ETIOLOGY AND MANAGEMENT OF CROWN AND ROOT ROTS OF WALNUT

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1 ETIOLOGY AND MANAGEMENT OF CROWN AND ROOT ROTS OF WALNUT Greg Browne, Ravi Bhat, Leigh Schmidt, Charles Leslie, Wes Hackett, Reid Robinson, Dan Kluepfel, Bob Beede, Janine Hasey, and Joe Grant ABSTRACT Our objectives for 2011/12 are to: 1) Complete Koch s postulates for the lethal (LPC) disease; 2) develop and validate detection methods for the causal agent(s) of LPC; 3) continue evaluations of resistance to Phytophthora in promising clonal hybrid rootstocks for English walnut; and 4) facilitate field research to monitor emerging soilborne diseases and test field performance of new rootstocks. In 2010, we reported preliminary evidence that Brenneria nigrifluens was associated with LPC, a root crown and trunk disease that kills walnut trees on rootstock in the San Joaquin and Sacramento Valleys. The bacterium was isolated from only one of more than 10 sampled LPC-symptomatic trees in an orchard with high incidence of the disease near Yuba City. Several of the B. nigrifluens isolates induced s in excised shoots. In 2011 we processed samples from an additional 12 orchards on rootstock. The necrotic bark was assayed on bacterial, fungal, and oomycete isolation media. Selected isolates were identified by rdna sequencing and tested for pathogenicity in excised shoots of walnut rootstocks. Brenneria nigrifluens was not detected in association with LPC among the 12 orchards sampled in 2011, but it was isolated from what resembled shallow bark s in cultivar Hartley in one of the orchards. A Phytophthora sp. (tentatively P. citricola, being confirmed by DNA sequencing) was detected in one of the 12 sampled orchards, both in Chandler scions and on rootstock, but the Phytophthorainfected s were distinctive from LPC s. Although several of the 12 surveyed orchards exhibited LPC, no single organism was consistently isolated from the LPC s. Some of the microorganisms isolated inconsistently from LPC s included yeast-like fungi, Geosmithia morbida (the Thousand Canker causal agent), other fungi, and bacteria. Some of the fungal isolates were pathogenic in excised walnut shoots. We evaluated resistance to P. cinnamomi and P. citricola in 17 new clonal rootstock selections with Juglans microcarpa parentage (open pollinated J. microcarpa as well as J. microcarpa J. regia Serr selections were included). Standards used in the test included AX1 (highly susceptible to the pathogens), RX1 (moderately resistant), Vlach (intermediate in susceptibility), and Chinese wingnut (highly resistant). The 17 selections with J. microcarpa parentage ranged from moderately susceptible to moderately resistant to each pathogen. We collaborated in field trials with J. Hasey and J. Grant. With Hasey, root systems of trees affected by Howard yellowing were excavated. Examination of the root systems revealed that trees in early stages of decline had no major symptoms of root or crown rot, although it is possible that they had fewer fine roots, compared to healthy trees. Trees in later stages of decline (i.e., trees with obviously chlorotic foliage, wilting, and defoliation) exhibited some decay of major roots. In samples from the yellowed trees, we detected no Phytophthora sp. or other known soilborne pathogen, S. Sudarshana detected no phytoplasmas, and B. Westerdahl detected only low lesion nematode counts. It seems important to determine whether: 1) the decline syndrome reappears in trees replanted where yellowed trees were removed (i.e., which would be consistent with involvement California Walnut Board 299 Walnut Research Reports 2011

2 of a soilborne pathogen), and 2) the decline syndrome can be avoided by use of certain clonal selections. With Grant, we confirmed that P. cinnamomi was associated with death of trees in his San Joaquin County rootstock trial established to compare performance of standard seedling and RX1 in a commercial orchard infested with the pathogen. In this trial, second-year tree mortality was 17% on seedling rootstock, compared to 0% on RX1 rootstock. All trees on RX1 were alive and growing well. Phytophthora cinnamomi was isolated from 63% of dead trees and 40% of poorly growing trees on seedling rootstock, indicating that P. cinnamomi infection was a principal cause of tree death and decline. INTRODUCTION Soilborne pathogens and pests are among the most serious challenges faced by walnut growers worldwide, especially since soil fumigation faces increasing restrictions. The ultimate goal of this project is to develop the knowledge base and rootstocks needed for long-term economical management of crown and root rots on California walnuts. In the case of Phytophthora crown and root rot, for which etiology is well documented, our research emphasizes collaborative development of desirable rootstocks with resistance to Phytophthora and other soilborne pathogens. In cases of walnut crown and root rot problems for which etiology is unclear, (i.e., a lethal (LPC) disease and a decline of cultivar Howard on rootstock (often referred to as Howard yellowing), our research emphasizes determining the unknown causal agents. Both LPC and Howard yellowing have been responsible for significant incidence of tree loss in many walnut orchards. LPC occurs in both the Sacramento and San Joaquin Valleys, whereas Howard yellowing occurs in the Sacramento Valley. Knowledge on etiology and ecology of these diseases ultimately will provide a strategic basis for their control. OBJECTIVES Our objectives for 2011/12 are to: 1. Complete Koch s postulates for the lethal disease. 2. Develop and validate detection methods for the causal agent(s) of the lethal. 3. Continue evaluations of resistance to Phytophthora species in promising clonal hybrid rootstocks for English walnut. 4. Facilitate field research to monitor emerging soilborne diseases and test field performance of new rootstocks. PROCEDURES Objective 1. Complete Koch s postulates for the lethal disease. In 2010, we reported preliminary evidence that Brenneria nigrifluens was associated with lethal (LPC), a root crown and trunk disease that kills walnut trees on rootstock in the San Joaquin and Sacramento Valleys (Browne et al., 2010 report to the California Walnut Board). The bacterium was isolated from only one of more than 10 sampled LPC-symptomatic trees in an orchard with high incidence of LPC near Yuba City. Several of the B. nigrifluens isolates from the LPC-affected tree induced s in excised shoots. In California Walnut Board 300 Walnut Research Reports 2011

3 2011 we collected and processed samples from an additional 12 orchards on rootstock in Butte, Glenn, Sutter/Yuba, Yolo, San Joaquin, and Fresno Counties. Details of the symptoms and their incidence were noted. Necrotic bark was removed from the s and cultured on bacterial, fungal, and oomycete isolation media (i.e., YDC and NBY for bacteria; water agar and PDA amended with ampicillin for fungi; and PARP for oomycetes). Selected isolates were identified by amplification and sequencing of their rdna and tested for pathogenicity in excised shoots of walnut rootstocks. In addition, DNA was extracted from selected LPC s collected in 2010 and 2011 and used for PCR with fungal specific primers, and, in some cases with primers specific for B. nigrifluens. Products of PCR with the fungal primers were cloned, and representative cloned fragments were sequenced to identify their source organisms. Selected isolates from LPC s were tested for pathogenicity in excised shoot segments. The shoots segments were ca. 20-cm long, and they were inoculated at their midpoints with an agar disk covered with the test pathogen. Control shoot segments were inoculated with sterile agar. For each selection of, three replicate shoot pieces were inoculated per inoculum treatment. The control and inoculated shoots were incubated for 1 to 3 weeks before pathogenicity evaluations (i.e., one replicate block of shoots was evaluated each week after inoculation). For each shoot, pathogenicity was assessed by measuring the length of necrotic tissue that was generated from the point of inoculation. Re-isolations were conducted to confirm that the inoculated pathogen was associated with necrosis. Objective 2. Develop and validate detection methods for the causal agent(s) of the lethal. Completion of this objective will require knowledge of the causal agent of LPC, but in the meanwhile we are using and modifying described procedures to detect the pathogens that have, up to now, been inconsistently associated with LPC. For example, in the case of B. nigrifluens, which was pathogenic in excised shoots but was detected from only one of many LPCaffected trees, we are experimenting with different isolation media and published B. nigrifluensspecific PCR detection methods to determine whether the inconsistent detection is due to a) inadequate diagnostics or b) the organism not being the LPC cause. We will use this confirmatory experimental approach for other organisms that are inconsistently associated with LPC and yet induce s in. 3. Continue evaluations of resistance to Phytophthora species in promising clonal hybrid rootstocks for English walnut. A small-plant greenhouse screening procedure was used to evaluate the resistance to P. cinnamomi and P. citricola in 17 new clonal selections with Juglans microcarpa parentage (i.e., two selections of open pollinated J. microcarpa and 15 selections of J. microcarpa J. regia Serr ). We included the following clonal rootstock selections as resistance standards : AX1 (J. californica J. regia, highly susceptible to P. cinnamomi and P. citricola) RX1 (J. microcarpa J. regia, moderately resistant to both pathogens), WN W (Pterocarya stenoptera, highly resistant to both pathogens) and Vlach (J. hindsii J. regia, intermediate in susceptibility to both pathogens). California Walnut Board 301 Walnut Research Reports 2011

4 All of the selections were micropropagated as shoots and then rooted and acclimatized under mist by the Walnut Improvement Program (WIP) using standard methods. The acclimatized plants were transplanted, first into Ray Leach Cone-tainers (RCL3, Stewe and Sons; Tangent, OR), then, after root trimming, into 6 cm 6 cm Anderson pots filled with Sunshine Mix (Sun Gro Horticulture, Belleview, WA). The plants were grown in a greenhouse until they all were 10 to 20 cm in height. Then the soil in each pot was inoculated with 30 ml of V8 juice-vermiculiteoat substrate (left sterile as a control or colonized by P. cinnamomi or P. citricola), and the soil was flooded once every 2 weeks for 48 h. The plants kept arranged in a split plot design. There were 5 replicate plants per rootstock in the control inoculum treatment and 10 replicate plants per combination treatment of rootstock and Phytophthora species. The inoculum treatments were randomized among main plots, and the rootstocks were randomized among subplots. Three months after soil infestation, the plants roots were washed free from the soil, and the root systems were evaluated visually for incidence and severity of root and crown rot. Objective 4. Conduct and facilitate field research to monitor emerging soilborne diseases and test field performance of new rootstocks. With J. Hasey, root systems of trees affected by Howard yellowing near Yuba City were excavated to gain insights into the disease. Howard yellowing is typified by relatively sudden yellowing, decline, and defoliation of Howard canopies on rootstock in the spring and summer. The decline often results in tree removal. With J. Grant, we confirmed that P. cinnamomi was associated with death of trees in his San Joaquin County rootstock trial that was established to compare performance of standard seedling and RX1 in a commercial orchard infested with the pathogen. Grant had established the rootstock trial as follows: Trees that had died from infection by P. cinnamomi in a commercial walnut orchard (our isolations confirmed the pathogen presence) were removed to accommodate a rootstock trial 2 years before the trial was planted. The orchard s soil was Archerdale Clay Loam. The vacant tree sites were pre-plant fumigated with 1 pound methyl bromide and then planted to RX1 clonal and seedling (J. hindsii J. regia per Burchell Nursery) rootstocks in a randomized complete block design. Each tree site was allocated an area of 28 28, according to the spacing of the previous trees. At each tree site, two rootstock plots were randomly allocated, one planted to RX1 and the other to seedling. The trees sharing a tree site were spaced about 2 apart and were similar in size. There were 10 replicate 10-tree-site blocks. All trees were hand-planted in March 2010 as bare-root ungrafted nursery whips and budded in August 2010 to Serr. Trees not successfully top-worked by this initial budding were grafted to Serr in spring 2011, and a small number of these trees were re-budded in August The trees were irrigated with impact sprinklers. Trees that had died in 2011 were removed for lab diagnostics. We cultured necrotic roots and root crown tissue in PARP isolation medium to determine if the tree death was associated with Phytophthora. California Walnut Board 302 Walnut Research Reports 2011

5 RESULTS AND DISCUSSION Objective 1. Complete Koch s postulates for the lethal disease. Brenneria nigrifluens was not detected in association with LPC among the 12 orchards we sampled in 2011, but it was isolated from what resembled shallow bark s in cultivar Hartley in one of the orchards (Table 1, Sutter/Yuba County results). The orchard with shallow bark s did not exhibit LPC. In another of the 12 sampled orchards, a Phytophthora sp. (tentatively P. citricola, being confirmed by DNA sequencing) was detected, both in Chandler scions and rootstock (Table 1, Glenn County results). The appearance of the Phytophthora-infected s was distinctive from that of LPC s in other orchards: although both Phytophthora and LPC s exuded black, viscous liquid, s yielding Phytophthora had more pointed, and irregular margins compared to s symptomatic of LPC. Also, LPC margins were relatively broad and hemispherical, and at their periphery they typically generated additional hemispherical lobes of new expansion. Another distinguishing feature of LPC has been its apparent lack of occurrence on Northern California black walnut rootstock, which is highly susceptible to crown and root rot caused by Phytophthora. Although several of the 12 surveyed orchards had trees with symptoms of LPC, no single organism was consistently isolated and identified from the s (Table 1). For example, organisms in the bacteria or yeast category were often but not always detected in significant numbers from LPC s (Table 1, second to last column). Bacteria were grouped with yeast in this category because many yeast colonies initially resembled bacterial colonies. When we subcultured isolates from the bacteria or yeast category we found that most of them were yeast (i.e., a group of fungi that make slimy bacterial-like colonies, at least under some conditions) and many fell within the genus Pichia. We are still completing identifications of the true bacteria. Some other microorganisms isolated inconsistently from LPC-affected tissues included yeastlike fungi (fungi that quickly generated hyphae from their initial slimy bacterial-like colonies), Geosmithia morbida (the Thousand Canker causal agent, a fungus), and other fungi (Table 1). Some of the fungal isolates, including G. morbida, Gibelulopsis nigrescens, and Pichia hampshirensis were pathogenic in excised walnut shoots (Fig. 1), especially in cultivar Burbank. When DNA was extracted from LPC s and used for culture-independent PCR with fungal specific primers, followed by cloning, then by sequencing of five to 12 cloned fragments per sample, no known pathogens were consistently detected (Table 2). However, one category of cloned DNA sequence, no similarity found, which indicated sequences that did not match any previously sequenced fragments in the NCBI DNA database, occurred in all DNA samples from LPC s. Cluster analysis revealed that the no similarity found sequences were all part of a single clade of sequences that were at least 95% genetically identical and that bore affinity to fungal sequences. We found that PCR primers described as specific for B. nigrifluens amplified DNA from our purified cultures of the pathogen and from the same DNA when it was used to spike LPC DNA extracts. However, without the artificial spiking, the same PCR primers did not detect the California Walnut Board 303 Walnut Research Reports 2011

6 target pathogen in any LPC samples, including the one that yielded B. nigrifluens in our bacterial isolation cultures in These results indicate that further work is needed before B. nigrifluens primers can be used to reliably test for the presence of the bacterium in LPC samples. Our continuing work to determine causes of LPC will include use of: 1) additional orchard surveys and diagnostic isolations for bacteria, fungi, and oomycetes, 2) experimentation with alternative approaches to detect and isolate B. nigrifluens (i.e., in case the reason we have not detected it in constant association with LPC because of inadequate isolation methods), 3) cultureindependent PCR diagnostics (i.e., in case the causal agent is not culturable), and 4) expanded pathogenicity tests on orchard trees, potted trees, and excised shoots. We hypothesize that orchard inoculations with test isolates that have been isolated inconsistently from LPC s would be very helpful in recognizing the true LPC pathogen by the distinctive disease symptoms it causes. However, such inoculations, if approved, would need to be done with care and under supervision of CDFA. Objective 2. Develop and validate detection methods for the causal agent(s) of the lethal. This objective has not been completed, due to the lack of a confirmed causal agent for LPC. As described above, our approach is to improve detection methods for the spectrum of possible causal agents, especially those for which we have demonstrated pathogenicity in rootstock. Objective 3. Continue evaluations of resistance to Phytophthora species in promising clonal hybrid rootstocks for English walnut. Among the 17 new rootstock selections with J. microcarpa in their parentage, several developed relatively small amounts of root and crown rot in soil infested with P. cinnamomi or P. citricola (Fig. 2). The J. microcarpa x J. regia Serr clones 29 JM7, 29 JMS3, and JMS5A all developed <30% crown length rotted and <30% root rot by both pathogens (Fig. 2). The J. microcarpa x J. regia Serr clones 29 JM8 and 29 JMS12 had <30% crown length rotted and <50% root rot with both pathogens. On the other hand, the J. microcarpa open pollinated selections, JMOP2 and JMS7, as well as some of the J. microcarpa J. regia Serr selections were more susceptible and developed >50% crown length rotted and/or >50% root rot with exposure to one or both pathogens. Although the rootstock standards AX1 and WN W were highly susceptible and highly resistant to both pathogens, respectively, as expected, RX1 exhibited high susceptibility to both pathogens, which was not expected (normally RX1 expresses moderate resistance to both pathogens). Reasons for the high levels of root and crown rot in RX1 are unknown, but one explanation may be that the plants were relatively small, compared to the other rootstocks, at the time of inoculation (Fig. 3). In fact, there was a significant negative correlation between stem diameter and severity of crown rot and root rot with one or both pathogens (Table 3). California Walnut Board 304 Walnut Research Reports 2011

7 The evaluations of the new rootstocks will need to be confirmed before they justify confidence. Nevertheless, the relatively low ratings of root and crown rot in several of the clones suggest that the items are worthy of further testing. Objective 4. Conduct and facilitate field research to monitor emerging soilborne diseases and test field performance of new rootstocks. The trees affected by Howard yellowing occurred in a spotty pattern and were scattered amongst healthy trees. Our tree excavations, conducted with a backhoe, indicated that yellowing trees began their decline before appearance of obvious major root system disease symptoms. Trees in early stages of decline (i.e., those showing relatively mild chlorosis and retaining most of their foliage) had no major symptoms of root or crown rot, although it is possible that they had fewer fine roots, compared to healthy trees. Trees in later stages of decline (i.e., trees with obviously chlorotic foliage, wilting, and defoliation) exhibited some decay of major roots. In samples from the trees, we detected no Phytophthora sp. or other known soilborne pathogen, and Dr. S. Sudarshana detected no phytoplasmas. It is considered important to determine whether: 1) the yellowing and decline symptoms reappear in trees replanted where yellowed trees were removed (i.e., which would be consistent with involvement of a soilborne pathogen in the yellowing symptoms), 2) whether the yellowing problem can be avoided by use of certain clonal selections (i.e., field observations suggest this may be so). In Joe Grant s field assessment of RX1 and seedling rootstocks at a commercial orchard site infested with P. cinnamomi, second-year tree mortality was 17% on seedling rootstock, compared to 0% on RX1 rootstock. All trees on RX1 were alive and growing well. Phytophthora cinnamomi was isolated from 63% of dead trees and 40% of poorly growing trees on seedling rootstock, indicating that P. cinnamomi infection was a principal cause of tree death and decline in the trial. It is interesting to note that pre-plant fumigation with MB did not prevent unacceptable levels of tree mortality in the seedling rootstock. To date the superior performance of RX1 in this trial tends to validate earlier evaluations of the rootstock as moderately resistant to P. cinnamomi. A similar trial has been initiated by J. Hasey in a commercial site infested with P. cinnamomi near Yuba City. California Walnut Board 305 Walnut Research Reports 2011

8 Table 1. Results of 2011 sampling and culture-based isolations to examine causes of lethal disease Number of trees yielding organism / number of trees sampled County Glenn Orchard GL-KA Phytophthora Pythium Aspergillus Penicillium Cladosporium Yeast-like Unknown Bacteria and yeast a Disease # of trees. symptoms (sample Sample source represented no.) Organisms identified a Healthy Chandler None /1 0/1 0/1 0/1 0/1 0/1 0/1 0/ GL-KA Healthy None /1 0/1 0/1 0/1 0/1 0/1 0/1 0/ GL-KA Canker on Chandler Phyt. 7293, 7298, /3 0/3 0/3 0/3 0/3 0/3 1/3 3(++++)/3 Phytophthora citricola (?) GL-KA Canker on Howard Phyt /1 0/1 0/1 0/1 0/1 0/1 0/1 1(++++)/ GL-KA Canker on & Chandler Phyt /1 0/1 0/1 0/1 0/1 0/1 0/1 1(++++)/1 P. citricola (?) Butte GL-KA BU-CR BU-MT Canker on Phyt /3 0/3 0/3 0/3 0/3 0/3 1/3 2(++++)/3 P. citricola (?) Canker on Uncertain 7290, /2 0/2 0/2 0/2 0/2 1/2 2/2 2(++++)/2 -- Healthy None /1 0/1 0/1 0/1 0/1 0/1 0/1 0/ BU-MT Le. 7303, /2 0/2 1/2 0/2 0/2 0/2 2/2 0/2 -- Sutter / Yuba SY-DD Le. 7068, /2 0/2 1/2 2/2 1/2 1/2 2/2 2(+)/2 Penicillium sp.* SY-CO Le /3 0/3 0/3 0/3 0/3 0/3 1/3 1(++), 1(++++) /3 Leptosphaeria sp.* SY-SA Le /5 0/5 0/5 0/5 0/5 4/5 4/5 2(++++)/5 Pichia hampshirensis* SY-WI Le. gb7239 0/5 0/5 0/5 2/5 2/5 5/5 3/5 1(++++), 2(++), 2(+) /5 Pichia hampshirensis*, Geosmithia morbida* SY-GR Hartley Shallow bk /3 0/3 0/3 0/3 0/3 0/3 0/3 1(++++)/3 Brenaria nigrafluens Yolo YO-WIP Le /4 0/4 0/4 1/4 2/4 3/4 4/4 1(+), 1(++++)/4 Pichia hampshirensis, Aureobasidium pullulans YO-ME Uncertain 7287, /2 0/2 0/2 0/2 0/2 2/2 0/2 1(+++), 1(++++) /2 Pichia mexicana, P. hampshirensis, Candida sp. San Uncertain /5 0/5 0/5 0/5 1/5 5/5 5/5 4(++++), Joaquin SJ-CH 1(++) /4 A. pullulans, Candida mycetangii, P. hampshirensis, Rhodotorula sp. Fresno FR-KI Healthy None gb7280 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/ FR-KI Le. 7281, 7283, /3 0/3 1/3 2/3 0/3 2/3 2/3 1(+)/3 -- a In column summarizing incidence of bacteria and yeast, Arabic numerator indicates number of trees yielding: 0=no colonies, + =<10 colonies, ++ = 10 to 20 colonies, +++ = 21 to 50 colonies, and ++++ = > 50 colonies per culture plate; and Arabic denominator indicates number of trees sampled. California Walnut Board 306 Walnut Research Reports 2011

9 Table 2. Results of 2011 culture-independent examinations to detect fungi associated with lethal disease Organisms detected by PCR, cloning, and sequencing of ITS2 rdna County Orchard Sample source Disease symptoms represented Sample number Alternaria mali Ascomycota sp. Aureobasidium sp. Chalara sp. Cladosporium langeronii Debaryomyces hansenii Didymella sp. Issatchenkia terricola Leptosphaeria sp. Mollisia dextrinospora No similarity found Scytalidium sp. Uncultured fungus SY-KA Canker on Lethal Sutter / Yuba SY-DD Canker on Lethal SY-SA Canker on Lethal Yolo YO-WIP Canker on Lethal Table 3. Correlations between initial stem diameters and disease response variables in the 2011 greenhouse experiment evaluating resistance to Phytophthora cinnamomi and P. citricola Disease response variable Percent crown length rotted Percent root rot Pathogen P. cinnamomi P. citricola P. cinnamomi P. citricola Value of r for correlation with initial stem diameter (and associated P value) a (0.11) (0.05) (0.01) (0.0003) Final disease severity rating P. cinnamomi P. citricola (0.0001) (<0.0001) a r=pearson correlation coefficient. All values were averaged within block before correlation analysis. California Walnut Board 307 Walnut Research Reports 2011

10 Canker length (mm) Burbank Vlach VX Control-MEA-MEA00 Control-PDA-PDA00 Ph.cinnamomi-FPC05 Aureobasidium-sp.-YPC08 Aureobasidium-sp.-YPC12 Bacteriamix-BPC01 Candida-mycentangii-YPC13 Candida-sp.-YPC14 Candida-sp.-YPC19 Candida-sp.-YPC20 Candida-sp.-YPC24 Cladosporium-colocasi-FPC02 Cladosporium-sp.-FPC01 Exophiala-sp.-FPC04 Geosmithia-morbida-YPC18 Gibellulopsis-nigresc-YPC17 Leptosphaeria-sp.-YPC26 Penicillium-sp.-YPC25 Pichia-hampshirensis-YPC05 Pichia-hampshirensis-YPC06 Pichia-hampshirensis-YPC07 Pichia-hampshirensis-YPC09 Pichia-hampshirensis-YPC15 Pichia-hampshirensis-YPC22 Pichia-mexicana-YPC21 Rhodotorula-sp.-YPC10 Rhodoturula-sp.-YPC16 Scytalidium-sp.-FPC03 Unknown-YPC01 Unknown-YPC02 Fig. 1. Pathogenicity of selected isolates from LPC s in excised shoot segments. The shoots segments were ca. 20-cm long, and they were inoculated at their midpoints with an agar disk covered with the test pathogen. Control shoot segments were inoculated with sterile agar. For each selection of, three replicate shoot pieces were inoculated per inoculum treatment. The control and inoculated shoots were incubated for 1 to 3 weeks before pathogenicity evaluations (i.e., one replicate block of shoots was evaluated each week after inoculation). For each shoot, pathogenicity was assessed by measuring the length of necrotic tissue that was generated from the point of inoculation. Reisolations were conducted to confirm that the inoculated pathogen was associated with necrosis. Vertical bars represent 95% confidence intervals. California Walnut Board 308 Walnut Research Reports 2011

11 Percent crown length rotted Percent root rot Disease severity rating No Phytophthora calxreg AX1 hinxreg PX1 micxreg RX1 ptero WNXW hinxreg Vlach micop JMOP2 micop JMS7 micxserr 29JM12 Combined means, P. cinnamomi & P. citricola micxserr 29JM22 micxserr 29JM7 micxserr 29JM8 micxserr JMS11 micxserr JMS12 micxserr JMS15 micxserr JMS19 micxserr JMS24 micxserr JMS3 micxserr JMS5 micxserr JMS5A micxserr STJM11 micxserr STJM4 No Phytophthora P. cinnamomi P. citricola calxreg AX1 hinxreg PX1 micxreg RX1 ptero WNXW hinxreg Vlach micop JMOP2 micop JMS7 micxserr 29JM12 micxserr 29JM22 micxserr 29JM7 micxserr 29JM8 micxserr JMS11 micxserr JMS12 micxserr JMS15 micxserr JMS19 micxserr JMS24 micxserr JMS3 micxserr JMS5 No Phytophthora P. cinnamomi P. citricola calxreg AX1 hinxreg PX1 micxreg RX1 ptero WNXW hinxreg Vlach micop JMOP2 micop JMS7 micxserr 29JM12 micxserr 29JM22 micxserr 29JM7 micxserr 29JM8 micxserr JMS11 micxserr JMS12 micxserr JMS15 micxserr JMS19 micxserr JMS24 micxserr JMS3 micxserr JMS5 micxserr STJM6 micxserr JMS5A micxserr STJM11 micxserr STJM4 micxserr STJM6 micxserr JMS5A micxserr STJM11 micxserr STJM4 micxserr STJM6 Fig. 2. Severity of disease caused by Phytophthora cinnamomi and P. citricola in 17 clonal walnut rootstock selections in 2011 greenhouse evaluation of resistance. A, percent crown length rotted, B, percent root rot, and C, disease severity rating. Lower-case abbreviations indicate parentage (cal reg=j. californica x J. regia, hinxreg= J. hindsii x J. regia, micxreg= J. microcarpa x J. regia, ptero= Pterocarya stenoptera, micop= J. microcarpa open pollinated, micxserr= J. microcarpa x J. regia Serr ) and uppercase letters following parentage indicate specific clone designations. Disease severity ratings were based on aboveground plant appearance using a score of 0 (healthy plant) to 5 (dead plant). Vertical bars are 95% confidence intervals. A B C California Walnut Board 309 Walnut Research Reports 2011

12 Stem diameter (mm) calxregax1 hinxregpx1 micxregrx1 pterownxw hinxregvlach micopjmop2 micopjms7 micxserr29jm12 micxserr29jm22 micxserr29jm7 micxserr29jm8 micxserrjms11 micxserrjms12 micxserrjms15 micxserrjms19 micxserrjms24 micxserrjms3 micxserrjms5 micxserrjms5a micxserrstjm11 micxserrstjm4 micxserrstjm6 Fig. 3. Average stem diameters at beginning of greenhouse experiment.. Lower-case abbreviations indicate parentage (cal reg=j. californica x J. regia, hinxreg= J. hindsii x J. regia, micxreg= J. microcarpa x J. regia, ptero= Pterocarya stenoptera, micop= J. microcarpa open pollinated, micxserr= J. microcarpa x J. regia Serr ) and upper-case letters following parentage indicate specific clone designations. Vertical bars are 95% confidence intervals. California Walnut Board 310 Walnut Research Reports 2011