Location: Crop Improvement and Protection Research
2018 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 report documents progress for project 2038-22000-016-00D.
Sub-objective 1.A Identify genes of Verticillium dahliae required for the initial stage of lettuce root infection: Gene deletion of a candidate gene involved in early stages of root infection was examined for pathogenicity on lettuce in order to test which of these genes that are highly differentially expressed may be important for interaction with the host. Surprisingly, the strain was pathogenic, and did not appear to lose virulence, although this requires further evaluation.
Sub-objective 1.B.1. Identify genes that are differentially expressed between downy mildew resistant and susceptible cultivars: A new spinach genome data set with new gene annotations was recently released by a university collaborator for sharing with the USDA, and hence required re-examination of gene sets that are significantly differentially expressed between resistant and susceptible infected spinach cultivars, using the newly available and better quality spinach genome.
Sub-objective 1.B.2. Develop a spinach leaf assay to assist in the development of resistance screens: Experiments were conducted based upon available research in similar organisms. The pathogen was inoculated on spinach seedlings for examination of downy mildew development under controlled conditions. Scientists were successful twice in getting full downy mildew disease development on spinach seedlings.
Sub-objective 1.C.1. Identify microbial predators of fungal pathogens for disease control: Lettuce plants were inoculated with potential biocontrol microorganisms and then inoculated with V. dahliae in a greenhouse, for testing of whether the biocontrol agents may be valuable to control Verticillium wilt as an alternative to soil fumigation. The current studies require replication and data analyses.
Sub-objective 1.C.2. Identify soil abiotic factors that reduce survival of Verticillium dahliae: Readings were taken of the survival rate of V. dahliae after six months in soil.
Sub-objective 1.C.4. Analyze pigment cluster genes of V. dahliae that contribute to long term survival: Work was completed on melanin biosynthesis gene cluster deletion mutants for analyses of differences from the wild type strain. Two were found to lack pigmentation, and scientist learned that these were required for increased survival following exposure to ultraviolet light. Understanding their functions may reveal a weakness in the pathogen because these genes are beneficial for long-term survival.
Sub-objective 1.D. Determine the correlation between genotype of Macrophomina phaseolina and virulence on strawberry: Over 98% of the isolates of the pathogen Macrophomina phaseolina recovered from strawberry are the same genotype. In contrast to isolates recovered from other hosts, isolates in this strawberry genotype tend to be aggressive in attacking strawberry while other genotypes do not or weakly attack strawberry. Trials are continuing to further evaluate this observation with a larger number of isolates.
Sub-objective 1.E. Assemble a high quality reference genome for Macrophomina phaseolina and identify genes associated with host specificity: The nuclear genomes of two isolates of M. phaseolina, one from strawberry and another that cannot attack this crop, have been assembled into nearly complete chromosomes. The regions of the genome coding for genes have been identified and the assemblies are currently being annotated. DNA sequences from 28 other isolates with a range in ability to infect strawberry were generated and investigations are under way to identify genes associated with host specificity towards strawberry.
Sub-objective 2.A.1. Develop in-field diagnostic test for Peronospora effusa: Sequences of DNA were examined for an in-field detection system for the spinach downy mildew pathogen, Peronospora effusa. An in-field detection system will help spinach growers to more quickly make disease management decisions.
Sub-objective 2.A.2. Identify population genetic molecular markers for the spinach downy mildew pathogen, Peronospora effusa: Markers have been identified to study how the population genetics of the spinach downy mildew pathogen is changing over time, and samples of the pathogen were collected and maintained in a freezer.
Sub-objective 2.B.1. Mitochondrial genomics project: Over 570 mitochondrial genomes from a group of plant pathogens referred to as Oomycetes have been assembled and comparisons among these genomes were done to identify possible regions that will be useful for development of molecular detection assays specific for particular taxa as well for amplification of specific genes that are useful for species identification and clarification of evolutionary relationships.
Sub-objective 2.B.2. Molecular diagnostics: Molecular diagnostic assays based on mitochondrial gene order differences have been developed for Oomycetes, including Phytophthora, Pythium and Aphanomyces (the latter two are currently being validated). Species-specific diagnostic markers have been developed that target unique putative open reading frames of selected genes in several downy mildew taxa (Pseudoperonospora cubensis, Pseudoperonospora humuli, Peronospora effusa, Bremia lactucae, Plasmopara obducens and Peronosclerospora philippinensis). The respective markers are, with the exception of Phytophthora and Bremia, currently being validated with a range of taxa to ensure their specificity and sensitivity.
Sub-objective 2.B.3. Oomycete phylogenetics: The assembled mitochondrial genomes provides a 35 mitochondrial genes that can be used for evaluating the evolutionary relationships among taxa. Scientists currently have mitochondrial genomes assembled for 157 taxa representing 10 genera of Oomycetes and are investigating the evolutionary relationships among them.
Sub-objective 2.B.4. Improved identification of Phytophthora species: Analysis of the different mitochondrial genes extracted for the assembled genomes has provided a rich resource for identification of loci useful for species identification. The genus specific amplicon between two genes (the locus used for the genus and species-specific diagnostic assay) works well for species identification. It is also useful for metagenomics studies of the genus. Another gene order difference has been identified that is highly conserved in Oomycetes but not present in plants or Eumycotan fungi, enabling amplification from environmental samples with minimal background amplification on nontarget sequences. This locus is particularly useful for identification of the downy mildew pathogens, as well as with Phytophthora and Pythium. Collaborations established with researchers from around the world to sequence several thousand isolates (including many downy mildews) seeks to develop a “bar code” database for isolate identification of all Oomycetes. The amplification primers have also been used in collaboration with another lab as a metagenomics locus for investigating Oomycete communities.
Sub-objective 2.C Develop molecular tools for detection and soil quantification of Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae: The nuclear genomes from 30 isolates of M. phaseolina and 130 isolates of F. oxysporum (including 64 of f. sp. fragariae) were sequenced by Illumina. The sequences for each pathogen species were compared and regions that were found only in the pathogenic strains were identified and used for developing two different types of molecular diagnostic assays for their detection. One approach used TaqMan real time polymerase chain reaction (PCR) to detect the pathogen and quantify its population in the soil. The other used an isothermal technology called recombinant polymerase amplification that allows detection from infected plant tissue directly in the field in as little as 10 minutes. The project with M. phaseolina has been completed and the ability to accurately quantify F. oxysporum f. sp. fragariae in field soil is currently under evaluation. A manuscript describing the M. phaseolina assay was recently published and a manuscript for the F. oxysporum f. sp. fragariae will be submitted shortly.
Accomplishments
1. Identification of a protein that affects the ability of the plant pathogenic fungus Verticillium dahliae to survive. Verticillium dahliae causes plant diseases, known commonly as Verticillium wilts, on many economically important crops 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. ARS researchers in Salinas, California, led a collaborative study with two scientists at two different institutions in China, and revealed the importance of two genes involved in melanin biosynthesis in the survival of V. dahliae. Knowledge of the types of genes and their protein products may be useful to devise environmentally friendly strategies to inhibit the fungus in the field.
2. Spinach pathogen in leaves detected prior to symptom development. Downy mildew disease of spinach, caused by the plant pathogenic microorganism Peronospora effusa, is a major disease constraint on spinach in the U.S. and worldwide. The period between plant infection and symptom development is known as the latent period. ARS researchers in Salinas, California, led the effort to detect DNA of the pathogen in the latent period in spinach leaves. Early detection of the pathogen in the leaves in the field will help to more effectively target fungicide applications prior to symptom development or to harvest organic crops earlier, and thereby help to prevent downy mildew epidemics.
3. Improved management of cucurbit downy mildew. Downy mildew can cause devastating losses to cucurbit and hops production. Development of an effective pathogen monitoring program would enable a more efficient spray program to prevent disease. ARS researchers in Salinas, California, and collaborators at North Carolina State University, developed a multiplexed molecular marker-based system for detecting and measuring the amounts of three downy mildew pathogens on cucurbits and hops. These markers have been shared with collaborators and will provide a means for rapid identification of the pathogen and improved management of fungicide spray programs for enhanced disease control.
4. Diagnostics for Macrophomina root and crown rot. Macrophomina phaseolina is an emerging pathogen in California strawberry production with the banning of methyl bromide for soil fumigation. ARS researchers in Salinas, California, and collaborators at University of California, developed two different types of molecular diagnostic tests that are specific for the Macrophomina types capable of infecting strawberry. The tests measure the amounts of the fungus in the soil as well as provide a means for rapid detection directly in the field. Isolates of the fungus that are highly infectious on strawberry are, with a few exceptions, genetically identical. These assays provide researchers with the tools they need to identify the pathogen and growers with the ability to determine risk prior to planting.
5. Diagnostics for Fusarium wilt. Fusarium oxysporum f. sp. fragariae 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 University of California developed two different types of molecular diagnostic tests specific for the fungal isolates capable of infecting strawberry. The tests measure the amounts of the fungus in soil, as well as provide a means for rapid detection directly in the field. Isolates of the fungus that are highly infectious on strawberry are, with a few exceptions, genetically similar. These assays provide researchers with the tools they need to identify the pathogen and growers with the ability to determine risk prior to planting.
Review Publications
Hao, W., Miles, T.D., Martin, F.N., Browne, G.T., Forster, H., Adaskaveg, J.E. 2018. Temporal occurrence and niche preferences of Phytophthora spp. causing brown rot of citrus in the Central Valley of California. Phytopathology. 108(3):384-391. https://doi.org/10.1094/PHYTO-09-17-0315-R.
Subbarao, C.S., Anchieta, A.G., Ochoa, L., Dhar, N., Kunjeti, S.G., Subbarao, K.V., Klosterman, S.J. 2018. Detection of latent Peronospora effusa infections in spinach. Plant Disease. https://doi.org/10.1094/PDIS-12-17-1956-RE.
Wang, Y., Hu, X., Fang, Y., Anchieta, A.G., Goldman, P.H., Hernandez, G., Klosterman, S.J. 2018. Transcription factor VdCmr1 is required for pigment production, protection from UV irradiation, and regulates expression of melanin biosynthetic genes in Verticillium dahliae. Microbiology. 164(4):685-696. https://doi.org/10.1099/mic.0.000633.
Crandall, S.G., Rahman, A., Quesada-Ocampo, L.M., Martin, F.N., Bilodeau, G.J., Miles, T.D. 2018. Advances in diagnostics of downy mildews: Lessons learned from other oomycetes and future challenges. Plant Disease. 102(2):265-275. https://doi.org/10.1094/PDIS-09-17-1455-FE.
Wang, J., Tian, L., Zhang, D., Short, D.P., Zhou, L., Song, S., Liu, Y., Wang, D., Kong, Z., Cui, W., Ma, X., Klosterman, S.J., Subbarao, K.V., Chen, J., Dai, X. 2018. SNARE-encoding genes VdSec22 and VdSso1 mediate protein secretion required for full virulence in Verticillium dahliae. Molecular Plant-Microbe Interactions. 32(6):651-664. http://doi.org/10.1094/MPMI-12-17-0289-R.
Rahman, A., Miles, T.D., Martin, F.N., Quesada-Ocampo, L.M. 2017. Molecular approaches for biosurveillance of the cucurbit downy mildew pathogen, Pseudoperonospora cubensis. Canadian Journal of Plant Pathology. 39:282-296. https://doi.org/10.1080/07060661.2017.1357661.
Li, N.Y., Zhou, L., Zhang, D.-D., Klosterman, S.J., Li, T.-G., Gui, Y.J., Kong, Z.-Q., Ma, X.-F., Short, D.P.G., Zhang, W.-Q., Li, J.-J., Subbarao, K.V., Chen, J.-Y., Dai, X.-F. 2018. Heterologous expression of the cotton NBS-LRR gene GbaNA1 enhances Verticillium wilt resistance in Arabidopsis. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2018.00119.
Choudhury, R.A., Garrett, K.A., Klosterman, S.J., Subbarao, K.V., Mcroberts, N. 2017. A framework for optimizing phytosanitary thresholds in seed systems. Phytopathology. 107(10):1219-1228. https://doi.org/10.1094/PHYTO-04-17-0131-FI.
Burkhardt, A.K., Ramon, M.L., Smith, B., Koike, S.T., Martin, F.N. 2018. Development of molecular methods to detect Macrophomina phaseolina from strawberry plants and soil. Phytopathology. https://doi.org/10.1094/PHYTO-03-18-0071-R.
