NEW APPROACHES TO CONTROL WALNUT BLIGHT. Milton Schroth, Joe osgood, Bill Olson, Art McCain, Mavis Chong, John Bahme, and Yang-An Lee.

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1 NEW APPROACHES TO CONTROL WALNUT BLIGHT December, 1988 Miltn Schrth, Je sgd, Bill Olsn, Art McCain, Mavis Chng, Jhn Bahme, and Yang-An Lee. ABSTRACT Last year's research prpsal presented a three year prgram t develp tw new appraches t cntrl walnut blight and t determine the extent and significance f cpper tlerance by the blight bacterium, Xanthmnas juqlandis. One apprach was t add additinal materials t fixed cpper bactericides t imprve the txicity f cpper t blight bacteria while nt increasing phyttxicity t the tree. The ther apprach was t find naturally ccuring rganisms that wuld be antagnistic t the blight bacteria and reduce their ppulatins in buds, the majr site fr verwintering. Bth appraches lk very prmising after the first year f research. Tw chemical additives, bth f which can be cnsidered safe, greatly increased the effectiveness f cpper in killing blight bacteria in labratry tests. It is thught that the tw materials, CB2 and CB3, affect the ability f the bacteria t take-up the cpper in. This als wuld suggest that a reduced amunt f cpper culd be used t cntrl blight which in turn wuld reduce the liklihd f cpper phyttxicity. The bilgical apprach als yielded interesting data in that many buds were clnized by a yeast and a whitish bacterium. Apprximately 20% f the buds that were clnized by these rganisms cntained n blight bacteria. Research n cpper tlerant blight bacteria suggested that this an imprtant factr affecting the efficacy f cpper materials but that the bacteria still culd be killed at higher dsages f cpper. It als appears that the cpper tlerance is smewhat transient and will nt remain fr lng perids f time. INTRODUCTION The effectiveness f cpper cmpunds in cntrlling blight bacteria and ther bacterial plant pathgens has remained relatively the same fr decades with sme minr differences depending upn the nature f the frmulatins. It has been difficult t imprve n the level f walnut blight cntrl. There are several reasns fr this. Past research supprted by the Walnut Industry revealed that bacteria clnize newly develping buds early in spring and then verwinter in thse sites until the buds develp int shts the fllwing year. These symptmless buds harbr ppulatins f blight bacteria f between 10, ,000 cellsper bud. Unfrtunately,thereis n bactericidethat appreciably affects the ppulatins f the bacteria nce they are within the buds. The ther prblem, which may turnut t be minr, is that cpper appears t induce tlerance. Cnsequently, after 120

2 , ~ the bacteria are cntinuusly expsed t cpper, f the free cpper in is required t kill them. a greater amunt The abve research findings prmpted us t lk fr ther methds t cntrl blight bacteria and t determine hw great a prblem cpper tlerance wuld cause. Firstly, we prpsed that ne way t imprve the efficacy f cpper wuld be t determine the mechanisms by which cpper affect bacterial grwth, r inversely hw the bacterium escaped txicity frm it. We hypthesized n the basis f ther wrk that susceptiblity and tlerance t the cpper in was related t the rganism's prductin f siderphres and that susceptibility culd be greatly imprved by adding materials. such as CB2 and CB3. This hypthesis appears t be crrect. {The full explanatin f the nature f CB2 and CB3 and the mechanisms that are invlved will be disclsed after the University has decided whether r nt the "inventin" shuld be prtected by a patent}. The examinatin f buds fr the presence f rganisms that wuld be antagnistic t blight bacteria was prmpted by research that revealed that buds were heavily clnized by varius micrrganisms. The thught was that perhaps these rganisms culd be used t bilgically cntrl blight bacteria by keeping them ut f the buds. The hypthesis is that this culd be dne by spraying develping buds with antagnists befre they becme clnized by blight bacteria. This apprach is analgus t the very successful bilgical cntrl f crwn gall f walnuts and varius ther plants. Cntrl is btained by treating wunds r nursery harvested plants with Agrbacterium radibacter K-84, an antagnist that prevents crwn gall bacteria frm making galls. Lastly, it is imprtant t fllw-up n ur research which revealed the ccurrence f cpper tlerant bacteria in walnut rchards. We need t knw just hw imprtant this is and hw best t deal with the prblem. The indicatins are that cpper tlerance may nt be a majr prblem. OBJECTIVES 1. Develp alternative appraches t cntrl walnut blight. a. Develp new frmulatins f cpper cmpunds using several envirnmentally safe materials that wuld enhance the efficacy f cpper by increasing uptake f the in int the bacterial cell. b. Reduce the ppulatin densities f walnut blight bacteria in buds and catkins by using naturally ccurring antagnists analgus t the bilgical cntrl f crwn gall. 2. Determine the significance f cpper tlerance by blight bacteria and devise stragegies t circumvent it. METHODS AND RESULTS 121

3 ImDrved codder txcitv t bliqht bacteria. The txicity f cpper t blight bacteria was tested in the labratry using standard biassay techniques. Different cncentratins f cpper sulfate was added t special media that wuld supprt grwth f blight bacteria. Cpper in this medium is nt chelated appreciably, thereby leaving the cpper ins free t act n the bacteria. ther substances can then be added t the grwth medium t determine if they will enhance cpper txicity. This was dne by adding cpper t the medium at a level that did nt kill the bacteria. The test substances were then incrprated int the medium and cmpared t media with cpper alne t determine effects n bacterial grwth. Fr example, when 5 ppm cpper was incrprated int the medium, blight bacteria grew at a rate cmparable t cntrls withut cpper. This kind f experiment led t the discvery that as little as 5 ppm f the substances CB2 r CB3 when added with the cpper resulted in death f the bacteria by increasing cpper txicity. Neither CB2 nr CB3 had any effect n bacterial grwth when tested alne. Natural antagnism t bl iqht bacteria. Tw hundred vegetative buds frm a Chic rchard were individually examined fr in,ternal ppulatins f blight bacteria in relatin t ppulatins f ptential antagnists. Only 9 f 200 buds were free f micrrganisms (table 1). Frty f the 200 buds cntained high ppulatins f blight bacteria, whereas the rest were heavily ppulated with yeast and a few miscellaneus bacteria. In previus labratry assays, the yeasts were inhibitry t blight bacteria. Althugh blight bacteria were fund in sme buds cntaining yeast, a significant finding was the number f buds that cntained nly yeast (table 2). Tlerance f bliqht bacteria t CODDer cmdunds. Many rchards have been mnitred fr cpper resistant blight bacteria. The fllwing are a summary f the results: --The level f tlerance varies greatly amng blight bacteria with mst being sensitive. Hwever, at the higher tlerance levels, standard fixed cpper treatments wuld nt effect the mst tlerant cells. --Tlerance has been fund as high as in abut 10% f the blight ppulatin. Usually, it is belw 1.0% --Blight bacteria inside the buds are mre sensitive t cpper than thse n the utside. Clnizatin f buds. --ppulatins f blight bacteria in buds ranged frm 10,000 t 100, Clnizatin f buds in 1988 did nt ccur until the first rains which were relative late. --Blight bacteria were first fund n the external surfaces and then later within the buds. --By winter time, clnizatin f buds with blight bacteria was typical f ther years. 122

4 Table 1. Occurrence f different micrrganism in walnut buds. Fifty buds were individually examined in each plt. Identity f micrrganisms in buds lts east bli ht bact. mucid ellw flures. nne ther 1. 47* * Means that 47 f 50 buds cntained a yeast and 20 cntained blight bacteria. Mucid=mucid like bacteria, yellw and flures.=yellwand fluescenttype bacteria, respectively., Table 2. Number f buds cntaining nly ne kind f micrrganism. Fifty buds were sampled per plt. Identity f micrrganisms in plts 1. yeast 18* bliaht bact. mucid 0 0 yellw * Means that ut f 50 buds, 18 cntained nly fund nly with blight bacteria. DISCUSSION buds flures yeast and ther nne were Bth new appraches t cntrl walnut blight appear t be very prmising and ffer the first alternativest standard practices in decades. There is every reasn t expect that the additives CB2 and CB3 that increased cpper txicity t bacteria in the labratry wuld d the same in the field. Anther additinal benefit f this ptentiallynew prduct is that it shuld als lead t a reductin in phyttxicityt the plants since the increased txicity f cpper is directlyrelatedt the susceptibilityf the blight bacteria. The prpsedmechanismsby which CB2 and CB3 wrk n the bacteria is very simple and is easily understandable. Hwever, as stated previusly, until the patent status f this discvery is knwn, the mechanism f actin cannt be discussed in public. Nw that tw additives have been fund that enhance cpper txicity, what is the next bjective? Since the discvery came late in the 1988 year, we need t wrk-ut the cncentratins f cpper and CB2 and CB3 which wuld be the mst effective t cntrl blight bacteria in the field. The next step cncerns frmulatin 123

5 f the new prduct and we may need sme help frm chemists. It is essential that CB2 and CB3 have slw release capabilities similar t the cpper frmulatin since the cpper in is slwly released frm fixed cpper frmulatins. Cnsequently, it is necessary t have sme CB2 r CB3 in a free frm n the plant s that it can interact with cpper and the bacterial cell.- If it were ttally water sluble, it wuld nt be present after the first rains. These are nt difficult prblems t research. The finding that yeasts heavily ppulate buds and that they are antagnistic t blight bacteria was an unexpected discvery. It suggests that there is a degree f natural bicntrl ccuring all f the time. The additinal finding that buds are nt clnized by micrrganisms until the first rain indicates that it might be pssible t greatly increase the effectiveness f the yeasts by spraying them n the develping buds, shuld further research crrbrate the hypthesis. Als, the yeasts appear t be very tlerant t cpper which is a psitive finding. It wuld nt be gd t eliminate all f the naturally ccurring micrrganisms by use, f bactericides