PREFACE TO CHAPTER 5. be expected to create different microenvironments for S. minor colonization and growth.

Transcription

1 PREFACE TO CHAPTER 5 Mowing is necessry culturl mngement prctice for turf mintennce nd mowing height could be vrible ffecting biocontrol system. Different mowing heights would be expected to crete different microenvironments for S. minor coloniztion nd growth. Periodic mowing t close heights could dd more stress, not only on weeds, but lso on turfgrss. In generl perennil plnts hve sesonl vritions for lloctions of crbohydrte reserves depending on their phenologicl stge nd environmentl conditions. Bsed on the bove fcts, the objective of Chpter 5 ws to investigte the performnce of S. minor under different mowing heights in both greenhouse nd field conditions. In greenhouse nd field experiments, the interction of mowing height with S. minor efficcy on dndelion ws studied by estimting boveground dmge, mesuring lef nd root biomss nd counting the number of dndelions recovered fter S. minor ppliction. A mnuscript of the results of Chpter 5 hs been ccepted for publiction in Biocontrol Science nd Technology (submitted August 1, 2005 ccepted for publiction November 7, 2005). The mnuscript is co-uthored by Professor Aln K. Wtson, my supervisor. I designed the experimentl set-up, performed the experiments nd the sttisticl nlysis, nd wrote the mnuscript. Professor Wtson supervised the work, provided finncil nd technicl resources, nd corrected the mnuscript. 80

2 CHAPTER 5 Effect of turfgrss mowing height on biocontrol of dndelion with Sclerotini minor MOHAMMED H. ABU-DIEYEH nd ALAN K. WATSON 1 Deprtment of Plnt Science, McGill University, 21,111 Lkeshore Rod, Ste.-Anne-de-Bellevue, Quebec, Cnd, H9X 3V9 1 Correspondence to: Aln Wtson, Tel: (+1) , Fx: (+1) , E-mil: ln.wtson@mcgill.c. A running title: Mowing effect on performnce of turf bioherbicide Biocontrol Science & Technology submitted August 1, 2005 ccepted for publiction November 7,

3 5.1. Abstrct The fungus Sclerotini minor Jgger is under development s bioherbicide for control of dndelion nd mny brodlef weeds in turfgrss environments. The effect of S. minor on dndelion survivl ws evluted under different mowing heights nd compred with Killex the commonly used herbicide. In the greenhouse, the onset of symptoms ws more rpid, folir dmge ws more severe, nd the reduction of boveground biomss nd root biomss ws greter for the bioherbicide thn the herbicide. The bioherbicide reduced root biomss 10 fold compred with untreted plnts. Under high weed infesttion levels in the field, S. minor cused greter initil reduction of dndelion density thn did the herbicide during the two-weeks-post ppliction period, lthough reductions were greter in herbicide treted plots by six weeks fter ppliction. Over the growing seson, S. minor nd the herbicide hd similr suppressive effects on dndelion density except under the closest mowing height (3-5 cm). After tretment, close mowing fvoured dndelion seedling recruitment nd the biocontrol hd no residul ctivity. Survivl of dndelion roots ws significntly less fter spring thn fll tretment of S. minor nd seson long mowing t the close height significntly reduce root survivl. Close mowing my be detrimentl for S. minor pplictions on hevily infested domestic lwns nd menity grsslnd res. Keywords: Bioherbicide; biologicl weed control; clipping; fungus; mowing; Sclerotini minor; Trxcum officinle; turfgrss; weed control 82

4 5.2. Introduction Brodlef weeds reduce the qulity nd quntity of turfgrss nd disrupt its visul uniformity (McCrty et l. 2001). Dndelion, Trxcum officinle Weber, is strong competitive perennil weed tht infests turfgrss environments nd is common in home lwns, turfgrss swrds, pstures, forges, golf courses, thletic fields, wooded res, nd rodside verges (Stewrt-Wde et l. 2002b). It is n undesirble plnt, cusing esthetic problems during flowering nd seed production. In 1992, survey of turfgrss specilists estimted tht there were 7.3 million hectres of lwn in the United Sttes (Elmore 1994). The lwn cre industry in North Americ hs been expnding t rte of 5-8% per yer (United Sttes Environmentl Protection Agency 1999). The need for regulr mngement nd mintennce of these res cretes mssive turfgrss industry in North Americ nd Europe. In 2001, 78 million households in the U.S. used home nd grden pesticides (U.S. Environmentl Protection Agency 2004). Herbicides ccounted for the highest usge of pesticides in the home nd grden sector with users spending 632 million dollrs for over 36 million kilogrms pplied on lwns nd grdens. 2,4-D (2,4- dichlorophenoxy cetic cid) ws the most widely used pesticide in the home nd grden sector. Repeted pplictions of phenoxy herbicides such s 2,4-D nd mecoprop ((±)-2-(4- chloro-2-methylphenoxy) propnoic cid), dicmb (3,6-dichloro-2-methoxybenzoic cid), or combintion products such s Killex hve been widely used for dndelion control (Anonymous 1997). Environmentl nd public helth concerns bout pesticides, especilly the use of chemicl herbicides in turfgrss for esthetics, hve led to the bnning or severe restrictions on the use of pesticides in mny regions of Cnd (Cisr 83

5 2004). Consequently, lterntives to chemicl herbicides re being sought (Neumnn & Bolnd 2002; Stewrt-Wde et l. 2002; Zhou et l. 2004). Sclerotini minor Jgger is n sporogenic scomycete plnt pthogen tht hs biocontrol potentil for dndelion in turfgrss, nd severl formultions of S. minor hve been shown to hve biologicl control ctivity (Ciotol et l. 1991; Riddle et l. 1991; Brière et l. 1992; Stewrt-Wde et l. 2002; Abu-Dieyeh & Wtson 2006: Chpter 5). S. minor mycelium in sodium lginte grnules (Brière et l. 1992) nd mycelilcolonized brley grits (Stewrt-Wde et l. 2002) effectively controlled dndelion nd brodlef plntin (Plntgo mjor L.) without dmge to turfgrss species. When pplied to turfgrss, S. minor IMI rrely produces scleroti (melnized survivl structures), nd these scleroti do not survive over winter. Myceli of S. minor IMI does not survive beyond 10 dys in the turfgrss environment (M.H. Abu-Dieyeh & A.K. Wtson, unpublished dt). Field nd greenhouse studies confirmed tht turfgrss species re not susceptible to S. minor IMI Independent toxicologicl studies hve estblished tht S. minor IMI is neither toxic nor pthogenic to humns, birds, fish, dphni, honey bees, erthworms, or wild nimls (A.K. Wtson personl communiction). Extension recommendtions for turfgrss often indicte tht dense, helthy turfgrss stnd is the best defence ginst weed coloniztion nd cn be mnged by proper mowing, wtering, nd fertiliztion (McCrty et l. 2001; Busey 2003). A sensible recommendtion but the methods re often stted in generlities nd re not bsed on scientific reserch (s stted by Busey 2003). Mowing is mjor culturl prctice for turf mintennce. Lower mowing heights cn cuse dditionl stress for certin grss species 84

6 nd/or brodlef weeds, especilly in summer (s reviewed by Busey 2003; Htcher & Melnder 2003), nd higher cnopies my mintin higher humidity levels close to the soil surfce for longer time periods, which cn be dvntgeous for pthogen ctivity (Gielser et l. 2000). When fungl pthogen ws combined with simulted mowing, weed suppression ws greter thn the fungus lone (Green et l. 1998; Kluth et l. 2003). In this reserch, the effect of turfgrss mowing height on the efficcy of grnulr formultion of S. minor to suppress dndelion in suburbn lwn environment ws evluted Mterils nd methods Production nd formultion of Sclerotini minor Sclerotini minor (IMI ) ws isolted from disesed lettuce plnts (Lctuc stiv L.) from southwestern Quebec in 1988 nd stock cultures mintined s scleroti t 4 o C. When required, scleroti from stock culture were wshed twice in sterile distilled wter, plced in 70% ethnol for 40 s, trnsferred to 1% hypochlorite solution for 3 min, rinsed twice with sterile distilled wter, nd set to dry on sterilized filter pper. The surfce sterilized scleroti were trnsferred septiclly onto potto dextrose gr (PDA, DIFCO Lbortories, Detroit, MI) pltes nd incubted for 4-5 dys t 20 ± 1 o C. Five gr plugs (5-mm dimeter), from the ctively growing mrgin of colonies on PDA were trnsferred to 100 ml of modified Richrd s solution (MRS) hving the following constituents per litre: 10 g of sucrose, 10 g of KNO 3, 5.0 g of KH 2 PO 4, 2.5 g of MgSO 4 7H 2 O, 0.02 g FeCl 3 6H 2 O, nd 150 ml V-8 juice (Cmpbell Soup Compny Inc.) in 250 ml Erlenmeyer flsks. Cultures were incubted for 5 dys on rotry shker 85

7 t 60 rpm t room temperture (20 ± 1 C). The grown mycelium ws collected into sterilized blender cup (Wring Commercil, Torrington, CT) nd homogenized gently with two 20 s bursts nd then inoculted onto utoclved brley (Hordeum vulgre L.) grits. For this, whole brley grins were ground nd sieved to mm dimeter grits. Three hundred g of brley grits were trnsferred into utoclvble bgs with brethble ptch 44 x 20.5 cm, 0.02 µm filter: 24 mm (SunBg, trnsprent, SIGMA-ALDRICH, Montrel, QC). Two hundred nd ten ml of distilled wter ws plced into ech of the bgs nd utoclved t 121 o C for 20 min. After utoclving the bgs were llowed to cool nd 15 ml liquot of the liquid S. minor mycelil culture ws trnsferred septiclly into ech bg. Inoculted bgs were incubted t 20 ± 1 o C in the drk nd shken on the 3 rd to 6 th dys of incubtion. The contents of ech bg were then dried seprtely by spreding the colonized brley grits onto mesh trys for 12 hr under lminr flow. The dried inocul ( w. 0.4) were plced in plstic bgs (PolyBgs, 17.5 x 40 x 7.5 cm, Gerrity Corrugted Pper Products, Concord, ON) nd the bgs were seled, nd stored t 4 o C. These S. minor grnulr formultions were used in our experiments fter two to four weeks of storge. The vibility of the S. minor preprtions ws verified by incubting ten grnules from ech bg on PDA pltes. Colony dimeters were mesured fter 24 nd 48 h of incubtion t 20 ± 1 o C in the drk. Additionlly, ten grnules from ech bg were plced onto excised dndelion leves mintined on moist sterile filter ppers in Petri dishes nd incubted t 20 o C in the drk; one grnule per lef. The dimeter of the lesions cused by the fungus ws mesured fter 24 nd 48 h of incubtion. Previous unpublished qulity control studies indicted vible btches to hve colony dimeter of mm fter 24 h nd mm fter 48 h nd virulent btches to 86

8 hve n verge lesion dimeter >15 mm fter 48 h of incubtion (Appendix-4). S. minor preprtions were only used in our experiments if they met these criteri Effect of S. minor nd mowing height on dndelion control in the greenhouse Dndelion seeds collected in spring 2002 from lwns on the Mcdonld cmpus, McGill University, Ste-Anne-de-Bellevue, QC nd stored t 4 o C were sown onto potting soil (2/3 blck psteurized soil nd 1/3 Pro-mix (Premier Promix, Premier Horticulture Ltee, Riviere-du-Loup, QC) in plstic continers (40L x 32W x 20D cm, 19 L cpcity; Sterilite Inc., Montrel, QC). One week fter germintion, seedlings were thinned to four equidistnt seedlings per continer. Two weeks fter germintion of the dndelion seeds, 2.5 g of commercil grss seed mixture [30% Kentucky bluegrss (Po prtensis), 40% creeping red fescue (Festuc rubr L. vr. rubr) nd 30% turf type perennil ryegrss (Lolium perenne L.), C.I.L. Golfgreen, Brntford, ON] ws scttered over the surfce of ech continer. Grss ws cut weekly with hedge shers (PlntSmrt, Wl*Mrt, Cnd), commencing three weeks fter grss sowing to height of 5, 10, 15 or 20 cm. Four weed control tretments were imposed: (1) untreted, (2) spot ppliction of 0.2 g plnt -1 non-colonized utoclved brley grits, (3) spot ppliction of 0.2 g plnt -1 brley grnulr formultion of S. minor, nd (4) brodcst folir ppliction of Killex t 1.7 kg.i h -1 (25 ml of the 0.6% originl concentrtion per continer). The herbicide ws pplied with 1.18 L vcuum spryer (Home nd Grden spryer. Model no RLF10-Mster Premium. Root-Lowell Mnufcturing Co, Lowell, MI). The weed control tretments were pplied three weeks fter initition of grss cutting heights nd thus 87

9 dndelion plnts were eight weeks old. Prior to weed control tretment pplictions, ll plnts were misted lightly with wter for two min. durtion to id brley grit dhesion with dndelion leves. The plnts were grown in the greenhouse t 20 ± 2 o C with 15 h of light per dy t minimum photon flux density of 350 ± 50 µmol m -2 s -1. Plnt continers received progrmmed drip irrigtion of 150 ml 3 x dy -1. To enhnce the estblishment of the plnts, 15:15:30 N-P 2 O 5 -K 2 O with micronutrients (Plntex, Plnt Product Co, Brmpton, ON) ws pplied t 3.5 g L -1 when the dndelions were five weeks old. Symptoms of dmge to dndelions were visully estimted weekly for four weeks fter ppliction using 0 to 10 visul scle compred to the control within the sme mowing height nd the sme block, where 0 = < 9%, 1 = 10-19%.9 = 90-99% nd 10 = 100% collpse of boveground biomss. Dt were converted bck to percentge for nlysis nd presenttion (fter Schnick et l. 2002). Plnt regrowth ws mesured s reduction in % dmge by estimting the biomss of new leves produced post inocultion compred to the control within the sme mowing height nd the sme block. Dmge estimtes of the four plnts in ech continer were verged nd nlyzed s one experimentl mesure. The number of post inocultion dndelion plnts tht survived ws recorded weekly for six weeks. Six weeks fter ppliction, ll of the dndelion plnts were crefully removed from the soil to extrct their entire tp root. The roots were thoroughly wshed nd dissected bove the crown, seprting bove ground nd below ground biomss. All lef or root biomss from ech continer ws bulked, plced in pper bgs, oven dried t 80 o C for 72 h, nd then weighed. The experiment ws split-plot design with five replictions nd ws conducted 88

10 twice through time (Jn 2003 nd Jn 2004). Min plots were weed control tretments nd subplots were grss heights. In the repet tril in 2004, the non-colonized utoclved brley grits tretment ws omitted nd excluded from the pooled nlysis s no significnt differences were obtined for ny of the studied prmeters when compred with the untreted tretment in the first tril. Dt for ech prmeter from the two experimentl trils were subjected to the Brtlett test of SAS (SAS Institute Inc., Cry, NC, 2002) to test for homogeneity of vrinces. Dt for ll prmeters were homogeneous, thus they were pooled. The min effects of weed control tretments, mowing heights, nd their interction were determined using ANOVA of SAS, nd the effects of time on boveground dmge, dt were nlyzed using SAS GLM procedure of repeted mesures. The mens were seprted using the Tukey test t P = 0.05 (SAS Institute Inc., Cry, NC, 2002) Effect of S. minor nd mowing height on dndelion control in the field Two field experiments were conducted, one in ech of two different sites on the Mcdonld Cmpus of McGill University, in Ste-Anne-de Bellevue, QC (45 o 25'N ltitude, 73 o 55'W longitude, m elevtion). The climte dt for the two yers of study (2003 nd 2004) re summrized in Tble 5.1. Study Site-1 (2003 experiment) ws pproximtely 600 m 2 on lomy snd soil (corse snd = 9%, fine snd 73%, silt= 12%, cly= 6%) with ph of 7.15 nd 8% orgnic mtter. The lwn ws estblished in 1973 nd received low mintennce mngement throughout its history except for repeted mowing during the growing seson (My to October). The grss swrd ws pproximtely 60% Kentucky blue grss, 30% perennil ryegrss, ptches of timothy 89

11 (Phleum prtense L.), nd rre occurrences of nnul blue grss (Po nnu L.). The lwn flor ws highly diversified with brodlef weeds (17 species were observed throughout the study period) nd the dominnt weed species ws dndelion (Trxcum officinle) with plnts m -2 recorded prior to the spring tretment ppliction. Study Site-2 (2004 experiment) ws locted in n open lwn re of pproximtely 600 m 2 with low humn disturbnce, estblished in The turf t site -2 ws in better visul qulity thn in Site-1 with minly Kentucky blue grss (~90%) nd 10% red fescue (Festuc rubr L.). Herbicides hd not been pplied for the pst 10 yers nd the mjor brodlef species were dndelion (60-70 plnts m -2 ), white clover (Trifolium repens L.) nd brodlef plntin (Plntgo mjor L.). The soil ws lomy snd (12% corse snd, 75% fine snd, 7% silt nd 6% cly) with ph of 7.2 nd 6.6% orgnic mtter. Both field experiments were estblished in My nd mintined until the end of October. Study sites (20 x 30 m) were mrked with metl posts nd plstic ropes nd the corners of ech plot were permnently mrked by wooden sticks to mintin plot integrity for the durtion of the study. The re of the experimentl unit (plot) ws one meter squre with 0.8 m lleys between plots. The distnce between ny two blocks ws 2-3 m. The experimentl design ws split plot with four replictions. Mowing heights were the min plots nd weed control tretments were the subplots. The three levels of mowing heights, 3-5 cm, 7-10 cm, nd cm were initited two weeks prior to weed control tretment pplictions. Plots were mowed weekly, except during the two-weekspost weed control tretment period, with gs powered rotry push mower. Grss clippings were returned during July nd August to ct s source of nitrogen (Kopp & 90

12 Guillrd 2002), but removed during other months during the six-weeks-post tretment periods to void cross contmintion between blocks. Four levels of weed control were imposed on 15-My nd on 15-September t ech study site: 1) untreted control, 2) brodcst folir ppliction of Killex t 1.7 kg.i. h -1 (200 ml m -2 of 0.6% of originl concentrtion), 3) brodcst ppliction of 60 g m -2 grnulr formultion of S. minor, nd 4) brodcst ppliction of 120 g m -2 grnulr formultion of S. minor. The herbicide ws pplied onto the grss surfce using 1.18 L vcuum spryer. The S. minor formultion ws pplied using 200 ml plstic bottle fitted with perforted lid (~10 mm dimeter) with suitble openings to pss the brley grits. If there ws no rinfll on the dy of ppliction or the grss ws not wet, the entire field ws sprinkler irrigted for two hours prior to lte fternoon tretment pplictions. No dditionl irrigtion or fertiliztion mngement ws pplied during the course of the study. The number of dndelions ws counted in ech plot the dy before weed control tretments, nd on single dy in the lst week of ech month therefter. In order to monitor post tretment recovery of dndelions, 10 dndelion plnts in ech of the fungl treted plots were rndomly mrked using white coloured pins prior to tretment pplictions. In Site-2, the number of dndelion seedling recruits versus mture plnts in ech plot ws counted every two weeks strting two weeks fter ppliction (30-My) nd continuing to the end of June. Dndelion density dt were djusted using the "Before-After, Control-Impct (BACI) eqution (Green 1979) to overcome sptil heterogeneity dt differences from pretreted time (15 My) nd lso to overcome the effect of progression in time. 91

13 BACI vlue = (At / Bt) / (Ac/Bc) x 100 (eqution 1) Where, B = dndelion density t the time of tretment (before the impct), A = dndelion density fter tretment (fter the impct), t = tretment, nd c = control. The control used for comprison ws the untreted plot t the sme mowing height in the sme block. To relize the effect of mowing heights on dndelion in untreted plots, the vlue obtined from the three mowing heights within the sme block were verged nd used s control in eqution 1. Dt for ech time period from the two yers (two study sites) were subjected to Levene procedure (SAS Institute Inc., Cry, NC, 2002) for testing homogeneity of vrinces. Becuse of differences in environmentl conditions between yers nd/or loctions, monthly dt were heterogeneous except for the spring pre-tretment ppliction (mid of My); two-weeks-post ppliction (end of My); six-weeks-post ppliction (end of June), nd for post control seson verge. Therefore, the seson verge, the two-weeks- nd six-weeks-post ppliction dt from the two experiments were pooled nd treted s one experiment. The 120 g m -2 S. minor tretment ws excluded from the nlysis s no significnt differences were obtined in comprison with the 60 g m -2 tretment for ll studied prmeters. Normlity for ech prmeter ws tested on model residuls using the Shpiro-Wilk test (SAS Institute Inc., Cry, NC, 2002). Dt were nlyzed using ANOVA to determine the significnt interctions mong mowing heights nd different weed control tretments. Differences in tretment mens were determined using Tukey's test (SAS) t P =

14 5.4. Results Effect of S. minor nd mowing height on dndelion control in the greenhouse Eight-week-old dndelions (6-8 leves) were highly susceptible to the S. minor ppliction. The bove ground biomss in ll treted plnts collpsed during the first week fter ppliction (Figure 5.1). Symptom expression ws more rpid with S. minor nd t 1-month-post ppliction it ws more effective thn the herbicide (Killex ). The herbicide nd the S. minor tretments cused highly significnt (P 0.01) bove ground biomss dmge to the dndelion plnts. S. minor cused significntly more dmge thn the herbicide over the entire study period (Figure 5.1). Mowing height hd no significnt effect on dndelion bove ground dmge in ny of the weed control tretments (Figure 5.1), but the bove ground nd root biomss of untreted dndelion were significntly reduced under the 5-10 cm compred with cm mowing heights (Figure 5.2A & B). At six-weeks-post ppliction, both S. minor nd the herbicide cused highly significnt (P 0.01) bove ground nd root biomss reduction compred with untreted plnts. Biomss reductions were greter for S. minor (Figure 5.2A & B). The plnts surviving the initil S. minor tretment hd short, wek roots nd few leves sprouted from the crown (Figure 5.3). Regrowth following S. minor tretments ws lest when combined with the closest mowing height (5 cm) (Figure 5.4) Effect of S. minor nd mowing height on dndelion control in the field The gretest effect of S. minor ws observed two weeks fter ppliction when dndelion densities declined by 65 to 95%. Under the 7-10 nd cm mowing heights, the effect of the S. minor (60 g m -2 ) ws significntly greter thn tht of the herbicide, but t 93

15 the 3-5 cm mowing height, the effect of S. minor nd the herbicide were similr two weeks fter ppliction (Figure 5.5A). Six weeks fter ppliction, dndelion densities within untreted plots were reduced by mowing t 3-5 cm compred to mowing t 7-10 or cm (Figure 5.5B). Dndelion popultion densities were reduced more by the herbicide thn by S. minor t ll mowing heights six weeks fter ppliction (Figure 5.5B). Under close mowing (3-5 cm), dndelion densities in S. minor-treted plots incresed from 35 to 80% between two nd six weeks post ppliction (Figure 5.5B). The BACI post ppliction verge of dndelion popultion densities over the entire growing seson (Figure 5.5C) illustrtes the recovery bility of the dndelion popultion nd lso the ccumulted ffect of mowing. While the effect of mowing heights ws conceled under the strong effect of the chemicl herbicide tretment, it ws pprently ble to interct significntly with sesonl verge dndelion density in untreted nd fungl treted plots (Figure 5.5C). Prior to the spring tretment, pproximtely 50% of the dndelion plnts in ll plots were new seedling recruits (Figure 5.6). In untreted plots, these recruits decresed grdully to pproximtely 10-20% of the popultion by six weeks fter ppliction (Figure 5.6) mostly due to rise in temperture (Tble 5.1). In herbicide treted plots, no seedlings were observed fter two- nd four-weeks-post ppliction nd very few t six weeks fter ppliction. In S. minor treted plots, the rtio of recruitments to mture plnts ws highly vrible ccording to time nd mowing fctors. Within the first twoweeks-post ppliction, no seedlings were observed under the 7-10 nd cm mowing heights but smll number (less thn 5 m -2 ) ws reported under the closest 94

16 mowing height (Figure 5.6). After four weeks, the lrge recruitment of seedlings under the closest mowing height resulted in significntly more mture plnts fter six weeks compred with the other mowing heights (Figure 5.6). Two weeks fter S. minor tretment, the percentge of dndelion plnts tht regenerted fter complete folir dmge ws significntly less in the spring (up to 15%) thn in the fll (up to 32%) tretments. Although the mowing height did not ffect the percentge of root regrowth fter the spring tretment of S. minor, there ws significntly less regrowth under the closest mowing height (22%) compred with the two higher heights (33%) fter the fll tretment (Figure 5.7). 95

17 Tble 5.1. Wether dt for Ste-Anne-de-Bellevue, Quebec during the two yers of study 2003 nd Environment Cnd Meteorologicl Dt. Ste-Anne-de-Bellevue Sttion. Temperture ( o C) Reltive humidity (%) Rinfll (mm) Month / yer Averge Min Mx Averge Min Mx Totl monthly My My June June July July August August September September October October

18 Figure 5.1. Effect of mowing heights nd weed control tretments on bove ground dmge (%) of dndelion in grss plnting. Within ech post ppliction time, the mens of the three weed control tretments re significntly different t the 1% level ccording to Tukey test. (1) : Spot ppliction of grnulr formultion of S. minor t 0.2 g plnt -1. (2) : Brodcst folir ppliction of Killex herbicide (2,4-D, mecoprop nd dicmb) t rte of 1.7 kg.i. h -1 % Above ground dmge of dndelion cm mowing height 10 cm 15 cm 20 cm Weeks post ppliction Sclerotini minor (1) Killex (2) Untreted 97

19 Figure 5.2. Effect of mowing heights nd weed control tretments on boveground (A) nd root (B) biomss of dndelion, six weeks fter tretment ppliction. Mowing heights were initited one month prior to tretment ppliction nd mintined throughout the study period. Within the sme grph, brs with common letter re not significntly different t the 5% level ccording to Tukey s test. (1) : Spot ppliction of grnulr formultion of S. minor t 0.2 g plnt -1. (2) : Brodcst folir ppliction of Killex herbicide, t rte of 1.7 kg.i. h

20 Untreted Sclerotini minor (1) Killex (2) Above ground dry mtter (g) (A) b b b b b b Root dry mtter (g) (B) bc de b bcd bcd cde 0.0 c c c c e e e e Mowing height (cm) 99

21 Figure 5.3. A comprison of the bove nd below ground biomss between untreted dndelion (two plnts bove) nd dndelion regrown six weeks fter inocultion with Sclerotini minor (three plnts below). Untreted Treted with S. minor 100

22 Figure 5.4. Effect of mowing height on dndelion root regrowth, six nd eight weeks fter S. minor inocultion. Within ech post ppliction time, brs with common letter re not significntly different t the 5% level ccording to Tukey s test weeks post inocultion 8 weeks post inocultion % of regrowth from roots b b b Mowing height (cm) 101

23 Figure 5.5. Effect of mowing heights nd weed control tretments on post ppliction dndelion density fter two weeks (A), six weeks (B), nd seson verge (C). Mowing heights were initited two weeks prior to spring ppliction nd mintined throughout the experiment. Within the sme grph, brs with common letter re not significntly different t the 5% level ccording to Tukey test. (1) : Brodcst folir ppliction of Killex herbicide, t rte of 1.7 kg.i. h -1. (2) : Brodcst of grnulr formultion of S. minor t 60 g m -2. (3) : Before-After, control-impct = (At / Bt) / (Ac /Bc) x 100, B = density before tretment; A= density fter tretment; t = treted nd c = control. 102

24 Dndelion density (BACI (3) vlues) A b b bc bc bcd d e e 3-5 cm 7-10 cm cm B b b c c d d d 3-5 cm 7-10 cm cm Dndelion density (BACI (3) vlues) C b c d d d d d 3-5 cm 7-10 cm cm Untreted Killex (1) Sclerotini minor (2) Mowing heights 103

25 Figure 5.6. Effect of mowing heights nd weed control tretments on seedling nd mture plnt densities of dndelion fter spring ppliction (15 My 2004). S. minor rte = 60 g m -2 ; Killex herbicide rte = 1.7 Kg.i./h. Within plnt stge in ech grph, brs lbelled with common letter re not significntly different t P = 0.05 ccording to Tukey s test. 104

26 No of plnts / m No of plnts / m 2 No of plnts / m 2 2 No of plnts / m Seedlings Mture plnts Untreted Herbicide S. minor Untreted Herbicide S. minor b r 3-5 cm Untreted Herbicide S. minor Untreted Herbicide S. minor s 3-5 cm Untreted Herbicide S. minor Untreted Herbicide S. minor bc st b u e t 3-5 cm f u c t b r d rs r r b rst 7-10 cm b 7-10 cm 7-10 cm f u de t u c u bc s de rs Untreted Herbicide S. minor b s cm Untreted Herbicide S. minor r cm Untreted Herbicide S. minor t Pre ppliction (15-My) cm 6-weeks post ppliction f 2-weeks post ppliction b u u c u 4-weeks post ppliction cd t bcd s e rst Untreted Herbicide S. minor Untreted Herbicide S. minor Untreted Herbicide S. minor 3-5 cm 7-10 cm cm 105

27 Figure 5.7. Effect of mowing heights on regrowth of dndelion roots, three-weeks-post tretment ppliction. Mowing heights were initited two weeks prior to spring ppliction nd mintined throughout the experiment. Brs with common letter re not significntly different t the 5% level ccording to Tukey s test cm 7-10 cm cm % of regrowth from roots bc c c b 0 Spring Fll 106

28 5.5. Discussion Previous studies on the virulence nd efficcy of S. minor on dndelion hve demonstrted its biocontrol potentil (Ciotol et l. 1991; Riddle et l. 1991; Brière et l. 1992; nd Stewrt-Wde et l. 2002). Our results support those studies nd indicte the importnce of correct mowing regimes on dndelion survivl rtes s influenced by S. minor. In the bsence of weed control, periodic mowing t ny of the studied levels ws not effective in controlling dndelion. However compred to other heights, periodic mowing t 5cm cused significnt reduction in root biomss nd field popultion density of dndelion, but close mowing height cused significnt increse in brodlef groundcover percentge nd diversity (Chpter 6). In nother study, mowing every two weeks eliminted field bindweed but did not prevent dndelion coloniztion (Timmons 1950). The high regenertive cpcity of dndelion roots (Stewrt-Wde et l. 2002b) is the min cuse of recoloniztion. The level of mowing height cpble of exerting significnt stress on plnts is highly vrible nd depends minly on the plnt species, the surrounding environment, the time of the yer, nd the frequency of mowing (Znoni et l. 1969; Meyer & Schmid 1999; Liu & Hung 2002 nd Nrr et l. 2004). Although close mowing my be hrmful to weeds, it my lso be hrmful to turfgrss species resulting in incresed weed infesttion by tipping the competitive blnce in fvor of the weeds (Busey 2003). The mjor turfgrss species in our fields were Kentucky bluegrss nd perennil ryegrss, nd in well mintined turf, both species re recommended to be mowed t medium height, ~5-7 cm (Turgeon 1985; Fry & Hung 2004). Therefore, our grss mowing regime t 5 107

29 cm my hve impcted the growth nd survivl of dndelions resulting in incresed coloniztion by competitive species with more resilience nd tolernce to close mowing. Under greenhouse conditions, the severe effect of the fungus on bove nd below ground biomss of dndelion ws pprent on the third dy fter ppliction nd ws complete within two weeks. Mowing height hd no effect on the efficcy of S. minor in the greenhouse experiment. This could be explined by the rpid destructive nture of S. minor, necrotrophic fungus cusing wilting, collpse nd deth of the infected plnt prts (Abwi & Grogn 1979; Melzer et l. 1997). This rpid destruction is chieved in high moisture conditions over wide rnge of temperture (5-25 o C) (Melzer & Bolnd, 1994). Therefore, the vrition mong the microenvironments within these mowing regimes likely did not ffect S. minor performnce. In our field experiments, two dtes were chosen, 15 My nd 15 September, to synchronize two fctors; 1) suitble climtic condition for S. minor to cuse disese, nd 2) high bundnce of dndelion during periods of recruitment. Averge dily men temperture, reltive humidity, nd dew point for the two-weeks-post ppliction period were 15 o C, 78% RH nd 10.6 o C, respectively. Under these conditions the fungus needs only 2-3 dys to germinte, invde nd colonize dndelion plnts. The mximum effect of the fungus occurs within dys nd then the fungus dies nd disintegrtes on the soil surfce (Appendix-2). Thus, the mximum effect of S. minor on dndelion density ws obtined two weeks fter ppliction. Folir turfgrss pthogens cn cuse more disese in microenvironments with higher cnopies (Fgerness & Yelverton 2001; Mrtin et l. 2001), but disese incidence of lettuce, mjor host of S. minor, ws not ffected by different microclimtes of the crop cnopy (Melzer & Bolnd 1994). 108

30 By two weeks fter ppliction in the field, the fungus destroyed most of the dndelion popultion in ll treted plots without being influenced by chnges in the microenvironment under the different mowing heights. In the 3-5 cm mowing height plots, bre soil ws exposed within the thin grss cnopy llowing greter sunlight interception t the soil surfce. Consequently, high dndelion seedling recruitment occurred fter the fungus lost vibility nd died. Full light is mjor requirement for germintion of dndelion seeds (Letchmo & Gosselin 1996) nd buried seeds re unble to germinte (Noronh et l. 1997). After the effect of S. minor declined, dndelion seeds strted to germinte nd seedling emergence continued until the end of June, nd then germintion declined. At mid June, significntly greter seedling emergence occurred in plots with the 3-5 cm mowing height compred with the 7-10 nd cm mowing heights. Therefore, t sixweeks-post ppliction, popultion density of dndelion incresed significntly in the 3-5 cm mowing height plots compred with other mowing heights nd becme similr to untreted plots. In plots treted with S. minor, dndelion re-estblished from seeds nd survived better under the 3-5 cm mowing height thn under the 7-10 cm nd the cm mowing heights. Consequently, dndelion biocontrol with the fungus t the two higher mowing heights ws s effective s the chemicl herbicide. The initil effect of the herbicide on dndelion density ws much slower thn tht of the fungus, but by six-weeks-post ppliction the density ws reduced by 90%, significntly lower thn S. minor effect. Mowing height did not interct with the herbicide effect. Seedling recruitment fter the herbicide tretment ws very low, due to persistence of the herbicide in the soil with hlf-life of dys reported (Cox 1999). 109

31 Treting dndelion t the reproductive stge with Killex reduced the germintion potentil down to 4.8% nd 18.4% fter spring nd fll tretments, respectively (Abu- Dieyeh et l. 2005). Moreover the herbicide tretment significntly reduced dndelion seed bnk (10 cm depth) compred with the untreted, control plots (Chpter 7). Root regenertion of dndelion is one of its competitive fetures nd mkes control difficult tsk (Stewrt-Wde et l. 2002b) since smll section of root cn propgte new plnt when covered by 5-10 cm of soil (Flkowski et l. 1989). Our greenhouse results indicted tht S. minor cused highly significnt reduction of root biomss, but 20-50% of the treted dndelions hd resprouted from the roots t six-weeks-post ppliction. Resprouted plnts were very wek lcking vigour nd chrcterized by tiny roots nd tiny lef shoots. Thus S. minor is not only ttcking boveground biomss of dndelion but lso ffecting the roots, leving them less likely to survive grss competition nd more prone to other biotic nd biotic stresses, especilly winter frost. Under greenhouse conditions, the closest mowing height cused significnt reduction of sprout percentge compred with the two higher mowing heights (15 nd 20 cm). There ws no difference in the number of resprouted dndelions obtined t eight-weekscompred with six-weeks-post ppliction. No signs of disese development by S. minor were observed on these new shoots, but the wekness of the root cused by the direct effect of the fungus on the mother plnts my diminish its survivl under the dditionl stress of grss competition. The extensive defolition stress cused by repeted mowing t the 5 cm level reduced the root biomss significntly in untreted plnts nd this my explin the reduction of regrowth under this mowing height. Similrly in other perennil plnts, repeted defolition reduced regrowth bility of Rnunculus cris L., fter 110

32 infection by the fungus S. sclerotiorum (Green et l. 1998) nd repeted cutting integrted with rust fungus, Puccini punctiformis exerted synergistic control effects on growth rte nd reproductive success of Cirsium rvense (Kluth et l. 2003). Mowing heights were initited two weeks prior to the spring ppliction, hence stress on the roots would not be strong, wheres the ccumultive effect of defolition stress by mowing t the 5 cm level over the growing seson prior to the fll tretment resulted in significntly less root regrowth compred with other heights. Dndelion, lloctes more resources for flowering nd vegettive growth in the spring (Cyr et l. 1990) while nitrogen resources re restored in the roots t the end of summer (Rutherford & Decon 1974) which my explin the increse in the percentge of regrowth in the fll. The mximum regrowth percentge reported in the field is lower thn greenhouse experiment which indictes tht field environments exert more ecologicl stress on dndelion leding to improved performnce of S. minor. In conclusion, in low-mintined cool-seson turf environment, integrting S. minor with pproprite mowing could be s effective s herbicide. Dndelion suppression ws the lest under close mowing due to the new opened environment which induced more germintion from the soil seed bnk. Extensive periodic defolition by mowing nd ppliction of S. minor on flowering dndelion (spring ppliction) might be the cuse of decresing dndelion root survivl through exhustion of the root crbohydrte nd nitrogen reserves. Understnding these physiologicl chnges in dndelion roots under different mowing heights will support successful deployment of S. minor for dndelion control. Additionlly, monthly monitoring of the weed species dynmics nd turfgrss qulity provides good portryl of the biocontrol system. 111

33 5.6. Acknowledgments The uthors thnk Philippe Seguin for his vluble help in sttisticl nlysis nd lso Miron Teshler for his help in field nd technicl work. The finncil support from Hshemite University, Zerk, Jordn nd from the Nturl Sciences nd Engineering Reserch Council of Cnd (NSERC) Ide to Innovtion (I2I) grnt re grtefully cknowledged. 112