Location: Sustainable Agricultural Systems Laboratory
2022 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 report covers work during all five years of the project (2017 – 2022). Over this period 26 peer-reviewed manuscripts and 10 reviews, book chapters, and research notes were published. They covered conventional and biological control of Pythium ultimum, Rhizoctonia solani, and Sclerotinia sclerotiorum; the rhizosphere and endophytic microbial community; biological control mechanisms; and genomics of the R. solani species complex and biological control agents.
There were also first disease reports. A Rhizoctonia pathogen (Waitea circinata var. prodigus) causing sheath spot disease on maize was identified in collaboration with scientists at Chaudhary Charan Sing Haryana Agricultural University (CCSHAU), India, and published in Plant Disease. Other pathogenic Rhizoctonia species, Thanatephorus cucumeris AG 2-1 and Ceratobasidium sp. AG-K isolates were shown to cause lily root rot in the US. Six fungal pathogens (Bipolaris zeicola, Curvularia papendorfii, C. siddiquii, C. sporobolicola, C. graminicola, Alternaria sp.) causing maize leaf blight diseases in India were identified in collaboration with Indian scientists at CCSHAU, ICAR-Institute of Maize Research, Banaras Hindu University, and National Bureau of Plant Genetic Resources. In collaborative research with North Dakota State University (NDSU), Rhizopus arrhizus, causing root rots in North Dakota and Minnesota; S. sclerotiorum, causing root rot and leaf blights in Minnesota; and Fusarium equiseti causing seedling death on sugar beet in Minnesota were identified and reported for the first time.
For Sub-objective 1A, microsatellite primers from the assembled R. solani AG2-2 IIIB genome sequence were identified using bioinformatics. However, microsatellite-based genome fingerprinting of this group of isolates was stopped because similar research had been recently published. Instead, in collaboration with Washington State University, isolates of Rhizoctonia tuliparum were identified, taxonomically characterized, and a quantitative PCR-based method for specific detection was developed and published. R. tuliparum causes Grey Bulb Rot of tulips and bulbous iris. In collaboration with CCSHAU, India, it was demonstrated that ITS2-based phylogenetic identification of W. circinata sub-species could be used instead of the entire ITS sequence (ITS1-5.8S-ITS2). Molecular identification work is important as R. solani species complex pathogens are morphologically similar but need different control measures. In collaboration with the Indian Institute of Maize Research, the Indian Agricultural Research Institute, and the University of Florida, the genetic diversity of Rhizoctonia pathogens causing banded leaf and sheath blight in maize was published.
For Sub-objectives 1B and 1C, genome sequencing, assembly, and annotation of 12 plant pathogenic isolates from the R. solani species complex covering 7 anastomosis groups (AGs) and selected subgroups [including AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3 (isolates AG3-PT, AG3-Rhs1A1, AG3-TB), AG4-HG-I (isolates Rs23A, R-118), AG5, AG6 (isolate 10EEA), AG8] was completed and supplemented with gene expression analysis to identify genes involved in pathogenesis, including secretomes and effectors. Using a pangenome comparative analysis of the 12 R. solani isolates and 15 other Basidiomycetes, unique and shared secretomes, CAZymes, and effectors across the AGs were identified. R. solani-derived factors potentially involved in determining AG-specific host preference, and the attributes distinguishing them from other Basidiomycetes were elucidated. Finally, the largest repertoire of R. solani genomes and their annotated components was presented as a comprehensive database, RsolaniDB, with tools for large-scale data mining, functional enrichment, and sequence analysis not available with other state-of-the-art platforms. From 02/01/2021 to 6/9/2022, RsolaniDB was accessed by researchers 368 times, and genomic contents downloaded 110 times, underscoring the usefulness of RsolaniDB. This work was published in Frontiers in Microbiology.
For Objective 2, it was demonstrated that several soybean root-nodule-associated, non-rhizobial bacterial endophytes were antagonistic to R. solani AG4 and S. sclerotiorum in vitro. Select nodule-inhabiting endophytic species also suppressed tomato wilt caused by Clavibacter michiganensis subsp. michiganensis, and bacterial speck caused by Pseudomonas syringae pv. tomato. Publication of this work established the foundation for further exploration of nodule-associated bacteria for plant protection and growth promotion. In collaboration with NDSU the novel fungus Penicillium pinophylum, associated with sugar beet taproots, was isolated, characterized, and control of Rhizoctonia root rot demonstrated in both the laboratory and greenhouse. Thymol, the key component of thyme oil, and six derivatives were evaluated for fungicide activity against R. solani. 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. In published research with the Agricultural Research Station, Banswara, Rajasthan, India and Lakshmi Bai Central Agricultural University, India, it was demonstrated that combined applications of Trichoderma harzianum and Pseudomonas fluorescens synergistically controlled rice blast and bacterial leaf blight in the field. In other published field trials with fungicides and antagonistic bacteria, it was found that Azoxystrobin 23 SC, Pseudomonas fluorescens DTPF-3, and Bacillus subtilis DTBS-5 were most effective in controlling Corynespora cassiicola on mungbean.
Also, for Objective 2, field research was continued and published in collaboration with the Oil Crops Research Institute (OCRI), People’s Republic of China, on biological control agents to apply at strategic points during the S. sclerotiorum disease cycle. Three Bacillus isolates were applied individually and in combinations as seed treatments and tested in field trials conducted at four locations with different soils. The treatment containing only B. subtilis BY-2 resulted in significant reductions in disease at all four locations. There was incremental, although not statistically significant, reduction in disease with increasing number of isolates in the treatments at three field locations. In plant growth promotion studies conducted in pots, the seed treatment containing all three isolates resulted in seed yield greater than the non-treated control in four of the five soils used. This plant growth promotion may be due to production of indole acetic acid (IAA) or increased phosphate availability as these strains produced IAA and increased soil phosphate levels. Further field studies demonstrated that isolate BY-2 significantly reduced Sclerotinia disease relative to the nontreated control when applied in a green-manure biofertilizer. This provides another application method for Bacillus biological control agents. Field experiments were also conducted at two locations with different soils to determine if combining different biological agents applied at different points in the disease cycle improves biological control of S. sclerotiorum. Seed treatment with three Bacillus isolates was combined with a spray application of the mycoparasite Aspergillus aculeatus Asp-4 to soil prior to planting the crop plant. The second year of field trials has been completed and the data is currently being analyzed and prepared for publication.
For Sub-objective 3A, in collaboration with the OCRI, ITS and 16S sequence analysis was used to detail the impact of a biofertilizer containing mycoparasitic Trichoderma sp. Tri-1 on the soil microbial community. In collaboration with USDA-ARS collaborators in Beltsville, Maryland, two manuscripts detailing the impact of different rice cultivars on the associated rhizosphere community were published in Soil Biology and Biochemistry and in Diversity.
For Sub-objective 3B, mechanisms underlying colonization and degradation of sclerotia of S. sclerotiorum by A. aculeatus Asp-4 were characterized using transcriptomics, proteomics, and qRT-PCR in collaboration with OCRI. Results indicate co-temporal up-regulation of genes and proteins involved in degradation of sclerotial compounds and mitigation of environmental stress by Asp-4 during colonization. A paper describing this work was published. Work demonstrated that a lacZ-tagged B. subtilis strain could enter and persist within root tissue for at least four months when applied as a seed treatment or in biofertilizer. Transcriptomic analysis of the impact of seed treatment with B. subtilis isolate BY-2 on the plant defense response was continued in collaboration with the OCRI. A large library of up-regulated and down-regulated plant genes is currently being analyzed to determine the plant defense-related genes regulated in response to colonization of internal/external plant tissues by 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 was analyzed in collaboration with scientists at the Unversidade Federal de Lavras, Brazil.
For Sub-objective 3C, works on sequencing and annotating the genomes of the biological control agents Serratia marcescens N4-5 and Bacillus velezensis were published in collaboration with the Universidade Federal de Lavras, Brazil. The N4-5 genome contained the complete pig cluster, the genes responsible for prodigiosin production, and biosynthetic genes for the surfactant serrawettin W1. A manuscript was published that detailed the role of prodigiosin in control of P. ultimum-damping-off of cucumber by ethanol extract of isolate N4-5. This is the first report indicating that prodigiosin can control a plant disease; providing a new chemistry to exploit in development of controls for oomycetes.
Accomplishments
1. Rhizoctonia solani species complex database (RsolaniDB) updated with new features and genomic information. Accurate identification of many Rhizoctonia pathogens of diverse genetic composition is essential for the selection of appropriate disease control tools as morphologically similar Rhizoctonia species complex isolates can vary in sensitivity to chemical or biological control measures. In a collaborative effort between USDA-ARS, Beltsville, Maryland, scientists and scientists at King Abdullah University of Science and Technology (KAUST), Saudi Arabia, six additional annotated R. solani genomes were added to RsolaniDB for a total of 18 genomes. RsolaniDB was also improved to make it more resourceful for pangenomic comparative investigations and for developing methods for identifying different isolates within the R. solani species complex. This database is a tremendous resource for plant pathologists, mycologists, and molecular biologists devising specific molecular detection methods for members of this morphologically similar but genetically different complex of economically important plant pathogens.
2. Specific quantitative detection method for tulip gray bulb rot pathogen. Gray bulb rot of tulips and bulbous iris, caused by the soil-borne fungus Rhizoctonia tuliparum (Rtul), is an economically important pathogen in the USA. In a collaborative effort between USDA-ARS, Beltsville, Maryland, scientists and scientists at Washington State University, a sensitive and specific quantitative PCR (qPCR) assay was developed for detection of Rtul on tulip and iris bulbs and in soil samples. Previously there were no reliable and rapid assay methods to assess inoculum levels of Rtul in the soil and on infected bulbs for early detection of gray bulb rot. Early detection being a key to the successful management of this disease. Moreover, these scientists clarified the taxonomy of Rtul using ribosomal internal transcribed spacer (ITS1-5.8S-ITS2) analysis. This quantitative pathogen identification method and the taxonomic characterization of the pathogen will be highly useful for fungicide selection and timing of disease management practices by extension plant pathologists and scientists.
Review Publications
Bhuiyan, Z.M., Lakshman, D.K., Mendoza, L., Mosher, P., Khan, M.F. 2021. Sclerotinia sclerotiorum (Lib) deBary causes root rot and necrosis in sugar beet in Moorhead, MN USA. Journal of Plant Protection Research. 61(4):384-391. https://doi.org/10.24425/jppr.2021.139247.
Bhuiyan, Z.M., Lakshman, D.K., Mosher, P., Khan, M.F. 2021. Identification of Rhizopus arrhizus (Fisher) causing root rot in sugar beet in North Dakota and Minnesota, USA. Journal of Plant Pathology. 104:357-362. https://doi.org/10.1007/s42161-021-00967-2.
Bhuiyan, Z.M., Lakshman, D.K., Mosher, P., Khan, M.F., Cymbaluk, T., Peters, D. 2021. Morphological and molecular characterization of sclerotinia sclerotiorum on sugar beet in Montana, USA. Plant Health Progress. https://doi.org/10.1094/PHP-11-21-0137-BR.
Singh, V., Lakshman, D.K., Roberts, D.P., Ismaiel, A.A., Abhishek, A., Hooda, K., Pal, I. 2021. Fungal species causing maize leaf blight in different agro-ecologies in India. Pathogens. 10:1621. https://doi.org/10.3390/pathogens10121621.
Coats, K., Debauw, A., Lakshman, D.K., Roberts, D.P., Ismaiel, A.A., Chastagner, G. 2022. Detection and molecular phylogenetic-morphometric characterization of Rhizoctonia tuliparum, causal agent of Grey Bulb Rot of tulips and bulbous iris. The Journal of Fungi. 8(2):163. https://doi.org/10.3390/jof8020163.
Kaushik, A., Roberts, D.P., Ramaprasad, A., Mfarrej, S., Nair, M., Lakshman, D.K., Pain, A. 2022. The pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solani and development of a comprehensive web resource: RsolaniDB. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2022.839524.
Khan, M.F., Bhuiyan, M.Z., Liu, Y., Lakshman, D.K., Bloomquist, M. 2021. First report of leaf blight of sugar beet (Beta vulgaris L.) caused by Sclerotinia sclerotiorum in Minnesota, USA. Plant Health Progress. 22:149-150. https://doi.org/10.1094/PHP-02-21-0048-BR.
Singh, V., Lakshman, D.K., Roberts, D.P., Hooda, K.S., Gogoi, R. 2021. Morphopathological and molecular morphometric characterization of Waitea circinata var. prodigus causing a novel sheath spot disease of maize in India. Plant Disease. 106:526-534. https://doi.org/10.1094/PDIS-05-21-0951-RE.
Bairwa, N.K., Jambhulkarv, P.P., Sushmita, V., Meenakshi, A., Manjunatha, N., Bajpai, R., Singh, D., Mani, C., Kumar, S., Chaturvedi, S.K., Lakshman, D.K. 2022. Evaluation of fungicides and bacterial antagonists for management of Corynespora leaf spot on mungbean (Vigna radiata L. Wilczek). Archives of Phytopathology and Plant Protection. 55:433-453. https://doi.org/10.1080/03235408.2022.2025686.
