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

Location: Crop Improvement and Protection Research

2020 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 Sub-objective 1A, deletion of a third candidate gene involved in early stages of root infection was examined for pathogenicity on lettuce. The strain requires further evaluation. We initiated the writing of a manuscript. Sub-objective 1B1 is to identify genes that are differentially expressed between downy mildew-resistant and susceptible cultivars. A manuscript on genes that are differentially expressed between downy mildew-resistant and susceptible cultivars subject was completed and published. Sub-objective 1B2 is to develop a spinach leaf assay to assist in the development of resistance screens. The spinach downy mildew pathogen was inoculated on spinach leaves in conjunction with a University of Arkansas collaborator for examination of disease symptoms. A manuscript was written on the subject, indicating that leaf assay shows promise for more quickly screening downy mildew resistance with less space requirements. Sub-objective 1C2 is to identify soil abiotic factors that reduce survival of Verticillium dahliae. Readings were taken of the survival rate of V. dahliae after a second year in soil. Sub-objective 1C4 is to analyze pigment cluster genes of V. dahliae that contribute to long term survival. We had previously completed work on melanin biosynthesis gene cluster deletion mutants for analyses of differences from the wild type strain, and in this period wrote a separate manuscript on the mechanisms associated with survival of V. dahliae in response to ultraviolet (UV) radiation, and published the findings. In Sub-objective 1D, pathogenicity tests identified pathogenic isolates. Preliminary trials with different levels of pathogen inoculum identified different virulence levels among isolates. In Sub-objective 1E, high quality reference genomes for a genotype virulent on strawberry and a genotype virulent on alfalfa have been assembled and annotated and a manuscript is in review. Genes present in the genotype virulent on strawberry, but absent in other genotypes, were identified. Additional efforts at identifying specific genes are underway. Sub-objective 2A1 is to develop an in-field diagnostic test for Peronospora effusa. Researchers conducted additional tests with samples collected from the field using a P. effusa-specific marker and we are now in the final stages of assay development and testing. Sub-objective 2A2 is to identify population genetic molecular markers for the spinach downy mildew pathogen, Peronospora effusa. Previously identified markers were tested on pathogen populations derived from field isolates. The markers developed to study how the population genetics of the spinach downy mildew pathogen is changing over time were used in continued analyses to assess their utility. Sub-objective 2B1 is related to the mitochondrial genomics project. Over 750 mitochondrial genomes representing 200 oomycete taxa have been assembled. These data have been used for developing a systematic approach for developing diagnostic markers for oomycetes and providing genes for use in phylogenetic studies. Sub-objective 2B2 is related to molecular diagnostics. Diagnostic markers were developed for five species of downy mildews and a genus/species specific assay for Aphanomyces. Markers for one 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. Sub-objective 2B3 relates to oomycete phylogenetics. Researchers are still extracting data from the assembled mitochondrial genomes for an expanded analysis of oomycete phylogeny. Sub-objective 2B4 relates to improved identification of Phytophthora species. Two mitochondrial loci work well for identification of Phytophthora, the spacer between atp9-nad9 (sequence database represents 146 species) and rps10. The rps10 locus has the advantage in that the conserved primers are in tRNA flanking the gene, a gene order that is found only in oomycetes. This locus has been developed as a new barcode marker for species identification and as a locus for metagenomics studies of oomycete communities. In Sub-objective 2C, researchers published a manuscript that describes a highly specific TaqMan assay for the genotype of Macrophomina phaseolina that is virulent on strawberry and its use for soil quantification. A TaqMan assay for Fusarium oxysporum f. sp. fragariae was developed and published. The marker works as a soil quantification assay but not to the level of sensitivity needed; additional work is in progress to improve DNA extraction from the soil in order to provide the sensitivity desired.

Accomplishments
1. Differentially expressed genes in downy mildew-resistant and susceptible spinach cultivars. Downy mildew is the main disease affecting spinach worldwide. The disease is particularly problematic in the organic market because there are no effective controls available for organic spinach, other than disease resistant plants. The downy mildew pathogen overcomes resistance very quickly, and thus additional understanding of spinach defense responses to this pathogen are required to more quickly produce disease resistant cultivars. A researcher in Salinas, California, led a collaborative project with the University of California, Davis, to sequence the expressed genes in spinach during resistant and susceptible interactions with the pathogen. The research provides insight on the defense mechanisms deployed by spinach to ward off the downy mildew pathogen. This can be useful to more quickly develop disease resistance by the selection of these markers in spinach populations.

2. Discovery of mechanisms utilized by a plant pathogenic fungus for protection against high temperature and ultraviolet (UV) light stress. The plant fungus Verticilium dahliae causes billions of dollars of annual losses on over 200 species of crops and ornamental plants worldwide. The fungus can survive for years in the field and infects a broad range of plant species, making this pathogen particularly difficult to control once it infests soil. An increased understanding of the mechanisms used by the pathogen for long term survival in the field may be helpful in efforts to eradicate this fungus in soil for disease control. A researcher in Salinas, California, participated in collaborative research with the Beijing Forestry University, China, to identify genes and their protein products in this fungus that are differentially expressed in response to UV light and high temperature stress. The results reveal insights into the biochemical and molecular mechanisms used by the fungus to cope with high temperature and UV stress. If these coping strategies can be inhibited, the findings from this study may be exploited to reduce the survival of the fungus in the field.

3. A new genomic resource for investigating lettuce isolates of a Verticillium wilt pathogen. Verticillium wilt disease on lettuce results in yield losses every year in organic and conventionally produced lettuce in the United States and worldwide. Obstacles to controlling the Verticillium wilt pathogen on lettuce include the ability of the pathogen to survive for long periods in the soil, attack different plant species and its ability overcome host plant resistance. Moreover, the genetic basis for rapid changes in the pathogen are not understood. A researcher in Salinas, California, participated in a collaborative study that sequenced the whole genome of an isolate of one race of this pathogen, and publicly released this information. This sequence resource will enable public and private researchers to determine the underlying genetic basis that enables the Verticillium wilt pathogen to attack so many different plant species, and to overcome host plant resistance. This resource also provides a means of identifying the pathogen variants and thereby enabling more rapid testing of spinach germplasm for resistance to the pathogenic variants.

4. Avirulence gene involved in race-specific resistance to Fusarium wilt of strawberry identified. Fusarium wilt of strawberry is an emerging soilborne disease in the post-methyl bromide era. The only known Fusarium wilt resistance gene (designated FW1) deployed in commercially available strawberry cultivars in California works when the corresponding avirulence gene is expressed by the pathogen. Researchers in Salinas, California, used population genomics to identify a candidate for the pathogen’s avirulence gene. This gene was knocked out by Agrobacterium-mediated transformation and multiple, independent knockout mutants gained virulence on FW1 cultivars. This information has motivated breeding companies to increase efforts to introgress additional sources of resistance into commercially available strawberry cultivars.

Review Publications
Burkhardt, A.K., Childs, K.L., Wang, J., Ramon, M.L., Martin, F.N. 2019. Assembly, annotation, and comparison of Macrophomina phaseolina isolates from strawberry and other hosts. BMC Genomics. 20:802. https://doi.org/10.1186/s12864-019-6168-1.
Dhar, N., Caruana, J., Erdem, I., Subbarao, K., Klosterman, S.J., Raina, R. 2020. The Arabidopsis senescence associated gene 13 regulates dark-induced senescence and plays contrasting roles in the defense against bacterial and fungal pathogens. Molecular Plant-Microbe Interactions. 33(5):754-766. https://doi.org/10.1094/MPMI-11-19-0329-R.
Tang, C., Li, T., Klosterman, S.J., Tian, C., Wang, Y. 2020. The bZIP transcription factor VdAtf1 regulates virulence by mediating nitrogen metabolism in Verticillium dahliae. New Phytologist. 226(5):1461-1479. https://doi.org/10.1111/nph.16481.
Kandel, S.L., Hulse-Kemp, A.M., Stoffel, K., Koike, S.T., Shi, A., Mou, B., Van Deynze, A., Klosterman, S.J. 2020. Transcriptional analyses of differential cultivars during resistant and susceptible interactions with Peronospora effusa, the causal agent of spinach downy mildew. Scientific Reports. 10. Article 6719. https://doi.org/10.1038/s41598-020-63668-3.
Li, J.-J., Zhou, L., Yin, C.-M., Zhang, D.-D., Klosterman, S.J., Wang, B.-L., Song, J., Wang, D., Hu, X.-P., Subbarao, K.V., Chen, J.-Y., Dai, X.-F. 2019. The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection. Environmental Microbiology. 21(12):4852-4874. https://doi.org/10.1111/1462-2920.14846.
Li, H., Dai, J., Qin, J., Shang, W., Chen, J., Zhang, L., Dai, X., Klosterman, S.J., Xu, X., Subbarao, K.V., Fan, S., Hu, X. 2020. Genome sequences of Verticillium dahliae defoliating strain XJ592 and nondefoliating strain XJ511. Molecular Plant-Microbe Interactions. 33(4):565-568. https://doi.org/10.1094/MPMI-11-19-0320-A.
McCoy, A.G., Miles, T.D., Bilodeau, G., Woods, P., Blomquist, C., Martin, F.N., Chilvers, M.I. 2020. Validation of a preformulated, field deployable, recombinase polymerase amplification assay for Phytophthora species. Plants. 9(4):466. https://doi.org/10.3390/plants9040466.
Wang, D., Tian, L., Zhang, D.-D., Song, J., Song, S.-S., Yin, C.-M., Zhou, L., Liu, Y., Wang, B.-L., Kong, Z.-Q., Klosterman, S.J., Li, J.-J., Wang, J., Li, T.-G., Adamu, S., Subbarao, K.V., Chen, J.-Y., Dai, X.-F. 2020. Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae. Molecular Plant Pathology. 21(5):667–685. https://doi.org/10.1111/mpp.12921.
Dhillon, B., Villarroel-Zeballos, M.I., Castroagudin, V.L., Bhattarai, G., Klosterman, S.J., Correll, J.C. 2020. Sporangiospore viability and oospore production in the spinach downy mildew pathogen, Peronospora effusa. Plant Disease. https://doi.org/10.1094/PDIS-02-20-0334-RE.
Dhar, N., Mamo, B.E., Subbarao, K.V., Koike, S.T., Fox, A., Anchieta, A.G., Klosterman, S.J. 2019. Measurements of aerial spore load by qPCR facilitates lettuce downy mildew risk advisement. Plant Disease. 104(1):82-93. https://doi.org/10.1094/PDIS-03-19-0441-RE.
Yu, J., Li, T., Tian, L., Tang, C., Klosterman, S.J., Tian, C., Wang, Y. 2019. Two Verticillium dahliae MAPKKKs, VdSsk2 and VdSte11, have distinct roles in pathogenicity, microsclerotial formation, and stress adaptation. mSphere. 4(4). https://doi.org/10.1128/mSphere.00426-19.
Fang, Y., Klosterman, S.J., Tian, C., Wang, Y. 2019. Insights into VdCmr1-mediated protection against high temperature stress and UV irradiation in Verticillium dahliae. Environmental Microbiology. 21(8):2977-2996. https://doi.org/10.1111/1462-2920.14695.
Kandel, S.L., Subbarao, K.V., Shi, A., Mou, B., Klosterman, S.J. 2019. Evaluation of biopesticides for managing downy mildew of spinach in organic production systems in 2017 and 2018. Plant Disease Management Reports. 13:V171.
Stravoradis, S., Leblanc, N., Marra, R.E., Crouch, J., Hulvey, J.P. 2019. Widespread occurrence of a CYP51A pseudogene in Calonectria pseudonaviculata. Mycobiology. 48(1):44-50. https://doi.org/10.1080/12298093.2019.1689600.
Salgado-Salazar, C., Leblanc, N., Wallace, E., Daughtery, M., Crouch, J. 2020. Peronospora bergamotii, Hyaloperonospora daughtreyae and H. iberidis: new species associated with downy mildew diseases affecting ornamental plants in the United States. European Journal of Plant Pathology. 157(1):311-326. https://doi.org/10.1007/s10658-020-01989-9.
Henry, P.M., Haugland, M., Lopez, L., Munji, M., Watson, D.C., Gordon, T.R. 2020. The potential for Fusarium oxysporum f. sp. fragariae, cause of Fusarium wilt of strawberry, to colonize organic matter in soil and persist through anaerobic soil disinfestation. Plant Pathology. https://doi.org/10.1111/ppa.13225.