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

Location: Crop Improvement and Protection Research

2019 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 1.A., deletion of a second candidate gene involved in early stages of root infection was examined for pathogenicity on lettuce. The strain appeared to be less virulent, although this requires further evaluation in another experiment. Sub-objective 1.B.1. is to identify genes that are differentially expressed between downy mildew-resistant and susceptible cultivars. New spinach sequence resources were released by a university collaborator for sharing with the USDA. Analyses of gene sets that are significantly differentially expressed between resistant and susceptible infected spinach cultivars were completed using the newly available and better-quality annotated spinach genome sequence. A manuscript on the subject is now in preparation. Sub-objective 1.B.2. is to develop a spinach leaf assay to assist in the development of resistance screens. The spinach downy mildew pathogen was inoculated on spinach seedlings for examination of downy mildew development under controlled conditions in four experiments. We now have the protocols developed to do this routinely on intact seedlings and will attempt a detached leaf assay. Sub-objective 1.C.2. is to identify soil abiotic factors that reduce survival of Verticillium dahliae. Readings were taken of the survival rate of V. dahliae after a year in soil. Sub-objective 1.C.4. is to analyze pigment cluster genes of V. dahliae that contribute to long term survival. We completed work on melanin biosynthesis gene cluster deletion mutants for analyses of differences from the wild type strain, wrote a manuscript on this subject, and published the findings. In Sub-objective 1.D., pathogenicity tests identified pathogenic isolates; preliminary trials with different levels of pathogen inoculum identified different virulence levels among isolates. In Sub-objective 1.E., 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 2.A.1. is to develop in-field diagnostic test for Peronospora effusa. Researchers conducted additional tests to determine pathogen detection specificity of an in-field detection system that will help spinach growers to more quickly make disease management decisions. This system with the specific marker is now deemed appropriate for the next step of final assay development and testing. Sub-objective 2.A.2. 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 in order to study how the population genetics of the spinach downy mildew pathogen is changing over time. The markers are yielding the appropriate size fragments for continued analyses. Sub-objective 2.B.1. is related to the mitochondrial genomics project. Over 550 mitochondrial genomes representing 150 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 2.B.2. is related to molecular diagnostics. Diagnostic markers were developed for five species of downy mildews. 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 2.B.3. relates to oomycete phylogenetics. Researchers are still extracting data from the assembled mitochondrial genomes for an expanded analysis of oomycete phylogeny. Sub-objective 2.B.4. 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 is under development as a new barcode marker for species identification and as a locus for metagenomics studies of oomycete communities. In Sub-objective 2.C., researchers published a manuscript that describes a highly specific TaqMan assay for the genotype of M. phaseolina that is virulent on strawberry and its use for soil quantification. A TaqMan assay for F. 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. Identification of a compound from a biocontrol organism affects survival of a soilborne plant pathogenic fungus. Verticillium (V.) dahliae is a fungus that causes plant diseases known commonly as Verticillium wilts, many of which are economically important in the U.S. and abroad. The long-term survival of this fungus in the soil often means that rotating to non-host crops or other plants does not curtail Verticillium wilt disease development in subsequent crops. A researcher in Salinas, California, working as part of a collaborative study with a scientist from China, revealed the mechanism by which a natural compound involved may limit the survival of V. dahliae in soil. Knowledge on the types of compounds may be useful to devise organic, environmentally friendly strategies to inhibit the fungus in the field.

2. A major plant defense-related gene also regulates flower development. Verticillium wilt disease causes extensive damage and yield losses in the U.S. in high value crops such as strawberry. This wilt disease is caused by the plant pathogenic fungus, Verticillium dahliae, and effective control for this pathogen is sometimes achieved by the use of cultivars resistant to this pathogen. A researcher in Salinas, California, led a study demonstrating that a gene (NDR1) required for disease resistance to the fungus that causes Verticillium wilt is also required to suppress early flowering in the model plant, Arabidopsis thaliana. When the gene is mutated, the Arabidopsis plants flower early and are very susceptible to the Verticillium wilt pathogen, and the early flowering is heavily dependent on the input or blockage of the plant hormone, gibberellic acid. The fungal pathogen and gibberellic acid independently stimulated earlier flowering in lettuce. This study emphasized the importance of understanding gene functions and regulatory activities of any gene product before targeting them as candidates for crop improvement.

3. Provided genomic sequence resources for the downy mildew pathogen of spinach. Downy mildew disease on spinach results in tremendous yield losses every year in organic spinach in the U.S. and worldwide. One of the major obstacles to controlling the downy mildew pathogen on spinach is that this pathogen quickly overcomes host plant resistance that has been incorporated in lengthy breeding programs. Secondly, the genetic basis for rapid changes in the pathogen are not understood. Researchers in Salinas, California, participated in a collaborative study to sequence the whole genomes of two variants of this pathogen, and to publicly release this information. These sequence resources enable public and private researchers to determine the underlying genetic basis that enables the spinach downy mildew pathogen to quickly overcome host plant resistance. This will provide a rapid, precise means of identifying the pathogen variants and thereby enabling more rapid testing of spinach germplasm for resistance to the newly arising pathogenic variants.

4. Characterized elements of the disease cycle of the spinach downy mildew pathogen. Peronospora effusa is a microscopic, plant pathogenic organism that causes downy mildew disease on spinach. The disease can result in 100% yield losses in organic fields. Researchers in Salinas, California, led a collaborative study to uncover critical aspects of the lifecycle of the spinach downy mildew pathogen. In addition to its established role as a foliar pathogen, the research revealed that the pathogen can penetrate spinach roots, and also resides in high numbers underneath the thick seed coat in some commercial spinach seeds. Uncovering the different phases of the spinach downy mildew pathogen disease cycle may be exploited for disease control by eliminating the pathogen at key points in the cycle, such as within seed.

5. New diagnostic tests for Macrophomina root and crown rot, and Fusarium wilt of strawberry. Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae are emerging pathogens in California strawberry production with the loss of methyl bromide for soil fumigation. An ARS researcher in Salinas, California, and collaborators at University of California, developed two different types of molecular diagnostic tests specific for the Macrophomina and Fusarium types capable of infecting strawberry that measure the amounts of each fungus in soil, as well as providing a means for rapid detection directly in the field. Isolates of the fungus that are highly infectious on strawberry are, with few exceptions, genetically identical. These assays provide diagnosticians and researchers with the tools they need to identify the pathogen and growers with the ability to determine risk prior to planting.

6. Assembly of the nuclear genome of Macrophomina phaseolina. Macrophomina phaseolina is an emerging pathogen in California strawberry production with the loss of methyl bromide for soil fumigation. ARS researchers in Salinas, California, and collaborators at Michigan State University, assembled and annotated high quality nuclear genomes for an isolate that attacks strawberry, and another from alfalfa, in order to provide an improved genetic resource for research on the genetics of host-pathogen interactions. Comparative analysis of the two respective genomes will provide information about host-specific differences for infection between these two strawberry and alfalfa isolates of the fungus. These assemblies provide researchers a valuable resource for understanding genome evolution in this clonally reproducing pathogen and help identify genes associated with pathogenicity on crop plants.

Review Publications
Fletcher, K., Klosterman, S.J., Derevnina, L., Martin, F.N., Bertier, L.D., Koike, S.T., Chin-Wo-Reyes, S., Mou, B., Michelmore, R. 2018. Comparative genomics of downy mildews reveals potential adaptations to biotrophy. BMC Genomics. 19:851. https://doi.org/10.1186/s12864-018-5214-8.
Zhang, D., Wang, J., Wang, D., Kong, Z., Zhou, L., Zhang, G., Gui, Y., Li, J., Huang, J., Wang, B., Liu, C., Yin, C., Li, R., Li, T., Wang, J., Short, D.P., Klosterman, S.J., Bostock, R.M., Subbarao, K.V., Chen, J., Dai, X. 2019. Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae. New Phytologist. 222(2):1012-1029. https://doi.org/10.1111/nph.15672.
Dhar, N., Short, D.P., Mamo, B.E., Corrion, A.J., Wai, C.M., Anchieta, A.G., Van Buren, R., Day, B., Ajwa, H., Subbarao, K.V., Klosterman, S.J. 2019. Arabidopsis defense mutant ndr1-1 displays accelerated development and early flowering mediated by the hormone gibberellic acid. Plant Science. 285:200-213. https://doi.org/10.1016/j.plantsci.2019.04.006.
Yu, D., Fang, Y., Tang, C., Klosterman, S.J., Tian, C., Wang, Y. 2018. Genome-wide transcriptome profiles reveal how Bacillus subtilis lipopeptides inhibit microsclerotia formation in Verticillium dahliae. Molecular Plant-Microbe Interactions. 32(5):622-634. https://doi.org/10.1094/MPMI-08-18-0233-R.
Kandel, S.L., Mou, B., Shishkoff, N., Shi, A., Subbarao, K.V., Klosterman, S.J. 2019. Spinach downy mildew: Advances in our understanding of the disease cycle and prospects for disease management. Plant Disease. 103:791-803. https://doi.org/10.1094/PDIS-10-18-1720-FE.
Li, T., Wang, B., Yin, C., Zhang, D., Wang, D., Song, J., Zhou, L., Kong, Z., Klosterman, S.J., Li, J., Adamu, S., Liu, T., Subbarao, K.V., Chen, J., Dai, X. 2019. The Gossypium hirsutum TIR-NBS-LRR gene GhDSC1 mediates resistance against Verticillium wilt. Molecular Plant Pathology. 20(6):857-876. https://doi.org/10.1111/mpp.12797.
Wang, Y., Deng, C., Tian, L., Xiong, D., Tian, C., Klosterman, S.J. 2018. The transcription factor VdHapX controls iron homeostasis and is crucial for virulence in the vascular pathogen Verticillium dahliae. mSphere. 3(5):e00400-18. https://doi.org/10.1128/mSphere.00400-18.
Li, T.-G., Zhang, D.-D., Zhou, L., Kong, Z.-Q., Hussaini, A.S., Wang, D., Li, J.-J., Short, D.P.G., Dhar, N., Klosterman, S.J., Wang, B.L., Yin, C.-M., Subbarao, K.V., Chen, J.-Y., Dai, X.-F. 2018. Genome-wide identification and functional analyses of the CRK gene family reveals that GbCRK18 confers Verticillium wilt resistance in Gossypium barbadense. Frontiers in Plant Science. 9:1266. https://doi.org/10.3389/fpls.2018.01266.
Martin, F.N., Zhang, Y., Cooke, D.E.L., Coffey, M.D., Grunwald, N.J., Fry, W.E. 2019. Insights into evolving global populations of Phytophthora infestans via new complementary mtDNA haplotype markers and nuclear SSRs. PLoS One. 14(1):e0208606. https://doi.org/10.1371/journal.pone.0208606.
Burkhardt, A.K., Koike, S.T., Henry, P.M., Gordon, T.R., Martin, F.N. 2019. Detection of Fusarium oxysporum f. sp. fragariae from infected strawberry plants. Plant Disease. 103:1006-1013. https://doi.org/10.1094/PDIS-08-18-1315-RE.
Munawar, M., Toljamo, A., Martin, F.N., Kokko, H. 2019. Recombinase Polymerase Amplification Assay for fast, sensitive and on-site detection of Phytophthora cactorum without DNA extraction. European Journal of Horticultural Science. 84:14-19. https://doi.org/10.17660/eJHS.2019/84.1.2.