EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT

Transcription

1 EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT J. E. Adskveg, H. Förster, D. Thompson, D. Felts, D. Cry, J. Connell, nd R. Buchner Cooperting: L. Wde, Aryst LifeScience ABSTRACT Although precipittion t mny loctions in the wlnut growing res of Cliforni ws very high in the spring of 2011, due to the lte occurrence of rin nd sub-optiml tempertures, the overll incidence of wlnut blight ws only moderte on the erly-blooming wlnut vrieties used in our studies. In field studies under nturl rinfll conditions, the efficcy of copper (e.g., Kocide 3000, Bdge X2, Cuev, nd n experimentl Isgro mteril)-mncozeb (e.g., Mnzte ProStick) tretments for reducing the incidence of wlnut blight ws compred to tht of copper or mncozeb lone nd to non-copper-mncozeb tretments. The ltter ones included two formultions of ksugmycin, the fungicides Quintec nd Cptn, the nturl products Regli nd Citrox/ProAlexin, the host defense ctivtor ReZist, the biocontrol Blossom Protect, nd selected mixtures. The results cn be summrized s follows: 1. Copper-Mnzte ws effective in ll trils conducted under low to moderte disese pressure. 2. Regli pplied by itself did not reduce the incidence of blight from tht of the control under modertely high disese pressure conditions (22.4 % disese in the control), only numericlly reduced the disese from the control in tril under lower disese pressure (14.4% disese in the control), nd ws very effective in nother tril where the lowest disese pressure (12.3% disese in the control) ws observed. Regli-Kocide 3000 ws sttisticlly similrly effective to Regli or Kocide 3000 lone in the first tril; ws sttisticlly similrly effective to Regli or copper mterils by themselves in the second tril; nd ws significntly more effective thn Regli or Kocide 3000 lone in the third tril. Thus, s previously documented, Regli cn be effectively used for wlnut blight mngement only under low disese pressure conditions. Regli-Kocide 3000 mixtures cn provide benefit over Regli by itself considering tht our trils in 2011 were conducted in orchrds with no or moderte copper resistnce levels in the pthogen popultion. 3. In comprison of copper tretments, Kocide 3000, Bdge X2, Cuev, nd n experimentl Isgro mteril were similrly effective when mixed with Mnzte. Among new tretments, mixtures of Ksumin with Mnzte, copper, or Cptn performed very well nd the efficcy ws similr to Kocide-Mnzte. The biocontrol Blossom Protect nd the nturl products Citrox/ProAlexin were very effective in one study where they were used nd these products will need continued evlution. Another biocontrol, Actinovte, numericlly, but not significntly reduced the disese from the control. 4. Field trils over the yers nd lso in 2011 demonstrted tht Ksumin mixed with copper or n EBDC showed consistent high efficcy for controlling wlnut blight. 5. Two new soil pplictions tht ffect stomtl opening of plnts were lso evluted for the mngement of blight. Four pplictions of RootZone were ineffective, wheres for CoMo Clssic, trend in disese reduction ws observed. 6. In n ppliction timing study, sprys were done either weekly, or before or fter predicted rins. Six weekly pplictions or four sprys pplied minly before rins were more effective Cliforni Wlnut Bord 265 Wlnut Reserch Reports 2011

2 thn pplictions timed fter rins. We previously documented the effectiveness of fter-rin pplictions. The lck of efficcy in this yer s tril cn be explined by the high mount of rinfll tht occurred over severl-dy period tht pprently ws highly conducive for disese development. 7. Vrietl susceptibility to wlnut blight ws gin evluted under simulted rin conditions. 8. Specific primers for X. rboricol pv. juglndis were developed nd were used in the identifiction of the pthogen in bud popultion studies. Pthogen popultion diversity ws evluted in severl orchrds. To dte, multiple strins of the pthogen hve been identified in our collections from wlnut growing res of Cliforni over number of yers. In 2011, some orchrd loctions hrbored single genotype, but in others mixed popultion of distinct genetic strins ws identified. INTRODUCTION Wlnut blight, cused by the bcterium Xnthomons rboricol pv. juglndis, continues to cuse crop losses in mny centrl nd northern Cliforni orchrds with vrying incidences from yer to yer. The pthogen ttcks ctkins, femle blossoms, fruit, green shoots, leves, nd buds of English wlnut. Fruit infections occur soon fter flowering nd these infections ccount for most of the economic loss. The extended period of host susceptibility is one of the min chllenges in controlling this disese. The pthogen survives from one yer to the next in twig lesions, buds (living nd ded), nd disesed fruit tht remin on the tree. The optimum temperture for bcteril growth is 28 to 32C nd the minimum is bout 1C, but lesions on immture fruit generlly do not occur below pproximtely 12C or bove 25C (Adskveg et l. 1994, 1998, 1999). Furthermore, disese increses with incresed wetness durtion within this temperture rnge (Adskveg et l. 1998, 1999) nd the disese does not increse when dily high tempertures exceed 27C regrdless of precipittion (Adskveg et l. 2009). Bsed on nlysis of wetness period durtion nd temperture dt from field wether sttions nd ctul disese progression, we developed XnthoCst s model to predict infection periods for wlnut blight to clculte the risk of disese. The website currently provides the bsic XnthoCst informtion with XnthoCst indices for wether sttions locted between Red Bluff nd Dvis. The XnthoClcultor llows individul web-bsed forecsting for different grower fields nd loctions. Thus XnthoCst system provides regionl forecsts, nd promotes judicil use of pesticides with disese control similr to clendr-bsed progrms. The UC-IPM website is still under development. One reserch gol is to use dew point temperture nd precipittion s n lterntive to lef wetness dt to identify lef wetness periods without ctully mesuring lef wetness. This chnge will offer considerble flexibility bsed on dt tht re vilble on CIMIS nd other dtbses. Our timing studies indicted tht erly femle flower emergence pplictions followed by pplictions bsed on XnthoCst provided the most consistent disese control. A ctkin bctericide tretment ws only needed when high precipittion occurred in n orchrd with history of high disese levels. Furthermore, incidence of blight ws significntly lower when bctericide tretments were bsed on clendr or on XnthoCst forecsts thn when only single erdiction or inoculum reduction bud brek tretments were pplied. Becuse we lso showed tht bcteril popultions cn exponentilly increse on disesed fruit under fvorble Cliforni Wlnut Bord 266 Wlnut Reserch Reports 2011

3 environmentl conditions nd cn rech very high levels within 3 to 6 hours, inoculum levels re not considered in the XnthoCst model fter the initil tretment for blight mngement. We pln to continue with development of the XnthoCst model with trils focused on timing of bctericide tretments in reltion to forecsted wether events. Specificlly, we will continue to look t pplictions of bctericides before nd immeditely fter mjor rin events, s well s excluding pplictions when dily high tempertures exceed 27C regrdless of rinfll. Our reserch project on the mngement of wlnut blight since 1994 hs ssisted in the development of copper-mneb (e.g., Mnex) tretments nd demonstrted the efficcy of zinc compounds nd zinc-mnex mixtures. We initited the rottion of copper- nd zinc-bsed bctericides nd use of copper-zinc mixtures (e.g., Kocide 20/20, Nordox 30/30) to minimize the development of copper-resistnt popultions of the pthogen. In ddition, new reduced-copper products (e.g., Kocide 3000, Bdge) were shown to be highly effective. From 2006 to 2010 we showed tht products contining mncozeb (Mnzte, Dithne, Penncozeb, etc.) insted of mneb (e.g., Mnex) re effective in mixtures with copper or the ntibiotic Ksumin. Although the EPA hs prompted the cncelltion of mneb in the United Sttes in , n emergency registrtion of Mnzte (DuPont sold this Product to UPI Ag Products in 2010) ws grnted by the EPA in 2010 nd A full registrtion is expected in the coming yers. Mnzte is formulted s flowble liquid (F) or dispersible grnulr (DF) mteril nd slight differences were observed between the formultions when mixed with copper products. Thus, n evlution of rin fstness of different mixtures is pproprite. Over the yers, we evluted mny possible lterntives. In 2005, we first identified the potentil for ksugmycin (Ksumin, Aryst Life Science) in wlnut blight mngement. This ntibiotic is used in griculture in other countries but not in humn or niml medicine. In Sept. 2005, food tolernce ws estblished for Ksumin on imported griculturl products. In 2007 IR-4 residue trils for ksugmycin were conducted nd the registrtion to EPA ws submitted in Jnury Currently, federl registrtion is trgeted for 2012 nd Cliforni stte registrtion for In our studies, Ksumin lone significntly reduced wlnut blight incidence nd, under low rinfll, it ws highly effective. Under high rinfll (simulted with overhed irrigtion or nturlly occurring), mixtures of the ntibiotic with EBDC or copper resulted in n incresed efficcy nd outstnding performnce. Moreover, in most trils, Ksumin-EBDC or Ksumincopper mixtures were similr or higher in efficcy to copper-ebdc mixtures. The registrnt is providing new formultions of the ntibiotic tht re more concentrted nd in 2011 we evluted Ksumin 8L. Thus, dditionl studies hve to be done with ksugmycin to ensure successful lunch on wlnuts. Additionl reserch is lso needed on lterntive tretments such s nturl compounds, biocontrols, nd enhncers of host resistnce, some of them demonstrted promising results before. These include ProAlexin nd Citrox, Actinovte, Blossom Protect, BioForge, nd ReZist. Actinovte ws registered for use in 2011 bsed on our efforts nd should provide copper lterntive for orgnic growers. The nti-trnspirnt Root Zone showed promise in 2010 nd ws evluted gin in 2011 together with similr compound, CoMo Clssic. These tretments pprently stimulte bscisic cid production tht triggers stomtl closure. Our overll gol is to ultimtely hve multiple tretments vilble tht re equl to or more efficcious thn trditionl copper-contining compounds nd tht could be used in orchrds with copper-sensitive nd copper-resistnt popultions of the wlnut blight pthogen. This would result in lower environmentl impct nd in lower chnce for resistnt popultions of the pthogen to develop nd spred. Cliforni Wlnut Bord 267 Wlnut Reserch Reports 2011

4 We lso continued to evlute wlnut vrieties for their nturl resistnce to blight nd exmined fruit for morphologicl chrcteristics tht re possible mechnisms of resistnce nd tht potentilly my be used s phenotypic mrkers in resistnce breeding. Studies on the moleculr identifiction of the pthogen nd on genetic vribility of the pthogen were lso done. This will provide us tools for popultion studies tht could led to better understnding of pthogen epidemiology nd disese development. OBJECTIVES I. New tretments - Evlute the toxicity of lterntive copper (i.e., the reduced-copper content products Kocide 3000 nd Bdge), non-copper bsed chemicls (e.g., the ntibiotic Ksumin, EBDC fungicides, biologicls (Actinovte, Blossom Protect), nd other experimentl mterils such s enhncers of plnt stress responses nd inducers of host defense (e.g., Citrox nd ProAlexin, BioForge, Root-Zone) ginst X. rboricol pv. juglndis nd evlute the efficcy of these mterils for mnging wlnut blight. A) Comprtive efficcy of new copper bctericides such s new products nd formultions in field trils under mbient nd simulted rin systems t the Kerney AgCenter (KAC), UC Dvis, nd in commercil orchrds in Butte or Sutter Co. Evlute copper persistence with nd without djuvnts under simulted rin B) Optimize the performnce of ksugmycin formultions (e.g., Ksumin 2L, 10L) in mixture rottions with copper nd EBDC fungicides (e.g., Mnzte, Dithne). Volume nd djuvnt studies different volumes (e.g., 200 vs. 100 gl/a) nd selected djuvnts Combintions of the ntibiotic with copper, EBDC, or biologicls (e.g., Actinovte) C) Investigte the use of biologicls (e.g., Actinovte, Blossom Protect) nd inducers of host defense mechnisms (e.g., Citrox, ProAlexin, BioForge) nd nti-trnspirnts (e.g., Root- Zone) s tretments for mnging wlnut blight. II. Epidemiology - Continue to evlute disese development throughout the spring nd monitor environmentl prmeters (e.g., lef wetness, precipittion, temperture, nd reltive humidity) tht re conducive to bcteril infection of wlnut tissues nd evlute popultions of the bcteril pthogen using moleculr pproches A) Develop nd evlute precipittion-temperture-bsed version of XnthoCst on University websites (ongoing) in coopertion with UC-IPM nd CIMIS progrms for utilizing temperture nd precipittion, dew point temperture, or reltive humidity dt insted of lef wetness for the model. B) Evlute spring timings before nd fter mjor rinfll or simulted rin events nd set upper temperture limits for bctericide pplictions (e.g., >27C). III. Host resistnce nd pthogen identifiction/diversity- A) Continue to evlute wlnut genotypes for nturl host resistnce to wlnut blight under simulted rinfll conditions t KAC nd continue to study microscopic morphologicl differences of fruit surfces. B) Evlute diversity within pthogen popultions nd rpid identifiction of the pthogen using moleculr methods. Cliforni Wlnut Bord 268 Wlnut Reserch Reports 2011

5 PROCEDURES Evlution of lterntive bctericides for mngement of wlnut blight in field studies. Trils were estblished in experimentl orchrds in Fresno Co. (Kerney Agriculturl Center - KAC) nd Solno Co. (UC Dvis), nd in commercil orchrds in Sutter nd Butte Co. In Fresno Co., trils were conducted on cvs. Chico, Chndler, nd Vin. Simulted rin ws pplied in these orchrds for 6-8 h fter ech of the four bctericide pplictions. In Solno Co., trils on cvs. Chico, Hrtley, nd Tulre were conducted under nturl rinfll conditions with bctericide pplictions bsed on XnthoCst indices. Trils in commercil orchrds were done on cvs. Vin nd Ashley. Tretment timings re indicted in Figs Tretments were pplied using n irblst spryer t 100 gl/a, except for CoMo Clssic nd RootZone. Incidence of disese ws bsed on the number of infected fruit of fruit evluted for ech of four or five single-tree replictions. Dt were evluted using nlysis of vrince or generl liner model procedures nd lest significnt difference (LSD) men seprtion procedures of SAS 9.1. Evlution of miniml spry progrms bsed on host phenology nd environmentl conditions (XnthoCst) using Kocide 3000-Mnzte. A tril under nturl rinfll conditions in Solno Co. on cv. Chico ws done to evlute nd improve timing progrms for bctericide tretments. Tretment timings for four to six pplictions of Kocide-Mnzte were bsed on host phenologicl stges (pistillte flower emergence) nd before or fter rin events. Disese incidence on nuts for ech of the four single-tree replictions ws evluted in lte June. Dt were evluted using nlysis of vrince nd LSD men seprtion procedures of SAS 9.1. Disese evlutions of wlnut genotypes in vriety block t the Kerney AgCenter. Wlnut genotypes from the wlnut breeding progrm under Dr. Chuck Leslie were evluted for disese incidence under simulted-rin conditions with six weekly 8-h simulted rin events using n impct sprinkler irrigtion system. These irrigtion periods provided lef wetness t ll stges of bloom (ctkins nd pistillte flowers) nd erly fruit development. Fifty to 130 fruit of ech of four to five single-tree replictions were evluted for blight. Bud popultions of the pthogen on pproximtely 50 buds for ech wlnut genotype were enumerted by plting onto gr medi. Bcteril colonies resembling X. rboricol pv. juglndis were verified for identity using newly designed PCR primers (see below). Additionlly, fruit from ten cultivrs were evluted for structurl differences tht possibly could relte to the observed susceptibility. For this, fruit were smpled in June nd tissue pieces of the stylr end were fixed for scnning electron microscopy (SEM) using stndrd procedures. Numbers of stomt nd trichomes (glndulr nd nonglndulr) were determined for n re of mm 2. Three sites on ech smpled piece (stem end, middle, stylr end) for ech of three fruit per vriety were evluted. Dt were nlyzed using ANOVA nd LSD men seprtion procedures of SAS. Development of specific PCR mplifiction primers for X. rboricol pv. juglndis nd studies on the genetic vribility within nd mong popultions in Cliforni. Specific primers were developed from rdna nd dnk sequences tht were obtined in For this, sequences were compred to published sequences of X. cmpestris, X. vesictori, nd X. xonopodis tht re ll closely relted to X. rboricol pv. juglndis. Primers were evluted for specificity using diverse collection of strins of the wlnut pthogen nd numerous other bcteril isoltes, including other yellow-pigmented strins obtined from wlnut tissues. Subsequently, the best primer pir ws used in rel-time SYBR green PCR rections to evlute their suitbility for popultion studies. To evlute genetic vribility within nd mong popultions, DNA of ech isolte ws mplified using rep-pcr primers BOX nd ERIC (Louws et l., 1994). PCR Cliforni Wlnut Bord 269 Wlnut Reserch Reports 2011

6 rections contined bcteril cells nd stndrd regent mixtures. Amplifiction rections were done t n nneling temperture of 55C, electrophoretic products were seprted in 1.5% grose gels, nd bnding ptterns were compred. Ech isolte ws mplified t lest twice. RESULTS AND DISCUSSION I. Field Efficcy Studies Although precipittion t mny loctions in the wlnut growing res of Cliforni ws very high in the spring of 2011, due to the lte occurrence of rin nd sub-optiml tempertures, the overll incidence of wlnut blight ws only moderte in the erly-blooming wlnut vrieties used in our studies. No dt could be obtined in our Fresno Co. trils t Kerney Ag Center nd in one tril in Sutter Co. In tril in Solno Co. where Kocide-Mnzte tretments were compred to Regli nd Regli-Kocide or -Mnzte tretments nd where disese pressure ws modertely high, Kocide reduced the incidence of disese to 6.4% s compred to the control with 22.4% (Fig. 1). The incidence mong the other tretments rnged from 11.5% (Kocide 3000-Mnzte) to 25.5% (Regli). Thus, four pplictions of Regli were not effective in reducing blight under moderte disese pressure. In tril in commercil orchrd in Sutter Co. with isoltes of the pthogen modertely resistnt to copper, the efficcy of 22 tretments ws compred. Disese pressure ws low with 12.3% incidence in the control. Incidence mong the tretments rnged from 0% (Kocide Mnzte) to 7.2% (Actinovte), nd most tretments performed sttisticlly similr (Fig. 2). The four copper mterils (Kocide 3000, Bdge, Cuev, Isgro) when mixed with Mnzte hd n incidence of between 0% nd 3.5% nd thus, performed very similr. Regli when pplied by itself (1.3% disese) ws very effective under these low-disese conditions. The other nturl products, Citrox/ProAlexin, the biocontrol Blossom Protect, nd the fungicides Cptn nd Quintec lso significntly reduced the disese. Mixtures of Ksumin with Bdge or Cptn resulted in disese incidence of less thn 1% nd thus, were mong the best tretments. Ksumin by itself, s in previous yers, ws less effective. The 8L formultion of Ksumin in combintion with Bdge ws sttisticlly similrly effective s the 2L formultion, but numericlly, disese incidence ws higher using the 8L formultion. In our studies on the mngement of other bcteril diseses with Ksumin, the 8L formultion performed better thn the 2L formultion. Thus, dditionl studies on wlnut blight will be done in In tril in commercil orchrd in Butte Co. with copper-sensitive popultions of X. rboricol pv. juglndis, disese pressure ws lower nd n incidence of 14.4% ws observed in the control. Regli-Kocide reduced the incidence to 1.0% s compred to the control with 14.4%, similr to Mnzte by itself with 1.3% disese (Fig. 3). Kocide 3000 lone ws lso very effective (3.9% incidence) wheres Regli by itself (8.7%) did not significntly reduce disese incidence from tht of the control, similr to the fungicide Quintec. Ksumin ws modertely effective (6.9% incidence) nd the ddition of Quintec did not increse the efficcy (9.7% incidence). Cliforni Wlnut Bord 270 Wlnut Reserch Reports 2011

7 In summry, over the yers Ksumin mixed with copper or n EBDC showed consistent high efficcy for controlling wlnut blight. As previously lso documented, Regli cn be effectively used for wlnut blight mngement only under low-disese pressure conditions. Regli-Kocide 3000 mixtures cn provide benefit over Regli by itself, but it hs to be considered tht our trils in 2011 were conducted in orchrds with no or moderte copper resistnce levels in the pthogen popultion. Thus, trils will hve to be conducted t sites with high levels of copper resistnce to evlute the potentil of the mteril to substitute the use of EBDCs. Two new soil pplictions tht ffect stomtl opening of plnts were lso evluted for the mngement of blight in Solno Co. In this tril, four pplictions of RootZone were ineffective in reducing the incidence of disese, wheres for CoMo Clssic, trend in disese reduction ws observed (Fig. 4). Thus, these dt my not support further evlution lthough RootZone hd shown very promising results in 2010 in first-yer evlution. In summry in these five field trils, we demonstrted tht: ) The EBDC fungicide mncozeb ws effectively used in mixtures with copper or Ksumin to mnge wlnut blight. b) Ksumin significntly reduced wlnut blight incidence when used by itself, but ws more effectively used in mixtures with n EBDC, copper bctericides, or with other fungicides. c) Regli cn be effectively used for wlnut blight mngement under low-disese pressure conditions. Mixtures of the mteril with copper cn be beneficil t higher disese pressure, but only when copper resistnce is not problem (bsed on our experiences over severl yers). Higher rtes need to be evluted similr to our initil studies with Milsn (the previous formultion of this nturl product). d) Among the other nturl products nd biocontrols, Citrox BC+ProAlexin hs shown consistent efficcy to suppress the disese over the lst two yers, wheres the efficcy of Actinovte hs been inconsistent. II. Epidemiology Evlution of ppliction timing of bctericide tretments with Kocide 3000-Mnzte strting t pistillte flower emergence. This tril ws done on cv. Chico in n orchrd in Solno Co. under nturl rinfll conditions. Four progrms were compred: 1) Applictions timed fter rin events; 2) Appliction t pistillte flower emergence nd timed before predicted rins; 3) Appliction t pistillte flower emergence nd timed fter rin events; nd 4) Appliction t pistillte flower emergence followed by four pproximtely weekly pplictions. Non-treted trees were used s controls nd disese ws evluted in lte June In this study, only the progrm with weekly pplictions nd the progrm with pplictions before predicted rins significntly reduced the disese from the untreted control where 44.7% of the fruit were disesed (Fig. 5). The four-spry progrm bsed on predicted fvorble conditions ws similrly effective s the six-spry weekly progrm. The two progrms with fter-rin tretments were not effective this yer. In contrst, studies of the lst two yers hd indicted tht post-rin pplictions cn be effective. This difference in results cn be explined by the fct tht high precipittion over n extended period between My 14 nd My 17, 2011, ws responsible for very fvorble disese conditions (Fig. 5). Thus, post- Cliforni Wlnut Bord 271 Wlnut Reserch Reports 2011

8 precipittion pplictions my only be effective for short durtion, low-rinfll events. Prerinfll pplictions re recommended s the most effective tretment strtegy nd post-rinfll pplictions my be beneficil for short, low rinfll periods tht were not nticipted. III. Host Resistnce to Wlnut Blight nd Pthogen Identifiction/Diversity Vrietl susceptibility. Wlnut genotype comprisons were conducted for lst yer in the old vriety orchrd t Kerney AgCenter. For this, we lso enumerted bud popultions of the pthogen in the genotypes evluted. Numerous bcteri were isolted from most bud smples, with inconsistency mong replictions of the sme genotype. Most bcteri were species other thn X. rboricol pv. juglndis. Although 10 buds were used for isoltions from ech of five trees per cultivr, the pthogen could only be recovered from cvs. Pyne nd Vin, but not from other cultivrs including Chndler, Hrtley, nd Serr. These ltter three cultivrs, however, hd between 4 nd 16% disese (see below) by lte spring nd erly summer. Thus, the use of bcteril bud popultions to generlly predict disese incidence is questionble for the genotypes nd cultivrs we evluted. Disese incidence mong 15 genotypes rnged from 19.5% in cv. Pyne to 0% in PI159568, Cheinovo, nd PI18256 (Fig. 6). In ech of the seven evlution yers, cv. Pyne ws the most susceptible nd PI followed by Cheinovo, 76-80, Sinensis 5, PI18256, nd Chse D9 selections were mong the lest susceptible. For other cultivrs, susceptibility mong the yers ws more vrible. In Fig. 6, the incidence of blight ws lso verged over the yers to show the overll performnce. With these evlutions, we re supporting our long-term gol of providing dt on blight susceptibility mong new nd old wlnut genotypes to ssist the breeding progrm in the Horticulture Deprtment, University of Cliforni, Dvis. We re concluding our evlution of stomtl number nd morphology s well s presence of trichomes on fruit of wlnut genotypes to potentilly identify chrcteristics tht determine levels of host susceptibility. Developing fruit were collected, nd tissue from selected fruit res ws prepred from scnning electron microscopy. Microscopic observtions were initited, but dt re currently pending. Pthogen identifiction nd diversity. Ten primer pirs from two gene sequences were evluted for their specificity to identify X. rboricol pv. juglndis using isoltes of the pthogen of diverse genetic bckground (s identified in 2010) nd of numerous other bcteri obtined from wlnut tissues including buds. Severl primer pirs were specific for X. rboricol pv. juglndis. As n exmple, mplifiction using primers Xj1F nd Xj1R designed from ribosoml DNA sequences is shown in Fig. 7A. Using ll primer pirs, however, primer-dimer bnds (from self-nneling of primers) were visible in the electrophoretic seprtion of products (Fig. 7A). Thus, primers were specific, but primer-dimer formtion will interfere with the detection of the pthogen by rel-time PCR. In dditionl studies, melt-curve nlyses of PCR mplicons with interclted SYBR green dye were done in rel-time PCR rections using the most specific primer pirs. Primers were confirmed to be specific. In the exmple in Fig. 7B with primers Xj1F nd Xj1R, the specific PCR product hs melt temperture of 83.5 C. Agin, primer-dimer bckground ws evident, nd the primer-dimer product hd melt temperture of 74 C in Fig. 7B. Thus, rel-time PCR Cliforni Wlnut Bord 272 Wlnut Reserch Reports 2011

9 method tht cn be used in popultion studies will need dditionl improvements. For this, we will modify our pproch nd develop TqMn probes in combintion with the primers we designed. This technology is more complex, but it is not ffected by the presence of primer dimers. Popultion diversity of the pthogen ws studied in three orchrds. Using two primers in PCR mplifictions tht trget repetitive sequences, multiple strins of the pthogen hve been identified in our collections from wlnut growing res of Cliforni over number of yers. In 2011, some orchrd loctions hrbored single genotype, but in others mixed popultion of two distinct genetic strins ws identified (Fig. 8). Thus, lthough geneticlly diverse throughout Cliforni, only limited number of genotypes of the pthogen is present within loction. CONCLUSIONS Among currently vilble tretments, copper-mncozeb (EBDC) combintions re the most consistent in controlling wlnut blight in Cliforni. In 2011, Ksumin ws gin the most promising new tretment. Although modertely effective by itself, it performed very well in combintion with copper or mncozeb. Ksugmycin (i.e., Ksumin) is currently pending US- EPA registrtion in Feb Clendr-bsed ppliction methods (e.g., weekly pplictions beginning t femle flower bud brek) my result in excessive pplictions tht my not improve disese control s compred to reduced-spry progrm nd my be costly. As in previous yers, our reserch demonstrted tht pplictions of copper-mncozeb t pistillte flower emergence followed by subsequent sprys bsed on temperture nd rinfll (s summrized in the XnthoCst model) is currently the most effective strtegy in mnging the disese. Thus, our reserch is lso compring timing of tretments before nd fter rinfll events tht my improve our understnding of bcteril invsion of host tissues, i.e., infection periods nd give growers options with non-nticipted rinfll prevent protective pplictions of bctericides. Potentilly in the future, ksugmycin-copper or -EBDC combintions in rottion with copper-ebdc tretments cn minimize the totl mount of ny bctericide being used while mintining high levels of disese control nd providing resistnce mngement by lternting different modes of ction. We found tht the enumertion of bud popultions t the beginning of the growing seson to estimte disese levels during the seson is difficult nd questionble due to the presence of numerous bcteril species colonizing buds, the reltively low inoculum levels of the pthogen, nd the potentilly rpid increse of the pthogen popultion under fvorble environmentl conditions. Thus, bud popultions my be n indiction of the disese potentil in some cultivrs but ctul disese levels vried drmticlly mong most genotypes evluted. Sttisticl smpling procedures need to be evluted to determine the proper number of buds to be smpled to estimte orchrd popultions of the pthogen t high confidence level (e.g., >95%). Additionlly, further development of rel-time PCR detection system for X. rboricol pv. juglndis will ssist in the ssessment of bud popultions, but moreover cn be used to study the response of the pthogen to environmentl chnges. Studies on pthogen popultion diversity indicted tht lthough multiple strins of the pthogen hve been identified in our collections from wlnut growing res of Cliforni over number of yers, individul orchrd loctions hrbored only one or two distinct genotypes. Cliforni Wlnut Bord 273 Wlnut Reserch Reports 2011

10 ACKNOWLEDGMENTS Specil thnks to the grower in Sutter Co. who llowed us to conduct our reserch in his orchrd nd to the chemicl industry representtives who cooperted with us with during this reserch. REFERENCES 1. Adskveg, J. E, et l Annul Wlnut Reports Wlnut Mrketing Bord of Cliforni. Scrmento, CA. 2. Christofferson, C., McGrnhn, G., Mills, N., nd Leslie, C Annul Wlnut Reports Wlnut Mrketing Bord of Cliforni. Scrmento, CA. 3. Conover, R. A. nd Grenhold, N. R Mixtures of copper nd mneb or mncozeb for control of bcteril spot of tomto (Xnthomons cmpestris pv. vesictori) nd their comptibility for control of fungus diseses. Proc. of Fl. Stte Hortic. Soc. 94: Lee, Y. -A., M. N. Schroth, M. Hendson, S. E. Lindow, X. -L. Wng, B. Olson, R. P. Buchner, nd B. Teviotdle Phytopthology 83: Louws, F.J., Fulbright, D.W., Tylor Stevens, C., nd de Bruijn, F.J Appl. Environ. Microbiol. 60: Miller, P. W. nd W. B. Bollen Oregon Agric. Exp. Stn. Tech. Bull pp. 7. Mulren, E. N. nd M. N. Schroth Phytopthology 71: Mulren, E. N. nd M. N. Schroth Phytopthology 72: Ogw, J. M. nd H. English Diseses of Temperte Zone Tree Fruit nd Nut Crops. University of Cliforni, Division of Agriculture nd Nturl Resources, Oklnd, CA. Publ. No pp. 10. Scortichini, M, Mrchesi, U, nd Di Prospero, P J. Phytopthology 149: Teviotdle, B. L., M. N. Schroth, nd E. N. Mulren In: Wlnut Orchrd Mngement. Ed. by E. Rmos. Univ. of Clif. Coop. Ext. Publ Young, J.M., Prk, D.-C., Shermn, H.M., nd Frgier, E Syst. Appl. Microbiol. 31: Cliforni Wlnut Bord 274 Wlnut Reserch Reports 2011

11 Fig. 1. Efficcy of copper nd Regli in combintion with nd mnzte for mnging wlnut blight on cv. Chico wlnut cused by copper-senstive popultions of Xnhomons rborico l pv. juglndis - Solno Co. 2011, nturl rinfll conditions - No copper resistnce Control Regli 0.25% Kocide lb Regli 0.25% + Kocide lb Regli 0.25% + Mnzte 2.4 lb Kocide lb + Mnzte ProStick 75DF 2.4 lb c bc bc b b Disese incidence (%) Applic tions were done were mde on 4-12, 4-20, 5-6, nd Dis es e ws ev luted in lte J une Inc idenc e of dis es e is bs ed on 100 fruit for ec h of 5 s ingle-tree replic tions. Fig. 2. Efficcy of new copper- nd non-copper-bsed tretments for mnging wlnut blight on cv. Vin cused by modertely copper-resistnt strins of Xnthomons rboricol pv. juglndis - Sutter Co., 2011, nturl rinfll conditions - Control Actinovte 12 oz + NuFilm 8 fl oz Ksumin 8L 100 ppm Regli 32 fl oz + Kocide lb Blossom Protect 1.34 lb - buffer 9.35 lb Cptn 80WP 5 lb Cuev 4 qt + Mnzte ProStick 75DF 2.4 lb Isgro 2.67 lb + Mnzte ProStick 75DF 2.4 lb Ksumin 8L 100 ppm + Bdge X2 4 lb Quintec 6 fl oz Bdge X2 4 lb + Mnzte ProStick 75DF 2.4 lb Citrox BC 0.1% + ProAlexin 0.1% Ksumin 8L 100 ppm + Mnzte F45 58 fl oz Bdge X2 4 lb Cuev 8 qt Bdge X2 4 lb + Rezist 16 fl oz Ksumin 2L 100 ppm + Quintec 6 fl oz Regli 32 fl oz Ksumin 8L 100 ppm + Cptn 80WP 5 lb Ksumin 2L 100 ppm + Bdge X2 4 lb Mnzte Prostick 75DF 2.4 lb Isgro 4 lb + Mnzte ProStick 75DF 2.4 lb Kocide lb + Mnzte ProStick 75DF 2.4 lb cde cde cde cde e bc b Disese incidence (%) Six pplic tions were done on 4-14, 4-21, 4-27, , nd Dis es e ws ev luted on Inc idenc e is bs ed on the number of dis es ed nuts of the totl of 100 nuts ev luted of ec h of four s ingle-tree replic tions. Cliforni Wlnut Bord 275 Wlnut Reserch Reports 2011

12 Fig. 3. Efficcy of copper nd non-copper tretments for mnging wlnut blight on cv. Ashley wlnut cused by copper-senstive popultions of Xnhomons rborico l pv. juglndis - Butte Co. 2011, nturl rinfll conditions - No copper resistnce Control Regli 0.25% Regli 0.25% + Kocide lb Kocide lb Regli 0.25% + Mnzte Pro-Stick 2.4 lb Mnzte Pro-Stick 2.4 lb Kocide lb + Mnzte Pro-Stick 58 fl oz Quintec 6 fl oz Ksumin 2L 100 ppm + Quintec 6 fl oz Ksumin 2L 100 ppm d cd bc b bc b b b b Disese incidence (%) All trees were s pry ed with c opper on Four s ubs equent pplic tions were done were mde on 4-13, 4-21, 4-26, nd Dis es e ws ev luted on J une 22, Inc idenc e of dis es e is bs ed on 100 fruit for ec h of 5 s ingle-tree replic tions. Fig. 4. Efficcy of new soil pplictions tht ffect stomtl opening for mnging wlnut blight on cvs. Hrtley nd Tulre cused by copper-sensitive strins of Xnthomons rboricol pv. juglndis* - Solno Co. 2011, nturl rin conditions - Control RootZone 1000 ml CoMo Clssic 16 fl oz Kocide lb + Mnzte DF 2.4 lb* Cv. Hrtley Cv. Tulre b b b b Disese incidence (%) RootZone nd CoMo Cls s ic were pplied to the s oil on 4-18 (pis tillte flower emergenc e), 5-4, 5-12, nd Applic tions were followed by 3-4-h s prink ler irrigtion. Koc ide-mnz te ws pplied s folir tretment on 4-17, 4-27, 5-4, 5-12, nd Dis es e ws ev luted in lte J une 2011 nd inc idenc e is bs ed on the ev lution of 100 nuts per s ingle-tree replic tion. Cliforni Wlnut Bord 276 Wlnut Reserch Reports 2011

13 Fig. 5. T iming study with Kocide 3000-Mnzte tretments in cv. Chico orchrd under nturl rin conditions Solno Co Temperture (C) Avg Air Temp ( C) Precip (mm) 5 2 Precipittion (mm) 0 3/25 4/1 4/7 4/14 4/21 4/28 5/2 5/7 5/14 5/21 5/28 Dte (2011) 0 Applictions Disese Incidence No. Progrm 4-4 (Pist.) (%) LSD 1 Control Pist. + After b 5 No Pist. + After Rin b 3 Pist. + Before b @ b Kocide 3000 Mnzte DF (4 lb-2.4 lb) ws pplied cv. Chico using n ir-blst spryer t 100 gl/a. Timings: Pistillte flower emergence 4/4/11 After rin events 4/19 to 4/20, nd 5/13 to 5/19 Before rin events 4/14, 5/5, nd 5/13 Progrm 1: Control Progrm 2: Appliction t pistillte flower emergence nd timed fter ech of 3 rin events. Progrm 3: No ppliction t pistillte flower emergence nd timed fter ech of 3 rin events. Progrm 4: Appliction t pistillte flower emergence nd timed before ech of 3 rin events. Progrm 5: Weekly (7-9 dy intervls) pplictions Disese ws evluted in June 22, Fig. 6. W lnut vriety susceptibility to blight in simulted rin-treted orchrd - Fresno Co PI Cheinovo PI Frnquette Chse D9 Sinensis Hrtley Adms 10 Chndler Tulre Serr Pyne d d d cd cd cd d bc 2011 b verge Fiv e to s ix week ly s imulted rin ev ents were pplied us ing high-ngle s prinkler s y s tem between April nd My in ec h y er. Cliforni Wlnut Bord 277 Wlnut Reserch Reports 2011

14 Fig. 7. Development of primers specific for Xnthomons rboricol pv. juglndis A B Amplifiction of DNA from strins of X.. pv. juglndis nd other bcteri obtined from wlnut buds nd developing fruit using primers Xj1F nd Xj1R designed from ribosoml DNA sequences. A. Gel with representtive strins. Lnes 1, 2, 17, nd 22 re positive for the pthogen; wheres ll other lnes of non- Xnthomons spp. re negtive. 0 = Stndrd moleculr ldder nd lne 30 is wter control. The blck rrow points to the specific mplicon for X.. pv. juglndis nd the white rrow points to the primer dimer bnd. B. Melt-curve nlysis of PCR mplicons using rel-time PCR rections of 6 strins of X.. pv. juglndis (83.5C melt temperture), 4 non-xnthomons spp., nd one wter control (74C melt tempertures represent primer dimers). Fig. 8. DNA electrophoretic ptterns of Xnthomons rboricol pv. juglndis strins fter rep-pcr using BOX primers. ML Orchrd 1 Orchrd 2 A A A A A B B B B B B B B B Strins were collected from two wlnut orchrds in northern Cliforni. Two genetic types designted A nd B were found in one orchrd; wheres in nother orchrd only the B genotype ws present. ML = moleculr ldder. Cliforni Wlnut Bord 278 Wlnut Reserch Reports 2011