Research Project: Biologically Based Technologies for Control of Soil-Borne Pathogens of Vegetables and Ornamentals

Location: Sustainable Agricultural Systems Laboratory

2020 Annual Report

Objectives
Objective 1. Develop diagnostics for detection and differentiation of soil-borne sclerotial fungi. Sub-objective 1A. Identify and differentiate Rhizoctonia (sensu lato) pathogens by developing genome fingerprint-based markers. Sub-objective 1B. Use functional omics approaches to discover and develop novel molecular markers for virulence, host specificity, and identification of Rhizoctonia solani. Sub-objective 1B1. Compare transcriptomes of Rhizoctonia solani anastomosis groups (AGs) to determine if differences and commonalities across and between AGs suggest clues to host-range and virulence. Sub-objective 1B2. Identify proteins involved in virulence through comparison of the proteomes of hypovirulent Rhizoctonia solani AG3 isolates with a virulent AG3 isolate. Sub-objective 1C. Develop a database of Rhizoctonia genome and transcriptome information. Objective 2. Develop control tactics for the soil-borne sclerotial fungi Rhizoctonia solani and Sclerotinia sclerotiorum and the soil-borne oomycete Pythium ultimum. Objective 3. Identify mechanisms involved in control of soil-borne sclerotial pathogens and the soil-borne oomycete Pythium ultimum by biological control agents and their natural products. Sub-objective 3A. Determine impact of multitactic disease control strategies on soil microbial communities. Sub-objective 3B: Use functional omics approaches to identify biological control mechanisms involved in control of sclerotial plant pathogens. Sub-objective 3C. Identify compounds in ethanol extract of S. marcescens responsible for control of damping-off of cucurbits caused by P. ultimum, other oomycetes, and fungi.

Approach
Omics (genomics, transcriptomics, proteomics) approaches will be employed to develop technologies for detection and identification of Rhizoctonia solani isolates so that appropriate control measures for specific R. solani isolates can be chosen for use in grower fields. Basic microbiology techniques will be used to develop new biologically based control measures, and combinations of control measures (biological controls, cover crops, chemical pesticides), for multiple pathogens (R. solani, Sclerotinia sclerotiorum, Pythium ultimum) over varied field conditions. Molecular biology and biochemistry approaches will be used to determine how existing biological controls control R. solani, S. sclerotiorum, and P. ultimum. Analysis of the rhizosphere microbiome using molecular techniques will determine the impact of these control measures on the rhizosphere microbial community. Transcriptomic and proteomic approaches will be used to identify genes and enzymes involved in degradation of sclerotia of S. sclerotiorum and other sclerotial pathogens by mycoparasitic biological control agents. Compounds in ethanol extract of Serratia marcescens responsible for control of damping-off of cucurbits caused by P. ultimum, other oomycetes, and fungi will be identified using biochemical and genetic approaches. Successful completion of this project will yield natural product chemistries, such as prodigiosin, for disease control and genes that can be used to screen for effective microbial biological control agents.

Progress Report
This project was initiated in June of 2017. A support scientist was transferred onto the project in FY2020 from another MU filling a long-term critical vacancy in a technical support position. Agreements are in place, or are planned, to increase progress after the long-time absence of permanent technical support. For Objective 1, we collected approximately 400 Rhizoctonia isolates from sugarbeet fields in eight states (Minnesota, North Dakota, Montana, Nebraska, Michigan, Oregon, Colorado, and Arizona) in collaboration with scientists at North Dakota State University and started assessment of genetic and morphological diversity of these isolates using the RAD-seq approach. We also constructed genomic and transcriptomic maps of 13 R. solani pathogens belonging to major Anastomosis Groups (AGs) and subgroups (AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3-PT-1AP, AG3-PT-1A1, AG3-TB-T5, AG4-HG-I-Rs23A, AG4-HG-I-R118-11, AG5, AG6-10EEA, AG6-18BWB, and AG8) of R. solani. Proteomic data resulting from these studies was generated and verification of proteomic data by Q-PCR is pending. A database containing this genomic, transcriptomic, and proteomic information was constructed and a manuscript detailing this database is in preparation. We are working to get this database accessible through AgCROS. R. solani AG-specific probes were designed using information in this database and validation of these probes for AG-specific identification of R. solani isolates is in progress (Objective 1). For Objective 2, we continued screening and identifying several plant secondary metabolites (PSMs) that suppressed growth of Rhizoctonia, Pythium, and Sclerotinia fungi. Terpenoid PSMs, with promising activity against these pathogens, will be utilized in multi-tactic disease control strategies. Moreover, we discovered and are characterizing a Trichoderma isolate which successfully controlled Rhizoctonia damping-off in the greenhouse. An additional potential benefit of this Trichoderma isolate is that it releases keratinase, an enzyme that degrades poultry feathers. Poultry feathers contain 12-14% nitrogen which could be utilized by this isolate. In collaboration with scientists at the Oil Crops Research Institute, Wuhan, People’s Republic of China, the second year of field research assessing the impact of spray application of the mycoparasite Aspergillus aculeatus Asp-4 to the field prior to sowing the crop, combined with seed treatment with Bacillus subtilis isolate BY-2 on Sclerotinia disease, is ongoing. We will evaluate the second-year results to determine if a third year of field work is needed (Objective 2). For Objective 3, we identified genes and proteins involved in colonization of sclerotia of Sclerotinia sclerotiorum ahead of schedule and reported the publication of this work in BMC Genomics in the FY2019 Annual Report. An ethanol extract of Serratia marcescens isolate N4-5 cell mass has been shown to control Pythium ultimum damping-off of cucumber. Construction of a library of transposon mutants of isolate N4-5 was completed. We screened ethanol extracts from mutants in this library for loss of control of damping-off of cucumber. Ethanol extracts from one mutant, S. marcescens Tn246, no longer controlled P. ultimum and did not contain the exolipid prodigiosin, correlating the presence of prodigiosin with disease control. Purified prodigiosin was shown to control this disease. A manuscript is being finalized detailing the role of prodigiosin in control of damping-off of cucumber by ethanol extract of isolate N4-5. This will be the first report indicating that prodigiosin can control a plant disease. A manuscript is also in preparation detailing the impact of a Trichoderma mycoparasite applied in a biological fertilizer on the soil microbial community. Transcriptomic profiling was continued in collaboration with scientists at the Oil Crops Research Institute and Purdue University to discern the impact of seed treatment with B. subtilis isolate BY-2 on the plant defense response by a crop plant. A large library of up-regulated and down-regulated plant genes is currently being analyzed to determine which plant defense-related genes are up-regulated or down-regulated in response to colonization of internal/external plant tissues by isolate BY-2 (Objective 3).

Accomplishments
1. Genomic database of the Rhizoctonia solani plant pathogen complex created. Genomes of 13 isolates from the Rhizoctonia solani plant pathogen complex were sequenced, assembled, and annotated – and a genome database was created (http://rsolanidb.kaust.edu.sa/RhDB/). This database provides information to scientists that is critical for development of identification methods for different isolates within the R. solani pathogen complex. Identification and differentiation of the many morphologically similar R. solani species within this pathogen complex is necessary as individual species vary in response to plant disease control measures.

Review Publications
Roberts, D.P., Mattoo, A.K. 2019. Sustainable crop production systems and human nutrition. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2019.00072.
De Jesus Silva, F., Carvalho Ferreira, L., Campos, V.P., Cruz-Magalhaes, V., Barros, A.F., Andrade, J.P., Roberts, D.P., De Souza, J.T. 2019. Complete genome sequence of the biocontrol agent Bacillus velezensis UFLA258 and its comparison with related species: Diversity within the commons. Genome Biology and Evolution. 11(10):2818-2823. https://doi.org/10.1093/gbe/evz208.
Lakshman, D.K., Kamo, K.K., Dasgupta, M.K. 2019. First report of a Ceratobasidium sp. AG-K isolate causing lily root rot in Oregon. Plant Disease. https://doi.org/10.1094/PDIS-05-19-1014-PDN.