WALNUT BLIGHT CONTROL USING INTEGRATED PEST MANAGEMENT TECHNIQUES

WALNUT BLIGHT CONTROL USING INTEGRATED PEST MANAGEMENT TECHNIQUES Richard P. Buchner, Steven E. Lindow, James E. Adaskaveg, Cyndi K. Gilles, and Renee Koutsoukis ABSTRACT Three years of surveying walnut blight and the causal pathogen in Tehama County and two years in Butte County revealing a linkage of a history of Xanthomonas arboricola pv juglandis populations in orchards with current season disease and temporal dynamics explained by differences in disease control measures. In the Tehama County orchards, walnut blight control has been very good and spray programs are maintaining bud pathogen populations at low levels. In the Butte County locations, although there are only two years of data, less effective spray programs have allowed disease damage and the initial inoculum overwintering in dormant buds appears to be increasing. Walnut blight damage differed greatly between different trees in a linear transect through a Vina orchard and disease was strongly related to the populations of the pathogen in the developing buds, suggesting that disease history of individual trees also varies, and is predictive of the risk of disease within an orchard. For trees with a history of high blight damage an aggressive spray program over two years greatly reduced pathogen abundance in over-wintering buds, and reduced blight damage to zero. Assessments of the population sizes of the pathogen in dormant buds is thus a good predictor of the risk of walnut blight in a given year since the walnut buds are the primary source of initial inoculum that can spread to susceptible leaf and nut tissues after they open.lastly,not all walnut buds open in a given season. Summer samples of these un-opened buds indicate that they contain pathogen. They most likely do not contribute to initial inoculum because they remain closed. INTRODUCTION Walnut blight caused by the bacterium Xanthomonas arboricola pv juglandis (Xaj) continues to be a serious disease, particularly in Northern California where conditions favor disease. Although the search continues for additional materials effective for Xaj control, the most effective materials currently available are copper products tank mixed with manganese ethylene bis-dithiocarbamate (EBDC) formulations. It is critical to have an effective control material when pathogen monitoring techniques indicate a spray application is necessary. Xanthocast developed by Adaskaveg et.al. predicts infection events based upon weather conditions, assuming that the pathogen is present. Buchner and Lindow have shown walnut blight damage is also strongly related to the presence and magnitude of Xaj overwintering under dormant bud scales. These Xaj populations serve as the initial inoculum for walnut blight infections. As buds open in the spring and young shoots emerge, walnut blight bacteria can move onto walnut flowers and developing nuts, presumably by rain splash, causing infection. Occasionally, under conditions particularly favorable for disease such as the occurrence of frequent rainfall, additional secondary infections may result from inoculum formed by earlier infections of the walnuts in the spring. The disease cycle is complete when bacteria again over- California Walnut Board 329 Walnut Research Reports 2012

winter in the dormant buds (Figure 1). Our work has focused on monitoring initial inoculum levels to predict the relative risk of disease in an orchard and using that information to reduce the number of in-season sprays to reduce the amount of copper/ebdc materials applied. Again, an effective control material is essential for walnut blight management. Growers interested in monitoring walnut blight bacteria in dormant buds can review how to sample dormant buds at cetehama.ucanr.edu. Click on orchard crops, click on walnut and click on sampling dormant walnut buds. Figure 1. Walnut Blight Disease Cycle. California Walnut Board 330 Walnut Research Reports 2012

OBJECTIVES FOR 2012 1) Evaluate how many bud samples are necessary to accurately represent pathogen abundance in an orchard. Since cost per sample is substantial and accuracy is critical, improving sampling techniques will help the economics and improve spray decisions based upon an integrated blight management approach. 2) Monitor pathogen populations in commercial orchards and utilize those data to make walnut blight spray decisions. Bud sampling would be the basis for an integrated blight management strategy. The goal is to establish comparisons of efficacy of reduced spray programs to standard spray programs in relation to estimates of early season pathogen abundance. 3) Use monitoring data to develop a walnut blight bud population history which would indicate if populations are increasing or decreasing in orchards differing in plant control strategies. 4) Support commercial labs that wish to offer bud population evaluations to walnut growers. 5) Improve our extension efforts to help walnut growers use population information to make management decisions. PROCEDURES Walnut Blight Survey Dormant buds in thirty walnut orchards in Tehama and Butte Counties were sampled to evaluate pathogen population. One hundred buds per orchard were grower sampled and evaluated by the Lindow lab at UC Berkeley and The California Seed and Plant Laboratory in Elverta, California. The lab analysis indicated the percent of buds with detectable pathogen and the pathogen population size in each bud. An average pathogen abundance could then be calculated (logarithm of the colony forming units,cfu). Spray programs for each orchard were compiled and blight damage was visually rated by counting 3000 to 4000 walnuts per orchard for blight symptoms. The data set includes a three year history for the Tehama orchards and a two year history for the Butte orchards. Blight Variation between trees and Associated Bud Populations A single row assessment of walnut blight was made in a transect of a Tehama County Vina orchard with an average disease incidence of 2.4% to 6.4% blighted walnuts in 2012. A single row of 69 trees east to west were rated for severity of walnuts dropped as a result of walnut blight infection. Individual trees were evaluated 7/13/12 by estimating the number of blighted walnuts on the ground. A 0 rating represented no blighted walnuts; L or low represented less than 30 dropped walnuts; M or medium represented 30 to 100 dropped walnuts and H or high indicates more than 100 dropped walnuts per tree. Composite bud samples were collected 11/13/12 by collecting 5 dormant buds from each tree for each rating category. In total, four dormant bud samples were evaluated by the Lindow lab at UC Berkeley and a frequency histogram of pathogen population size in individual buds was determined for each of the four disease severity classes. Blight Suppression and History Following Artificial Inoculation In 2010, a group of seven walnut trees were artificially spray inoculated with a suspension of Xaj about one week after first flowers were visible. Xaj agar plates cultured in the Lindow lab at UC Berkeley were the source of inoculum. Plates were gently scraped into one gallon of water and poured into 30 gallons of California Walnut Board 331 Walnut Research Reports 2012

water in a spray tank with the agitator running. The final X.aj concentration was one million colony forming units (cfu) per ml. Trees were sprayed to run off by hand gun at 250 psi. Spray coverage was excellent. Walnut blight was estimated 6/16/10; the artificial inoculation resulted in 61.41 percent blighted walnuts. A bud sample taken 12/1/10 and evaluated in the Lindow lab indicated that 73.3% of the buds were infested with an average population of 10 2.92 cfu. No walnut blight control sprays were applied in 2010 to the artificially inoculated trees. During the spring of, artificially inoculated trees received five grower applied walnut blight sprays by speed sprayer and three hand gun applied sprays. On 4/10, 4/17 and 4/23 the machine spray mix was 8 lbs. Nu-Cop 50 DF plus 2.4 lbs. Manzate Prostick plus 4 oz. Sylgard plus 1.0 lb. zinc sulfate. On 5/4 and 5/13 the spray mix was changed to 6 lbs. Nu-Cop 50 DF plus 2.4 lbs. Manzate Pro-stick plus 4 oz. Sylgard and 5 lbs. potassium nitrate. Hand gun sprays were applied 4/22, 5/5 and 5/13/11 using 4 lbs. Kocide 3000 plus 2.4 lbs. Manzate Prostick. In all, 8 total sprays were applied and the last spray on 5/13 was both grower and handgun applied to ensure good spray coverage. During the spring of 2012, artificially inoculated trees received six grower applied half sprays by speed sprayer and four hand gun applied sprays. On 4/2/12, 4/9/12 and 4/15/12, the speed sprayer mix was 8 lbs/ac Nu-Cop 50DF plus 2.4 lbs/ac Manzate plus 4 oz/100 Sylgard and 1 lb/ac Zinc Sulfate. ON 4/23/12, 5/1/12 and 5/10/12 the machine mix was changed to 6 lbs/ac Nu- Cop 50DF plus 2.4 lbs/ac Manzate plus 4 oz/100 Sylgard plus 5 lbs/ac Potassium Nitrate. Hand gun applications were made on 4/17/12, 4/24/12, 5/4/12 and 5/16/12 using 4 lbs/ac Kocide 3000 plus 2.4 lbs/ac Manzate ProStick. The goal in and 2012 was to apply an aggressive disease control program and observe how long it would take to drive the bud populations down and eliminate blight infections on walnuts. Blight was visually rated by counting 400 walnuts per tree. Pathogen Population in Unopened Buds To answer the question whether viable Xaj over summer in walnut buds that remain unopened throughout the spring, un-opened buds of Chandler that had formed in previous years were collected 7/16/12 and evaluated at the Lindow lab at UC Berkeley. The percent of buds with pathogen and the pathogen population in each bud sample (logarithm of the colony forming units) are reported. RESULTS AND DISCUSSION Walnut Blight Survey Walnut blight population survey information for the Tehama County orchards are listed in Figure 2(2010), Figure 3 () and Figure 4 (2012). Each orchard designation is the same over the three year sample period. For example: Orchard 1 is a Howard variety having 0.4% blighted walnuts in 2010, 0.13% blighted walnuts in and 0.17% blighted walnuts in 2012. The fourteen Tehama orchards each achieved very good walnut blight suppression and maintained low bud populations suggesting that the spray applications and timing used were effective. Chandler orchard #7 had 70 percent buds with pathogen in 2010, 93 percent of buds with pathogen in and no buds with pathogen in 2012. The high values in the first two years were a result of blight research conducted in that orchard. Notice that it took two years in orchard #7 to substantially reduce the bud population or initial inoculum. California Walnut Board 332 Walnut Research Reports 2012

Orchard 2010 % Buds with Pathogen 2010 Population Log (cfu/g) 1) Howard 23 3.079 2010 Spray Mix 8lbs. Nu-Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 1 lb. Zinc Sulfate 4/6, 4/17, 4/23 2010 Spray Mix 6 lbs. Nu- Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 5 lbs. Potassium Nitrate 2010 Spray Mix 6 lbs. Nu- Cop 50DF, 1 qt. Regalia, 4 oz. Sylgard, 5 lbs. Potassium Nitrate 2010 Spray Mix 6 lbs. Nu- Cop 50DF, 4 oz. Sylgard, 5 lbs. Potassium Nitrate 5/5 5/13 0.4 2010 % Blight 2) Howard 3 3.183 4/6 1.8 3) Chandler 0 0 4/10, 4/17 4/22 5/5 5/13 0 4) Hartley 0 0 5) Chandler 0 0 6) Howard 0 0 7) Chandler 70 3.547 8) Howard 10 2.739 9) Howard 0 0 10) Chandler 0 0 11) Chandler 0 0 4/16, 4/23, 5/5 4/10, 4/18, 4/24 4/8, 4/10, 4/18 4/9, 4/17, 4/23 4/9, 4/17, 4/23 4/10, 4/19, 4/24 4/7, 4/16, 4/23 4/8, 4/16, 4/25.03 5/5 5/15 0 4/25 5/6 5/14 0 5/5 5/14.16 5/6 5/14.83 5/6 5/14.56 5/3 5/13 0 5/6 5/13 0 Figure 2. 2010 Walnut blight population survey information for eleven orchards in Tehama County. The % buds with pathogen and the average pathogen population in the total sample (logarithm of the colony forming units) were taken March 16, 2010 prior to bud break. The spray program and the resulting percent blight are shown in the remaining two columns. All sprays were half sprays (every other row) alternating by ground application. Blight was visually rated 7/8 to 7/13/10 by randomly counting 3000 walnuts per orchard. California Walnut Board 333 Walnut Research Reports 2012

Orchard % Buds with Pathogen Population Log (cfu/g) avg. Spray Mix 8lbs. Nu-Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 1 lb. Zinc Sulfate Spray Mix 6 lbs. Nu-Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 5 lbs. Potassium Nitrate % Blight 1) Howard 13.43 4/12, 4/20, 5/2 5/12, 5/24.13 2) Howard 70 2.56 4/12, 4/20.07 3) Chandler 20.75 4/16, 4/23, 5/4 5/12, 5/24.37 4) Hartley 3.09 4/14, 4/20, 5/2 5/11, 5/23.10 5) Chandler 3.07 4/17, 4/21, 5/5 5/11, 5/25 0 6) Howard 10.30 4/12, 4/19, 5/4 5/10, 5/28 0 7) Chandler 93 3.59 4/16, 4/23, 5/5 5/9, 5/26.77 8) Howard 3.10 4/14, 4/22, 5/6 5/12, 5/28 0 9) Howard 0 0 4/11, 4/21, 5/4 5/13, 5/24 0 10) Chandler 0 0 4/16, 4/23, 5/5 511, 5/28 0 11) Chandler 10.30 4/15, 4/22, 5/4 5/13, 5/27 0 12) Howard 6.17 4/16, 4/20, 5/3 5/10, 5/25 0 13) Chandler 10.43 4/19, 4/23, 5/5 5/12, 5/24 0 14) Chandler 30 1.21 4/19, 4/23, 5/5 5/12, 5/24.02 Figure 3. Walnut blight population for the same eleven Tehama County orchards as shown in Figure 2. Orchards, 12, 13 and 14 were added in. The percent buds with pathogen and the average pathogen population in the total samples (logarithm of the colony forming units) were taken March 15, prior to bud break. The spray program and the resulting percent blight are shown in the remaining two columns. All sprays were half sprays (every other row alternating) by ground application. Blight was visually rated 6/14 to 6/22/11 by randomly counting 3000 walnuts per orchard. California Walnut Board 334 Walnut Research Reports 2012

Orchard 2012 % Buds with Pathogen 2012 Population Log (cfu/g) avg. 2012 Spray Mix 8lbs. Nu-Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 1 lb. Zinc Sulfate 2012 Spray Mix 6 lbs. Nu-Cop 50DF, 2.4 lbs. Manzate Pro-stick, 4 oz. Sylgard, 5 lbs. Potassium Nitrate 1) Howard 6.17 4/16, 4/24, 4/30 5/9.17 2) Howard 10.43 4/16, 4/24, 4/30 5/9.07 3) Chandler 30 1.07 4/19, 4/27, 5/10.33 4) Hartley 0 0 4/16, 4/23, 5/1 5/10.03 5) Chandler 10.24 4/21, 4/27, 5/3 5/8.03 6) Howard 10.24 4/17, 4/25, 5/1 5/11 0 7) Chandler 0 0 4/19, 4/26, 5/8.93 8) Howard 13.45 4/18, 4/24, 5/2 5/15.07 9) Howard 0 0 4/21, 4/25, 5/7 5/16 0 10) Chandler 0 0 4/21, 4/26.03 11) Chandler 0 0 4/19, 4/309, 5/8 0 12) Howard 4/16, 4/24, 4/28 5/9 0 13) Chandler 4/18*, 4/26, 5/8 0 14) Chandler 4/20, 5/8.33 2012 % Blight Figure 4. 2012 walnut blight population survey information for the same fourteen orchards in Tehama County as shown in figures 2 and 3. The percent buds with pathogen and the average pathogen population in the total sample (logarithm of the colony forming units) were taken March 14, 2012 prior to bud break. The spray program and the resulting percent blight are shown in the remaining two columns. All sprays were half sprays (every other row alternating) by ground application. In orchard 13 (*) 14.5 oz of pristine was added to the first spray on 4/18. Blight was visually rated 6/13 to 6/25/12 by visually counting 3000 walnuts per orchard. California Walnut Board 335 Walnut Research Reports 2012

Orchard % Buds with Pathogen Population Log (cfu/g) Avg. Spray Schedule % Blight 1) Hartley 16.53 4/15(A) 4/22(A) NC+PS 5/5(A) 5/16(G) 6/2(A).11 2) Chandler 0 0 4/15(G) 4/21(G) 5/5(G) 5/14(G) 5/27(G) 6/3 (G) 2.59 3) Chandler 73 3.20 4/15(A) 4/21(G) 5/5(G) 5/13(G) 5/24(G) 6/3(G) 3.94 4) Chandler 56 2.19 4/14(G) 4/19(G) 4/28(G) 5/13(G) 5/23(G) 6/2(G) 1.44 5) Ashley 0 0 3/31(A) NC+PS 4/6(A) 4/14(G) 4/21(A) 5/9(G) 5/20(A) 6/2(G) 5.24 6) Howard 20.67 4/12(G) 4/18(G) 4/26(G) 5/11(G) 5/21(G) 5/31(G).18 7) Chandler 46 1.77 4/15(A) 4/22(G) 5/5(A) 5/14(G) 5/24(G) 6/3(G) 1.76 8) Howard 40 1.38 4/13(G) B_PS 4/19(G) 4/27(G) 5/11(G) NC+PS 5/20(G) 5/28(G).79 9) Howard 16.76 4/12(G) 4/16(G) 4/27(G) 5/12(G) 5/20(G) 5/28(G).68 10) Vina 60 2.50 4/16(A) 4/12(A) 4/21(A) 5/10(G) 5/20(A) 6/2(A) 2.60 11) Howard 53 1.93 4/14(G) 4/19(G) 4/27(G) 5/12(G) 5/23(G) 6/2(G).41 12) Howard 40 1.29 4/14(G) 4/19(G) 4/27(G) 5/12(G) 5/23(G) 6/2(G) 4.82 13) Tulare 0 0 4/12(G) 4/15(G) 4/21(G) 5/6(G) 5/19(G) 5/30(G).03 14) Vina 3.12 4/6(A) 4/12(A) 4/22(A) 5/6(G) 5/21(A) 6/2(A).52 15) Chandler 83 2.81 4/14(G) 4/20(G) 4/28(G) 5/13(G) 5/23(G) 6/2(G) 2.09 Figure 5. walnut blight population survey information for 15 orchards in Butte County. The percent walnut buds with pathogen and the average pathogen population in the total sample (logarithm of the colony forming units) were sampled in March prior to bud break. The spray program and the resulting percent blight damage are shown in the remaining two columns. (G) or (A) indicates ground or air application. All ground applications were ½ sprays (every other row alternating) except the 5/16 spray on orchard #1. K=Kentan at 6.0 lbs/ac, NC=Nu-cop at 6.0 lbs/ac, PS=Pro-stick at 2.4 lbs/ac, KC=Kocide 2000 at 6.0 lbs/ac 2000 and B=Badge at 3, 4 or 5 lbs/ac. Buds were sampled 3/12/11 and blight was visually rated 6/23 to 6/27/11 by randomly counting 4000 nuts per orchard. California Walnut Board 336 Walnut Research Reports 2012

Orchard 2012 % Buds with Pathogen 2012 Population Log (cfu/g) Avg. 2012 Spray Schedule 2012 % Blight 1) Hartley 0 0 4/16(A),S 4/24(G) 5/1(G) 5/23(G).18 2) Chandler 46 1.51 4/16(G) 4/20(G) 4/30(G),S, Z 5/23(G) 2.50 3) Chandler 43 1.41 4/16(G) 4/20(G) 5/1(G) 5/23(G) 10.25 4) Chandler 33 1.03 4/17(G) 4/23(G) 5/3(G) 5/24(G) 6/5(G) 2.68 5) Ashley 70 1.95 3/23(G),S, Z 3/30(A) K2,M,S 4/7(G) 4/16(A),S 4/24(G) 5/25(G) 17.84 6) Howard 16.53 4/10(G) 4/18(G) 4/24(G) 5/22(G) 1.31 7) Chandler 30.98 4/16(G) 4/20(G) 5/1(G) 5/22(G) 13.73 8) Howard 6.27 4/9(G) 4/18(G) 4/24(G) 5/23(G) 1.40 9) Howard 4/11(G) 4/18(G) 4/24(G) 5/21(G) 1.42 10) Vina 43 1.36 4/2(A) 4/9 (G) 4/16(A),S 4/24(G) 5/25(G) 16.17 11) Howard 20.67 4/8(G) 4/17(G) 4/23(G) 5/24(G) 1.41 12) Howard 23.78 4/8(G) 4/17(G) 4/23(G) 5/24(G) 6/6 (G) 5.45 13) Tulare 0 0 4/5(G) 4/15(G) 4/22(G) 5/21(G).03 14) Vina 3.14 4/2(A),S 4/9(G) 4/16(A),S 4/24(G) 5/24(G).87 15) Chandler 50 1.93 4/7(G) 4/23(G) 5/2(G) 5/24(G) 6.00 Figure 6. 2012 Walnut blight population survey information for the same 15 orchards in Butte County as shown in figure 5. The percent of buds with pathogen and the average pathogen population in the total sample (logarithm of the colony units) were taken in February 2012 prior to bud break. The spray program and the resulting percent blight damage are shown in the remaining two columns. (G) or (A) indicates ground or air applications. All ground applications were half sprays (every other row alternating) except the 4/24 and 5/1 spays in orchard #1. B=Badge at 4lbs/Ac. In orchards 5, 6 and 9 the Badge rate was reduced to 3.5lbs/Ac. K2=Kocide 2000 at 6lbs/Ac. M=Manzate at 2.4 lbs/ac. S=Freeway at 4oz/Ac and Z=Zinc Sulfate 36% at 1lb/Ac. Buds were sampled 2/23/12 and blight was visually rated 6/19 and 6/20/12 by randomly counting 4000 nuts per orchard. California Walnut Board 337 Walnut Research Reports 2012

The incidence of walnut blight in fifteen orchards in Butte County are presented in Figure 5 () and Figure 6 (2012). It is clear that blight control in the Butte orchards is not as good as compared to the Tehama sites. Despite the fact that only two years of data are available, it appears that walnut blight damage is increasing with time in many of these orchards. For example, in the Hartley 2012 (#1) and Tulare (#13) orchards, pathogen populations remain low and damage from walnut blight is correspondingly low. The two Vina orchards, #14 having 3% infected buds and 0.87% blight damage and orchard #10 with 43% infested buds and 16.17% blighted walnuts in 2012 reveal how pathogen populations are predictive of disease later in the spring. The Ashley orchard (#5) having 70% infested buds and 17.84% blight damage also reveals how high pathogen populations led to high disease compared to other orchards. The five Chandler orchards (#2, #3, #4, #7 and #15) had moderately high bud populations (ranged from 30% to 50%) as well as blight damage (2.5% to 13.7% infection). The five Howard orchards (#6, #8, #9, #11 and #12) had lower pathogen populations in the buds compared to Chandler as well as less blight damage. Howard bud populations ranged from 6% to 23% of the buds with pathogen while blight damage ranged from 1.31% to 5.45%. Orchards with a high percentage of infected buds also had larger pathogen population sizes within their buds. It appears that bacterial populations are not being substantially reduced by the spray programs being used, and these programs did not adequately control blight damage given the 2012 spring weather conditions. Orchard Location and Variety % Buds with pathogen Population Log (cfu/g) % Blight 2012 % Buds with pathogen 2012 Population Log (cfu/g) 2012 Spray Schedule 4 lbs/ac Badge plus 2.4 lbs/ac Manzate 1) Upper, Vina 3.08 1.58 _ 2) Lower South, Vina 16.50 1.49 30 1.10 4/16 & 5/2 3.50 3) Lower Middle, Vina 23.82 1.23 10 0.35 4/16 & 5/2 6.49 4) Lower North, Vina 6.23 2.00 16 0.63 4/16 & 5/2 2.42 2012 % Blight Figure 7. Two years of walnut blight survey information for a Vina variety walnut orchard in Northern Tehama County. The table starts on the left showing the walnut blight history followed by the 2012 bud population information followed by the 2012 spray program and the resulting walnut blight damage. Sprays were full coverage (every row) using ground application equipment. For 2012, dormant buds were sampled 3/15/12 and blight was visually rated 6/13/12 by counting 1,000 walnuts per location. Figure 7 shows two years of blight history for a Vina variety orchard in Northern Tehama County. This orchard is another example of an orchard exhibiting increased blight damage in 2012 compared to the previous year. Blight damage in was 1.23% to 2.00% with 3-23 percent of the buds with pathogen compared to 2.42% to 6.49% blighted walnuts with 10-30 percent of the buds with pathogen in 2012. The timing and/or frequency of bactericide sprays made to this orchard appear to have been inadequate to prevent the moderate amount of pathogen inoculum present in over-wintering buds from causing disease in 2012. California Walnut Board 338 Walnut Research Reports 2012

Blight Variation and Associated Bud Populations within trees in a given orchard. It has always been assumed that while the severity of disease is quite variable between different orchards (probably as a result of different spray histories that have led to different abundances of pathogen in the buds) that the variation in pathogen abundance and hence disease is relatively small within an orchard. We explored this assumption by examining differential nut drop due to walnut blight infection from tree to tree in the same orchard. All trees received the same walnut blight control spray program. Results suggest substantial tree to tree variation in the amount of disease in 2012. Furthermore, the abundance of pathogen in buds that formed on those trees in 2012 (and sampled in November, 2012) differed greatly, and were directly related to the amount of disease which had occurred in 2012. (Figure 8). Of the 69 sampled trees, 13 trees had no dropped walnuts ( no disease), 28 trees had less than 30 dropped walnuts (low disease), 15 trees had between 30 and 100 dropped walnuts(moderate disease) and 9 trees had over 100 dropped walnuts ( heavy disease). The trees are in a single row, with tree #69 being on the west end and tree #1 on the east end. Trees 56 to 63 represent a cluster of trees with relatively more blighted walnuts on the ground compared to the others. For the rest of the row, the disease appeared to be more random, varying between none, low and medium with a possible gradient with increasing damage to the west. There were many more cells of the pathogen in buds from trees which tended to have dropped nuts than those which did not. Except for orchard variation, the survey trees were relatively similar so there may be a legacy effect of disease from one year to the next. The blight pattern suggests it is very important for growers to take their dormant bud samples for pathogen assessment in the winter from widely scattered locations throughout the orchard; sampling of several buds form a given tree would not provide a good average estimate of pathogen abundance. The current suggestion of one bud from each of 100 trees appears to be an acceptable sampling strategy. California Walnut Board 339 Walnut Research Reports 2012

Tree # Rating Tree # Rating 69 L 34 M 5.0 4.0 Xaj Histogram for Zero Blight Rating 68 M 33 L 67 L 32 L Log (Cfu/bud) 3.0 2.0 1.0 66 L 31 M 65 M 30 M 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Bud # 64 L 29 L 63 H 28 L 62 H 27 L 61 H 26 L 60 H 25 Replant 59 H 24 L Log (Cfu/bud) 6.0 5.0 4.0 3.0 2.0 1.0 Xaj Histogram for Low Blight Rating 58 H 23 M 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 57 H 22 L Bud # 56 H 21 L 55 M 20 0 54 M 19 L 6.0 Xaj Histogram for Medium Blight Rating 53 L 18 L 52 L 17 L 51 M 16 Replant 50 0 15 0 49 M 14 0 Log (Cfu/bud) 5.0 4.0 3.0 2.0 1.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Bud # 48 L 13 0 47 M 12 L 46 M 11 0 45 M 10 L 44 L 9 0 43 M 8 0 42 H 7 0 41 Replant 6 0 Log (Cfu/bud) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Xaj Histogram for High Blight Rating 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 40 L 5 L Figure 8. Walnut blight transect for a Vina orchard in Tehama County. Blight drop under each tree was 39 0 4 L visually rated 7/13/2012. 0 represents no blighted 38 M 3 Replant walnuts on the ground. L is less than 30 dropped 37 36 35 L L L 2 1 0 0 walnuts. M represents 30 to 100 dropped walnuts. H indicates more than 100 dropped walnuts per tree. All trees were sprayed on 4/16 and 5/2/12 with 4lbs/ac Badge plus 2.4lbs/ac Manzate. The histograms represent walnut blight populations in bud samples taken 11/13/12 for the no blight, low blight, medium and high blight dropped nut trees. Bud # California Walnut Board 340 Walnut Research Reports 2012

Blight Suppression and History Following Artificial Inoculation A group of seven Chandler trees were artificially inoculated with Xaj on 4/16/10. Three frequency histograms (Figures 9, 10 and 11) illustrate the progressive reduciotn in both the incidence and extent of colonization of the buds over several years following aggressive bactericide spray programs to drive the bud populations down. Figure 9 reveals a high starting bud population following artificial inoculation on 4/16/10 and with no subsequent blight control sprays. On 12/1/10 73.3 percent of the buds were infected with an average log of cfu of 2.92. Bud pathogen populations had decreased11/28/11 after 8 walnut blight sprays were applied in the spring (Figure 10); 30% of the buds were infested and the average log cfu was 1.28. By the third year when Figure 11 shows the bud population profile for the same trees on 11/13/12. By the third year, when ten blight sprays were applied in the spring, only 10% of the buds were infested with an average log cfu of 0.44. Thus the 2 years of aggressive blight control spray programs were quite effective in getting the bud pathogen to a low level. One more year of sampling in 2013 should confirm additional population size reduction. Visual blight counts on the inoculated trees compared to grower treated trees are shown in Figure 12. No blight was observed on the inoculated trees in 2012 suggesting that the two years of elevated bactericide application were effective in getting the disease back under control. 10 Xaj Treated Walnut Buds (Year 1) Avg. log cfu = 2.92 73.3 % infected # of samples 8 6 4 2 0 0 2.5 3 3.5 4 4.5 More Log cfu/sample Figure 9. 2010 walnut blight population histogram from bud samples taken on 12/1/10. The histogram represents bud populations from 61.41% blight damage following artificial inoculation on 4/16/10 and no disease control sprays in 2010. This population level is the initial infection level prior to an aggressive clean up effort in and 2012. California Walnut Board 341 Walnut Research Reports 2012

# of Samples 40 30 20 10 0 0 0.5 Xaj Treated Walnut Buds (Year 2) Avg. log (cfu/bud) = 1.28 30% Infected 1 1.5 2 2.5 3 3.5 4 4.5 5 More Log (CFU/bud) Figure 10. walnut blight population histogram for Chandler walnuts artificially inoculated on 4/16/10 and aggressively sprayed 4/10 to 5/13/11. Spray applications on 4/10, 4/17, 4/22, 4/23, 5/4, 5/5, 5/13 (grower) and 5/13 (handgun). Eight walnut blight sprays reduced blight incidence to 1.46% blighted walnuts. Buds were sampled 11/28/11 and evaluated in the Lindow Lab at UC Berkeley. Log (Cfu/bud) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Xaj Treated Walnut Buds (Year 3) Avg. log (cfu/bud = 0.44) 10% Infected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Bud # Figure 11. 2012 walnut blight population histogram for Chandler walnuts artificially inoculated on 4/16/10 and aggressively sprayed 4/10/11 to 5/13/11. Spray applications by speed sprayer 4/2, 4/9, 4/15, 4.23, 5/1 and 5/10. Handgun applications 4/17, 4/14, 5/4 and 5/16. Blight observation on 6/13/12 indicated no blighted walnuts. Bud samples were taken on 11/13/12 and evaluated in the Lindow lab at UC Berkeley. California Walnut Board 342 Walnut Research Reports 2012

Treatment Variety 2010 % Blight % Blight 2012 % Blight Inoculated Trees Chandler 61.41 1.46 0.00 Grower Trees Chandler 2.67 0.00 0.07 Figure 12. 2010, and 2012 walnut blight counts for Chandler walnut in Tehama County. Inoculated trees were sprayed once on 4/16/10 with a Xaj solution of one million cfu per gallon. Initial blight was 61.41%. An aggressive spray program in using copper plus Manex reduced blight to 1.46%. A second (2012) aggressive blight control program using copper plus Manzate Pro-stick reduced the blight damage to zero. Blight Population in Unopened Buds Not all walnut buds open and leaf out each season. These unopened buds could be a source of inoculum for future infections if they were to open later. To check that possibility, unopened buds from Chandler trees were sampled 7/16/12. A high incidence of infestation of the buds with Xaj were found; (Figure 13); 43% of the buds were infested with an average log cfu of 1.80 cfu/bud. It appears as though unopened buds could be a source of inoculum but they would most likely have to open and leaf out for the bacteria to infect green walnut tissue. Xaj Pathogen levels in unopened walnut buds Avg. log (cfu/bud) = 1.80 43% infected Log (cfu/bud) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 # of buds Figure 13. Walnut bight population histogram for unopened chandler walnut buds sampled 7/16/12. Buds were evaluated at the Lindow lab at UC Berkeley. Pathogen levels suggest walnut blight bacteria can survive in buds that do not open and could be a future source of inoculum. California Walnut Board 343 Walnut Research Reports 2012

Temperature F Date Rainfall (in) Max Min 3/01/12.02 59 39 3/13/12.55 53 49 3/14/12.15 58 52 3/15/12.11 61 55 3/16/12.07 62 44 3/22/12.02 65 41 3/24/12.69 50 43 3/25/12.38 54 41 3/27/12.89 52 47 3/28/12.01 63 53 3/31/12.42 58 46 4/04/12.02 61 35 4/10/12.46 56 47 4/11/12.07 63 49 4/12/12.46 56 47 4/13/12.29 51 45 4/25/12.04 66 57 5/03/12.02 65 51 6/04/12.16 71 55 4.83 Figure 14. 2012 Rainfall and maximum/minimum temperature for the Gerber (CIMIS #8) weather station in Tehama County. 40% prayer stage was estimated on 4/4/12 for Vina and 40% prayer occurred for Chandler on 4/17/12. Forty percent of the open buds at prayer stage signals the first spray application. California Walnut Board 344 Walnut Research Reports 2012