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

Location: Sustainable Agricultural Systems Laboratory

2021 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. This report covers work during the fourth year of the project. For Objective 1, the genomes of 13 plant pathogenic isolates from the Rhizoctonia solani species complex covering 7 anastomosis groups (AGs) and selected subgroups [AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3 (isolates AG3-PT, AG3-Rhs1A1, and AG3-TB), AG4-HG-I (isolates Rs23A and R-118), AG5, AG6 (isolate 10EEA and 18BWB), and AG8] were sequenced, assembled, and annotated. The draft manuscript on the Rhizoctonia pangenome analysis showing unique features of each pathogenic group, including molecular factors potentially involved in determining AG-specific host preference, has been uploaded in the Pre-print server BioRxiv (https://doi.org/10.1101/2020.12.18.423518) and is currently under peer-review in Nucleic Acids Research - Genomics and Bioinformatics. For the first time, a user-friendly and publicly accessible genome database of 18 Rhizoctonia solani (Teleomorph: Thantephorus cucumeris) isolates and six previously published Rhizoctonia genomes are presented at one site (http://rsolanidb.kaust.edu.sa/RhDB/). This database is a comprehensive web resource and stands as a valuable platform for formulating new hypotheses by hosting annotated genomes, with tools for functional enrichment, orthologs, and sequence analyses, currently not available with other accessible state-of-the-art platforms hosting Rhizoctonia genome sequences. Between 02/01/2021 and 5/27/2021, the RsolaniDB was accessed by other researchers 197 times, and the genomic contents were downloaded 78 times, underscoring the usefulness of this database to plant pathologists, mycologists, and molecular biologists. In collaborative research with North Dakota State University scientists, we identified two new pathogens (Fusarium exquisite and Sclerotinia sclerotiorum) associated with sugar beetroot rots in Minnesota. The manuscript identifying the Fusarium pathogen has been published, while the manuscript on the Sclerotinia pathogen has been accepted for publication in a peer-reviewed journal. Recently we identified and characterized the morpho-pathogenic and molecular morphometric properties of Waitea circinata var. prodigus, a new pathogen of maize in India; the manuscript detailing this work is under peer-review at the journal Plant Disease. For Objective 2, we demonstrated that several soybean root-nodule-associated non-rhizobial bacterial endophytes are antagonistic to soilborne plant pathogens Rhizoctonia solani AG4 isolate Rs23A, and Sclerotinia sclerotiorum in vitro; we also demonstrated that selected nodule endophytic species suppress soilborne wilt disease of tomato caused by Clavibacter michiganensis subsp. michiganensis, and bacterial speck disease caused by Pseudomonas syringae pv. tomato. Isolation and identification of beneficial nodule-associated bacteria established the foundation for further exploration of potential nodule-associated bacteria for plant protection and growth promotion. This work was published in the journal Plants. In collaborative research with North Dakota State University scientists, we isolated and characterized a novel fungus Penicillium pinophylum associated with sugar beet taproots, proved its antagonism to R. solani AG2-2IIIB in vitro, and further demonstrated that P. pinophylum controls Rhizoctonia root rot, both in the laboratory and in the greenhouse. This work has been published in Sugar Tech. A study on methods of delivery of Bacillus subtilis BY-2 in biological fertilizer for control of Sclerotinia sclerotiorum was completed and published. In collaboration with scientists at the Oil Crops Research Institute, Wuhan, People's Republic of China, the third year of field research assessing the impact of spray application of the mycoparasite Aspergillus aculeatus Asp-4 to the field before sowing the crop, combined with seed treatment with Bacillus subtilis isolate BY-2 on Sclerotinia disease, was completed. We are currently compiling and analyzing the data from the third year of the field trial. Research results from the second year of field trials were lost due to the Covid-19 pandemic. For Objective 3, work on sequencing and annotating the genome of Serratia marcescens N4-5 was published. Seed treatment with extracts of S. marcescens N4-5 was previously shown to control damping-off of cucumber caused by P. ultimum and seed treatment with chemical pesticide in some soils. Transposon insertion sites in mutants that were decreased or deficient in control of P. ultimum damping-off of cucumber were mapped to genes within the S. marcescens N4-5 genome. A manuscript was published that detailed the role of prodigiosin in controlling the damping-off of cucumber by ethanol extract of isolate N4-5. This is the first report indicating that prodigiosin can control plant disease, potentially providing new chemistry to exploit in developing controls for oomycetes. A manuscript is also in preparation detailing the impact of a Trichoderma mycoparasite applied in a biological fertilizer on the soil microbial community. In collaboration with USDA-ARS collaborators in Beltsville, Maryland, two manuscripts describing related work on the impact of rice cultivars on the associated rhizosphere community were published in Soil Biology and Biochemistry and in the journal Diversity. 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. Data from an RNA Seq study on the impact of cucumber root exudate on gene expression by the biocontrol agent Psuedomonas protegens PF-5 has been analyzed in collaboration with scientists at the Universidade Federal de Lavras, Lavras, Brazil. A manuscript detailing this work is in preparation.

Accomplishments
1. Genomic database of the Rhizoctonia solani species complex (RsolaniDB) updated with new features and information, making it more user-friendly and resourceful. In a collaborative effort between USDA-ARS, Beltsville, Maryland, scientists and scientists at King Abdullah University of Science and Technology (KAUST), Saudi Arabia, six new R. solani genomes were added to the RsolaniDB making a total of 18 genomes. The database was improved to make it more resourceful to the fungal research community for pangenomic comparative investigations and for developing methods for identifying different isolates within the R. solani species complex. The many morphologically similar R. solani plant pathogens within this species complex makes easy identification and differentiation necessary for selection of appropriate disease control tools. Between 02/01/2021 and 5/27/2021, the updated RsolaniDB was accessed 197 times and the genomic contents downloaded 78 times, highlighting the usefulness of the genomic database to plant pathologists, mycologists, and molecular biologists.

2. Nodule-associated beneficial bacteria for plant protection and growth promotion. USDA-ARS scientists in Beltsville, Maryland, in collaboration with scientists at the University of Nebraska, Lincoln demonstrated that soybean root nodules harbor many non-Rhizobium endophytic bacteria such as Proteus hauseri, Enterobacter cloacae, Ochrobactrum species, Acinetobacter calcoaceticus, and several species of Pseudomonas. Certain of these bacteria were antagonistic to plant pathogenic Clavibacter michiganensis subsp. michiganensis (Cmm), Pseudomonas syringae pv. tomato (Pst), Rhizoctonia solani, and Sclerotinia sclerotiorum in vitro. They also effectively controlled wilt of tomato caused by Cmm and bacterial speck of tomato caused by Pst; and resulted in growth promotion of tomato in the greenhouse. Isolation and identification of beneficial nodule-associated bacteria established the foundation for further exploration of nodule-associated bacteria for plant protection and growth promotion.

Review Publications
Tokgoz, S., Lakshman, D.K., Gozlan, M., Pinar, H., Roberts, D.P., Mitra, A. 2020. Soybean nodule-associated non-rhizobial bacteria inhabit plant pathogens and induce growth promotion in tomato. Plants. https://doi.org/10.3390/plants9111494.
Roberts, D.P., Short, N.M., Jr., Sill, J., Lakshman, D.K., Hu, X., Buser, M.D. 2021. Precision agriculture and geospatial techniques for sustainable disease control. Indian Phytopathology. https://doi.org/10.1007/s42360-021-00334-2.
Haque, M.E., Lakshman, D.K., Aiming, Q., Khan, M.F. 2021. Penicillium pinophilum has the potential to reduce damping-off caused by Rhizoctonia solani in sugar beet. Sugar Tech. https://doi.org/10.1007/s12355-021-00958-8.
Khan, M.F., Bhuiyan, M.Z., Liu, Y., Lakshman, D.K., Liu, Z., Zhong, S. 2021. First report of Fusarium equiseti causing seedling death on sugar beet in Minnesota, USA. Plant Disease. https://doi.org/10.1094/PDIS-10-20-2102-PDN.
Roberts, D.P., Vandenberg, B., Mirsky, S., Buser, M., Reberg-Horton, C., Short, N., Shrestha, S. 2020. How to feed the world. In: Wright, D.J., Harder, C., editors. Applying Mapping and Spatial Analytics. GIS for Science. Redlands, CA:Esri Press. p. 110-123.
Li, Y., Qin, L., Roberts, D.P., Hu, X., Xie, L., Gu, C., Shen, X., Liao, X., Han, P., Liao, X. 2020. Application of Bacillus subtilis BY-2 in a biological fertilizer for control of Sclerotinia sclerotiorum on oilseed rape. Crop Protection. 138:105340. https://doi.org/10.1016/j.cropro.2020.105340.
Ghozlan, M.H., El-Argawy, E., Tokgoz, S., Lakshman, D.K., Mitra, A. 2020. Plant defense against necrotrophic pathogens. American Journal of Plant Sciences. 11(12):2122-2138. https://doi.org/10.4236/ajps.2020.1112149.
Ferreira, L.A., Maul, J.E., Viana, M.V., De Sousa, T.J., Azevedo, V.A., Roberts, D.P., De Souza, J.T. 2020. Complete genome sequence of Serratia marcescens strain N4-5, a biological control agent of soil-borne plant pathogens. Brazilian Journal of Microbiology. 52:245-250. https://doi.org/10.1007/s42770-020-00382-2.
Roberts, D.P., Selmer, K.J., Lupitskyy, R., Rice, C., Buyer, J.S., Maul, J.E., Lakshman, D.K., De Souza, J. 2021. Seed treatment with prodigiosin controls damping-off of cucumber caused by Pythium ultimum. Applied Microbiology and Biotechnology Express (AMB Express). 11:10. https://doi.org/10.1186/s13568-020-01169-2.
Fernandez-Baca, C.P., Rivers, A.R., Maul, J.E., Kim, W., McClung, A.M., Roberts, D.P., Reddy, V., Barnaby, J.Y. 2021. Rice plant-soil microbiome interactions driven by root and shoot biomass. Diversity. https://doi.org/10.3390/d13030125.
Fernandez-Baca, C.P., Rivers, A.R., Kim, W., McClung, A.M., Roberts, D.P., Reddy, V., Barnaby, J.Y. 2021. Changes in rhizosphere soil microbial communities across plant developmental stages of high and low methane emitting rice genotypes. Soil Biology and Biochemistry. http://doi.org/10.1016/j.soilbio.2021.108233.
Vega, F.E., Emche, S.E., Shao, J.Y., Simpkins, A., Summers, R., Mock, M., Ebert, D., Infante, F., Aoki, S., Maul, J.E. 2021. Cultivation and genome sequencing of bacteria isolated from the coffee berry borer (Hypothenemus hampei), with emphasis on the role of caffeine degradation. Frontiers in Microbiology. 12:644768.