PABA treted inoculted leves between symptomtic (i.e., bcteril speck lesions) nd symptomtic lef tissue indicted tht symptomtic tissue hd Pst popultions three orders of mgnitude higher (5.12 x 10 8 CFU per cm 2 lef tissue) thn symptomtic tissue (1.12 x 10 5 CFU per cm 2 lef tissue) from the sme leflets (p = , sem = 0.645). Thus, lesions mde the gretest contribution to totl Pst popultion in leflet. Even though PABA tretment reduced the incidence of bcteril speck lesions per cm 2 of lef 49

73 tissue (Figure 2.7), men lesion size in PABA treted plnts ws 94% lrger thn those on control plnts (Figure 2.7b). However, the fewer number of these lrger lesions resulted in no significnt difference in the percentge of totl lef surfce re with lesions between the PABA nd control tretments (Figure 2.7d). Lesion circumference ws lso clculted since popultion growth of Pst, which is hemibiotroph, is likely highest ner lesion edges. Men lesion circumference ws 28% greter when PABA ws pplied compred to lesions from control plnts (Figure 2.7c). These results suggest tht PABA is suppressing initil infections so tht fewer lesions develop, but fter initil infection, the lesions of PABA treted plnts reched lrger size by five DPI Field evlution In 2014, PABA ws pplied to folige 2, 8 or 9 times, or one time s plug seedling sok, to tomto cv. H5108. No symptoms of phytotoxicity were observed when PABA ws pplied to folige, but phytotoxicity ws observed fter drench pplictions of 1 mm PABA. Affected plnts were rndomly distributed within tretment plots nd hd noticebly purple colour with stunting. Erly seson bcteril speck incidence ws mesured from mid-june to erly July s the percentge of symptomtic leves in 1.24 m 2 re in ech plot. None of the PABA tretments reduced disese incidence during the erly seson ssessment period compred to the non-treted control on ny of the ssessment dtes (Figure 2.8), nor ws there ny significnt effect on erly seson AUDPC (Tble 2.6). The CuOH stndrd lso showed no differences from non-treted control on ny ssessment dte (Figure 2.8) or erly seson AUDPC (Tble 2.6). Lte seson disese severity ws mesured s the percent defolition per plot from mid-july to lte-aug. Similr to the results with erly seson disese severity, there ws no effect of PABA on lte seson disese severity compred to the non-treted control on ny ssessment dte (Figure 2.9), or for lte seson AUDPC (Tble 2.6). The CuOH stndrd ws lso ineffective. While bcteril speck incidence on tomto fruit with ll the methods of folir PABA pplictions were numericlly lower thn the non-treted control, there were no significnt differences mong tretments (Tble 2.7). The CuOH stndrd ws lso ineffective compred to the non-treted control. Bcteril spot (X. grdneri) symptoms on tomto fruit were lso detected, nd even though the nine PABA pplictions resulted in 55% of the disese incidence of the control, there were no significnt differences mong PABA tretments nd the non-treted control. This ws relted to high vribility mong plots. The CuOH stndrd ws once gin ineffective. The presence of bcteril spot on fruit is strong indictor tht some of the bcteril disese symptoms on tomto folige in the tril were cused by X. grdneri s well s Pst. Reltive chlorophyll ws mesured 24, 30 nd 36 DAT. None of the PABA tretments hd ny 50

74 effect on reltive chlorophyll content compred to the non-treted control, nd the CuOH stndrd tretment lso did not ffect reltive chlorophyll content compred to the non-treted control (Figure 2.10). Similrly, there ws no significnt effect of ny PABA tretments on totl, red, green or rotten tomto fruit yield compred to the non-treted control (Tble 2.8). There ws trend towrd higher green fruit yield compred to the non-treted control with the PABA seedling sok, but this ws likely due to symptoms of phytotoxicity in the PABA seedling sok tretment tht delyed growth. The CuOH stndrd ws lso ineffective in ltering totl, red, green or rotten tomto fruit yield compred to the nontreted control. 51

75 Tble 2.1 Effect of PABA concentrtion on growth of P. syringe pv. tomto (Pst) in filter disc ssy. Filter discs soked in 0, 1, 9, 18, nd 27 mm pr-minobenzoic cid (PABA) dissolved in wter, nd 0, 18, nd 72 mm PABA dissolved in 70% ethnol were plced on tryptic soy gr covered thoroughly with Pst, incubted t room temperture, nd ssessed for zones fter three dys. Zone of Inhibition (mm) PABA (mm) Wter Ethnol (70%) c b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with 10 replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 52

76 NS Disese Incidence (lesions/cm 2 ) NS NS PABA (mm) Figure 2.1 Effect of different pr-minobenzoic cid (PABA) concentrtions nd ppliction volumes on the systemic cquired resistnce response in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). A pipette ws used to pply 10 ( ), 20 ( ), or 40 ( ) ml PABA to the root zone 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Disese incidence five dys post inocultion is shown. Dt points with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 53

77 ) b) c) Figure 2.2 Phytotoxicity symptoms on tomto ) true lef, nd b) nd c) cotyledon lefs coted to runoff with 18 mm PABA t 10 nd 15 dys fter seeding. 54

78 4.5 y = x, R 2 = 0.53, p < Disese Incidence (lesions/cm 2 ) LOG PABA concentrtion (mm) Figure 2.3 Effect of different pr-minobenzoic cid (PABA) concentrtions on the systemic cquired resistnce response in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). Plnts were coted with 0, 0.01, 0.1, 0.5, 1, 4, 9, or 18 mm PABA (LOG + 1 = 1, , , , , , 1.000, mm PABA) 10 nd 15 dys fter seeding (DAS), nd then plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Disese incidence five dys post inocultion is shown. Errors brs represent stndrd error of the men. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 55

79 5.0 Disese Incidence (lesions/cm 2 ) b b b 0.0 H2401 H5108 H9553 H9909 TSH33 TSH4 Cultivr Figure 2.4 Incidence of bcteril speck symptoms on the second nd third youngest leves of six tomto cultivrs treted with pr-minobenzoic cid (PABA). Plnts were coted with 18 mm PABA 10 nd 15 DAS. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto (Pst) 20 dys fter seeding (DAS). Disese incidence five dys post inocultion is shown for the nontreted control ( ) nd PABA ( ). Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for cvs. H2401, H9553, TSH33, nd TSH4 with five replictions of ech tretment, nd five independent trils for cv. H5108 nd cv. H9909 with five or four replictions of ech tretment, ws pooled together becuse ANOVA showed no tretment x tril interction. One outlier ws removed ech dt set for cv. H9909 nd cv. H

80 Tble 2.2 Folir, root, nd totl dry weight of six tomto cultivrs treted with pr-minobenzoic cid (PABA). Plnts were coted with 18 mm PABA 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto 20 DAS. Dry weight of plnts five dys post inocultion is shown. Dry weight (mg/plnt) Folige Roots Totl Cultivr No PABA PABA sem No PABA PABA sem No PABA PABA sem H b H H H b TSH b b b 22.1 TSH sem = stndrd error of the men for ll ls mens in the sme row for the sme vrible. b Mens in the sme row nd group followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 57

81 5.0 ) Disese Incidence (lesions/cm 2 ) b b b b) Disese Incidence (lesions/cm 2 ) # Applictions Figure 2.5 Incidence of bcteril speck symptoms on the second nd third youngest leves of tomto cultivrs ) cv. H5108, nd b) cv. TSH33 treted with zero, one (15 dys fter seeding (DAS)), two (10 nd 15 DAS), or three (10, 12, nd 15 DAS) pplictions of pr-minobenzoic cid (PABA). Folir PABA tretments (18mM) were pplied by coting fine mist on plnts. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Disese incidence five dys post inocultion is shown. Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Dt for cv. H5108 ws log trnsformed to meet ssumptions of ANOVA. 58

82 Tble 2.3 Folir, root, nd totl dry weight of tomto cv. H5108 nd cv. TSH33 treted with zero, one, two, or three pplictions of prminobenzoic cid (PABA). One, two, or three folir PABA tretments (18mM) were pplied by coting fine mist on plnts 10, 12, nd/or 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Dry weight of plnts five dys post inocultion is shown. Dry weight (mg/plnt) Folige Roots Totl H5108 TSH33 H5108 TSH33 H5108 TSH33 Appliction Timing No PABA b DAS 89.3 b b b nd 15 DAS 94.4 b b b , 12, nd 15 DAS 88.1 b b b 99.3 sem c One outlier ws removed from the cv. TSH33 folir dry weight dt nd totl dry weight dt. There ws significnt tril*trt interction for cv. TSH33 pooled folir nd totl dry weight, however, dt ws pooled becuse tretment effects within trils were wek nd inconsistent. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. c sem = stndrd error of the men for ll ls mens in the sme column. 59

83 5.0 A Disese Incidence (lesions/cm 2 ) b B z z Pst inocultion timing (# dys fter lst PABA ppliction) Figure 2.6 Incidence of bcteril speck symptoms on the second nd third youngest leves of tomto cv. H5108 inoculted with P. syringe pv. tomto (Pst) five, seven, or 10 dys fter pr-minobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five, seven, or 10 dys fter the second PABA ppliction. Disese incidence five dys post inocultion is shown for the nontreted control ( ) nd PABA ( ). Errors brs represent stndrd error of the men. Dt points with the sme letter for the sme inocultion timing re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. One outlier ws removed from the nlysis for the dt from seven dys fter the lst PABA ppliction. 60

84 Tble 2.4 Folir, root, nd totl dry weight of tomto cv. H5108 inoculted with P. syringe pv. tomto (Pst) five, seven, or 10 dys fter prminobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 dys fter seeding (DAS)). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five, seven, or 10 dys fter the second PABA ppliction. Dry weight of plnts five dys post inocultion is shown. Inocultion timing (# dys fter lst PABA ppliction) Dry weight (mg/plnt) Folige Roots Totl No PABA PABA sem No PABA PABA sem No PABA PABA sem b sem = stndrd error of the men for ll ls mens in the sme column. b Mens in the sme row nd group followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 61

85 Tble 2.5 Incidence of bcteril speck symptoms, popultion of P. syringe pv. tomto (Pst) per cm 2, nd per lesion on the third youngest terminl leflet of tomto cv. H5108 fter pr-minobenzoic cid (PABA) tretment. Two folir PABA tretments (18 mm) were pplied by coting fine mist on plnts beginning t the cotyledon stge (10 nd 15 dys fter seeding (DAS)). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst five dys fter the second PABA ppliction. Tretment Disese incidence (lesions/cm 2 ) Popultion (CFU/cm 2 ) Popultion (CFU/lesion) No PABA x x 10 1 b PABA 3.2 b 1.55 x x 10 2 sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. There were two missing plots for the control due to poor germintion nd smpling error. b sem = stndrd error of the men (LOG10) for ll ls mens in the sme column, except for No PABA, which ws 0.37, 0.12, nd 0.16 for disese incidence, popultion per cm 2, nd popultion per lesion. 62

86 Disese Incidence (lesions/cm 2 ) ) b Lesion size (mm 2 ) b) b 0.0 control PABA 0.00 control PABA Lesion circumference (mm 2 ) c) b Lesion surfce re (% of lef surfce) d) 0.00 control PABA 0.0 control PABA Figure 2.7 The effect folir pplictions of pr-minobenzoic cid (PABA) on ) the incidence of bcteril speck symptoms, b) men lesion size, c) men lesion circumference, nd d) percent lef re with lesions on the third youngest terminl leflet of tomto cv. H5108. Folir PABA tretments (18mM) were pplied 10 nd 15 dys fter seeding (DAS) by coting fine mist on plnts. Plnts were coted with fine mist of 2 x 10 7 CFU/ml of P. syringe pv. tomto five dys fter the lst PABA ppliction. Disese incidence nd men lesion size of 10 to 15 lesions is 63

87 shown for the nontreted control ( ) nd PABA ( ). Error brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with 10 (tril 1) nd seven (tril 2) replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 64

88 50 Leves with Lesions (%) Trnsplnting Inocultion #1 NS NS 10 NS 0 19-My 02-Jun 16-Jun 30-Jun 14-Jul 28-Jul Figure 2.8 Erly seson disese progress of bcteril speck symptoms in tomto cv. H5108 treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON. The percentge of leves with disese symptoms in the nontreted control ( ), eight pplictions of CuOH t 7-dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5-dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Tble 2.6. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 65

89 100 NS NS 80 Defolition (%) NS 20 NS NS 0 14-Jul 21-Jul 28-Jul 04-Aug 11-Aug 18-Aug 25-Aug Figure 2.9 Lte seson disese progress of bcteril speck symptoms in tomto cv. H5108 treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON. Defolition in the nontreted control ( ), eight pplictions of CuOH t 7-dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5-dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in whole plots is shown. The corresponding re under the disese progress curve is shown in Tble 2.6. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 66

90 Tble 2.6 Are under the disese progress curve (AUDPC) for erly nd lte disese in tomto cv. H5108. Erly seson disese ws mesured by clculting the percentge of leves with disese symptoms nd lte seson disese ws mesured by estimting defolition in plnts treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, in No. of Appliction AUDPC Tretment pplictions intervl (dys) Erly seson Lte seson Nontreted control b 1250 CuOH PABA PABA PABA PABA 1 seedling sok sem c All tretments were pplied to tomto folige except for the seedling sok. Tomto seedlings in the sok tretment were soked in PABA solution for one hour just prior to trnsplnting. CuOH ws pplied t 69 mm for the first four pplictions nd 46 mm for the finl four pplictions becuse of incresing wter volume. Folir PABA ws pplied using 18 mm nd the seedling sok using 1 mm. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. c sem = stndrd error of the men for ll ls mens in the sme column. 67

91 Tble 2.7 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. H5108, hrvested from plots treted with CuOH or pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted. No. of Appliction Incidence (%) Tretment pplictions intervl (dys) Speck Spot Nontreted control b 18.0 CuOH PABA PABA PABA PABA 1 seedling sok sem c All tretments were pplied to tomto folige except for the seedling sok. Tomto seedlings in the sok tretment were soked in PABA solution for one hour just prior to trnsplnting. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. c sem = stndrd error of the men for ll ls mens in the sme column. 68

92 NS NS SPAD chlorophyll reding NS Jun 23-Jun 30-Jun 07-Jul 14-Jul Figure 2.10 Reltive chlorophyll mesured 24, 30, nd 36 dys fter trnsplnting in tomto cv. H5108 treted with CuOH nd pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto in Ridgetown, ON, SPAD redings in the nontreted control ( ), eight pplictions of CuOH t 7- dy intervls ( ), eight pplictions of PABA t 7-dy intervls ( ), two pplictions of PABA t 5- dy intervls ( ), nine pplictions of PABA t 5-dy intervls ( ), nd seedlings soked in PABA for one hour before trnsplnting ( ) in whole plots is shown. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 69

93 Tble 2.8 Totl, red, green, nd rotten fruit yield in 2m section of tomto cv. H5108 treted with CuOH nd pr-minobenzoic cid (PABA) nd inoculted with P. syringe pv. tomto, Ridgetown, ON, in No. of Appliction intervl pplictions (dys) - - Yield (kg) Totl Red Green Rotten Tretment Nontreted control b CuOH PABA PABA PABA PABA 1 seedling sok sem c All tretments were pplied to tomto folige except for the seedling sok. Tomto seedlings in the sok tretment were soked in PABA solution for one hour just prior to trnsplnting. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. c sem = stndrd error of the men for ll ls mens in the sme column. 70

94 2.4 Discussion This study demonstrted tht folir pplictions of PABA cn be effective in reducing disese incidence of bcteril speck when tomtoes were grown in growth room conditions. This confirms the effectiveness of PABA under controlled conditions previously reported for vriety of diseses of tomto (Tzhoor, 2014), tobcco (Yng et l., 2011b) nd pepper (Song et l., 2013). However, PABA ws ineffective in controlling bcteril speck under field conditions, unlike the field control reported with PABA ginst P. striiformis in whet (Kelmn & Cook, 1977) nd Xe/Xp in pepper (Song et l., 2013). This study lso demonstrted tht the bility of PABA to suppress bcteril speck of tomto ws gretly ffected by number of fctors, such s PABA concentrtion, ppliction method, host genotype, plnt ge nd inocultion timing. Thus, comprison mong studies is difficult to mke unless such fctors re nlyzed nd then the conditions optimized for PABA efficcy Direct ntimicrobil effects of PABA The solubility of PABA in wter is 43 mm t 25 C versus 911 mm in lcohol (Anonymous, 2013). Thus, 18.0 mm should be soluble in wter, wheres 72.0 mm should not. In wter, no toxicity to Pst ws observed with up to 27 mm PABA, but in ethnol, 18 nd 72 mm PABA produced zones of inhibition greter thn the control, which lso inhibited the pthogen. This provides the first evidence tht PABA directly ffects Pst growth probbly in dose dependent mnner. However, the extent of inhibition, which ws 23% for 18 mm nd 33% for 72 mm ws slight. There re reports in the literture tht PABA hs ntimicrobil effects ginst other bcteri. For exmple, growth of Listeri monocytogenes, Slmonell sp., nd E. coli ws inhibited t 9 to 24 mm PABA dissolved in brin hert infusion broth (Richrds et l., 1995) nd 37 to 111 mm PABA dissolved in edible films (Cgri et l., 2001). Direct ntimicrobil effects ginst Xe/Xp were not observed t 0.1 to 10 mm, but the solvent ws not specified (Song et l., 2013). It is likely tht the mode of ction observed in vivo in this study is result of induced resistnce becuse the dose of 18 mm PABA in wter cused no direct growth inhibition of Pst in vitro. This is lso supported by the ppliction of PABA to different leves thn were ssessed for bcteril speck incidence, nd so there ws no direct contct between PABA nd the Pst popultions in lef tissue exmined PABA concentrtions nd ppliction methods PABA pplied s soil drench ws toxic to tomto seedlings t 18 mm, but not t 1 to 9 mm. However, there ws no effect on disese incidence using 1 to 9 mm PABA s soil drench, which is equivlent to 1.4 nd 49.4 mg PABA per seedling. This contrdicts Song et l. (2013), who showed tht 71

95 0.1 to 13.7 mg PABA per seedling pplied to gr growth medium reduced the severity of disese cused by Xe/Xp in peppers, nd Yng et l. (2011b), who found tht to mg PABA per seedling pplied to gr growth medium ws effective ginst P. crotovotrum subsp. crotovotrum in tobcco. Both Song et l. (2013) nd Yng et l. (2011b) did not report ny phytotoxicity due to PABA. Thus, similr or even higher mount of PABA pplied per seedling in the current study ws ineffective ginst Pst in tomtoes, unlike Xe/Xp in pepper nd P. crotovotrum subsp. crotovotrum in tobcco. Although host response to plnt ctivtors cn vry mong plnts species (Azmi-Srdooei et l., 2010), this result is surprising since ctivtors re generlly effective cross rnge of plnt species (Boubkri et l., 2016, Durrnt & Dong, 2004). One difference is tht the tomtoes were grown in pet-bsed soilless potting mix in growth rooms in this work, wheres Song et l. (2013) nd Yng et l. (2011b) grew plnts on MS gr under septic conditions. Agr plte growing systems provide low cost mens of producing sterile culture of plnts (Conn et l., 2013), nd re chemiclly inert (Best et l., 2014). However, plnt growth cn be ltered, such s with light exposure to roots. Light exposure cn increse root biomss, root length nd root hir density nd reduce primry root length (Xu et l., 2013) nd induce plnt stress response (Yokw et l., 2014). Also, septic conditions lter the expression of SA nd JA mrker genes in A. thlin compred to non-septiclly grown plnts (Crvlhis et l., 2013). Although Song et l. (2013) observe pepper response to PABA under field conditions, the seedlings were lso rered in septic conditions. The pet-bsed soilless potting mix used in the current study is similr to tht used by tomto growers to produce seedlings. Petbsed soilless mixes re used becuse they hve good dringe nd ertion nd re low in soluble slts (Mynrd & Hochmuth, 2007), lthough there cn be high vribility mong btches (Conn et l., 2013). The efficcy of PABA could hve been reduced by interctions with components of these mixes s tht hs been reported for phytohormones nd phytohormone inhibitors pplied to soil (Best et l., 2014). However, using higher PABA concentrtion resulted in phytotoxicity, nd thus, it ws not possible to find concentrtion tht provided disese control without plnt deth using soil drench pplictions of PABA. In contrst, two folir pplictions of either 9 or 18 mm PABA were effective ginst Pst lthough they did cuse some phytotoxicity. PABA concentrtion ws inversely relted to disese incidence demonstrting tht the PABA response ws dose dependent. One folir ppliction of 18 mm PABA ws lso effective ginst Pst in one tomto breeding line (Tzhoor, 2014), but there re no other reports for induced resistnce by PABA for folir pplictions. For pepper seedlings, growth medi pplictions less thn 0.1 mm were not effective, wheres pplictions of 0.1, 1, nd 10 mm PABA effectively reduced the severity of Xe/Xp; however, it ws not cler if the effect ws dose dependent (Song et l., 2013). In tobcco seedlings, growth medi pplictions of 1.0 nd 18.0 mm PABA reduced 72

96 the severity of P. crotovotrum subsp. crotovotrum with 18 mm being more effective (Yng et l., 2011b). Dose-dependent responses re lso reported for other ctivtors. For exmple, there ws dosedependent inverse response to ASM for X. perforns bcteril spot of tomto (Hung et l., 2012), nd riboflvin for the TMV disese of tobcco, A. lternt lef spot of tobcco nd Peronospor prsitic downy mildew of Arbidopsis (Dong & Beer, 2000). Although the PABA response ws dose dependent, one, two, or three pplictions of 18 mm PABA ws redily soluble in wter, effective, reproducible nd cused limited phytotoxicity to the leves Host genotype effect There re numerous reports of the effectiveness of plnt defense ctivtors being ffected by plnt genotype (Tzhoor, 2014, Dnn et l., 1998, Hijwegen & Verhr, 1994, Ishikw et l., 2007). Among six commercil processing tomto cultivrs in this study, three cultivrs, H5108, H9553 nd H9909, were responsive to PABA, while three cultivrs, H2401, TSH33 nd TSH4, were not responsive to PABA. All the responsive cultivrs re from Heinz Seed ( Although they my shre some common ncestry, full informtion bout their bckgrounds is not publiclly vilble (G. Collier, personl communiction). One of the non-responsive cultivrs is lso from Heinz Seed. Cultivr H2401 crries the Pto gene for resistnce to Pst rce 0, indicting the isolte used to inoculte tomtoes in this study is rce 1. The other two non-responsive cultivrs re from Tomto Solutions ( nd thus likely shre limited ncestry with cvs. H5108, H9553 nd H9909. Cultivrs TSH33 nd TSH4 hve different prents, but both reportedly crry tolernce to Pst by limiting reproduction of the pthogen on folige in non-rce specific mnner (J. Dick, personl communiction). However, only non-treted cv. TSH4 ppered tolernt in this study, perhps becuse of the growing conditions nd Pst popultion t the time of inocultion. PABA responsiveness ws lso observed mong tomto breeding lines, which hd vrious wild tomtoes in the pedigrees (Tzhoor, 2014). Among eight breeding lines, only one line, which hd linege from Solnum lycopersicoides showed significnt induced resistnce ginst Pst following single folir PABA ppliction. In contrst, Song et l. (2013) nd Yng et l. (2011b) only exmined single cultivrs of pepper nd tobcco, respectively, following PABA pplictions. Other exmples of genotype effect on induced disese resistnce include INA nd ASM tht were effective in the soyben cultivrs Elgin 87 nd Willims 82, but less effective in cultivrs Corsoy 79 nd NKS19-90 ginst S. sclerotiorum (Dnn et l. 1998), nd INA tht ws effective in cucumber cultivr Flmingo but not cultivrs Coron nd Lnge Groene Gignt ginst Spherothec fuligine (Hijwegen nd Verhr 1994). In tomto, SAR ginst F. oxysporum f. sp. lycopersici ctivted by vlidmycin A vried from over 80% to less thn 30% effectiveness mong 20 tomto cultivrs (Ishikw 73

97 et l., 2007). One explntion for this vrition is differences in the response of defense genes to defense ctivtors. The expression pttern of cidic nd bsic PR1 (PR1b) vried mong three tomto cultivrs fter ppliction of ASM (Hermn et l., 2007). The differentil response to PABA in tomto breeding lines reported by Tzhoor (2014) ws ssocited with fster nd stronger increse in SlPR1 expression fter Pst inocultion in the responsive breeding line, which suggests tht the lines differ in how defense genes re ffected by PABA ppliction. Thus, the pedigree of the tomto plys criticl role in determining the effectiveness of PABA. Future studies re needed to determine if the different response of the commercil tomto cultivrs in this study my be relted to greter increses in defense gene expression. Phytotoxicity symptoms were observed on tomto leves treted with the effective concentrtions of PABA in the current study, nd so one possibility is tht the wounding nd not PABA itself induced resistnce ginst Pst. Although SAR hs been ssocited with tissue dmge in response to necrotizing or HR-inducing pthogens, plnt tissue necrosis is not required for SAR (Mishin & Zeier, 2007). Tzhoor (2014) observed similr response to Pst in tomto without phytotoxicity symptoms using single PABA ppliction. Furthermore, phytotoxicity ws observed on ll tomto cultivrs tested, including cultivrs tht were non-responsive to PABA pplictions, nd ws observed with 4.0 mm PABA in PABA responsive cultivr tht did not result in significnt reduction in bcteril speck symptoms. This suggests the response ws not solely becuse of necrosis induced by PABA. This study nd tht of Tzhoor (2014) shows tht first screening cultivrs for their response to PABA is necessry for ny prcticl ppliction of PABA s disese mngement tool, since significnt number of cultivrs my not respond to PABA s defense ctivtor. There re mny steps from ppliction of defense ctivtor until disese resistnce is expressed (Pieterse et l., 2009). Further work is needed to determine which step(s) vry between tomto genotypes to PABA, such s differences in PABA uptke, ROS production, SA defense signling pthwys, specific trnscription fctors nd/or other fctors Prmeters ffecting protection nd the durtion of protection by PABA The effect of ltering the number of PABA pplictions in responsive nd non-responsive cultivr ws evluted to determine if this ffected the PABA response. Incresing the number of PABA pplictions from one to three did not induce resistnce in the non-responsive cultivr TSH33, nd it lso did not lter the induced resistnce in the responsive cv. H5108. This is the first report on the effect of ppliction number for the level of induced resistnce cused by PABA or the other B vitmins thimine nd riboflvin. One reson tht multiple pplictions of PABA my not hve improved its effectiveness is becuse of its mode of ction. For the two pplictions in this study, PABA use ws seprted by five 74

98 dys (10 nd 15 DAS), nd for three pplictions, PABA use ws seprted by two nd three dys (10, 12, nd 15 DAS), nd ll tretments were pplied prior to Pst inocultion. In previous reserch in tomto the PR1 expression pttern fter single PABA ppliction in responsive tomto breeding line ws not immeditely greter thn expression in control plnts (Tzhoor, 2014). Rther, expression ws greter thn the control only eight DPT, which ws three dys fter inocultion with Pst (Goodwin et l., 2017). Song et l. (2013) lso observed n increse in SA-relted gene expression in pepper only fter pthogen chllenge. This suggests tht PABA primes host defenses vi SA-dependent signling, but unlike ASM, it does not result in the immedite ctivtion of defense genes within few dys of ppliction in the bsence of pthogen (Hermn et l., 2007). Priming is physiologicl stte whereby plnt cells respond fster nd stronger thn non-primed cells to stimulus (Conrth, 2011). In SAR, priming is ssocited with the ccumultion of mrna nd dormnt MPK3 nd MPK6 for ASM nd A. thlin (Beckers et l., 2009), nd ROS- nd C2+-signling dependent pthwys for MPK3 nd MPK6 ctivtion for riboflvin nd A. thlin (Nie & Xu, 2016). The priming mechnisms for PABA re unknown, but since previous reserch ssocites induced resistnce by PABA with SA-signling, it my lso be MPK3 nd MPK6 dependent. In this study, one to three pplictions of PABA were mde prior to pthogen chllenge with no effect on disese severity, thus it is possible tht priming threshold ws reched with single PABA ppliction, t lest within the time period PABA ws pplied. Preliminry experiments using single ppliction of PABA in this study were inconsistent, which is why further tests were completed using two PABA pplictions; however, these experiments were completed using different cultivrs nd the disese ssessment ws completed seven DPI insted of five DPI, nd lter experiments showed efficcy with one to three pplictions were equivlent. For ASM, pplictions every eight to 10 dys re superior to four or 14 dy intervls nd reppliction of ASM improves induced resistnce ginst Xnthomons spp. in tomto (Hung et l., 2012, Pontes et l., 2016). In field study of gene expression in tomto cultivrs fter induction with multiple pplictions of ASM to tomto in the field resulted in 3.6 fold increses in cidic PR1 expression in the cultivr Supersonic fter the first ASM ppliction, while fter the second ppliction the increse ws 9.2 fold (Hermn et l., 2007). The difference in the ASM nd PABA response my be becuse ASM both primes nd ctivtes defense gene expression, or other differences in the moleculr mechnisms of induced resistnce of ASM compred to PABA. For exmple, ASM, which is n nlogue of SA, functions by inhibiting the enzymes ctlse, scorbte peroxidse, nd mitochondril oxidse, which results in the production of ROS nd incresed phenolic compounds (vn der Merwe & Dubery, 2006, Wendehenne et l., 1998), but the moleculr mechnisms for induced resistnce by PABA remin lrgely unknown. 75

99 Song et l. (2013) reported tht single PABA ppliction to roots of seedlings resulted in induced resistnce to Xe/Xp nd CMV in peppers for 77 dys, indicting tht PABA provides very longlsting protection. PABA ws effective when tomto plnts were inoculted with Pst five nd seven dys, but not 10 dys, fter the lst PABA ppliction. Thus, PABA remined effective for t lest seven dys fter the lst ppliction. However, this ws clerly less thn tht described by Song et l. (2013) nd this could be due to differences in host nd pthogen biology. However, confounding fctor is tht the susceptibility of tomto to Pst my be dependent on developmentl stge, lso known s ge-relted resistnce (Develey-Rivière & Glin, 2007) s the disese incidence in the control plnts dropped from pproximtely 5 to 2 lesions/cm 2 of lef tissue from seven to 10 DPT. Susceptibility of Arbidopsis to Pst decreses 10- to 100-fold s the plnt becomes older, which ws ssocited with the ccumultion of SA (Kus et l., 2002). In tomto, ge-relted resistnce hs been described for P. infestns (Shh et l., 2015), C. michignensis subsp. michignensis (Shrbni et l., 2013), nd Cldosporium fulvum (Pnter et l., 2002) Effect of PABA on bcteril speck lesion incidence versus Pst popultion The Pst popultion in lef tissue is normlly relted to bcteril speck severity s mesured by the percentge of leves with symptoms in A. thlin (Vlld et l., 2003) or severity disese index bsed on the number of lesions per leflet in tomto (Bysl et l., 2007, Scrponi et l., 2001). Unexpectedly, the Pst popultion per cm 2 of lef tissue did not differ mong tretments, despite reductions in disese incidence. Reductions in bcteril pthogen popultions in plnt tissues re common for plnt defence ctivtors. For exmple, lef discs from PABA treted plnts contined one order of mgnitude fewer Xe/Xp CFUs thn lef discs from control plnts (Song et l., 2013). Similrly, resistnce induced by thimine resulted in Pst popultion per grm of fresh weight in A. thlin tht were lower thn control plnts (Ahn 2007), nd ASM pplictions reduced Xe/Xp popultions in lef tissue by lmost two orders of mgnitude (Louws et l., 2001). However, not ll cses of induced resistnce involve reduction in pthogen popultion (Hmmerschmidt, 2009, Summermtter et l., 1995). For exmple, resistnce induced by P. s. pv. syringe through n incomptible interction on lower leves resulted in reduced necrosis in upper leves fter lter chllenge in A. thlin, but the popultions of P. syringe pv. syringe remined unchnged (Summermtter et l., 1995). Similrly, inocultion with Xcv, virulent Xcv, or Pst resulted in induced resistnce ginst subsequent chllenge with Xcv or Pst in helthy leves on the sme plnt bsed on lower levels of necrosis, increses in the expression of PR1 nd PR1b, nd reductions in ion lekge s n indictor of tissue dmge, but popultions of Xcv nd Pst in those leves were not ffected (Block et l., 2005). For the ltter exmple, the term systemic cquired tolernce ws used to describe reduction in symptoms but no reduction in pthogen popultion. The host response to 76

100 PABA differs from systemic cquired tolernce in tht the number of lesions but not the totl mount of necrosis observed ws reduced by PABA. The lck of correltion between disese incidence nd Pst popultion in this study could be due to more bcteri outside of the lesion or more bcteri per lesion in PABA treted plnts. To ddress the first possibility, the Pst popultion in different lef res ws exmined, nd popultions outside of the lesion in the symptomtic res on the sme leflets were more thn three orders of mgnitude lower thn res of the lesions. Thus, by fr the grestest contributor to popultion per lef re ws lesion re. To ddress the second possibility tht the Pst popultion per lesion in PABA treted plnts ws higher thn lesions in control plnts, Pst popultion per lesion ws exmined. Lesion size nd circumference in PABA treted plnts were lrger thn the control, nd thus lthough PABA is suppressing initil infections so tht fewer lesions develop, fter initil infection, the lesions of PABA-treted plnts enlrge fster. This is in contrst to the effect of ASM on bcteril speck, which reduced lesion dimeter with ASM incresing concentrtion in tomto (Scrponi et l., 2001). The effect of PABA on reducing lesion incidence but incresing their size my be explined by its mode of ction. Induced resistnce by PABA is dependent on SA-signling pthwys (Song et l., 2013, Yng et l., 2011b, Tzhoor, 2014), which re ssocited with control of biotrophs s opposed to JA/ET signling pthwys, which re ssocited with control of necrotrophs (Ton et l., 2006, Pieterse et l., 2009). For the hemibiotrophic pthogen Pst, the first opportunity for induced resistnce by PABA to ffect infection is during penetrtion through wounds or nturl openings such s stomtl closure. Stomtl closure is resistnce mechnism due to PTI (Melotto et l., 2006, Melotto et l., 2008). However, Pst cn suppress stomtl closure using the T3SS effector AvrRpt2 nd the phytotoxin corontine (Melotto et l., 2006). If one effect of PABA ws to induce resistnce by incresing stomtl closure or some other spect of penetrtion, then the bility of Pst to enter the poplst of the substomtl cvity would be reduced resulting in fewer number of infection sites. After successful entry, Pst initilly multiplies in the poplstic fluid s biotroph (Rico & Preston, 2008), where key element is the relese of T3SS effectors to suppress PTI llowing for prolonged feeding on the host cells (Preston, 2000, Cunnc et l., 2009, Munkvold et l., 2009). At this stge, the effect of PABA could be to increse resistnce mechnisms so tht the bility of these T3SS effectors to suppress PTI in the poplst is limited, thus resulting in much less biotrophic growth nd fewer infection sites where the Pst popultion could increse in size to trnsition to necrotrophic growth resulting in lesion development. However, the induction of SA-relted defenses by PABA my benefit necrotrophic growth of Pst resulting in lrger lesions, since SA cn suppress JA/ET defense signling (Gimenez-Ibnez & Solno, 2013). As result, Pst could possibly crete lrger lesions with less JA/ET-medited defenses. There is evidence for this with PABA s expression of the JA signling mrker gene CPIN2 nd the ET signling mrker gene 77

101 CTIN1 ws down-regulted 6 hours fter Xe/Xp inocultion in PABA-treted peppers (Song et l., 2013). Goodwin et l. (2017) lso observed the down-regultion of the JA-dependent gene, SlPin2, nd the ET-dependent gene, SlPR2b, compred to the wter control t one nd seven dys fter Pst inocultion, respectively. Evidence tht suppression of JA/ET defense signling by SA cn increse disese severity comes from n increse in symptoms for the necrotrophic fungus, Alternri brssiccol, which occurred when resistnce ws induced in A. thlin by prior inocultion with Pst. This inocultion induced SA-relted defense genes, such s PR1 nd suppressed JA/ET-relted defense genes, such s PDF1.2, HEL, nd CHI-B (Spoel et l., 2007). Suppression of the JA signling pthwy ws lso observed when combintion of the defense ctivtors, ASM, BABA nd cis-jsmone ws pplied to brley resulting in the downregultion of the JA biosynthesis gene LOX2 nd n increse in necrotic symptoms of Rmulri collo-cygni (Wlters et l., 2011). A second possibility is tht the lef dmge observed on leves spryed directly with PABA creted sub-lethl stress tht interfered with the bility of plnts to defend themselves ginst necrotroph. Abiotic stresses increse ROS concentrtion in plnt cells, which is ssocited with chnges in the expression of detoxifiction enzymes (Hernández et l., 2001, Alscher et l., 2002) nd hormone signling pthwys (Ben Rejeb et l., 2014). Wlters et l. (2011b) observed tht prior biotic stress due to infection of brley with R. seclis resulted in filure of the combintion of ASM, BABA, nd cisjsmone to induce resistnce in new leves s well s reduce expression of PR1 nd defense-relted enzymes. This exmple shows how prior stress resulting in necrosis cn ffect the bility of defence ctivtor to subsequently induce resistnce Effects of PABA on bcteril speck incidence in the field A field tril in 2014 ws done using concentrtion of PABA (18 mm) nd tomto cultivr (cv. H5108) tht hd resulted in significnt reductions in disese incidence in the growth room. Although folir pplictions were mde from 2 to 9 times s well s soil sok ppliction, no significnt effects of PABA were observed for ny Pst disese vribles, unlike in the growth room. This is in contrst to Song et l. (2013), who found tht PABA worked similrly in the growth room nd the field for pepper ginst Xe/Xp. One possible explntion for the results in this study is tht PABA reduced bcteril speck lesion incidence but not Pst popultions in growth room studies, nd thus reservoir of the pthogen remined in the lef. Since bcteril speck is polycyclic disese, it is possible tht even if PABA initilly reduced lesion numbers, the lesions would expnd more rpidly relesing the pthogen for future infections thus compromising the effect of PABA for sustined disese suppression over the seson. Another possibility is tht the PABA ws rpidly degrded or wshed off the plnts in the field. PABA is n orgnic compound nd hs been found to be degrded by bcteri, such s Alcligenes species isolted from soil 78

102 (Hung et l., 1981). PABA is lso known to undergo photodegrdtion prticulrly when exposed with dissolved orgnic mtter tht cts s photosensitizer (Zhou et l., 2013). Another fctor is tht compounds pplied to leves in the field, such s conventionl pesticides, cn be lost due to being wshed off by rin (Oliver & Hewitt, 2014). However, one might hve expected some degree of disese control when nine pplictions were distributed over the growing seson. There my hve been severl resons for loss of PABA pplied to the folige in the field, nd future tests my require the ddition of UV stbilizers to prevent photodegrdtion or dhesives to stick onto the folige (Oliver & Hewitt, 2014), such s those used for conventionl pesticides. Finlly, notble difference between the growth room nd field ws tht phytotoxicity ws not observed in the field, which indictes tht PABA did not induce n biotic stress response, nd thus n increse in ROS or induction of hormone signling pthwys my not hve occurred or ws gretly reduced, which possibly contributed to the resistnce observed in the growth room experiments. However, Song et l. (2013) obtined resistnce for n entire field seson in peppers with only one PABA ppliction to roots of seedlings, in which cse, it seems unlikely tht increses in ROS or hormones could hve persisted for severl months Effects of PABA on plnt growth nd development In ddition to inducing disese resistnce, there re severl reports of PABA ltering plnt growth. PABA t 7 to 14 mm pplied by seed soking stimulted seed germintion in winter whet nd winter brley (Bekusrov et l., 2013). In A. thlin grown on MS gr, the ddition of 0.02 to 2 mm PABA reduced root length with incresing concentrtion nd incresed lterl root length t low concentrtions (Crisn et l., 2014). PABA t 1 mm pplied to soil of pepper seedlings incresed yield (Song et l., 2013). In this study, 18 mm PABA generlly hd no effect on tomto root or shoot biomss in the growth room, except for reducing folir nd root weight for the non-responsive cv. TSH33, nd reducing root weight in the responsive cv. H9909 in one set of experiments. The effect of plnt defense ctivtors on plnt growth hs been most studied for ASM. Reductions in plnt growth nd development by ASM is believed to be due to resources being diverted from plnt growth to sustined defense responses (Wlters & Heil, 2007, Durrnt & Dong, 2004). This could be similr for PABA, however, Tzhoor (2014) detected significntly higher levels of PAL5 expression just prior to Pst infection in PABA treted tomtoes but not non-treted controls in PABA responsive breeding line, which indictes induction of gene expression in the bsence of the pthogen nd is ssocited with fitness costs, like with ASM. Also, expression of PR1 incresed in PABA treted tomtoes only fter Pst inocultion, which is indictive of priming, nd priming is ssocited with fewer fitness costs thn immedite induction of resistnce (Conrth et l., 2006). In the field, PABA hd no effects on tomto yield, which contrdicts the results of Song et l. 79

103 (2013) in pepper, where PABA ppliction resulted in yield increse. However, tht my hve been due to disese control, nd there my hve been no effect on yield in this study becuse PABA did not reduce bcteril speck nd bcteril spot severity. Reltive chlorophyll ws mesured s n indictor of N sttus (Muñoz-Huert et l., 2013), since plnt fitness costs re ssocited with deficiency in energy nd nutrients (Wlters & Heil, 2007). There ws no difference in reltive chlorophyll in PABA treted nd control tomtoes, indicting tht chlorophyll levels nd N sttus were not ffected by PABA. Thus, there is no evidence for improved plnt growth prmeters from the field pplictions of PABA in this study, nd the only detection of growth impcts were negtive effects in the growth room, which were not consistently observed Conclusions This study demonstrtes tht PABA cn decrese disese incidence of bcteril speck of tomtoes in the growth room bsed on lesion numbers, lthough the popultion of the pthogen nd the overll plnt surfce re ffected by bcteril speck were not ffected by PABA. Unlike other studies, ppliction of PABA to roots hd no effect on disese incidence, possibly becuse of differences in methods of plnt culture. However, folir pplictions were effective in the growth room, which ws dependent on PABA concentrtion nd host genotype. The timing of Pst inocultion fter the lst PABA ppliction my influence effectiveness, but this requires further investigtion due to confounding effects of plnt ge nd possible developmentl resistnce to Pst. Surprisingly, other fctors, such s the number of PABA pplictions prior to Pst inocultion did not ffect efficcy, indicting tht single PABA ppliction my hve sustined effect tht mximizes defense gene expression over t lest seven dys. Despite selecting fctors tht provided disese resistnce in the growth room, there ws no evidence tht PABA ws effective in one field experiment. The usefulness of PABA s disese mngement tool in tomto is limited unless the fctors cusing PABA to be ineffective under field conditions re discovered. However, even if PABA cn be mde effective in the field, there re concerns with its use s PABA did not reduce Pst popultions or necrotic lef surfce re in treted plnts. There could be lrge reservoirs of the pthogen in treted plnts, nd thus even more severe disese outbreks could occur if PABA tretments re stopped or become ineffective during the growing seson. Further study on the moleculr mechnisms of the effect of PABA on lesion number nd size re needed to determine if PABA my ctully be counterproductive for disese control in crops such s tomto, which re ttcked by rnge of different pthogens tht re biotrophic, hemibiotrophic nd necrotrophic. 80

104 3 Chpter 3: Effects of the plnt growth regultor uniconzole with the plnt defense ctivtor cibenzolr-s-methyl on incidence nd severity of bcteril speck (P. syringe pv. tomto) nd bcteril spot (X. grdneri) in tomto 3.1 Introduction Bcteril spot, cused by one or more members of the BSX (Xe, Xv, Xp nd Xg) nd bcteril speck cused by Pst re the two importnt folir diseses of tomto. When estblished shortly fter trnsplnting, they hve cused losses s high s 75% for speck during the cool nd riny seson in Isrel (Yunis et l., 1980) nd 100% for spot during the wrm nd riny seson in Florid (Ritchie, 2000). In the wrm nd humid growing seson in Ontrio, losses up to 60% hve been reported s result of speck nd spot epidemics (LeBoeuf et l., 2009). The pthogens infect folige resulting in the development of smll (<5 mm), drk brown, irregulr lesions tht reduce photosynthetic cpcity, ccelerte defolition, nd cuse premture fruit ripening tht reduce yield nd disrupt hrvest scheduling. Spot nd speck lesions on fruit enlrge to 5 to 8 mm nd 1 to 2 mm in dimeter, nd when severe, reduce the bility of processors to effectively peel tomtoes for whole pck nd diced mrkets (LeBoeuf et l., 2009, Jones, 1991, Jones, 1991b, Koike et l., 2007). Copper bctericides lone or in combintion with the EBDC fungicide mncozeb re the primry chemicl option for controlling bcteril spot nd speck. However, copper hs often proven to be ineffective, possibly becuse of the ppernce of copper tolernt strins of Pst nd BSX in mny prts of the world (Griffin et l., 2017), including Florid (Mrco & Stll, 1983), North Crolin (Ritchie & Dittpongpitch, 1991), Arizon nd Mexico (Adskveg & Hine, 1985), Cliforni (Bender & Cooksey, 1986, Bender & Cooksey, 1985, Cooksey et l., 1990, Cooksey & Grhm, 1989), Ohio, Tiwn nd Argentin (Cooksey, 1990) nd Ontrio (Cuppels & Elmhirst, 1999, Abbsi et l., 2015). Alterntive products, such s ksugmycin (Ksumin), B. subtilis QST 713 (Serende Mx), nd extrct of R. schlinensis (Regli Mxx), re registered for suppression of bcteril spot in Cnd nd the USA, but their performnce hs been inconsistent (Roberts et l., 2008, Truemn, 2015, Miller et l., 2005, Miller & Mer, 2008, Vlld & Hung, 2011). Another option for chemicl control is the plnt defence ctivtor ASM, which is mrketed s Actigrd in North Americ nd Bion in Europe. ASM ctivtes the plnt defense response known s SAR resulting in incresed resistnce for mny crops ginst vriety of pthogens (Kunz et l., 1997). In tomto, for exmple, ASM incresed disese resistnce to bcteril spot (Abo-Elyousr & El-Hendwy, 2008, Louws et l., 2001), bcteril speck (Byrne et l., 2005, Hermn et l., 2007), grey mould (Achuo et l., 2006), corky root rot (Bubici et l., 2006) nd bcteril cnker (Bysl et l., 2003), s well s incresed 81

105 insect pest resistnce to two-spotted spider mite (Choh et l., 2004), silverlef whitefly (Nombel et l., 2005) nd green pech phid (Boughton et l., 2006). However, the level of control by ASM cn be highly vrible in the field. Louws et l. (2001) reported tht n undefined number of ASM pplictions on 7 or 10-dy intervl reduced folir severity of bcteril spot rnging from 17 to 85% in 13 of 14 experiments nd bcteril speck from 23 to 100% in ll seven experiments compred to the nontreted controls showing high vribility. Similrly, Roberts et l. (2008) reported no effect of 11 ASM ppliction compred to nontreted control in one experiment but reductions in folir bcteril spot disese severity of 66% in nother experiment, nd no effect of six ASM pplictions in one experiment but 67% reduction in disese in one other experiment. Wilson et l. (2002) reported folir bcteril speck reductions of 35 to 70% in three experiments with 8 to 10 ASM pplictions, nd Obrdovic et l. (2004) reported folir bcteril spot disese reductions of 18 to 37% in three experiments with 6 ASM pplictions. Although Hung et l. (2012) reported tht weekly pplictions of ASM effectively reduced Xp severity with n inverse reltionship between ASM rtes nd disese severity, the inverse reltionship ccounted for only 37 nd 35% of the vrition observed, suggesting tht other fctors lso influenced the effectiveness of ASM in the field. One explntion for the vribility of ASM effectiveness could be relted to it hving host fitness costs (Durrnt & Dong, 2004, Wlters & Heil, 2007). During the SAR response, ASM, which is functionl nlog of SA, inhibits mitochondril NADH:ubiquinone oxidoreductse of complex I of the electron trnsport chin (vn der Merwe & Dubery, 2006) s well s the H 2 O 2 scvenging enzymes ctlse nd scorbte peroxidse (Wendehenne et l., 1998), resulting in incresed levels of ROS nd thus possibly plnt stress. The ccumultion of ROS nd SA triggers systemic response tht upregultes the expression of genes encoding defense compounds, like PR proteins, tht require plnt nutrients nd energy tht would otherwise be llocted to plnt growth nd development nd stress resistnce (Pieterse et l., 2009, Spoel & Dong, 2012). In some cses, yield losses hve been ssocited with ASM ppliction to tomto under field conditions (Dmicone & Trent, 2003, Lnge & Smrt, 2005, Louws et l., 2001, Pontes et l., 2016). However, the concentrtions used in those studies were either more thn two times higher thn the Cndin lbel rte or included more thn the recommended number of pplictions per seson (PMRA, 2011), wheres no yield reductions were observed in tomtoes t concentrtions closer to the Cndin lbel rte (Alexnder & Wldenmier, 2003, Grves & Alexnder, 2002, Lnge et l., 2007, Lewis Ivey et l., 2004, Miller et l., 2002, Roberts et l., 2008). Folir ASM pplictions to grfted field tomto lso resulted in reduction in yield compred to grfted control plnts growing in R. solncerum infested soils using concentrtion tht ws 25% higher thn the Cndin lbel rte (Kunwr et l., 2017). Nevertheless, the observtion tht ASM-treted plnts cn occsionlly lower yields indictes plnt stress. 82

106 One possible wy to minimize the fitness costs of ASM is to provide the plnt with other tretments tht lter the plnt s physiology for incresed stress tolernce. Appliction of the trizole-type PGR UNI, which is mrketed s Sumgic in North Americ, inhibits the synthesis of GA in plnts by blocking the cytochrome P-450-dependent monooxygenses which prevents the oxidtion of ent-kurene into ent-kurenoic cid in the GA biosynthesis pthwy (Rdemcher, 2000). It is used in commercil tomto seedling production to prevent stem elongtion nd limit the production of over-sized plnts tht re difficult to trnsplnt into the field (Zndstr et l., 2006). In ddition, UNI ppliction increses stress tolernce. UNI reduced symptoms of chilling injury in tomto, slt stress in Dtur spp., nd drought stress in soyben nd whet, which ws linked to incresed ntioxidnt levels, protein contents, photosynthetic rtes, nd chlorophyll content, s well s chnges in other phytohormone levels (Al- Rumih & Al-Rumih, 2007, Dun et l., 2008, Senrtn et l., 1988, Zhng et l., 2007). Combining ASM with pclobutrzol, nother trizole-type PGR structurlly similr to UNI, reduced symptoms of Pst on tomto leves under greenhouse conditions to greter extent thn ASM lone, but the effect on subsequent disese development nd plnt growth in the field ws not evluted (Mhesniy, 2002). The combintion of ASM nd pclobutrzol pplied to tomto seedlings ws evluted for effects on disese severity in field trils completed between 2002 nd 2004 t the University of Guelph, Ridgetown Cmpus, but results were inconsistent nd the BSX species used to inoculte in the field is unknown (Dr. Ron Pitbldo unpublished). As previous results using the combintion of ASM nd trizole-type PGR hve been inconsistent nd/or not well described, the potentil benefits of pplying ASM to trizole-type PGRtreted tomto seedlings were explored in this chpter. UNI ws chosen s the trizole-type PGR insted of pclobutrzol becuse pclobutrzol ws not pproved for use on tomto seedlings in Cnd by the PMRA, wheres UNI ws pproved for use in Cnd in 2011 (J. LeBoeuf, pers. communiction). The objective of this reserch ws to determine if n ASM nd UNI combintion might result in reducing ASM induced fitness costs nd improve the consistency of bcteril spot nd bcteril speck suppression in processing tomtoes under Ontrio field conditions. This ws tested by evluting seson-long effects on bcteril speck nd spot development nd tomto growth with one greenhouse ppliction of UNI followed by six field pplictions of ASM or one greenhouse ppliction of UNI followed by three greenhouse pplictions of ASM nd no field pplictions of ASM. 3.2 Mterils & Methods Wether conditions Outdoor minimum nd mximum dily temperture nd rinfll during the study period ech yer ws obtined from the Environment Cnd ( wether sttion locted t 83

107 Ridgetown Cmpus. Ten yer verges for dily minimum nd mximum temperture nd monthly precipittion were clculted for For periods with missing vlues, dt from the New Glsgow wether sttion locte 20 km northest ws used insted Greenhouse tretment with UNI followed by field tretment with ASM Experimentl design nd tretments Tomto cv. TSH4 ws seeded in commercil trnsplnt production greenhouse locted pproximtely 25 km from Ridgetown, Ontrio, Cnd on 5 April 2011, 4 April 2012, nd 8 April 2013, nd the seedlings were produced using norml grower prctices. The tretments were non-treted control, 0.02 mm UNI pplied s Sumgic (Vlent Cnd, Guelph, ON; 0.055% UNI), 0.14 mm ASM pplied s Actigrd (Syngent Cnd, Guelph, ON, CA; 50% ASM), nd 0.14 mm ASM mm UNI (Tble 3.1). Tretment with UNI occurred once by sprying tomto folige in the greenhouse on 27 April 2011 (22 DAS), 23 April 2012 (19 DAS), nd 6 My (32 DAS) t the two-lef stge. UNI pplictions were mde using hnd mist spryer with 100 ml of solution pplied per 288-cell try (Tble 3.1). ASM tretments were performed in field plots estblished t the Ridgetown Cmpus ( N, W). The tomto seedlings were trnsplnted into twin-rows in the field using mechnicl trnsplnter on 31 My 2011 (56 DAS), 18 My 2012 (44 DAS) nd 22 My 2013 (44 DAS). Individul rows within ech set of twin rows were 50 cm prt with plnts within rows on 33 cm spcing. Ech set of twin-rows ws spced 1.5 m prt. Ech plot consisted of one 7 m twin-row. Tretments were rrnged in rndomized complete block design with four replictions per tretment. ASM tretment ws done using six pplictions beginning 2 or 3 DAT to the field t 7-dy intervls, except in 2011 when poor wether delyed some tretment pplictions (Tble 3.1). ASM ws pplied t 280 L/H for the first two pplictions one nd two, 467 L/H for third nd fourth pplictions, nd 655 L/H for the fifth nd sixth pplictions in ccordnce with the Cndin product lbel (Syngent Cnd 2012). ASM ws pplied using hnd-held 2 m CO 2 boom spryer with ULD nozzles (Pentir Ltd., New Brighton, MN, USA) t pressure of 241 kp. Preventtive fungicide pplictions of Qudris (zoxystrobin, Syngent Cnd, Guelph, ON, CA) were mde for erly blight (Alternri solni) on 10 July (96 DAS) nd Brvo (chlorothlonil, Syngent Cnd, Guelph, ON, CA) on 24 July nd 2 Aug 2012 (110 nd 123 DAS). Applictions of Brvo were lso pplied for prevention of erly blight nd lte blight on 19 June, 4, 17, nd 30 July 2013 (73, 89, 102, nd 115 DAS). All insecticide nd fungicide pplictions were pplied using lbelled Cndin product rtes Pthogen inocultions 84

108 Pst isolte 06T2 nd Xg isolte DC00T7A were obtined from Dr. Dine Cuppels, Agriculture nd Agri-Food Cnd, London, Ontrio, Cnd. These were originlly isolted from tomtoes in southwestern Ontrio in 2006 nd 2000, respectively. Isoltes were grown seprtely in tryptic soy broth overnight with shking t 150 rpm, nd the concentrtion of ech djusted to pproximtely 1 x 10 6 CFU/mL in distilled wter with surfctnt, 0.025% Sylgrd 309 (siloxylted polyether; Dow Corning, Georgetown, ON, CA). Pst isolte 06T2 nd Xg isolte DC00T7A were mixed to obtin equl popultions nd pplied using hnd-held 2 m CO 2 boom spryer with ULD nozzles t pressure of 241 kp t rte of 200 L wter/h on 16 nd 30 June 2011 (72 nd 86 DAS), 30 My 2012 (55 DAS), nd 29 My nd 5 June 2013 (51 nd 58 DAS) to tomtoes post-trnsplnting. Only one inocultion ws mde in 2012 becuse symptoms were clerly visible within seven dys fter the first inocultion dte Disese nd plnt growth ssessments Disese incidence ws determined by counting the number of compound leves with symptoms of smll necrotic drk brown lesions indicting bcteril spot nd speck in 1.24 m 2 re (five plnts) in ech plot on 22, 30 June, 8, 19 nd 27 July 2011 (78, 86, 94, 105, nd 113 DAS), 7, 15, 21 June, nd 4 July 2012 (63, 71, 77, nd 90 DAS), nd 11, 20 nd 29 June 2013 (57, 66, nd 75 DAS). In 2013, the totl number of leves in the re ws lso counted nd the percentge of leves with symptoms ws clculted. Lef counts did not continue beyond these dtes s the mount of folige present mde it impossible to ccurtely ssess ll leves present in the re. Disese incidence ws lso determined by estimting the percent defolition for the entire re of ech plot t the times when leves with lesions were counted, s well s 8, 18 nd 23 Aug 2011 (125, 135, nd 140 DAS), 11, 23 July, 7 Aug 2012 (97, 109, nd 124 DAS), nd 29 June, 7, 16, 24 July, 1, 13, nd 19 Aug 2013 (75, 83, 92, 100, 108, 120, nd 126 DAS). The disesed lef number nd percent defolition vlues were used to clculte n erly nd lte seson AUDPC using the following eqution: AUDPC = [((Y i + Y i-1 ) (X i X i-1 ))/2]. Y i is number of infected leves or percent defolition t dy X i, nd Y i-1 is number of infected leves or percent defolition t dy X i-1. Plnt growth stges for the five plnts used for lef counts were lso recorded on ech disese ssessment dte. Growth stges were: seedling (no brnches), vegettive growth (brnched plnt with no flowers), flowering (minimum first inflorescence developed), or ripening (one or more fruit with breker colour). Reltive plnt chlorophyll levels on one newly emerged leflet from ech of the 5 plnts used for disese ssessment ws mesured using Minolt SPAD-502 Chlorophyll Meter (Konic Minolt, Osk, Jpn) t 18, 36 nd 56 DAT ech yer (74, 92 nd 112 DAS in 2011, nd 62, 80, nd 100 DAS in 2012 nd 2013). Tomtoes were hrvested from ll four replicted blocks on 24 nd 25 Aug 2011 (141 nd

109 DAS) nd 13 Aug 2012 (130 DAS). In 2013, tomtoes from three replicted blocks were hrvested on 23 Aug (130 DAS), but the fourth replicted block ws not hrvested becuse of poor plnt stnd due to frost dmge in My. A 2 m section of twin-row ws hrvested in ech plot, nd the red, green nd rotted fruit were weighed. Fifty green fruit were rndomly selected nd evluted for incidence of bcteril spot (drk brown lesions 3 to 6 mm in dimeter) nd bcteril speck (drk brown lesions 1 to 2 mm in dimeter). Fifty red fruit were lso rndomly selected nd evluted similrly for the incidence of bcteril spot nd speck, nd the red fruit were then stored t room temperture for three dys nd ssessed second time for the severity of nthrcnose (sunken lesions with concentric rings usully t lest 1 cm in dimeter) using the following scle: 0 = no nthrcnose, 1 = one lesion, 2 = two lesions, 3 = three lesions, 4 = four or more lesions. A disese severity index for nthrcnose ws clculted using the following eqution (Kobriger nd Hgedorn 1983): [(clss no.)(no. of fruit in ech clss)] DSI = x 100 (totl no. fruit per smple)(no. clsses -1) Fifty dditionl red fruit were wshed nd submitted for processing tomto qulity ssessment by the Ridgetown Cmpus tomto breeding lb of S. Loewen. Soluble solids, pulp ph, nd tomto colour were mesured s described by Vn Eerd & Loewen (2009) Sttisticl nlysis Sttisticl nlysis ws completed using SAS v9.4 (SAS Institute, Cry, NC, USA). Dt were tested for normlity using the Shpiro-Wilk sttistic. Outliers were identified using Lund s test of stndrdized residuls (Lund 1975). Anlysis of vrince ws completed using Proc Mixed with tretment s fixed effect nd repliction s rndom effect. Dt were nlyzed s rndomized complete block design. Mens comprisons were performed when P 0.05, nd mens were seprted using Tukey s HSD. A preliminry nlysis combining dt from ll yers reveled tretment x yer interctions, nd therefore it ws decided to nlyse dt seprtely from ech yer Greenhouse tretment with UNI followed by greenhouse tretment with ASM or CuOH Experimentl design nd tretments Tomto cv. TSH4 nd cv. H9909 were seeded in commercil trnsplnt production greenhouse locted pproximtely 25 km from Ridgetown, Ontrio, Cnd on 5 April 2011, 4 April 2012, nd 8 April 2013, nd the seedlings were produced using norml grower prctices. Greenhouse plots consisted of one-hlf of 288-cell try rrnged in rndomized split-plot design with four replictions, with cultivr s the split-plot nd chemicl tretment s the min plot. The tretments were non-treted control, 17.6 mm CuOH pplied s Kocide 2000 (E.I. du Pont Cnd Co, Missisug, ON; 53.8% 86

110 CuOH), 17.6 mm CuOH mm UNI pplied s Sumgic, 0.14 mm ASM pplied s Actigrd, nd 0.14 mm ASM mm UNI (Tble 3.2). An dditionl tretment, UNI (0.02 mm) lone, ws included in UNI tretments were pplied using registered lbel ppliction timing nd rtes to tomto folige in the greenhouse t the two-lef stge on 27 April 2011 (22 DAS), 23 April 2012 (19 DAS), nd 6 My 2013 (32 DAS). The pplictions were mde using the sme methods s described for greenhouse pplictions in section CuOH tretment ws done by sprying folige six times in 2011 nd 2012 nd five times in 2013 on 5 to 8 dy intervl beginning 20 April 2011 (15 DAS), 21 April 2012 (17 DAS), nd 26 April 2013 (18 DAS). ASM tretment ws done sprying folige on 28 April 6 nd 17 My 2011 (23, 31, nd 42 DAS), 25 April, 4 nd 14 My 2012 (21, 30, nd 40 DAS), 30 April nd 1 nd 20 My (22, 33, nd 42 DAS). CuOH nd ASM were pplied using hnd mist spryer using wter volume of 1000 L/H. The seedlings were then trnsplnted to field locted t the Ridgetown Cmpus, University of Guelph on 31 My 2011 (56 DAS) nd 21 My 2012 (47 DAS). For 2013, the trnsplnting occurred on 5 June 2013 (57 DAS), s n erlier trnsplnting on 22 My (44 DAS) ws discrded due to serious frost event on 25 nd 26 My cusing pproximtely 80% plnt loss. The field plots were estblished using the methods described in section Fungicide nd insecticide pplictions were mde on the sme dtes s those listed in section , except n dditionl ppliction of Brvo ws pplied 22 Aug 2013 (135 DAS) Pthogen inocultions Tomto plnts were inoculted with Pst isolte 06T2 nd Xg isolte DC00T7A using the methods described in section on 16, 30 June, nd 9 July 2011 (72, 86, nd 95 DAS), 30 My nd 12 June 2012 (55 nd 67 DAS), nd 11 nd 19 June 2013 (63 nd 71 DAS). However, the inocultion of 9 July 2011 (95 DAS) ws done by soking collection of infected leves from grower's field locted pproximtely 150 km from Ridgetown in wter, filtering through cheesecloth, nd pplying the solution t rte of 100 ml slurry/l. This ws done becuse disese symptoms were slow to develop Disese nd plnt growth ssessments Trnsplnt cnopy height ws mesured t 14, 22, 28, 35, 41 nd 52 DAS (19, 25 April, 1, 7 nd 18 My 2011, 18, 24, 30 April, 6 nd 17 My 2012, nd 22, 28 April, 4, 10 nd 21 My 2013) t five loctions in ech plot from the soil line to the top of the cnopy. Stem dimeter t the bse of five seedlings ws mesured using digitl clipers t 35 nd 41 DAS in ech yer s well s t 52 DAS in 2011 nd Root nd folir dry weight ws mesured t 56 DAS in 2011 (22 My) nd 44 DAS in 2012 nd 2013 (9 nd 13 My), which ws just prior to trnsplnting. Dry weights were obtined from roots nd folige from 5 plnts per replicte tht were rinsed to remove medi nd then dried in 87

111 greenhouse for 1 to 2 weeks. Disese, plnt growth, nd yield evlutions were completed using the methods described in Section Plnt lef counts were performed 22, 30 June, 8, 19, nd 27 July 2011 (78, 86, 94, 105 nd 113 DAS), 8, 15, 21 June, nd 5 July 2012 (63, 70, 76 nd 90 DAS), nd 22 June, 1 nd 8 July 2013 (74, 83, nd 90 DAS). Percent defolition ws estimted on ll dtes where lesions were counted, s well s 8, 18 nd 29 Aug 2011 (125, 135 nd 146 DAS), 11, 23 July, 7 nd 17 Aug 2012 (102, 114, 129, nd 139 DAS), nd 16, 23 July, 1, 13 nd 23 Aug 2013 (98, 105, 114, 126 nd 135 DAS). All four replicte blocks of tomtoes were hrvested on 30 Aug 2011 (147 DAS), 21 Aug 2012 (143 DAS), nd 30 Aug 2013 (TSH4) (142 DAS), but only three out of four replicte blocks were hrvested on 3 Sept 2013 (cv. H9909) (146 DAS). The fourth replicte block of cv. H9909 ws hrvested on 11 Sept (153 DAS) s there ws dt entry error for this block on 3 Sept. TSH4 ws hrvested before cv. H9909 in 2013 becuse they mtured erlier Sttisticl nlysis Sttisticl nlysis ws completed using the sme methods described in section A preliminry nlysis combining dt from ll yers nd cultivrs reveled numerous tretment x yer, tretment x cultivr, nd yer x cultivr interctions, therefore it ws decided to nlyse dt seprtely from ech yer nd cultivr for ech vrible. 88

112 Tble 3.1 Tretment descriptions nd ppliction timings for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Further detils on pplictions methods re described in the text. Appliction Timing (# dys) Tretment DAS DAT DAS DAT DAS DAT 1. Control UNI (0.02 mm) b ASM (0.14 mm) - 3, 15, 21, 34, 42, 49-2, 10, 17, 24, 31, 38-2, 9, 16, 23, 30, UNI (0.02 mm) ASM (0.14 mm) , 15, 21, 34, , 10, 17, 24, 31, 38 42, 49 DAS = dys fter seeding; DAT = dys fter trnsplnting. b UNI pplictions were mde when seedlings were t the two-lef stge. - 2, 9, 16, 23, 30, 37 89

113 Tble 3.2 Tretment descriptions nd ppliction timings for tomto cv. TSH4 nd H9909 treted with uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Further detils on pplictions methods re described in the text. Tretment Appliction Timing (DAS) Control CuOH (17.6 mm) 15, 23, 27, 32, 36, 43 17, 22, 27, 33, 37, 42 18, 27, 33, 38, UNI (0.02 mm) b CuOH (17.6 mm) 22 15, 23, 27, 32, 36, , 22, 27, 33, 37, , 27, 33, 38, ASM (0.14 mm) 23, 31, 42 21, 30, 40 22, 37, UNI (0.02 mm) ASM (0.14 mm) 22 23, 31, , 30, , 37, UNI (0.02 mm) DAS = dys fter seeding. b UNI pplictions were mde when seedlings were t the two-lef stge. 90

114 3.3 Results Wether Conditions Sesonl vrition in mximum nd minimum dily tempertures in 2011, 2012 nd 2013 tended to follow the 10-yer historicl trend with tempertures peking in lte July. However, there were dys bove 30 C in erly June 2011 nd multiple dys over 30 C from lte My to lte July in 2012 (Figure 3.1-d). Frost events on 25 nd 26 My dmged tomto plnts in 2013 (Figure 3.1c). Averge monthly mximum nd minimum tempertures in 2011 were similr to the verge, except for the mximum dily verge being 2.2 C bove the 10-yer verge in July (Figure 3.2), nd minimum dily verge being 1.1 nd 2.2 C bove the 10-yer verge in My nd July (Figure 3.2b). Monthly mximum nd minimum dily tempertures in 2012 rnged from 0.5 to 2.5 C nd 0.7 to 1.5 C degrees higher thn the 10-yer verge in My, June, nd July. Averge mximum dily tempertures in June, July, nd Aug 2013 were 1.6, 1.3, nd 0.9 C below the 10-yer verge. Thus, tempertures were generlly bove verge in the erly prt of the seson in 2011 nd most of the seson in 2012, nd below verge most of the seson in Dily rinfll records showed tht hevy rin events (>25 mm) occurred twice in My nd once in June in 2011, once in June, four times in July, nd once in Aug in 2012, nd once in My, twice in June, nd once in July nd Aug in 2013 (Figure 3.1-c). Two of the July rin events in 2012 exceeded 50 mm of precipittion. Totl rinfll in 2011 ws similr to the 10 yer monthly verge, except tht the hevy rinfll events in My resulted in totl monthly rinfll 50 mm greter thn the 10-yer verge (Figure 3.2c). Rinfll in 2012 ws 60% below the 10-yer verge in My, but ws 179% bove the 10-yer verge in July, minly due to two rinfll events tht exceeded 50 mm of precipittion. Rinfll in 2013 ws below the 10-yer verge in ll months except June. Thus, monthly rinfll went from bove verge to similr to the 10-yer verge in 2011, below verge to bove the 10-yer verge in 2012, nd generlly below the 10-yer verge in Greenhouse tretment with UNI followed by field tretment with ASM In this section of the study, UNI ws pplied once in the greenhouse before trnsplnting to the field using the recommended ppliction timing for plnt height control, nd ASM ws pplied six times fter trnsplnting in the field in order to evlute the effect of ASM on disese development during the growing seson following the currently registered use pttern of ASM in Cnd Effect of greenhouse UNI followed by field ASM on bcteril speck nd bcteril spot Erly seson bcteril speck nd spot incidence on cv. TSH4 ws mesured s the number of 91

115 symptomtic leves in 1.24 m 2 re in ech plot, which ws ssessed from lte June to lte July in 2011, erly June to erly July in 2012 nd mid-june to erly July in 2013 (Figure 3.3). In 2011, pplictions of ASM first reduced disese incidence (74% fewer symptomtic leves) compred to the nontreted control on 19 July (105 DAS) nd then by 68 nd 51% compred to the nontreted control nd UNI on 27 July (113 DAS) in 2011 (Figure 3.3). UNI nd ASM+UNI tretments reduced erly seson disese compred to the nontreted control on 27 July (113 DAS). Erly seson AUDPC in 2011 (22 June to 27 July) ws 65% lower in the ASM tretment thn the nontreted control (Figure 3.4). In 2012, pplictions of ASM reduced erly seson disese incidence compred to the nontreted control on 4 July (90 DAS) (Figure 3.3b). Erly seson disese for the UNI tretment ws never significntly lower thn the nontreted control, but ASM+UNI tretment ws significntly lower thn the nontreted control strting on 15 June (71 DAS) (86% fewer symptomtic leves) nd then ws 32 nd 28% lower thn the nontreted control nd UNI, respectively, on 4 July (90 DAS). Erly seson AUDPC in 2012 (7 June to 4 July) ws 38 nd 42% lower in tretments with ASM nd ASM+UNI thn the nontreted control (Figure 3.4b). In 2013, erly seson disese incidence for ll the tretments (ASM, UNI nd ASM+UNI) ws significntly lower thn the nontreted control on 20 June 2013 (66 DAS) (Figure 3.3c). Erly seson AUDPC in 2013 (11 June to 29 June) ws 31 nd 50% lower in the ASM nd ASM+UNI tretments thn the nontreted control (Figure 3.4c). Thus, ASM pplictions resulted in reduction in erly seson AUDPC compred to the nontreted control in ll yers but UNI+ASM only in 2012 nd Lte seson bcteril speck nd spot incidence ws mesured s the percent defolition per plot, which ws ssessed from lte July to lte Aug in 2011 nd lte June to mid-aug in 2012 nd 2013 (Figure 3.5). In 2011, ASM, UNI nd UNI+ASM tretments ll hd less defolition thn the nontreted control on 27 July, but by 23 Aug (140 DAS), only defolition in the ASM tretment ws lower thn the nontreted control (Figure 3.5). Lte seson AUDPC in 2011 (27 July to 23 Aug) with ASM ws the only tretment tht ws significntly lower thn the nontreted control (by 58%) (Figure 3.6). In 2012 nd 2013, there ws no difference mong tretments for defoliiton (Figure 3.5b & Figure 3.5c, Figure 3.6b & Figure 3.6c). Thus, ASM provided some reduction in defolition in one of three yers compred to the nontreted control nd there ws no effect of UNI on defolition, indicting limited consistent benefits of either tretment in preventing premture tomto defolition. The incidence of bcteril speck on red fruit rnged from 2.0 to 10.5% from 2011 to 2013, while the incidence of bcteril spot on red fruit rnged from 9.0 to 24.2% from 2011 to 2013 (Tble 3.3). Xg ws the predominte red fruit pthogen in 2012 nd 2013, but ws similr in incidence to Pst in There ws no significnt reduction in either bcteril speck or spot symptoms mong tretments on red (Tble 3.3) or green fruit (Tble A.3). There ws no effect on the incidence or severity of nthrcnose on tomto fruit in ny yer (Tble A.4). 92

116 Effect of greenhouse UNI followed by field ASM on tomto growth, yield, nd qulity In 2011, reltive chlorophyll content of the nontreted control t 18 DAT (74 DAS) ws equivlent to ASM but ws 8.6 nd 10.2 SPAD units lower thn UNI nd UNI+ASM, respectively (Figure 3.7). In 2012, reltive chlorophyll content mong tretments t 18, 36 nd 56 DAT ws not different (Figure 3.7b). In 2013, reltive chlorophyll content of the nontreted control t 18 DAT (62 DAS) ws equivlent to ASM but ws 5.7 nd 7.6 SPAD units lower thn UNI nd UNI+ASM, respectively (Figure 3.7c). There were no differences mong tretments t 36 DAT in 2013 but UNI ws higher thn the control nd other tretments t 56 DAT. Thus, ASM did not lter chlorophyll content compred to the nontreted control, wheres UNI or UNI+ASM pplictions incresed chlorophyll content t 18 DAT in two of three yers. The number of dys to rech first inflorescence, fruit set, nd ripening ws not ffected by tretment (Tble A.5). Totl red, green nd rotten fruit tomto yield ws 22% higher in the ASM+UNI tretment thn the nontreted control in 2011; however, ASM nd UNI lone did not significntly ffect red, green or rotten tomto yield in 2011 (Tble 3.4). There were no significnt differences mong tretments in 2012 nd There ws no difference mong tretments for tomto fruit colour, ph nd soluble solids, except in 2011 when tretment UNI hd 0.05 higher ph units thn the control (Tble 3.5) Greenhouse tretment with UNI followed by greenhouse tretment with ASM In this section of the study, UNI ws pplied once in the greenhouse before trnsplnting to the field using the recommended ppliction timing for plnt height control, nd ASM ws pplied pplied three times in the greenhouse before trnsplnting in order to evlute long term effects of ASM on disese development nd plnt growth post-trnsplnting. Greenhouse tretment with ASM ws exmined to determine if the effects of ASM persisted for long periods of time, since three pplictions from lte April to mid-my were done for the greenhouse ASM tretment versus six pplictions from lte My to erly July ws done for the field ASM tretments. Assessments were completed using similr methods nd similr timings s those described in the previous section for greenhouse tretment with UNI followed by field tretment with ASM. However, cv. H9909 ws included s well s cv. TSH Effect of greenhouse UNI followed by greenhouse ASM on bcteril speck nd bcteril spot For cv. TSH4, there ws no difference mong tretments for erly seson folir disese incidence in 2011 (22 June to 27 July; Figure 3.8) or the erly seson AUDPC (Figure 3.9). In 2012, ASM nd CuOH+UNI hd no effect on erly seson incidence compred to the nontreted control t ny time point 93

117 (8 June to 4 July), but ASM+UNI tretment ws 55% lower thn the control on 5 July (91 DAS) (Figure 3.8b). The ASM+UNI tretment lso hd 52, 57, nd 51% lower totl erly seson AUDPC thn the control, CuOH nd CuOH+UNI (Figure 3.9b). In 2013, there ws significnt differences between tretments only on 22 June with ASM+UNI hving lower incidence thn the nontreted control nd CuOH, nd UNI nd CuOH+UNI hving lower incidence thn the nontreted control (Figure 3.8c). However, there ws no difference mong tretments for totl erly seson AUDPC in 2013 (Figure 3.9c). Thus, compred to the nontreted control, CuOH+UNI nd UNI (2013 only) provided dditionl benefits, but only on specific ssessment dtes. The only tretment with lower erly seson AUDPC thn the control ws ASM+UNI which reduced erly seson AUDPC compred to the nontreted control in one of three yers. For cv. H9909 in 2011, ASM tretment ws equivlent to the nontreted control on ll ssessment dtes (22 June to 27 July; Figure 3.8d) nd for totl erly seson AUDPC (Figure 3.8b). The CuOH+UNI nd ASM+UNI tretments hd fewer erly seson symptomtic leves thn the control nd n equivlent number to CuOH tretment on 30 June, 8 nd 19 July (86, 94 nd 105 DAS). Totl erly seson AUDPC for 2011 ws lowest for CuOH nd CuOH+UNI tretments, which were both significntly lower thn the control but equivlent to the ASM+UNI tretment. In 2012, pplictions of ASM, CuOH+UNI, nd ASM+UNI were equivlent to the nontreted control on ll ssessment dtes (8 June to 4 July; Figure 3.8e), nd for erly seson AUDPC (Figure 3.9b). Similrly, there ws no effect in 2013 of ASM, UNI, CuOH+UNI or ASM+UNI tretments on erly seson disese incidence or totl erly seson AUDPC (Figure 3.8f nd Figure 3.9c). Thus, ASM or ASM+UNI did not reduce totl erly seson disese compred to the nontreted control in ny yer in cv. H9909, which differed from cv. TSH4 where pplictions of ASM+UNI provided benefits bsed on AUDPC in one yer. Lte seson defolition ws ssessed in the sme mnner s the section on field pplictions of ASM. In 2011 for cv. TSH4, pplictions of ASM nd ASM+UNI resulted in defolition levels tht were equivlent to the control nd CuOH on ll ssessment dtes (27 July to 29 Aug; Figure 3.10) nd for totl lte seson AUDPC (Figure 3.11). Applictions of CuOH+UNI resulted in reduction in defolition compred to the control on 8 nd 18 Aug. Lte seson AUDPC ws lower thn the nontreted control for CuOH+UNI. In 2012, ASM nd ASM+UNI hd similr effects with the tretments resulting in significnt reduction in defolition compred to the control on 23 July, nd compred to the control nd CuOH on 7 nd 18 Aug (5 July to 18 Aug; Figure 3.10c). Applictions of CuOH+UNI did not reduce defolition compred to the control or CuOH on ny ssessment dte. Lte seson AUDPC ws lower for ASM nd ASM+UNI thn the control, CuOH nd CuOH+UNI tretments (Figure 3.11b). In 2013, pplictions of ASM, UNI, CuOH+UNI nd ASM+UNI hd no effect on defolition or totl lte seson AUDPC (1 July to 23 Aug; Figure 3.10e nd Figure 3.11c). Thus, tretments with ASM hd lower lte 94

118 seson AUDPC compred to the nontreted control in only one of three yers with cv. TSH4. For cv. H9909, there ws no difference mong tretments for lte seson defolition in 2011 (27 July to 29 Aug; Figure 3.10b nd Figure 3.11), including AUDPC. In 2012, ASM tretment hd higher defolition thn the nontreted control nd CuOH+UNI tretment on Aug 18 (Figure 3.10d). The ASM+UNI tretment lso hd higher defolition thn these tretments on Aug 18, nd hd higher totl lte seson AUDPC thn CuOH+UNI (Figure 3.11b). In 2013, pplictions of ASM, UNI, CuOH+UNI, nd ASM+UNI hd defolition levels tht were equivlent to the nontreted control nd CuOH (Figure 3.10f nd Figure 3.10c). Thus, tretments including ASM incresed defolition in one of three yers nd hd no effects on defolition in the other two yers. UNI hd little effect on defolition, except when UNI ws combined with ASM, where incresed defolition ws lso observed in one of three yers. This differed from the response in cv. TSH4, where pplictions of ASM nd ASM+UNI provided mrginl benefits in the sme yer tht ASM incresed defolition in cv. H9909. None of the tretments reduced the incidence of bcteril speck nd spot on red or green fruit for either cultivr in ny yer, except ASM+UNI in cv. TSH4 in 2012 (Tble 3.6, Tble A.6). The incidence of bcteril spot nd bcteril speck on fruit followed similr pttern to tht observed in the ASM field pplictions discussed erlier nd suggests tht Xg ws lso the predominnt pthogen with the greenhouse ASM pplictions in 2012 nd 2013, but tht both Xg nd Pst cused similr levels of dmge to fruit in There ws no tretment effect on the incidence or severity of nthrcnose (Tble A.7) Effect of greenhouse UNI followed by greenhouse ASM on tomto growth, yield nd qulity Tretment effects on trnsplnt height were mesured t weekly intervls beginning two weeks fter seeding up until the time of trnsplnting. For cv. TSH4 in 2011, plnt height in the ASM tretment ws equivlent to the control nd CuOH on ll ssessment dtes (Figure 3.12). For CuOH+UNI tretment, plnts were shorter thn the control nd CuOH t 35 nd 41 DAS (13 nd 19 dys fter UNI ppliction). ASM+UNI plnt height ws less thn CuOH t 28 DAS nd less thn CuOH nd the control t 35 nd 41 DAS. In 2012, pplictions of ASM reduced plnt height compred to the control t 28 nd 35 DAS, but were equivlent to CuOH on ll dtes (Figure 3.12b). CuOH+UNI nd ASM+UNI treted plnts hd lower height thn the control nd CuOH t 28, 35 nd 41 DAS (nine, 16 nd 22 dys fter UNI ppliction). In 2013, plnt height with ASM tretment ws lower thn the control nd CuOH t 35 DAS (Figure 3.12c). Applictions of UNI, CuOH+UNI nd ASM+UNI resulted in shorter plnt height thn the control t 35, 41 nd 52 DAS (three, nine nd 20 dys fter UNI ppliction). Thus, tretments including UNI consistently reduced plnt height in cv. TSH4 in ll yers within t lest 16 dys fter ppliction. For cv. H9909,plnt height in the ASM tretment ws equivlent to the control nd CuOH 95

119 tretments on ll ssessment dtes in ll yers (Figure 3.12d). Plnt heights with CuOH+UNI nd ASM+UNI tretments were shorter thn the control nd CuOH tretments t 28, 35 nd 41 DAS (six, 13 nd 19 dys fter UNI ppliction) in In 2012, CuOH+UNI nd ASM+UNI pplictions resulted in lower plnt height t 22, 28, 35 nd 41 DAS (three, nine, 16, nd 22 dys fter UNI ppliction; Figure 3.12e). In 2013, the UNI, CuOH+UNI, nd ASM+UNI hd lower plnt height thn the control nd CuOH tretments t 35, 41 nd 52 DAS (three, nine, nd 20 dys fter UNI ppliction; Figure 3.12f). UNI pplictions to cv. H9909 resulted in shorter plnts in ll three yers within t lest 10 dys fter UNI ppliction, regrdless of whether plnts were lso treted with ASM or CuOH during the study period. UNI pplictions reduced plnt height by 32 to 34% in cv. TSH4 nd 26 to 32% in cv. H9909, suggesting slightly greter effect of UNI on cv. TSH4. Stem dimeter ws mesured on tomto seedlings t 35 nd 41 DAS. In cv. TSH4 in 2011, stem dimeters in plnts treted with ASM ws equivlent to the control nd CuOH tretments (Tble 3.7). However, stem dimeters with the CuOH+UNI nd ASM+UNI tretments ws lower thn the control nd CuOH t 35 DAS, nd 35 nd 41 DAS, respectively. In 2012, stem dimeter with ASM tretment ws equivlent to the control nd CuOH on both ssessment dtes. CuOH+UNI tretment hd lower stem dimeters thn the control, nd ASM+UNI tretment hd lower stem dimeter thn the control nd CuOH 35 DAS, but there were no differences mong these tretments by 41 DAS. In 2013, stem dimeter with ASM tretment ws lso equivlent to the control nd CuOH t 35 nd 41 DAS. However, UNI treted plnts hd lower stem dimeter thn CuOH treted plnts, nd pplictions of CuOH+UNI nd ASM+UNI resulted in lower stem dimeters thn with nontreted control nd CuOH ppliction t 41 DAS. Thus, pplictions of ASM or CuOH lone did not ffect stem dimeter, wheres UNI sometimes but not lwys resulted in lower stem dimeter thn tretments tht did not receive UNI. Stem dimeter in cv. H9909 in 2011 ws equivlent for the nontreted control, CuOH nd ASM treted plnts, which were ll higher thn CuOH+UNI nd ASM+UNI treted plnts, except for ASM t 41 DAS (Tble 3.7). In 2012, there were no differences mong tretments for stem dimeter. In 2013, there were no differences mong tretments for stem dimeter t 35 DAS, but by 41 DAS, ppliction of UNI nd ASM+UNI hd the lowest stem dimeters followed by ASM nd CuOH+UNI ppliction. The nontreted control hd the lrgest stem dimeter, equivlent to CuOH tretment. Unlike cv. TSH4, cv. H9909 hd reduced stem dimeters in one yer with ASM lone but CuOH lone ws never significntly different from the control. The effect of UNI on reducing stem dimeter in cv. H9909 ws not s consistent from yer to yer s in cv. TSH4, but lso generlly resulted in decresed stem dimeter compred to tretments where no UNI ws pplied. Reltive chlorophyll ws mesured t 18, 37 nd 56 DAT in ech yer (74, 93 nd 112 DAS in 2011 nd 62, 81 nd 100 DAS in 2012 nd 2013) (Figure 3.13). In cv. TSH4 in 2011, there ws no 96

120 tretment effect on reltive chlorophyll content (Figure 3.13), but in 2012, ASM tretment hd chlorophyll content equivlent to the nontreted control nd CuOH t 18 nd 37 DAT (Figure 3.13b). At 56 DAT, reltive chlorophyll ws lso equivlent to the control but ws lower thn the CuOH tretment. Both CuOH+UNI nd ASM+UNI hd higher reltive chlorophyll thn the control nd CuOH t 18 DAT, but levels were equivlent to these tretments t 37 nd 56 DAT. In 2013, the ASM tretment hd chlorophyll content equivlent to the nontreted control nd CuOH t 18 nd 37 DAT, but by 56 DAT, the reltive chlorophyll ws lower thn the CuOH tretment (Figure 3.13c). The CuOH+UNI tretment hd higher reltive chlorophyll thn the control nd CuOH only t 18 DAT. For ASM+UNI, reltive chlorophyll ws higher thn the control nd CuOH t 18 DAT, nd higher thn CuOH t 56 DAT. The UNI tretment, which ws only included in 2013, hd higher reltive chlorophyll thn the nontreted control nd CuOH t 18 DAT but ws equivlent to these tretments t 37 DAT nd lower thn CuOH tretment, but not the control, t 56 DAT. Thus, in two of three yers, pplictions of UNI resulted in higher reltive chlorophyll content t lest t 18 DAT nd sometimes ASM treted plnts hd lower reltive chlorophyll thn CuOH, CuOH+UNI nd ASM+UNI treted plnts. For cv. H9909 in 2011, ASM tretment hd reltive chlorophyll content equivlent to the control nd CuOH t 18, 37 nd 56 DAT (Figure 3.13d). Both CuOH+UNI nd ASM+UNI treted plnts hd higher reltive chlorophyll content thn the control nd CuOH treted plnts t 18 DAT, but these tretments were equivlent t 37 nd 56 DAT. In 2012, the ASM nd ASM+UNI tretments were equivlent to the control nd CuOH tretments on ll ssessment dtes (Figure 3.13e). Applictions of CuOH+UNI resulted in higher reltive chlorophyll content thn the control nd CuOH treted plnts t 18 DAT, but hd no effect on chlorophyll content t 37 nd 56 DAT. In 2013, reltive chlorophyll in the ASM tretment ws equivlent to the control nd CuOH tretments on ll ssessment dtes (Figure 3.13f). Applictions of UNI nd CuOH+UNI resulted in higher reltive chlorophyll content thn the CuOH tretment t 18 DAT, wheres the ASM+UNI tretment ws equivlent to both CuOH nd the control tretments on the sme ssessment dte. For cv. H9909, tretments tht included UNI frequently but not lwys resulted in higher reltive chlorophyll content 18 DAT in ll three yers, but there ws no effect of UNI on reltive chlorophyll content t 37 or 56 DAT, indicting tht the effects of UNI were no longer pprent by 37 DAT. ASM or CuOH lone tretments hd reltive chlorophyll contents equivlent to the nontreted control in ll three yers. These results were similr to those observed for cv. TSH4. There were no effects on the number of dys to rech first inflorescence, fruit set, nd fruit ripening for either cultivr, except for cv. H9909 in 2013, where the number of dys to rech fruit set ws five dys fewer for the UNI thn the ASM tretment, but neither ws different thn the nontreted control (Tble A.8). For cv. TSH4 in 2011, there were no differences mong tretments for totl, red, green or rotten 97

121 tomto yield (Figure 3.14). In 2012, red tomto yield ws 29% higher with ASM nd ASM+UNI tretments compred to the nontreted control nd CuOH+UNI, but were equivlent to the CuOH tretment (Figure 3.14b). In 2013, there were gin no differences mong tretments for tomto yield (Figure 3.14c). Thus, in one of three yers ASM ppliction hd positive effect on tomto yield, but this effect ws not ugmented with the inclusion of UNI into the ASM ppliction. For cv. H9909 in 2011, tomto yield in the ASM tretment ws equivlent to the control nd CuOH tretments, but totl nd red tomto yield ws 31 nd 26% lower thn the yield obtined in the CuOH+UNI tretment (Figure 3.14d). Yields in the CuOH+UNI nd ASM+UNI tretment were equivlent to ll other tretments. In 2012, ASM nd ASM+UNI reduced totl yield by 24% compred to the control, CuOH, nd CuOH+UNI tretments (Figure 3.14e). Red tomto yield in tretment ASM ws lso 24 nd 22% lower thn CuOH nd CuOH+UNI tretments. In 2013, there were no differences mong tretments for yield (Figure 3.14f). Thus, in two of three yers, negtive effects of ASM on yield were observed t some level compred to the best tretment, CuOH+UNI. This even included less yield thn the nontreted control in This result is in contrst to the results in cv. TSH4 in the sme yer, where ASM tretment incresed yield. There were no tretment effects on tomto qulity prmeters including colour, ph, nd soluble solids in cv. TSH4 in ny yer (Tble 3.8). Tretments with ASM nd ASM+UNI hd lower soluble solids thn the control in cv. H9909 in 2012, but not in 2011 nd However, soluble solids for ll tretments were reltively high for Ontrio growing conditions in 2012, possibly becuse of hot nd dry growing conditions. Juice ph in tretment ASM+UNI ws lso higher thn the control for cv. H9909 in Overll, tretments only hd effects on cv. H9909, which would be considered negtive, but the effects on the qulity prmeters tested were slight. 98

122 40 ) b) Temperture ( C) Rinfll (mm) Temperture ( C) Rinfll (mm) My Jun Jul Aug 0 0 My Jun Jul Aug 0 40 c) d) Temperture ( C) Rinfll (mm) Temperture ( C) Rinfll (mm) My Jun Jul Aug 0 0 My Jun Jul Aug 0 Figure 3.1 Dily mximum ( ) nd minimum ( ) tempertures, nd totl dily rinfll ( ) for ) 2011, b) 2012, c) 2013, nd d) 10-yer verge ( ) t Ridgetown Cmpus, University of Guelph. 99

123 35 30 ) Men dily mximum temp ( C) My June July Aug b) Men dily minimum temp ( C) My June July Aug 250 c) 200 Totl monthly rinfll (mm) My June July Aug Figure 3.2 Men monthly ) mximum temperture, b) minimum temperture, nd c) monthly rinfll in 2011 ( ), 2012 ( ), 2013 ( ) nd 10-yer verge ( ) ( ) t the Ridgetown Cmpus, University of Guelph. 100

124 120 ) # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 NS 0 16-My 30-My 13-Jun 27-Jun 11-Jul 25-Jul Inocultion #2 NS NS b b b b bc c # symptomtic leves / 1.24 m sq b) Trnsplnting Inocultion #1 NS b b b NS 21-My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul b bc c 120 c) # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 Inocultion #2 NS b bc c b b 20-My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul b Figure 3.3 Erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011, b) 2012, c) The number of leves with disese symptoms in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.2. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 101

125 1200 ) 1000 AUDPC - erly seson b b b Control UNI ASM ASM + UNI 1200 b b) 1000 AUDPC - erly seson bc c Control UNI ASM ASM + UNI c) AUDPC - erly seson b b Control UNI ASM ASM + UNI Figure 3.4 Are under the disese progress curve (AUDPC) for erly seson disese. Erly seson disese ws mesured by the number of leves with disese symptoms (shown in fig. 2) for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (22 June-27 July), b) 2012 (7 June-4 July), c) 2013 (11 June-29 June). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. 102

126 100 ) 80 Defolition (%) Jun 04-Jul 18-Jul 01-Aug 15-Aug 29-Aug b b b b b b b b b b b c 100 b) 80 NS Defolition (%) NS 20 NS NS 0 25-Jun 09-Jul 23-Jul 06-Aug 20-Aug 100 c) 80 NS Defolition (%) NS 20 NS NS NS NS NS 0 24-Jun 08-Jul 22-Jul 05-Aug 19-Aug Figure 3.5 Lte seson progress of bcteril spot nd speck on tomto cv. TSH4 treted with uniconzole (UNI_ in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011, b) 2012, c) Defolition in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.4. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 103

127 1000 ) AUDPC - lte seson b b Control UNI ASM ASM + UNI 1000 b) AUDPC - lte seson Control UNI ASM ASM + UNI 1000 c) AUDPC - lte seson Control UNI ASM ASM + UNI Figure 3.6 Are under the disese progress curve (AUDPC) for lte seson disese. Lte seson disese ws mesured by the percent defolition (shown in fig. 4) for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (27 July-23 Aug), b) 2012 (4 July-27 Aug), c) 2013 (29 June-19 Aug). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. 104

128 Tble 3.3 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted. Bcteril speck on fruit (%) Bcteril spot on fruit (%) Tretment Control UNI ASM ASM + UNI sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. b sem = stndrd error of the men for ll ls mens in the sme column. 105

129 75 70 ) SPAD chlorophyll reding Trnsplnting b b NS NS My 30-My 13-Jun 27-Jun 11-Jul 25-Jul b) SPAD chlorophyll reding Trnsplnting NS NS NS My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul c) SPAD chlorophyll reding Trnsplnting b b NS b b b 20-My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul Figure 3.7 SPAD chlorophyll redings mesured 18, 36, nd 56 dys fter trnsplnting in tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in the nontreted control ( ), UNI ( ), ASM ( ), nd ASM + UNI ( ) tretments ) 2011, b) 2012, c) Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 106

130 Tble 3.4 Totl, red, green, nd rotten fruit yield in 2m section of tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Yield (kg) Totl Red Fruit Green Fruit Rotten Fruit Tretment Control b UNI b ASM b ASM + UNI sem b Mens in the sme column grouping followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. b sem = stndrd error of the men for ll ls mens in the sme column. 107

131 Tble 3.5 Soluble solids, Agtron colour redings, nd juice ph of ripe tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws processed. Soluble solids (%) Agtron colour ph Tretment Control b UNI ASM b ASM + UNI b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. b sem = stndrd error of the men for ll ls mens in the sme column. 108

132 120 ) 120 b) # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 Inocultion #2 Inocultion #3 # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 Inocultion # My 30-My 13-Jun 27-Jun 11-Jul 25-Jul 0 21-My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul # symptomtic leves / 1.24 m sq c) Trnsplnting Inocultion #1 Inocultion #2 NS b b b b b b bc c cd d 20-My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul # symptomtic leves / 1.24 m sq d) Trnsplnting Inocultion # My 30-My 13-Jun 27-Jun 11-Jul 25-Jul Inocultion #2 NS Inocultion #3 * * b b b b NS 120 e) f) 120 NS # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 NS NS Inocultion #2 b b b b NS # symptomtic leves / 1.24 m sq Trnsplnting Inocultion #1 Inocultion #2 NS NS 0 21-My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul 0 20-My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul Figure 3.8 Erly seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) H9909, nd f) 2013-H9909. The number of leves with disese symptoms in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments in 1.24 m 2 re is shown. The corresponding re under the disese progress curve is shown in Figure 3.9. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. An * indictes differences mong tretments which re discussed in the text. 109

133 1000 ) 800 AUDPC - erly seson A B B A AB Control CuOH CuOH + UNI ASM ASM + UNI 1000 b) 800 AUDPC - erly seson b A A b A bc A c A Control CuOH CuOH + UNI ASM ASM + UNI AUDPC - erly seson A c) ) A A A A A Control CuOH CuOH + UNI ASM ASM + UNI UNI Figure 3.9 Are under the disese progress curve (AUDPC) for erly seson disese. Erly seson disese ws mesured by the number of leves with disese symptoms (Figure 3.8) for tomto cv. TSH4 (blck brs) nd cv. H9909 (grey brs) treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (22 June-27 July), b) 2012 (8 June-5 July), c) 2013 (22 June-8 July). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. 110

134 Defolition (%) ) NS b b b b b b b b NS 0 20-Jun 04-Jul 18-Jul 01-Aug 15-Aug 29-Aug Defolition (%) b) NS NS 25-Jun 09-Jul 23-Jul 06-Aug 20-Aug b b b b b b b bc c c) NS d) Defolition (%) NS Defolition (%) NS 20 NS NS NS NS NS 20 NS NS NS 0 24-Jun 08-Jul 22-Jul 05-Aug 19-Aug 0 20-Jun 04-Jul 18-Jul 01-Aug 15-Aug 29-Aug e) NS b b b f) NS Defolition (%) NS NS NS 25-Jun 09-Jul 23-Jul 06-Aug 20-Aug Defolition (%) NS NS NS NS NS b b b b b 24-Jun 08-Jul 22-Jul 05-Aug 19-Aug Figure 3.10 Lte seson progress of bcteril spot nd speck symptoms in tomto cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011-TSH4, b) TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Defolition in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments in whole plots is shown. The corresponding re under the disese progress curve is shown in Figure Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 111

135 ) 1200 AUDPC - lte seson A b A b A b A b A Control CuOH CuOH + UNI ASM ASM + UNI AUDPC - lte seson ABB AB B B b AB b b) A Control CuOH CuOH + UNI ASM ASM + UNI c) AUDPC - lte seson A A A A A A Control CuOH CuOH + UNI ASM ASM + UNI UNI Figure 3.11 Are under the disese progress curve (AUDPC) for lte seson disese. Lte seson disese ws mesured by the percent defolition (shown in fig. 8) for tomto cv. TSH4 (blck brs) nd cv. H9909 (grey brs) treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011 (27 July-29 Aug), b) 2012 (5 July-8 Aug), c) 2013 (1 July-23 Aug). Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. 112

136 Tble 3.6 The incidence of bcteril speck nd bcteril spot on red tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws evluted. TSH4 H9909 Bcteril speck Bcteril spot Bcteril speck Bcteril spot Tretment Control b CuOH CuOH + UNI b ASM b ASM + UNI b b UNI 0.5 NT NT 19.0 NT NT 3.0 NT NT 25.0 NT NT sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. NT = not tested. b sem = stndrd error of the men for ll ls mens in the sme column, except for the control tretment, cv. H9909, 2012 the sem is 2.21 nd the CuOH tretment, cv. H9909, 2011 the sem is

137 18 16 ) b) Cnopy height (cm) NS b b b b b b b b b b b b Cnopy height (cm) NS NS b b c c b b c c b b No. dys fter seeding No. dys fter seeding Cnopy height (cm) c) NS NS NS b cd cd d b b b b b b Cnopy height (cm) d) NS NS b b b b b b No. dys fter seeding No. dys fter seeding Cnopy height (cm) e) NS b b b b c c b b b b Cnopy height (cm) f) NS NS NS b b b b b b b b b No. dys fter seeding No. dys fter seeding Figure 3.12 Height of tomto seedlings cv. TSH4 nd H9909 treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse, Ridgetown, ON, in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 114

138 Tble 3.7 Stem dimeter nd folir nd root dry weight of tomto seedlings, cvs. TSH4 nd H9909, treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse, Ridgetown, ON, TSH4 H9909 Tretment Stem dimeter (35 DAS) Control 1.83 b CuOH CuOH + UNI 1.61 b 1.79 b b ASM ASM + UNI 1.71 b 1.71 b b UNI NT NT 1.73 NT NT 1.85 sem c Stem dimeter (41 DAS) Control b 2.51 b CuOH b CuOH + UNI 1.96 b cd 2.16 bc b ASM bcd 2.47 bc bc ASM + UNI 2.00 b d 2.10 c c UNI NT NT 1.97 bcd NT NT 2.04 c sem Folir dry weight (g/plnt) d Control CuOH b b b b CuOH + UNI bc bcd b bc ASM b b bc b b ASM + UNI c b d b c UNI NT NT cd NT NT b sem Root dry weight (g/plnt) d Control e CuOH b CuOH + UNI b ASM b ASM + UNI UNI NT NT NT NT sem DAS = dys fter seeding. b Mens in the sme row followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. NT = not tested. b sem = stndrd error of the men for ll ls mens in the sme row. c Dry weight of 5 seedlings per plot ws mesured 56 DAS in 2011, nd 44 DAS in 2012 nd d Mens in this row were trnsformed for ANOVA using log trnsformtion. The bck-trnsformed mens re shown here. The sem is for the trnsformed mens. 115

139 SPAD chlorophyll reding ) Trnsplnting NS NS NS SPAD chlorophyll reding b) Trnsplnting b b b b b b b b b bc c My 30-My 13-Jun 27-Jun 11-Jul 25-Jul 08-Aug 22-Aug My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul 13-Aug 27-Aug SPAD chlorophyll reding c) Trnsplnting b b b NS b b bc bc c SPAD chlorophyll reding d) Trnsplnting b b b NS NS My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul 12-Aug 26-Aug My 30-My 13-Jun 27-Jun 11-Jul 25-Jul 08-Aug 22-Aug 70 e) b NS 70 f) SPAD chlorophyll meter Trnsplnting bc bc c NS SPAD chlorophyll reding Trnsplnting b b b b NS NS My 04-Jun 18-Jun 02-Jul 16-Jul 30-Jul 13-Aug 27-Aug My 03-Jun 17-Jun 01-Jul 15-Jul 29-Jul 12-Aug 26-Aug Figure 3.13 SPAD chlorophyll redings mesured 18, 37, nd 56 dys fter trnsplnting in tomto cv. TSH4 nd cv. H9909 treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in the nontreted control ( ), CuOH ( ), CuOH + UNI ( ), ASM ( ), ASM + UNI ( ), nd UNI ( ) tretments ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Error brs represent stndrd error of the men. Dt points on the sme dte with the sme letter re not significntly different t P 0.05, Tukey s HSD. NS = no significnt difference. 116

140 30 ) b) 30 Yield (kg) 20 A A A A A Yield (kg) 20 B AB B A A Control z Z z Z CuOH CuOH + UNI z Z ASM z Z ASM + UNI z Z 0 Control z z z Z Z Z CuOH CuOH + UNI z Z ASM ASM + UNI z Z c) d) Yield )kg) A A A A A A Yielf (kg) b AB b AB A b B b AB 10 z z z z z z 10 z z z z z 0 Control Z CuOH Z CuOH + UNI Z ASM Z ASM + UNI Z UNI Z 0 Control Z CuOH Z CuOH + UNI Z ASM Z ASM + UNI Z Yield (kg) e) f) AB A AB b b C BC Yield (kg) A A A A A A Control z zy zy y zy Z Z Z Z Z CuOH CuOH + UNI ASM ASM + UNI 0 Control z Z CuOH z Z CuOH + UNI z Z ASM z Z ASM + UNI z Z UNI z Z Figure 3.14 Totl ( ), red ( ), green ( ), nd rotten ( ) fruit yield in 2m section of tomto cv. TSH4 nd cv. H9909 treted with CuOH, uniconzole (UNI), nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, in ) 2011-TSH4, b) 2012-TSH4, c) 2013-TSH4, d) 2011-H9909, e) 2012-H9909, nd f) 2013-H9909. Error brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. 117

141 Tble 3.8 Soluble solids, Agtron colour redings, nd juice ph of ripe tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit hrvested in 2m section of ech plot ws processed. TSH4 H9909 Soluble solids (%) Agtron colour ph Soluble solids (%) Agtron colour ph Tretment Control b 4.27 CuOH b b 4.28 CuOH + UNI b b 4.29 ASM b b 4.24 ASM + UNI b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. b sem = stndrd error of the men for ll ls mens in the sme column. 118

142 3.4 Discussion For these experiments, the combintion of ASM nd UNI for bcteril speck nd spot control ws ssessed by pplying them together in two different sequences over three yers. In the first sequence of the UNI+ASM combintion, UNI ws pplied in the greenhouse nd ASM ws pplied in the field, while in the second sequence of the combintion, both UNI nd ASM were pplied in the greenhouse. For the first ASM+UNI sequence, tretments with ASM lone, UNI lone or nontreted control were included over the three yers. Experiments with the second ASM+UNI sequence included tretments ech yer with ASM lone, CuOH nd CuOH+UNI s well s nontreted control. In the finl yer for the second ASM+UNI sequence, UNI lone tretment ws lso included. ASM+UNI ws compred to the nontreted control to determine if the combintion controlled disese, nd compred to ASM lone nd UNI lone to see if the ddition of UNI to ASM ffected its level of disese control. Tretment with CuOH ws included to compre ASM+UNI to stndrd control, nd CuOH+UNI ws lso exmined to compre ASM+UNI to UNI modified stndrd control. Typiclly, field trils evluting bcteril speck nd spot mngement only involve mesurements of lte seson disese severity (i.e. defolition or percent lef re ffected) nd yield (Louws et l., 2001, Hung et l., 2012, Roberts et l., 2008). However, in this work, erly seson disese incidence ws lso ssessed to better cpture differences in disese development during erly phses of the field epidemic ech yer, since mny of the tretment pplictions occurred during the greenhouse seedling stge nd it ws unknown how long potentil effects would be detectble. Those results could then be compred to the stndrd lte seson disese severity ssessments for defolition. In ddition to yield mesurements, this work lso mesured tomto fruit qulity s both UNI nd ASM re known to impct tomto physiology Greenhouse UNI with field ASM ppliction Appliction of UNI to greenhouse tomto seedlings followed by ASM in the field suppressed erly seson AUDPC compred to the nontreted control in two of three yers, but hd no effect on lte seson AUDPC, fruit yield or qulity in ny yer, except for yield in However, ASM without UNI suppressed erly seson AUDPC in ll three yers, nd lte seson AUDPC in one of three yers. Thus, the ddition of UNI to ASM reduced the period of ASM effectiveness in one yer, mking ASM effectiveness more vrible. UNI ppliction lone did not reduce erly or lte seson AUDPC, yield or fruit qulity compred to the control in ny yer. Bsed on these results, UNI did not pper to hve ny disese control effectiveness lone; however, UNI did provide lower disese incidence nd percent defolition thn the control on 27 July in 2011, nd lower disese incidence on 20 June in

143 Therefore, ppliction of UNI lone did ffect disese, but this ws slight nd inconsistent. This is the first report of ny effect of UNI lone on bcteril spot or speck severity in tomto nd suggests tht there re long-term effects of UNI on tomto growth nd development. This is consistent with reported longterm effects of UNI on plnt growth nd development in bedding plnts (Blnchrd & Runkle, 2007). Tken together, the results indicte tht the ddition of greenhouse pplictions of UNI with field pplictions of ASM my reduce the reltive effectiveness of ASM in some yers, nd tht UNI lone hs little effect on bcteril speck nd spot. It ws expected tht reductions in disese would be reflected in higher yield nd greter tomto qulity, since bcteril speck nd spot re considered economiclly importnt field diseses tht ffect fruit in Ontrio (LeBoeuf et l., 2009, Jones, 1991, Jones, 1991b, Koike et l., 2007). The ASM+UNI combintion hd n effect on yield compred to the control in only one of the three yers, 2011, when significnt increse ws observed. However, this did not correspond to reductions in erly or lte seson AUDPC in the sme yer, nd so my hve been due to fctor not relted to bcteril speck or spot. Applictions of ASM lone or UNI lone did not result in ny yield benefit compred to the control in ny yer. This suggests tht pplictions of UNI with ASM my hve synergistic effect on yield in some yers. Since both erly nd lte seson AUDPC with ASM nd ASM+UNI were equivlent in 2011, this effect ppers to be unrelted to disese intensity. ASM+UNI resulted in higher reltive chlorophyll content on 18 DAT thn ASM lone in 2011, suggesting higher plnt N sttus my hve plyed role in the yield increse, but the sme chlorophyll content response ws observed in 2013 with ASM+UNI nd no yield benefit ws observed, not supporting this hypothesis. Thus, the mechnism of the yield benefit by ASM+UNI in 2011 is unknown. Future studies exmining the effect of the greenhouse pplictions of UNI nd field pplictions of ASM combintion on yield in the bsence of disese my provide dditionl clues with regrds to the mechnism of this response. UNI is ssocited with stress tolernce in other plnt systems (Al-Rumih & Al-Rumih, 2007, Dun et l., 2008, Senrtn et l., 1988, Zhng et l., 2007), but it is not cler if similr mechnisms were t ply here. Applictions of UNI lone did not ffect yield compred to the nontreted control. A previous report from Ontrio ssocited UNI ppliction with erlier bloom, dvnced fruit mturity, nd incresed biomss (Zndstr et l., 2006). Few detils on experimentl methods re provided by Zndstr et l. (2006), but the uthors reported tht higher rtes of fertilizer were used during seedling production thn in the nontreted control plnts. Fertilizer regimes were not ltered for UNI treted plnts in this study to void confounding fctors in comprisons mong tretments. Reltive chlorophyll content in the ASM+UNI tretment ws higher thn the nontreted control nd ASM lone t 18 DAT in 2011 nd 2013, nd t 56 DAT in 2013, but were equivlent to UNI lone on the sme dtes. Applictions of ASM hd no effect on chlorophyll levels. These results suggest tht 120

144 while UNI ppliction cn increse reltive chlorophyll content, field pplictions of ASM lone or in combintion with UNI hve no effect on reltive chlorophyll content. The combintion of greenhouse pplictions of UNI nd field pplictions of ASM did not ffect tomto colour, ph, or soluble. Applictions of UNI lone or ASM lone lso did not ffect these results with the notble exception of UNI lone resulting in higher tomto juice ph thn the control in However, the vlue of ph 4.25 mesured in the UNI tretment ws not of concern becuse it is still within n cceptble rnge for processing tomtoes (Monti, 1980) Greenhouse UNI nd greenhouse ASM ppliction In the second series of trils, UNI nd ASM were pplied to seedlings in the greenhouse, nd cv. H9909 ws included s well s cv. TSH4. Combining UNI nd ASM pplictions reduced erly nd lte seson AUDPC nd fruit with bcteril spot in cv. TSH4 in 2012, but not in 2011 or ASM nd ASM+UNI pplictions lone did not reduce erly seson AUDPC, but ASM+UNI did reduce lte seson AUDPC compred to the nontreted control in There ws no effect of ASM lone on fruit disese incidence in ny yer, except tht it resulted in n increse in the incidence of bcteril speck on green fruit in 2012 compred to ll other tretments. Thus, the ddition of UNI to ASM incresed ASM effectiveness for cv. TSH4 ginst the nontreted control compred to ASM lone, but only in Applictions of the ASM+UNI combintion to cv. H9909 did not result in ny reductions in erly nd lte seson AUDPC or disesed fruit, except for reduction in the incidence of green fruit with bcteril speck in The sme ws true for ppliction of ASM lone including the reduction in the incidence of green fruit with bcteril speck in In fct, the lte seson AUDPC in 2012 for ASM+UNI ws higher thn CuOH+UNI, suggesting some negtive impct of ASM+UNI on disese control. Thus, cv. H9909 ws lrgely unresponsive to the combintion of ASM+UNI pplied in the greenhouse. Greenhouse ASM+UNI pplictions incresed red tomto yield compred to the nontreted control for cv. TSH4 in 2012, but not in 2011 or ASM lone lso hd higher red tomto yield only thn the nontreted control in 2012, nd so the ddition of UNI did not further increse yield compred to ASM lone. In contrst, greenhouse pplictions of ASM+UNI with cv. H9909 resulted in lower red tomto yield in The yield response for ASM+UNI nd ASM were equivlent, with ASM lone lso hving lower totl yield thn the control nd stndrd tretments. Similrly, totl yield in the ASM-only tretment for cv. H9909 in 2011 ws lower thn the modified stndrd CuOH+UNI tretment, suggesting ASM nd not UNI s the min driver of this yield effect. The increse in disese with greenhouse pplictions of ASM in cv. H9909 is consistent with observtions of higher disese levels in the sme 121

145 tretments in 2012, wheres in cv. TSH4 there ws lower disese nd higher yields with the sme greenhouse ASM tretments, suggesting genotype dependent effect. Reltive chlorophyll redings in the greenhouse ASM+UNI tretment in cv. TSH4 were equivlent to the nontreted control in 2011, but higher thn the nontreted control nd ASM only tretments 18 DAT in 2012 nd This indictes tht UNI increses reltive chlorophyll for severl weeks fter ppliction. Reltive chlorophyll in the ASM-only tretment hd lower reltive chlorophyll content thn the CuOH tretment in cv. TSH4 in 2012 nd DAT. For cv. H9909, the impct of ASM+UNI on reltive chlorophyll ws less pprent. This tretment ws equivlent to the control 18 DAT in ll three yers but hd higher reltive chlorophyll 18 DAT thn ASM lone in 2011 nd Similr to the previous discussion on the results of field pplictions of ASM, yield differences did not pper to be ssocited with differences in reltive chlorophyll content, which tended to be higher cross both cultivrs when UNI ws pplied regrdless of the negtive yield effects in cv. H9909 in 2011 nd 2012 or positive yield effects in cv. TSH4 in These results support the observtion tht ASM cn hve long term impcts on tomto physiology. In most cses, greenhouse ASM combined with UNI did not hve negtive impct on tomto qulity. Tretments including ASM hd lower soluble solids thn the control in cv. H9909 in However, levels of soluble solids greter thn 4.5% re desirble in processing tomto production soluble solids, nd ll tretments were higher thn this vlue in ll yers (Monti, 1980). Juice ph in tretment ASM+UNI ws higher thn the control for cv. H9909 in 2012 when ASM ws pplied to seedlings, but juice ph for ll tretments except the control ws higher thn the cceptble limit of ph Overll, plnt response to ASM nd UNI when both products were pplied t the greenhouse seedling stge vried from the results of the first ppliction sequence when ASM ws pplied in the field. Although the effect ws mrginl, greenhouse pplictions of ASM+UNI to cv. TSH4 resulted in more reductions in disese severity compred to the nontreted control thn ASM lone, which is in contrst to the result from field only pplictions, where ASM combined with UNI reduced the effectiveness of ASM in some yers. However, this effect ws not noted in cv. H9909. The effect of ASM on yield lso vried in 2012 with no yield response to field only pplictions but positive yield response to greenhouse ASM ppliction with cv. TSH4 nd negtive response to greenhouse ASM ppliction with cv. H Greenhouse UNI with greenhouse CuOH ppliction A stndrd tretment, CuOH, nd modified stndrd, CuOH+UNI were lso pplied in the greenhouse s control. The greenhouse tretment of CuOH ws chosen insted of field pplictions of CuOH so tht the greenhouse tretments could be directly compred to ech other. CuOH+UNI ws 122

146 included to observe if it my hve n effect on CuOH since t the time of the experiments CuOH ws widely used nd it ws expected tht UNI my be widely dopted in greenhouse production in Ontrio. The greenhouse UNI tretment ws dded in 2013 s control to compre the response of UNI treted plnts to tht of CuOH+UNI treted plnts. Greenhouse pplictions of CuOH+UNI ws the only tretment to hve lower lte seson AUDPC thn the nontreted control for cv. TSH4 in 2011, but ws equivlent to the nontreted control in ll other yers. Furthermore, this tretment ws equivlent to the nontreted control for erly seson AUDPC in ll yers, nd hd the equivlent erly seson AUDPC s ASM+UNI in Similrly, pplictions of CuOH resulted in erly nd lte seson AUDPC equivlent to the nontreted control in ll yers, nd in 2012 erly seson AUDPC ws higher with CuOH thn ASM lone. For cv. H9909, CuOH+UNI ws the only tretment to hve lower lte seson AUDPC thn ASM+UNI in Erly seson AUDPC ws lower with CuOH+UNI nd CuOH lone thn the nontreted control nd ASM lone in 2011, but in ll other yers, erly nd lte seson AUDPC for these tretments ws equivlent to the nontreted control, ASM lone or ASM+UNI. Thus, the effect of the ddition of UNI to CuOH is similr to tht of the ddition of UNI to ASM, which indicted occsionl slight benefits of UNI to tomto helth, except under conditions of drought stress for cv. H9909 in When greenhouse UNI ws included in 2013, AUDPC for erly nd lte seson disese ws equivlent to CuOH+UNI in both cultivrs, indicting tht the ddition of CuOH to UNI provided no benefits. Greenhouse pplictions of CuOH rrely resulted in ny benefit in field control of bcteril spot nd speck. Greenhouse pplictions of CuOH nd CuOH+UNI resulted in yields equivlent to the nontreted control in ll yers. It is not surprising tht pplictions of CuOH provided few dvntges for disese mngement or tomto growth, since the product ws only pplied to tomto seedlings, nd CuOH is contct bctericide. Other studies show limited benefit of CuOH pplictions for long-term, consistent mngement of bcteril speck nd spot, even when pplictions re mde in the field (Griffin et l., 2017). This my be due to copper tolernce, s reported by Griffin et l. (2017). However, this is unlikely s the Xg isolte used in this study is not copper tolernt (Abbsi et l., 2015), nd thus demonstrtes limited long term benefits of greenhouse CuOH. As previously discussed, Zndstr et l. (2006) reported benefits in erliness nd tomto yield with the use of UNI, but these effects were not observed here, possibly becuse fertilizer progrms were not incresed s Zndstr et l. (2006) hd done. Greenhouse pplictions of UNI frequently incresed reltive chlorophyll content compred to plnts tht were not treted with UNI in both cultivrs, which ws response similr to tht observed for ASM+UNI. Higher reltive chlorophyll suggests higher N sttus in UNI-treted tomtoes, however s discussed bove this did not result in consistent increse in yield. Higher N sttus could be one fctor tht explins the slight benefit in bcteril disese tolernce observed in this tril, however, reltive 123

147 chlorophyll content 18 DAT, when the response to UNI ws most consistent mong site yers, ws not consistently correlted with tomto yield or disese intensity (dt not shown). Tomto qulity ws not ffected by tretments of CuOH lone, UNI lone or CuOH+UNI Improvement of ASM effectiveness It ws hypothesized tht the combintion of greenhouse UNI with field or greenhouse ASM pplictions would improve the consistency of disese suppression with ASM both in folige nd fruit. Previous reports hve shown inconsistency of field ASM pplictions for mngement of lte seson disese severity nd fruit yield losses in the field due to bcteril spot nd speck in tomto. Lte seson disese severity (Obrdovic et l. 2004) nd seson-long AUDPC (Roberts et l. 2008) in ASM treted plots were not consistently lower thn the nontreted control nd only occsionlly lower thn stndrd copper + EBDC tretments in field tomtoes. ASM lso did not reduce the severity of bcteril spot on tomto folige in one of 14 nd 10 of 14 trils bsed on seson-long AUDPC nd finl disese severity rtings (Louws et l. 2001). However, Louws et l. (2001) found tht seven out of seven trils hd reduced folir disese severity of bcteril speck with ASM compred to the nontreted control. For fruit disese incidence, only five out of eight trils ginst spot nd one out of three trils ginst speck showed significnt reductions (Louws et l., 2001). Similrly, there were few effects on fruit disese incidence when ASM ws tested in Ontrio from 2010 to 2012 (Truemn, 2015). Roberts et l. (2008), Lewis Ivey et l. (2004), nd Grves nd Alexnder (2002) ll reported percent mrketble yield fter ASM ppliction s opposed to fruit disese incidence. However, it is difficult to determine if the results were only due to control of bcteril spot nd speck becuse they include evlutions of other diseses, such s nthrcnose, or were completed using fresh mrket tomtoes, where tolernce for fruit lesions is zero. Mhesniy (2002) reported improved control of bcteril speck on tomto seedlings tretments with ASM nd pclobutrzol, however, disese ssessments were completed t the tomto seedlings stge nd disese intensity in the field ws not evluted. This study ws undertken under the ssumption tht pplictions of ASM cn induce fitness costs in the host plnt due to the re-lloction of resources from growth nd development to plnt defence pthwys. However, reltive chlorophyll redings in ASM-only tretments were not significntly lower thn the nontreted control when ASM ws pplied in the field, suggesting no fitness costs ssocited with ASM ppliction nd plnt nitrogen sttus. In two of three yers UNI treted plnts hd higher reltive chlorophyll thn ASM nd the control, but this ws not correlted with higher tomto yield or disese tolernce (dt not shown). Ginquinto et l. (2006) found reltive chlorophyll 29 to 92 DAT ws positively correlted with tomto yield, but there is lck of dditionl informtion in the literture on the reltionship between reltive chlorophyll nd yield erlier in the growing seson. In ddition, no negtive 124

148 effects of ASM on tomto yield were observed when it ws pplied in the field nd ccording to current lbel directions, despite conditions tht included limited precipittion nd irrigtion for severl weeks in 2012 nd frost dmge in In the cses where ASM-induced yield loss in tomto is reported by Louws et l. (2001), Dmicone nd Trent (2003), nd Lnge et l. (2007) ppliction rtes were 26.3 to 35.0 g ASM/H, which re more thn two times higher thn the rte of 12.5 g/h used in this study, included ppliction on grfted plnts (Kunwr et l., 2017), or included more thn eight pplictions in single seson (Pontes et l., 2016). Thus, t the ppliction rtes nd timings used, fitness cost potentil of ASM my hve been limited Environmentl impcts on disese nd control mesures Tomto response to ASM nd UNI ppliction vried mong yers nd my be relted to environmentl conditions, since effects of ASM on greenhouse tomto nd Pst re generlly reported s being very consistent (Hermn et l., 2008, Lnn-Filho et l., 2017). Severl biotic stress fctors could hve ffected tomto response to ASM nd UNI pplictions including trnsplnting delys in 2011 due to wet field conditions, unusully hot nd dry field conditions in 2012, nd low temperture nd frost events in 2013 tht occurred within dys of trnsplnting. The ASM differing response mong genotypes observed in 2012 my be relted to differences in SAR induction, bility to compenste for growth chnges induced by ASM, or bility to tolerte het nd wter stress. This is consistent with Dietrich et l. (2005) who reports tht the bility of A. thlin to compenste for reductions in growth immeditely fter ASM ppliction is influenced by environmentl conditions. Plnt response to biotic stress is complex, prticulrly when multiple biotic stresses occur simultneously or in combintion with biotic stress such s pthogen infection (Suzuki et l., 2014). For exmple, wter stress in tomtoes involves 19 different metbolic pthwys, including those relted to production of plnt secondry metbolites, PR genes, hormones such s GA nd SA (Gong et l., 2010). Wter stress incresed susceptibility to B. cinere nd reduced efficcy of ASM in one greenhouse study in tomto (Mymoune et l., 2015), but reduced susceptibility to the sme pthogen in nother greenhouse study (Achuo et l., 2006). In A. thlin, number of hormones including ABA, JA, GA, nd ET ffected wter stress-relted gene expression (Hung et l., 2008). Furthermore, ABA is reported to lter JA/ET-dependent gene expression nd JA biosynthesis in n A. thlin-pythium irregulr system (Adie et l., 2007), nd suppress SAR induction in n A. thlin-pst system (Mohr & Chill, 2007). Hung et l. (2012) reported tht ASM concentrtion explined only 35 nd 37% of the vrition in AUDPC for bcteril spot in two Florid field trils. UNI pplictions re ssocited with reductions in chilling injury in tomto when UNI ws pplied four dys prior the chilling stress (Senrtn et l., 1988), slt stress in Dtur spp.(al-rumih & Al-Rumih, 2007), nd drought stress in soyben (Zhng et l., 2007) nd whet (Dun et l., 2008), but 125

149 UNI ppered ineffective t reducing plnt stress in the present study for cv. H9909. These findings re importnt s positive or negtive yield effects hve importnt implictions for industry, even if the risk vries from yer to yer. Differences in yield response in cv. TSH4 nd cv. H9909 support the hypothesis tht ASM pplictions to tomto seedlings cn induce fitness costs in tomto, but this effect is dependent on the environment nd host genotype. In other field trils completed t the sme loction, pplictions of ASM to cv. H9909 in the field did not negtively ffect tomto yield (Truemn, 2015), suggesting fitness costs my lso be relted to plnt ge t the time of ASM ppliction Durtion of ASM effectiveness In 2012, greenhouse pplictions of ASM lone or in combintion with greenhouse pplictions of UNI for cv. TSH4 hd lower lte seson AUDPC thn the nontreted control CuOH nd CuOH+UNI. The finl greenhouse ASM ppliction in 2012 occurred 15 dys prior to the first inocultion with Pst nd Xg, suggesting tht ASM pplied within the first 6 weeks of emergence cn hve long-term effects on host immunity nd other components of host physiology under certin conditions. Previous reserch hs demonstrted tht ASM is trnslocted nd degrded in tomto leves within 72h of ppliction in tomtoes t the 5 to 6 true lef stge (Scrponi et l., 2001), nd increses in PR1 gene expression in field tomtoes re pprent within 1 dy of ppliction (Hermn et l., 2007), but less is known bout fctors ffecting the durtion of the response. Appliction of ASM to pproximtely 22-week old tomto plnts induced PR1 gene expression in ll three cultivrs tested in New York field tril, nd lthough the pttern of expression vried mong the three cultivrs tested, expression returned to bseline levels within seven dys of ctivtion (Hermn et l., 2007). Tht result ws supported by observtions tht 14- dy intervl field pplictions of ASM were ineffective t reducing bcteril spot severity in Florid (Hung et l. 2012) nd tht the optiml levels of disese control ws chieved with 8 to 10 intervl in Brzil (Pontes et l., 2016). Tomto seedlings treted with ASM t 15 DAS with nd without Xv chllenge hd higher levels of peroxidse nd ctlse compred to controls with nd without pthogen chllenge 12 dys fter ASM ppliction (Cvlcnti et l., 2006). Similrly, peroxidse nd chitinse ctivity in tomto seedlings treted with ASM with nd without chllenge by Clvibcter michignensis ssp. michignensis remined higher thn the wter nd wter nd pthogen control for 10 dys fter ASM ppliction (Bysl et l., 2003), nd tomto seedlings grown from ASM-treted seeds showed differentil gene expression t 12 to 22 DPT compred to non-treted control (Goodwin et l., 2017b). In tobcco, ASM induced SAR genes including PR1 were induced up to 20 dys fter ppliction in 8- week old greenhouse grown plnts (Friedrich et l., 1996), nd ASM ppliction to cnol 21 dys prior to inocultion with P. syringe pv. mculicol still resulted in reduction in pthogen growth nd 126

150 incresed PR1 nd PR2 gene expression (Potlkyl et l., 2007). This indictes there re long term effects of ASM on host defense in tomto nd other plnt species Comprison of disese ssessment methods Disese incidence in the erly prt of the seson ws completed by counting the number of leves with ny bcteril speck or spot symptoms in n re of 1.24 m 2, which represents the pproximte re occupied by five tomto plnts. The incidence vlues therefore represent the density of symptomtic leves in n re. No visul differences in plnt size were observed mong tretments in the trils in ny yer. An lterntive method for clculting incidence ws lso implemented in both trils in 2013, where the totl number of leves in 1.24 m 2 nd the number of symptomtic leves ws counted, so tht the percentge of leves with symptoms could be clculted. A comprison of totl lef counts in the trils when ASM ws pplied in the field showed no difference in totl lef counts mong tretments when erly seson disese ssessments were completed (Figure A.2). Mens seprtion differed slightly from the symptomtic lef counts per 1.24 m 2, but the conclusion tht ASM nd ASM+UNI provided equivlent levels of bcteril disese suppression remins the sme. For the tril when ASM ws pplied to seedlings, there ws lso no difference mong tretments for the number of leves in 1.24 m 2 for cv. H9909, nd no chnge in mens seprtion when the percent symptomtic leves ws nlysed compred to the number of symptomtic leves per 1.24 m 2 (Figure A.3). For TSH4, the control hd fewer totl leves thn ASM+UNI on the finl erly seson ssessment dte (8 July, 90 DAS), nd there were some slight differences in mens seprtion on the first two ssessment dtes (22 June/74 DAS nd 1 July/83 DAS). Percent symptomtic lef nlysis showed tht the ASM nd ASM+UNI tretments were equivlent on 22 June (74 DAS), wheres the nlysis using the number of symptomtic leves per 1.24 m 2 showed the ASM+UNI tretment hd lower disese incidence thn ASM. The nlysis of percent symptomtic leves lso showed tht on 1 July (83 DAS) the ASM+UNI tretment hd fewer infected leves thn the control, wheres the nlysis using the number of symptomtic leves per 1.24 m 2 showed no difference mong tretments. Overll, there ws little difference in the two ssessment methods nd the overll conclusions of the study were not ffected. Disese intensity ws evluted using different methods in the erly compred to the lte prt of the growing seson. Although this could be considered disdvntge becuse disese progress over the course of the seson could not be directly compred or summrized, the erly seson ssessment method llowed for ssessment of tretment effects t the beginning of disese onset, but prior to the beginning of defolition symptoms, so tht ny erly tretment effects could be detected. It ws not possible to continue using the erly seson disese ssessment throughout the entire seson due to the quntity of plnt biomss produced by the tomto plnts nd difficulty in counting leves without dmging plnts. 127

151 Defolition ws ssessed on percent scle using 5% increments which is demonstrted to be equivlent to the commonly used Horsfll-Brrtt when disese severity is greter thn 1% (Ching et l., 2014). Symptoms of other folir plnt diseses such s erly blight, Septori lef spot, nd lte blight were not observed in the trils in ny yer, nd preventtive fungicide pplictions were pplied to ll tretments in 2012 nd 2013, confirming tht ny tretment differences observed were result of bcteril speck nd spot Conclusions This reserch explored the possibility tht tomtoes treted with UNI t the seedling stge combined with ASM t either the greenhouse seedling or field stge will hve higher tolernce to bcteril speck nd spot under field conditions in Ontrio, Cnd. The results indicte tht under the best cse scenrio, pplictions of UNI my occsionlly provide mrginl benefits for suppression of these diseses when combined with or without ASM. Due to the limited benefits nd inconsistent response to UNI for bcteril disese mngement, use of UNI should be for trnsplnt height mngement only with the understnding tht, in some yers, its use combined with ASM in the field my be beneficil. ASM pplied to tomto seedlings cn result in reduction in bcteril speck nd spot intensity severl weeks fter tretment in some yers; however, in certin genotypes nd under certin environmentl conditions there is the potentil for incresed disese susceptibility nd lower yield. Commercil ppliction of crop protection tools requires predictble response under field conditions so tht products cn be used efficiently nd effectively to limit losses to plnt disese. Plnt defense inducers hve the potentil to be prt of n integrted solution for mngement of bcteril speck nd spot in Ontrio, but dditionl reserch on the long term effects of plnt defense inducers on host immunity, physiology, growth, nd host interctions with environmentl stresses is required to relize the full potentil of these tools in commercil tomto production. 128

152 4 Chpter 4: Use of bcteril endophytes to control the incidence of bcteril speck disese (Pseudomons syringe pv. tomto) nd lter plnt growth in processing tomto seedlings 4.1 Introduction Bcteril endophytes of plnts re bcteri tht reside in surfce sterilized plnt tissue with neutrl or beneficil effects on their host (Hllmnn et l. 1997). Mny re culturble, but there re lso vible but not culturble bcteril endophytes tht re identified s the set of microbil genomes locted inside plnt orgns (Giero et l., 2013, Bulgrelli et l., 2013). The mjor sources of endophytes re seeds, propgtive mteril, the phylloplne nd soil, but the ltter is considered the most importnt inoculum source t lest for root endophytes (Hllmnn et l., 1997, Hrdoim et l., 2008, Rosenblueth & Mrtinez- Romero, 2006). Txonomiclly, bcteril endophytes re minly members of the Actinobcteri, Bcteroidetes, Firmicutes nd Proteobcteri, which is less diverse thn rhizosphere bcteri (Berg et l., 2005, Bulgrelli et l., 2013). Bsed on their dpttions for endophytism, Hrdoim et l. (2008) divided bcteril endophytes into pssenger endophytes, which hve few dpttions nd re present in the outer root cortex tissue only by chnce, opportunistic endophytes, which hve more dpttions but re still confined to root cortex tissues, nd competent endophytes, which re the most dpted to the plnt environment llowing for higher popultions in tissues, vsculr coloniztion nd spred inside the plnt for ner complete plnt coloniztion. Competent endophytes re believed to hve been under selection pressure becuse they provide benefits to the host in the interction. For tomto, bcteril endophyte species pper to be reltively diverse. Cultures of endophytic Pseudomons oleovorns, Pseudomons plecoglossicid, P. nntis, Citrobcter freundii, Stphylococcus hominis, Sphingobcterium multivorum, Enterobcter cloce, Arthrobcter globiformis nd Rhizobium rdiobcter were obtined from tomto roots (Upreti & Thoms, 2015). In nother exmple, Feng et l. (2013) used non-culture-dependent method (PCR- restriction frgment length polymorphism nd 16S rdna sequencing) to identify Sphingomons ynoikuy, Pseudomons pseudolcligenes, S. mrcescens, B. megterium, Penibcillus polymyx, B. pumilus, B. cereus, P. fluorescens nd A. globiformis s endophytes from roots nd stems of two tomto cultivrs. However, mss sequencing of 16S-ribosoml RNAs demonstrted tht the endophyte popultion in tomto is even more diverse s 80 opertionl txonomic units were found for tomto lef bcteril endophytes, which were dominted by members of the Proteobcteri with reltively smll numbers in the Actinobcteri, Plnctomycetes, Verrucomicrobi nd Acidobcteri (Romero et l., 2014). In nother study, the diversity of 41 bcteril endophyte isoltes cultured from tomto stems ws less cler, s only two of them, Stphylococcus epidermidis nd B. myloliquefciens, were identified (Nwngsih et l., 2011). Two importnt benefits tht bcteril endophytes provide plnts re induced disese resistnce 129

153 nd plnt growth promotion (Hrdoim et l., 2015). Endophytes mostly induce resistnce vi ISR, which involves priming of ET nd JA dependent signlling pthwys nd typiclly induces resistnce ginst hemibiotrophs nd necrotrophs, but some endophytes could be considered to induce SAR, which involves SA dependent signling pthwys nd typiclly induces resistnce ginst biotrophs nd hemibiotrophs (Pieterse et l., 2009, Ton et l., 2006). Bcteril endophytes cn promote plnt growth by incresing the vilbility of nitrogen nd phosphorous, nd modulting levels of phytohormones including IAA, cytokinins nd ET (Hrdoim et l., 2015). Prt of the plnt growth promotion is lso due to competition with plnt pthogens through production of ntimicrobil compounds, nutrient competition nd siderophores, which cn ct in ddition to induced resistnce (Hllmnn et l. 1997; Lugtenberg nd Kmilov 2009; Rosenblueth nd Mrtinez-Romero 2006). For tomto, number of bcteril endophytes hve been shown to be beneficil. For exmple, endophytic Bcillus nd Arthrobcter species from tomto tht produced IAA nd siderophores nd solubilized inorgnic phosphte, cused tomto growth promotion (Amresn et l., 2012). A. johnsonii, S. mrcescens, Sinorhizobium sp. nd B. megterium induced resistnce ginst erly blight (A. solni) nd bcteril speck, nd lso incresed plnt height (Brretti et l., 2009). Since the endophytes were pplied to cut stems nd the pthogens were inoculted on leves, it is likely tht induced resistnce ws involved. Isoltes of B. pumilus nd B. myloliquefciens from tomto induced resistnce ginst bcteril speck in tomto, which ws ssocited with induction of peroxidse, polyphenol oxidse nd phenyllnine mmoni-lyse ssocited with resistnce (Lnn-Filho et l., 2017). P. oleovorns nd Agrobcterium tumefciens, which were lso isolted from tomto, incresed seedling vigour nd reduced tomto bcteril wilt, lthough induced resistnce ws not estblished (Thoms & Upreti, 2016, Upreti & Thoms, 2015). B. pumilus SE34 induced resistnce ginst F. oxysporum f. sp. rdicis-lycopersici by inducing ultrstructurl chnges in tomto, such s the production of root wll ppositions nd electrondense substnces tht re ssocited with resistnce (Benhmou et l., 1998). Thus, there is mple evidence for tomto bcteril endophytes s providing both plnt growth promotion nd induced resistnce. Bcteril endophyte popultions in plnts cn be influenced by number of fctors including host species (McInroy & Kloepper, 1995), cultivr (Hrdoim et l., 2011, Rsche et l., 2006) nd plnt development stge (Andreote et l., 2010, Monteiro et l., 2011). The effect of plnt development, for exmple, ws observed with dizotrophic bcteril popultions of vetiver tht were more common fter trnsplnting t 3, 6 nd 12 months thn 1 month (Monteiro et l. 2011), nd popultions of phosphte solubilising bcteri in soyben were higher in roots t the vegettive thn the flowering nd senescence stges (Kuklinsky-Sobrl et l., 2004). The effect of plnt genotype, for exmple, ws observed with more IAA-producing bcteril endophytes in n erly ripening thn lte-ripening soyben cultivr 130

154 (Kuklinsky-Sobrl et l., 2004), nd the diversity of tomto root endophytes ws greter in bcteril wilt resistnt thn wilt susceptible cultivr, which ws relted to there being more bcteri with ntgonistic ctivity ginst R. solncerum in the resistnt cultivr (Upreti & Thoms 2015). Developing new bcteril endophytes for tomto disese protection is ppeling becuse endophytes re lredy dpted to colonizing the plnt environment nd my lso promote plnt growth (Mercdo-Blnco & Lugtenberg, 2014). However, their effectiveness when pplied to plnts is determined by mny fctors, such s the inoculum density, environment nd plnt genotype (Pilly & Nowk, 1997). Thus, developing bcteril endophytes for disese control nd plnt growth promotion in tomto requires not just obtining promising endophyte but lso exmining mny spects of the system. The objective of this study ws to screen bcteril endophytes from cultivted tomto (S. lycopersicum), wild tomto (S. rcnum, S. chmieleskii nd S. cheesmnie) nd N. benthminum for their bility to induce resistnce ginst Pst nd then optimize conditions for inducing resistnce ginst bcteril speck through n exmintion of endophyte inocultion timing, inocultion method nd concentrtion, nd the impct of host genotype. 4.2 Mterils & Methods Isoltion of endophytes from tomto nd wild tomto species Seeds of tomto cv. Tiny Tim nd TSH4 were obtined from the A.E. McKenzie Co, Brndon, MB nd Tomto Solutions, Chthm, ON, respectively, nd seeds of the wild tomto species, S. rcnum LA2152, S. chmielewskii LA1327 nd LA1306, S. cheesmnie LA1040, nd S. glpgense LA1508 were obtined from the C.M. Rick Tomto Genetics Resource Centre, University of Cliforni t Dvis, CA, USA. Soil ws collected from rnge A7 (sndy lom with more thn 30 yers of vegetble production) or grss buffer strip locted ner the edge of wood lot t the Ridgetown Cmpus, University of Guelph ( N, W) nd psteurized in drying oven for 11 min per L of soil t 60 C to obtin n internl temperture of 60 C for 30 minutes. The psteurized soil ws mixed with Ffrd germintion mix (Ffrd et Frères, St. Bonventure, PQ) nd fine grnulted wshed snd (Alltret Frms, Arthur, ON) t rtio of 40:40:20 nd plced in 4-inch pots. Tomto cv. Tiny Tim or TSH4 were seeded in the rnge A7 soil nd S. rcnum LA2152, S. chmielewskii LA1327 nd LA1306, S. cheesmnie LA1040, nd S. glpgense LA1508 were seeded in the grss buffer strip soil. The second soil source ws included in lter experiments to try nd increse the diversity of endophytes isolted from tissues, s there ws no history of crop production in this soil. Plnts were grown in growth chmber t 22 C with 16 h photoperiod with light intensity of pproximtely 95 µmol/m 2 /s for four to six weeks to obtin roots for endophyte isoltion. Tomto cv. TSH4 ws lso grown in the field for pproximtely three months fter trnsplnting in rnge A7 using stndrd prctices for processing tomtoes in Ontrio. 131

155 Roots were seprted from the soil nd gently but thoroughly wshed in tp wter, surfce sterilized for 60 sec in 70% ethnol, 120 sec in 10% blech, rinsed three times in sterile distilled wter, nd then blot dried on sterile filter pper. The fresh weight of ech root mss ws recorded, nd then roots were mcerted using mortr nd pestle until no root pieces were visible. Approximtely 2 ml sterile distilled wter with 0.02% Tween 80 ws dded to the mcerted roots, which ws then serilly diluted using sterile distilled wter with 0.02% Tween ul of the mcerted solution of ech dilution nd the wsh wter from the third root rinse ws plted on TSA in duplicte nd incubted t room temperture (22 C). The number nd type of colonies ws recorded fter two dys, nd representtive single colonies of common phenotypes were streked on TSA to obtin pure cultures. Long term storge cultures of ech isolte were prepred by inoculting LB broth from single colony, growing overnight, nd mixing with sterile glycerol to rech finl concentrtion of 15% glycerol. Cultures were stored t - 80 C Screening of endophytes for induced resistnce nd plnt growth effects Twenty bcteril endophytes from tomto roots isolted t Ridgetown nd seven from Nicotin benthmin roots isolted in the lbortory of P. H. Goodwin, University of Guelph were screened for their bility to induce resistnce ginst Pst using n overnight seedling root sok + soil drench ssy (Vlenzuel-Soto et l. 2010). Seeds of tomto cv. TSH4 were plnted in Ffrd germintion mix in 288-cell plug try (pproximtely 14 ml per cell). Endophytes were grown for 72 hrs on TSA, trnsferred to 10 mm MgCl 2 using sterile swb, vortexed, djusted to bsorbnce 1.0 (+/ ; OD = 600) nd then diluted 10-fold. Nine dys fter plnting, seedlings were crefully removed from the soil, nd the roots were plced in the endophyte solution nd shken for 30 min t 50 rpm. The seedlings were then trnsplnted into 4-inch (500 ml) round pots filled with 50:50 mix of psteurized soil from the sme buffer strip described previously nd Ffrd germintion mix. 10 ml of the endophyte solution used to sok the seedling roots onto the soil surfce of ech pot. Control tretments included equl volumes of 10 mm MgCl 2 in plce of ll endophyte solutions. For this study, the tomto, bcteril root endophyte nd folir bcteril speck (Pst) system ws chosen s ppliction of bcteril endophytes cn induce resistnce in tomto (Benhmou et l., 1998, Lnn-Filho et l., 2017), nd Pst cn be controlled by both SAR nd ISR (Hermn et l., 2008). Pst cuses bcteril speck, which cuses folir lesions, defolition, erly fruit ripening nd sunscld in commercil tomtoes (Preston 2000; Young et l. 1978). Plnts were inoculted with pproximtely 2 x 10 7 CFU/mL of Pst DC06T2-4 in distilled wter with 0.01% Sylgrd 309 t 20 DAS. The upper lef surfce of ech plnt ws spryed with inoculum until just before runoff using hnd-held mist pplictor, nd then the plnts were covered with trnslucent plstic continer for 24 hours in growth 132

156 chmber. No fertilizer ws pplied during the study period. The number of lesions on ll compound leves on ech plnt ws recorded seven DPI with Pst. Lef re ws determined by detching compound leves, trcing the circumference of ech leflet on cler cette sheet, scnning the imge, nd determining the number of pixels using Imge J v.1.47 ( Pixel number ws converted to re (cm 2 ) using the following eqution: re = ((0.0171*no. of pixels) ) * The eqution ws developed by mesuring the numbers of pixels mesured for circles with known res nd creting stndrd curve of pixel number versus re. At seven DPI, plnt height from the soil line to the growing point, folir fresh weight nd root fresh weight were determined, nd the reltive chlorophyll the first leflet of ech compound lef ws recorded using Minolt SPAD-502 Chlorophyll Meter (Konic Minolt, Tokyo Jpn). Folir nd root dry weights were recorded fter fresh smples were plced in greenhouse for 1 to 2 weeks until dry. After one experiment using 27 endophytes, endophytes R9, R17, R19, R20, R21 nd Serende Mx (B. subtilis QST713) (Byer CropScience, Clgry, AB) were pplied using the seedling sok ssy s described bove. The purpose of these preliminry experiments ws to further evlute the selected endophytes s cndidtes for future experiments, nd to refine the experimentl method, thus experimentl methods vried. Serende Mx ws ment to serve s possible positive control for disese suppression. The seedlings were grown in 50:50 Promix PGX (Premier Tech Horticulture, Rivière-du- Loup, PQ) with psteurized rnge A7 soil in experiment one nd Promix PGX only in experiment two. Also, Promix PGX Biofungicide (B. subtilis MBI600) (Premier Tech Horticulture, Rivière-du-Loup, PQ) ws pplied by growing the seedlings in the Promix PGX potting medi mixed 50:50 with psteurized rnge A7 soil in experiment one nd non-psteurized rnge A7 soil in experiment two. The purpose of using the Promix PGX Biofungicide ws to determine its utility s positive control. Inocultion with Pst ws the sme s described for the initil screening, except inoculum ws pplied to both the upper nd lower lef surfces. The experiment ws rndomized complete block with four replictions per tretment nd one pot per replicte. In ddition, seed sok + seed drench + seedling drench inocultion method ws developed nd tested for endophytes R9, R19 nd R20 in three experiments, nd R17 nd R21 in four experiments, ech in rndomized complete block design with 4 replictions per tretment. Seeds of tomto cv. TSH4 were surfce sterilized in 2% sodium hypochlorite for 10 min nd rinsed three times in distilled wter for five minutes (Tyburski & Tretyn, 2004). Bcteril endophyte solutions were prepred s previously described. For the seed sok portion of the ppliction, tomto seeds were dded to the endophyte solution nd incubted overnight t 150 rpm. Seeds for the control were plced in 10 mm MgCl 2 only. After 14 to16 h the seeds were crefully removed from the solution, plced on pper towels, sown in 3.5-inch squre pots (530 ml) filled with Ffrd germintion mix, covered with vermiculite, nd plced in growth chmber 133

157 t 24 C nd 16 h photoperiod. For the seed drench portion of the ppliction, pipette ws used to pply 4.95 ml of the endophyte solution tht hd been used to sok the seeds overnight to the surfce of the germintion mix. For the seedling drench portion of the ppliction, fresh solution of ech endophyte solution ws prepred s bove nd pplied to the surfce of the germintion mix t 10 DAS using the sme method s the seed drench. No fertilizer ws pplied during the study period. Pst inocultion nd plnt growth nd disese ssessments were completed s previously described Stndrd curve of CFU versus A600 A stndrd growth curve ws developed for R17 nd R21 by plting dilutions of the bcteri grown on TSA for 72 h nd then diluting in sterile distilled wter to 0.2, 0.4, 0.6, 0.8, nd 1.0 bsorbnce (OD = 600). Seril dilutions t ech bsorbnce level were plted onto TSA nd counted t 48 hours fter incubtion t pproximtely 22 C. From this, formul ws developed for R17: CFU/mL = [(9x10 7 ) (bsorbnce)] - 1x10 7, nd formul ws developed for R21: CFU/mL = [(2x10 9 ) (bsorbnce)] - 2x10 8. Bsed on popultions obtined from the seril dilutions t 1.0 bsorbnce, the pproximte popultion of R17 nd R21 used in the previous trils ws clculted to be pprox. 8 x 10 6 nd 3 x 10 8 CFU/mL. The formuls were used to clculte OD = 600 required for the R17 nd R21 cells to be djusted to 1 x 10 7 nd 1 x 10 8 CFU/mL for R17 nd R21 for ll dditionl experiments. Only bsorbnce mesurements t 600 nm were used t < to void errtic mesurements t higher bsorbnce levels Direct ntimicrobil effects of R17 nd R21 The direct ntimicrobil effects of R17 nd R21 were evluted using n gr overly ssy nd modified cross-streking ssy (Jinhu et l., 2002). For the gr overly ssy, TSA ws prepred using stndrd methods, nd 2 cm dimeter circle trced on the bottom of ech plte. The re inside ech circle ws then streked with endophyte solution, nd the pltes were incubted t room temperture for two dys. TSA ws prepred (1.50% gr), nd 5 ml dded to sterile test tubes nd incubted in 51 C wter bth. The molten gr ws then inoculted with 0.1 ml of 2 x 10 7 CFU/mL Pst 06T2-4, inverted three times to mix, nd then slowly poured over the endophyte inoculted TSA plte. The pltes were incubted t room temperture nd ssessed for zone of inhibition round the endophyte inoculted re two dys fter Pst inocultion. The overly ssy ws repeted three times with eight replictions per tretment. A cross streking ssy ws completed by streking single endophyte on TSA to form single liner strek cross the centre of ech plte. Pltes were incubted for two dys t room temperture, nd then single colony of Pst 06T2-4 ws streked t 90 ngle from the edge of the plte to the edge of 134

158 the endophyte strek. Cre ws tken to finish the strek s close s possible to the endophyte without touching it. Pltes were incubted for two dys t which time the strek of Pst ws well grown nd pltes could be ssessed for zone of inhibition. The Pst strek width t the rim end of the plte nd the center of the plte where the strek stopped ws lso mesured nd used to clculte the percent reduction in strek width. Ech ssy ws repeted three times with five replictions per tretment Optimiztion of bcteril endophyte tretment To compre the effects of bcteril culture medi, R17 nd R21 were grown on TSA, 0.1 TSA, nd LB gr for three dys nd then prepred s previously described for seed sok + seed drench + seedling drench inocultion in one experiment. To compre the effects of soil fertility, the seed sok + seed drench + seedling drench inocultion ws done with R17 nd R21 fter growth on 0.1 TSA nd TSA s previously described, but plnts were grown with no dded fertilizer versus ddition of 20 ml (three experiments) or 80 ml (one experiment) of Plnt-Prod Ultimte fertilizer (Plnt Products Co Ltd, Brntford, ON, CA) t 11, 16 nd 21 DAS. The fertilizer ws micronutrients fertilizer solution mixed t 1.26 g/l. The incidence of bcteril speck lesions, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined s previously described, except ssessments were completed t five DPI nd the number of lesions per cm 2 on only the second nd third youngest mture leves on ech plnt ws clculted. The second youngest lef ws identified using the definition of the youngest developing lef s the first lef from the picl meristem with terminl leflet midrib length of 1.5 cm or longer. To evlute the effects of the endophyte ppliction method, R17 nd R21 were pplied s described bove for seed sok, seed drench, seedling drench, seed sok + seed drench or seed sok + seed drench + seedling drench. Applictions of 10 mm MgCl 2 t the time of the different endophyte tretments were used s controls. Plnts were grown s bove with 20 ml of fertilizer in the first tril, nd 80 ml fertilizer in the second nd third trils, nd disese nd plnt growth ssessments were tken t five DPI. Lesion counts nd lef re were evluted for the second nd third youngest leves of ech plnt. To determine the effect of rw nd pelleted seed with different ppliction methods, the seed sok, seed drench or seed sok + seed drench ppliction methods for endophyte R17 ws repeted in two trils with seven replictions s bove. At five DPI, the incidence of bcteril speck lesions on the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined. A comprison of five commercil tomto cultivrs on the effectiveness of endophyte R17 ws done with tomto cv. H2401, cv. H5108, nd cv. H9553 (H.J. Heinz, Lemington, ON), nd cv. TSH33 nd cv. TSH4 (Tomto Solutions, Chthm, ON). The effect of different doses of R17 on induced 135

159 resistnce in cv. TSH4 ws determined by evluting five concentrtions of R17 (1x10 5, 1x10 6, 1x10 7, 1x10 8, nd 1x10 9 CFU/mL) nd nontreted control in cv. TSH4 using the seed drench method. The durtion of the plnt response to R17 ws determined by Pst inocultion performed 15, 20 nd 25 DAS on cv. TSH4. Except for the dose experiment, R17 ws pplied t 1 x 10 7 cfu/ml in10 mm MgCl 2 using the seed drench method, nd the plnts were fertilized t 10, 15 nd 21 DAS with 80 ml fertilizer solution, except tht plnts inoculted 25 DAS in the timing of induction trils were lso fertilized t 26 DAS. Control plnts received 10 mm MgCl 2. At five dpi, the incidence of bcteril speck lesions on the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined Effect of R17 inocultion on popultions in plnt Spontneous rifmpicin resistnt strins of R17 were developed using method similr to Andreote et l. (2008). Briefly, n overnight culture of R17 ws grown in tryptic soy broth with shking. A 4 ml smple ws removed from the overnight culture, centrifuged t 6000 rpm for 15 minutes, the superntnt removed nd the bcteril pellet resuspended in 1 ml sterile distilled wter. The bcteri were then spred on TSA mended with 50 µg/ml rifmpicin nd incubted t room temperture for four dys. Single colonies were picked from the rifmpicin-mended pltes, grown overnight in TS broth, nd stored in 15% glycerol t -80 C. To confirm the rifmpicin resistnt R17 strins hve the sme disese suppression chrcteristics s the wt R17, three rifmpicin resistnt strins (R17-RfpA, R17-RfpB, nd R17-RfpC) were compred to the wt R17 (R17-Wt) using cv. TSH4. Three trils with five or six replictions per tretment were completed using the seed drench ppliction method nd effects on disese suppression nd plnt growth were ssessed s previously described. The bility of R17-RfpC to colonize the rhizosphere nd plnt tissues ws exmined t 10 nd 25 DAS. R17-RfpC ws pplied s seed drench nd plnts were grown s previously described, except ech replicte consisted of five pots with 10 plnts per replicte t 10 DAS (2 plnts/pot) nd five plnts per replicte t 25 DAS (1 plnt/pot). Cotyledon leves were used for folir smples t 10 DAS becuse there ws little or no true lef tissue present. At 25 DAS, the terminl leflet on the second nd third youngest leves were collected. Root tissue ws collected by gently digging out roots from pots nd removing the Ffrd germintion mix by rinsing in tp wter. Prior to rinsing in tp wter, the germintion mix from round the roots ws collected for rhizosphere smples. Root nd lef tissue ws wrpped in pper towel, plced in plstic bg, nd stored t mbient temperture until processing, which ws within four hours of smple collection. Rhizosphere smples were collected in plstic centrifuge tubes to void moisture loss prior to processing. To ensure R17 ws not present in the germintion mix 136

160 prior to drenching, smples were collected directly from the Ffrd germintion mix bg nd processed using the sme method s the rhizosphere smples. No colonies resembling R17 were detected. Root smples were processed s previously described for endophyte isoltion, except only 1.5 ml sterile distilled wter with 0.02% Tween 80 ws dded to mcerted roots, which ws directly plted onto 10 pltes of TSA mended with 50 µg/ml rifmpicin. The method for folir smples ws the sme s the root tissue except the tissue ws surfce sterilized for 90 seconds in 2-3% sodium hypochlorite, followed by 60 seconds in 70% ethnol, nd rinsing in sterile distilled wter. For rhizosphere smples, 1 g of germintion mix ws mixed with 10 ml sterile distilled wter with 0.02% Tween 80, mcerted in mortr nd pestle to reduce prticle size, nd incubted t room temperture for 30 to 40 minutes. The solution ws then vortexed nd plted s described bove for root nd folir smples. Dry weight of the rhizosphere smples ws determined by using n equl subsmple of the germintion mix, nd the percent moisture of rhizosphere smples nd popultion of R17-RfpC per g of dry soilless mix clculted. Colony types mtching R17 were counted t six dys fter plting, nd the presence of R17 ws confirmed by streking rndom single colonies on TSA nd submitting smples to Lbortory Services, University of Guelph for identifiction using 16S nd gyrb DNA sequencing described below Effect of R17 inocultion on Pst popultions nd lesion size in plnt Tomto cv. TSH4 ws grown nd inoculted with R17 with the seed drench method. On the dy of disese ssessment (five DPI), the third terminl leflets of control nd R17 inoculted plnts were excised, nd the lef re of ech terminl leflet ws determined s previously described. The fresh weight of ech smple ws recorded nd surfce steriliztion ws completed by immersing ech leflet in 3.1% sodium hypochlorite solution for 90 seconds, 70% lcohol for 60 seconds, nd then rinsing in sterile distilled wter for 30 seconds. Leflets were blot dried on filter pper nd homogenized in 2 ml of sterile distilled wter using mortr nd pestle. A dilution series ws completed nd 100 µl from dilutions 10-2 through 10-6 were plted on VBTr, semi-selective medi for Pst (Cuppels & Elmhirst, 1999). The efficcy of the surfce steriliztion method ws confirmed by plting rinse wter from ech smple on TSA. The number of colonies on ech plte ws recorded fter incubtion t room temperture for four dys. The number of CFU per cm 2 of lef tissue nd per lesion ws then clculted. The tril ws repeted three times with three (experiments 1 nd 2) or two (experiment 3) replictions per tretment in ech experiment. To determine the effect of R17 inocultion on lesion size nd circumference, tomto cv. TSH4 ws grown s previously described, nd the terminl leflet on the third youngest tomto lef collected on the dy of ssessment (five DPI). A Nikon D300s cmer (Nikon Cnd Inc., Mississug, ON) set to JPEG fine qulity ws used to tke photo (12.2 MB, 4288 x 2824 pixels) of ech leflet. A ruler ws 137

161 lso included within ech imge. Photos were uploded to PowerPoint 2010 (Microsoft, Redmond, Wshington, USA), incresed in size to llow for trcing of the lesion circumference, printed in colour, nd the circumference of 12 to 15 lesions per leflet trced using fine point permnent mrker. Lesions were selected by trcing ll lesions within rndomly plced 225 mm 2 qudrnt, nd then 12 to 15 lesions were rndomly selected. The lesion circumferences were then scnned nd the number of pixels determined s previously described for lef re, except the size of ech imge ws stndrdized by clibrting ech imge. This ws completed in Imge J by using the line tool to drw stright line representing distnce of 2 cm on the ruler included in the originl imge, clicking nlyze nd set scle, setting the distnce in pixels to 40 nd the known distnce to 2 cm. Lesion circumference ws clculted using the eqution C = 2π [ (men lesion re/π)]. The percent lef re covered in lesions ws clculted using the eqution re = (# lesions/cm 2 * men lesion re (cm 2 )) * 100. The experiment ws repeted twice with seven (experiment 1) nd six (experiment 2) replictions per tretment Identifiction of endophytes R17 nd R21 The 16S DNA prtil sequences of R17 nd R21 were mplified with primers 27f (5 GCYTAACACATGCAAGTCGA-3 ) nd 1495r (5 -GTGTGTACAAGNCCCGGGAA-3 ) (Lbortory Services, University of Guelph), where Y designtes C or T, nd N designtes ny nucleotide, to obtin 1265 bp mplicon of ech endophyte. In ddition, the 16S sequence of R17 ws lso determined using primers 8F (5 -AGAGTTTGATCCTGGCTCAG -3 ) nd universl primer 515F (5 -GTGCCAGCMGCCGCGGTAA -3 ), where M designtes A or C (Lne, 1991). For R21, rpob gene frgment ws lso mplified using primers Vic3 (5'-GGCGAAATGGCWGAGAACCA-3') nd Vic2 (5'- GAGTCTTCGAAGTTGTAACC-3') (Puw et l., 2008), where W designtes A or T to obtin 951 bp mplicon. For R17, primers gyrb560f (5 -ACTCGTATGCGTGAGTTGGC-3 ) nd gyrb1840r (5 - CAAGTTTCCCTTCAAGATGCA-3 ) (Lbortory Services, University of Guelph) were used to mplify nd sequence 1134 bp product of the gyrb sequence. Primers were synthesized by Lbortory Services, University of Guelph. Pure cultures were submitted to the Pest Dignostic Clinic, University of Guelph, for DNA extrction nd sequencing. To design the gyrb primers, gene sequences were obtined of CP for B. mycoides nd CP for B. weihenstephnensis from the NCBI nr dtbse nd ligned with ClustlW. Conserved regions for ll species were identified tht would result in n mplicon between 1000 nd 1400 bp. The primer melting tempertures were determined to ensure both forwrd nd reverse tempertures were similr nd greter thn 52 C. In order to determine the phylogenetic reltionship of R17 nd R21 to other species of bcteri, BLASTn serch of the 16S sequences were done ginst the GenBnk 16S ribosoml RNA gene dtbse 138

162 ( nd BLASTn of the rpob nd gyrb sequences were done using the Genbnk nr dtbse. Sequences from different species with e-vlues of 0.0 were collected. The sequences of the overlpping regions of ech endophyte nd selected mtches were ligned by ClustlW v.1.83 (Kyoto University Bioinformtics Centre, Kyoto, Jpn) multiple sequence lignment tool ( Neighbour-joining trees for gene sequences of ech endophyte were constructed in ClustlW with 1000 bootstrp replictes nd visulized with TreeView v1.6.6 (Glsgow University, Glsgow, UK). The bility of R17 to grow t cold tempertures ws evluted by streking single colony on TSA nd incubting t 4 C for six dys. Motility ws determined in wet mount from n overnight culture grown in TSB using light microscope (100x) (Clery et l., 2002, Chester & Poulos, 1980) Sttisticl nlysis Sttisticl nlysis ws completed using SAS v9.4 (SAS Institute, Cry, NC, USA). Dt were tested for normlity using the Shpiro-Wilk sttistic. Outliers were identified using Lund s test of stndrdized residuls (Lund 1975). Anlysis of vrince ws completed using Proc Mixed with tretment s fixed effect nd repliction s rndom effect. Dt from different trils ws pooled together when sttisticl nlysis showed no tretment x tril interction (P 0.05), except where noted in some tbles nd figures. Tril ws treted s fixed effect. Dt were nlyzed s rndomized complete block design. Mens comprisons were performed when P 0.05, nd mens were seprted using Tukey s HSD. Regression nlysis for endophyte concentrtion (LOG10) ws completed using Proc Reg to determine model significnce nd Proc Glm to obtin prmeter estimtes. 4.3 Results Bcteril endophytes isolted from tomto nd wild tomto species A totl of 21 phenotypiclly different bcteril endophytes were collected from roots of tomto nd wild tomto species, including seven from cv. Tiny Tim, eight from cv. THS4, two from S. rcnum LA2152, three from S. chmieleskii LA1327, nd one from S. cheesmnie LA1040 (Tble 4.1). Most isoltes were smooth, trnslucent, entire, circulr, nd crem in colour Screening of bcteril endophytes for induced resistnce nd plnt growth effects Initil screening of 20 endophytes from tomto nd wild tomto, seven bcteril endophytes isolted from N. benthmin, B. subtilis QST 713, nd B. subtilis MBI600 ws completed using the overnight seedling root sok + soil drench ssy (Vlenzuel-Soto et l. 2010) with the concentrtions of 139

163 endophytes djusted to OD600=1.0 t lest once for ll isoltes except R4, which could not be evluted due to contmintion of the long term storge culture. There ws no effect of endophyte inocultion on the number of bcteril speck lesions per cm 2 of lef tissue t seven DPI, reltive chlorophyll, plnt height, folir dry weight, or root dry weight compred to the MgCl 2 control (Tble 4.2 nd Tble 4.3). However, there ws numericl trend towrd lower disese incidence for endophytes R9, R17, R19, R20 nd R21. These endophytes were exmined gin using modified version of the Vlenzuel-Soto et l. (2010) method, where the seed rther thn seedling roots were soked nd the soil ws drenched t the time of plnting nd then once the seedling hd developed. This ws done to reduce dmge to the roots, nd the method ws designted the seed sok + seed drench + seedling drench inocultion method. Inocultion with this method significntly reduced the number of bcteril speck lesions per cm 2 of lef tissue t seven DPI for R9, R17 nd R21 compred to the MgCl 2 control (Tble 4.4). However, there ws significnt tretment x tril interction with R9. While R9 significntly reduced lesion numbers by 41% in one experiment, it hd no significnt effect in nother experiment. Therefore, R9 ws considered inconsistent. Lesion numbers were reduced by 27% with both R17 nd R21compred to the control. None of the endophytes ffected reltive chlorophyll, plnt height, or dry root weight (Tble 4.5). Folir dry weight ws not ffected by ny endophyte, except for n increse with R17, which ws inconsistent s it ws significntly higher thn the control in experiment one but equivlent to the control in the finl two experiments. Bsed on these results, only R17 nd R21 were further exmined Direct ntimicrobil effects of R17 nd R21 The direct ntgonistic effects of R17 nd R21 ginst Pst were evluted in cross-streking nd gr overly ssys. In the cross-streking ssy, R17 produced zone of inhibition with the Pst strek width ner R17 being reduced by 90.2% compred to strek width t the edge of the petri plte distnt from R17 (Tble 4.6). In the gr overly ssy, the zone of inhibition of Pst by R17 ws 8 mm compred to none for the control (Tble 4.7). R21 produced zone of inhibition in the cross-streking ssy with the Pst strek width ner R17 being reduced by 46.5% compred tht t the opposite end of the petri plte (Tble 4.6). However, no zone of inhibition of Pst by R21 ws observed in the gr overly ssy (Tble 4.7). The results demonstrte both R17 nd R21 hve ntgonistic effects ginst Pst in vitro, but R21 hd only slight effects on Pst growth becuse it only slightly reduced growth in the cross-strek ssy nd produced no zones of inhibition in the overly ssy Optimiztion of bcteril endophyte tretment 140

164 To compre the effects of bcteril culture medi, the seed sok + seed drench + seedling drench inocultion ws done with R17 nd R21 tht were grown on TSA, 0.1 TSA, nd LB gr. The level of induced resistnce using R17 nd R21 grown on 0.1 TSA ws numericlly better thn 1.0 TSA or LB, nd so for future experiments, the endophytes were grown on 0.1 TSA (dt not shown). To compre the effects of soil fertility, the plnts either hd no plnt fertilizer or 20 ml or 80 ml fertilizer progrm. Preliminry results suggested more consistent response using the 80 ml fertilizer progrm, nd so the 80 ml fertilize progrm ws chosen (dt not shown). To determine the endophyte concentrtion for inocultion, stndrd curve of R17 popultion versus bsorbnce ws mde (Figure A.4). Bsed on tht, the endophyte concentrtions were djusted to 1 x 10 7 CFU/ml for R17 nd 1 x 10 8 CFU/ml for R21 becuse the concentrtions of R17 nd R21 used in the previous trils ws clculted to be pprox. 8 x 10 6 nd 3 x 10 8 CFU/mL, respectively. Also, it ws decided to ssess plnts for disese incidence t five DPI insted of seven DPI to mke it esier to distinguish individul bcteril speck lesions, nd to count bcteril speck lesions on only the second nd third youngest leves insted of ll compound leves to reduce vribility ssocited with bscised or dying leves. Using the bove conditions, seed sok + seed drench + seedling drench tretment with R17 or R21 reduced the number of bcteril speck lesions per cm 2 of lef tissue t five DPI by 33 nd 38 %, respectively. There ws non-trget effect of both endophyte tretments reducing plnt height by 0.5 to 0.7 cm, but neither endophyte tretment hd n effect on reltive chlorophyll or folir nd root dry weight (Tble 4.8). These results suggest tht under the conditions tested, both R17 nd R21 induced host resistnce in tomto cv. TSH4 ginst Pst, reduced plnt height, nd hd no effect on reltive chlorophyll nd plnt biomss ccumultion. Different endophyte ppliction methods of R17 nd R21 were compred: seed sok, seed drench, seedling drench, seed sok + seed drench, nd seed sok + seed drench + seedling drench. Although ll endophyte tretments resulted in numericlly lower number of bcteril speck lesions per cm 2 of lef tissue, only the seed sok nd seed drench tretments with R17 resulted in significnt reduction in disese incidence (Tble 4.9). There were no other effects of endophyte inocultion on reltive chlorophyll or plnt growth. Bsed on this, only R17 ws further studied, even though R21 hd previously been effective (Tble 4.8). The seed sok nd seed drench ppliction methods for R17 were lso evluted on both rw nd pelleted seed. Disese incidence with rw seed ws never different from pelleted seed for ny of the ppliction methods (Tble 4.10). The gretest reduction in the number of bcteril speck lesions per cm 2 of lef tissue occurred when R17 ws pplied s seed drench or seed sok + seed drench, similr to the previous set of trils tht evluted endophyte ppliction method, there were no significnt effect of R17 ppliction on plnt height, or folir nd root dry weight. The seed drench ppliction method ws selected for future experiments becuse of its consistency nd becuse it 141

165 ws less lbour intensive thn the seed sok tretment, which required n overnight seed sok. Four commercil processing tomto cultivrs were evluted to determine if the host response to R17 ppliction ws consistent mong different host genotypes. A reduction in the number of bcteril speck lesions per cm 2 of lef tissue ws only observed in cv. TSH4 (Figure 4.1). Another commercil processing tomto cultivr, H2401, ws lso tested, but it ws inconsistent between experiments, with disese incidence being lower thn the control in experiment one but not in experiments two nd three (Tble A.9). Reltive chlorophyll ws higher in R17 treted plnts thn the control for cv. TSH4, but there ws no effect on cv. H5108, TSH33, or H2401 (Figure 4.1b, Tble A.9). Plnt height nd folir nd root dry weight were not ffected by R17 ppliction (Figure 4.1c-e), except for cv. H2401 where the response for folir nd dry root weight ws gin inconsistent mong experiments (Tble A.9). These results suggest tht the disese suppression response to R17 is genotype dependent. Future experiments were completed using cv. TSH4 becuse this ws the only cultivr where R17 ppliction resulted in consistent reduction in bcteril speck incidence. The dose response of R17 seed drench ppliction ws determined using concentrtions of 1 x 10 5 to 1 x 10 9 CFU/mL. There ws significnt qudrtic response to R17 concentrtion: disese incidence = (LOG10 (R17 concentrtion)) (LOG10 (R17 concentrtion)) 2 (Tble 4.11). Pek disese suppression ws chieved t pproximtely 1 x 10 7 CFU/mL. There ws no dose response observed for reltive chlorophyll, plnt height, or folir nd root dry weight. These results confirmed tht pplictions of R17 t concentrtion of 1 x 10 7 CFU/mL provided the best opportunity for bcteril speck suppression. To determine the durtion of the plnt response to R17, tomtoes were inoculted with Pst t 15, 20, nd 25 dys fter R17 seed drench ppliction. A reduction in the number of bcteril speck lesions per cm 2 of lef tissue occurred in R17 treted plnts when Pst inocultion occurred t 15 nd 20 DAS, but not t 25 DAS (Figure 4.2). Susceptibility of cv. TSH4 to bcteril speck ppered to decline between 15 nd 25 DAS, indicting potentil ge-relted host resistnce to Pst. The results for plnt growth nd reltive chlorophyll were inconsistent with previous results t 20 DAS (Figure 4.2b) Effect of R17 inocultion on Pst popultions nd lesion size in plnt The influence of R17 ppliction on Pst popultion in symptomtic leves ws ssessed by determining disese incidence nd Pst popultion on the third youngest terminl leflet. R17 ppliction did not reduce totl Pst popultion per cm 2 of lef tissue when there ws reduction in the number of bcteril speck lesions per cm 2 of lef tissue (Tble 4.12). However, the men Pst popultion per lesion in R17 treted plnts ws 75% higher compred to the men popultion in control plnts. This is n indiction tht bcteril speck lesions ppering in R17 treted plnts contin more bcteri thn those in 142

166 control plnts. Additionl experiments were completed to further exmine bcteril speck lesion size mong R17 treted nd control plnts. Lesion re nd lesion circumference were 45 nd 18% higher in R17 treted plnts (Figure 4.3-c). However, there ws no chnge in the totl lef tissue surfce re covered with lesions (Figure 4.3d). These results suggest tht R17 is suppressing initil infections so tht fewer lesions develop, but fter initil infection, the lesions of R17 treted plnts reched lrger size by five DPI thn those of control plnts. For contorol plnts, comprison of Pst popultions in symptomtic (i.e., bcteril speck lesions) nd symptomtic lef tissue showed tht symptomtic tissue hd Pst popultions three orders of mgnitude higher (5.12 x 10 8 CFU per cm 2 lef tissue) thn symptomtic tissue (1.12 x 10 5 CFU per cm 2 lef tissue) from the sme leflet. Thus, lesions pper to hve mde the gretest contribution to totl Pst popultion in leflet Effect of R17 inocultion on popultions in plnt Rifmpicin resistnt mutnts R17-RfpA, R17-RfpB, nd R17-RfpC were developed in order to determine the coloniztion pttern of R17 in tomto 10 nd 25 DAS. These mutnts suppressed the number of bcteril speck lesions per cm 2 of lef tissue to the sme level s the R17 wt (Tble 4.13). The R17-wt nd the Rfp mutnts lso did not ffect reltive chlorophyll, plnt height, or folir nd root dry weight. Thus, it ws ssumed tht the Rfp mutnts lso colonize cv. TSH4 in similr mnner to R17-wt. R17-RfpC ws never observed in leves or roots of buffer treted plnts, but low popultion of colony type mtching R17 with rifmpicin resistnce ws observed in the rhizosphere soil (Tble 4.14). At 10 DAS, colonies mtching R17-RfpC were recovered in reltively lrge mount from rhizosphere soil, pproximtely four orders of mgnitude less from roots nd six orders of mgnitude less from leves. This indictes tht the bcteri were ble to infect roots nd cotyledon leves. At 25 DAS, the popultion in rhizosphere hd declined by pprox. hlf from 10 DAS. However, the popultion in root tissue t 25 DAS ws two orders of mgnitude lower thn root tissue t 10 DAS, nd the popultion in true leves ws undetectble. Thus, coloniztion of plnt tissues by R17-RfpC declined over time in ll smples. Rndomly picked colonies from the rhizosphere nd different tissues were confirmed to be R17 bsed on gyrb sequencing described below. This included the rhizosphere colonies from buffer treted plnts Identifiction of R17 nd R21 The reltionship of endophyte R21 to other bcteri ws determined by prtilly sequencing 16S DNA nd rpob. To crete the R21 16S neighbour-joining tree, representtive sequences of ll Enterobcter sp. nd ll other species with 98% nt mtch or higher in BLASTn serch were selected 143

167 for multiple lignment. For the R21 rpob neighbour-joining tree, representtive Enterobcter, Citrobcter nd Leclerci sp. in the top 500 mtches nd ll Enterobcter ludwigii in the top 1000 mtches were included in the multiple lignment. When multiple sequences of species were vilble, preference ws given to published sequences. Enterobcter soli ws selected s the outgroup becuse it ws the Enterobcter sp. with the lowest mtch in the 16S query tht is not member of the E. cloce complex, nd sequences for both 16S nd rpob were vilble. R21 clustered most closely with ll 11 sequences of E. ludwigii, three of nine sequences of E. cloce, nd one of three sequences of Pntoe gglomerns in the 16S neighbour-joining tree (Figure 4.4), nd ll five sequences of E. ludwigii nd three of eight sequences of E. cloce in the rpob neighbour-joining tree (Figure 4.5). P. gglomerns did not pper within the top 1000 mtches of the BLASTn serch for rpob. These results suggest tht R21 is member of the E. cloce complex nd most closely relted to ll E. ludwigii nd some E. cloce isoltes entered in the BLASTn dtbse. To crete the R17 16S neighbour-joining tree, selected representtive sequences with 98% nt mtch or higher in BLASTn were used including ll vilble B. mycoides nd B. weihenstephnensis sequences. The outgroup Bcillus thermomylovorns ws selected becuse it ws the best mtching Bcillus species tht is not member of the B. cereus group. Attempts to produce prtil sequence of the gyrb region of R17 using primers Up-1S nd Up-2Sr (Ymmoto & Hrym, 1995), Up1-F nd Up2-R (Ymmoto & Hrym, 1995), gyrb293f nd gyrb1494r (Lbortory Services, University of Guelph), nd gyrb560f nd gyrb1840r (Lbortory Services, University of Guelph) were mde. Only primers gyrb560f nd gyrb1840r resulted in the successful mplifiction of gyrb gene sequences. For the R17 gyrb neighbour-joining tree, ll vilble B. mycoides nd B. weihenstephnensis were included s well s up to five sequences of species tht lso ppered in the 16S neighbour-joining tree. Published sequences were given priority when more thn five sequences of given species were vilble. The outgroup Bcillus bombysepticus ws selected becuse it ws the best mtching Bcillus species tht is not member of the B. cereus group. R17 clustered most closely with both sequences of B. weihenstephnensis nd ll three B. mycoides in the 16S neighbour-joining tree (Figure 4.6), nd ll six sequences of B. weihenstephnensis nd two of three sequences of B. mycoides in the gyrb neighbourjoining tree (Figure 4.7). Thus, R17 is most likely member of the B. cereus group nd most closely relted to sequences identified s B. mycoides nd B. weihenstephnensis. R17 ppered to be pschychrotolernt s it grew on TSA t 4 C nd motile, s it demonstrted movement in TSB when cells from n overnight culture were observed under the microscope. 144

168 Tble 4.1 Source nd colony description of endophytes isolted from roots of S. lycopersici, S. rcnum, S. chmielewskii, S. cheesmnie, nd N. benthmin fter growth on tryptic soy gr t room temperture (~22 C) for two dys. Endophytes were isolted from mcerted roots of plnts growing outdoors in the field t Ridgetown Cmpus, in medi contining 40 to 50% psteurized field soil mixed with snd under controlled conditions (Ridgetown R isoltes), or in 1:1 mixture of psteurized soil from the Guelph Turfgrss Institute (Guelph, ON) nd potting mix (Guelph G isoltes). Growing Phenotypic description Isolte Code Host loction Soil source Colour Surfce Opcity Mrgin Form R1-LR S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Trnslucent Entire Circulr R2-SWR S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Trnslucent Entire Circulr R3-Y S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Yellow Smooth Trnslucent Entire Circulr R4-LC S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Very Entire Circulr trnslucent R5-LR S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Trnslucent Entire Circulr R6-LR S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Trnslucent Entire Circulr R7-LR S. lycopersici cv. Tiny Tim Controlled RCUG-A7 Crem Smooth Trnslucent Entire Circulr R8-SWR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R9-VLR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R10-Y S. lycopersici cv. TSH4 Field RCUG-A7 Yellow Smooth Trnslucent Entire Circulr R11-Y S. lycopersici cv. TSH4 Field RCUG-A7 Yellow Smooth Trnslucent Entire Circulr R12-LR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R13-SWR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R14-VLR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R15-LR S. lycopersici cv. TSH4 Field RCUG-A7 Crem Smooth Trnslucent Entire Circulr R PY S. rcnum LA2152 Controlled RCUG-B Ple yellow Smooth Trnslucent Entire Circulr R B S. rcnum LA2152 Controlled RCUG-B Crem Rough Opque Entire Circulr R PY S. chmieleskii LA1327 Controlled RCUG-B Ple yellow Smooth Trnslucent Entire Circulr R B S. chmieleskii LA1306 Controlled RCUG-B Crem Rough Opque Entire Circulr R LR S. chmieleskii LA1327 Controlled RCUG-B Crem Smooth Trnslucent Entire Circulr R LR S. cheesmnie LA1040 Controlled RCUG-B Crem Smooth Trnslucent Entire Circulr G1-LW7 (Pseudomons N. benthmin Controlled GTI Crem Smooth Trnslucent Entire Circulr 145

169 sp.) G2-LW1(CW1) (Bcillus cereus) G3-SW1 (Pseudomons lcligenes) G4-LbW(cbW) (Bcillus simplex) G5-LW(CW2) (Bcillus megterium) G7-LY (Bcillus mrisflvi) G8-DC (Bcillus N. benthmin Controlled GTI Crem Rough Opque Entire Circulr N. benthmin Controlled GTI Crem Smooth Trnslucent Entire Circulr N. benthmin Controlled GTI Light brown Smooth Trnslucent Entire Circulr N. benthmin Controlled GTI Crem Smooth Trnslucent Entire Circulr N. benthmin Controlled GTI Yellow Smooth Trnslucent Entire Circulr N. benthmin Controlled GTI Crem Smooth Very trnslucent Entire Circulr pumilis) RCUG-B = grssed buffer strip on edge of woodlot t Ridgetown Cmpus, University of Guelph; RCUG-A7 = griculturl reserch rnge A7 t Ridgetown Cmpus, University of Guelph; GTI = Guelph Turfgrss Institute, University of Guelph, Guelph, ON. 146

170 Tble 4.2 Disese incidence of bcteril speck on non-fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes listed in Tble 4.1, Serende Mx (B. subtilis QST713) or Promix PGX (B. subtilis MBI600). Endophytes were inoculted following the Vlenzuel-Soto et l. (2010) method using solution of ech endophyte with A600=1.000 then diluted 1:10 in 10 mm MgCl 2, except for MBI600 which ws included in the potting mix (1 x 10 7 CFU/mL) nd QST713 which ws pplied t 1 x 10 6 CFU /ml. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto t 20 dys fter seeding, nd disese incidence ssessed t seven dys post inocultion. Disese ws ssessed on ll leflets of ech plnt. Disese incidence (lesions/cm 2 ) MgCl 2 Endophyte Endophyte treted sem b No. Trils No. Reps R1 2.4 c R R R R R R R R R R R R R R R R R R R G G G G G G G MBI600 d QST713 d R4 ws not tested due to problems with contmintion. For endophytes R9, R17, R19, R20, R21, MBI600, nd QST713 one tril ws completed with plnts growing in Promix potting mix nd not Ffrd germintion mix. b sem = stndrd error of the men for ll ls mens in the sme column. The sem for MBI600 nd QST713 for the control tretment ws This vlue is for the ls mens clculted from log trnsformtion. The sem differs from the inoculted tretments due to missing plots. c Mens in the sme row followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from different trils ws pooled together becuse ANOVA showed no tretment x tril interction. d Dt in these rows ws trnsformed using log trnsformtion, the bck trnsformed mens re shown here. 147

171 Tble 4.3 Growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of non-fertilized tomto cv. TSH4 following inocultion with the endophytes listed in Tble 4.1, Serende Mx (B. subtilis QST713) or Promix PGX (B. subtilis MBI600). Endophytes were inoculted following the Vlenzuel-Soto et l. (2010) method using solution of ech endophyte with A600=1.000 tht ws diluted 1:10 in10 mm MgCl 2, except for MBI600 which ws included in the potting mix (1 x 10 7 CFU/mL) nd QST713 which ws pplied t 1 x 10 6 CFU/mL. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were ssessed t 27 dys fter seeding on the sme dy s ssessment of disese incidence. Endophyte Reltive Chlorophyll Height (cm) Dry weight (mg/plnt) No. No. Folige Roots Trils Reps MgCl2 Endophyte sem b MgCl2 Endophyte sem MgCl2 Endophyte sem MgCl2 Endophyte sem treted treted treted treted R c R R R R R R R R R R R R R R R R R R R G G G G G G G MBI b QST R4 ws not tested due to problems with contmintion. For endophytes R9, R17, R19, R20, R21, MBI600, nd QST713 one tril ws completed with plnts growing in Promix potting mix nd not Ffrd germintion mix. 148

172 b sem = stndrd error of the men for ll ls mens in the sme column. The sem for MBI600 nd QST713 for the control tretment ws 1.12 for reltive chlorophyll, 0.34 for height, for folir dry weight, nd for root dry weight. c Mens in the sme row nd group followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from different trils ws pooled together becuse ANOVA showed no tretment x tril interction. There ws significnt tretment x tril interction for folir dry weight for MBI600 nd QST713 but there ws no difference mong these tretments nd the control in either tril nd so the pooled results re presented. 149

173 Tble 4.4 Disese incidence of bcteril speck on non-fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes R9, R17, R19, R20 nd R21. Endophytes were inoculted using the seed sok + seed drench + seedling drench inocultion method (A600=1.000 then diluted 1:10 in10 mm MgCl 2 ). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with Pst t 20 dys fter seeding, nd disese incidence ssessed t seven dys post inocultion on ll leflets of ech plnt. Disese incidence (lesions/cm 2 ) Endophyte MgCl 2 Endophyte treted sem No. Trils No. Reps R9 7.5 b 4.4 b R R b R R R b sem = stndrd error of the men for ll ls mens in the sme row. The sem for R9 for the control tretment ws 0.39 nd for R b Mens in the sme row nd group followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from different trils ws pooled together becuse ANOVA showed no tretment x tril interction. There ws significnt tretment x tril interction for disese incidence for R9 nd results re presented seprtely due to different tretment response in ech tril. In one tril, R17 ws grown on 0.1 tryptic soy gr (TSA) insted of TSA. 150

174 Tble 4.5 Growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of non-fertilized tomto cv. TSH4 following inocultion with the endophytes R9, R17, R19, R20 nd R21. Endophytes were inoculted using the seed sok + seed drench + seedling drench inocultion method (A600=1.000 diluted by fctor of 10 in10 mm MgCl 2 ). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were ssessed t 27 dys fter seeding on the sme dy s ssessment of disese incidence. Dry weight (mg/plnt) Reltive Chlorophyll Height (cm) Folige Roots MgCl 2 Endophyte MgCl 2 Endophyte MgCl 2 Endophyte MgCl 2 Endophyte No. No. Endophyte treted sem treted sem treted sem treted sem Trils Reps R b R b R R R sem = stndrd error of the men for ll ls mens in the sme column. The sem for R9 for the control tretment ws 0.15 for reltive chlorophyll nd 0.51 for plnt height, nd for R19 for the control tretment ws 0.96 for reltive chlorophyll, 0.35 for plnt height, for folir dry weight, nd for dry root weight. b Mens in the sme row nd group followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from different trils ws pooled together when ANOVA showed no tretment x tril interction. 151

175 Tble 4.6 In vitro inhibition of P. syringe pv. tomto (Pst) strin DC06T2-4 by co-incubtion with endophytes R17 nd R21 using cross streking ssy. Ech endophyte ws streked in single line cross the centre of plte of tryptic soy gr nd incubted for two dys. Pst ws then streked in stright line from the edge of the plte to the endophyte strek t 90 ngle. Pltes were ssessed two dys fter streking Pst. Endophyte Zone of Inhibition (mm) Strek width reduction (%) Control 0.0 b b 12.0 c R R b 46.5 b sem c Percent reduction in Pst strek width t the rim end nd the centre of the plte where the strek stopped. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. c sem = stndrd error of the men for ll ls mens in the sme column. 152

176 Tble 4.7 In vitro inhibition of P. syringe pv. tomto (Pst) strin DC06T2-4 by co-incubtion with endophytes R17 nd R21 using n gr overly ssy. The overly ws tryptic soy gr (TSA) (1.50% gr) (50 C tht ws spiked with 0.1 ml of 2 x 10 7 CFU/mL Pst. This ws poured over 2-cm dimeter circle of endophyte tht hd grown for 48 hours on hrd TSA gr bse (1.50% gr), incubted for 48 hours, nd ssessed for the presence of zone of inhibition. 1.50% gr bse with 1.50% gr overly with Pst Zone of Inhibition (mm) endophyte Agr only Agr only 0 Agr only Pst 0 R17 Agr only 0 R17 Pst 8 R21 Agr only 0 R21 Pst 0 sem b 0.2 Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with eight replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 153

177 Tble 4.8 Disese incidence of bcteril speck nd growth prmeters (reltive chlorophyll, plnt height, nd root nd folige weight) of fertilized tomto cv. TSH4 following inocultion with buffer or the endophytes R17 nd R21. Plnts were fertilized 10, 15 nd 21 dys fter seeding (DAS) with 20 (three experiments) or 80 ml (one experiment) of 1.26 g/l micronutrients fertilizer solution. Endophytes were inoculted using the seed sok + seed drench + seedling drench inocultion method t concentrtions of 1 x 10 7 CFU/ml (R17) or 1 x 10 8 CFU/ml (R21). Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS. The incidence of bcteril speck lesions on ll leflets on the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst. Disese incidence Reltive Dry weight (mg/plnt) Tretment (lesions/cm 2 ) chlorophyll Height (cm) Folige Roots MgCl R b b R b b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with four replictions (two trils) or 10 replictions (one tril) of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 154

178 Tble 4.9 The effect of seed sok, seed drench nd/or seedling drench ppliction methods of R17 nd R21 on disese incidence of bcteril speck nd growth prmeters of fertilized tomto cv. TSH4. Plnts were inoculted with the seed sok, seed drench, seedling drench, seed sok + seed drench or seed sok + seed drench + seedling drench methods using 1 x 10 7 CFU/ml (R17) or 1 x 10 8 CFU/ml (R21) in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15 nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS. The incidence of bcteril speck lesions on ll leflets of the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst. Seed sok tretment (0 DAS) Seed drench tretment (0 DAS) Seedling drench tretment (10 DAS) Disese incidence (lesions/cm 2 ) Reltive chlorophyll Height (cm) Dry weight (mg/plnt) Folige Roots MgCl 2 MgCl 2 MgCl R17 MgCl 2 MgCl b MgCl 2 R17 MgCl b MgCl 2 MgCl 2 R b R17 R17 MgCl b R17 R17 R b R21 MgCl 2 MgCl b MgCl 2 R21 MgCl b MgCl 2 MgCl 2 R b R21 R21 MgCl b R21 R21 R b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Dry weight for folige nd root weight is from two independent trils with five replictions of ech tretment becuse smples from one tril were destroyed. b sem = stndrd error of the men for ll ls mens in the sme column. 155

179 Tble 4.10 The effect of rw nd pelleted seed with seed sok nd/or seed drench ppliction methods of R17 tretment on disese incidence of bcteril speck nd growth prmeters of fertilized tomto cv. TSH4. Plnts were inoculted using the seed sok, seed drench, or seed sok + seed drench method using 1 x 10 7 CFU/ml in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. Plnts were inoculted with P. syringe pv. tomto (Pst) t 20 DAS. The incidence of bcteril speck lesions on ll leflets of the second nd third youngest leves, reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were determined t five dys post inocultion with Pst. Seed sok tretment (0 DAS) Seed drench tretment (0 DAS) Disese incidence (lesions/cm 2 ) Reltive chlorophyll Height (cm) Dry weight (mg/plnt) Folige Roots Seed type MgCl 2 MgCl 2 Rw MgCl 2 MgCl 2 Pellet 2.1 b R17 MgCl 2 Rw 1.7 bc R17 MgCl 2 Pellet 1.6 bc R17 R17 Rw 1.5 c R17 R17 Pellet 1.5 c MgCl 2 R17 Rw 1.5 c MgCl 2 R17 Pellet 1.4 c sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with seven replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 156

180 Disese Incidence (lesions/cm 2 ) ) b) b Reltive chlorophyll b 0 H5108 H9553 TSH33 TSH4 0 H5108 TSH33 TSH4 Cultivr Cultivr 10 Root dry weight (mg/plnt) c) d) Height (cm) H5108 H9553 TSH33 TSH4 0 H5108 H9553 TSH33 TSH4 Cultivr Cultivr e) Folir dry weight (mg/plnt) H5108 H9553 TSH33 TSH4 Cultivr Figure 4.1 Comprison of four commercil processing tomto cultivrs for the effect of R17 seed drench tretment on the ) incidence of bcteril speck lesions, b) reltive chlorophyll, c) plnt height, d) dry root weight, nd e) folir dry weight. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench t 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with P. syringe pv. tomto t 20 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves. Dt for ech vrible is shown for the nontreted control ( ) 157

181 nd R17 ( ). Errors brs represent stndrd error of the men. Brs with the sme letter for the sme cultivr re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with six replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Results for cv. H2401 (most vribles) nd cv. H9553 (reltive chlorophyll) were inconsistent nd re presented in Tble A

182 Tble 4.11 Effect of different doses of R17 s seed drench tretment on the incidence of bcteril speck disese incidence nd growth prmeters (reltive chlorophyll, plnt height, folir dry weight, nd root dry weight) of fertilized tomto cv. TSH4. R17 ws pplied s seed drench t zero dys fter seeding (DAS) t 0, 1x10 5, 1x10 6, 1x10 7, 1x10 8 or 1x10 9 cfu/ml in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were fertilized 10, 15 nd 21 DAS with 80 ml of micronutrients fertilizer solution mixed t concentrtion of 1.26 g/l. Plnts were inoculted with P. syringe pv. tomto t 20 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves. R17 concentrtion (cfu/ml) Vrible 0 1x10 5 1x10 6 1x10 7 1x10 8 1x10 9 Regression eqution R 2 P sem Disese incidence y = x < (lesions/cm 2 ) 0.01x 2 Reltive y = x chlorophyll Height (cm) y = x Folir dry weight y = x (mg/plnt) Root dry weight (mg/plnt) y = x Regression nlysis ws completed in SAS proc reg nd proc glm using dt from three independent tril with six replictions per tril. X = LOG10 (R17 concentrtion). 159

183 Disese Incidence (lesions/cm 2 ) ) b) b b Reltive chlorophyll b Timing (dys post R17 tretment) Timing (dys post R17 tretment) Height (cm) Folir dry weight (mg/plnt) c) d) Timing (dys post R17 tretment) Pst inocultion timing (# dys fter R17 ppliction) e) Root dry weight (mg/plnt) Pst inocultion timing (# dys fter R17 ppliction) b Figure 4.2 The durtion of the plnt response to R17 ( ) or10 mm MgCl 2 ( ) seed drench tretment s mesured by the ) incidence of bcteril speck lesions, b) reltive chlorophyll, c) plnt height, d) dry root weight, nd e) folir dry weight in fertilized tomto cv. TSH4. Plnts were fertilized 10, 15, 21 nd 26 DAS (plnts inoculted with P. syringe pv. tomto t 25 dys fter seeding (DAS) only) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with Pst 15,

184 or 25 DAS, nd disese incidence ssessed t five dys post inocultion on ll leflets of the second nd third youngest leves. Dt points with the sme letter t the sme time point re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with six replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. Error brs represent stndrd error of the men. 161

185 Tble 4.12 The effect of R17 seed drench tretment on the incidence of bcteril speck lesions, P. syringe pv. tomto (Pst) popultion (LOG CFU) per cm 2 nd Pst popultion (LOG CFU) per lesion on terminl leflets of the third youngest leves of fertilized tomto cv. TSH4. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of micronutrients fertilizer solution mixed t concentrtion of 1.26 g/l. R17 ws pplied s seed drench 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Plnts were inoculted with Pst 20 DAS, nd disese incidence ssessed t five dys post inocultion. Plnts receiving 10 mm MgCl 2 were used s control. Tretment Disese incidence (lesions/cm 2 ) Popultion/cm 2 (LOG10 CFU) Popultion/lesion (LOG10 CFU) MgCl b R b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with three (two trils) or two (one tril) replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column on LOG10 scle. 162

186 Disese Incidence (lesions/cm 2 ) ) b Lesion size (mm 2 ) b) b 0.0 Control R Control R17 c) d) 8.00 Lesion circumference (mm 2 ) b Lesion surfce re (% of lef surfce) Control R Control R17 Figure 4.3 The effect of R17 seed drench tretment on ) the incidence of bcteril speck symptoms, b) men lesion size, c) men lesion circumference, nd d) percent lef re with lesions on the terminl leflet of the third youngest lef of fertilized tomto cv. TSH4. Disese incidence nd men lesion size of 10 to 15 lesions is shown for the nontreted control ( ) nd R17 ( ). Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench 0 DAS t 1 x

187 CFU/mL in10 mm MgCl 2. Control plnts received 10 mm MgCl 2. Plnts were inoculted with P. syringe pv. tomto 20 DAS, nd disese incidence ssessed t five dys post inocultion. Errors brs represent stndrd error of the men. Brs with the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with seven (experiment 1) nd six (experiment 2) replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. 164

188 Tble 4.13 Comprison of R17 seed drench tretments with wt nd rifmpicin-resistnt strins on disese incidence of bcteril speck nd growth prmeters of fertilzed tomto cv. TSH4. Plnts were fertilized 10, 15, nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17 ws pplied s seed drench t 0 DAS t 1 x 10 7 CFU/mL in10 mm MgCl 2. Plnts receiving 10 mm MgCl 2 were used s control. Plnts were inoculted with P. syringe pv. tomto (Pst) 20 DAS, nd disese incidence ssessed t five dys post inocultion (DPI) on ll leflets of the second nd third youngest leves. The reltive chlorophyll content, plnt height, dry root weight nd folir dry weight were lso determined t five DPI. Tretment Disese incidence Reltive Height Dry weight (mg/plnt) (lesions/cm 2 ) chlorophyll (cm) Folige Roots MgCl R17-wt 2.6 b R17-RfpA 2.9 b R17-RfpB 2.7 b R17-RfpC 2.7 b sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils for with five or six replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. For disese incidence dt, dt ws log trnsformed to meet ssumptions of ANOVA nd the bck trnsformed mens re shown. One outlier ws removed for the disese incidence dt. b sem = stndrd error of the men for ll ls mens in the sme column. 165

189 Tble 4.14 Popultions of rifmpicin resistnt R17 mutnt R17-RfpC in leves, roots nd rhizosphere of tomto cv. TSH4. Plnts were fertilized t 10, 15 nd 21 dys fter seeding (DAS) with 80 ml of 1.26 g/l micronutrients fertilizer solution. R17-RfpC ws pplied s seed drench t 0 DAS t 1 x 107 CFU/mL in10 mm MgCl2. Plnts receiving 10 mm MgCl2 were used s control. R17-RfpC popultions in rhizosphere growing medi, roots nd cotyledon leves t 10 DAS or the rhizosphere growing medi, roots nd terminl leflet on the second nd third youngest leves t 25 DAS. Smples were from 10 (10 DAS) or five (25 DAS) plnts per replicte. Leves (CFU/g fresh tissue) Roots (CFU/g fresh tissue) Rhizosphere (CFU/g dry soil) Tretment 10 DAS 25 DAS 10 DAS 25 DAS 10 DAS 25 DAS MgCl 2 0 b 0 0 b 0 b 2.5 b 2.8 b R17- RfpC x x x 10 6 sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from three independent trils with four replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men (LOG10) for ll ls mens in the sme row. 166

190 Figure 4.4 Neighbour joining tree of prtil 16S sequences (1160 bp) from representtive members of the E. cloce complex, other Enterobcter sp., closely relted Enterobctere, nd R

191 Figure 4.5 Neighbour joining tree of prtil rpob sequences (934 bp) from representtive members of the E. cloce complex, other Enterobcter sp., closely relted Enterobctere, nd R

192 Figure 4.6 Neighbour joining tree of prtil 16S sequences (1234 bp) from representtive members of the B. cereus group, other Bcillus sp., nd R

193 Figure 4.7 Neighbour joining tree of prtil gyrb sequences (696 bp) from representtive members of the B. cereus group, other Bcillus sp., nd R

194 4.4 Discussion Screening bcteril endophytes from tomto resulted in the isoltion of two strins with puttive induce resistnce ginst Pst. The most effective ws B. mycoides/weihenstephnensis strin R17, which could be pplied to the soil resulting in reduction of the incidence of bcteril speck in tomtoes in growth room conditions by up to 51%. Mny species of Bcillus hve been shown to induce resistnce in plnts, including B. mycoides (Choudhry & Johri, 2009). In tomto, bcteril endophytes tht hve induced resistnce ginst Pst re B. pumilus nd B. myloquefciens tht reduced lesion incidence by 56% nd 62%, respectively (Lnn-Filho et l., 2017), B. pumilus SE34 nd B. myloliquifciens IN937 tht reduced lesion incidence by 63% nd 49%, respectively (Ji et l., 2006), nd B. megterium tht reduced lesion incidence by 54% (Brretti et l., 2009). These levels of induced resistnce re similr or slightly higher thn tht chieved in this study. Mny bcteril endophytes cn ffect plnt growth in tomto (Pilly & Nowk, 1997, Amresn et l., 2012, Brretti et l., 2009, Lnn-Filho et l., 2017), nd ppliction of strin R17 ffected plnt growth bsed on increses in folir dry weight in two experiments, reductions in plnt height in one experiment, chnges in reltive chlorophyll in two experiments nd increses in root dry weight in one experiment. However, these effects were inconsistent mong experiments, nd thus no firm conclusions bout growth promotion or inhibition cn be mde. Bcteril endophytes in tomto re not lwys ssocited with plnt growth promotion, such s B. pumilus nd B. myloquefciens, which reduced plnt height nd totl dry weight in greenhouse ssys (Lnn-Filho et l., 2017). As this study demonstrtes, identifiction of n endophyte with puttive induced resistnce is only the strting point. The effectiveness of strin R17 ws influenced by number of fctors including the inocultion timing, inocultion method, nd bcteril concentrtion nd host genotype. This shows tht using n endophyte is dependent on number of fctors relted to both the bcteril endophyte nd its host Isoltion of endophytes from domestic nd wild tomto Screening for phenotypiclly diverse endophytes from roots of the dwrf cv. Tiny Tim nd the commercil processing cv. TSH4 yielded only two colony types, with some vrition in colony size within ech group. However, higher diversity of colonies ws expected s Xi et l. (2015) obtined 32 endophyte species from n unspecified tomto cultivr nd Upreti nd Thoms (2015) found 27 different endophyte species from cvs. Ark Abh nd Ark Viks. Root endophytes typiclly infect roots from the soil, nd soil trits cn gretly ffect root endophyte popultions (Long et l., 2010). The low endophyte diversity in this study my be due to the use of field soil from n griculturl reserch rnge tht is 171

195 hevily mnged resulting in different totl soil crbon, soil nitrogen, nd C:N rtio thn found in nturl system, which re key elements ffecting microbil diversity (Trivedi et l., 2016). Another fctor ws tht the soil ws psteurized to obtin n internl temperture of 60 C for 30 min, which ws done s the mjority of emerging cv. Tiny Tim seedlings died with symptoms of dmping off in the growth room grown in unpsteurized soil. Psteuriztion will reduce inoculum of soil borne plnt pthogens nd other microbes in the soil, selecting for het tolernt bcteri, such s Bcillus spp., which re ble to withstnd tempertures of up to 100 C (Bker, 1962, Bker & Olsen, 1964), Dwson et l. (1965). However, soil psteuriztion does not explin the low diversity of endophytes obtined from cv. TSH4 roots growing in the sme soil directly in the field. Another possible explntion for low diversity is tht some endophyte species did not survive tissue surfce steriliztion during isoltion. Perhps the method of Upreti nd Thoms (2015) to use N 2 S 2 O 3 to remove residues of the sterilizer, chlormine, from root tissue prior to mcertion would hve helped to remove residul blech in the current study. In ddition, seril dilutions nd spred plting ws used, which is commonly used for recovery of culturble endophytes from plnts (Rsche et l., 2006, Goodwin & Go, 2017, Mhffee & Kloepper, 1997). However, Thoms et l. (2012) demonstrted tht spred plting of endophytes cn be vrible resulting in reduced nd inconsistent recovery compred to spotting dilutions nd then tilting the plte to spred the solution without spreder. In order to increse the diversity of bcteril endophytes, isoltions were lso mde from the wild tomto species S. rcnum, S. chmieleskii nd S. cheesmnie grown in non-cultivted soil (i.e., grss buffer strip). Non-griculturl soils with perennil plnts my hve more totl soil crbon nd nitrogen with different C:N which cn increse bcteril diversity nd bundnce (Trivedi et l., 2016). While the sme colony types were obtined s with cvs. Tiny Tim nd TSH4, there were lso other colony phenotypes. While this study ws not designed to evlute the effect of host species or soil on bcteril endophyte diversity of tomto, this indictes tht more diverse tomto genotypes nd soil types provide greter chnce to obtin different bcteril endophytes. This is consistent with reports of orgniclly cultivted soils hving higher diversity of endophytes in corn, tomto, melon, nd pepper compred to soils in conventionl griculturl production (Xi et l., 2015), nd reports of differences in plnt physiology, such s ET production, ffecting the bcteril endophyte community in tobcco roots (Long et l., 2008) Screening for ctivity of endophytes for induced resistnce nd plnt growth promotion Bcteril endophytes isolted from domesticted nd wild tomto were screened for induced resistnce nd plnt growth promotion in the commercil processing tomto cv. TSH4. In ddition, collection of endophytes known to induce resistnce ginst Colletotrichum orbiculre in N. benthmin 172

196 (Goodwin & Go, 2017) ws included, s well s commercil formultions of B. subtilis QST713 nd B. subtilis MBI600. B. subtilis QST713 is mrketed s Serende Mx nd B. subtilis MBI600 is mrketed s Promix PGX for disese suppression nd plnt growth promotion (Boriss, 2016, Nv-Diz, 2006). In initil screening tests, no response for disese or plnt growth promotion vribles ws observed for ny endophytes or the two commercil products. It is possible tht the endophytes effective ginst fungl pthogen of N. benthmin will not function ginst bcteril pthogen of tomto. For Serende Mx, folir pplictions of the bcterium for bcteril spot nd speck control in tomto were inconsistent (Roberts et l., 2008, Truemn, 2015), nd this orgnism my ct more by directly ttcking pthogens rther thn s n elicitor of resistnce. For Promix PGX, it my lso ct primrily by directly ttcking pthogens s it is sold s prt of growing medium nd is described s controlling root infections by cused by Fusrium, Pythium nd Rhizoctoni. Growth promotion by B. subtilis MBI600 ws observed in tomto in field experiments but not greenhouse experiments, nd thus its response my lso vry with environmentl conditions (Nv-Diz, 2006). Although there were no significnt differences, some of the endophytes in the initil screenings ppered promising despite high level of vribility in the incidence of bcteril speck symptoms nd plnt growth. One source of vribility could hve been the endophyte inocultion method which involved dipping extrcted bre root seedlings in the bcteril suspension nine DAS. This my hve triggered mechnicl stress response which could ffect endophyte coloniztion or the host response. Mechnicl stress in plnts is ssocited with increses in ET biosynthesis in plnts (Druege, 2006), nd chnges in ET sttus ffect the plnt defense response (Pieterse et l., 2009) nd endophyte coloniztion (Iniguez et l., 2005). Five endophytes with the gretest numeric reductions in disese were selected for further evlution by soking the seed in the bcteri nd then pplying the bcteril solution s soil drench fter seeding nd gin 10 DAS (seed sok + seed drench + seedling drench) without removl of seedlings from the growing medium. Using the modified endophyte inocultion technique, endophytes R17 from S. rcnum nd R21 from S. cheesmnie reduced the incidence of bcteril speck symptoms, lthough it did not result in ny ny consistent ltertions in plnt growth. Inocultion of tomto seeds nd seedlings with bcteril endophytes from oilseed rpe nd grpe, wtermelon nd ppy, respectively, reduced the severity of wilt symptoms cused by F. oxysporum f. sp. lycopersici (Nejd & Johnson, 2000) or R. solncerum (Thoms & Upreti, 2014). While the reduction in disese incidence ws greter with the root dip method compred to the seed sok + seed drench + seedling drench method (45% versus 27% for R17, nd 42% versus 27% for R21), the vribility ws less with the ltter method. The root dip method my hve been more effective becuse of better penetrtion into the roots. Zkri et l. (2008) nd Bressn nd Borges (2004) 173

197 demonstrted tht higher bcteril endophyte densities re chieved with bre root inocultion compred to rhizosphere inocultion in rice, nd root pruning inocultion compred to seed, soil drench, folir, nd seed + soil drench inocultion in mize, respectively, possibly becuse of incresed entry vi wounding. Another fctor tht my hve mde the results less vrible with the seed sok + seed drench + seedling drench compred to the root dip method is tht it llowed for 20 dys insted of 10 dys for endophyte coloniztion prior to chllenge with Pst. In other studies of bcteril endophytes on tomto, Pst chllenge occurred 10 dys fter 24 h seed sok endophyte inocultion (Lnn-Filho et l., 2017), but 2 weeks fter seedling drench endophyte inocultion (Fujit et l., 2017) nd up to 7 weeks fter seed sok + seedling drench endophyte inocultion (Ji et l., 2006) Identifiction of strins R17 nd R21 Sequencing the 16S rdna region of endophyte R21 resulted in the closest mtches being E. ludwigii, E. cloce, nd P. gglomerns. One concern with 16S sequence dt is tht there re very lrge numbers of such sequences submitted to GenBnk resulting in mny unvlidted 16S rdna sequences with incorrect identifictions (Woo et l., 2008). Also, 16S sequencing my not lwys provide enough informtion to identify mny bcteril species (Woo et l., 2008). The rpob gene encoding the RNA polymerse bet subunit ws lso sequenced for R21 s it is considered more informtive sequence for identifiction of species within the E. cloce complex (Puw et l., 2008, Yousf et l., 2011). The rpob sequence nlysis showed tht R21 is most closely relted to ll E. ludwigii nd some E. cloce isoltes entered in GenBnk, which re both members of the E. cloce complex. Enterobcter species fll within clss Gmmproteobcteri of the Proteobcteri, which is common phylum contining bcteril endophytes (Bulgrelli et l., 2013, Rosenblueth & Mrtinez-Romero, 2006, Hrdoim et l., 2008, Hllmnn & Berg, 2006). E. ludwigii is reltively new species, being first described by Hoffmnn et l. (2005), nd ws formerly known s the genovr cluster V in the E. cloce complex. It is ssocited with humn clinicl specimens s well s plnts (Hoffmnn et l., 2005). In plnts, E. ludwigii ws isolted from the rhizosphere of Lolium perenne nd provided plnt growth promotion fter seed inocultion nd lso hd in vitro ntgonistic effects ginst F. solni (Shoebitz et l., 2009), nd ws isolted s n endophyte from roots of tomto cv. Ark Anny nd hd ntgonistic ctivity ginst R. solncerum (Upreti & Thoms, 2015) E. ludwigii ws lso isolted s n endophyte in roots nd shoots of Itlin ryegrss nd birdsfoot trefoil nd ws ble to degrde hydrocrbons fter coloniztion of the rhizosphere nd plnts tissues of Itlin ryegrss, birdsfoot trefoil, nd lflf growing in soil spiked with diesel fuel (Yousf et l., 2011). E. cloce is ssocited with bulb rot of onion (Schroeder et l., 2009) nd mulberry (Zhu et l., 2010). However, E. cloce cn lso be beneficil to plnts, nd it is possible tht E. cloce isoltes described before Hoffmnn et l. (2005) my now be clssified s E. ludwigii. E. 174

198 cloce ws isolted from the rhizosphere of cotton, nd root inocultion resulted in incresed biomss of tomto seedlings (Myk et l., 2001), nd n endophytic E. cloce from roots of tomto cv. Ark Abh exhibited ntgonism ginst the cusl gent of bcteril wilt, R. solncerum, in vitro (Upreti & Thoms, 2015). Thus, it is not surprising tht E. ludwigii could be isolted S. rcnum nd tht it could induce resistnce ginst Pst when inoculted into domesticted tomto. Sequencing the 16S rdna region of endophyte R17 gve the closest sequence mtches to B. mycoides nd B. weihenstephnensis, which re both members of the B. cereus group. For the resons described previously for R21, dditionl sequencing ws done to identify R17 to species. In this cse, the gene chosen ws gyrb encoding the DNA gyrse B subunit, which is considered reltively effective t distinguishing between species of the B. cereus group (L Duc et l., 2004, Wng et l., 2007). The gyrb sequencing confirmed tht R17 is very closely relted to both B. mycoides nd B. weihenstephnensis but did not provide ny evidence tht R17 is more closely relted to one species thn the other. The cretion of B. weihenstephnensis s seprte species within the B. cereus group is reltively recent nd is bsed on clustering of psychrotolernt strins of Bcillus sp., which cn grow t 4 to 7 C nd hd 2 nt differences in the 16S rdna region (Lechner et l., 1998). The sequence including the 2 nt of R17 mtches tht of the psychrotolernt B. weihenstephnensis type strin 10204, nd R17 does grow t 4 C. More recently, severl B. mycoides nd B. cereus strins confirmed to grow t 7 C nd contining specific signture sequences in the 16S rrna, cspa, glpf, gmk, purh nd tpi chrcteristic of B. weihenstephnensis, were proposed for reclssifiction to B. weihenstephnensis (Soufine & Côté, 2013). However, bsed on whole-genome sequence-bsed Genome BLAST Distnce Phylogeny, Liu et l. (2015) proposed tht B. mycoides nd B. weihenstephnensis re not geneticlly distinct nd should be considered the sme species. Another reson for questioning the cretion of B. weihenstephnensis ws tht group of bcteril strins isolted from the tomto rhizosphere were clssified s B. weihenstephnensis bsed on the 16S sequence nd multi-locus sequence typing of seven housekeeping genes but showed mesophilic growth (Hollensteiner et l., 2016). Thus, whether B. mycoides nd B. weihenstephnensis should be considered seprte species remins uncler, nd so R17 ws clssified s B. mycoides/weihenstephnensis. The colony morphology of B. mycoides is described s chins of cells forming curving rdil filments on gr, lso known s rhizoid or mycoidl growth (Di Frnco et l., 2002). Lechner et l. (1998) stted tht B. mycoides hd rhizoidl growth nd B. weihenstephnensis hd non-rhizoidl growth. However, isoltes of B. mycoides from pet bog in Germny hd motile cells nd non-rhizoid colony morphology (von Wintzingerode et l., 1997), nd isoltes of B. weihenstephnensis from the tomto rhizosphere hd two colony morphologies with one type being rhizoidl nd similr to B. mycoides nd the other type being non-rhizoidl hving circulr to wekly irregulr colonies with entire or undulte 175

199 edges similr to B. thuriengiensis (Hollensteiner et l., 2016). Psychrotolernt B. weihenstephnensis isoltes from soil in Denmrk hd colony morphology like tht of B. weihenstephnensis insted of B. mycoides (Thorsen et l., 2006). Logn nd De Vos (2009) stted tht the bility to form rhizoid colonies by B. mycoides my be lost, indicting tht it is n unrelible txonomic chrcteristic, nd Soufine nd Côté (2013) speculted tht genes responsible for colony morphology in B. mycoides my be trnsferred through horizontl gene trnsfer. Strin R17 showed circulr to wekly irregulr colonies with entire or undulte edges nd ws motile in TSB. B. mycoides is widespred in the environment, including the rhizosphere (Buyer, 1995) nd phyllosphere (Brgbus et l., 2002), wheres B. weihenstephnensis is described from diry products nd refrigerted food (Soufine & Côté, 2013, Meer et l., 1991, Lrsen & Jørgensen, 1997) but more recently from soil (Hollensteiner et l., 2016). This is the first report of B. mycoides or B. weihenstephnensis s n endophyte, nd it my be tht motility nd colony morphology is ffected by source mteril. Future reserch needs to determine if B. mycoides nd B. weihenstephnensis isoltes colonizing plnt tissues (i.e., ded sphgnum moss in bog or live plnt roots) differ from those coming from other environments. Bcillus sp. fll within clss Bcilli within phylum Firmicutes, which is common phylum contining bcteril endophytes tht cn induce disese resistnce (Bulgrelli et l., 2013, Rosenblueth & Mrtinez-Romero, 2006, Hrdoim et l., 2008, Hllmnn & Berg, 2006). B. mycoides isolte J, which ws isolted from the phyllosphere of sugr beet, reduced symptoms of Cercospor lef spot (C. beticol) in sugr beet by pproximtely 70% when pplied to leves, but effects on growth promotion were not reported (Brgbus et l., 2004). In tomto, commercil product contining B. mycoides isolte J, mrketed s LifeGrd, ws recently registered in Cnd for folir ppliction to suppress Xe, A. solni, nd P. infestns, nd prtilly suppress Pst (Brgbus et l., 2003, Brgbus et l., 2002, FRAC, 2017, Certis, 2017b). Gene expression nlysis ws not completed but form of induced resistnce is implicted due to incresed ctivity of oxidtive burst, chitinse nd B-glucnse ws reported for the isolte on sugr beets (Brgbus et l., 2003). For B. weihenstephnensis, three strins from ginseng rhizosphere reduced the severity of P. cctorum when pplied to soil, but effects on growth promotion were not evluted (Lee et l., 2015). Similrly, some isoltes of B. weihenstephnensis from the tomto rhizosphere were ntgonistic ginst V. dhlie nd Verticillium longisporum in vitro, but effects on induced resistnce nd plnt growth were not evluted (Hollensteiner et l., 2016). Reports of Bcillus sp. implicted in induced resistnce in tomto include B. pumulis ginst Pst nd F. oxysporum f. sp. rdices-lycopersici (Lnn-Filho et l., 2017, Ji et l., 2006, Benhmou et l., 1998), B. myloliquefciens ginst Pst (Lnn-Filho et l., 2017, Ji et l., 2006) nd B. megterium ginst A. solni nd Pst (Brretti et l., 2009). None of these studies report positive effects of these endophytes on plnt growth, lthough these mesurements of plnt growth were only reported by Lnn-Filho et l. (2017) nd Brretti et l. 176

200 (2009). Thus, R17 belongs to genus of bcteri commonly ssocited with induced resistnce but not plnt growth promotion in tomto Optimiztion of bcteril endophyte tretment To increse the effectiveness of seed sok + seed drench + seedling drench ppliction of strins R17 nd R21, three pplictions of fertilizer insted of no fertilizer ws used with the tomto seedlings becuse low nitrogen in tissues ws reported to reduce constitutive levels of defense relted enzymes nd the induced level of peroxidse nd chitinse in A. thlin treted with the SAR-inducer ASM (Dietrich et l., 2004) nd lso reduced the effectiveness of mycorrhiz-induced resistnce by the rbusculr mycorrhizl fungus, Rhizophgus irregulris, ginst B. cinere in tomto (Snchez-Bel et l., 2016).With fertilized tomto seedlings, the disese reduction with R17 nd R21 ws 33% nd 38%, respectively, which ws higher thn the 27% reduction observed in non-fertilized plnts, indicting tht incresed control ws possible. However, comprison of the level of induced resistnce in non-fertilized versus fertilized tomtoes cnnot be mde becuse disese ssessments were mesured t five DPI in fertilized plnts insted of seven DPI in non-fertilized plnts. This ws done to reduce the vribility becuse it ws more ccurte to count lesions t five DPI becuse fewer lesions hd colesced, thus providing more ccurte number. However, pplictions of fertilizer did not ffect plnt growth promotion by either endophyte. Plnt height ws reduced with R17 nd R21 inocultion compred to the MgCl 2 control, lthough this ws not ssocited with reduction in folir or root biomss. Host fitness costs re most often ssocited with SAR becuse plnt resources re llocted immeditely to defense response (Wlters & Heil, 2007), s opposed to ISR, which is ssocited with priming of host defenses for pthogen ttck (vn Hulten et l., 2006, Conrth et l., 2006). However, Lnn-Filho et l. (2017) observed reductions in plnt height, totl dry weight, nd growth rte in five week old tomto grown from seed inoculted with endophytes B. pumilus nd B. myloliquefciens, but these effects were not s strong s similr effects cused by the ppliction of ASM. The current study ws only 3.5 weeks in durtion, which is short for mesuring growth rte, nd thus the longer term effects of R17 nd R21 were not evluted. The first spect exmined to increse the effectiveness of the endophytes ws to further modify the endophyte inocultion technique. For strin R17, the most effective reduction in disese incidence ws s seed sok or seed drench tretment compred to inocultion s seedling drench, seed sok + seed drench or seed sok + seed drench + seedling drench. Thus, it ppers tht multiple pplictions re not beneficil for inducing resistnce s well s ltter pplictions once the seedling hs developed. R17 my be better dpted to colonize tomto plnts t the time of seed germintion reltive to other developmentl stges. During seed germintion, the spermosphere, which is the environment surrounding the seed under 177

201 the influence of seed crbon deposition, releses root exudtes tht ttrct some microorgnisms, including Bcillus sp., within few hours of sowing (Nelson, 2004). Also, endophyte community diversity in other plnt species chnges with host developmentl stge (Andreote et l., 2010, Monteiro et l., 2011, Kuklinsky-Sobrl et l., 2004), which my be due to chnges in host defense mechnisms tht ffect coloniztion (Andreote et l., 2010) or other fctors, such s the level of soluble crbohydrtes, clcium nd phenolic compounds (Hunter et l., 2010) or strch content (Inceoglu et l., 2010). It my be tht R17 is better t responding to root exudtes during seed germintion (Budoin et l., 2002) or less ble to colonize tomto tissues during lter stges due to selection by the host for bcteri tht re required for growth during lter developmentl stges (Qio et l., 2017). Seed soking or seed drench pplictions in tomto using bcteril endophytes lso reduced the severity of Pst in other studies (Lnn- Filho et l., 2017, Ji et l., 2006). However, soil drench inocultion of one-week tomto seedlings with n endophytic Azospirillum sp. resulted in induced resistnce to Pst in tomto (Fujit et l., 2017), nd multiple pplictions of B. pumilus SE34 by seed tretment nd soil drenches to young tomto seedlings lso resulted in induced resistnce ginst Pst (Ji et l., 2006). There re no reports of induced resistnce by inocultion with bcteril endophytes pst the seedling stge (i.e. > 6 weeks). This indictes tht endophytes my differ s to which inocultion method is best, nd ech resercher must determine this empiriclly. Although the inocultion method ffected the level of disese incidence, there ws no effect on plnt growth using ny inocultion method. In contrst, strin R21 showed no significnt control of Pst incidence regrdless of the endophyte inocultion technique. The lck of efficcy of ll R21 tretments indicte tht the efficcy of R21 ws inconsistent even using the seed sok + seed drench + seedling drench technique where it ws effective in only one set of experiments. Hermn et l. (2008) lso reported inconsistent effects of the mixture of the PGPR B. subtilis GB03 nd endophyte B. myloliquefciens IN937 for Pst control in tomto, which ws ssocited with inconsistent induction of PR1 nd PR1b fter pthogen inocultion nd n increse in Pin2 expression beginning 12h prior to pthogen inocultion. The inconsistency justified discontinuing studies on strin R21. As ppliction to seeds ws most effective, but commercil tomto seeds re typiclly coted with proprietry mterils, such s combintions of cly nd snd nd other inter mterils to improve hndling nd ct s crriers for pesticide seed tretments (Sundstrom, 2002), the seed sok nd seed drench inocultion methods for R17 were evluted gin compring rw nd pelleted seed. No differences were observed. This ws expected becuse the seed cotings generlly contin inert mterils such s snd nd cly, which would not be expected to hve ntimicrobil properties, nd no known pesticides tht could diffuse with the root exudtes during the seed soking or when the seed is germinting in the soil nd negtively ffect bcteril popultions, including the R17 cells pplied to the 178

202 seed or soil. Other reports of microorgnisms in tomto using inocultion t the time of seedling do not specify the use of rw or pelleted seed (Lnn-Filho et l., 2017, Ji et l., 2006, Hermn et l., 2008), nd so this ppers to be the first study to show tht commercil seed coting does not ffect the bility of endophytes to reduce disese, t lest in tomto. There ws lso no effect of seed coting on plnt growth, which ws lso similr to previous experiments. Compring different doses of R17 using the seed drench method showed tht the reltionship between inoculum dose nd the level of disese reduction ws qudrtic rther thn liner, nd there ws no significnt reltionship for inocultion concentrtion nd ny of the plnt growth vribles mesured. In contrst, there ws significnt liner reltionship between the initil popultion density of the rhizobcteri nd endophyte B. pumilus SE34 nd rhizobcteri P. fluorescens 89B61 inoculums (rnging from 10 3 to 10 9 CFU/g soil mix) nd tomto seedling growth nd induced resistnce ginst lte blight disese (Yn et l., 2000). The optimum concentrtion of pproximtely 1 x 10 7 CFU/mL in this study ws generlly lower thn tht used in severl other studies of endopytes reducing disese in tomto. By comprison, 3 x 10 8 to 7 x 10 8 CFU/mL ws optimum for plnt growth promotion in tomto using the endophyte Pseudomons sp. PsJN (i.e., B. phytofirmns PsJN) pplied s seedling root sok in gnotobiotic system (Pilly & Nowk, 1997). For induced resistnce in tomto ginst Pst, Azospirillium sp. 510 ws pplied s seedling soil drench t 1 x 10 9 CFU/ml (Fujit et l., 2017), B. pumulis nd B. myloquefciens were pplied s seed sok t 1 x 10 8 CFU/ml (Lnn-Filho et l., 2017), nd B. pumilus SE34 nd B. myloliquifciens IN937 were pplied s seed tretment t 1 x 10 9 CFU/ml (Ji et l., 2006). However, (Ji et l., 2006) lso used B. pumilus SE34 nd B. myloliquifciens IN937 s seedling soil drench of tomto t 1 x 10 7 CFU/ml to induce resistnce ginst Pst, nd Li et l. (2016) found tht the reltively low concentrtion of 1.21 x 10 6 CFU/ml ws most effective for growth promotion by n endophytic Bcillus sp. pp02 in hybrid pennisetum when pplied s soil drench. Thus, the concentrtion used in this study is t lest within the rnge used by others. Most studies do not describe how the endophyte inoculum concentrtions were chosen. However, Pilly nd Nowk (1997) compred severl inoculum concentrtions of the well studied endophyte strin, Pseudomons sp. PsJN (i.e., B. phytofirmns PsJN) in tomto nd showed tht root surfce popultions incresed linerly with incresing inoculum densities between 10 7 to 10 8 CFU/ml, while root endophyte popultions did not increse linerly with inoculum concentrtion but were highest t pproximtely CFU/ml. Shoot endophyte popultion were not ffected by the inoculum concentrtion. Inoculum concentrtion my thus ply less of role in ltter stges s the endophyte invdes the plnt, nd other fctors become more importnt, which my explin why there ws limited rnge of popultions ( CFU/g tissue) inside roots nd shoots. The popultion size within tissues ws proposed to result from 179

203 limittions of nutrients nd microniches for the bcteri inside the plnt. The inoculum concentrtions tht best resulted in coloniztion of inner roots gve best plnt growth promotion, nd Pilly nd Nowk (1997) proposed stimultion threshold of the endophyte popultion ws needed for the plnt response. Thus, the optimum concentrtion for inocultion with bcteril endophytes ppers to be vrible nd my depend on the host, bcteril endophyte, inocultion method nd growing environment. A screening of five commercil processing tomto cultivrs reveled tht R17 hd some effects on prticulr prmeters of plnt growth for certin cultivrs unlike previous experiments. Further work is needed to discover why certin plnt growth prmeters were sometimes ffected by R17. Kloepper et l. (1989) stted tht inconsistencies in promoting plnt yield with PGPR results from the effects of mny spects of the environment on the interction between the introduced bcteri nd the plnt nd soil microflor. Thus, similr inconsistencies with host-endophyte interctions for plnt growth promotion my be t ply in this work. Among the tomto cultivrs, R17 cused reduction in bcteril speck symptoms in cv. TSH4, nd not in cvs. H5108, H9553, H2401 nd TSH33. The differentil response of the tomto cultivrs to inocultion with R17 nd chllenge with Pst is not surprising. The responses of A. thlin nd whet to induced resistnce by rhizobcteri re genotype dependent (Ton et l., 2001, Ton et l., 2002, Mketon et l., 2012). In tomto, growth promotion nd induced resistnce to B. cinere following inocultion with the rhizosphere fungi T. hrzinum T22 nd T. troviride P1 differed between two inbred processing tomto lines, the lndrce Corbrino, the dvnced breeding line SM26 nd the wild tomto S. hbrochites (Tucci et l., 2011). In the bove cses, the differences in response were relted to different expression of host defense genes. Thus, if induced resistnce is the mode of ction of R17, cv. TSH4 my hve stronger induction of defense genes in response to R17 thn the other cultivrs tested. Another possibility is tht cv. TSH4 is better dpted for coloniztion by R17. Endophyte coloniztion cn vry depending on tomto genotype. For exmple, more diverse popultion of endophytes colonize the bcteril wilt resistnt cv. Ark Abh compred to the susceptible cv. Ark Viks (Upreti & Thoms, 2015). Similrly, coloniztion of the spermosphere by 24 seed inoculted B. cereus strins differed mong inbred tomto lines (Simon et l., 2001). There is limited informtion vilble on the pedigrees of the cultivrs included in this study. For the Heinz cultivrs, there is possibility tht they shre some common ncestry; however, full informtion on their bckgrounds is not publiclly vilble (G. Collier, personl communiction). The responsive cv. TSH4 nd non-responsive cv. TSH33 re from Tomto Solutions ( nd re from different prents but no dditionl informtion on their bckgrounds is publiclly vilble (J. Dick, personl communiction). Since cv. TSH4 ws the only responsive cultivr identified in this study, it would be useful to know if it hs ny unique ncestors in its bckground tht llow for better coloniztion of R17, unique host defense response or both. 180

204 The durtion of the induced resistnce response in cv. TSH4 by R17 showed tht it declined from 15 to 20 DPT until there ws no disese reduction by 25 DPT. For induced resistnce ginst Pst in tomto, this is longer thn the 10 DPT produced by the endophytes, B. pumulis nd B. myloquefciens, (Lnn-Filho et l., 2017) but shorter thn the 7 weeks post tretment due to the endophyte, B. subtilis (Hermn et l., 2008). A fctor ffecting the results from 25 DPT in this work ws tht the host susceptibility to Pst progressively declined from 15 to 25 DPT, perhps due to ge-relted resistnce. Age-relted resistnce hs been described in mny plnt-pthogen interctions (Develey-Rivière & Glin, 2007). For Pst, it hs been noted in A. thlin nd is independent of ISR or SAR, but ws dependent upon SA (Kus et l., 2002). In tomto, this is the first report for Pst, but it hs been reported for C. michignensis subsp. michignensis (Shrbni et l., 2013), P. infestns (Shh et l., 2015) nd C. fulvum (Pnter et l., 2002). The decline in the durtion of the disese response by R17 could be due to the popultion of R17 diminishing over time, which ws demonstrted in the study of R17 coloniztion fter inocultion. Pilly nd Nowk (1997) proposed tht endophyte popultions need to rech stimultion threshold to chieve plnt response for improved growth, nd root coloniztion t 10 4 CFU/g fresh weight of cucumber by P. fluorescens G8-4 (89B61) ws ssocited with induced resistnce to C. orbiculre on leves t 14 dys fter emergence (Kloepper et l., 1992). However, the extent of plnt coloniztion required by endophytes to reduce disese hs not been well estblished (Hrdoim et l., 2015). For exmple, while seed inocultion of cucumbers with the PGPRs, S. mrcescens nd P. putid 89B-27, resulted in greter reduction in disese symptoms by C. orbiculre t the fifth lef stge compred to the first lef stge, the popultion of the bcteri colonizing plnt roots dropped from pproximtely 10 8 nd CFU/g fresh weight t seven dys fter plnting for 89B-27 nd , respectively, to pproximtely 10 3 CFU/g fresh weight t 28 dys fter plnting for both bcteri (Liu et l., 1995). As rhizosphere bcteri cn induce resistnce without coloniztion of internl tissues (Kloepper & Ryu, 2006, Bent, 2006), it is lso possible tht internl coloniztion is not even required by endophytes to induce resistnce. In the cse of R17, popultions declined over time both inside tomto tissues nd the rhizosphere. Another reson for the decline in R17 effectiveness over time could be tht the effects of R17 on the host decline over time. For exmple, induced resistnce by endophytes nd PGPRs is most often ssocited with ISR involving priming of defense gene expression (i.e., stronger nd fster induction following infection) medited by ET nd JA-dependent signling pthwys, which is usully considered to be longer lsting thn tht medited by SA-dependent signling pthwys (Pieterse et l., 2009, Ton et l., 2006). For exmple, protection lsted up to five weeks ginst C. orbiculre in cucumber fter seed inocultion with the PGPRs, S. mrcescens nd P. putid 89B-27 (Liu et l., 1995). However, the number of biotic nd biotic stress genes showing up- nd down-regultion of expression using 181

205 microrrys of Theobrom cco t three, seven nd 14 dys fter inocultion with the endophytic fungus Colletotrichum tropicle demonstrted tht the resistnce response cn chnge over time (Mejí et l., 2014). Much-Mni et l. (2017) hypothesize tht the durtion of defense priming my be dependent on the frequency nd intensity of the stress pressure, which indictes tht signling needs periodic stimultion to be mintined t similr levels. While plnts cn induce JA signling by stresses, they re lso cpble of suppressing it by switching off JA signling for subset of genes through the conversion of JA to 12-hydroxyjsmonic cid (Miersch et l., 2008). In ddition, SA ppers to be importnt for induced resistnce cused by some PGPRs (Murhofer et l., 1998). Lnn-Filho et l. (2017) lso ssocited induced resistnce ginst Pst in tomto due to the endophytes, B. pumilus nd B. myloliquefciens, with SA-dependent signling pthwys bsed on increses in defense enzymes including PAL up to four dys prior to pthogen chllenge. Induction of gene expression vi SA is well known to decline over time. For exmple, folir pplictions of the SAR ctivtor ASM to tomto resulted in incresed expression of the defense genes PR1 nd PR1b, but this declined to bseline levels within one week (Hermn et l., 2007). Thus, it is possible tht the decline in the durtion of the response to R17 ws relted to chnges in the defense signling response to the endophyte, which could lso be combined with the effect of the declining popultion of R17 possibly reducing the frequency nd intensity of the ctivtion of the resistnce Direct ntimicrobil effects of promising endophytes It is possible tht the bility of R17 to reduce disese incidence is due to direct ntimicrobil effects ginst Pst, since R17 showed some in vitro ctivity ginst Pst using two in vitro ssys. However, due to the limited bility of R17 to colonize lef tissue s n endophyte, the likelihood is low tht the ntimicrobil compounds were the primry explntion for it reducing the incidence of Pst. This is the first report of direct ntimicrobil effects of strin of B. mycoides/weihenstephnensis ginst Pst. The production of ntibiotics by Bcillus sp. group is reported for mny species ginst 80 bcteri or fungl orgnisms, including mny bcteri nd plnt pthogens (Shfi et l., 2017). Lipopeptides belonging to the surfctin, iturin nd fengycin (or plipsttin) fmilies re the most common ntibiotics produced by Bcillus sp., nd hve shown ctivity ginst mny plnt pthogenic bcteri, fungi nd oomycetes (Ongen & Jcques, 2008). Lipopeptides hve been described from B. mycoides including cerexin A 1 (Cochrne et l., 2015) s well s bcteriocin-like substnces (Abriouel et l., 2011). Genome nlysis of 13 strins of B. weihenstephnensis reveled gene clusters for bcteriocin nd microcin production, nd some strins hd gene clusters for other peptides including lnthipeptides nd lssopeptides (Hollensteiner et l., 2016). The ntifungl ctivity of some B. weihenstephnensis isoltes 182

206 from the tomto rhizosphere ws ssocited with the siderophore bcillibctin nd mycolytic chitinses (Hollensteiner et l., 2016). Given the widespred bility of Bcillus sp. to suppress the growth of other microorgnisms, it is most likely tht the ntimicrobil effects of B. mycoides/weihenstephnensis strin R17 re result of severl ntibcteril compounds. The direct ntimicrobil effects of the other disese resistnce ctivting strin in this study, R21, ginst Pst were less cler s it only showed ctivity using one of the two in vitro ssys. However, tht ws sufficient to conclude tht this is the first report of direct ntimicrobil effects of E. cloce/ludwigii ginst Pst. The ntimicrobil effects of Enterobcter sp. re lso widely reported. For exmple, the endophyte E. cloce subsp. cloce from pepper hs ntgonistic ctivity ginst Collectotricum cpsici, S. sclerotiorum, Alternri sp., D. bryonie, F. oxysporum, nd R. solncerum (Liu et l., 2013), E. cloce nd E. ludwigii from tomto re ntgonistic ginst R. solncerum (Upreti & Thoms, 2015), nd E. ludwigii from L. perenne decresed spore germintion nd produced zone of inhibition ginst F. solni in vitro (Shoebitz et l., 2009). The ntimicrobil ctivity of E. cloce subsp. cloce ws ssocited with the bcteriocin, colicin V, s well s siderophores, nd chitinses (Liu et l., 2013), nd the ntimicrobil ctivity of E. cloce ws linked to the presence of hydrogen cynide (Upreti & Thoms, 2015). Species of Enterobcter from soil lso produce ntimicrobil lipopeptides (Mndl et l., 2013). Thus, there re vriety of potentil ntimicrobil fctors being produced by E. ludwigii strin R Coloniztion of R17 inside tomto tissues To confirm tht R17 colonizes tomto tissue s n endophyte, rifmpicin resistnt R17 mutnts were creted, which ppered to be identicl to the originl strins reltive to reducing bcteril speck incidence nd not ffecting plnt growth. In roots, the popultion of R17-RfpC did not exceed 1.3 x 10 2 CFU/g fresh tissue, which is reltively low compred to other studies which found root bcteril endophyte popultions rnging from 10 2 to CFU/g fresh weight (Hllmnn & Berg, 2006). However, tht popultion dropped by two orders of mgnitudes between 10 nd 25 DPT. In the rhizosphere, the R17-RfpC popultion ws pproximtely 10 6 CFU/g dry soil, which is lower thn tht of the totl popultion of rhizobcteri of 10 7 to CFU/g dry soil in brley, whet, nd cnol (Lupwyi et l., 2004), nd 10 8 CFU/g soil or higher in wtermelon (An et l., 2011). However, the percentge of culturble Bcillus sp., rnged from 17% in 70-dy old plnts to 36% in 28-dy old plnts in the cucumber rhizosphere (Mhffee & Kloepper, 1997), nd pproximtely 10% of the potto rhizosphere were Firmicutes (Berg et l., 2005). Thus, the R17-RfpC popultion in the rhizosphere s component of the totl popultion of rhizobcteri is resonble. Between 10 nd 25 DPT, the R17-RfpC popultion dropped by 50% in the rhizosphere indicting tht it ws out-competed by the endemic rhizobcteri. 183

207 These declines would not however preclude R17 from inducing resistnce. The endophytes B. pumilus SE34 nd rhizobcteri P. fluorescens 89B61, which induced resistnce ginst lte blight of tomto (Yn et l., 2000), were incorported into the growing medium t the time of plnting, but their popultions dropped in the tomto rhizosphere by pproximtely one hlf to one order of mgnitude, similr to R17, by 28 dys fter plnting (Yn et l., 2003). As R17 both colonize plnt tissues nd the rhizosphere, one possibility is tht the induced resistnce by R17 my hve occurred due to its rhizosphere coloniztion thus cting more like PGPR, insted of its internl root coloniztion, lthough both my hve been involved. In leves, R17-RfpC ws only detected in cotyledon leves t 10 DPT but not true leves t 25 DPT. Thus the bcterium hs limited bility to spred nd persist in bove ground tissues. The bsence of R17 in lef tissue t 25 DPT is consistent with Romero et l. (2014) who found few endemic Firmicutes present in tomto lef tissues t 30 DAS. Since R17-RfpC ws found in cotyledon leves t low numbers t 10 DPT, it is possible tht R17-RfpC colonized the cotyledons directly when the germinting seed ws treted, rther thn infecting the roots nd then spreding internlly through the plnt like tht of competent endophyte (Hrdoim et l., 2008). On the other hnd, verticl spred of B. polymyx nd B. subtilis in plnt tissues ws reported in spruce (Shishido et l., 1999) nd cco (Shishido et l., 1999), indicting tht they were competent endophytes. Thus, lthough coloniztion through the xylem vsculr is thought to tke severl weeks (Hrdoim et l., 2015), it is lso possibility for coloniztion of the cotyledons by R17. Another possible reson for the decline in R17 popultions inside plnt tissues following inocultion is chnges in the physiology of the host. Endophyte coloniztion is dependent on series of interctions with the host plnt including the host recognition of MAMPs nd the elicittion of MTI, ETS nd ETI (Zmioudis nd Pieterse 2012). Thus, the host cn hve significnt impct, nd severl studies hve shown tht endophyte coloniztion is influenced by plnt developmentl stge. For exmple, in soyben root endophyte popultions were higher t the lef senescence stge compred to the flowering stge, which ws higher thn the vegettive stge (Kuklinsky-Sobrl et l., 2004). In potto, the popultion of Pseudomons spp. nd Actinobteri ws lower t plnt senescence thn flowering or juvenile stges (vn Overbeek nd vn Elss 2008), nd 73% of the vrition observed in endophyte genetic fingerprints ws explined by plnt growth stge. Totl endophyte popultion in cucumbers roots incresed during the first three weeks post-emergence, but then stbilized t 10 5 CFU/g fresh weight (Mhffee & Kloepper, 1997), nd in whet, popultions of B. subtilis peked before heding nd then stedily declined, unlike popultions of other endophytes tht remined reltively constnt throughout the whet life cycle (Comby et l., 2016). Thus, R17 popultions my hve been declined due to chnges in the interction of endophytes with tomto s the plnts grew. 184

208 4.4.7 Effect of R17 on bcteril speck lesion incidence versus Pst popultion Typiclly, the Pst popultion in lef tissues is relted to the percentge of leves with symptoms, which hs been demonstrted in A. thlin (Vlld et l., 2003), or the Pst popultion is severity disese index bsed on the number of lesions per leflet, which occurs in tomto (Bysl et l., 2007, Scrponi et l., 2001). However, in this study the Pst popultion per cm 2 in R17 inoculted plnts ws equivlent to the control, even though the number of lesions per leflet ws reduced. This ws relted to higher Pst popultion per lesion in R17 treted plnts thn the control. This is in contrst the endophytes B. pumilis nd B. myliloquefciens, which reduced Pst popultion on tomto lef surfces by three to four orders of mgnitude nd reduced the number of lesions per leflet, but Pst popultions inside the leves were not mesured (Lnn-Filho et l., 2017), nd the endophyte Azospirillum sp. B510 which reduced both Pst popultion growth in plnt nd disese severity in tomto (Fujit et l., 2017). However, sometimes reductions in plnt disese symptoms re not ssocited with reductions in pthogen popultion (Hmmerschmidt, 2009, Summermtter et l., 1995). Inocultion of P. syringe pv. syringe induced resistnce in A. thlin through n incomptible interction on lower leves, which resulted in reduced necrosis in upper leves fter lter chllenge, but the popultions of P. syringe pv. syringe in the chllenged leves remined unchnged (Summermtter et l., 1995). In ddition, induced resistnce resulting in lower levels of necrosis by Xcv or Pst on chllenged leves ctivted by Xcv, virulent Xcv, or Pst on different leves on the sme plnt ws not relted to reduction in popultions of Xcv nd Pst on the chllenged leves (Block et l., 2005). Block et l. (2005) described reduction in symptoms without n ssocited reduction in pthogen popultion s systemic cquired tolernce. It ppers tht R17 in the current study cuses rection similr to systemic cquired tolernce, except the number of lesions nd not totl necrosis, is reduced. The lck of correltion between disese incidence nd Pst popultion in this study ppered to be due to the lesions on R17 inoculted plnts being lrger thn lesions on control plnts. This is similr to wht ws found for PABA in Chpter 2, nd R17 my suppress initil infections so tht fewer lesions develop, but then lesion development occurs more rpidly post-infection. Although lesion size for Pst infected plnts is rrely reported, this ws surprising since induced resistnce by the SAR inducer ASM reduced bcteril speck lesion dimeter in tomto (Scrponi et l., 2001). The mechnism of disese reduction by R17 is unknown; however, induced resistnce ssocited with endophytes is mostly relted to ISR, which involves control of necrotrophs through JA nd ET signlling pthwys, unlike SAR, which generlly controls biotrophs nd is ssocited with SA signlling pthwys (Pieterse et l., 2009, Ton et l., 2006). In the current study, one explntion for the fewer lesions in R17 treted plnts could be becuse R17 primes or triggers defense response prior to Pst entry 185

209 into the plnt. Pst must penetrte the lef tissue through wounds or nturl openings, such s stomt. Prt of the defense response ginst folir infection by bcteri includes stomtl closure, which is triggered by bcteril PAMPs, positively regulted by SA versus negtively regulted by JA nd cn be suppressed by the pthogen using mechnisms, such s the T3SS effector AvrRpt2 nd the phytotoxin corontine (Melotto et l., 2008, Melotto et l., 2017). Therefore, it is possible tht R17 prevents Pst from entering the poplst by incresing stomtl closure if the induced resistnce mechnism involved SA, which would reduce the number of infection sites nd result in fewer lesions. This ide is supported by the finding tht the rhizobcterium B. subtilis FB17 ccelerted stomtl closure in A. thlin ginst Pst for 48 h post-inocultion in n ABA nd SA-dependent mnner (Kumr et l., 2012). Following penetrtion, the biotrophic phse of Pst occurs s the pthogen grows in the poplstic fluid nd releses T3SS effectors to suppress PTI (Preston, 2000, Cunnc et l., 2009, Munkvold et l., 2009). Induced resistnce due to R17 could hve triggered defenses relted to limiting biotrophic growth of Pst, nd thus prevented infection tht would subsequently develop into lesions. Induction of SA signlling cn suppress JA/ET defense signling (Gimenez-Ibnez & Solno, 2013). For exmple, infection by Pst induced SA-medited defenses in A. thlin resulting in the suppression of expression of the JA/ET dependent genes nd incresing susceptibility to the necrotroph, A. brssiccol (Spoel et l., 2007). Therefore, if incresed resistnce due to R17 is medited through SA trgeting Pst biotrophic growth, then this could suppress JA-relted defenses ginst necrotrophic growth result in lrger lesions, which is due to Pst necrotrophic growth,. Like stomtl resistnce, this explntion implies tht R17 induces SA-dependent defence signlling. Although less frequent thn ssocitions with ISR, there is evidence for Bcillus sp. with SA-dependent defense signling. B. pumulis nd B. myloquefciens induced higher PAL ctivity fter Pst inocultion (Lnn-Filho et l., 2017), which is ssocited with SAR, Ahn et l. (2002) found tht B. myloquefciens induces both SA nd JA-pthwys by mesure of PR1, PDF1.2 nd PAL gene expression, nd Niu et l. (2011) found tht B. cereus AR156 primes enhnced expression of SA, JA nd ET-dependent genes in A. thlin chllenged with Pst. To ssess the role of SA with R17, the bility of R17 to induce resistnce in SA-deficient plnts, like nhg tomtoes (Brding et l., 2000), should be determined to see if the level of induced resistnce is ffected Conclusions In this study, the bility of two bcteril endophytes, B. mycoides/weihenstephnensis strin R17 nd E. ludwigii/cloce strin R21, to reduce symptoms of bcteril speck in tomto ws demonstrted. The endophytes, which were isolted from two wild species of tomto, re members of gener often ssocited with endophytes (Bulgrelli et l., 2013, Rosenblueth & Mrtinez-Romero, 2006, Hrdoim et l., 2008, Hllmnn & Berg, 2006) nd in the cse of B. mycoides/weihenstephnensis, induced resistnce 186

210 (Bulgrelli et l., 2013, Rosenblueth & Mrtinez-Romero, 2006, Hrdoim et l., 2008, Hllmnn & Berg, 2006). R17 nd R21 lso demonstrted ntimicrobil effects ginst Pst in vitro, which were first reports for these orgnisms ginst this plnt pthogen, lthough both species hve shown ntibcteril ctivity ginst rnge of other microorgnisms. However, the physicl seprtion of R17 nd R21, which were pplied to seed nd soil, from the leves tht were ssessed for bcteril speck incidence suggested tht induced resistnce ws the mechnism of disese suppression. This should be confirmed with dditionl reserch on the moleculr mechnisms of this response. Neither endophyte showed consistent effects on plnt growth promotion showing tht endophytes beneficil for disese re not lwys linked to plnt growth promotion (Lnn-Filho et l., 2017). Mny fctors ffected the bility of R17 to suppress bcteril speck. Among the fctors tested, endophyte inocultion method, host genotype, endophyte concentrtion nd durtion of resistnce, the most criticl ones were host genotype nd inocultion method. No disese reduction occurred unless prticulr genotype ws used. This is potentilly very limiting fctor in the ppliction of R17 s mny tomto genotypes hve been developed for specific commercil purposes, such s high soluble solids content, mturtion time, pest resistnce nd fruit colour (Berry & Uddin, 1991, Bi & Lindhout, 2007). Durtion of the effect ws lso fctor tht ws criticl to chieve ny level of suppression nd ppers to be pprox. 3 weeks. The impliction from this is tht repeted pplictions of R17 my be needed to mintin the effect, which would increse costs. Despite exmining number of fctors, the mximum level of Pst symptom reduction ever chieved ws 51%. This would not provide commercilly cceptble level of control in commercil production s bcteril speck is polycyclic disese with multiple infection cycles during the seson. In polycyclic disese cycles, disese ccumultes in n exponentil mnner nd so it is unlikely tht reducing infections by only hlf would hve significnt impct on totl disese over growing seson (Arneson, 2001). To help explin the disese suppression, R17 coloniztion of the tomtoes following tretment nd Pst popultions in treted nd non-treted plnts were exmined. R17 coloniztion declined in the tomto rhizosphere, root nd folir tissue over time. While it is uncler how endophyte coloniztion is relted to the effect observed here (Hrdoim et l., 2015, Liu et l., 1995b, Kloepper & Ryu, 2006, Bent, 2006), rpidly declining popultions my men tht R17 would need to be repplied in the field. The prcticl use of R17 s disese mngement tool my lso be limited becuse lthough it reduced the number of bcteril speck lesions, these lesions were lrger thn those in control plots, nd s result, did not reduce the popultions of Pst or the totl lef surfce re with lesions. To control polycyclic diseses like bcteril speck, control gent must be ble to limit the rte of reproduction or the durtion of the epidemic, s discussed bove. R17 tretments would likely not limit the inoculum for subsequent infections or dely the epidemic by delying initil infections nd thus ultimtely not control field 187

211 epidemics of the disese. Overll, while R17 cn suppress Pst, it hs limited potentil for field use unless issues, such s the level of disese control, durtion of disese control nd inbility to reduce Pst popultions, cn be ddressed. 188

212 5 Chpter 5: Generl Discussion Bcteril speck nd bcteril spot remin serious diseses of field tomtoes, especilly in production yers with frequent or intense rin events. Copper is the trditionl tool for mngement of these diseses. However, in review of 45 studies evluting copper efficcy for bcteril spot or speck control, 56% of the studies reported less thn 50% disese reduction, while only 38% of studies reported more thn 60% disese reduction (Griffin et l., 2017). The ctivity of copper in field tomtoes my be limited by poor coverge, which cn result when there is hevy cnopy, nd/or the presence of copper tolernt bcteri. Widespred tolernce to copper by Pst, Xg, nd Xp is reported in mjor tomto growing res, including Ontrio (Abbsi et l., 2015, Cuppels & Elmhirst, 1999, Griffin et l., 2017). This hs motivted the serch for more effective disese mngement tools. Active ingredients currently registered nd vilble for use ginst bcteril speck or bcteril spot on field tomtoes in Cnd include ksugmycin (Ksumin 2L), B. subtilis QST 713 (Serende Opti, Serende Mx, Cese), copper hydroxide (Coppercide WP, Kocide 2000, Prsol WG), copper octnote (Cuev), extrct of gint knotweed (Regli MAXX) nd B. mycoides isolte J (LifeGrd) (OMAFRA, 2014, Certis, 2017b, Byer, 2017, Byer, 2014, Bioworks, 2016). The registrtion of these products, including the induced resistnce ctivtors B. subtilis QST 713, B. mycoides isolte J, nd extrcts from R. schlinensis (Regli MAXX) suggests tht mny compnies consider them to be promising tools for bcteril speck nd spot mngement in tomto (PMRA, 2011, PMRA, 2016d, Certis, 2017b, Byer, 2017, Byer, 2014). However, the efficcy of B. subtilis QST 713, extrcts from R. schlinensis nd ASM hs proven to be quite vrible under field conditions (Obrdovic et l., 2004, Roberts et l., 2008, Wilson et l., 2002, Truemn, 2015, Louws et l., 2001), while there is no publiclly vilble dt on the efficcy of B. mycoides isolte J. Furthermore, ASM ppliction sometimes results in host fitness costs (Wlters & Heil, 2007, Lnn-Filho et l., 2017, Louws et l., 2001, Dmicone & Trent, 2003, Lnge & Smrt, 2005, Pontes et l., 2016, Kunwr et l., 2017), nd the registrnt for the commercil formultion of ASM withdrew the product from the Cndin mrket becuse of declining sles in 2016 (Filots, 2015). The need for more effective control products re cler from survey tht showed tht copper use hs declined (J. LeBoeuf, pers. communiction) nd defense ctivtors hve not been widely dopted by Ontrio processing tomto growers for bcteril spot nd speck mngement becuse of lck of perceived benefits due to poor or inconsistent field performnce (J. LeBoeuf, pers. communiction). Pesticide ppliction, including copper nd defense ctivtors, re not expected to hve significnt use in mnging tomto bcteril disese in Ontrio becuse of copper tolernce nd poor performnce in field trils (Truemn & LeBoeuf, 2015). Thus, while induced resistnce holds promise s n lternte disese mngement tool, more informtion is needed to mke defense ctivtors effective in the field. 189

213 In this thesis, n ttempt ws mde to improve the efficcy of ASM by first treting tomtoes with the PGR UNI, which is lso ssocited with incresing stress tolernce in plnts (Al-Rumih & Al- Rumih, 2007, Dun et l., 2008, Senrtn et l., 1988, Zhng et l., 2007). The hypothesis ws tht ASM is sometimes ineffective becuse of excess stress creted by the compound, nd tht stress could be reduced by UNI to llow for induced resistnce. However, there ws little evidence of fitness costs by ASM, nd when evidence for fitness costs ws observed, UNI did not prevent yield loss. There my be other wys to mke ASM more consistently effective ginst speck nd spot on field tomtoes in Ontrio. One lterntive would be to combine ASM with biologicl ISR ctivtors, like B. subtilis GB03 nd B. myloliquefciens IN937 for bcteril speck in tomto, where control ws better thn ASM lone in one of three greenhouse experiments (Hermn et l., 2008). In this cse, ASM could be combined with B. mycoides/weihenstephnensis strin R17, which ws puttively found to be effective in inducing resistnce in this thesis. Another possibility is to combine ASM with copper bsed nnomterils, such s core-shell copper, multi-vlent copper nd fixed qurternry mmonium copper, since the nno sized copper mterils re toxic to copper tolernt strins of Xp (Stryer-Scherer et l., 2017). Another wy to possibly improve the effectiveness of ASM is to use the product only on tomto genotypes tht cn respond with incresed resistnce considering tht only one out of eight tomto breeding lines showed incresed resistnce to Pst following seed pplied ASM (Goodwin et l., 2017b). Future work could exmine dditionl tomto genotypes, both with ASM lone nd ASM combined with UNI, B. mycoides/weihenstephnensis strin R17 nd copper bsed nnomterils. Yet nother wy to mke ASM more effective would be to increse the time between pplictions more thn the Cndin Actigrd lbel recommendtion of seven dys (PMRA, 2011). The intervl period my be relted to the durtion of induced defense gene expression s Hermn et l. (2007) showed tht defense gene expression induced by ASM returns to bseline levels fter seven dys in three fresh mrket cultivrs. However, Pontes et l. (2016) showed tht slightly longer intervl of eight to 10 dys provided the mximum bcteril spot reduction using response-surfce curve nlysis for processing tomtoes. These differences my be becuse Pontes et l. (2016) used the processing tomto cv. H9992 compred to the fresh mrket cultivrs Supersonic, Rutgers, nd Rio Grnde used by Hermn et l. (2007). A longer intervl for ASM my hve been better using cvs. TSH4 nd H9909 in this thesis. Defense gene expression induced by ASM mong different tomto genotypes cn vry in intensity nd durtion (Goodwin et l., 2017b, Hermn et l., 2007), nd thus future work needs to exmine defense gene expression in cvs. TSH4 nd H9909 following ASM ppliction to help determine whether seven dy intervl is pproprite. While combining ASM with other compounds nd defense ctivtors, limiting the rnge of cultivrs on which it should be pplied nd djusting ppliction intervls for ech cultivr my limit the use of ASM, t lest it could give greter confidence of success in those cultivrs where it is effective. 190

214 Abiotic fctors cn lso influence gene expression nd host fitness effects of ASM (Dietrich et l., 2004, Dietrich et l., 2005) nd there is limited bility to control most biotic stresses in the field, thus, ny disese mngement system using ASM would hve to be sufficiently robust to be effective in reltively wide rnge of environments. Future studies need to exmine ll the prmeters included in this thesis under rnge of environmentl conditions tht might resonbly be expected by field tomtoes in Ontrtio. ASM will likely only chieve cceptnce by tomto growers if the risks ssocited with its use is gretly reduced. Mny other chemicl compounds, such s tidinil, thimine nd PABA, re lso reported to induce resistnce ginst plnt pthogens but hve not been developed s commercil products (Schreiber & Desveux, 2008, Song et l., 2013). In this thesis, PABA ws selected for evlution ginst Pst in tomto becuse it reduced the severity of Xe/Xp in peppers nd P. crotovorum subsp. crotovotrum in tobcco (Song et l., 2013, Yng et l., 2011b). Although PABA pplictions reduced the incidence of bcteril speck lesions in growth chmber experiments, the mximum level of control ws 43% reduction in lesion incidence, nd there ws no effect on the totl lef surfce re with bcteril speck or Pst popultion or disese severity or tomto yield in one field experiment. This occurred despite exmining ppliction method, timing of Pst inocultion fter ppliction, number of pplictions, concentrtion nd host genotype to increse PABA performnce. A limittion of the growth chmber experiments in this thesis is tht only young plnts were exmined, nd so dditionl reserch should be undertken to better understnd the effectiveness of PABA in older tomto plnts. In ddition, only six tomto genotypes were tested for their response to PABA, nd future work could exmine mny more genotypes s well s breeding for stronger response to PABA pplictions (Michelmore et l., 2017). Field performnce of PABA in the current study my hve been reduced becuse non-formulted version of PABA ws used. Pesticides re exposed to vrible environmentl conditions in the field, including hevy dews, rin, het, nd UV light, tht cn reduce their effectiveness, which would not be the cse in the protected environment of growth chmber (Oliver & Hewitt, 2014). Typiclly, formultions of pesticides spryed onto plnts contin UV stbilizers to prevent photodegrdtion, dhesives to lengthen the durtion of effectiveness nd surfctnts to improve coverge (Oliver & Hewitt, 2014). Future experiments should exmine different formultions of PABA under field conditions. In ddition to chemicl ctivtors, there re mny reports of using living orgnisms s biologicl ctivtors to induce plnt disese resistnce (Bent, 2006, Hrdoim et l., 2015). Bcteril endophytes were studied in this thesis s potentil biologicl ctivtors for mngement of bcteril speck. While PGPRs re vilble s commercil biologicl ctivtors of resistnce, such s B. subtilis QST 713 (Serende Mx, Serende Opti, Cese) nd B. subtilis GB03 nd B. myloliquefciens (BioYield) (Fousi et l., 2016, Hermn et l., 2008, Byer, 2017, Byer, 2014, Bioworks, 2016), bcteril endophytes re 191

215 ppeling becuse they cn internlly colonize the plnts where they re protected from the environment nd potentilly single infection cn led to endophyte estblishment for the life of the plnt. Two bcteril endophytes, E. cloce/ludwigii R21 nd B. mycoides/weihenstephnensis R17, from wild tomto species were identified s cusing puttive induced resistnce becuse ppliction resulted in reduction in bcteril speck lesion incidence. R17 ws the most consistent in growth room experiments. However, the mximum level of control ws 51% reduction in lesion incidence despite exmining mny fctors, including inocultion timing, inocultion method, bcteril concentrtion nd host genotype, to increse the level of control. Future work tht could possibly increse tht level of control would include exmining more tomto cultivrs to find ones tht show greter level of response since R17 induced response ginst Pst in only one of five commercil cultivrs tested. Unlike chemicls, biologicl ctivtors need to be ble to grow nd persist with the crop. R17 coloniztion in growth chmber experiments declined within few weeks of inocultion, but it ws not cler if coloniztion is required for the response to occur, or if the response in older seedlings declined for other resons such s developmentl resistnce. If coloniztion of internl tissues by R17 is required to elicit n effect, then the bility of R17 to reduce bcteril speck symptoms my be limited over longer periods. In ddition, studies re needed on the bility of R17 to induce response in tomto t different growth stges s only young plnts were tested in the growth room, nd confirm induced resistnce s the mechnism of the response. This should include field trils. Considering tht the level of disese control ws similr between PABA nd B. mycoides/weihenstephnensis R17 in the growth room but PABA ws ineffective in the field, B. mycoides/weihenstephnensis R17 my not be effective in the field. If it were to be effective, then for bcteril ctivtor to be commercilized, it must lso be evluted for their environmentl nd toxicologicl impct, bility to be produced on commercil scle nd suitblity for long term storge (Montesinos, 2003). There re reports of B. mycoides being pthogenic to frmed chnnel ctfish (Goodwin et l., 1994), but regultory gencies in the USA nd Cnd did not identify ny other concerns regrding environmentl or humn helth impcts in review of B. mycoides isolte J (PMRA, 2016b). However, work is still needed to confirm the sfety of B. mycoides/weihenstephnensis R17. B. mycoides isolte J hs been commercilized s LifeGrd nd this could serve s model for future work on lrge scle production (Certis, 2017b). For storge, Bcillus spp., such s R17, re spore forming nd thus cn survive for long periods s well s enbling the bcteri to be resistnt to environmentl stress mking it esier to formulte in commercil product (Rhmn, 2016). Future work could exmine endospore production by R17. Finlly, it ws unexpected tht the only bcteril endophytes tht ffected disese incidence in this study originted from wild tomto species. This suggests tht more work should be done exmining wild tomto species s sources of bcteril endophytes tht my provide benefits to 192

216 domesticted tomto. In this study, disese incidence ws mesured in growth chmber trils by clculting the number of lesions per unit re of tomto lef tissue. It ws initilly ssumed tht this mesure would correlte with reduction in the pthogen popultion in lef tissues; however, for both PABA nd R17, there ws no reduction in Pst popultion per unit re of lef. This effect ws ssocited with fewer but lrger lesions resulting in higher Pst popultion per lesion thn control plnts. Such results my occur becuse PABA nd R17 my limit initil infections of Pst (i.e. reduce the number of lesions) but then hve little effect on the subsequent increse in pthogen popultion nd lesion size. Since Pst enters plnts vi nturl openings or wounds, dditionl reserch on stomtl defense in the Pst-tomto pthosystem would help elucidte the mechnisms of this response. Recent reserch shows tht stomtl defense is dependent on MAMPs, PAMPs nd hormonl signlling, but it cn be counterblnced by pthogen effectors nd phytotoxins, s well s environmentl fctors (Melotto et l., 2017). There is derth of informtion on the role of plnt defense ctivtors in stomtl defense, nd thus further reserch on PABA nd R17 on stomtl closure in tomto would further our understnding of this response s it reltes to induced resistnce. One possible reson tht Pst popultions nd lesion size incresed fster in plnts treted with PABA or R17 thn in control plnts could be relted to cross-tlk between the SA nd JA/ET signling pthwys resulting in decline in JA/ET-relted defenses if SA-relted defenses re ctivted nd vice vers (Gimenez-Ibnez & Solno, 2013, Song et l., 2013, Goodwin et l., 2017, Spoel et l., 2007, Wlters et l., 2011). The effect of PABA is clerly relted to SA in plnts (Song et l., 2013, Tzhoor, 2014, Goodwin et l., 2017, Yng et l., 2011b), nd so future work needs to determine if tht my be suppressing JA/ET-relted defenses. One pproch is to exmine the effects of PABA nd R17 on lesion number, lesion size nd Pst popultion in tomto mutnt lines deficient in SA, JA or ET signling, such s ETR mutnts deficient in ET perception (Lshbrook et l., 1998), NhG trnsformnts in which SA is degrded (Brding et l., 2000, Di et l., 2017), nd def1 mutnts in which JA signlling is compromised (Di et l., 2017). This would help uncover if the lesion number response is dependent on prticulr pthwy. Since Pst DC3000 infects A. thlin, which hs mutnts nd combintions of mutnts for lrge numbers of genes, then the nture of PABA or R17 ctivted resistnce could be very extensively exmined if PABA nd R17 hve similr effects on Pst in tht pthosystem. The lck of reltionship observed between lesion density nd Pst popultion for R17 nd PABActivted resistnce in this study indictes need for this to be considered in future reserch on Pst nd possibly other diseses where resistnce is evluted bsed on lesion number. Growth chmber nd greenhouse studies on plnt defense ctivtors nd Pst normlly evlute disese severity using vriety of methods including visully with photos (Fujit et l., 2017, Block et l., 2005), rting scles bsed on 193

217 severity or the number of lesions per leflet (Hermn et l., 2008) or the bsolute number of lesions per leflet (Lnn-Filho et l., 2017). However, those do not ccount for vrition in leflet size nd the difference in lesion size observed in this work ws so smll tht it required very creful imge nlysis. Few studies determine if defense ctivtors ctully reduce pthogen popultion or growth in plnt tissues s opposed to just symptom development (Lnn-Filho et l., 2017, Hermn et l., 2008). In future reserch, it my be informtive to test severl methods of ssessment for disese incidence or severity. Becuse R17 nd PABA-ctivted resistnce do not reduce Pst popultions, then they my not even hve n impct on bcteril speck epidemiology in the field unless they induced very high levels of resistnce. This is becuse the polycyclic nture of bcteril speck would still llow the pthogen popultion to build over time in the field even if symptom reductions were observed. This could possibly even be observed in growth room experiments if continued for prolonged time with overhed irrigtion or misting to simulte rinfll. Thus, possible future experiment would be to grow tomtoes inoculted with Pst until mturity in growth rooms with overhed irrigtion nd then use methods of disese ssessment like in this thesis to test if R17 nd PABA reduce disese progress over time. Although effects regrding the ctivity nd optimiztion of the ctivtors ASM, PABA, R17, nd R21 were found in this study, prcticl mngement of bcteril speck nd spot remins chllenge. In recent yers, only products lbelled for suppression, which is defined s hving commercil benefit (i.e. 60 to 80% reduction), or prtil suppression, which is defined s less thn 60% reduction hve been registered for bcteril speck or spot control in tomto (PMRA, 2011, Byer, 2017, Byer, 2014, Bioworks, 2016, Certis, 2017b, PMRA, 2016d, Certis, 2017, PMRA, 2016f). Bsed on the results of field efficcy tests of defense ctivtors in Cnd nd the USA, it is unlikely tht such products lone will provide economiccontrol of bcteril speck nd spot (Truemn, 2015, Roberts et l., 2008). However, plnt defense ctivtors could be prt of true systems pproch tht involves ll tennts of integrted pest mngement (IPM). Furthermore, the incresing demnd by consumers for orgnic or sustinbly produced food (Lmichhne et l., 2015, Dewen et l., 2017), the withdrwl or proposed withdrwl of long pproved pesticides becuse of helth nd sfety or environmentl concerns in Cnd nd Europe (Lmichhne, 2017, Hillocks, 2012, PMRA, 2016e, PMRA, 2016c) nd shifts in corporte nd government policy towrd mndtory IPM (Lmichhne et l., 2015, Bonduelle, 2015) support this pproch. Reserch is still needed to improve bcteril disese mngement in field tomto. Trditionl breeding pproches hve yielded few cultivrs with commercilly cceptble levels of host resistnce ginst multiple rces of Pst or rces nd species of BSX. However, new opportunities in plnt breeding include breeding for non-blighting trits, identifiction of resistnce quntittive trit loci, nd the introduction of resistnce genes using CRISPR technology (USDA, 2015). Breeding for specific effector 194

218 trgets tht re conserved cross Pst rces or BSX rces nd species, my lso led to the development of tomto cultivrs with more durble resistnce (Timilsin et l., 2016). Snittion is nother option. Reserch on the role of contminted seedling trnsplnt trilers nd trnsplnting equipment in bcteril spot epidemics in Ontrio includes the evlution of clening nd snittion tools to prevent spred of BSX (OPVG, 2017). It my lso be possible to control bcteril speck nd spot by modifying the plnt environment by introducing mulch cover crops s this incresed the yield of commercil grde sqush by reducing the popultion of pthogenic P. syringe in plots (Toussint et l., 2012), or with the strtegic use of windbreks, s suggested to limit spred of citrus cnker (Xnthomons cmpestris pv. citri) in Florid (Tmng et l., 2010). Despite these efforts, more work is needed on gents tht cn control Pst nd Xg infections in the field. The use of ntibiotics in commercil plnt griculture is effective, but unlikely to be pproved in Cnd nd hs resulted in reltively rpid evolution of resistnce in bcteril pthogens in other jurisdictions (Ritchie & Dittpongpitch, 1991, Arujo et l., 2012). Therefore, defense ctivtors will continue to be investigted s one option, lthough this thesis hs shown some of the chllenges in developing nd using these tools. 195

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254 7 Appendix Tble A.1 Effect of different pr-minobenzoic cid (PABA) concentrtions nd ppliction volumes on the reltive chlorophyll content in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). PABA ws pplied to the root zone with pipette 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Reltive chlorophyll ws mesured t five dys post inocultion. PABA (mm) Volume pplied (ml) PABA pplied (mg) Reltive chlorophyll b b b b b b b b b sem b 0.96 Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 231

255 Tble A.2 Effect of different PABA concentrtions on the reltive chlorophyll content in tomto cv. H9909 inoculted with P. syringe pv. tomto (Pst). Plnts were coted with 0, 0.01, 0.1, 0.5, 1, 4, 9, or 18 mm pr-minobenzoic cid (PABA) 10 nd 15 dys fter seeding (DAS). Plnts were coted with fine mist of 2 x 10 7 CFU/ml of Pst 20 DAS. Reltive chlorophyll ws mesured t five dys post inocultion. PABA (mm) Reltive chlorophyll sem b 0.94 Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. Dt from two independent trils with five replictions of ech tretment ws pooled together becuse ANOVA showed no tretment x tril interction. b sem = stndrd error of the men for ll ls mens in the sme column. 232

256 Tble A.3 The incidence of bcteril speck nd bcteril spot on green tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 green fruit hrvested in 2m section of ech plot ws evluted. Tretment Bcteril speck on fruit (%) Bcteril spot on fruit (%) Control UNI ASM ASM + UNI sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. b sem = stndrd error of the men for ll ls mens in the sme column. 233

257 Tble A.4 Incidence nd severity of nthrcnose symptoms on red tomto fruit, cv. TSH4, hrvested from plots treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit ws collected from ll red fruit hrvested in 2m section of ech plot, stored for three dys t room temperture, nd then ssessed for nthrcnose symptoms. Incidence (%) DSI Tretment Control 10.7 b UNI ASM ASM + UNI sem c DSI = disese severity index nd ws clculted using the eqution the following eqution: DSI = [(clss no.) (no. of fruit in ech clss)] / [(totl no. fruit per smple) (no. clsses -1)] *100. Fruit were sorted into the following clsses: 0 = no lesions, 1 = one lesion, 2 = two to three lesions, 3 = four or more lesions. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. c sem = stndrd error of the men for ll ls mens in the sme column. 234

258 Tble A.5 Number of dys fter trnsplnting to begin inflorescence, fruit set, nd ripening for tomto cv. TSH4 treted with uniconzole (UNI) in the greenhouse nd cibenzolr-s-methyl (ASM) in the field nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Five plnts per plot were monitored t seven to 12 dy intervls fter trnsplnting. First inflorescence First fruit set First fruit ripening Tretment Control 35 b UNI ASM ASM + UNI sem c First inflorescence, fruit set, nd fruit ripening were defined s plnts with open flowers, visible fruit pproximtely 1 cm in dimeter, nd ny pink or red colourtion on fruit, not including fruit with blossom end rot. b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. c sem = stndrd error of the men for ll ls mens in the sme column. 235

259 Tble A.6 The incidence of bcteril speck nd bcteril spot on green tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 green fruit hrvested in 2m section of ech plot ws evluted. TSH4 H9909 Bcteril speck on fruit (%) Bcteril spot on fruit (%) Bcteril speck on fruit (%) Bcteril spot on fruit (%) Tretment Control b CuOH c CuOH + UNI bc ASM ASM + UNI b UNI 5.5 NT NT 32.0 NT NT 4.0 NT NT 29.5 NT NT sem b Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. NT = not tested. b sem = stndrd error of the men for ll ls mens in the sme column. 236

260 Tble A.7 Incidence nd severity of nthrcnose symptoms on red tomto fruit, cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, A subsmple of 50 red fruit ws collected from ll fruit hrvested in 2m section of ech plot, stored for three dys t room temperture, nd then ssessed for nthrcnose symptoms. TSH4 H9909 Incidence (%) DSI Incidence (%) DSI Tretment b Control 13.5 c CuOH CuOH UNI ASM ASM UNI UNI 8.5 NT NT 3.0 NT NT 4.5 NT NT 1.8 NT NT sem d DSI = disese severity index nd ws clculted using the eqution the following eqution: DSI = [(clss no.) (no. of fruit in ech clss)] / [(totl no. fruit per smple) (no. clsses -1)] *100. Fruit were sorted into the following clsses: 0 = no lesions, 1 = one lesion, 2 = two to three lesions, 3 = four or more lesions. b Dt in this column were squre root trnsformed to meet ssumptions of ANOVA. The bck-trnsformed mens re presented here. c Mens in the sme column followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. d sem = stndrd error of the men for ll ls mens in the sme column, except for the DSI for tretment Control, TSH4, 2012 the sem is

261 Tble A.8 Number of dys fter trnsplnting to begin inflorescence, fruit set, nd ripening for tomto cv. TSH4 nd H9909, hrvested from plots treted with CuOH, uniconzole (UNI) nd cibenzolr-s-methyl (ASM) in the greenhouse nd inoculted with P. syringe pv. tomto nd X. grdneri, Ridgetown, ON, Five plnts per plot were monitored t seven to 12 dy intervls fter trnsplnting. Growth stge Cultivr Yer First TSH4 inflorescence First fruit set First fruit ripening H9909 TSH4 H9909 TSH4 Control CuOH Tretment CuOH + UNI ASM ASM + UNI UNI sem b c NT NT NT NT NT NT NA NT NT NA b 35 b 34 b b 31 b NT NT NA H NT NT NA First inflorescence, fruit set, nd fruit ripening were defined s plnts with open flowers, visible fruit pproximtely 1 cm in dimeter, nd ny pink or red colourtion on fruit, not including fruit with blossom end rot. b sem = stndrd error of the men for ll ls mens in the sme row. NA = not nlysed becuse ll observtions were the sme. c Mens in the sme row followed by the sme letter re not significntly different t P 0.05, Tukey s HSD. 238

262 Figure A.1. Lesion size ws determined by ) tking photo (12.2 MB, 4288 x 2824 pixels) of ech leflet ws tken using Nikon D300s cmer with ruler for reference, b) uploding, enlrging nd printing ech photo in colour, c) trcing lesion circumference using fine point mrker on cette, nd d) scnning lesion circumference imges nd nlyzing using Imge J. 239