EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT

Transcription

1 EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT J. E. Adaskaveg, H. Förster, D. Thompson, D. Cary, K. Nguyen, J. Connell, and R. Buchner Cooperating: L. Wade, Arysta LifeScience ABSTRACT With low to moderate rainfall in the spring of 2013, the incidence of walnut blight was also low to moderate at most locations. For blight management, we evaluated copper products (i.e., Cuprofix Ultra Disperss, Badge X2, Kocide 3000, NUP 12032, AgCop 74) alone or in mixtures with Manzate and/or lime, antibiotics (Kasumin, Fireline), biocontrols (Actinovate, Botector, Double Nickel 55, Serenade Optimum), stimulators of plant host defense mechanisms (Quintec, PM1, Actigard) in mixtures with other materials, and other products (e.g., K-Phite - monopotassium phosphate/dipotassium phosphite, Prophyt, Ko-Phite potassium phosphite) with potential direct or indirect effects on disease development. Mancozeb received full registration for use on walnuts in In research trials, copper-manzate was highly effective. In some commercial orchards, however, a reduced efficacy of this treatment was reported by growers and reduced sensitivity was confirmed in the lab. Badge when used by itself was not effective in the presence of copperresistant populations of the pathogen, but very effective when mixed with Manzate. In a comparison of copper products mixed with Manzate, Cuprofix Ultra Disperss, Badge X2, Kocide 3000, and NUP were statistically similarly effective, but the lowest disease incidence was observed with NUP (1.9% as compared to the control with 17.3% disease). Kocide 3000 and AgCop 75 were similar in efficacy at another location. Rates of Cuprofix (5 and 8 lb) with Manzate (1.8 and 2.4 lb) were compared at two locations and there was no consistent rate effect (i.e., low rates of copper and mancozeb were as effective as high rates of both compounds). In two trials, the addition of lime to copper-manzate did not improve efficacy but increased persistence as measured by copper residues. Kasumin used by itself showed high or moderate efficacy. When mixed with other products such as Quintec or copper, the efficacy was improved and was similar to copper-manzate. Among other alternative treatments, Actinovate at 12 oz/a resulted in a 60% reduction in disease as compared to the control in one trial. Other treatments with intermediate efficacy included Fireline, and Serenade. Botector and Double Nickel 55 were not effective. K- Phite resulted in a reduction of disease in one of the two trials where it was included. Copper resistance in pathogen populations was found in 27 of 47 orchards tested. Using a modified in vitro sensitivity assay with copper and mancozeb, we identified isolates of Xaj with reduced sensitivity to copper-mancozeb at nine locations, indicating that a shift to reduced mancozeb sensitivity may be occurring in California populations of the pathogen. In epidemiological studies on bacteria colonizing walnut buds, we again found that Xaj commonly occurs together with other yellow-pigmented and non-yellow-pigmented bacteria. Of the 35 orchard locations sampled, buds from 27 locations yielded Xaj and other yellow-pigmented bacteria. On average, 67.7% of the yellow-pigmented bacteria were confirmed as Xaj. Non-Xaj bacteria were also recovered from buds after surface-sterilization. In studies on the prediction of walnut blight disease risk and disease levels in the current season, disease levels in the previous season were a more accurate predictor than the size of Xaj populations in walnut buds collected in the previous fall season, but error rates were still high and need to be reduced. The best strategy to optimize management efforts is to identify high-risk orchards based on disease levels in the previous season and to use weather forecasts (i.e., XanthoCast) in the current season after the pistillate-flower-emergence application of a bactericide. California Walnut Board 291 Walnut Research Reports 2013

2 OBJECTIVES I. New treatments - Evaluate the toxicity of alternative copper and non-copper based materials against Xaj in the laboratory and the efficacy of these materials for managing walnut blight under ambient and simulated rain conditions at Kearney Ag Research and Extension Center (KARE), UC Davis, and in commercial orchards. A) Comparative the efficacy of new copper bactericides such as reduced-mce products (Kocide 3000, Badge X2) and other copper products Support the full-registration of mancozeb in copper mixtures for the management of walnut blight Evaluate copper persistence with and without adjuvants under simulated rain conditions B) Optimize the performance of kasugamycin in mixture rotations with copper, EBDC compounds (e.g., Manzate, Dithane) and other fungicides (e.g., captan, Quintec, polyoxin-d) or systemic acquired resistance materials (e.g., Actigard) for sustained usage and stewardship of the antibiotic. C) Investigate the use of novel chemistries and biologicals in mixtures and rotations to inhibit the pathogen and protect trees for extended periods. Biofilm inhibitors, sanitizers (e.g., Citrox, Perasan), and residual oxidizers (e.g., AgriTitan) Biocontrols (e.g., Actinovate, Blossom Protect, Double Nickel 55) Inducers of host defense mechanisms (e.g., Actigard, ProAlexin, phosphonates) 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 and evaluate populations of the bacterial pathogen using molecular approaches A) Develop and evaluate a new version of XanthoCast on University websites (ongoing) in cooperation with UC-IPM and CIMIS programs that utilizes temperature and precipitation, dew point temperature, or relative humidity data instead of leaf wetness for the model. B) Evaluate and characterize bacterial species in walnut buds (commensals and pathogens) to determine if Xaj populations or early summer disease levels can be used to forecast disease risk in orchards in the subsequent spring season. C) Evaluate diversity within Xaj populations and rapid identification and quantification of the pathogen using molecular methods (e.g., qpcr). III. Natural host resistance of new walnut genotypes against walnut blight. New genotypes will be evaluated including Forde, Ivanhoe, and others at KARE. SIGNIFICANT FINDINGS With low to moderate rainfall in the spring of 2013, the incidence of walnut blight was also low to moderate at most locations. We obtained data on new walnut blight treatments from two commercial and three experimental orchards (UC Davis and UC KARE). For blight management, we evaluated copper products (i.e., Cuprofix Ultra Disperss, Badge X2, Kocide 3000, NUP 12032, AgCop 75) alone or in mixtures with Manzate and/or lime, antibiotics (Kasumin, Fireline), biocontrols (Actinovate, Botector, Double Nickel 55, Serenade Optimum), stimulators of plant host defense mechanisms (Quintec, PM1, Actigard) in mixtures with other materials, and other products with potential direct or indirect effects on disease development (e.g., K-Phite - California Walnut Board 292 Walnut Research Reports 2013

3 monopotassium phosphate/dipotassium phosphite, Prophyt, Ko-Phite potassium phosphite). Captan was also mixed with other treatments, as well as other selected mixtures. The results can be summarized as follows: 1. Copper-Manzate was highly effective in all trials conducted although a reduced efficacy of this treatment has been reported by growers at some locations. Mancozeb received full registration for use on walnuts in Badge, as a representative copper product, when used by itself was not effective in the presence of copper-resistant populations of the pathogen, but very effective when mixed with Manzate. In a comparison of copper products mixed with Manzate, Cuprofix Ultra Disperss, Badge X2, Kocide 3000, and NUP were statistically similarly effective, but the lowest disease incidence was observed with NUP (1.9% as compared to the control with 17.3% disease). Kocide 3000 and AgCop 75 were similar in efficacy at another location. Rates of Cuprofix (5 and 8 lb) mixed with Manzate (1.8 and 2.4 lb) were compared at two locations and there was no consistent rate effect (i.e., low rates of copper and mancozeb were as effective as high rates of both compounds in mixtures). In two trials, the addition of lime to copper-manzate did not improve efficacy but copper residues on walnut leaves were greater than for copper-manzate mixtures over a 5-week period. Additionally, residues were higher in copper-manzate or copper- Manzate-oil mixtures than when only copper was used (i.e., not in mixtures). 2. Kasumin used by itself showed high or moderate efficacy. When mixed with other products such as Quintec or copper, the efficacy was improved and was similar to copper-manzate. 3. Among other alternative treatments, Actinovate at 12 oz/a resulted in a 60% reduction in disease as compared to the control in one trial. Other treatments with intermediate efficacy included Fireline, and Serenade. Botector and Double Nickel 55 were not effective. K-Phite resulted in a reduction of disease in one of the two trials where it was included. Copper resistance in pathogen populations was found in 27 of 47 orchards tested. Using a modified in vitro sensitivity assay with copper and mancozeb, we identified isolates of Xaj with reduced sensitivity to copper-mancozeb at nine locations, indicating that a shift to reduced mancozeb sensitivity may be occurring in California populations of the pathogen. In epidemiological studies on bacteria colonizing walnut buds, we again found that Xaj commonly occurs together with other yellow-pigmented and non-yellow-pigmented bacteria. Of the 35 orchard locations sampled, buds from 27 locations yielded Xaj and other yellow-pigmented bacteria. On average, 67.7% of the yellow-pigmented bacteria were confirmed as Xaj. Non-Xaj bacteria were also recovered from buds after surface-sterilization. In studies on the prediction of walnut blight disease risk and disease levels in the current season, disease levels in the previous season were a more accurate predictor than the size of Xaj populations in walnut buds collected in the previous fall season, but error rates were still high and need to be reduced. Thus, neither method is advised at this time for predicting disease levels in the current season and using this prediction alone for designing management practices. The best strategy to optimize treatment timings and treatment intensity for managing the disease is to identify high-risk orchards based on disease levels in the previous season and to use weather forecasts (i.e., XanthoCast) in the current season after the pistillate-flower-emergence application of a bactericide. California Walnut Board 293 Walnut Research Reports 2013

4 INTRODUCTION Walnut blight caused by the bacterium Xanthomonas arboricola pv. juglandis (Xaj) is a continuous threat for California walnut growers, especially in central and northern locations where precipitation in the springtime is generally higher. The incidence of disease varies widely among years and crop Disease Cycle of Walnut Blight Pathogen is heterogenetic with multiple strains Primary inoculum Pathogen enters through stomata Catkin expansion Pistill. flower emergence End blight Only Xaj found on diseased nuts Xaj and other yellow bacteria Monocyclic Secondary inoculum Diagram 1. Disease cycle of walnut blight. Pathogen and numerous other bacteria live between bud scales Environmental Conditions Polycyclic Environment Side blight Life Cycle by J. E. Adaskaveg losses can be very high. The pathogen infects catkins, female blossoms, fruit, green shoots, leaves, and buds of English walnut and survives from one year to the next in twig lesions, living and dead buds, and diseased fruit that remain on the tree. Fruit infections occur soon after flowering and these infections account for most of the economic loss. The extended period of host susceptibility and the availability of few effective treatments are the main challenges in controlling walnut blight (see Diagram 1). Knowledge of environmental requirements of the pathogen, analysis of wetness period duration and temperature data from field weather stations, as well as actual disease progression led to the development of XanthoCast as a model to predict infection periods for walnut blight and calculate the risk of disease. Inoculum levels after the initial treatment for blight management are not considered in the model because bacterial populations can rapidly and exponentially increase to very high levels on diseased fruit under favorable environmental conditions. The model has been continuously refined based on new insights on the epidemiology of the disease. The website provides the basic XanthoCast information with XanthoCast indices for weather stations located between Red Bluff and Davis. Our bactericide application timing studies indicate that early female flower emergence applications followed by treatments based on XanthoCast provide the most consistent disease control. A catkin bactericide treatment is only needed when high precipitation occurs in an orchard with a history of high disease levels. XanthoCast provides regional forecasts and promotes judicial use of pesticides with disease control similar to calendar-based programs. The UC-IPM website for XanthoCast for utilization of CIMIS data was still under evaluation in California Walnut Board 294 Walnut Research Reports 2013

5 For forecasting disease pressure and the intensity of bactericide management programs in the coming season, others have proposed that the magnitude of female bud pathogen populations as determined between July and late winter of the previous year can be used. We are challenging this concept based on our epidemiological understanding of the disease where development of epidemics is more dependent on environmental conditions than on inoculum levels. We are suggesting that disease levels in the previous season (that can be can much more easily obtained than levels of bud populations) can be a reliable indicator for disease risk in the coming season, and that management programs have to be based on environmental conditions. To experimentally support this strategy, we have been comparing bud population sizes and actual disease levels from a large number of orchards as predictors for disease risk. In this research objective, we also determined the diversity of bacterial populations in walnut buds to test the assumption of other researchers that all yellow-pigmented bacteria obtained from bud samples are Xaj. Data for this ongoing objective are presented in this report. Our research on the management of walnut blight since 1994 has assisted in the development of copper-ebdc treatments and demonstrated the efficacy of zinc compounds and zinc-ebdc mixtures that can be used to minimize the development of copper-resistant populations of the pathogen. We evaluated numerous copper compounds including copper hydroxide, cuprous oxide, and copper oxychloride, as well as reduced-copper products (e.g., Kocide 3000, Badge). More recently, we showed that products containing mancozeb (Manzate, Dithane, Penncozeb, etc.) instead of maneb are effective in mixtures with copper or the antibiotic Kasumin. An important outcome of this is the recent full registration of mancozeb for use on walnuts. Copper-mancozeb applications are currently the most effective treatment for walnut blight. In recent years, some growers suffered high disease losses although copper-mancozeb treatments were applied. This led to an investigation if shifts in sensitivity or even resistance to mancozeb could have developed in some pathogen populations and in 2013 we continued our surveys on bactericide activity against Xaj. Over the years, we evaluated many possible alternatives to copper-ebdc treatments. Among them, kasugamycin (Kasumin, Arysta Life Science) was found to have the greatest potential in walnut blight management. This antibiotic is used in agriculture in other countries but not in human or animal medicine. Kasumin alone significantly reduced walnut blight incidence and, under low rainfall, it was highly effective. Under high rainfall (simulated with overhead irrigation or naturally occurring), mixtures of the antibiotic with EBDC or copper resulted in an increased efficacy and outstanding performance. Moreover, in most trials, Kasumin-EBDC or Kasumincopper mixtures were similar to or higher in efficacy than copper-ebdc mixtures. Currently, federal registration is targeted for early 2014 and a California registration possibly later that year. Ideally, we would like to have some research-authorized demonstrations this coming growing season once a federal registration has been established on walnut. Additional research is still needed on other possible alternative treatments such as natural compounds (e.g., Regalia, Citrox evaluated in previous years; Serenade Optimum), biocontrols (e.g., Actinovate, Blossom Protect, Double Nickel 55, Botector), enhancers of host resistance (e. g., Actigard, Quintec), antibiotics registered for other bacterial diseases (e.g., oxytetracycline Fireline), and phosphonates (e.g., ProPhyt, K-phite). Some of these compounds demonstrated promising results before. Thus, Actinovate was registered for use in 2011 based on our efforts and is a copper alternative for organic growers. Our overall goal for management of walnut blight is to ultimately California Walnut Board 295 Walnut Research Reports 2013

6 have multiple treatments available that provide equal or improved efficacy to that obtained with traditional copper-containing compounds. New treatments will need to be effective against coppersensitive and -resistant populations of the walnut blight pathogen. This strategy will result in a lower usage and decreased environmental impact of any one material and a lower potential for selecting resistant populations of the pathogen. PROCEDURES Isolation of Xaj from diseased walnut fruit and determination of in vitro sensitivities against copper, mancozeb, and new bactericides. Samples of buds and blighted walnut fruit were collected by farm advisors, PCAs, and chemical industry representatives from 55 orchard locations in the spring of Tissue samples were suspended in sterile water, aliquots were streaked onto yeastdextrose-calcium carbonate (YDC) agar amended with cycloheximide, cephalexin, 5-fluorouracil, and tobramycin and single colonies were obtained. Colonies were characterized by PCR using our Xanthomonas-specific primers and for their sensitivity to copper and mancozeb. Copper sensitivity was determined by streaking isolates onto nutrient agar amended with 10 ppm or 50 ppm copper ion using copper sulfate pentahydrate. Growth at 50 ppm copper after 2 days of incubation at 25C was considered copper-resistant. Mancozeb sensitivity was tested using the spiral gradient dilution method. Bacterial suspensions were streaked radially onto amended nutrient agar plates and incubated for 2 days at 25C. Insensitivity to copper-mancozeb was tested by applying a mancozeb gradient to CuSO 4 -amended agar plates. Sixty-seven isolates from one location are pending mancozeb sensitivity evaluation. Inhibitory concentrations where a reduction of bacterial growth was observed were determined by measuring radial growth and then using a computer program with an algorithm based on the molecular weight of the test substance and diffusion constants. Several derivatives of a new chemical were evaluated in vitro as potential new bactericides and were coded ATT, TDDT, and TDK. They were evaluated on spiral gradient dilution plates by themselves and in combination with copper sulfate as described above. For evaluation of the biofilm inhibitor 3,4-dehydro-L-proline, a suspension of Xaj (copperresistant) was spread uniformly onto nutrient agar plates. Four filter paper disks (6 mm in diameter) were placed onto the agar surface, and 10 µl of test solution was added. Test solutions included streptomycin, oxytetracycline, kasugamycin, and copper sulfate by themselves and in combination with 3,4-dehydro-L-proline at 1000 ppm. After 24 h of incubation, inhibitory zones around the filter paper disks were measured and inhibition of the bactericides by themselves was compared to that of the mixtures. Evaluation of alternative bactericides for management of walnut blight in field studies. Trials were established in experimental orchards at KARE (Fresno Co.; cv. Vina; Xaj copper-sensitive) and UC Davis (Solano Co.; cv. Vina; Xaj copper-sensitive), and in commercial orchards in Solano (cv. Tulare; Xaj partially copper-resistant) and Sutter/Yuba Co. (cv. Tehama; Xaj copperresistant). The Fresno Co. trial was conducted with simulated rain applied for 6 h after each of the four bactericide applications. The other trials were conducted under natural rainfall conditions with bactericide applications based on XanthoCast, and four or five treatments were done with treatment timings indicated in Figs California Walnut Board 296 Walnut Research Reports 2013

7 Copper compounds evaluated included the microencapsulated copper hydroxide Kocide 3000 (DuPont Chemical Co.), copper hydroxide (NUP 12032; NuFarm Americas, Inc.), a pre-mixture of copper hydroxide/oxychloride (Badge X2; Isagro, Inc.), basic copper sulfate (Cuprofix Ultra Disperss; United Phosphorus), and cuprous oxide (AgCop 75; American Chemet Corp.). The EBDC Manzate ProStick 75DF, the phthalimide Captan (Arysta Life Science), the antibiotics kasugamycin (Kasumin; Arysta Life Science) and oxytetracycline (Fireline; AgroSource, Inc.), and the phosphonates Prophyt (Luxembourg Industries) and K-Phite (Plant Food Systems, Inc.) were used either alone or in selected mixtures. Natural products/biocontrols evaluated included Actinovate (a fermentation product of Streptomyces lydicus; Natural Industries, Inc.); Botector (Aureobasidium pullulans; Westbridge Agricultural Products), Double Nickel 55 (Bacillus amyloliquifaciens; Certis USA), and Serenade Optimum (Bacillus subtilis; Bayer Crop Science). Stimulators of plant host defense mechanisms evaluated were Quintec (quinoxyfen; DowAgro), Actigard (acibenzolar-s-methyl; Syngenta Crop Protection), and the experimental PM1. Incidence of disease was based on the number of infected fruit of fruit evaluated for each of four or five single-tree replications. Data were evaluated using analysis of variance or general linear model procedures and least significant difference (LSD) mean separation procedures of SAS 9.1. Develop and evaluate a new version of XanthoCast on University websites. The UC-IPM website for XanthoCast was still under evaluation in To use the CIMIS system, we developed datasets for XanthoCast with dew point temperature, relative humidity, and precipitation as parameters to identify leaf wetness periods without actually measuring leaf wetness. These data were then compared to archived data sets where leaf wetness data was obtained with disease incidence data. The CIMIS data was regional data and not for the specific location where disease data was obtained. Regressions were evaluated for disease incidence and for selected environmental parameters or combinations of selected parameters using statistical procedures of SAS 9.1. Evaluation of predictors of disease risk in the current season: Xaj population sizes in walnut buds in the fall of the previous season vs. actual disease in the previous season. Thirty to fifty female flower buds were collected in November of 2012 and 2013 by PCAs in 35 orchards that had low (<5%), moderate (5-20%), or high (>20% diseased nuts) disease levels in early summer of 2012 and Buds were split in half to be used for bacterial isolation and future qpcr amplifications. For bacterial isolation, from each location three bud halves from each of 10 branches were thinly sliced, placed in 0.5 ml of sterile water, and vigorously shaken for 5 min. A subset of samples was also surface-sterilized in 400 ppm sodium hypochlorite before slicing and incubating in water. The solution was diluted with sterile water (1:100) and then spiral-plated onto YDC agar as described above. Plates were incubated at 25C for 3-4 days and then evaluated for bacterial growth. Growth of Xaj, other yellow bacteria, and non-yellow bacteria was rated using a scale from 0 = no growth, 1 = fewer than 100 colonies, 2 = colonies, and 3 = plates containing more than 200 colonies. Representative colonies of putative Xaj were verified for species identity using specific PCR primers. These ratings were categorized as low (rating <1), moderate (1-1.7), or high (>1.7). Bacterial contaminants were rated using the same scale. These ratings were done for each isolation plate and were then averaged for each location. The incidence of Xaj among the total of yellow bacteria recovered was then calculated. A tabular comparison was done for the two disease predictors (i.e., Xaj bud population sizes and disease levels in the previous season). The total number of mis-matches (anytime when low, medium, or high disease was not predicted accurately) and an adjusted number of mis-matches (only those mis-matches were considered where low predicted disease was actually medium or California Walnut Board 297 Walnut Research Reports 2013

8 high, or medium predicted disease was actually high) were counted. Percent error was then calculated based on the number of mis-matches of the total number of orchards in each disease category. Disease evaluations of walnut genotypes in a variety block at the Kearney AgCenter. Trees in the new walnut variety orchard at Kearney Ag Center with new commercial cultivars and genotypes from the walnut breeding program under Dr. Chuck Leslie were evaluated in early summer for the incidence of walnut blight. RESULTS AND DISCUSSION Walnut blight incidence in California in 2013 and sensitivity of Xaj isolates to copper, coppermancozeb, and new bactericides. With low to moderate rainfall in the spring of 2013, the incidence of walnut blight was also low to moderate at most locations. High disease levels in 2012 presumably created high inoculum levels in flower buds. With less favorable environmental conditions and timely applications of bactericides, however, the disease was mostly well managed in Still, some growers noted a reduced efficacy of copper-mancozeb treatments over the last two years. We followed up this observation with in vitro sensitivity assays for new isolations of Xaj. A total of 55 orchards were sampled and Xaj was obtained from 48 locations. To date, 234 isolates were evaluated for copper and copper-mancozeb sensitivity (data are pending for 67 isolates from one location). Copper resistance was found at 27 locations with an incidence of 16.7% to 100% among orchard populations (Table 1). Copper-resistant isolates from 9 locations showed a reduced sensitivity to copper-mancozeb mixtures. In our assay using spiral gradient dilution plates with a gradient of mancozeb concentrations applied to a uniform concentration of copper, most isolates of Xaj showed no or little growth and minimum inhibitory concentration values for mancozeb in the presence of 50 ppm copper ion were less than 0.06 ppm. Less sensitive isolates, however, were able to grow at 0.12 to 0.37 ppm mancozeb with the same level of copper present. Thus, it appears that a 2X to 6X shift to reduced copper-mancozeb sensitivity is occurring in California populations of Xaj and this will be further investigated in future studies. A slight shift towards reduced sensitivity may lead to further decreased sensitivity. The activity of mancozeb that was finally fully registered on walnut in California in 2013 needs to be protected after over twenty years of emergency registrations for copper-edbc mixtures. In Florida, where copper-mancozeb was used since the early 1980s, copper-edbc mixtures are ineffective against copper-resistant strains in some locations. Thus, we are continuing our evaluation of alternative bactericides. With kasugamycin as the best treatment identified over multiple years, the Walnut Board of California is currently exploring emergency registration options with the California Department of Food and Agriculture for this antibiotic that is only used in agriculture and not in human medicine. Residue data for kasugamycin developed by IR-4 has been reviewed by EPA and full registration is pending. California Walnut Board 298 Walnut Research Reports 2013

9 Table 1. Survey for copper and manzate sensitivity in populations of Xanthomon as arboricola pv. juglandis from walnut orchards in California 2013 Number of orchards No. of isolates tested Incidence of Cu-resistance + Incidence of Curesistance reduced sensitivity to mancozeb Orchards with no resistance Orchards with Cu resistance only % 0 Orchards with Cu resistance and reduced sensitivity to mancozeb Total % % * - Copper sensitivity was tested using CuSO 4 -amended nutrient agar plates. An isolate was considered Curesistant when growth was not inhibited in the presence of 50 mg Cu ion/l (200 mg/l CuSO 4 ). Manzate sensitivity was tested using the spiral gradient dilution plate method. Double insensitivity was tested by applying a mancozeb gradient to CuSO 4 -amended agar plates. 67 isolates from one location are pending mancozeb sensitivity evaluation. Three members of a chemical group currently not used in agriculture in the US were evaluated in vitro as potential new bactericides. None one of them was highly inhibitory when used by itself. All three compounds increased the sensitivity of copper-resistant isolates to copper, but ATT was the most active (Fig. 1). Thus, ATT has a similar effect as mancozeb when used in combination with copper and has potential as a walnut blight treatment. This group of chemicals has low human toxicity (ED 50 values >5000 ppm). It is used for control of bacterial diseases in some countries and its registration potential in the US will need to be explored. S MR MR HR HR MR HR HR A B C Control Cu 50 ppm Cu 50 ppm + gradient of ATT ( ppm) Fig. 1. In vitro sensitivity of copper and a mixture of copper and ATT against one copper-sensitive strain (S) of Xaj, two strains moderately resistant to copper (MR), and four strains highly resistant to copper (HR). (A) shows growth of the bacterial strains on an non-amended control plate. (B) For copper sensitivity, nutrient agar was amended with 50 ppm copper ion. (C) ATT was spiral plated onto the agar plate, resulting in a concentration gradient from 0.4 to 40 ppm. Growth of MR strains is almost completely inhibited in the presence of copper and ATT, whereas growth of HR strains is reduced as compared to copper alone. The biofilm inhibitor 3,4-dehydro-L-proline showed no activity against a copper-resistant isolate of Xaj when a solution of 10,000 ppm was added to filter paper disks on the surface of agar plates with a bacterial lawn. Additionally, the compound at 1,000 ppm mixed with streptomycin, oxytetracycline, kasugamycin, or copper sulfate did not increase the toxicity of these materials. Biofilms are produced by bacterial pathogens and are thought to help protect bacteria from harsh environments. Inhibition of biofilm formation and by keeping bacteria in a planktonic (single-cell) California Walnut Board 299 Walnut Research Reports 2013

10 state has been suggested to make bacteria more sensitive to chemical treatments. Our results with 3,4-dehydro-L-proline were not supportive of this, but potentially other biofilm inhibitors may have better activity. Evaluation of alternative bactericides for management of walnut blight in field studies. Data on new walnut blight treatments were obtained from two commercial and two experimental orchards (UC Davis and UC KARE). Copper-Manzate was highly effective in all trials conducted (Figs. 2-4) although a reduced efficacy of this treatment has been reported by growers at some locations (see above). Badge, as a copper representative, when used by itself was not effective when populations of the pathogen were copper-resistant (Fig. 2). Still, this copper product was very effective at this trial location when mixed with Manzate. Mancozeb received full registration for use on walnuts in In a comparison of copper products that were mixed with Manzate, Cuprofix Ultra Disperss, Badge X2, Kocide 3000, and NUP were statistically similarly effective, but numerically the lowest disease incidence was observed with NUP (1.9% disease as compared to the control with 17.3% disease) (Fig. 2). Kocide 3000 and AgCop 75 were similar in efficacy at another trial location (Fig. 3). Rates of Cuprofix (5 and 8 lb) with Manzate (1.8 and 2.4 lb) were compared at two locations. There was no consistent rate effect and low rates of Cuprofix or Manzate were sometimes more effective than higher rates (Figs. 2, 4). In two trials, the addition of lime to copper-manzate did not improve efficacy (Figs. 2, 3) but copper residues on walnut leaves had greater persistence over a 5-week period (Table 2). Additionally, residues were higher when copper was used with mancozeb than when only copper was used (i.e., not in mixtures). Mancozeb has its own toxicity against the pathogen but apparently also maintains the availability of free copper ions on plant surfaces, resulting in an increased activity of copper in reducing walnut blight. Kasumin used by itself showed high (Fig. 2) or moderate (Figs. 3,5) efficacy in reducing the incidence of blight. When mixed with other products such as Quintec or copper, the efficacy of Kasumin was improved and was similar to copper-manzate (Figs. 3,5). Thus, over the years, Kasumin mixed with copper, an EBDC, or other products such as Quintec has shown consistent high efficacy for controlling walnut blight. Registration of Kasumin is pending in California Walnut Board 300 Walnut Research Reports 2013

11 Xaj copper resistant Treatment Rate 3/28 4/2 4/11 4/17 @ Cuprofix Ultra Disperss + Manzate ProStik 75DF 5 lb + @ K-phite 96 @ Badge X2 @ Cuprofix Ultra Disperss + Manzate ProStik 75DF 8 lb + @ Kasumin 2L + Manzate ProStik 75DF 100 ppm + @ Prophyt 96 @ Cuprofix Ultra Disperss + Manzate ProStik 75DF 8 lb + @ Rotation Badge X2 + Captan 80WDG 5 lb + 5 Kasumin 2L + Captan 80WDG 100 ppm + 5 lb --- Kasumin 2L + Badge X2 100 ppm + @ Badge X2 + Manzate ProStik 75DF + Lime 5 lb lb + @ Kasumin 2L + Badge X2 80 ppm + @ Cuprofix Ultra Disperss + Manzate ProStik 75DF 5 lb + @ Kasumin 2L @ Badge X2 + Manzate ProStik 75DF 5 lb + @ Kocide Manzate ProStik 75DF 5 lb + @ NUP WDG + Manzate ProStik 75DF 5 lb + @ Table 2. Copper residues on walnut leaves collected from field trials on the management of walnut blight on cv. Tehama walnut in Yuba Co Disease incidence (%) g abcdef bcdef cdefg cdefg defg defg fg efg efg ab abc abcd abcde abcdef bcdef a Fig. 2. Efficacy of new copper- and non-copper-based treatments for managing walnut blight on cv. Tehama walnuts under natural rainfall conditions - Yuba-Sutter Co Disease was evaluated in late May. Incidence of disease is based on 100 fruit for each of 4 single-tree replications. Copper residue on June 11, 2013 (ppm) Treatment* Trial 1 Trial 2 Control 40 ND Copper Copper-Manzate Copper-Manzate-Lime 863 ND Copper-Manzate-Lime-Zinc ND 702 Copper-Manzate-Vintre ND 628 * - 5 applications were done between late March and early May. Copper used in the first trial was Badge X2 (5 lb/a) and in the second trial Kocide 3000 (5.25 lb/a). Other rates used were: Manzate 2.4 lb/a, lime 4 lb/a, zinc 2 lb/a, and Vintre 2 qt/a. Copper residues were determined by the ANR Analytical Lab at UC Davis. ND = Not determined. California Walnut Board 301 Walnut Research Reports 2013

12 Xaj partially copper resistant Treatment* Rate 4/1 4/9 4/18 5/7 Botector Double Nickel 55 Kasumin 2L + Actigard 100 ppm + K-phite 96 Kasumin 2L Actinovate + NF-P Kasumin 2L + Fireline 100 ppm + Serenade Optimum Fireline Actinovate + Kasumin 2L + NF-P 12 oz + Actinovate + Kocide oz + Badge X2 + Quintec 5 lb + 6 Actinovate + NF P AgCop 75 + Manzate ProStik 75DF 5 lb + Kasumin 2L + Badge X2 80 ppm + Kasumin 2L + Quintec 100 ppm + 6 Kocide Manzate ProStik 75DF + Lime 5 lb lb + Kocide Manzate ProStik 75DF 5 lb + Disease incidence (%) bcd bcdef bcde bcde bcdef cdef cdef cdef cdef def ef f ab abc abc bcd bcde bcde a Fig. 3. Efficacy of new copper- and non-copper-based treatments for managing walnut blight on cv. Tulare walnuts under natural rainfall conditions - Solano Co Disease was evaluated in late May. Incidence of disease is based on 100 fruit for each of 4 single-tree replications. Xaj copper sensitive Treatment* Rate 4/1 4/10 4/18 4/25 5/6 Control Kasumin 2L + Manzate ProStick 75DF Cuprofix Ultra Disperss + Manzate ProStick 75DF Kocide Manzate ProStick 75DF Cuprofix Ultra Disperss + Manzate ProStick 75DF Cuprofix Ultra Disperss + Manzate ProStick 75DF Cuprofix Ultra Disperss + Manzate ProStick 75DF 100 ppm + @ 8 lb + @ 5 lb + @ 5 lb + @ 8 lb + @ 5 lb + @ Disease incidence (%) c c c bc bc b a Fig. 4. Efficacy of new copper- and non-copper-based treatments for managing walnut blight on cv. Vina walnuts under natural and similated rainfall conditions - Fresno Co Simulated rain was applied for 6 h each one day after each bactericide application. Disease was evaluated in June. Incidence of disease is based on 100 fruit for each of 4 single-tree replications. California Walnut Board 302 Walnut Research Reports 2013

13 Xaj copper sensitive Treatment* Rate 4/18 5/3 5/15 @ Double Nickel @ Serenade Optimum 48 @ Kasumin 2L @ Kasumin 2L + PM1 100 ppm + @ Kasumin 2L + Quintec 100 ppm + 6 @ Kasumin 2L + Badge X2 80 ppm + @ Disease incidence (%) Among other alternative treatments, Actinovate at 12 oz/a resulted in a 60% reduction in disease as compared to the control in one trial (Fig. 3). Other treatments with intermediate efficacy included Fireline, and Serenade (Figs. 3, 5). Botector and Double Nickel 55 were not effective (Fig. 3). K-phite resulted in a reduction of disease in one of the two trials where it was included (Figs. 2, 3). In conclusion of the management studies, among currently available treatments, copper-mancozeb (EBDC) combinations were fully registered (Section 3) in 2013 and are the most consistent in controlling walnut blight in California. Kasumin was again a promising new treatment. Although moderately effective by itself, it performed better in combination with copper or mancozeb and similar to copper-mancozeb combinations. As in previous years, our research demonstrated that applications of copper-mancozeb at pistillate flower emergence followed by subsequent sprays based on temperature and rainfall (as summarized in the XanthoCast model) is currently the most effective strategy in managing the disease. Potentially in the future, kasugamycin-copper or - EBDC combinations in rotation with copper-ebdc treatments can minimize the total amount of any bactericide being used while maintaining high levels of disease control and providing resistance management by alternating different modes of action. Previously we stated that Regalia-copper combinations can be effective under low to moderate disease levels and for organic production of walnuts where use of mancozeb is not allowed. Neither Regalia nor Kasumin can be considered potential replacements of mancozeb because copper-mancozeb mixtures have a twenty-year history of proven consistent performance for managing walnut blight under low to highly favorable conditions for disease. Considering shifts in copper-mancozeb sensitivity in sub-populations of the pathogen, alternatives are should be registered immediately to reduce the total number of any treatment in a given season. Develop and evaluate a new version of XanthoCast on University websites. The UC-IPM website for XanthoCast was still under evaluation in The main problem is lack of leaf wetness data on CIMIS. Our goal is to prepare XanthoCast for the next generation of computerized systems that push data from automated computer models using real time-data in final form to end-users such as growers and PCAs. To use the CIMIS system, XanthoCast will need to use dew point temperature, possibly relative humidity, and precipitation to identify leaf wetness periods without actually measuring leaf wetness. Changing parameters of the model require considerable evaluations and simulations of archived data. To date, we have not been able to obtain the same level of statistical confidence in using dew point and relative humidity. A possible problem is using regional CIMIS b b ab a ab a a Fig. 5. Efficacy of new copper- and noncopper-based treatments for managing walnut blight on cv. Vina walnuts under natural rainfall conditions - Solano Co Disease was evaluated in June. Incidence of disease is based on fruit for each of 4 singletree replications. California Walnut Board 303 Walnut Research Reports 2013

14 data on archived disease data originally obtained using leaf wetness sensors with dataloggers on the same trees where disease data was obtained. Thus, dew point, relative humidity, and precipitation data needs to be measured in orchards where disease data is collected. The original model can still be accessed on CIMIS with user defined data sets (e.g., temperature and leaf wetness) but this requires some effort and needs to be automated if it will be utilized in the future. Table 3. Recovery of Xanthomonas arboricola pv. juglandis (Xaj ) and other bacteria from walnut buds collected in the fall of 2013 at 35 locations* Rating for recovery of Xaj (0-3) Rating for recovery of yellow contaminants (0-3) Rating for recovery of non-yellow contaminants (0-3) Incidence of Xaj among yellow bacteria recovered (%) 0-3 (1.3) (0.3) (0.3) (67.7) * - From each location, three buds from each of 10 branches were cut in half. Halves were thinly sliced and placed in 0.5 ml of sterile water and vigorously shaken for 5 min. The solution was diluted with sterile water (1:100) and then spiral-plated onto YDC agar amended with cycloheximide, cephalexin, 5-fluorouracil, and tobramycin. Plates were incubated at 25C for 3-4 days and then evaluated for bacterial growth. Growth of Xaj, other yellow bacteria, and nonyellow bacteria was rated using a scale from 0 = no growth, 1 = low number of colonies, 2 = medium number of colonies, to 3 = high number of colonies. Numbers in parentheses are the average of the 35 locations. Representative colonies of putative Xaj were verified for species identity using specific PCR primers. Isolation of Xaj from walnut buds and prediction of walnut blight. In isolations from walnut buds, as in last year s studies, a range of yellow-pigmented and other bacteria were recovered from surface-sterilized and non-surface-sterilized buds indicating that a range of microorganisms can colonize and survive on the protected tissues between the bud scales. Among yellowpigmented bacteria, Xaj was identified by cultural characteristics and by using specific primers in PCR amplifications. Xaj was recovered in 28 of the 35 samples. Twenty-seven of the bud populations contained non-xaj yellow-pigmented bacteria and 18 had other bacterial contaminants. There was a wide range of percent recovery of Xaj among other bacteria, and on average, 67.7% of yellow-pigmented bacteria were identified as Xaj (Table 3). Thus, on average 32.3% of the yellow bacteria from buds was not the pathogen. Moreover, extreme variability occurred with a range of 0 to 100% of the yellow bacteria from buds being the pathogen. Expressed as a frequency, the pathogen was present at an occurrence of >90% of the yellow bacteria only 25% of the time (data not shown). Altogether this indicates that quantification of Xaj populations in walnut buds is difficult considering the complexity of bacterial communities present. To predict walnut blight disease risk and disease levels in the current season, disease levels in the previous season and size of Xaj populations in walnut buds collected in the previous fall were evaluated as possible predictors. Both predictors resulted in errors for the 35 orchard locations. Using the previous year s disease level as a predictor, and considering only those errors where a low or medium disease level was forecasted, but actual disease was higher (thus, potentially using a less stringent management program which would be insufficient to prevent losses in a highdisease situation), there were errors of 20%, 37.5% and 0% for the low-, medium-, and highdisease prediction groups (Table 4). Total error for the disease method was 5 mismatches out of 35 or 14.3%. Using fall bud population sizes as a predictor, error rates were higher and were 35.7%, 77.8%, and 0%, respectively, for these disease groups. Total error for the bud population California Walnut Board 304 Walnut Research Reports 2013

15 method was 12 mismatches out of 35 or 34.3%. Under these stipulations, the error will always be 0% for the high-disease group because a maximum of disease is already present that cannot increase any more. Thus, both prediction methods resulted in an error, but actual observed disease levels were a more accurate predictor. Bud populations that were determined in the fall of 2013 and 2013 disease levels will again be used to calculate errors in actual disease outcome in A comparison of both methods is the best way to come to a scientific conclusion as to the method with the lowest error. When considering the value of the crop, error rates greater than 5% (or possibly 10%) are considered statistically and economically unacceptable. Table 4. Evaluation of disease level in the previous season or bud population size of Xanthomonas arboricola pv. juglandis in the previous fall as predictors for disease level in the following season for 35 commercial walnut orchards. Comparison 1: Blight incidence 2012 vs. Blight incidence 2013 Blight No. of orchards Blight No. of orchards incidence within each incidence Low, Medium, summer category summer 2013* or High** 2012* No. of mismatches total % error No.of mismatches*** % error Low 10 Low Low Medium 2 Low High 0 Medium 8 Low Medium Medium 4 Medium High 3 High 17 Low High Medium 5 High High 11 Comparison 2: Bud populations of X. arboricola pv. juglandis fall 2012 vs. blight incidence 2013 Bud population fall 2012**** No. of orchards within each category Blight incidence summer 2013* No. of orchards Low, Medium, or High** No. of mismatches total % error No.of mismatches*** % error Low 14 Low Low Medium 4 Low High 1 Medium 9 Low Medium Medium 2 Medium High 7 High 12 Low High Medium 5 High High 6 * - Disease in the orchard was evaluated in early summer and was rated as low (<5% disease), moderate (5-20% disease, or high (>20% disease). ** - Orchards within each of the three fruit disease ratings in summer 2012 or within the three fall 2012 bud population ranges. ***- Only mismatches considered where Low disease actually was Medium or High, or Medium disease was High. **** - Isolation plates from walnut bud isolations were rated after 3 days of incubation at 25C based on a scale from 0 = no colonies to 3 = high density of Xaj colonies. In summary, neither method is advised at this time for predicting disease levels in the current season and using this information alone for designing management practices. Still, determining incidence of disease in a several-hundred nut sample in June or July is inexpensive and much easier than collecting buds, shipping, and culturing of bacteria from buds. Currently, the best strategy to optimize treatment timings and treatment intensity (i.e., materials, frequency, rates, California Walnut Board 305 Walnut Research Reports 2013

16 etc.) is to identify high-risk orchards based on disease levels in the previous season and to use weather forecasts (i.e., XanthoCast) in the current season (after the pistillate-flower-emergence application of a bactericide). Varietal susceptibility. Disease was evaluated in our new variety orchard at Kearney Agricultural Research and Extension (KARE) Center, however, disease incidence was again extremely low. This is a relatively new orchard that was planted two years ago. Thus, favorable conditions for walnut blight development will be continued to be provided in the coming seasons by applying overhead irrigation to simulate rainfall. This was our strategy in the previous variety orchard where disease increased from very low levels to high levels over three to four seasons using the same strategy. ACKNOWLEDGMENTS Special thanks to the growers in Solano and Sutter/Yuba Co. who allowed us to conduct our research in their orchards and to the chemical industry representatives and PCAs who cooperated with us with during this research. REFERENCES 1. Adaskaveg, J. E, et al Annual Walnut Reports Walnut Marketing Board of California. Sacramento, CA. 2. Christofferson, C., McGranahan, G., Mills, N., and Leslie, C Annual Walnut Reports Walnut Marketing Board of California. Sacramento, CA. 3. Conover, R. A. and Grenhold, N. R Mixtures of copper and maneb or mancozeb for control of bacterial spot of tomato (Xanthomonas campestris pv. vesicatoria) and their compatibility for control of fungus diseases. Proc. of Fla. State Hortic. Soc. 94: Lee, Y. -A., M. N. Schroth, M. Hendson, S. E. Lindow, X. -L. Wang, B. Olson, R. P. Buchner, and B. Teviotdale Phytopathology 83: Lee, Y.-A., Sung, A.-N., Liu, T.-F., and Lee, Y.-S Appl. Environm. Microbiol. 75: Louws, F.J., Fulbright, D.W., Taylor Stevens, C., and de Bruijn, F.J Appl. Environ. Microbiol. 60: Miller, P. W. and W. B. Bollen Oregon Agric. Exp. Stn. Tech. Bull pp. 8. Schaad, N.W., Jones, J.B., and Chun, W Laboratory Guide for Identification of Plant Pathogenic Bacteria. Third Edition. APS Press, St. Paul, MN. 9. Scortichini, M, Marchesi, U, and Di Prospero, P J. Phytopathology 149: Teviotdale, B. L., M. N. Schroth, and E. N. Mulrean In: Walnut Orchard Management. Ed. by E. Ramos. Univ. of Calif. Coop. Ext. Publ Young, J.M., Park, D.-C., Shearman, H.M., and Fargier, E Syst. Appl. Microbiol. 31: California Walnut Board 306 Walnut Research Reports 2013