Location: Crop Improvement and Protection Research
2017 Annual Report
Objectives
The long-term objectives of this project are to develop disease management strategies for diseases of economic importance of strawberries and vegetables. The two overall objectives of the current project extend from the need to deliver and evaluate alternative approaches for management of these important pathogens, as well as to develop and deploy molecular diagnostic tools for their management. The project subobjectives examine cultural, biological, and genetic approaches for management of plant pathogenic fungi and oomycetes, including Verticillium dahliae, Peronospora effusa, and Macrophomina phaseolina, and provide molecular diagnostic tools to monitor populations of Fusarium oxysporum f. sp. fragariae, P. effusa, Phytopthora species, and M. phaseolina. We will focus on these following major objectives and subobjectives during the next five years.
Objective 1: Optimize delivery and evaluate performance of cultural and biological methods, management practices, and genetic approaches for management of pathogens, including those currently mediated by soil fumigation.
Subobjective 1A: Identify genes of Verticillium dahliae required for the initial stage of lettuce root infection.
Subobjective 1B: Identify genetic alternatives for resistance to downy mildew of spinach caused by Peronospora effusa.
Subobjective 1C: Identify edaphic factors that influence long term or reduced survival of soilborne fungi.
Subobjective 1D: Determine the correlation between genotype of Macrophomina phaseolina and virulence on strawberry.
Subobjective 1E: Assemble a high quality reference genome for M. phaseolina and identify genes associated with host specificity.
Objective 2: Develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of strawberries and vegetables, and use these tools in the development of disease management strategies.
Subobjective 2A: Identify population genetic markers, diagnostic markers and develop tests for rapid identification of Peronospora effusa, the downy mildew pathogen of spinach.
Subobjective 2B: Develop molecular tools for identification and detection of Oomycete plant pathogens.
Subobjective 2C: Develop molecular tools for detection and soil quantification of Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae.
Approach
1.A: Identify genes of V. dahliae required for lettuce root infection. Hypothesis: Genes identified as up-regulated in V. dahliae in the rhizosphere but not in contact with plant roots are required for the initial stage of infection. Approach: Genes identified as upregulated in response to lettuce roots deleted for analysis. Lettuce inoculated with deletion mutant strain of the pathogen and mock-inoculated control. 1.B.1: Identify genes differentially expressed between resistant and susceptible. Hypothesis: Downy mildew resistance and susceptibility is associated with differentially expressed genes. Approach: RNA-Seq analysis. 1.B.2: Develop a spinach leaf assay. Goal: Develop assay to allow routine screening. Approach: Analyses of the infection of different spinach downy mildew races assessed by inoculating spinach leaves of different spinach cultivars in plastic containers, in a single chamber. 1.C.1: Identify microbial predators of fungal pathogens for disease control. Goal: Isolate and identify individual bacterial strains from soils using pathogen baiting techniques. Approach: A Petri-dish based baiting method will be used to enrich for and isolate microbes that are able to feed on Verticillium microsclerotia. 1.C.2: Identify soil abiotic factors that reduce survival of V. dahliae. Goal: Assess effect of soil type, moisture levels, and temperature on long-term survival of V. dahliae. Approach: V. dahliae microsclerotia-infested microcosms will be maintained with different soil types and monitored over time. 1.C.3: Analyze biotic factors that affect survival of V. dahliae or reduced infections. Hypothesis: Root biome-derived bacteria will degrade or otherwise reduce the survival of the microsclerotia of V. dahliae and protect plant hosts. Approach: Microsclerotia-infested microcosms inoculated with bacterial strains. Subobjective 1.C.4: Analyze pigment cluster genes of V. dahliae that contribute to long-term survival. Hypothesis: Genes in the melanin biosynthesis cluster of V. dahliae required for long-term survival. Approach: Analyze three cluster genetic mutants for survival over time, on growth media. Subobjective 1D: Evaluate genotype of M. phaseolina and virulence. Goal: Genotype isolates of the pathogen in California and evaluate differences in their virulence on a susceptible strawberry cultivar. Approach: Plant a susceptible cultivar in a greenhouse into soil amended with M. phaseolina and evaluate disease. 1E: Assemble genome for M. phaseolina and identify genes. Goal: Identify host specificity genes. Approach: DNA sequencing and mapping. 2.A.1: Develop in-field diagnostic test for P. effusa. Goal: Develop a quick diagnostic test. Approach: Recombinase polymerase amplification. 2.A.2: Identify and deploy population genetic markers. Hypothesis: DNA sequences are different between populations. Approach: Simple sequence repeat marker analysis. Subobjective 2.B.1: Mitochondrial genomics. 2.B.2: Molecular diagnostics. Subobjective 2.B.3: Oomycete phylogenetics. Subobjective 2.B.4: Improved identification of Phytophthora. Approach and Goal for 2.B.1-2.B.4: Sequence and develop molecular techniques for diagnostics.
Progress Report
This report documents progress for project 2038-22000-016-00D, which started in March, 2017 and continues research from project 2038-22000-015-00D, "Detection and Management of Pathogens in Strawberry and Vegetable Production Systems."
Substantial progress was made in FY 2017 in support of Objective 1.
Under Subobjective 1.A, analysis of differentially expressed genes and translated products was initiated using bioinformatics approaches in order to determine which of the genes that were highly differentially expressed may be important for interaction with the host. A DNA construct for a gene deletion of a candidate gene involved in early stages of root infection was prepared.
Under Subobjective 1.B.1, bioinformatics approaches were used to determine the gene sets that are significantly differentially expressed between resistant and susceptible infected spinach cultivars, although we are requesting the new gene annotation of the spinach genome sequence to complete this work.
Under Subobjective 1.B.2, experiments were planned, based upon available research in similar organisms, and the pathogen was inoculated on spinach seedlings for examination of downy mildew development under controlled conditions. Natural infections were maintained in nearby outdoor microplots.
Under Subobjective 1.C.1, lettuce plants were infected with V. dahliae in a greenhouse, and the long-term survival structures of the pathogen, also known as microsclerotia, were collected from leaf tissue. This generated the pathogen inoculum derived from natural conditions for experimentation.
Under Subobjective 1.C.2, a standard operating procedure was developed for inoculation of soils with V. dahliae microsclerotia in order to conduct experiments to determine how long the microsclerotia, can survive under different conditions for potential use in development of disease control techniques.
Under Subobjective 1.C.4, examination of melanin biosynthesis gene cluster deletion mutants for differences from the wild type strain, revealed two that lack pigmentation. Pigment is thought to be beneficial for long-term survival, and thus understanding its production may reveal a weakness or point of control in the pathogen.
Under Subobjective 1.D, we genotyped nearly 500 isolates of Macrophomina phaseolina that were recovered from California and strawberry isolates that were recovered from other production areas around the world with 24 simple sequence repeat (SSR) loci. Pathogenicity tests were conducted with isolates that represented a wide range of genotypes. The isolates recovered from strawberry, with a few exceptions, represent a single genotype. These data helped guide the selection of isolates to sequence for development of diagnostic assays targeting the genotypes important for strawberry production.
Under Subobjective 1.E, high quality PacBio assemblies were completed for two isolates of Macrophomina phaseolina, one representing the aggressive genotype that attacks strawberry and another that does not infect this host (90% of the genome represented in the largest 15-20 contigs). An optical map of one of the isolates and Hi-C analysis of both isolates is in progress and should help to scaffold the contigs into chromosomes and fill in the gaps of coverage. We are working with a collaborator at Michigan State University on annotation of the assembly and posting the results on a genome browser. An additional 15 isolates have been sequenced with Illumina thus far and comparative genomics has been used to identify unique coding regions.
Progress continued in FY 2017 in support of Objective 2.
Under Subobjective 2.A.1, sequences of Peronospora effusa DNA were identified for potential usage for an in-field diagnostic test for the spinach downy mildew pathogen, Peronospora effusa. An in-field detection system will help spinach growers to more quickly make disease management decisions.
Under Subobjective 2.A.2, to determine changes in spinach downy mildew pathogen populations over time, samples of infected leaves were collected and stored in a freezer as a first step.
Under Subobjective 2.B.1, mitochondrial genomes for over 500 isolates representing 100 taxa and 13 genera have been assembled and annotated. This includes genomes from the Select Agent Peronosclerospora philippinensis and other tropical downy mildews of corn using DNA/sequence data provided by collaborators at the University of the Philippines, ARS in Ft. Detrick, Maryland and the University of California, Davis.
Under Subobjective 2.B.2, TaqMan real time polymerase chain reaction (PCR) assays were developed for Pythium and Aphanomyces using comparative genomics of the mitochondrial data for a systematic approach for designing genus- and species-specific markers. Comparative genomics identified unique sequences in downy mildew taxa that has facilitated design of TaqMan assays for five taxa thus far. We are currently working on P. philippinensis.
Under Subobjective 2.B.3, we provided sequence data for 34 mitochondrial genes that are being used for phylogenetic analysis of oomycete taxa. Of particular interest has been tropical downy mildews that infect corn, especially the select agent P. philippinensis, since there is taxonomic confusion over whether or not it and P. sacchari are members of the same species.
Under Subobjective 2.B.4, sequence data for four mitochondrial genes and the Phytophthora genus-specific amplicon were generated for 25 recently described taxa. Comparative genomics of the mitochondrial genomes identified gene orders that are unique for oomycetes (not present in Eumycotan fungi or plants) that have been useful for designing primers for metagenomic studies. The oomycete-specific primers are currently being evaluated with an ARS collaborator in Oregon. While these primers will provide a good overview of the taxa present, a detailed picture of the community structure of oomycetes should be attainable when they are used in conjunction with the genus-specific loci developed for Phytophthora, Pythium, and Aphanomyces in a hierarchical sampling.
Under Subobjective 2.C, three phylogenetic lineages of Fusarium oxysporum formae specialis fragariae have been identified. The TaqMan real time PCR assay for M. phaseolina has been validated against 51 isolates representing all subgroupings of genotypes and other fungi and found to be specific. Soil plating techniques were developed in conjunction with our University of California Cooperative Extension for quantification of M. phaseolina; soil plate counts and TaqMan assay results were highly correlated. The Taqman assay has sensitivity to 3 microsclerota per gram of soil. The assay for F. oxysporum f. sp. fragariae has been validated for specificity against a range of other F. oxysporum f. sp. and found to be specific.
Accomplishments
1. Identified genes required for pigment production in the fungus Verticillium dahliae. Melanin, a dark pigment, is thought to help long-term survival of the fungus Verticillium dahliae in soil in the absence of host plants. An ARS researcher in Salinas, California compared gene mutants for several of the pigment genes in V. dahliae with the wild type strain and found two that lacked pigmentation. These genes are required for pigment production under normal circumstances. Thus, understanding the genetic control of pigment production may reveal a genetic weakness in the pathogen that can be exploited for control of this fungus that causes devastating losses to a wide array of crops, including lettuce and strawberry.
2. Detection of the lettuce downy mildew pathogen for more judicious use of fungicides. Downy mildew of lettuce is a disease of economic importance in central coastal California, where the disease development is favored by the cool coastal climate. Two ARS researchers in Salinas, California, in collaboration with University of California personnel and commercial lettuce growers, deployed a DNA-based detection assay for the downy mildew pathogen of lettuce. The assay predicted disease risk based on spore load in the environment, and this information was used to reduce the number of fungicide applications. There was an average reduction of 1.7 fungicide applications for the three field experiments in this study.
3. Quantification of the emerging strawberry pathogen Macrophomina phaseolina. Macrophomina phaseolina is an emerging pathogen in California strawberry production with the transition from methyl bromide fumigation to alternative fumigants. An ARS researcher in Salinas, California, along with a University of California Cooperative Extension collaborator, developed an assay for measuring the amount of the pathogen in soil. A gene-based assay specific for strains of the pathogen that infect strawberry was also developed and validated for rapid assessment of pathogen density. These assays will assist growers in determining disease risk in specific fields, and will provide researchers a rapid and accurate means for evaluation of disease control in the development of improved disease management strategies.
