EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHTwalnutresearch.ucdavis.edu/2011/2011_265.pdf · for...

EPIDEMIOLOGY AND MANAGEMENT OF WALNUT BLIGHT

J. E. Adaskaveg, H. Förster, D. Thompson, D. Felts, D. Cary, J. Connell, and R. Buchner

Cooperating: L. Wade, Arysta LifeScience

ABSTRACT

Although precipitation at many locations in the walnut growing areas of California was very high

in the spring of 2011, due to the late occurrence of rain and sub-optimal temperatures, the overall

incidence of walnut blight was only moderate on the early-blooming walnut varieties used in our

studies. In field studies under natural rainfall conditions, the efficacy of copper (e.g., Kocide

3000, Badge X2, Cueva, and an experimental Isagro material)-mancozeb (e.g., Manzate

ProStick) treatments for reducing the incidence of walnut blight was compared to that of copper

or mancozeb alone and to non-copper-mancozeb treatments. The latter ones included two

formulations of kasugamycin, the fungicides Quintec and Captan, the natural products Regalia

and Citrox/ProAlexin, the host defense activator ReZist, the biocontrol Blossom Protect, and

selected mixtures. The results can be summarized as follows:

1. Copper-Manzate was effective in all trials conducted under low to moderate disease pressure.

2. Regalia applied by itself did not reduce the incidence of blight from that of the control under

moderately high disease pressure conditions (22.4 % disease in the control), only numerically

reduced the disease from the control in a trial under lower disease pressure (14.4% disease in

the control), and was very effective in another trial where the lowest disease pressure (12.3%

disease in the control) was observed. Regalia-Kocide 3000 was statistically similarly

effective to Regalia or Kocide 3000 alone in the first trial; was statistically similarly effective

to Regalia or copper materials by themselves in the second trial; and was significantly more

effective than Regalia or Kocide 3000 alone in the third trial. Thus, as previously

documented, Regalia can be effectively used for walnut blight management only under low

disease pressure conditions. Regalia-Kocide 3000 mixtures can provide a benefit over

Regalia by itself considering that our trials in 2011 were conducted in orchards with no or

moderate copper resistance levels in the pathogen population.

3. In a comparison of copper treatments, Kocide 3000, Badge X2, Cueva, and an experimental

Isagro material were similarly effective when mixed with Manzate. Among new treatments,

mixtures of Kasumin with Manzate, copper, or Captan performed very well and the efficacy

was similar to Kocide-Manzate. The biocontrol Blossom Protect and the natural products

Citrox/ProAlexin were very effective in one study where they were used and these products

will need continued evaluation. Another biocontrol, Actinovate, numerically, but not

significantly reduced the disease from the control.

4. Field trials over the years and also in 2011 demonstrated that Kasumin mixed with copper or

an EBDC showed consistent high efficacy for controlling walnut blight.

5. Two new soil applications that affect stomatal opening of plants were also evaluated for the

management of blight. Four applications of RootZone were ineffective, whereas for CoMo

Classic, a trend in disease reduction was observed.

6. In an application timing study, sprays were done either weekly, or before or after predicted

rains. Six weekly applications or four sprays applied mainly before rains were more effective

California Walnut Board 265 Walnut Research Reports 2011

than applications timed after rains. We previously documented the effectiveness of after-rain

applications. The lack of efficacy in this year’s trial can be explained by the high amount of

rainfall that occurred over a several-day period that apparently was highly conducive for

disease development.

7. Varietal susceptibility to walnut blight was again evaluated under simulated rain conditions.

8. Specific primers for X. arboricola pv. juglandis were developed and were used in the

identification of the pathogen in bud population studies. Pathogen population diversity was

evaluated in several orchards. To date, multiple strains of the pathogen have been identified

in our collections from walnut growing areas of California over a number of years. In 2011,

some orchard locations harbored a single genotype, but in others a mixed population of

distinct genetic strains was identified.

INTRODUCTION

Walnut blight, caused by the bacterium Xanthomonas arboricola pv. juglandis, continues to cause

crop losses in many central and northern California orchards with varying incidences from year to

year. The pathogen attacks catkins, female blossoms, fruit, green shoots, leaves, and buds of

English walnut. Fruit infections occur soon after flowering and these infections account for most of

the economic loss. The extended period of host susceptibility is one of the main challenges in

controlling this disease. The pathogen survives from one year to the next in twig lesions, buds

(living and dead), and diseased fruit that remain on the tree.

The optimum temperature for bacterial growth is 28 to 32C and the minimum is about 1C, but

lesions on immature fruit generally do not occur below approximately 12C or above 25C

(Adaskaveg et al. 1994, 1998, 1999). Furthermore, disease increases with increased wetness

duration within this temperature range (Adaskaveg et al. 1998, 1999) and the disease does not

increase when daily high temperatures exceed 27C regardless of precipitation (Adaskaveg et al.

2009). Based on analysis of wetness period duration and temperature data from field weather

stations and actual disease progression, we developed XanthoCast™ as a model to predict infection

periods for walnut blight to calculate the risk of disease. The website www.agtelemetry.com

currently provides the basic XanthoCast™ information with XanthoCast indices for weather

stations located between Red Bluff and Davis. The XanthoCalculator allows individual web-based

forecasting for different grower fields and locations. Thus XanthoCast system provides regional

forecasts, and promotes judicial use of pesticides with disease control similar to calendar-based

programs. The UC-IPM website is still under development. One research goal is to use dew point

temperature and precipitation as an alternative to leaf wetness data to identify leaf wetness periods

without actually measuring leaf wetness. This change will offer considerable flexibility based on

data that are available on CIMIS and other databases.

Our timing studies indicated that early female flower emergence applications followed by

applications based on XanthoCast provided the most consistent disease control. A catkin

bactericide treatment was only needed when high precipitation occurred in an orchard with a

history of high disease levels. Furthermore, incidence of blight was significantly lower when

bactericide treatments were based on calendar or on XanthoCast forecasts than when only single

“eradication” or inoculum reduction bud break treatments were applied. Because we also showed

that bacterial populations can exponentially increase on diseased fruit under favorable


environmental conditions and can reach very high levels within 3 to 6 hours, inoculum levels are

not considered in the XanthoCast model after the initial treatment for blight management. We

plan to continue with development of the XanthoCast model with trials focused on timing of

bactericide treatments in relation to forecasted weather events. Specifically, we will continue to

look at applications of bactericides before and immediately after major rain events, as well as

excluding applications when daily high temperatures exceed 27C regardless of rainfall.

Our research project on the management of walnut blight since 1994 has assisted in the

development of copper-maneb (e.g., Manex) treatments and demonstrated the efficacy of zinc

compounds and zinc-Manex mixtures. We initiated the rotation of copper- and zinc-based

bactericides and use of copper-zinc mixtures (e.g., Kocide 20/20, Nordox 30/30) to minimize the

development of copper-resistant populations of the pathogen. In addition, new reduced-copper

products (e.g., Kocide 3000, Badge) were shown to be highly effective. From 2006 to 2010 we

showed that products containing mancozeb (Manzate, Dithane, Penncozeb, etc.) instead of maneb

(e.g., Manex) are effective in mixtures with copper or the antibiotic Kasumin. Although the EPA

has prompted the cancellation of maneb in the United States in 2008-09, an emergency registration

of Manzate (DuPont sold this Product to UPI Ag Products in 2010) was granted by the EPA in

2010 and 2011. A full registration is expected in the coming years. Manzate is formulated as a

flowable liquid (F) or a dispersible granular (DF) material and slight differences were observed

between the formulations when mixed with copper products. Thus, an evaluation of rain fastness of

different mixtures is appropriate.

Over the years, we evaluated many possible alternatives. In 2005, we first identified the potential

for kasugamycin (Kasumin, Arysta Life Science) in walnut blight management. This antibiotic is

used in agriculture in other countries but not in human or animal medicine. In Sept. 2005, a food

tolerance was established for Kasumin on imported agricultural products. In 2007 IR-4 residue

trials for kasugamycin were conducted and the registration to EPA was submitted in January 2010.

Currently, federal registration is targeted for 2012 and a California state registration for 2013. In

our 2007-11 studies, 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 Kasumin-

copper mixtures were similar or higher in efficacy to copper-EBDC mixtures. The registrant is

providing new formulations of the antibiotic that are more concentrated and in 2011 we evaluated

Kasumin 8L. Thus, additional studies have to be done with kasugamycin to ensure a successful

launch on walnuts.

Additional research is also needed on alternative treatments such as natural compounds, biocontrols,

and enhancers of host resistance, some of them demonstrated promising results before. These

include ProAlexin and Citrox, Actinovate, Blossom Protect, BioForge, and ReZist. Actinovate was

registered for use in 2011 based on our efforts and should provide a copper alternative for organic

growers. The anti-transpirant Root –Zone showed promise in 2010 and was evaluated again in 2011

together with a similar compound, CoMo Classic. These treatments apparently stimulate abscisic

acid production that triggers stomatal closure. Our overall goal is to ultimately have multiple

treatments available that are equal to or more efficacious than traditional copper-containing

compounds and that could be used in orchards with copper-sensitive and copper-resistant

populations of the walnut blight pathogen. This would result in a lower environmental impact and in

a lower chance for resistant populations of the pathogen to develop and spread.


We also continued to evaluate walnut varieties for their natural resistance to blight and examined

fruit for morphological characteristics that are possible mechanisms of resistance and that

potentially may be used as phenotypic markers in resistance breeding. Studies on the molecular

identification of the pathogen and on genetic variability of the pathogen were also done. This will

provide us tools for population studies that could lead to a better understanding of pathogen

epidemiology and disease development.

OBJECTIVES

I. New treatments - Evaluate the toxicity of alternative copper (i.e., the reduced-copper content

products Kocide 3000 and Badge), non-copper based chemicals (e.g., the antibiotic Kasumin,

EBDC fungicides, biologicals (Actinovate, Blossom Protect), and other experimental materials

such as enhancers of plant stress responses and inducers of host defense (e.g., Citrox and

ProAlexin, BioForge, Root-Zone) against X. arboricola pv. juglandis and evaluate the efficacy

of these materials for managing walnut blight.

A) Comparative efficacy of new copper bactericides such as new products and formulations

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) Optimize the performance of kasugamycin formulations (e.g., Kasumin 2L, 10L) in

mixture rotations with copper and EBDC fungicides (e.g., Manzate, Dithane).

Volume and adjuvant studies – different volumes (e.g., 200 vs. 100 gal/A) and selected

adjuvants

Combinations of the antibiotic with copper, EBDC, or biologicals (e.g., Actinovate)

C) Investigate the use of biologicals (e.g., Actinovate, Blossom Protect) and inducers of host

defense mechanisms (e.g., Citrox, ProAlexin, BioForge) and anti-transpirants (e.g., Root-

Zone) as treatments for managing walnut blight.

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 precipitation-temperature-based version of XanthoCast on

University websites (ongoing) in cooperation with UC-IPM and CIMIS programs for

utilizing temperature and precipitation, dew point temperature, or relative humidity data

instead of leaf wetness for the model.

B) Evaluate spring timings before and after major rainfall or simulated rain events and set

upper temperature limits for bactericide applications (e.g., >27C).

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 KAC and continue to study microscopic morphological

differences of fruit surfaces.

B) Evaluate diversity within pathogen populations and rapid identification of the pathogen

using molecular methods.


PROCEDURES

Evaluation of alternative bactericides for management of walnut blight in field studies. Trials

were established in experimental orchards in Fresno Co. (Kearney Agricultural Center - KAC)

and Solano Co. (UC Davis), and in commercial orchards in Sutter and Butte Co. In Fresno Co.,

trials were conducted on cvs. Chico, Chandler, 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.

Chico, Hartley, and Tulare were conducted under natural rainfall conditions with bactericide

applications based on XanthoCast indices. Trials in commercial orchards were done on cvs. Vina

and Ashley. Treatment timings are indicated in Figs. 1-4. Treatments were applied using an air-

blast sprayer at 100 gal/A, except for CoMo Classic and RootZone. 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 or general linear model procedures

and least significant difference (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 done to evaluate and improve timing programs for bactericide treatments.

Treatment timings for four to six applications of Kocide-Manzate were based on host

phenological stages (pistillate flower emergence) and before or after rain events. Disease

incidence on 100-200 nuts for each of the four single-tree replications was evaluated in late June.

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 were evaluated for disease

incidence under simulated-rain conditions with 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. Bud populations of the pathogen on

approximately 50 buds for each walnut genotype were enumerated by plating onto agar media.

Bacterial colonies resembling X. arboricola pv. juglandis were verified for identity using newly

designed PCR primers (see below). 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. 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.

Development of specific PCR amplification primers for X. arboricola pv. juglandis and

studies on the genetic variability within and among populations in California. Specific primers

were developed from rDNA and dnaK sequences that were obtained in 2010. For this, sequences

were compared to published sequences of X. campestris, X. vesicatoria, and X. axonopodis that

are all closely related to X. arboricola pv. juglandis. Primers were evaluated for specificity using

a diverse collection of strains of the walnut pathogen and numerous other bacterial isolates,

including other yellow-pigmented strains obtained from walnut tissues. Subsequently, the best

primer pair was used in real-time SYBR green PCR reactions to evaluate their suitability for

population studies. 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.

RESULTS AND DISCUSSION

I. Field Efficacy Studies

Although precipitation at many locations in the walnut growing areas of California was very high

in the spring of 2011, due to the late occurrence of rain and sub-optimal temperatures, the overall

incidence of walnut blight was only moderate in the early-blooming walnut varieties used in our

studies. No data could be obtained in our Fresno Co. trials at Kearney Ag Center and in one trial

in Sutter Co.

In a trial in Solano Co. where Kocide-Manzate treatments were compared to Regalia and

Regalia-Kocide or -Manzate treatments and where disease pressure was moderately high, Kocide

reduced the incidence of disease to 6.4% as compared to the control with 22.4% (Fig. 1). The

incidence among the other treatments ranged from 11.5% (Kocide 3000-Manzate) to 25.5%

(Regalia). Thus, four applications of Regalia were not effective in reducing blight under

moderate disease pressure.

In a trial in a commercial orchard in Sutter Co. with isolates of the pathogen moderately resistant

to copper, the efficacy of 22 treatments was compared. Disease pressure was low with 12.3%

incidence in the control. Incidence among the treatments ranged from 0% (Kocide 3000-

Manzate) to 7.2% (Actinovate), and most treatments performed statistically similar (Fig. 2). The

four copper materials (Kocide 3000, Badge, Cueva, Isagro) when mixed with Manzate had an

incidence of between 0% and 3.5% and thus, performed very similar. Regalia when applied by

itself (1.3% disease) was very effective under these low-disease conditions. The other natural

products, Citrox/ProAlexin, the biocontrol Blossom Protect, and the fungicides Captan and

Quintec also significantly reduced the disease. Mixtures of Kasumin with Badge or Captan

resulted in a disease incidence of less than 1% and thus, were among the best treatments.

Kasumin by itself, as in previous years, was less effective. The 8L formulation of Kasumin in

combination with Badge was statistically similarly effective as the 2L formulation, but

numerically, disease incidence was higher using the 8L formulation. In our studies on the

management of other bacterial diseases with Kasumin, the 8L formulation performed better than

the 2L formulation. Thus, additional studies on walnut blight will be done in 2012.

In a trial in a commercial orchard in Butte Co. with copper-sensitive populations of X. arboricola

pv. juglandis, disease pressure was lower and an incidence of 14.4% was observed in the control.

Regalia-Kocide reduced the incidence to 1.0% as compared to the control with 14.4%, similar to

Manzate by itself with 1.3% disease (Fig. 3). Kocide 3000 alone was also very effective (3.9%

incidence) whereas Regalia by itself (8.7%) did not significantly reduce disease incidence from

that of the control, similar to the fungicide Quintec. Kasumin was moderately effective (6.9%

incidence) and the addition of Quintec did not increase the efficacy (9.7% incidence).


In summary, over the years Kasumin mixed with copper or an EBDC showed consistent high

efficacy for controlling walnut blight. As previously also documented, Regalia can be effectively

used for walnut blight management only under low-disease pressure conditions. Regalia-Kocide

3000 mixtures can provide a benefit over Regalia by itself, but it has to be considered that our

trials in 2011 were conducted in orchards with no or moderate copper resistance levels in the

pathogen population. Thus, trials will have to be conducted at sites with high levels of copper

resistance to evaluate the potential of the material to substitute the use of EBDCs.

Two new soil applications that affect stomatal opening of plants were also evaluated for the

management of blight in Solano Co. In this trial, four applications of RootZone were ineffective

in reducing the incidence of disease, whereas for CoMo Classic, a trend in disease reduction was

observed (Fig. 4). Thus, these data may not support further evaluation although RootZone had

shown very promising results in 2010 in a first-year evaluation.

In summary in these five field trials, we demonstrated that:

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

to manage walnut blight.

b) Kasumin significantly reduced walnut blight incidence when used by itself, but was more

effectively used in mixtures with an EBDC, copper bactericides, or with other fungicides.

c) Regalia can be effectively used for walnut blight management under low-disease pressure

conditions. Mixtures of the material with copper can be beneficial at higher disease

pressure, but only when copper resistance is not a problem (based on our experiences over

several years). Higher rates need to be evaluated similar to our initial studies with Milsana

(the previous formulation of this natural product).

d) Among the other natural products and biocontrols, Citrox BC+ProAlexin has shown

consistent efficacy to suppress the disease over the last two years, whereas the efficacy of

Actinovate has been inconsistent.

II. Epidemiology

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 rain

events; 2) Application at pistillate flower emergence and timed before predicted rains; 3)

Application at pistillate flower emergence and timed after rain events; and 4) Application at

pistillate flower emergence followed by four approximately weekly applications. Non-treated

trees were used as controls and disease was evaluated in late June 2011. In this study, only the

program with weekly applications and the program with applications before predicted rains

significantly reduced the disease from the untreated control where 44.7% of the fruit were

diseased (Fig. 5). The four-spray program based on predicted favorable conditions was similarly

effective as the six-spray weekly program.

The two programs with after-rain treatments were not effective this year. In contrast, studies of

the last two years had indicated that post-rain applications can be effective. This difference in

results can be explained by the fact that high precipitation over an extended period between May

14 and May 17, 2011, was responsible for very favorable disease conditions (Fig. 5). Thus, post-


precipitation applications may only be effective for short duration, low-rainfall events. Pre-

rainfall applications are recommended as the most effective treatment strategy and post-rainfall

applications may be beneficial for short, low rainfall periods that were not anticipated.

III. Host Resistance to Walnut Blight and Pathogen Identification/Diversity

Varietal susceptibility. Walnut genotype comparisons were conducted for a last year in the old

variety orchard at Kearney AgCenter. For this, we also enumerated bud populations of the

pathogen in the genotypes evaluated. Numerous bacteria were isolated from most bud samples,

with inconsistency among replications of the same genotype. Most bacteria were species other

than X. arboricola pv. juglandis. Although 10 buds were used for isolations from each of five

trees per cultivar, the pathogen could only be recovered from cvs. Payne and Vina, but not from

other cultivars including Chandler, Hartley, and Serr. These latter three cultivars, however, had

between 4 and 16% disease (see below) by late spring and early summer. Thus, the use of

bacterial bud populations to generally predict disease incidence is questionable for the genotypes

and cultivars we evaluated.

Disease incidence among 15 genotypes ranged from 19.5% in cv. Payne to 0% in PI159568,

Cheinovo, and PI18256 (Fig. 6). In each of the seven evaluation years, cv. Payne was the most

susceptible and PI159568 followed by Cheinovo, 76-80, Sinensis 5, PI18256, and Chase D9

selections were among the least susceptible. For other cultivars, susceptibility among the years

was more variable. In Fig. 6, the incidence of blight was also averaged over the years to show

the overall performance. With these evaluations, we are supporting our long-term goal of

providing data on blight susceptibility among new and old walnut genotypes to assist the

breeding program in the Horticulture Department, University of California, Davis. We are

concluding our evaluation of stomatal number and morphology as well as presence of trichomes

on fruit of walnut genotypes to potentially identify characteristics that determine levels of host

susceptibility. Developing fruit were collected, and tissue from selected fruit areas was prepared

from scanning electron microscopy. Microscopic observations were initiated, but data are

currently pending.

Pathogen identification and diversity. Ten primer pairs from two gene sequences were

evaluated for their specificity to identify X. arboricola pv. juglandis using isolates of the

pathogen of diverse genetic background (as identified in 2010) and of numerous other bacteria

obtained from walnut tissues including buds. Several primer pairs were specific for X. arboricola

pv. juglandis. As an example, amplification using primers Xaj1F and Xaj1R designed from

ribosomal DNA sequences is shown in Fig. 7A. Using all primer pairs, however, primer-dimer

bands (from self-annealing of primers) were visible in the electrophoretic separation of products

(Fig. 7A). Thus, primers were specific, but primer-dimer formation will interfere with the

detection of the pathogen by real-time PCR.

In additional studies, melt-curve analyses of PCR amplicons with intercalated SYBR green dye

were done in real-time PCR reactions using the most specific primer pairs. Primers were

confirmed to be specific. In the example in Fig. 7B with primers Xaj1F and Xaj1R, the specific

PCR product has a melt temperature of 83.5°C. Again, primer-dimer background was evident,

and the primer-dimer product had a melt temperature of 74°C in Fig. 7B. Thus, a real-time PCR


method that can be used in population studies will need additional improvements. For this, we

will modify our approach and develop TaqMan probes in combination with the primers we

designed. This technology is more complex, but it is not affected by the presence of primer

dimers.

Population diversity of the pathogen was studied in three orchards. Using two primers in PCR

amplifications that target repetitive sequences, multiple strains of the pathogen have been

identified in our collections from walnut growing areas of California over a number of years. In

2011, some orchard locations harbored a single genotype, but in others a mixed population of

two distinct genetic strains was identified (Fig. 8). Thus, although genetically diverse throughout

California, only a limited number of genotypes of the pathogen is present within a location.

CONCLUSIONS

Among currently available treatments, copper-mancozeb (EBDC) combinations are the most

consistent in controlling walnut blight in California. In 2011, 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) is currently pending US-

EPA registration in Feb. 2012. 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 as compared to a reduced-spray program and may be costly. 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. Thus, our

research is also comparing timing of treatments before and after rainfall events that may improve

our understanding of bacterial invasion of host tissues, i.e., infection periods and give growers

options with non-anticipated rainfall prevent protective applications of bactericides. 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.

We found that the enumeration of bud populations at the beginning of the growing season to

estimate disease levels during the season is difficult and questionable due to the presence of

numerous bacterial species colonizing buds, the relatively low inoculum levels of the pathogen,

and the potentially rapid increase of the pathogen population under favorable environmental

conditions. Thus, bud populations may be an indication of the disease potential in some cultivars

but actual disease levels varied dramatically among most genotypes evaluated. Statistical

sampling procedures need to be evaluated to determine the proper number of buds to be sampled

to estimate orchard populations of the pathogen at a high confidence level (e.g., >95%).

Additionally, further development of a real-time PCR detection system for X. arboricola pv.

juglandis will assist in the assessment of bud populations, but moreover can be used to study the

response of the pathogen to environmental changes. Studies on pathogen population diversity

indicated that although multiple strains of the pathogen have been identified in our collections

from walnut growing areas of California over a number of years, individual orchard locations

harbored only one or two distinct genotypes.


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.

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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

Regalia 0.25%

Kocide 3000 4 lb

Regalia 0.25% + Kocide 3000 4 lb

Regalia 0.25% + Manzate 2.4 lb

Kocide 3000 4 lb + Manzate ProStick 75DF 2.4 lb 0 5 10 15 20 25 30

Applications were done were made on 4-12, 4-20, 5-6, and 5-13-11. D isease was evaluated in late June 2011. Inc idence of disease is based on 100 fruit for each of 5 s ingle-tree replications .

Fig. 1. Efficacy of copper and Regalia in combination with and manzate for managing walnut blight on cv. Chico walnut caused by copper-senstive populations of Xanhomonas arborico la pv. juglandis

- Solano Co. 2011, natural rainfall conditions -

Disease incidence (%)

a

ab

bc

c

Fig. 2. Efficacy of new copper- and non-copper-based treatments for managing walnut blight on cv. Vina caused by moderately copper-resistant strains of Xanthomonas arboricola pv. juglandis

- Sutter Co., 2011, natural rainfall conditions -

Six applications were done on 4-14, 4-21, 4-27, 5-10. 5-16, and 5-26-11. D isease was evaluated on 6-29-11. Inc idence is based on the number of diseased nuts of the total of 100 nuts evaluated of each of four s ingle-tree replications .

ControlActinovate 12 oz + NuFilm 8 fl oz

Kasumin 8L 100 ppmRegalia 32 fl oz + Kocide 3000 4 lb

Blossom Protect 1.34 lb - buffer 9.35 lbCaptan 80WP 5 lb

Cueva 4 qt + Manzate ProStick 75DF 2.4 lbIsagro 2.67 lb + Manzate ProStick 75DF 2.4 lb

Kasumin 8L 100 ppm + Badge X2 4 lbQuintec 6 fl oz

Badge X2 4 lb + Manzate ProStick 75DF 2.4 lbCitrox BC 0.1% + ProAlexin 0.1%

Kasumin 8L 100 ppm + Manzate F45 58 fl ozBadge X2 4 lb

Cueva 8 qtBadge X2 4 lb + Rezist 16 fl oz

Kasumin 2L 100 ppm + Quintec 6 fl ozRegalia 32 fl oz

Kasumin 8L 100 ppm + Captan 80WP 5 lbKasumin 2L 100 ppm + Badge X2 4 lb

Manzate Prostick 75DF 2.4 lbIsagro 4 lb + Manzate ProStick 75DF 2.4 lb

Kocide 3000 4 lb + Manzate ProStick 75DF 2.4 lb

0 5 10 15 20Disease incidence (%)

aab

bcd

e

bc

cde

No copper resistance

ab

abc

bcdbcd

bcd

bcde

cde

cdecde

bcde

bcdebcdebcde

bcdebcdebcdebcdebcdebcde


0 10 20 30 40

Control

RootZone 1000 ml

CoMo Classic 16 fl oz

Kocide 3000 4 lb + Manzate DF 2.4 lb*

0 10 20 30 40

Fig. 4. Efficacy of new soil applications that affect stomatal opening for managing walnut blight on cvs. Hartley and Tulare caused by copper-sensitive strains of Xanthomonas arboricola pv. juglandis*

- Solano Co. 2011, natural rain conditions -


a

RootZone and CoMo C lass ic were applied to the soil on 4-18 (pis tillate flower emergence), 5-4, 5-12, and 5-20-11. Applications were followed by a 3-4-h sprink ler irr igation. Koc ide-Manzate was applied as a foliar treatment on 4-17, 4-27, 5-4, 5-12, and 5-20-11. D isease was evaluated in late June 2011 and inc idence is based on the evaluation of 100 nuts per s ingle-tree replication.

a

Cv. Hartley

ab

b

Control Regalia 0.25%

Regalia 0.25% + Kocide 3000 4 lb Kocide 3000 4 lb

Regalia 0.25% + Manzate Pro-Stick 2.4 lb Manzate Pro-Stick 2.4 lb

Kocide 3000 4 lb + Manzate Pro-Stick 58 fl oz Quintec 6 fl oz

Kasumin 2L 100 ppm + Quintec 6 fl oz Kasumin 2L 100 ppm

0 5 10 15 20

All trees were sprayed w ith copper on 4-1-11. Four subsequent applications were done were made on 4-13, 4-21, 4-26, and 5-10-11. D isease was evaluated on June 22, 2011. Inc idence of disease is based on 100 fruit for each of 5 s ingle-tree replications .

Fig. 3. Efficacy of copper and non-copper treatments for managing walnut blight on cv. Ashley walnut caused by copper-senstive populations of Xanhomonas arborico la pv. juglandis

- Butte Co. 2011, natural rainfall conditions -


aab

bc

d

No copper resistance

abab

b

b

bc

cd

Cv. Tulare

a

a

ab

b


Fig. 6. Walnut variety susceptibi l i ty to bl ight in a s imulated rain-treated orchard - Fresno Co. 2005-2011 -

PI159568Cheinovo

PI1825676-80

FranquetteChase D9Sinensis 5

91-28-1Hartley

90-28-30Adams 10Chandler

TulareSerr

Payne0 5 10 15 20 25

bc

bcd

Five to s ix week ly s imulated rain events were applied us ing a high-angle sprink ler sys tem between April and May in each year.a

bcdbcd

bcdcd

b

d

2011

a

ddd

cdcd

0 5 10 15 20 25

2005-2011 average

No. Program 4-4 (Pist.) 4-12 4-14 4-19 4-20 4-27 5-5 5-9 5-13 5-19 (%) LSD1 Control - - - - - - - - - - 44.7 a2 Pist. + After Rain @ - - @ - - - @ - @ 36.1 ab5 No Pist. + After Rain - - - @ - - - @ - @ 36.1 ab3 Pist. + Before rain @ - @ - - - @ - @ - 20.5 b4 Weekly @ @ - - @ @ @ - @ - 19.0 b

Disease IncidenceApplications

3/25 4/1 4/7 4/14 4/21 4/28 5/2 5/7 5/14 5/21 5/28

Date (2011)

0

5

10

15

20

25

Tem

pera

ture

(C)

0

2

4

6

8

10

Precipitation (mm

)

Avg Air Temp (°C)Precip (mm)

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

Timings: Pistillate flower emergence – 4/4/11 After rain events – 4/19 to 4/20, and 5/13 to 5/19Before rain events – 4/14, 5/5, and 5/13

Program 1: ControlProgram 2: Application at pistillate flower emergence and timed after each of 3 rain events. Program 3: No application at pistillate flower emergence and timed after each of 3 rain events.Program 4: Application at pistillate flower emergence and timed before each of 3 rain events.Program 5: Weekly (7-9 day intervals) applications

Disease was evaluated in June 22, 2011.

Fig. 5. T iming s tudy with Koc ide 3000-Manzate treatments in a cv. Chico orchard under natural rain conditions – Solano Co. 2011


A

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0 25 26 27 28 29 0 30

Fig. 7. Development of primers specific for Xanthomonas arboricola pv. juglandis

Amplification of DNA from strains of X. a. pv. juglandis and other bacteria obtained from walnut buds and developing fruit using primers Xaj1F and Xaj1R designed from ribosomal DNA sequences. A. Gel with representative strains. Lanes 1, 2, 17, and 22 are positive for the pathogen; whereas all other lanes of non-Xanthomonas spp. are negative. 0 = Standard molecular ladder and lane 30 is a water control. The black arrow points to the specific amplicon for X. a. pv. juglandis and the white arrow points to the primer dimer band. B. Melt-curve analysis of PCR amplicons using real-time PCR reactions of 6 strains of X.a. pv. juglandis (83.5C melt temperature), 4 non-Xanthomonas spp., and one water control (74C melt temperatures represent primer dimers).

Orchard 1 Orchard 2

A A A A A B B B B B B B B B Strains were collected from two walnut orchards in northern California. Two genetic types designated “A” and “B” were found in one orchard; whereas in another orchard only the “B” genotype was present. ML = molecular ladder.

Fig. 8. DNA electrophoretic patterns of Xanthomonas arboricola pv. juglandis strains after rep-PCR using BOX primers.

ML


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