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

Location: Crop Improvement and Protection Research

2021 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
In support of Sub-objective 1B, a manuscript was published on the identification of genes that are differentially expressed between downy mildew resistant and susceptible spinach cultivars. The research also yielded insights on genes expressed in the downy mildew pathogen in the susceptible cultivar. Also in support of Sub-objective 1B, we developed and published a research manuscript on a spinach leaf assay that may be useful to assist in downy mildew disease resistance screening. Such a method would allow screening more varieties in a smaller space under a strict set of environmental conditions required for pathogen infection and colonization of spinach. In support of Sub-objective 1C, soil factors were examined that reduce survival of Verticillium dahliae. Soils were sampled for Verticillium dahliae survival by plating assays, revealing survival of microsclerotia in one sandy soil type, but not another clay type soil. In support of Sub-objective 2A, one more small field trial was conducted in which leaves were selected and tested for the presence of the pathogen, Peronospora effusa, and we are now in the final stages of writing the manuscript on an in-field diagnostic test for Peronospora effusa. In further support of Sub-objective 2A, we identified population genetic molecular markers for the spinach downy mildew pathogen, Peronospora effusa. Specifically, DNA sequences were further analyzed on all the leaf samples collected from years one through three and additional samples were collected in year four. Testing of the available sequences from different isolates of the pathogen from different locations revealed that some of these markers do not give adequate information on population variability, and therefore we have tested additional potential markers and narrowed the pool of available markers for usage. In support of Sub-objective 2B, over 800 mitochondrial DNA sequences representing 250 oomycete taxa have been assembled. These data have been used for developing a systematic approach for developing diagnostic markers for oomycete pathogens and providing genes for use in phylogenetic studies that differentiate these organisms. In further support of Sub-objective 2B, diagnostic markers were developed for seven species of downy mildews and a genus/species specific assay for Aphanomyces. Markers for four species have been published to date; the others are in later stages of validation. Diagnostic markers for Pythium based on gene order differences have been validated and the manuscript is in preparation. Also in support of Sub-objective 2B, researchers are still extracting data from the assembled mitochondrial genomes for an expanded analysis of oomycete phylogeny. A collaborator in Canada was lead author on a manuscript in the final stages of revision on a phylogenomics approach supporting the reclassification of the genus Pythium into four genera. Additionally in support of Sub-objective 2B, to improve identification of Phytophthora species, DNA sequences were deposited and maintained in the database, and work was done to add other oomycete taxa to the database.

Accomplishments
1. Spore trapping downy mildew pathogens at two different valleys in California. Downy mildews are major diseases of spinach and lettuce, and routine chemical sprays are required to prevent downy mildew damage. Accurate means of disease forecasting are necessary to reduce the numbers of pesticide sprays required, which are often administered on a weekly basis without knowing whether the pathogens are present or not. An ARS researcher in Salinas, California, in collaboration with the University of California, Riverside, deployed spore traps in the Salinas and Coachella Valleys of California and assessed airborne inoculum loads of downy mildews at different times of the year. The research identified different patterns of the accumulation of airborne inoculum which will be useful for improving the accuracy of disease forecasting models.

2. Identified a predicted pathogenicity chromosome in F. oxysporum f. sp. fragariae. Fusarium oxysporum f. sp. fragariae causes Fusarium wilt of strawberry, a disease that has become widespread in California. The genes controlling host specificity for this strawberry pathogen are unknown. Through comparative genomics and transcriptomics, ARS scientists in Salinas, California, identified a predicted pathogenicity chromosome that has been horizontally transferred at least four times. This information is a major step toward identifying genes that are required for virulence on strawberry, which will fundamentally improve understanding of this pathosystem and could lead to biotechnology-based strategies for disease control.

3. Assembled and analyzed the genomes of F. oxysporum f. sp. apii and F. oxysporum f. sp. coriandrii. Fusarium wilt diseases of celery and cilantro (caused by F. oxysporum f. sp. apii and F. oxysporum f. sp. coriandrii, respectively) have become increasingly common in California. ARS scientists in Salinas, California, determined that the strains of Fusarium oxysporum causing disease on these crops are very closely related and primarily differ in only approximately 5% of the total genome length. The high levels of relatedness presented an obstacle for differentiation of these pathogens with molecular tools. However, the scientists developed a multi-locus polymerase chain reaction (PCR) assay to distinguish these pathogens without recourse to a pathogenicity test. The celery and cilantro industry will benefit from methods that enable them to distinguish between these pathogens.

4. Green manure organic soil amendments alter activity of soil microbes and increase vegetable yield. Incorporation of crop residue into soil as green manures can improve soil and crop health. Microbes in soil play an important but poorly understood role in this process. An ARS researcher in Salinas, California, demonstrated that different kinds of green manures increase the activity of specific types of soil microbes while increasing or decreasing vegetable yield. This research improves our understanding of which soil microbes are responsible for the beneficial impacts of green manures and will help optimize this practice for conventional and organic agriculture.

5. Analyzed biocontrol agents for control of Verticillium wilt. The soilborne plant pathogenic fungus, Verticillium dahliae, causes billions of dollars of losses annually worldwide. Control of this fungus is difficult without the use of soil fumigation, and fumigation practices with synthetic chemicals are becoming increasingly less acceptable to the public. Therefore, alternative approaches are required to control this soilborne pathogen. An ARS researcher in Salinas, California, teamed up with the University of California, Davis, to investigate the use of two biological control organisms within the same genus as V. dahliae. The results revealed Verticillium klebahnii and V. isaacii isolates exhibit host specificity in biological control of Verticillium wilt caused by V. dahliae. The use of these two biological control agents may be useful to reduce the input of synthetic chemicals in the soil for plant disease control.

6. Genomic sequencing and analysis of members of the genus Pythium. The soilborne plant pathogenic genus, Pythium, is ubiquitous and causes pre- and post-emergence death of a wide range of economic crop plants, as well as reduced yield due to reduced host vigor. Because of overlapping morphological features, it is difficult to identify isolates to a species level. The taxonomy of the genus is in a state of confusion and the phylogenetic relationship among species lacks clarity. To address this, a researcher in Salinas, California, has collaborated with a Agriculture and Agri-Food Canada scientist to assemble genomic sequence data from every described species and conduct a broad-scale phylogenetic analysis of the genus to provide a comprehensive resource to researchers and regulatory personnel. This sequence resource will lead to simplified species identification, a comprehensive evaluation of taxonomic classifications and evolutionary relationship among species, and will support development of diagnostic markers.

7. Development of a new locus for identification of oomycetes. Identification of a group of plant pathogens referred to as oomycetes can be difficult due to overlapping morphological features and the inability to grow some taxa in culture. While the DNA sequence of the gene cox1 has been used for species identification, it has limitations when working with environmental samples. An ARS scientist in Salinas, California, found that oomycetes have a unique gene order a that has been useful for designing highly specific primers for amplification from a wide range of oomycete taxa with limited background amplification. Sequence differences in this region among taxa work well for identification purposes. Collaborations have been established with researchers in the United States, Canada, Austria, and Australia to develop a comprehensive sequence database publicly available on a project website and further validate the use of the locus as a means for species identification. The completion of this project will simplify identification of oomycete plant pathogens.

Review Publications
Henry, P.M., Kaur, S., Pham, Q.A.T., Barakat, R., Brinker, S., Haensel, H., Daugovish, O., Epstein, L. 2020. Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii. BMC Genomics. 21. https://doi.org/10.1186/s12864-020-07141-5.
Henry, P.M., Pincot, D.D.A, Jenner, B.N., Borrero, C., Aviles, M., Nam, M., Epstein, L., Knapp, S.J., Gordon, T.R. 2021. Horizontal chromosome transfer and independent evolution drive diversification in Fusarium oxysporum f. sp. fragariae. New Phytologist. 230(1):327-340. https://doi.org/10.1111/nph.17141.
Dhar, N., Chen, J.-Y., Subbarao, K.V., Klosterman, S.J. 2020. Hormone signaling and its interplay with development and defense responses in Verticillium-plant interactions. Frontiers in Plant Science. 11. Article 584997. https://doi.org/10.3389/fpls.2020.584997.
Chen, J.-Y., Zhang, D.-D., Huang, J.-Q., Wang, D., Hao, S.-J., Li, R., Puri, K.D., Yang, L., Tong, B.-Z., Xiong, K.-X., Simko, I., Klosterman, S.J., Subbarao, K.V., Dai, X.-F. 2020. Genome sequence of Verticillium dahliae race 1 isolate VdLs.16 from lettuce. Molecular Plant-Microbe Interactions. 33(11):1265-1269. https://doi.org/10.1094/MPMI-04-20-0103-A.
Liu, S., Zhang, X., Xiao, S., Ma, J., Shi, W., Qin, T., Xi, H., Nie, X., You, C., Xu, Z., Wang, T., Wang, Y., Zhang, Z., Li, J., Kong, J., Aierxi, A., Yu, Y., Lindsey, K., Klosterman, S.J., Zhang, X., Zhu, L. 2021. A single-nucleotide mutation in a glutamate receptor-like gene confers resistance to Fusarium wilt in Gossypium hirsutum. Advanced Science. 8(7). Article 2002723. https://doi.org/10.1002/advs.202002723.
Zhang, Y., Zhang, Y., Chen, J., Huang, J., Zhang, J., Liu, L., Wang, D., Zhao, J., Song, J., Li, R., Yang, L., Kong, Z., Klosterman, S.J., Subbarao, K.V., Dai, X., Zhang, D. 2021. Genome sequence data of MAT1-1 and MAT1-2 idiomorphs from Verticillium dahliae. Phytopathology. https://doi.org/10.1094/PHYTO-01-21-0012-A.
Puri, K.D., Hu, X., Gurung, S., Short, D.P., Sandoya, G.V., Schild, M., Zhang, Y., Zhao, J., Anchieta, A.G., Klosterman, S.J., Subbarao, K.V. 2021. Verticillium klebahnii and V. isaacii isolates exhibit host specificity in biological control of Verticillium wilt caused by V. dahliae. Phytofrontiers. https://doi.org/10.1094/PHYTOFR-01-21-0001-R.
Clark, K.J., Feng, C., Dhillon, B., Kandel, S.L., Poudel, B., Mou, B., Klosterman, S.J., Correll, J.C. 2020. Evaluation of spinach cultivars for downy mildew resistance in Yuma, AZ 2020. Plant Disease Management Reports. 14. Article V146.
Chen, J.-Y., Klosterman, S.J., Hu, X.-P., Dai, X.-F., Subbarao, K.V. 2021. Key insights and research prospects at the dawn of the population genomics era for Verticillium dahliae. Annual Review of Phytopathology. 59. https://doi.org/10.1146/annurev-phyto-020620-121925.
Xiao, S., Hu, Q., Shen, J., Liu, S., Yang, Z., Chen, K., Klosterman, S.J., Javornik, B., Zhang, X., Zhu, L. 2021. GhMYB4 downregulates lignin biosynthesis and enhances cotton resistance to Verticillium dahliae. Plant Cell Reports. 40:735-751. https://doi.org/10.1007/s00299-021-02672-x.
Pincot, D.D., Hardigan, M.A., Cole, G.S., Famula, R.A., Henry, P.M., Gordon, T.R., Knapp, S.J. 2020. Accuracy of genomic selection and long-term genetic gain for resistance to Verticillium wilt in strawberry. The Plant Genome. 13(3). Article e20054. https://doi.org/10.1002/tpg2.20054.
Gordon, T.R., Henry, P.M., Jenner, B.N., Scott, J.C. 2021. Spontaneous changes in somatic compatibility in Fusarium circinatum. Fungal Biology. 12(9):725-732. https://doi.org/10.1016/j.funbio.2021.04.008.
LeBlanc, N.R., Cubeta, M., Crouch, J.A. 2020. Population genomics trace clonal diversification and intercontinental migration of an emerging fungal pathogen of boxwood. Phytopathology. 111:184-193. https://doi.org/10.1094/PHYTO-06-20-0219-FI.
Yang, X., Mcmahon, M.B., Ramachandran, S.R., Garrett, W.M., Leblanc, N.R., Crouch, J., Shishkoff, N., Luster, D.G. 2021. Comparative analysis of extracellular proteomes reveals effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata. Bioscience Reports. 41(3):BSR20203544. https://doi.org/10.1042/BSR20203544.
Castroagudín, V., Weiland, J.E., Baysal-Gurel, F., Cubeta, M., Daughtrey, M., Gauthier, N., Lamondia, J., Luster, D.G., Hand, F., Shishkoff, N., Williams-Woodward, J., LeBlanc, N., Yang, X., Crouch, J.A. 2020. One clonal lineage of Calonectria pseudonaviculata is primarily responsible for the boxwood blight epidemic in the United States. Phytopathology. 110(11):1845-1853. https://doi.org/10.1094/PHYTO-04-20-0130-R.
Crandall, S.G., Ramon, M.L., Burkhardt, A.K., Bello, J.C., Adair, N., Gent, D.H., Hausbeck, M.K., Quesada-Ocampo, L.M., Martin, F.N. 2021. A multiplex TaqMan qPCR assay for detection and quantification of clade 1 and clade 2 isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli. Plant Disease. https://doi.org/10.1094/PDIS-11-20-2339-RE.