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

Location: Crop Improvement and Protection Research

2022 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 is the final report for the project 2038-22000-016-000D, Management of Pathogens for Strawberry and Vegetable Production Systems, which terminated in March 2022. This has been replaced by the new project 2038-22000-019-000D, Disease Management and Improved Detection Systems for Control of Pathogens of Vegetables and Strawberries. Substantial results were realized over the five years of project 2038-22000-016-000D. For Objective 1, the performance of cultural and biological methods, management practices, and genetic approaches for management of pathogens, including those mediated by soil fumigation, were optimized for delivery and evaluated. Two biological control organisms within the same genus as Verticillium dahliae were investigated, revealing that Verticillium klebahnii and Verticillium isaacii isolates exhibit host specificity in biological control of Verticillium wilt caused by the soilborne fungus Verticillium dahliae. Genes of Verticillium dahliae were examined to determine their involvement in lettuce root infection. Though none of the candidate genes identified were important for virulence on lettuce, one candidate gene was identified as important for pathogenicity in another system, on cotton. Additional genomic resources were published for Verticiillium dahliae that are useful in assessing race-specific differences between isolates of this species. A publication was also produced describing a key regulator of melanin biosynthetic genes in V. dahliae which is also required for protection against ultraviolet (UV) light. Pigment is thought to be beneficial for long term survival, and thus understanding its production may reveal a weakness in the pathogen. Using new spinach genome data, a publication was produced describing gene sets significantly differentially expressed between resistant and susceptible infected spinach cultivars that were identified, and which provide insight into how spinach defends against downy mildew. These data are therefore useful to plant breeders for developing resistance. Likewise, publication of a spinach leaf assay during this reporting period assists in the development of resistance screening methods for downy mildew. Deployment of spore traps in the Salinas and Coachella Valleys of California were used to assess 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. A chromosome that influences pathogenicity in the strawberry pathogen that causes wilt of strawberry, Fusarium oxysporum f. sp. fragariae, was identified, and is useful for diagnostics and in plant breeding research for disease resistance. Additional work demonstrated that different kinds of green manures increase the activity of specific types of soil microbes while altering 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. Substantial progress was also made on Objective 2, to develop rapid and accurate molecular diagnostic tools for the identification of emerging diseases of strawberries and vegetables, and to use these tools in the development of disease management strategies. Mitochondrial genomes for over 800 isolates were assembled and many of these were annotated. These include genomes from the Select Agent Peronosclerospora philippinensis and other tropical downy mildews using DNA sequence data provided by collaborators. The genomic sequence data from every described species of soilborne Pythium was prepared, and phylogenetic analysis was conducted, providing a comprehensive resource to researchers and regulatory personnel alike. The resource is especially important since soilborne plant pathogens of genus Pythium are ubiquitous and cause pre- and post-emergence damping off disease on a wide range of economic crop plants as well as reduced yield due to reduced host vigor. Collaborations were established with researchers in Canada, Austria, and Australia to develop a comprehensive sequence database publicly available on all related species and for testing a diagnostic DNA target as a universal means of distinguishing between species. The completion of this project will simplify identification of these types of plant pathogens. A multiplexed molecular marker-based system was developed for detecting and measuring the amounts of three downy mildew pathogens on cucurbits and hops. These markers were shared with collaborators and provide a means for rapid identification of the pathogen and improved management of fungicide spray programs for enhanced disease control. Sensitive soil detection assays were also developed for both soilborne pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae that cause charcoal rot and wilt of strawberry, respectively. The structure and function of spinach seed microbiomes were characterized in commercial spinach seed lots originating from Europe and New Zealand that were infested or not infested with two different pathogens that affect spinach. Assays were developed and published to detect DNA of the pathogen Peronospora effusa in the latent period in spinach leaves. Early detection of the pathogen in the leaves in the field will help to target fungicide applications more effectively prior to symptom development or to harvest organic crops earlier, and thereby help to prevent downy mildew epidemics. Overall, this project evaluated performance of cultural and biological methods, and genetic approaches for management of pathogens, including those mediated by soil fumigation, and new diagnostic tools and approaches were developed. These tools and approaches will streamline usage of fungicides for both soilborne and airborne pathogens and optimize delivery of control measures.

Accomplishments
1. New diagnostic method to detect a spinach pathogen in leaves. Downy mildew disease of spinach, caused by the plant pathogenic microorganism Peronospora effusa, is the major disease constraint on spinach in the United States and worldwide. ARS researchers in Salinas, California, led the effort to develop a species-specific detection system that can be used in the field. The system is DNA-based and can be deployed from a pickup truck, detecting the pathogen within three to four hours. This new early detection technology will help growers to target fungicide applications more effectively prior to symptom development, and thereby help to prevent downy mildew epidemics. Since nearly 45% of the U.S. spinach is organic, the technology is especially helpful to organic growers who may choose to harvest organic crops earlier, avoiding symptom development that renders their product unmarketable.

2. Characterization of microbiomes in commercial spinach seeds. The worldwide distribution of plant seeds can disseminate beneficial and plant-pathogenic microorganisms. This phenomenon is of particular concern where seed production is geographically isolated from crop production, as is the case with spinach in the United States. ARS researchers in Salinas, California, characterized the structure and function of microbiomes in commercial spinach seed lots originating from Europe and New Zealand that were infested or noninfested with two different pathogens that affect spinach. The findings revealed differences in the composition of microbiomes depending on both geographic origin and whether the seeds were infested or noninfested. These results indicate the extent of movement of beneficial and plant-pathogenic microorganisms on commercial spinach seeds and has implications for disease control. Spinach growers in the United States and worldwide may use the findings to predict health of seeds.

3. Identification of beneficial bacteria as primary components of strawberry root microbiomes. Beneficial Pseudomonas bacteria can improve crop health by increasing plant growth and resistance to soilborne plant diseases. An ARS researcher in Salinas, California, demonstrated that Pseudomonas spp. are among the most common bacteria that live inside strawberry plant roots. This study found multiple Pseudomonas spp. can colonize the same strawberry plant and that these bacteria display higher abundance in strawberry roots compared to above ground tissue. These results demonstrate strawberry plants are naturally colonized by bacteria that have potential to improve strawberry growth and resistance to soilborne disease. These findings are useful to strawberry growers to ward off certain types of plant pathogens without the use of pesticides.

4. Characterization of bacterial communities in soil of commercial strawberry production systems. Bacterial communities in soil play multiple ecological roles that have positive and negative impacts on crop health. An ARS researcher in Salinas, California, characterized the diversity and composition of bacterial communities in soil of commercial strawberry production systems at two locations in the Salinas Valley, California. Results of this study demonstrated bacterial communities were influenced by spatial location of soil samples within a field and soil pH at one of the two locations. Outcomes of this research indicate factors influencing bacterial communities in commercial strawberry production systems are inconsistent among different locations. Future efforts to manage soil microbiomes to improve strawberry health at a new location will require prior characterization of bacterial communities in soil. These findings can be used by strawberry growers to predict health of soils.

5. 1,3-dichloropropene significantly contributes to soil disinfestation of Macrophomina phaseolina. The soil fumigant 1,3-dichloropropene was historically considered a nematicide with poor fungicidal properties. An ARS scientist in Salinas, California, discovered that fumigant mixtures containing 1,3-dichloropropene and chloropicrin were significantly better than chloropicrin alone at disinfesting Macrophomina phaseolina from soil. The fungal pathogen M. phaseolina causes significant yield losses in strawberry production each year, and these results will help growers to maintain plant health in the absence of methyl bromide, an ozone depleting substance that was phased out of use by the Montreal Protocol.

6. Evaluation of genomic sequences in Pythium supports splitting the genus into 4 different genera. 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 evolutionary relationship among species lacks clarity. To address this, an ARS researcher in Salinas, California, collaborated with an Agriculture and Agri-Food Canada scientist on a broad-scale phylogenomic 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, support development of diagnostic markers and has supported dividing Pythium into four distinct genera.

7. Improved DNA extraction from soil for enhanced disease risk assessment. The ability to quantify soilborne fungal plant pathogens in a field prior to planting would help growers assess risk and make management decisions to reduce losses. However, for many types of fungi, techniques to facilitate this are not available. An ARS scientist in Salinas, California, has developed molecular assays capable of quantifying several of the important pathogens of strawberry and vegetable crops often grown in rotation, as well as an improved soil DNA extraction procedure that results in purified DNA from larger amounts of soil than previously used. These accomplishments have improved the ability to accurately quantify lower levels of the pathogens and thereby provide more accurate data to growers.

8. A complete genome resource of the spinach downy mildew pathogen. Genome assemblies are important resources for determining the molecular basis for changes in virulence resulting in the emergence of new pathotypes and to provide informative loci for population studies. Peronospora effusa is the major spinach pathogen in the United States and worldwide. Previous genomic investigations have yielded fragmented and repeat-sparse genome assemblies of several isolates of this pathogen. An ARS scientist in Salinas, California, participated in work that provided a 17-chromosome assembly for P. effusa that was generated using PacBio high fidelity reads. Sixteen telomere-to-telomere gapless contigs and one telomere-to-telomere scaffold were assembled. Due to its completeness, the sequence assembly is a landmark for oomycete genomics and will be valuable for comparative genomics studies dissecting the genetic basis of virulence in this pathogen and other related pathogens.

Review Publications
Kandel, S.L., Anchieta, A.G., Shi, A., Mou, B., Klosterman, S.J. 2022. Crustacean meal elicits expression of growth and defense-related genes in roots of lettuce and tomato. Phytofrontiers. 2(1):10-20. https://doi.org/10.1094/PHYTOFR-03-21-0017-R.
Kandel, S.L., Henry, P.M., Goldman, P.H., Mou, B., Klosterman, S.J. 2022. Composition of the microbiomes from spinach seeds infested or noninfested with Peronospora effusa or Verticillium dahliae. Phytobiomes Journal. 6(2):169-180. https://doi.org/10.1094/PBIOMES-05-21-0034-R.
LeBlanc, N.R. 2021. Bacteria in the genus Streptomyces are effective biological control agents for management of fungal plant pathogens: A meta-analysis. Biocontrol. 67:111-121. https://doi.org/10.1007/s10526-021-10123-5.
LeBlanc, N.R. 2022. Green manures alter taxonomic and functional characteristics of soil bacterial communities. Microbial Ecology. 85:684–697. https://doi.org/10.1007/s00248-022-01975-0.
Aiello, D., Guarnaccia, V., Vitale, A., LeBlanc, N.R., Shishkoff, N., Polizzi, G. 2022. Impact of Calonectria diseases on ornamental horticulture: Diagnosis and control strategies. Plant Disease. 106(7):1773-1787. https://doi.org/10.1094/PDIS-11-21-2610-FE.
Leblanc, N.R., Martin, F.N., Castroagudin, V.L., Crouch, J. 2021. Mitochondrial loci enable specific quantitative real-time PCR detection of the pathogen causing contemporary impatiens downy mildew epidemics. Plant Disease. 106(1):144-150. https://doi.org/10.1094/PDIS-05-21-0933-RE.
Chen, J., Zhang, D., Huang, J., Li, R., Wang, D., Song, J., Puri, K.D., Lin, Y., Kong, Z., Tong, B., Li, J., Huang, Y., Simko, I., Klosterman, S.J., Dai, X., Subbarao, K.V. 2021. Dynamics of Verticillium dahliae race 1 population under managed agricultural ecosystems. BMC Biology. 19. Article 131. https://doi.org/10.1186/s12915-021-01061-w.
Liu, L., Zhang, Y., Zhang, D., Zhang, Y., Wang, D., Song, J., Li, R., Kong, Z., Klosterman, S.J., Dai, X., Subbarao, K.V., Zhao, J., Chen, J. 2021. Biological characteristics of Verticillium dahliae MAT1-1 and MAT1-2 strains. International Journal of Molecular Sciences. 22(13). Article 7148. https://doi.org/10.3390/ijms22137148.
Lai, M., Cheng, Z., Xiao, L., Klosterman, S.J., Wang, Y. 2022. The bZip transcription factor VdMRTF1 is a negative regulator of melanin biosynthesis and virulence in Verticillium dahliae. Microbiology Spectrum. 10(2). Article e02581-21. https://doi.org/10.1128/spectrum.02581-21.
Wang, D., Zhang, D., Usami, T., Liu, L., Yang, L., Huang, J., Song, J., Li, R., Kong, Z., Li, J., Wang, J., Klosterman, S.J., Subbarao, K.V., Dai, X., Chen, J. 2021. Functional genomics and comparative lineage-specific region analyses reveal novel insights into race divergence in Verticillium dahliae. Microbiology Spectrum. 9(3). Article e01118-21. https://doi.org/10.1128/Spectrum.01118-21.
Fletcher, K., Shin, O.H., Clark, K.J., Feng, C., Putman, A.I., Correll, J.C., Klosterman, S.J., Van Deynze, A., Michelmore, R.W. 2022. Ancestral chromosomes for family Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa. Molecular Plant-Microbe Interactions. 35(6):450-463. https://doi.org/10.1094/MPMI-09-21-0227-R.
Geng, Q., Li, H., Wang, D., Sheng, R.C., Zhu, H., Klosterman, S.J., Subbarao, K.V., Chen, J.Y., Chen, F.M., Zhang, D.D. 2022. The Verticillium dahliae Spt-Ada-Gcn5 acetyltransferase complex subunit Ada1 is essential for conidia and microsclerotia production and contributes to virulence. Frontiers in Microbiology. 13. Article 852571. https://doi.org/10.3389/fmicb.2022.852571.
Clark, K.J., Anchieta, A.G., da Silva, M.B., Kandel, S.L., Choi, Y., Martin, F.N., Correll, J.C., Van Denyze, A., Brummer, C.E., Klosterman, S.J. 2022. Early detection of the spinach downy mildew pathogen in leaves by recombinase polymerase amplification. Plant Disease. 106(7):1793-1802. https://doi.org/10.1094/PDIS-11-21-2398-RE.
Tang, C., Li, W., Klosterman, S.J., Wang, Y. 2021. Transcriptome variations in Verticillium dahliae in response to two different inorganic nitrogen sources. Frontiers in Microbiology. 12. Article 712701. https://doi.org/10.3389/fmicb.2021.712701.