EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHTwalnutresearch.ucdavis.edu/2010/2010_195.pdf · Our...

EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT J. E. Adaskaveg, H. Förster, D. Thompson, and J. Enns, J. Connell, and R. Buchner Cooperating: L. Wade, Arysta LifeScience ABSTRACT 1. Field studies included evaluations of the efficacy of mixtures of copper (e.g., Kocide 3000,

Badge) with mancozeb (e.g., Manzate Pro Stick and Manzate Flowable), copper or mancozeb used alone, kasugamycin (Kasumin), as well as natural products and a biocontrol under ambient and simulated-rain conditions for reducing the incidence of walnut blight.

• The EBDC fungicide mancozeb was effectively used in mixtures with copper and

Kasumin to manage walnut blight. This supports the first emergency registration of mancozeb as Manzate and supports full registration of EBDC products (e.g, Dithane, Manzate, Penncozeb) as replacements of maneb (e.g., Manex) for walnut blight control.

• The antibiotic Kasumin significantly reduced walnut blight incidence, but was most effectively used in mixtures with EBDC or copper bactericides. The efficacy was equivalent or better than copper-EBDC treatments in orchards with copper resistance. Kasugamycin was submitted to the IR-4 program in 2006, the federal registration is expected in 2011, and the California registration in 2012. Kasugamycin represents the first new antibiotic in US agriculture in more than 40 years and the first effective alternative to copper in the management of walnut blight.

• Mancozeb by itself reduced disease incidence, but was not as effective as in combinations with copper or Kasumin.

• The natural product Regalia alone or in combination with copper was not effective in an orchard with moderate disease pressure and copper-resistant populations of the pathogen.

• The biocontrol Actinovate (Streptomyces lydicus) in combination with NuFilm P effectively reduced walnut blight under moderate disease pressure, similar to last year when another adjuvant was added. The natural product mixture Citrox BC+ProAlexin also suppressed the disease and thus, further research is warranted for these biologicals.

• The plant growth regulators Bioforge and Biozyme (the latter used in combination with Kasumin), as well as the anti-transpirant Root-Zone that stimulates abscisic acid production reduced disease and should be continued to be evaluated.

2. Minimal spray programs with Kocide-Manzate starting at female bud-break and treatments before and after rainfall events were evaluated. The most effective programs used a pistillate flower emergence application followed by subsequent applications timed around rainfall events. Pre- and post-rainfall treatments were found to be effective.

3. In studies on walnut varietal susceptibility to walnut blight, in each of the six years where we conducted these evaluations, cv. Payne was the most susceptible followed by cv. Tulare. Cv. PI159568 followed by cvs. Cheinovo, 76-80, Sinensis 5, PI18256, and Chase D9 were among the least susceptible. Although there were some differences in the ranking of the remaining cultivars over the years, overall, results were quite consistent. Selected varieties were

California Walnut Board 195 Walnut Research Reports 2010

evaluated for fruit surface stomatal and trichome density to possibly provide an explanation for the differential susceptibility.

4. Based on repetitive polymerase chain reaction (rep-PCR), the degree of genetic diversity found among California populations of Xanthomonas arboricola pv. juglandis was much higher than that reported by others who compared populations from several continents. Isolates from the same location in our study were more homogeneous. Sequencing of three gene loci from representative isolates of the rep-PCR groups revealed close relationships among the isolates and they clustered with reference isolates of X. arboricola. Based on this information, possibly specific primers can be developed for the detection of X. arboricola pv. juglandis that could be used in population studies.

INTRODUCTION Walnut blight, caused by Xanthomonas arboricola pv. juglandis, is a major disease of walnut in central and northern California. The pathogen infects catkins, female blossoms, green shoots, leaves, buds, and fruit of English walnut with fruit infections accounting for most of the economic loss. Infections commonly occur in the spring under wet conditions. The bacterium survives from one year to the next in healthy and diseased buds, diseased fruit that remain on the tree, and possibly in twig lesions (Miller and Bollen 1946; Mulrean and Schroth 1982; Teviotdale et al. 1985, Ogawa and English 1991). For management of walnut blight, short-term goals include properly timed bactericidal treatments including copper, EBDCs, kasugamycin, and biological products, whereas long-term goals include the use of blight resistant cultivars selected in breeding programs. Cultivars that are naturally resistance to the disease or cultivars that are late-blooming and thus, escape favorable environments for disease development are two strategies that can be part of a breeding program. Both types of goals have been a focus of our research over the years. Perpetual, over-usage of copper-based products for walnut blight management over several decades has resulted in copper-resistant populations of the pathogen and environmentally unsustainable programs with higher usage rates and more frequent applications. Thus, there is need for alternative treatments and improved application methods. The introduction of copper-maneb treatments by Conover and Genhold (1981) with the application of two active ingredients has improved the efficacy of fixed copper treatments against copper-resistant pathogen populations. New effective copper-based and non-copper-based bactericides have been identified. Thus, Kocide 3000 and Badge products, contain lower metallic copper equivalents (30 and 28%), respectively, and can be applied at lower rates per acre than previous products, but still are highly efficacious. Thus, these treatments are a step in reducing copper usage and allowing walnut blight management to have less impact on the environment. Our disease forecasting model XanthoCast™ (available at Agtelemetry.com), an accumulation model that utilizes wetness period duration and temperature parameters obtained from weather stations, assists in the proper timing of treatments by predicting infection periods for walnut blight. Using this model, the number of applications can often be reduced while maintaining the same level of control as a calendar-based program. In ongoing studies each year, the model is being validated under different climatic conditions, e.g. highly conducive and less favorable disease conditions. Currently, our recommendations are that in seasons with low rainfall and


unfavorable temperatures, one or two early-season (bloom) applications that are based on blossom phenology (e.g., pistillate flower emergence “PFE”) should be followed up with a mid- to late season application around a favorable environmental event (i.e., as predicted by XanthoCast) to provide disease control and to reduce pathogen populations for the following season. In seasons with more rain and favorable temperatures, additional post-bloom applications (based on XanthoCast) are necessary. Thus, protective bloom and subsequent treatments during fruit development are based on predicted imminent rain or wetness events and favorable temperatures to prevent new infections. We demonstrated that a single low-gallonage (100 gal/A) bud-break application with Kocide-EBDC (with or without Breakthru) does not

satisfactorily control the disease and resulted in epidemics especially when wetness occurred after bloom, i.e., mid- to late spring rains.

The incidence of walnut blight in 2010 was moderate at most locations because with high rainfall, but cool temperatures, environmental conditions in the spring were moderately conducive for the disease. In our field trials with or without the application of simulated rain, data were obtained on the evaluation of new bactericide treatments for walnut blight management and on the timing of applications. Copper compounds evaluated included a micro-encapsulated copper hydroxide (Kocide 3000 30% MCE; DuPont Chemical Co.), copper hydroxide (Kocide 2000 36% MCE; DuPont Chemical), pre-mixtures of copper hydroxide/oxychloride (Badge SC and X2 20% and 28% MCE; Isagro, Inc.), and an experimental copper (GWN 4620; Gowan Co.). Additionally, the EBDC fungicides, Manzate Pro Stick 75DF or Manzate Flowable F45, were compared by either using them alone or in selected tank mixtures. These trials and trials in the last few years with mancozeb products supported the first-year emergency registration of Manzate in 2010. Numerous potential alternatives to copper materials have been evaluated by us over the years with results ranging from ineffective or inconsistent, weakly to moderately effective; or the material could not be registered. The antibiotic kasugamycin (Kasumin® - Arysta Life Science) is the most promising material. It is registered for agricultural use for managing plant diseases caused by fungi and bacteria in Japan. This class of antibiotics is not being used in human medicine or in animal agriculture, has a mode of action different from other antibiotics such as streptomycin or oxytetracycline, and there is no cross-resistance known to occur. In our previous years’ trials we obtained very good to excellent results with kasugamycin against walnut blight. Based on our research, kasugamycin entered the IR-4 program and was submitted for federal registration in Jan. 2010. California registration is expected in 2012. This antibiotic was again incorporated into our 2010 trials using two formulations (2L and 10L) either by itself or in mixtures with mancozeb, copper, or organic products. In addition, we evaluated several products that could potentially be used in organic production. These include natural compounds such as Regalia (an extract of the plant Reynoutria sachalinensis; Marrone Bio Innovations), Cerebrocide (an extract of a mushroom; Arysta Life Science), Citrox BC + ProAlexin (Citrox, Ltd.), and Actinovate (Natural Industries, Inc.) – a biocontrol that contains the actinomycete Streptomyces lydicus. Additionally, the plant stress response inducers BioForge (Stoller-USA) and Biozyme (Arysta Life Science) were evaluated. We also tested Moisturin and Root-Zone (both GSI Horticultural) which are both anti-transpirants. Moisturin is applied foliar and aims to plug stomatal openings with a film. Root-


Zone is used as a soil application and is thought to stimulate the tree to produce abscisic acid, a plant hormone that induces closing of stomata. Thus, these treatments can regulate stomatal function and change the microclimate. Because stomata are primary entry points for plant pathogenic bacteria, regulation of stomatal opening could have an effect on disease incidence. In a walnut variety plot we have been ranking cultivars and genotypes for their disease susceptibility over several years and this information is made available to walnut breeding programs. A secondary goal of these evaluations is to find phenotypic markers associated with disease susceptibility that could facilitate selection of breeding lines. Additionally, we have initiated studies on the population structure of X. arboricola pv. juglandis in California. One goal of these studies is to develop molecular markers at the species level that could be used in population studies. These studies may lead to a better understanding of host susceptibility and genetic diversity of the pathogen that could lead to integrated management practices that minimize pesticide use with more disease resistant cultivars and prevent the selection of resistant strains of the pathogen to agricultural chemical treatments. OBJECTIVES

I. New treatments - Evaluate the toxicity of alternative copper (i.e., Kentan, Badge, and the low-copper-concentration product Kocide 3000) and non-copper based chemicals (e.g., the antibiotic Kasumin, EBDC fungicides, new natural products – Regalia), biologicals (Actinovate), and other experimental materials such as enhancers of plant stress responses (e.g., BioForge, ReZist) and defense mechanisms (e.g., ProAlexin) against X. juglandis and evaluate the efficacy of these materials for managing walnut blight.

A) Comparative efficacy of new bactericides (synthetic such as new formulations of copper,

biologicals such as Actinovate, and natural products such as Regalia, ProAlexin) using air-blast spray application methods in field trials under ambient and simulated rain systems at the Kearney AgCenter (KAC), UC Davis, and in commercial orchards in Butte or Sutter Co. • Evaluate copper persistence with and without adjuvants under simulated rain

B) Studies using mixtures of kasugamycin with copper or EBDC fungicides (e.g., Manzate, Dithane). • Volume study – high vs. low (e.g., 200 vs. 100 gal/A) • Adjuvant study – with and without a selected adjuvant (e.g., spreader/sticker) • Studies on combinations of the antibiotic – copper, EBDC, or biological (e.g., Actinovate)

C) Investigate the use of ProAlexin as a bactericide and inducer of host defense mechanisms along with BioForge and ReZist.

II. Epidemiology - Continue to evaluate disease development throughout the spring and monitor environmental parameters (e.g., leaf wetness, precipitation, temperature, and relative humidity) that are conducive to bacterial infection of walnut tissues using dataloggers and CIMIS data. (This will be done in orchards with other ongoing blight research programs).


A) Develop and evaluate a precipitation-temperature-based version of XanthoCast on University websites (ongoing) in cooperation with UC-IPM and CIMIS programs for utilizing rainfall and dew point temperature (i.e., dew point periods) data instead of leaf wetness for the model.

B) Evaluate early, mid-, and late-spring timings (e.g., male (catkin) vs. female (pistillate) flower emergence or delayed emergence), as well as mid- to late spring season timings) under natural and simulated rain environments using upper temperature limits (e.g., >27C).

C) Evaluate the benefits of applications of bactericides before and after major rainfalls III. Host resistance and pathogen identification/diversity-

A) Continue to evaluate walnut genotypes for natural host resistance to walnut blight under simulated rainfall conditions at the KAC and to continue to study morphological differences among fruit.

B) Evaluate diversity within pathogen populations and identify species using molecular methods.

PROCEDURES Evaluation of alternative bactericides for management of walnut blight in field studies. Five trials were established in experimental orchards in Fresno Co. (Kearney Agricultural Center - KAC) and Solano Co. (UC Davis), and in a commercial orchard in Yuba-Sutter Co. In Fresno Co., trials were conducted on cvs. Chico and Vina. Simulated rain was applied in these orchards for 6-8 h after each of the four bactericide applications. In Solano Co., trials on cvs. Hartley and Vina were conducted with three bactericide applications following XanthoCast indices. Seven calendar-based applications were done in the commercial orchard on cv. Vina. Treatment timings are indicated in Figs. 1-4, and treatments were applied using an air-blast sprayer at 100 gal/A, except for Moisturin and Root Zone. Moisturin was applied as a foliar treatment at 200 gal/A in the Yuba-Sutter Co. trial. Root-Zone was applied as a soil treatment at the Fresno Co. trial with two applications of 13 L for a total of 26 L per treatment per weekly application for four weeks. The soil treatments were watered in after each application. Incidence of disease was based on the number of infected fruit of 50-150 fruit evaluated for each of four or five single-tree replications. Data were evaluated using analysis of variance and least significant difference mean separation procedures or general linear model and LSD mean separation procedures of SAS 9.1. Evaluation of minimal spray programs based on host phenology and environmental conditions (XanthoCast) using Kocide 3000-Manzate. A trial under natural rainfall conditions in Solano Co. on cv. Chico was set up to evaluate and improve timing programs and to reduce the total number of applications of bactericide treatments. Treatment timings for three to six applications of Kocide-Manzate were based on host phenological stages (pistillate flower emergence) and before or after rain events. Disease incidence was evaluated in late May based on evaluation of 100-200 nuts for each of the four single-tree replications. Data were evaluated using analysis of variance and LSD mean separation procedures of SAS 9.1 .


Disease evaluations of walnut genotypes in a variety block at the Kearney AgCenter). Walnut genotypes from the walnut breeding program under Dr. Chuck Leslie and planted in a variety block at KAC were evaluated for disease incidence under simulated-rain conditions. Natural host resistance to walnut blight was evaluated after six weekly 8-h simulated rain events using an impact sprinkler irrigation system. These irrigation periods provided leaf wetness at all stages of bloom (catkins and pistillate flowers) and early fruit development. Fifty to 130 fruit of each of four to five single-tree replications were evaluated for blight. Additionally, fruit from ten cultivars were evaluated for structural differences that possibly could relate to the observed susceptibility. For this, fruit were sampled in June and tissue pieces of the stylar end were fixed for scanning electron microscopy (SEM) using standard procedures. Numbers of stomata and trichomes (glandular and non-glandular) were determined for an area of 0.283 mm2. For this, three sites on each sampled piece (stem end, middle, stylar end) for each of three fruit per variety were evaluated. Data were analyzed using ANOVA and LSD mean separation procedures of SAS version 9.1. Initial studies on the genetic variability within and among populations of X. arboricola pv. juglandis in California and species identity. To evaluate genetic variability within and among populations, DNA of each isolate was amplified using rep-PCR primers BOX and ERIC (Louws et al., 1994). PCR reactions contained bacterial cells and standard reagent mixtures. Amplification reactions were done at an annealing temperature of 55C, electrophoretic products were separated in 1.5% agarose gels, and banding patterns were compared. Each isolate was amplified at least twice. For species identification, dnaK, rpoD and rDNA genes were amplified according to Young et al. (2008). Reference sequences were obtained from GenBank and sequence alignments were done using ClustalW. For each gene, a Neighbor-Joining tree was constructed to visualize genetic relationships using ClustalW. RESULTS AND DISCUSSION Evaluation of alternative bactericides for management of walnut blight. In trials in experimental orchards with copper-sensitive or -resistant strains of the pathogen, applications of Kasumin, Kasumin-copper, copper, or copper-mancozeb mixtures significantly reduced the incidence of walnut blight as compared to the control (Figs. 1-4). The copper product GWN 4620 and mixtures of different copper products (e.g., GWN 4620, Badge X2, Kocide 2000, and Kocide 3000) with Manzate Pro Stick were similarly effective, reducing disease from 15% in the control to less than 5.6% (3.9-5.6%) among the treatments (Fig. 1). In the orchard with copper-resistant strains, GWN 4620 reduced the disease from 42.5% to 36.8%. Copper products mixed with Manzate Pro Stick reduced the disease to less than 23.2% in a reduced three-spray program (Fig. 2). In another trial in Fresno Co. with no copper resistance, the Kasumin-Manzate F45 mixture was among the best treatments, reducing disease incidence from 32.3% in the control to 2.8%, similar to the tank mixture of Kocide 3000-Manzate Pro Stick 75DF, the tank mixture of Kasumin-Badge X2, and the Badge X2 treatments that had 4.3%, 3.3%, and 4.3% disease, respectively (Fig. 3). Kasumin alone or with a sticker (e.g., NuFilm P), Citrox, Root-Zone, and Kasuran (Kasumin-copper pre-mixture) also significantly reduced the disease to less than 9.3% in this trial. In the second trial in Solano Co., Kasumin–Manzate F45, Kasumin mixed with Badge X2,


and Badge X2 resulted in less than 4.3% disease incidence as compared to 16.8% disease in the untreated control. Bioforge reduced the disease to 6.8% in this trial (Fig. 4). The use of the anti-transpirant Root-Zone and the plant stress response inducer BioForge are novel approaches in disease control that target plant growth regulators by stimulating abscisic acid production (that causes stomatal closure during infection periods, i.e., rain events) or reducing ethylene, respectively. In the Sutter-Yuba Co. trial in a commercial orchard with copper resistance present in the pathogen population, high rainfall during the 2010 spring season favored disease development. Seven applications of bactericides were made from late March to early May. The plot was evaluated for disease twice (Fig. 4). In late April, 9.6% and by mid-May 25.9% disease incidence developed in the non-treated control. In this second evaluation, no significant differences were found among the following treatments and the non-treated control: Moisturin, Regalia, Regalia-Badge, and Kocide 3000 treatments. Kasumin+Badge SC also was not an effective treatment in contrast to Kasumin-Badge XT (Figs. 3,4). Possibly, this could indicate an incompatibility between Kasumin and the Badge SC formulation. This needs to be investigated in 2010 by including mixtures of Kasumin with both Badge formulations in the same trial. Kocide numerically reduced the disease to 14.3% but the treatment did not provide satisfactory control in managing the copper-resistant strains of the pathogen. Badge X2 or Kasumin 2L or 10L mixed with Manzate F45 reduced the disease to the lowest levels (<6% disease incidence). Manzate by itself reduced the disease to 8.5% incidence. Treatments with Kasumin, Kasumin+Cerebrocide, and Citrox BC+Proalexin had an intermediate efficacy with of disease incidence from 9.8 to 12.5%. Interestingly, the biological control Actinovate + NuFilm P performed well with 8.3% incidence. In summary, in these five field trials we demonstrated that:

a) The EBDC fungicide mancozeb was effectively used in mixtures with copper and Kasumin

to manage walnut blight. This data justifies and supports the emergency registration of mancozeb.

b) Kasumin (also using the new 10L formulation) significantly reduced walnut blight incidence but was most effectively used in mixtures with EBDC or copper bactericides.

c) Mancozeb treatments by themselves reduced disease incidence but were not as effective as in combinations with copper or Kasumin.

d) Of the natural products, Regalia alone or in combination with copper was not effective in an orchard with moderate disease pressure and copper-resistant populations of the pathogen.

e) Actinovate+NuFilm P effectively reduced the disease under moderate disease pressure, similar to last year when another adjuvant was added. Citrox BC+Prolexin at relatively low rates also suppressed the disease and thus, further research is warranted for these biologicals.

f) The plant growth regulators Bioforge and Biozyme (the latter used in combination with Kasumin) as well as the anti-transpirant Root-Zone reduced disease and should continue to be evaluated.

g) Pending additional evaluations, these biological products could be of interest for organic walnut production in providing rotation treatments to organically approved copper products where copper-resistant populations have developed.


Evaluation of application timing of bactericide treatments with Kocide 3000-Manzate starting at pistillate flower emergence. This trial was done on cv. Chico in an orchard in Solano Co. under natural rainfall conditions. Four programs were compared: 1) Applications timed after 3 rain events; 2) Application at pistillate flower emergence and timed before 2 rain events; 3) Application at pistillate flower emergence and timed after 3 rain events; and 4) Application at pistillate flower emergence and timed before and after

rain events. Non-treated trees were used as controls and disease was evaluated in late May 2010. All of the programs significantly reduced disease from 29.8% in the control to less than 13% in Programs Nos. 1 and 2 and to less than 4.8% in Programs Nos. 3 and 4 (Fig. 6). The most effective programs used a pistillate flower emergence application followed by subsequent applications timed around rainfall events. Program No. 3 used only four applications. The last three applications were made only after rainfall events. In Program No. 4, treatments were applied before and after rainfall for a total of six applications. Program No. 1 had three applications following rainfall events, and No. 2 had a pistillate flower application with three applications before rainfall events. As previously shown, this demonstrates that the early applications may be important in reducing inoculum levels but are not self-standing for the entire season when additional rainfall may occur. Additionally, the success of post-rainfall applications of bactericides may indicate that bacterial invasion into plant tissue probably does not occur during rain events. The copper-mancozeb treatment is a protective treatment and is not known to be systemic in plant tissue. In order to obtain a high level of efficacy as a post-rainfall application, the treatment would have to be reducing bacterial populations on the plant surface before infections occur. Thus, infections mostly like occur after rain events. In summary, combination programs that include female flower (pistillate) emergence applications followed by additional applications based on environmental conditions have provided the best level of disease control. The XanthoCast environmental model is based on this principle and can be used effectively to provide consistent disease control. Research should continue on post-rainfall applications to determine if the rules used in the model can be improved. Additionally, it warrants the investigation of a comparison of rain-fastness among bactericide treatments.

Timing of walnut blight treatments to date can be summarized as follows: • Early applications at catkin and female flower emergence may be important in reducing

inoculum levels but are not self-standing for the entire season when additional rainfall may occur.

• Combination programs that include delayed female (pistillate) flower emergence applications followed by applications based on the XanthoCast environmental model proved the most consistent disease control over the years.

• Occasionally, starting a program at catkin flowering can be beneficial in orchards with a history of the disease and under high rainfall conditions.. This is most likely explained by bud populations of the pathogen and environmental conditions at the beginning of the season that allow for high bacterial populations at early female flowering (pistillate flower emergence).

• Subsequent applications during fruit development reduce fruit infections and minimize pathogen populations from increasing and thus, these treatments reduce disease from mid-to late spring and minimize re-contamination of developing buds by the pathogen (thus, lower the initial amount of inoculum in the following spring).


• Post-rain bactericide applications can be beneficial, especially if a pre-rain treatment was not done.

• Once daily high temperatures exceed 27C, additional bactericide applications may not be necessary even when precipitation is forecasted.

Disease evaluations of walnut genotypes in a variety block at the Kearney AgCenter. Walnut genotype comparisons were conducted in a variety orchard in Fresno Co. where simulated rain was applied. Disease incidence among 15 genotypes ranged from 17.4% on cv. Payne to 0.7% on PI159568 (Fig. 7). In each of the six years where we conducted these evaluations, cv. Payne was the most susceptible of the cultivars evaluated, whereas PI159568 followed by Cheinovo, 76-80, Sinensis 5, PI18256, Franquette, and Chase D9 were among the least susceptible. Although there were some differences in the ranking of the remaining cultivars over the years, overall, results were quite consistent. Thus, our long-term goal is to provide data on blight susceptibility among new and old walnut genotypes to assist the breeding program in the Horticulture Department at UC Davis. To possibly find phenotypic markers associated with disease susceptibility that could facilitate selection of breeding lines, we started to evaluate surface characteristics of developing fruit of selected varieties and we performed a quantitative analysis of stomata and trichomes (Fig. 8). Fruit surfaces of cvs. Payne, Cheinovo, Chandler, Tulare, Hartley, and Franquette as visualized by SEM are shown in Figs. 9, 10, 11. The stylar end of the fruit was investigated because here primary infections are most commonly initiated. Three regions of the stylar end (stylar, middle, and stem ends) were compared. The most susceptible cultivar in seven years of disease evaluations, cv. Payne, had the highest amount of trichomes and the highest combined number of trichomes and stomata per unit area at the stylar end. Payne also had the highest total number of trichomes at the three regions evaluated. Similar to cv. Payne in the number of stylar-end trichomes and total number of trichomes were cvs. Chandler and Hartley. Among the early varieties, cv. Payne had the highest disease incidence whereas Sinensis 5 had significantly lower disease and lower stylar-end trichome numbers. Overall, cvs. Franquette, Chase D9, Adams 10, and Sinensis 5 had the lowest disease and significantly lower stylar-end and total trichome numbers than cv. Payne. It is possibly that trichomes are involved with the survival of epiphytic bacterial pathogen populations by increasing the duration of surface wetness or by releasing exudates that support bacterial growth. Moreover, in addition to stomata, they may provide entry points for infection when injured. Previously, in codling moth susceptibility studies conducted by Christofferson et al. (1999), fruit without glandular trichomes had a significantly greater level of insect establishment compared to pubescent fruit. Thus, nut trichomes may have an inverse effect on insect and walnut blight susceptibility. Initial studies on the genetic variability within and among populations of X. arboricola pv. juglandis in California and species identity. Examples of rep-PCR fragment analyses for selected isolates are shown in Fig. 12. Based on these data, the degree of genetic diversity found among 40 isolates of X. arboricola pv. juglandis from California that were collected at different locations and over several years was much higher than that reported by others (Scortichini et al., 2001) who compared populations from several continents using the same PCR assays. Thus, in this latter research, the similarity among isolates was >78%, and the authors note that the seven isolates from California that they analyzed all had a unique banding pattern. In our study, at least


five major distinct genetic groups were identified among the 40 isolates and similarity between these groups was often <30% (a detailed quantitative analysis has not been done). Isolates from the same location often were more homogeneous. Because this high diversity was unexpected, we did additional investigations to evaluate if these groups belong to the same species, X. arboricola. Using representative isolates, a DNA sequence analysis of three loci was done and sequences were compared to those of published sequences of X. arboricola and several other closely related species. The analyses demonstrated that for two of the loci (i.e., dnaK - coding for a chaperon protein and rpoD - coding for a RNA polymerase) our walnut isolates clustered most closely with X. arboricola and for the rDNA locus the walnut isolates were in a cluster with X. arboricola, X. gardneri, and X. vesicatoria, species that are known to be closely related (Fig. 13). This confirms the species identity of the diverse California populations as X. arboricola (pathovar differentiation is based on very minor genetic differences that cannot be resolved using molecular information from basic metabolic enzymes). Based on the sequence information and the close clustering of California walnut isolates with reference sequences of X. arboricola, it may be possible to design specific primers for the detection of the walnut blight pathogen that could be used in bacterial population studies. CONCLUSIONS This year's studies indicated that copper-mancozeb (EBDC) combinations are the most consistent treatments in controlling walnut blight in California. This information will continue to support emergency and full registrations of mancozeb (e.g., Manzate) on walnut. New formulations of copper such as Kocide 3000 and new copper products (e.g., Badge products) that can be applied at much lower metallic copper equivalents (MCE), provided similar disease control levels as compared to other formulations that require higher amounts of copper. Thus, these treatments are a step in reducing copper usage while maintaining a high level of disease control. Still, for consistent results, ca. 1.2 lb of copper (metallic ion equivalent) per acre is needed when using the newer copper formulations. Among new treatments evaluated, Kasumin was again the most promising new treatment. Although moderately effective by itself, it performed very well in combination with copper or mancozeb. Kasugamycin (i.e., Kasumin) was submitted to the IR-4 program and the registration package submitted to the US-EPA in Jan. 2010. A federal registration is expected for 2011 and a California registration for 2012. Additionally, we continue to explore and develop new treatments such as the organically approved Actinovate (walnut label expected in spring 2011) and plant growth regulator treatments that potentially can alter host physiology and reduce walnut susceptibility to blight. As in previous years, our research demonstrated that applications of copper-Manex at catkin or bud break could not satisfactorily control the disease when additional rainfall occurs during the season. Calendar-based application methods (e.g., weekly applications beginning at female flower bud break) may result in excessive applications that may not improve disease control and may be costly. Additionally, our research is investigating optimal timing of treatments and may improve our understanding of bacterial invasion of host tissues, i.e., infection periods. Thus, we will continue to evaluate programs that use a combination of pistillate flower emergence i.e., bud break (initiation of the model based on host phenology) and the epidemiological model (i.e., XanthoCast) to optimize timing of bactericides with environmental conditions and disease control while minimizing the total number of applications. Thus, walnut blight can be effectively managed with properly timed in-season applications of copper-EBDC bactericides and


potentially with kasugamycin-copper or EBDC bactericide combinations in rotation with copper-EBDC treatments that minimize the total amount of any material being used while maintaining high levels of disease control and preventing the selection of pesticide resistance within the bacterial pathogen population. ACKNOWLEDGMENTS Special thanks to the grower in Sutter Co. who allowed us to conduct our research in his orchard and to the chemical industry representatives who cooperated with us with during this research. REFERENCES 1. Adaskaveg, J. E, et al. 1998-2009. Annual Walnut Reports – 1998-2009. Walnut Marketing

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6. Miller, P. W. and W. B. Bollen. 1946. Oregon Agric. Exp. Stn. Tech. Bull. 9. 107 pp. 7. Mulrean, E. N. and M. N. Schroth. 1981. Phytopathology 71: 336-339. 8. Mulrean, E. N. and M. N. Schroth. 1982. Phytopathology 72: 434-438. 9. Ogawa, J. M. and H. English. 1991. Diseases of Temperate Zone Tree Fruit and Nut Crops.

University of California, Division of Agriculture and Natural Resources, Oakland, CA. Publ. No. 3345. 461 pp.

10. Scortichini, M, Marchesi, U, and Di Prospero, P. 2001. J. Phytopathology 149:325-332. 11. Teviotdale, B. L., M. N. Schroth, and E. N. Mulrean. 1985. In: Walnut Orchard Management.

Ed. by E. Ramos. Univ. of Calif. Coop. Ext. Publ. 21410. 12. Young, J.M., Park, D.-C., Shearman, H.M., and Fargier, E. 2008. Syst. Appl. Microbiol.

31:366-377.


Control

GWN 4620 4 qt

GWN 4620 4 qt + Manzate ProStick 75DF 2.4 lb

Badge X2 4 lb + Manzate ProStick 75DF 2.4 lb

Kocide 3000 4 lb + Manzate ProStick 75DF 2.4 lbKocide 2000 6 lb + Manzate Prostick 75DF 2.4 lb

0 4 8 12 16

Four applications were done on 4-6, 4-12, 4-20, and 4-28-10. Simulated rain applied on: 4-8, 4-15, 4-22, 4-29-10. Disease was evaluated on June 20. Incidence of disease is based on 160-200 fruit for each of 5 single-tree replications.

Fig. 1. Efficacy of copper treatments on cv. Vina walnut with simulated rain applied - Fresno Co. 2010 -

Disease incidence (%)

ab

bb

bb

Three applications were done on 3-30, 4-16, and 4-29-10. Disease was evaluated in early June. Incidence of disease is based on 50-120 fruit evaluated for each 5 single-tree replications.

Control

GWN 4620 4 qt

Badge SC 2.27 67.5 fl oz + Manzate ProStick 75DF 2.4 lb

Badge X2 4 lb + Manzate ProStick 75DF 2.4 lbGWN 4620 4 qt + Manzate ProStick 75DF 2.4 lb

Kocide 2000 6 lb + Manzate Prostick 75DF 2.4 lb

Kocide 3000 4 lb + Manzate ProStick 75DF 2.4 lb0 10 20 30 40 50


ab

c

bc

cc

Fig. 2. Efficacy of copper treatments on cv. Vina walnut with simulated rain applied - Solano Co. 2010 -

bc

Fig. 3. Efficacy of new bactericide treatments on cv. Chico walnut with simulated rain applied - Fresno Co. 2010 -

Four applications were made on 4-15, 4-22, 4-30, and 5-10-10. Root-Zone was applied weekly, twice on the same day as a soil treatment. Simulated rain was applied for 6-8 h on 4-16, 4-24, 5-3, and 5-12-10. Disease was evaluated on 5-27-10. Incidence is based on the number of diseased nuts of the total of 100 nuts evaluated of each of four single-tree replications.

ControlKasumin 10L 100 ppm

Kasumin 10L 100 ppm + Badge X2 4 lbCitrox BC 0.1%

Kasumin 10L 100 ppm + NuFilm P 8 fl ozKocide 3000 4 lb + Manzate F45 58 fl oz

Root-Zone 10%Kasuran 1.67 lb

Kasumin 2L 100 ppm + Badge X2 4 lbKocide 3000 4 lb + Manzate ProStick 75DF 2.4 lb

Badge X2 4 lbKasumin 10L 100 ppm - Manzate F45 58 fl oz

0 10 20 30 40 50Disease incidence (%)

ab

b

b

bb

bb

b

bbb

ControlBioforge 1 pt/0.5 pt

Kasumin 10L 100 ppm+ NuFilm P 8 fl ozKasumin 10L 100 ppm

Kasumin 10L 100 ppm - Manzate F45 58 fl ozKasumin 10L 100 ppm + Badge X2 4 lbKasumin 2L 100 ppm + Badge X2 4 lb

Badge X2 4 lb

Fig. 4. Efficacy of new bactericide treatments on cv. Hartley walnut under natural rainfall conditions- Solano Co. 2010 -

0 5 10 15 20 25Disease incidence (%)

bbc

a

Three applications were made on 4-9, 4-26, and 5-12-10. Disease was evaluated in early June. Incidence of disease is based on 100 fruit for each of 5 single-tree replications.

bc

c

bcbc

bc

No copper resistance



Copper resistance


0 5 10 15 20 25 30

ControlRegalia 0.25% + Badge 67.5 fl oz

Regalia 0.5 %Kasumin 2L 100 ppm + Badge SC 67.5 fl oz

Moisturin 10%Kocide 3000 3.5 lb

Citrox BC 133 ml + ProAlexin 133 ml

Kasumin 2L 100 ppmKasumin 10L 100 ppm + Cerebrocide 0.5%

Manzate F45 58 fl ozActinovate 12 oz + NuFilm P 8 fl oz

Badge SC 67.5 fl oz + Manzate F45 58 fl ozKocide 3000 3.5 lb + Manzate F45 58 fl ozKasumin 10L 100 ppm + Biozyme 32 fl oz

Badge X2 4 lb + Manzate F45 58 fl ozKasumin 2L 100 ppm + Manzate F45 58 fl oz

Kasumin 10L 100 ppm + Manzate F45 58 fl oz0 3 6 9 12 15

Fig. 5. Efficacy of selected copper treatments on cv. Vina walnut under natural rainfall conditions - Yuba-Sutter Co. 2010 -


a

Seven applications were done on 3-24 and 3-29 (catkin emergence), 4-6, 4-15, 4-22, 4-29, and 5-11-10. Incidence of disease is based on 100 fruit evaluated for each 4 single-tree replications.

ef

bcde

bcd

cdef

abc

bcd

f

d

defdef

Evaluation 4-29-10 Evaluation 5-11-10

cdab

cd

cd

cdcd

cd

cd

cdcd

cdcd

aab

abcdabcabc

abc

bcdbcd

cdefcdef

cdef

Copper resistance

Fig. 6. Timing study with Kocide 3000-Manzate treatments in a cv. Chico orchard under natural rain conditions – Solano Co. 2010

Kocide 3000 – Manzate DF (4 lb-2.4 lb) was applied using an air-blast sprayer at 100 gal/A.

Timings: Pistillate flower emergence - 3-29-10. After rain events - 4-6, 4-14, and 4-29-1Before rain events - 4-9 and 4-18-10

Program 1: Applications timed after 3 rain events.Program 2: Application at pistillate flower emergence and timed before 2 rain events.Program 3: Application at pistillate flower emergence and timed after 3 rain events.Program 4: Application at pistillate flower emergence and timed before and after rain events.

Disease was evaluated in late May 2010. California Walnut Board 207 Walnut Research Reports 2010

0 10 20 30 40 500 10 20 30 40 50

Fig. 7. Walnut variety susceptibility to blight in a simulated rain-treated orchard - Fresno Co. 2005-2010 -

PI159568

Franquette

Cheinovo

76-80

Sinensis 5

PI18256

Chase D9Hartley

Serr

Chandler

91-28-1

Adams 10

90-28-30

Tulare

Payne

0 10 20 30 40 50


cdefg

b

g

bc

bcd

Five to six weekly simulated rain events were applied using a high-angle sprinkler system between April and May.

a

b

bcd

bcdebcde

bcdebcde

bcde

bcde

cde

de

e

a

ababcd

abcdedefg

abcdea

abcbcdef

cdefgefg

gefg

2005 2006

bc

bc

bc

bc

0 10 20 30 40 50

2007g

defgefgdefg

cdefef

bcd

efbc

bcde

efcdefgcdef

a

ab

2008

c

a

b

bc

bcbc

bcbc

c

c

c

0 10 20 30 40 50

cd2009

b

bc

bcd

a

b

bcd

c

d

bc

bcd

bcdbcd

bcd

d

0 10 20 30 40 50

cde

2010

ab

abc

abcd

a

abcbcde

cde

def

bcde

bcde

ef

f

cdecde

0 4 8 12 16 200 4 8 12 16 20 0 4 8 12 16 20 0 4 8 12 16 200 4 8 12 16 200 4 8 12 16 20

Fig. 8. Average walnut variety susceptibility to blight during 2005 to 2010 and quantitative analysis of stomata and trichomes on walnut fruit at three fruit locations

Cheinovo

Chase D9

Franquette

Sinensis 5

Hartley

Adams 10

Chandler

Tulare

Serr

Payne

0 20 40 60 80 100

cde

e

Data for relative disease incidence are the average of the 2008-2010 seasons of Fig. 10. Relative disease incidence was calculated for each variety based on disease incidence for cv. Payne as 100%. The number of stomata and trichomes were determined by scanning electron microscopy for an area of 0.283 mm2. For this, fruit were sampled in June and tissue pieces of the stylar end were fixed for scanning electron microscopy (SEM) . Numbers of stomata and trichomes were determined for an area of 0.283 mm2. For this, three sites of each sampled piece (stem end, middle, stylar end) for each of three fruit per variety were evaluated. Colors of each data bar represent bloom dates of the cultivars from early (black shading), to mid (dark gray shading), to late (light gray shading), to very late (white shading).

a

ab

abcd

bcd

cd

bcd

a

abc

cd

bc

d

bc

de

2005-2010 Stem end Middle Stylar end Stem end Middle Stylar end

Stomata Trichomes

ab ab

a a abab

ab cdaa

ab abc a ab bcd abc

abab abc

ab abab aa abc

ab aaa abc

bcd bcda bc abc

abab abc de

c d c cd e cde

Relative disease incidence (%)

Number of stomata

Number of stomata

Number of stomata

Number of trichomes

Number of trichomes

Number of trichomes


A B

C D

E F

Fig. 9. Scanning electron micrographs of surfaces of developing walnut fruit of cultivars Payne (A, C, E) and Cheinovo (B, D, F). The stylar end (A,B), the mid-portion (C,D), and the stem end (E,F) are shown.


Fig. 10. Scanning electron micrographs of surfaces of developing walnut fruit of cultivars Chandler (A, C, E) and Tulare (B, D, F). The stylar end (A,B), the mid-portion (C,D), and the stem end (E,F) are shown.


Fig. 11. Scanning electron micrographs of surfaces of developing walnut fruit of cultivars Hartley (A, C, E) and Franquette (B, D, F). The stylar end (A,B), the mid-portion (C,D), and the stem end (E,F) are shown.

E F

C D

A B


A

B

Fig. 12. Electrophoretic patterns of Xanthomonas arboricola pv. juglandis strains collected from California walnut orchards after rep-PCR using (A) ERIC and (B) BOX primers. Collection numbers of each strain are indicated across the top of the gels. Molecular ladder (ML) is shown in the left lane.


X-125 X-140X-844-22-11-7EU498750 X. arboricolaEU498793 X. arboricola

EU498793 X. axonopodis

A

EU498774 X. vesicatoriaEU498774 X. campestris

B AF123088 X. vesicatoriaX-140

AF123093 X. gardneriX-1254-22-1X-841-7AF209752 X. arboricola

AF209755 X. campestrisAF209766 X. axonopodis

C X-125 4-21-7

EU499070 X. arboricolaEU499121 X. arboricolaX-1402-1

EU499137 X. campestrisEU499099 X. vesicatoria

EU499071 X. axenopodis

Fig. 13. Phylogenetic analysis using the Neighbor-Joining method of Clustal-W for DNA sequence data of Xanthomonas arboricola pv. juglandisisolates collected in California and of reference species based on (A) dnaK(Chaperon protein), (B) rDNA(ribosomal DNA), and (C) rpoD (RNA polymerase) genes. Sequences with species assignments are f rom GenBank, coded sequences are f rom walnut blight isolates f rom California.

X-84


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