Location: Sustainable Agricultural Systems Laboratory
2018 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 plan was implemented approximately 12 months ago in June 2017. The long-term critical vacancy of a technical support position hampered work objective 1. Agreements are in place, or are planned, to achieve progress in the absence of permanent technical support. RNA transcriptome sequencing of Rhizoctonia solani isolates from various AG groups was initiated in collaboration with scientists at the King Abdullah University of Science and Technology, Thuwal, Saudia Arabia (Objective 1B). Proteomics work to decipher virulence and hypovirulence of R. solani AG3 isolates was initiated in collaboration with scientists at Texas Tech University, Lubbock, Texas and the Indian Institute of Maize Research (IIMR), New Delhi (Objective 1B). In work associated with Objective 1 we detected and identified a new pathogen causing lily root rot. In collaboration with scientists at IIMR we 1) developed a long-term preservation method for non-spore forming fungi such as R. solani; 2) developed a seed treatment method for evaluation of virulence of R. solani and for cross protection; and 3) evaluated morphological and molecular variability among Indian isolates of R. solani causing banded leaf and sheath blight in maize (Objective 1). Screening treatment combinations for control of damping-off of cucumber caused by Pythium ultimum was completed using a number of soils differing in texture, organic matter, and pH. Treatments containing ethanolic extract of Serratia marcescens N4-5 in combination with Trichoderma virens GL21 provided the most consistent control of this disease over three soils (Objective 2). In collaboration with scientists at the University of Nebraska several soybean nodule-associated bacteria were identified that displayed in vitro antagonism against plant pathogenic bacteria and fungi including R. solani. Thymol, the key component of thyme oil, and its derivatives were evaluated for fungicide activity against R. solani in collaboration with scientists at Beltsville, Maryland. Of the six thymol derivatives, thymol acetate was the most commercially viable due to its superior efficacy and low phytotoxity. These findings will be useful for eco-friendly management of Rhizoctonia diseases (Objective 2). Field research at four locations was conducted in collaboration with scientists at the Oil Crops Research Institute, Wuhan, People’s Republic of China. This research demonstrated that Bacillus subtilis isolate BY-2 provided consistent reduction of Sclerotinia disease when applied as a seed treatment in four different soils and that combining other Bacillus isolates with BY-2 in seed treatments provided an incremental improvement in disease reduction while also promoting plant growth. Results were detailed in a paper submitted to Plant Disease (Objective 2). Further evaluation of isolate BY-2 applied in a biological fertilizer was conducted at two locations over two years. Data from these experiments are currently being analyzed. Field research with scientists at the Oil Crops Research Institute was initiated to assess spray application of the mycoparasite Aspergillus aculeatus Asp-4 to the field prior to sowing the crop along with seed treatment with isolate BY-2. Disease control efficacy and consistency of treatments are being assessed at multiple locations with different soil types (Objective 2). Mechanisms underlying degradation of sclerotia of S. sclerotiorum by A. aculeatus Asp-4 were characterized using a combined transcriptomic and qRT-PCR approach in collaboration with scientists at the Oil Crops Research Institute. Genes functioning in degradation of sclerotial components and genes functioning in response to environmental conditions were up-regulated. Proteomic analysis of Asp-4 growing on this sclerotial material identified 26 up-regulated and 6 down-regulated proteins relative to the control. Certain proteins with increased abundance had putative functions in degradation of polymeric components of sclerotia and the mitigation of environmental stress. Our results suggest co-temporal up-regulation of genes involved in degradation of sclerotial compounds and mitigation of environmental stress by A. aculeatus Asp-4 during colonization of sclerotia of S. sclerotiorum. A paper describing this work was published in BMC Genomics (Objective 3B). 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 was collected. We are in the process of initiating bioinformatic analysis of this library of genes (Objective 3B). We characterized the ethanolic extract of S. marcescens using TLC-MS and LC-MS/MS and found the extract to contain prodigiosin, the surfactant serrawettin W1, and at least 14 additional seratomolides. We purified prodigiosin and conducted disease control assays with treatments containing seed treated with purified prodigiosin to demonstrate that this compound was involved in control of damping-off by this extract. We continued construction of a transposon mutant library of S. marcescens and screening of ethanolic extracts of these mutants for mutants decreased or deficient in control of damping-off of cucumber. All mutants decreased or deficient in prodigiosin production were also decreased or deficient in disease control corroborating our work with purified prodigiosin. The genome of S. marcescens isolate N4-5 was sequenced and annotated in collaboration with scientists at Universidade Federal de Lavras, Lavras, Brazil. A paper describing the isolate N4-5 genome is being finalized for submission to the journal Standards in Genomic Sciences. We are currently identifying genes inactivated by transposon insertion in this mutant library (Objective 3C).
Work focused on the management of Botrytis on several ornamental bulb crops was continued in collaboration with scientists at Washington State University. A manuscript entitled “Survey reveals a broad range of fungal pathogens on peonies in the United States” was submitted to Plant Disease and a manuscript entitled “Development of a Quantitative PCR Assay to Detect and Quantify Rhizoctonia tuliparum” is being prepared. Four scientific papers were published by Washington State University scientists in the past year. A new Fact Sheet relating to tobacco rattle virus in peonies (http://extension.wsu.edu/publications/wp-content/uploads/sites/54/publications/fs284e.pdf) and a management guide relating to anthracnose on peonies were also published (https://pnwhandbooks.org/plantdisease). Seven field trials on tulips, iris, daffodils, lilies and peonies were established to examine the effectiveness of new “reduced-risk” and biopesticides in controlling foliar diseases on these crops.
Accomplishments
1. Seed treatment improves control of cucumber disease. Controlling soil-borne plant pathogens with methyl bromide alternatives is promising but can provide inconsistent performance in different soils. ARS researchers in Beltsville, Maryland, demonstrated that a combination treatment containing ethanolic extract of the bacterium Serratia marcescens and the plant-beneficial fungus Trichoderma virens isolate GL21 improved disease control performance relative to individual application of these treatment components in several soils. Strategies such as this that improve efficacy and consistency of disease control will enhance grower acceptance of biologically based disease control treatments.
2. Controlling soil-borne pathogens in conventional and organic crop production systems. ARS researchers in Beltsville, Maryland, demonstrated that seed treatment with cell-free ethanolic extract of the bacterium Serratia marcescens was as effective as the commercial pesticide Thiram. These same researchers have determined that this ethanolic extract contains large amounts of the compound prodigiosin and that treating seeds with purified prodigiosin controlled the cucumber disease “damping-off.” Prodigiosin is potentially useful for controlling damping-off disease on cucurbits in conventional and organic production systems.
3. Thymol acetate is a commercially viable plant-based fungicide. ARS scientists in Beltsville, Maryland, screened thymol, the key component of thyme oil, and its derivatives for fungicidal activity against the plant pathogen Rhizoctonia solani, which is one of the major pathogens causing the disease “damping-off” on seedlings. Of six thymol derivatives, thymol acetate has superior disease control efficacy and low phytotoxity. Compounds such as thymol acetate could provide environmentally friendly control of this important soil-borne plant pathogen in conventional and organic cropping systems.
Review Publications
Lakshman, D.K., Kamo, K.K. 2018. First report of lily root rot caused by Thantephorus cucumeris AG 2-1 in the United States. Plant Disease. https://doi.org/10.1094/PDIS-09-17-1497-PDN.
Chauhan, K.R., Le, T.C., Chintakunta, P., Lakshman, D.K. 2017. Phyto-fungicides: Structure activity relationships of the thymol derivatives against Rhizoctonia solani. Journal of Agricultural Chemistry and Environment. 6:175-185.
Lakshman, D.K., Chauhan, K.R., Pandey, R., Choudhury, B. 2017. Evaluation of plant-based antifungal chemicals and control of damping-off caused by Rhizoctonia solani. Biopesticides International. 13(1):21-34.
Hu, X., Qin, L., Roberts, D.P., Lakshman, D.K., Gong, Y., Maul, J.E., Xie L., Yu, C., Li, Y., Hu, L., Liao, X., Liao, X. 2017. Characterization of mechamisms underlying degradation of sclerotia of Sclerotinia sclerotiorum by Aspergillus aculeatus Asp-4 using a combined qRT-PCR and proteomic approach. BMC Genomics. 18:674. https://doi.org/10.1186/s12864-017-4016-8.
Roberts, D.P., Mattoo, A.K. 2018. Sustainable Agriculture - Enhancing environmental benefits, food nutritional quality and building crop resilience to abiotic and biotic stresses. Agriculture. 8:8. https://doi.org/10.3390/agriculture8010008.
