Progress 07/13/07 to 05/08/12
Outputs
Progress Report Objectives (from AD-416): 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. Approach (from AD-416): 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. This is a final report for Proj. 5306-22000-014-00D (terminated 05/08/12) which was replaced with Proj. 5306-22000-015-00D. Development of sustainable control strategies for key soil borne diseases of tree & vine crops is a major focus of our research program, addressing Obj#1. Contributed to these objectives in 2012 included identification of crown gall (CG) and Phytophthora- resistant host genotypes under greenhouse & field conditions. Wild walnut species were identified with resistance to CG disease and Phytophthora crown rot. To identify the genetic loci mediating CG resistance, F1 progeny generated from crosses between CG resistant female parents and CG susceptible pollen donors were screened for CG resistance. The progeny exhibited a 1:4 inheritance ratio of resistant: susceptible progeny. A genetically diverse collection of Prunus rootstocks is being examined in 3 field trials for resistance to CG & Prunus replant disease (PRD). Using culture-independent and culture-dependent approaches, we 1)examined the association of specific soilborne microbial communities with PRD to determine etiology and 2)characterized soilborne microbial communities associated with the inhibition of CG under field conditions. Pathogenicity of diverse microorganisms associated with PRD was tested. We continued existing orchard replant studies and established 2 new ones to evaluate efficacy of fumigant & non-fumigant based alternatives to fumigation with methyl bromide. We optimized a greenhouse bioassay for;1)predicting incidence of PRD & 2)use as an experimental "tool" to facilitate rapid progress in determining etiology of the disease. In the assay, plant age, ratios of field soil : sand mixtures, and soil pre-treatments (e.g. microfumigation)were examined. Work under obj#3, i.e. development of molecular detection methods for 4 phytopathogenic microbial agents was completed. Work on obj#4 was continued to examine etiology and characterize graft & pollen transmissible pathogens in fruit & nut trees. The virus that causes walnut black line disease was cloned & sequenced, which facilitated extensive comparative analysis. Markers used for hypersensitivity to the causal virus were used to identify germplasm with putative hypersensitivity to the virus. Almond brownline disease, a serious disease of almond, is being examined to identify the causative agent. Phytoplasma specific primers were used to screen both brownline symptomatic and asymptomatic trees. The sudden oak death pathogen Phytophthora ramorum propagates clonally, yet displays diverse phenotypes due to host-induced epigenetic alteration. Compared with P. ramorum isolates originating from bay laurel, those from oak display irregular colony morphology, reduced virulence, and diverse global gene/transposon expression profiles. This modification of fungal gene expression & transposon activation, as a function of host colonization, is being characterized using deep sequencing. ARS scientists(Davis, CA)are examining the molecular mode of action of phosphonate fungicide using a genetically tractable host and pathogen with well-annotated genomes to understand molecular microbial interactions in this system. Accomplishments 01 Fumigant and non-fumigant based methyl bromide alternatives. Tree growt and yield data were collected from 10 orchard replant trials to assess efficacy of alternatives to pre-plant soil fumigation with methyl bromid (MB). Broadcast, strip, and GPS-controlled spot fumigation treatments wi 1,3-dichloropropene, chloropicrin, or mixtures of the two fumigants all provide equal or superior control of Prunus replant disease (PRD), compared to soil fumigation with MB. Pre-plant spot treatments with stea and pre- and post-plant treatments with various fungicides (all applied tree planting sites) were ineffective, whereas short-term pre-plant suda grass improved cumulative peach yield. 02 Rootstock resistance to Phytophthora. Twenty-two standard and prospecti rootstocks for almond and peach were evaluated for resistance to PRD and Phytophthora niederhauseri. Among the rootstock selections, tolerance to PRD was detected in several peach x almond hybrids and a plum hybrid; resistance to P. niederhauseri was detected in several plum hybrids. Seventeen clonal selections of potential walnut rootostocks representing Juglans microcarpa and J. microcarpa x J. regia parentage were evaluated for resistance to P. cinnamomi and P. citricola using a small-plant assa method. Collaborative field testing confirmed resistance of �RX1�, a patented J. microcarpa x J. regia hybrid, to P. cinnamomi. 03 Etiology of Prunus replant disease (PRD). Culture-dependent isolations, culture-independent rDNA �fingerprinting�, and pathogenicity testing wer continued to examine pathogen-PRD associations. Results implicated Cylindrocarpon macradidymum as a PRD contributor in multiple replanted orchards. Pythium spp. and Thieleviopsis sp., although not always associated with PRD, were determined to be pathogenic on Nemaguard peach rootstock. Additional unidentified fungi were found associated with PRD rDNA fingerprinting. 04 PRD Bioassays. Experiments were established to develop and optimize a greenhouse bioassay for PRD etiology and PRD prediction. Planting materi (10-cm-tall clonal plants vs. sprouting seedlings of Nemaguard rootstock percentage of field soil tested in volumetric mixtures with sterile sand and soil pre-treatment methods were tested as factors influencing expression of PRD. Use of seedlings and micro-fumigation appear to dramatically improve the potential of the assay for use in PRD etiology and PRD prediction in commercial orchards. 05 Identification of crown gall resistant walnut genotypes. Agrobacterium tumefaciens is a soil-borne bacterium that causes crown gall (CG), a serious disease on walnut. ARS scientists in Davis, CA identified walnut wild species (Juglans microcarpa) which exhibit elevated levels of CG resistance. Crown gall resistant J. microcarpa genotypes were crossed wi a CG susceptible English walnut cultivar and one in five of the progeny from these crosses exhibited CG resistance. These new hybrid genotypes m provide the industry with a viable control measure for crown gall diseas which afflicts most walnut rootstocks currently used in the industry. 06 Enhanced sanitation strategies for Agrobacterium tumefaciens control. AR scientists in Davis, CA identified a positively charged surfactant (detergent) which effectively controlled pathogen contamination of bud wood and grafting tools in the field. In the presence of high organic matter concentrations, the surfactant was more effective at disinfecting solid surfaces than standard bleach solutions. 07 Host induced/modified gene expression in Phytophthora ramorum. Isolates originating from coastal live oak (PrOak) and those from California bay laurel foliage (PrBay) are genetically indistinguishable but show significant difference in colony morphology and pathogenicity. Our preliminary experiments have shown PrBay isolates artificially inoculate into boles of canyon live oak, and re-isolated 4 months after inoculatio display signs of phenotypic conversion. As much as 20% of re-isolates showed colony phenotypes and mRNA expression pattern resembled to those PrOak. In particular, transposable elements are upregulated and genes in crinkler effector family are downregulated in PrOak in comparison to PrB DNA and RNA samples obtained from the original PrBay isolate and two re isolates from canyon live oak are being subjected to next generation sequencing. This study provides some of the first experimental evidence epigenetic control in fungal pathogens which will enhance investigations into the study of host-pathogen co-evolutionary processes. 08 Fungicidal activity of Phosphonate; identification of the genes which mediate this process. Two pathosystems, each comprised of a combination of a host and its natural pathogen, with publicly-available genome sequences, have been found to yield reproducible results in terms of phosphonate protection. The first is Arabidopsis thaliana and its downy mildew pathogen Hyaloperonospora arabidopsidis. Rich sources of genetic and molecular biology in the host-pathogen interactions make this system extremely powerful. The other is tomato and Phytophthora capsici pathosystem. Exploitation of these systems will advance the use of phosphonate in the tree crop industry. 09 Plum necrotic union. Repeated patch grafts, root to root and scion to scion, of affected pluot trees from an orchard in Kings County failed to reproduce the symptoms. Several pluot trees obtained from a commercial nursery for inoculation experiments showed declining symptoms and necrot union. Results obtained so far do not indicate the involvement of an infectious agent. A genetic incompatibility between the scion and rootstock appears to be the reason for this disorder. 10 Evaluation of Marianna 2624 rootstock for tolerance to Xylella fastidios Trunk diameter of almond trees and mean kernel weight were measured in year 4 after inoculation with X. fastidiosa. Trunk diameter of almond grown on plum rootstock was not affected by X. fastidiosa infection. The same trend was observed for Nonpareil almond trees on Titan hybrid rootstock. This was not the case with Peerless almond on plum rootstock. The light interception was lowered by X. fastidiosa infection across all combinations and no clear benefit of having plum rootstock was noticed. Kernel weight of almond produced on infected trees was not influenced by plum rootstock. 11 Walnut blackline disease. The complete genome of a walnut strain of Cherry leaf roll virus (CLRV) was sequenced. RNA-1 and RNA-2 consist of 7915 and 6361 nucleotides, respectively, and contain a polyA tail. The R 1 shares identity of 78% and 85% with CLRV strains of Cherry and Rhubarb respectively, while RNA-2 shares identity of and 77% and 83%, respective with cherry and rhubarb strains of CLRV. A binary vector containing cDN sequence corresponding to 700 nucleotides of 3�-untranslated region of R 1 and RNA-2, has been cloned into a binary vector and transformed into EHA105 strain of Agrobacterium tumefaciens.
Impacts
(N/A)
Publications
- Yakabe, L.E., Maccree, M.M., Sudarshana, P., Mcclean, A.E., Parker, S.R., Wechter, W.P., Presting, G., Kluepfel, D.A. 2012. Novel primers for detection of genetically diverse virulent Agrobacterium tumefaciens bv1 strains. Journal of General Plant Pathology. DOI: 10.1007/s10327-012-0364- z.
- Luz, G., Kasuga, T., Glass, N., Taylor, J. 2011. Array CGH Phylogeny: How accurate are Comparative Genomic Hybridization-based trees?. Biomed Central (BMC) Genomics. 12:487.
- Al Rwahnih, M., Rowhani, A., Smith, R.J., Uyemoto, J.K., Sudarshana, M.R. 2012. Grapevine necrotic union, A newly recognized disease of unknown etiology in grapevines grafted on 110 Richter rootstock in California. Journal of Plant Pathology. 94(1), 149-156.
- Luz, G., Lee, C., Kasuga, T., Taylor, J. 2011. Array Comparative Genomic Hybridizations: Assessing the ability to recapture evolutionary relationships using an in silico approach. Biomed Central (BMC) Genomics. 12:456.
- Loper, J.E., Hassan, K.A., Mavrodi, D., Davis, E.W., Lim, C., Shaffer, B.T. , Elbourne, L.H., Stockwell, V.O., Hartney, S.L., Breakwell, K., Henkels, M.D., Tetu, S.G., Rangel, L.I., Kidarsa, T.A., Wilson, N.L., Van Mortel, J. , Song, C., Blumhagen, R., Radune, D., Hostetler, J.B., Brinkac, L.M., Durkin, A., Kluepfel, D.A., Wechter, W.P., Anderson, A.J., Kim, Y., Pierson Iii, L.S., Pierson, E.A., Lindow, S.E., Raaijmakers, J.M., Weller, D.M., Thomashow, L.S., Allen, A.E., Paulsen, I.I. 2012. Comparative genomics of plant-associated Pseudomonas spp.: Insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genetics. 8(7):e1002784.
- Kluepfel, D.A., Aradhya, M.K., Browne, G.T., Mckenry, M.V., Leslie, C.A., Mcclean, A.E., Moersfelder, J.W., Velasco, D.M., Baumgartner, K. 2012. The quest to identify disease resistance in the USDA-ARS Juglans germplasm collection. Acta Horticulturae. 948:105-111.
- Al Rwahnih, M., Osman, F., Sudarshana, M.R., Uyemoto, J.K., Minafra, A., Martelli, G., Rowhani, A. 2012. Detection of Grapevine Leafroll-associated virus 7 using real-time qRT-PCR and conventional RT-PCR. Journal of Virological Methods. 179:383-9.
- Ziliang, F., Weihua, W., Hildebrand, A., Kasuga, T., Zhang, R., Xiaochao, X. 2012. A novel biochemical platform for fuels and chemicals production from cellulosic biomass. PLoS One. 7(2).
- Kasuga, T., Bui, M.Q. 2011. Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms. Fungal Genetics Reports. 58:4-13.
- Kasuga, T., Kozanitas, M., Bui, M.Q., Huberli, D., Rizzo, D., Garbelotto, M. 2012. Phenotypic diversification is associated with host-induced transposon derepression in the Sudden Oak Death pathogen Phytophthora ramorum. PLoS One. 7:e34728.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. Approach (from AD-416) 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. We continued our field, greenhouse and laboratory trials to: examine the etiology and develop sustainable control strategies for crown gall disease (CG) and black line of walnut, Phytophthora crown rot of almond and walnut, and replant disease of almond and peach. Crown gall- and Phytophthora-resistant breeding material continue to be identified in wild relatives of cultivated walnut. Crown gall resistant black walnut species were crossed with English walnut pollen to generate novel hybrid disease-resistant genotypes. In addition, we examined how A. tumefaciens infects walnut hybrid seeds used to generate rootstocks and identified sources of A. tumefaciens inoculum which infects and colonizes walnut hybrid seed under commercial conditions. Sustainable management practices to minimize crown gall incidence are now possible. An enhanced semi selective growth media for A. tumefaciens also was developed aiding all our CG disease control efforts. A planting of 25 genetically diverse Prunus rootstocks in a prunus replant disease field site was established. Pathogenicity assays and metagenomic (culture-independent) analyses of microbial communities associated with almond replant disease is continuing in an effort to determine etiology of the disease. We developed a binary plasmid containing the highly conserved sequences of RNA-1 and RNA-2 from Cherry leaf roll virus and inserted into Agrobacterium tumefaciens EHA105. Our UCD collaborators have generated walnut callus obtained from somatic embryos and we can now initiate transformation. Fourth annual survey for almond brown line disease in an almond orchard on plum rootstock in Sutter County was completed. New infections only were noticed in replanted trees. None of the older trees showed symptoms. It appears almonds may be a dead end host for the phytoplasma associated with the disease. We demonstrated this phytoplasma is genetically different from the Pear decline and Peach yellow leaf roll phytopalsmas with which it shares 98% identity at the rDNA locus. We developed primers and a SYBRgreen based qPCR system to detect this phytoplasma. The Sudden Oak Death pathogen, Phytophthora ramorum exhibits the hallmarks of an invasive plant pathogen as its genetic uniformity is consistent with a genetic bottleneck. Genetic diversity within the dominant NA1 clonal lineage in California is low yet the pathogen shows diverse phenotypic variations in colony morphology, senescence and virulence. We demonstrated this variation is associated with originating host species and disease types. Global mRNA profiling detected large differences in gene expression between isolates originating from coastal live oak (PrOak) and those from California bay laurel foliage (PrBay). It is noteworthy that PrOak and PrBay isolates are genetically indistinguishable. This observation suggests the phenotypic variation observed in P. ramorum isolates derived from oak and bay laurel is governed by gene expression, i.e. epigenetic imprinting associated with host environments. These are essential first steps in defining the genetic mechanisms underlying its broad host range and adaptability to new environments. Accomplishments 01 Resistance to crow gall disease in walnut. Agrobacterium tumefaciens is soil-borne bacterium that causes crown gall (CG) a serious disease on walnut. There is an urgent need to develop commercially available CG resistant walnut rootstocks. ARS scientists at Davis, CA, are focusing o search for durable CG resistance on Juglans microcarpa (Texas black waln genotypes. Approximately 25% of the open pollinated seedlings from J. microcarpa mother trees at the USDA repository are showing high levels o resistance to A. tumefaciens. Clonal material (rooted dormant cuttings) propagated from this material is continuing to express CG resistance. To facilitate detection and tracking of A. tumefaciens during these germpla screening, a semi-selective growth medium has been developed to detect agrobacterium populations in native soil down to 100 cells/gram soil. Resistant rootstocks will improve walnut production. 02 Crown gall disease of walnuts-transmission through seeds. Cost effectiv control measures for crown gall disease require understanding of how the causal agent Agrobacterium tumefaciens is transmitted. ARS scientists in Davis, CA, identified the effect of early Agrobacterium infection of walnut hybrid seed on CG development 12 to 24 months post exposure to A. tumefaciens. A. tumefaciens was detected in the husk and shell surface but never found in/on the embryo or cotyledons in seeds collected using industry standards or in/on seeds harvested directly from mother trees. Seeds colonized by A. tumefaciens and planted in MeBr fumigated soil, under greenhouse conditions, rapidly developed extensive tumors and facilitated the systemic movement of A. tumefaciens in both symptomatic and asymptomatic trees. This is the first documented case of A. tumefaciens moving systemically in walnut seedlings. Combined, this knowledge resulted in modifications to grafting tool sanitation frequenc and methods, and aids future development of cost effective control measures. 03 Quirum sensing in Brenneria disease of walnut. Microorganisms sense oth microbes present in the external environment using a variety of mechanis One of the best studied chemical communication systems in bacteria is quorum sensing (QS), a type of intercellular communication which depends on the production of, and response to, low molecular weight inducers in cell-density dependent manner. ARS scientists in Davis, CA, discovered a characterized a QS system in the phytopathogenic bacterium Brenneria rubrifaciens which causes a debilitating disease on walnut trees. We identified the low molecular weight compounds (acyl homoserine lactones) that mediate this communication and demonstrated their ability to facilitate communication between three Brenneria species. Understanding the role of QS in B. rubrifaciens is providing insights into the long latency period of this disease which has been poorly understood for decades resulting in ineffective disease control strategies. 04 Sudden Oak Death. A single clonal lineage of the sudden oak death pathogen, Phytophthora ramorum dominates Californian forests yet the pathogen displays a wide range of virulence and colony morphology. ARS scientists in Davis, CA, determined multilocus genotypes of 47 isolates and investigated associations between genotype and phenotype. We found virulence and fungal colony morphology are tightly associated with the originating host plant species and not with fungal genotype. We then fou global mRNA expression patterns in P. ramorum were associated with originating host species which demonstrates, epigenetic variation is the source of phenotypic diversity in P. ramorum. This study provides some o the first experimental evidence on epigenetic control in fungal pathogen which will enhance investigations into the study of host-pathogen co- evolutionary processes. 05 Use of new molecular probes to screen for virus resistance in walnut. I California, black line disease caused by a virus adversely impacts nut production and longevity of some orchards. U.C. Davis scientists incorporated a resistance gene from black walnut into English walnuts an developed virus resistant backcrosses for release in areas with high incidence of black line disease. A molecular marker associated with blackline resistance was optimized and used to identify virus resistant germplasm. In collaboration with University of California at Davis, ARS scientists at Davis, CA, screened more than 1200 saplings from different back crosses and trees potentially resistant to the virus have been identified. Genetic resistance to black line disease will improve walnut production. 06 Almond brown line disease. A graft transmissible disorder in almonds grown on plum rootstock in orchards on marginal land was discovered in California in the 1990s. When the trees are infected by Peach yellow lea roll phytoplasma, a bacterium without cell walls, they develop a brown necrotic line at the graft union resulting in tree death. It has been difficult to prove the association of phytoplasma in infected almond tre because of non-availability of a suitable detection assay. ARS scientist in Davis, CA, developed a molecular assay and successfully detected this phytoplasma in almond extracts. They also found evidence that this phytoplasma is genetically different from peach yellow leaf roll phytoplasma and have developed a method to detect this phytoplasma in nucleic acid extracts from symptomatic almond trees. Management strategi for brown line disease will be based on knowing the causal agent.
Impacts
(N/A)
Publications
- Alkowni, R., Zhang, Y., Rowhani, A., Uyemoto, J.K., Minafra, A. 2011. Biological, molecular, and serological studies of a novel strain of grapevine leafroll associated virus 2. Virus Genes. DOI 10.1007/s11262-011- 0607-7.
- Browne, G.T., Grant, J.A., Schmidt, L.S., Leslie, C.A., Mcgranahan, G.H. 2011. Resistance to phytophthora and graft compatibility with persian walnut among seedlings of chinese wingnut from different sources. HortScience. 46:371-376.
- Browne, G.T., Schmidt, L.S., Prichard, T.J., Krueger, W.H. 2011. Evaluation of phosphonate treatments for control of phytophthora crown rot of walnut. Plant Health Progress. doi:10.1094/PHP-2011-0601-01-RS.
- Browne, G.T., Schmidt, L.S., Bhat, R., Leslie, C., Hackett, W., Beede, R., Hasey, J. 2011. Etiology and management of crown and root rots of walnut. Walnut Research Conference. pp.225-236.
- Sudarshana, M.R., Mahmood, N., Rowhani, A. 2011. Characterization of R genes involved in resistance to Cherry leaf roll virus in paradox hybrids. Walnut Research Conference. pp. 245-251.
- Caprile, J., Mcgranahan, G., Leslie, C., Sudarshana, M.R. 2011. Evaluation of blackline resistant (hypersensitive) cultivars. Walnut Research Conference. pp. 41-46.
- Kluepfel, D.A., Aradhya, M.K., Maccree, M., Moersfelder, J.W., Mcclean, A. E., Hackett, W. 2011. Evaluation of wild juglans species for crown gall resistance. Walnut Research Conference. pp. 239-242.
- Moyne, A., Sudarshana, M.R., Blessington, T., Koike, S.T., Cahn, M.D., Harris, L.J. 2011. Fate of Escherichia Coli 0157:H7 in field-inoculated lettuce. Food Microbiology. doi:10.1016/j.fm.2011.02.001.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. Approach (from AD-416) 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. FORMERLY CRIS PROJECT #5306-22000-013-00D We continued our field, greenhouse and laboratory trials on etiology and sustainable control strategies for soil borne diseases of fruit and nut crops, including crown gall disease (CG) of walnut and Phytophthora crown rot of almond and walnut, and replant disease of almond and peach. We estimate the economic impact of these diseases to be greater than $20M in annual losses in California. This does not include significant nursery losses that also occur each year. CG- and Phytophthora-resistant breeding material were identified in wild relatives of cultivated walnut, and a Phytophthora-resistant walnut hybrid rootstock, RX1, was patented (joint UC/ARS patent). Disease-resistant walnut relatives will be useful in breeding disease resistant rootstocks. In addition, we are examining the ecology of the CG bacterium and determining how it infects walnut hybrid seeds used to generate rootstocks used in the industry. Cost effective strategies to limit early A. tumfaciens infection at the nursery have been identified. Pathogenicity assays and culture-independent analyses of microbial communities associated almond replant disease were conducted to examine etiology of the disease. Interdisciplinary orchard replant trials were conducted to evaluate chemical and cultural alternatives to MB. We are characterizing of causative agents associated with graft union disorders in almonds, stone fruits, and walnuts and replant disease in almonds and stone fruits. Sustainable management practices to minimize losses caused by these agents were tested. In several counties of California, blackline disease, caused by Cherry leafroll virus, continues to be a menace in the production of walnuts. Because of the demands by stake holders, we have initiated a project to examine the potential of genomics approaches to manage this disease. Last year, an outbreak of almond brown line disease occurred in a young California almond orchard on plum rootstock. Preliminary evidence pointed to involvement of a phytoplasma, a species of bacteria without cell wall and difficult to culture on artificial medium. We were able to detect a phytoplasma in one of the diseased trees last fall using molecular bioassays and, based on the sequence identity, identified it as Pear decline phytoplasma. We will soon provide UCD Foundation Plant Services suitable primers and sampling times for the routine detection of this phytoplasma in almond foundation germplasm. Sudden Oak death is caused by a fungus-like organism whose genome has been sequenced. Using comparative genomics, protein motif search and gene expression analysis, we are validating approx 16,000 computer-predicted gene models. In addition we conducted Illumina RNA-Seq on cDNA libraries derived from 4 distinct life stages of the pathogen and detected c.a. 2,500 putative transcripts not previously identified. This work will facilitate our ability to define genes that mediate such parameters as virulence, host range, and survival in both soil and leaf surfaces there by enhancing disease control and pathogen quarantine efforts. Accomplishments 01 Identification of disease resistant walnut germplasm. Crown gall (CG) i an important disease in walnut and resistant germplasm is needed. ARS scientists in Davis, CA found, under greenhouse conditions, durable crow gall resistance is present in Juglans microcarpa, J. ailantifolia, J. mandischurica and Pterocarya accessions. Over 1500 individual greenhou propogated Juglans seedlings were screening for CG resistance. Approximately 15% of the rooted dormant cuttings from mother trees exhibiting CG resistance continue to exhibit CG resistance. New directed crosses were made generating interspecific hybrids between male and fema trees which exhibit various degrees of CG resistance. Development of CG resistant stock would save $20 million in annual losses. 02 The genetic diversity of A. tumefaciens. Agrobacterium tumefaciens is a soil-borne bacterium that causes crown gall disease on walnut. ARS scientists in Davis, CA continued efforts to define the genetic diversit of A. tumefaciens as a function of geography, host origin and most importantly, nursery origin. Using BOX-PCR these scientists have enhance our forensic ability to identify sources of inoculum which aids the industry as it moves towards the cultivation of A. tumefaciens free planting stock and reduction of post plant infections leading to crown gall formation. Identifying the source of infection facilitates development of cost effective control measures. 03 Sudden Oak Death. Sudden Oak death is caused by a fungus-like organism whose genome has recently been sequenced. The majority of the genes sequenced in Phytophthora ramorum do not have experimental evidence supporting their identification. To validate the gene models, and make t genome data more reliable and useful, Researchers at the Crops Pathology/Genetics Research Unit in Davis, CA conducted Illumina RNA- sequencing on cDNA libraries from 4 life stages of the fungus which provided experimental data to 75% of predicted gene models. However, wh transcripts were detected within the genic regions, inconsistency was identified in half of the intron-exon junctions as we detected c.a. 2,50 genes which were not identified by gene prediction programs. Homologs fo these putative transcripts were rarely recognized in other Phytophthora genomes and known protein motifs, such as pfam domains, were seldom recognized in these transcripts. Identifying and characterizing these putative transcripts, in addition to the correction of predicted gene models are pivotal to understanding the genetic peculiarity of P. ramoru 04 Use of new molecular probes to screen for virus resistance in walnut. I California, black line disease (caused by a virus) adversely impacts nut production and longevity of some orchards. ARS scientists at Davis, CA have incorporated a resistance gene from black walnut into English walnu and developed virus resistant backcrosses for release in areas with high incidence of black line disease. A marker had been found associated wit resistance. We developed and optimized a molecular assay, based on this marker, to identify virus resistant germplasm. In a collaboration with University of California at Davis, more than 700 saplings from different back crosses were screened and trees potentially resistant to the virus have been identified for field testing. 05 Identification of the causal agent associated with the almond brownline disease. Almond brown line disease was discovered in California in the 1990s as a graft union disorder in almonds grown on plum rootstock in orchards on marginal land. When the trees are infected by Peach yellow leafroll phytoplasma, a bacterium without cell walls, they develop a bro necrotic line at the graft union resulting in tree death. It has been difficult to prove the association of phytoplasma in infected almond tre because of non-availability of a suitable detection assay. ARS scientis in Davis, CA developed a molecular assay and successfully detected this phytoplasma in almond extracts. This assay can be used to monitor the trees in a commercial orchard impacted by almond brown line disease.
Impacts
(N/A)
Publications
- Kluepfel, D.A., Yakabe, L., Parker, S., Mcclean, A.E., Maccree, M.M. 2010. DETECTION AND IMPLICATIONS OF EARLY AGROBACTERIUM TUMEFACIENS INFECTION OF PARADOX SEEDS AND SEEDLINGS. Acta Horticulturae. 861:497-500.
- Hasey, J., Lampinen, B., Anderson, K., Grant, J., Caprile, J., Beede, R., Kluepfel, D.A. 2010. CROWN GALL INCIDENCE: SEEDLING PARADOX WALNUT ROOTSTOCK VERSUS OWN-ROOTED ENGLISH WALNUT TREES. Acta Horticulturae. 861:453-455.
- Yakabe, L., Parker, S., Kluepfel, D.A. 2010. Effect of pre-plant soil fumigants on Agrobacterium tumefaciens, pythiaceous species, and subsequent soil recolonization by A. tumefaciens. Crop Protection. 29:583- 590.
- Mcelrone, A.J., Grant, J.A., Kluepfel, D.A. 2010. THE ROLE OF TYLOSES IN CROWN HYDRAULIC FAILURE OF MATURE WALNUT TREES AFFLICTED WITH APOPLEXY DISORDER. Tree Physiology. 30:761-772.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. Approach (from AD-416) 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. FORMERLY CRIS PROJECT #5306-22000-013-00D Significant Activities that Support Special Target Populations We continued our field, greenhouse and laboratory trials to identify sustainable control strategies for crown gall disease (CG) and phytophthora crown rot of walnut trees in both orchard and nursery conditions. We estimate the economic impact of these diseases to be $20M annual loss in CA alone. This does not include the significant nursery losses that also occur each year. We have focused our efforts in two areas. First,CG- and Phytophthora-resistant breeding material was identified in wild relatives of cultivated walnut, and a Phytophthora- resistant walnut hybrid rootstock was identified. A joint UC/ARS patent has been sought for the rootstock. The disease-resistant walnut relatives will be useful in breeding disease resistant rootstocks. Second, we are examining the ecology of the CG bacterium and determining how it infects the walnuts that are planted and used to generate the trees used in the industry. Our goal is to develop disease resistant rootstocks combined with sustainable disease management strategies for the walnut industry. We continued our characterization of causative agents associated with graft union disorders in almonds, stone fruits, and walnuts and replant disease in almonds and stone fruits. Sustainable management practices to minimize losses caused by these agents were tested. In several counties of California, blackline disease, caused by Cherry leafroll virus, continues to be a menace in the production of walnuts. Because of the demands by stake holders, we have initiated a project to examine the potential of genomics approaches to manage this disease. Last year, an outbreak of almond brown line disease occurred in a young California almond orchard on plum rootstock. Preliminary evidence pointed to involvement of a phytoplasma, a species of bacteria without cell wall and difficult to culture on artificial medium. We were able to detect a phytoplasma in one of the diseased trees last fall using molecular bioassays and, based on the sequence identity, identified it as Pear decline phytoplasma. We will soon provide UCD Foundation Plant Services suitable primers and sampling times for the routine detection of this phytoplasma in almond foundation germplasm. Sudden Oak death is caused by a fungus-like organism whose genome has recently been sequenced. Using a combination of comparative genomics, protein motif search and gene expression analysis, we are validating the identification of approx 16,000 genes. In addition we are using a functional genomic approach to predict gene function in about 1800 genes that were not previously identified. This work will facilitate our ability to clearly define the genes that mediate such important parameters as virulence, host range, and survival in both soil and leaf surfaces there by enhancing disease control and pathogen quarantine efforts.
Impacts
(N/A)
Publications
- Mcclean, A.E., Kluepfel, D.A. 2009. GENETIC LOCI INVOLVED IN RUBRIFACINE PRODUCTION IN THE WALNUT PATHOGEN BRENNARIA RUBRIFACIENS. Phytopathology. 99:145-151.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) 1. Improve management strategies for key soil borne diseases of tree fruit and nut crops. 1A. Develop economical alternatives to preplant use of methyl bromide (MB). 1B. Identify and characterize available walnut and almond rootstock germplasm for resistance to key soil borne pathogens. 2. Characterize the etiology of key soil borne diseases of tree fruit and nut crops, and examine the molecular microbial ecology of both pathogenic and beneficial plant-associated microorganisms. 2A. Determine the etiology of Prunus replant disease (PRD). 2B. Characterize the genetic diversity and ecology of Agrobacterium tumefaciens. 3. Develop enhanced traditional and culture-independent molecular detection methods for key pathogens of fruit and nut crops. 3A. Develop enhanced detection protocols for Agrobacterium tumefaciens. 3B. Develop enhanced detection protocols for Brennaria rubrifaciens. 3C. Develop enhanced detection protocols for Phytophthora cactorum. 4. Examine etiology, epidemiology, and control strategies for systemic/graft transmissible pathogens of fruit and nut trees. 4A. Identify and characterize graft-transmissible pathogens(GTP)in fruit and nut trees with an emphasis on determining the etiology of emerging diseases such as Tieton cherry stunt (TCS) and Plum necrotic union (PNU). 4B. Epidemiology and molecular characterization of PBNSPaV. 4C. Evaluate Marianna 2624 rootstock for tolerance to Xyllela fastidiosa in almond. 5. Examine the structural and functional genomics of Phytophthora ramorum, the causal agent of Sudden Oak Death. 6. Identify and characterize rhizobacterial genes whose expression is mediated by plant root exudates and evaluate involvement of these genes in such ecologically important phenotypes as root colonization, infection, competition, and persistence in the environment. 7. Characterize the biology, genetic diversity, and ecology of key plant pathogenic agents of fruit/nut trees including A. tumefaciens strains, and examine the ecology and significance of molecular microbe-microbe and plant-microbe interactions of key plant associated microorganisms. Approach (from AD-416) 1. Chemical and crop-rotation-based alternatives to methyl bromide will be tested for control of Prunus replant disease (PRD), and genetic resistance to Phytophthora spp. and Agrobacterium tumefaciens will be explored in promising almond and walnut rootstocks. 2. Examine shifts in soil borne microbial communities associated with PRD incidence using culture-based and culture-independent approaches and complete Koch's postulates for organisms linked to the disease. Examine the molecular microbial ecology of A. tumefaciens under both orchard and nursery conditions in an effort to design effective crown gall control strategies. Examine Ti-plasmid ecology under commercial orchard conditions. Assess the genetic diversity of A. tumefaciens in California. 3. Described and novel PCR primers will be tested for specific amplification of ribosomal and mitochondrial DNA from P. cactorum. Enhance described and develop novel PCR primers for species-specific detection and quantification of A. tumefaciens in soil and inplanta. Develop PCR primers and define the extraction and cycling parameters for the detection and quantification of Brennaria rubrifaciens. 4. Epidemiological studies and molecular characterization of Plum bark necrosis-stem pitting associated virus (PBNSPaV) will be performed. Discarded and healthy almond trees on M2624 plum and peach rootstocks will be compared for tree performance, nut set, nut quality and decline symptoms as a function of X. fastidiosa infection. 5. Structural and functional genomics and/or a proteomics approaches will be used to examine the biochemical and molecular mechanisms of pathogenesis. Analysis of the P. ramorum genome also will focus on gene identification, assignment of putative gene function, and comparative genomics studies using the currently sequenced genomes of Phytophthora spp. FORMERLY CRIS PROJECT #5306-22000-013-00D Significant Activities that Support Special Target Populations Field trials were established at 4 locations to evaluate fumigant and non- fumigant alternatives to methyl bromide for pre-plant soil fumigation for almond and other stone fruits. Novel spot treatments using GPS-controlled shanks and site-directed drip tubing as well as conventional strip and broadcast treatments were tested. Two trials examined the use of short term fallowing or crop rotation to supplement alternative fumigation treatments. Greenhouse trials indicate that durable crown gall (CG) resistance is present in Juglans microcarpa, J. ailantifolia, J. mandischurica and Pterocarya accessions. We demonstrated that latent infection with A. tumefaciens occurs. This complicates interpretation of CG resistance screening and prolongs the observation period to 10 months. Greenhouse trials were completed to evaluate resistance to P. cinnamomi and P. citricola in walnut rootstocks, and resistance to P. megasperma in almond rootstocks. Root and soil samples were collected from healthy and replant-disease- affected Nemaguard peach trees (in fumigated and non-fumigated soil; or sterile and non-autoclaved soil). Microbial communities were characterized using culture-based and culture-independent detection methods. Data matrices of bacterial, fungal, and oomycete incidence � health status of the host were prepared. Redundancy analysis identified shifts in the microbial communities associated with incidence of replant disease. The BOX-PCR profiles of 190 virulent A. tumefaciens biovar1 isolates were analyzed using UPGMA analysis software GelCompar II. Distinct genetic profiles were clustered according to similarity. Analysis of a subset of 93 virulent biovar 1 isolates revealed genotypes unique to; geography, isolate and nursery source. In addition we identified 1 genotype that occurred in at least 4 counties. Refined DNA extraction-purification protocols to facilitate PCR diagnostics for P. cactorum in diseased strawberry plants. Incubation of the tissue in a detergent solution, followed by embedding and electrophoretic purification in agarose significantly reduced concentrations of PCR inhibitors. PCR primers were developed and validated to selectively amplify rDNA of P. cactorum. Diseased tissue from plants naturally and artificially infected with P. cactorum were used to examine the relative accuracy/sensitivity of the PCR detection protocol. PCR detection was more rapid, and just as sensitive as culture- based sampling. Our A. tumefaciens specific PCR primers (Tip4) were refined and improved standard and real-time cycling parameters were developed. The new primers Tip 5 and 6, enhance sensitivity and reduce PCR costs. Dr. Sudarshana is a new SY hired to initiate a plant virus research program. His lab has been set up to provide diagnostics and conduct research on molecular biology of viruses and graft-transmissible agents. Dr Kasuga is a new SY hired to initiate a program on the structural- functional genomics of P. ramorum, the causal agent of sudden oak death. A new office and lab have been established, equipment has been purchased and initial collaborations are in place. This research addresses NP303 plant disease component 4 & 4A.
Impacts
(N/A)
Publications
- Fennimore, S.A., Duniway, J.M., Browne, G.T., Martin, F.N., Ajwa, H.A., Westerdahl, B.B., Goodhue, R.E., Haar, M., Winterbottom, C.Q. 2008. Methyl bromide alternatives for california strawberry nurseries. California Agriculture. April-June 2008:62-67
- Shrestha, A., Browne, G.T., Lampinen, B.D., Schneider, S.M., Simon, L., Trout, T.J. 2008. Perennial crop nurseries treated with methyl bromide and alternative fumigants: effects on weed seed viability, weed densities, and time required for hand weeding. Weed Technology. 22:267-274
- Shaw, D.V., Hansen, J., Browne, G.T., Shaw, S.M. 2008. Components of genetic variation for resistance of strawberry to phytophthora cactorum estimated using segregating seedling populations and their parent genotypes. Plant Pathology. 57:210-215.
- Mcclean, A.E., Sudarshana, P., Kluepfel, D.A. 2008. Enhanced detection and isolation of the walnut pathogen brennaria rubrifaciens: causal agent of deep bark canker. European Journal of Plant Pathology. 10.1007/s 10658-008- 9308
- Bhat, R.G., Browne, G.T. 2007. Genetic diversity in populations of phytophthora citricola associated with horticultural crops in california. Plant Disease. 91:1556-1563
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