Research Project: Management of Pathogens for Strawberry and Vegetable Production Systems

Location: Crop Improvement and Protection Research

Project Number: 2038-22000-016-000-D
Project Type: In-House Appropriated

Start Date: Mar 30, 2017
End Date: Mar 29, 2022

Objective:
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.