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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #426096

Research Project: Detection and Management of Pathogens in Strawberry and Vegetable Production Systems

Location: Crop Improvement and Protection Research

2014 Annual Report


1a. Objectives (from AD-416):
The overall objective of this project is to develop diagnostic tools for detection, quantification, and identification of soilborne plant pathogens currently controlled by chemicals including methyl bromide and to develop technically and economically feasible alternatives to chemical control for high value crops, such as strawberries and vegetables. Objective 1: Optimize delivery and evaluate performance of cultural and biological control, management practices, and genetic alternatives for management of pathogens currently mediated by soil fumigation. Sub-objective 1.a. Evaluate alternatives to replace current fumigation strategies for management of soilborne pathogens. Sub-objective 1.b. Identify genes associated with pathogenicity of Verticillium dahliae to support the development of alternative control procedures. Sub-objective 1.b.1. Identify genes associated with pathogenicity of Verticillium dahliae based on a previous insertional mutagenesis study. Sub-objective 1.b.2. Evaluate expression of candidate pathogenicity-related genes in resistant and susceptible lettuce-Verticillium dahliae interactions. Sub-objective 1.c. Identify emerging diseases and their etiology, and evaluate germplasm for resistance to diseases of strawberry, lettuce, and vegetables. Sub-objective 1.c.1. Evaluate spinach germplasm for new sources of Verticillium dahliae resistance using an enhanced screening technique. Sub-objective 1.c.2. Evaluate primary inoculum sources of Peronospora effusa, the cause of downy mildew of spinach. Sub-objective 1.c.3. Identify pathogens of emerging and established bacterial diseases of strawberry and vegetables. Sub-objective 1.c.4. Identification and characterization of lettuce and crucifer germplasm resistant to bacterial pathogens. Sub-objective 1.d. Identify factors associated with production, maintenance, and the degradation of inoculum to elucidate potential alternatives for pathogen control. Sub-objective 1.d.1. Identify and evaluate microbial predators of fungal and bacterial pathogens for disease control. Sub-objective 1.d.2. Functionally analyze candidate genes identified as up-regulated in microsclerotia development in Verticillium dahliae. Objective 2: Develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of vegetables and strawberries, and use these tools in the development of management strategies as alternatives to methyl bromide. Sub-objective 2.a. Develop molecular markers for detection and quantification of bacterial plant pathogens. Sub-objective 2.b. Develop molecular diagnostic tools for detection, quantification, and characterization of isolates of Peronospora effusa. Sub-objective 2.c. Develop molecular tools for identification and detection of Pythium and Phytophthora species. Sub-objective 2.c.1. Molecular diagnostics. Sub-objective 2.c.2. Improved identification of Phytophthora species. Sub-objective 2.c.3. Mitochondrial genomics project.


1b. Approach (from AD-416):
Develop integrated management approaches including crop rotation, biological control, selection of disease resistant varities, organic production, other biological practices, and combinations of biological practices to control diseases. Identifying useful biological agents as well as new agents that will help to improve disease management. Molecular tools will be developed for plant pathogen identification, evaluation of the efficacy of pathogen management and modeling population dynamics of plant pathogens and beneficial microbes.


3. Progress Report:
This is the first report for this project which began in December of 2013, and is replacing the bridging project 505-22000-014-00D, "Detection and Management of Pathogens in Strawberry and Vegetable Production Systems". Please see the previous project for additional information. Sub-Objective 1.a: Evaluate alternatives to replace current fumigation strategies for management of soilborne pathogens. The objective to finish analysis of pathogen/pest survival under different fumigation treatments has not been completed because collection of additional data is necessary. Sub-Objective 1.b.1: Identify genes associated with pathogenicity of Verticillium dahliae based on a previous insertional mutagenesis study. A gene knockout construct for a major facilitator superfamily gene is under preparation and the milestone for this sub-objective was substantially met. The confirmation of the gene deletion and analyses of phenotype are the next steps. Sub-Objective 1.b.2: Evaluate expression of candidate pathogenicity-related genes in resistant and susceptible lettuce-Verticillium dahliae interactions: Primers were prepared to assess pathogenicity-related gene expression in lettuce for future analyses. In the coming year, we will evaluate pathogenicity-related gene expression in lettuce in response to the fungus, Verticillium dahliae. Sub-objective 1.c.1: Evaluate spinach germplasm for new sources of Verticillium dahliae resistance using an enhanced screening technique: Resistance to Verticillium wilt in spinach accessions was correlated with reduced infected seed and reduced pathogen nucleic acid quantities, and a research manuscript is currently in preparation describing this correlation. Sub-objective 1.c.2: Evaluate primary inoculum sources of Peronospora effusa the cause of downy mildew of spinach: Dozens of leaf samples were collected from spinach fields infected with downy mildew, although microscopy analyses indicated that the samples are all negative for oospore production. Additional spinach seed samples were also examined for the presence of oospores, and none were detected. We could not detect the spinach downy mildew pathogen in soil samples six months after collection. The sampling and analysis of plant material and soil for oospores is ongoing. Sub-objective 1.c.3: Identify pathogens of emerging and established bacterial diseases of strawberry and vegetables: We have determined the etiology of several bacterial diseases of vegetables and small fruit. Bacterial pathogens of basil are being further investigated for their relationship to other pathogens that are similar to opportunistic plant pathogen Pseudomonas viridiflava. Likewise, the taxonomy of the Pseudomonas syringae strains causing blight on raspberry leaves needs further refinement because the research demonstrated that the pathogen was more closely related to. P. syringae pathovar (pv.) aceris and P. syringae pv. solidagae than to P. syringae pv. syringae as previously reported by other researchers. The host range data of pathogens of the Apiaceae and radicchio was summarized and additional sampling or experiments needed were outlined. Additionally, the first draft of a manuscript describing Multi Locus Sequence Typing (MLST) for fluorescent pseudomonads was written. Sub-objective 1.c.4: Identification and characterization of lettuce and crucifer germplasm resistant to bacterial pathogens: We have drafted a manuscript that describes the genetic diversity of Xanthomonas campestris pv. vitians and complete resistance in lettuce genotypes. We have two additional manuscripts ready for submission describing the genetics of resistance in lettuce to one of the genotypes of the pathogens and the restriction of pathogen populations on lettuce due to a hypersensitive response (HR). Methods for screening arugula for resistance to Pseudomonas cannabina pv. alisalensis have been developed and tested. Germplasm was gathered from the Germplasm Resources Information Network (GRIN) and will be evaluated in the coming year. Sub-objective 1.d.1: Identify and evaluate microbial predators of fungal and bacterial pathogens for disease control: Microsclerotia of Verticillium dahliae were produced in lettuce leaves and a standard operating procedure (SOP) was prepared for subsequent experiments. We have decided to evaluate inhibition of myxobacteria at the same time that we are checking viability of our bacterial collection. Thus an SOP for inhibition of Verticillium dahliae was generated and methods will be evaluated this summer. Bacterial phage stocks are being regenerated in order to evaluate the ability of various phage to lyse the diverse strains of X. campestris pv. vitians. Sub-objective 1.d.2: Functionally analyze candidate genes identified as up-regulated in microsclerotia development in Verticillium dahliae: A gene encoding a hypothetical gene that we had previously identified as up-regulated in the microsclerotia of Verticillium dahliae was successfully knocked out. Initial analyses of the mutant did not reveal a difference relative to the wild-type. An additional gene knockout from the up-regulated gene set is being prepared for study. Sub-Objective 2.a: Develop molecular markers for detection and quantification of bacterial plant pathogens: A paper that established a polymerase chain reaction (PCR) protocol for detection and quantification of Sphingomonas suberifaciens was published. Most of the primers available for detection and quantification of P. cannabina pv. alisalensis have been evaluated for specificity. An additional two primer pairs are being optimized prior to testing the protocols developed for detection and quantification from environmental samples. In order to find pathovar and genotype-specific sequences for the development of detection methods and to further understand diversity in X. campestris pv. vitians, the genomes of 10 strains of the pathogen and close relatives were sequenced with Illumina technology. Assembly of the sequences is scheduled for this fall and will be followed by comparative analysis. Sub-objective 2.b: Develop molecular diagnostic tools for detection, quantification, and characterization of isolates of Peronospora effusa: A manuscript that describes real time PCR detection of Peronospora effusa, the cause of downy mildew on spinach, was completed. Additional downy mildew samples were collected and nucleic acid was extracted for further validation of the real time PCR assay. The samples were stored for the next phase of the research. Additional molecular markers were examined for their diagnostic utility. Sub-objective 2.c.1: Molecular Diagnostics: A manuscript that describes a real time PCR assay for Phytophthora that is multiplexed for genus and species specific detection was published. Species-specific markers for 14 species (several of them invasive quarantine species) have been validated and the data suggest that markers for at least an additional 70 species can be developed. To support technology transfer, sequences used for marker design and putative markers for other species have been deposited at the journal website. More recently, diagnostic markers were validated for 21 invasive Phytophthora species that are of concern to USDA Animal and Plant Health Inspection Service (APHIS). The technology was transferred to California Department of Food and Agriculture (CDFA) and USDA APHIS Plant Protection and Quarantine (PPQ) Center for Plant Health Science and Technology (CHPST) for their use. A Phytophthora genus and species-specific isothermal detection assay was developed that does not need DNA extraction, is as sensitive as TaqMan real time PCR and results can be obtained in as little as 15 minutes. We have been working with the manufacturer of a battery powered data collection unit run by an application on a smart phone via a blue tooth connection to optimize the software for running this assay. This detection system is fully field portable, and with the ability to obtain rapid results provides a point-of-sample collection diagnostics capability. This will reduce the cost of sample processing and enhance regulatory and containment capabilities. Sub-objective 2.c.2: Phytophthora Database: We finished uploading sequence data, updated the molecular diagnostics section of database and generated a species page and sequence data for newly described Phytophthora species. Sub-objective 2.c.3: Mitochondrial genomics for development of molecular markers and phylogenetic purposes: Collaborative projects have been established internationally where raw genomic sequence data are provided and mitochondrial genomes are assembled in Salinas. Over 160 genomes have been assembled from a wide range of genera representing plant pathogenic oomycetes. Comparative genomics identified loci useful for development of diagnostic markers for Pythium and downy mildews, genes for broader scale phylogenetic analyses, mitochondrial haplotypes for population studies and provides insight on mechanisms driving mitochondrial genome evolution.


4. Accomplishments
1. Identified two genes required for the fungus Verticilium dahliae to cause disease. The plant pathogenic fungus Verticillium dahliae causes economically important wilt diseases on crops in the U.S. and worldwide. Yet, there remains a lack of knowledge on the full complement and function of genes in Verticillium dahliae that enable the organism to cause disease. An ARS researcher in Salinas, California, completed collaborative work with researchers in Spain to identify two genes that are essential for pathogenicity on lettuce. Identification of the gene products responsible for pathogenicity on lettuce and other crops provides a basis for the development of new technologies for disease control.

2. Gene expression regulates long-term survival in a soilborne plant pathogen. Verticillium dahliae is a soilborne plant pathogen that causes destructive wilt diseases on numerous crops worldwide. The pathogen can persist in the soil for 15 years as survival structures known as microsclerotia. An ARS researcher in Salinas, California, completed collaborative work with researchers in China to analyze genes that are differentially expressed at multiple stages of microsclerotia development. Expression analyses of genes that regulate and execute the processes of microsclerotia development provide insights on targets for the control of this fungus in the soil.

3. Deployment of tools to quantify an airborne plant pathogen in the field. Downy mildew on spinach is the primary disease constraint on US spinach production. The pathogen produces oospores that are capable of survival in the soil but also airborne spores that can infect healthy plants under cool, wet conditions. ARS researchers at Salinas, California, led the development of a DNA-based assay to analyze levels of this airborne pathogen and validated its usage in the field. The assay can be useful to predict disease risk based on spore load in the environment, and therefore may be useful to reduce the number of fungicide applications for disease control.

4. Validated and published methods for quantification and detection of Sphingobium suberifaciens. Research on Sphingobium suberifaciens is limited because the pathogen is not amenable to classical detection and quantification methods. An ARS researcher in Salinas, California, validated and published polymerase chain reaction (PCR) protocols for detection and quantification of the pathogen that causes corky root in lettuce. These methods were used to detect and quantify the pathogen from soil and crop samples. Producers can detect and quantify the pathogen before planting susceptible crops.

5. Identified complete resistance in lettuce to bacterial leafspot. The hypersensitive response (HR) in lettuce confers complete resistance to bacterial leafspot incited by specific genotypes of Xanthomonas campestris pv. vitians. ARS researchers in Salinas, California, described five distinct genotypes of Xanthomonas campestris pv. vitians. HR-based resistance in lettuce was shown to be effective against four of the five genotypes of the pathogen described to date. These results allow us to target further germplasm screening to identify resistance to the fifth pathogenic genotype. This work enables USDA and public seed company breeders to develop lettuce cultivars resistant to strains that overcome the currently available resistance and allows for methods to distinguish California genotypes from others as they move into the state.

6. Identified bacterial plant pathogens that cause diseases new to the region on Basil, raspberry, bok choy, mustard cover crops, and napa cabbage. An ARS researcher in Salinas, California, determined the cause of several emerging plant pathogens from vegetables and small fruit. Molecular and phenotypic characters were used to identify bacteria isolated from diseased tissue and pathogenicity was confirmed. Identification of Pseudomonas cannabina pv. alisalensis from symptomatic tissue of the mustard cover crops bok choy and napa cabbage extends the natural host range of the pathogen. Determination of the cause of bacterial diseases and descriptions of the host ranges allow producers and other agricultural professionals to design crop rotation strategies to prevent subsequent outbreaks.

7. Web site to support Phytophthora research. The genus is responsible for many diseases of crop plants worldwide and consists of approximately 127 species that are difficult to identify. ARS researchers at Salinas, California, and other ARS locations, and university researchers developed a web-based database for Phytophthora research to enhance the understanding of the genus, simplify species identification and stimulate further research on the genus. This database will serve as a resource for researchers working on the genus, a means to simplify sequence based identification of species, as well as a repository for future work. Updating of content is a continuous process.

8. Molecular diagnostics of Phytophthora. To facilitate detection of the broad host range plant pathogen Phytophthora, an ARS researcher at Salinas, California, published a new real time polymerase chain reaction (PCR) diagnostic marker system that can detect the pathogen at a genus and species-specific level. The system allows for a systematic development of species-specific markers for over 85% of described species, and markers have been validated for 21 species on the Animal and Plant Health Inspection Service (APHIS) species of concern list. DNA sequence data used for designing the marker system for nearly all Phytophthora spp. and a list of putative TaqMan probes for 85 species have been posted on the journal website to facilitate adoption of the technology by other labs. Publication of this research facilitates diagnostic efforts with this pathogen and provides tools for APHIS to detect invasive species.

9. Clarified the phylogenetic relationships in Phytophthora. The genus Phytophthora includes a large number of economically important plant pathogens that are difficult to identify with certainty due to similar morphology, and which may, therefore, result in misidentification and inappropriate regulatory action. An ARS researcher at Salinas, California, used four mitochondrial and three nuclear loci to investigate the phylogeny of 109 taxa of Phytophthora to better understand the evolutionary relationships and clarify species classifications within the genus Phytophthora. The analysis revealed the same phylogenetic clades as a prior analysis based on nuclear loci; addition of the mitochondrial loci enabled clarification of the basal relationships among the clades. A more detailed analysis of species from clades 2 and 8 identified seven additional phylogenetic lineages that warrant further investigation to determine if they represent distinct species. These results provided additional genes useful for phylogenetic analyses, helped clarify species boundaries, and identified new taxa in need of species descriptions.


Review Publications
Xiong, D., Wang, Y., Ma, J., Klosterman, S.J., Xiao, S., Tian, C. 2014. Deep mRNA sequencing reveals stage-specific transcriptome alterations during microsclerotia development in the smoke tree vascular wilt pathogen, Verticillium dahliae. BMC Genomics. 15:324.

Klosterman, S.J., Anchieta, A.G., Mcroberts, N., Koike, S.T., Subbarao, K.V., Voglmayr, H., Choi, Y., Thines, M., Martin, F.N. 2014. Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii). Phytopathology. 104:1349-1359.

Bull, C.T., Goldman, P.H., Martin, K.J. 2014. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ. Molecular and Cellular Probes. DOI: 10.1016/j.mcp.2014.03.001.

Wechter, W.P., Keinath, A.P., Smith, J.P., Farnham, M.W., Bull, C.T., Schofield, D.A. 2014. First report of bacterial leaf blight on mustard greens (Brassica juncea) caused by pseudomonas cannabina pv. alisalensis in Mississippi. Plant Disease. 98:1151.

Muramoto, J., Gliessman, S.R., Koike, S.T., Shennan, C., Schmida, D., Stephens, R., Swezey, S., Bull, C.T., Klonsky, K. 2014. Integrated biological and cultural practices can reduce crop rotation period of organic strawberries. Agroecology and Sustainable Food Systems. 38:603-631. DOI: 10.1080/21683565.2013.878429.

Bull, C.T., Coutinho, T.A., Denny, T.P., Firrao, G., Fischer-Le Saux, M., Li, X., Saddler, G.S., Scortichini, M., Stead, D.E., Takikawa, Y. 2014. List of new names of plant pathogenic bacteria (2011-2012). Journal of Plant Pathology. 96:223-226.

Martin, F.N., Blair, J.E., Coffey, M.D. 2014. A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora. Fungal Genetics and Biology. 66:19-32.

Bilodeau, G., Martin, F.N., Coffey, M.D., Blomquist, C. 2014. Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order. Phytopathology. 104:733-748.