Location: Sustainable Agricultural Systems Laboratory
2019 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. Work on Objective 1 was hampered by the long-term critical vacancy in a technical support position. Agreements are in place, or are planned, to achieve progress in the absence of permanent technical support. We made progress identifying, in silico, microsatellite (MS) regions in the genome of a Rhizoctonia solani AG2-2IIIB plant pathogenic fungus isolate previously sequenced in our laboratory. In collaboration with scientists at North Dakota State University, we collected approximately 400 Rhizoctonia isolates from sugarbeet fields in eight states (Minnesota, North Dakota, Montana, Nebraska, Michigan, Oregon, Colorado, and Arizona) for assessment of genetic diversity using a MS-based genome fingerprinting assay. We constructed genomic and transcriptomic maps of major pathogenic AGs (AG1-IC, AG2-2IIIB, AG3 NT, AG4, AG5, AG6, and AG8) of R. solani, and a database has been constructed. In addition, both up-, and down-regulated proteins from virulent and hypovirulent R. solani AG3-PT isolates have been identified using 2-D Differential Gel Electrophoresis (DIGE) assays. In collaboration with the Easter Lily Research Foundation, using molecular and pathological diagnostic approaches we determined, for the first time, that a Ceratobasidium sp., AG-K isolate causes lily bulb and root rot disease in Oregon. A manuscript has been submitted describing this work (Objectives 1.A, 1B., and 1C). We screened and identified several plant secondary metabolites (PSMs) that suppressed growth of Rhizoctonia, Pythium, and Sclerotinia fungi. The discovered terpenoid bio-pesticides will be utilized in multitatactic disease control strategies against soilborne plant pathogens (Objective 2). Field research conducted in collaboration with scientists at the Oil Crops Research Institute, Wuhan, People’s Republic of China, was completed that demonstrated that Bacillus subtilis isolate BY-2 provided consistent reduction of Sclerotinia disease at multiple field sites when applied as a seed treatment. This research was published in the journal Biological Control. Results published in this paper also demonstrated that combining this isolate with two other Bacillus isolates enhanced growth promotion relative to each isolate applied in the seed treatment alone (Objective 2). Further field studies completed at three separate locations demonstrated that isolate BY-2 provided a significant reduction in Sclerotinia disease relative to the nontreated control when applied in a green-manure biological fertilizer. This provides another method for applying Bacillus biological control agents compatible with the cropping system. A manuscript regarding this work has been submitted for publication in the journal Crop Protection. In collaboration with scientists at the Oil Crops Research Institute, the first 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 isolate BY-2 on Sclerotinia disease has been completed. A second year of work will be initiated in the fall of 2019 (Objective 2). A manuscript is in preparation detailing the impact of a Trichoderma mycoparasite applied in a biological fertilizer on the soil microbial community (Objective 3A). Work with a lacZ-tagged isolate BY-2 strain demonstrated that isolate BY-2 could enter plant roots and persist within root tissue for at least four months when BY-2 was applied as a seed treatment or in the biological fertilizer. Transcriptomic analysis of the impact of seed treatment with B. subtilis isolate BY-2 on the plant defense response by a crop plant was continued in collaboration with scientists at the Oil Crops Research Institute. 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 3B). Due to the retirement of a long-time technician, work with the ethanol extract from S. marcescens was redirected from host-range screening to biochemical and genetic analysis of compounds in the extract important for control of damping-off of cucumber caused by P. ultimum. We continued to screen ethanolic extracts from transposon mutants of S. marcescens to identify compounds in the extract which correlate with disease control. In addition to prodigiosin, the presence of a slow migrating surfactant on TLC plates was correlated with disease control. Ethanolic extracts of one of the transposon mutants, strain Tn246, was characterized by LC-MS/MS and found to be deficient in prodigiosin, an unidentified serratomolide, and disease control, providing further circumstantial evidence that prodigiosin is involved in disease control. We are determining if the unidentified serratomolide is the slow-migrating surfactant detected by TLC. The genome of S. marcescens isolate N4-5, previously sequenced, was found to contain the complete pig cluster, the genes responsible for prodigiosin production. Biosynthetic genes for serrawettin W1 were also identified. We continue to mine the genome for other genes possibly involved in disease control in collaboration with scientists at Universidade Federal de Lavras, Lavras, Brazil. A paper describing the isolate N4-5 genome has been submitted to the journal MicrobiologyOpen. In collaboration with scientists at Universidade Federal de Lavras, Lavras, Brazil, a second paper regarding a genus-wide comparative analysis of Serratia genomes is in preparation (Objective 3C).
Accomplishments
1. Biological control for white mold. The soil-borne plant pathogenic fungus Sclerotinia sclerotiorum can cause devastating losses to numerous crops in the United States and elsewhere in the world. Additional methods for control of diseases caused by this plant pathogen are needed as the commonly used pesticides for control can be environmentally damaging, and in some cases, are losing effectiveness. ARS scientists in Beltsville, Maryland, in collaboration with scientists at the Oil Crops Research Institute in Wuhan, China, tested seed treated with Bacillus isolates and found them to be as effective as the recommended commercial pesticide treatment. Additionally, combinations of Bacillus isolates applied as seed treatments increased crop yield in the absence of disease. This research provides an additional method for disease control and improvement of crop yield.
2. Development of a low-temperature preservation (cryo-preservation) method for non-sporulating fungi. Long-term preservation of experimental fungi without loss of genetic and pathological properties is critical for plant pathology investigations. Existing methods can be cumbersome, hazardous, expensive, and often not suitable for long-term storage of fungi that do not easily produce spores under laboratory conditions. ARS scientists in Beltsville, Maryland, developed a method for preservation of non-spore-forming fungi in commercially available porous beads (MicrbankTM) under low-temperature (-80 degrees C) conditions without loss of morphological, pathological and genetically identifiable characteristics. Research findings demonstrated the utility of low-temperature preservation in Microbank beads as a convenient method for long-term storage of a wide group of fungi for plant pathology investigations. This information will be useful to scientists investigating the biology and management of sterile pathogenic fungi.
Review Publications
Singh, V., Amaradasa, B.S., Karjagi, C.G., Lakshman, D.K., Hooda, K.S., Kumar, A. 2018. Morphological and molecular variability among Indian isolates of Rhizoctonia solani causing banded leaf and sheath blight in maize. European Journal of Plant Pathology. 152:45-60. https://doi.org/10.1007/s10658-018-1447-2.
Singh, V., Karjagi, C.G., Lakshman, D.K., Kumar, A., Mehra, R., Shekher, M. 2018. Phytotoxic and cross-protective effects of culture filtrates of Rhizoctonia solani isolates on Zea mays. Annals of Plant Protection Sciences. 26:153-159. https://doi.org/10.5958/0974-0163.2018.00033.2.
Lakshman, D.K., Cloyd, R.A., Chastagner, G.A. 2019. Integrated management of diseases and pests on ornamental geophytes: Challenges and progress. Acta Horticulturae. 1237:13-31.
Hu, X., Roberts, D.P., Xie, L., Qin, L., Li, Y., Liao, X., Han, P., Yu, C., Liao, X. 2019. Seed treatment containing Bacillus subtilis BY-2 in combination with other Bacillus isolates for control of Sclerotinia sclerotiorum on oilseed rape. Biological Control. 133:50-57.
Buyer, J.S., Vinyard, B.T., Maul, J.E., Selmer, K.J., Lupitskyy, R., Rice, C., Roberts, D.P. 2019. Combined extraction method for metabolomic and PLFA analysis of soil. Applied Soil Ecology. 135:129-136.
