2013 Annual Report
1a.Objectives (from AD-416):
Develop molecular tools to detect, identify, characterize, and counteract the pathogenicity of soilborne pathogens, such as Rhizoctonia solani in ornamental crops and turfgrasses. Examine episomal and chromosomal genetic elements affecting biology or virulence of R. solani. Analyze gene expression of important soilborne pathogens, such as R. solani, to understand the virulence of the organism. Evaluate transgenic plants, including ornamentals for resistance to fungal pathogens. Improve the efficacy and consistency of biological control agents for important soilborne pathogens (Rhizoctonia, Fusarium and Ralstonia) through combination with organic amendments, new and safer chemicals, composts, and reduced-risk fungicide(s). Screen plant extracts and reduced-risk chemicals with broad spectrum properties against soilborne pathogens. Study structure/activity relationships of potential reduced-risk chemicals from plant extracts for understanding biocidal effects on R. solani or other pathogens. Investigate the combined effectiveness of bio-fumigation and/or soil-treatment with botanical extracts, antagonistic microbe(s), reduced-risk chemicals, or compost made from pine needles to control soilborne pathogens.
1b.Approach (from AD-416):
Utilize molecular approaches such as UP-PCR, rDNA sequencing, etc. to distinguish pathogenic Rhizoctonia isolates and to group them. Construct expressed gene cDNA libraries of virulent and hypovirulent Rhizoctonia isolates, and analyze to identify differentially expressed genes. Develop transformation system for R. solani. Evaluate transgenic gladiolus for resistance against Fusarium oxysporum, fsp gladioli. Screen to identify plant extracts inhibitory to R. solani and other soilborne plant pathogens. Evaluate antagonistic fungi, bacteria, or other microbes to check their effectiveness alone or in combination with biorationals or soil amendments in controlling soilborne pathogens of ornamental crops.
This is the final report for ARS project 1230-22000-029-00D, which expired on May 19, 2013. Progress on the new project, 1230-22000-039-00D is documented in a separate report. Significant progress was made on both objectives of this Project.
In Objective 1A, we compared the ITS sequences with genome fingerprints generated by UP-PCR of Rhizoctonia solani, the causal agent of blight in many turf species. Both methods of clustering corresponded well and also indicated the presence of a new group of Waitea species in the transition zone of USA. In addition, our collaborator at Washington State University developed a sensitive Q-PCR assay to detect Rhizoctonia tuliparum from gladiolus bulbs and infested soil. Additional information is provided in the report for Project 1230-22000-039-01S. Also in Objective 1A, we initiated a project to sequence the genome of selected isolates of Rhizoctonia solani and Cylindrocladium pseudonaviculatum in order to characterize genes that could contribute to identification, pathogenesis and significant biological processes. Additional information is provided in the report of Project 1230-22000-039-02S.
In Objective 1C, we bio-assayed by root infection of several transgenic gladiolus lines expressing putative fungal resistance genes against Fusarium oxysporum f. sp. Gladioli (Fog). We used both wild type Fog as well as cyan fluorescent marker transformed Fog to check the degree of infestation in various transgenic gladiolus lines by the pathogen.
In Objective 2C, we screened poultry feather waste for Trichoderma, which may be useful as biocontrol agents and also to assist in slow release of nitrogen from the feathers. We identified four Trichoderma isolates that degraded feather fibers in culture. In greenhouse studies, three of the isolates effectively controlled Rhizoctonia solani infection of cucumber seedlings. Also in Objective 2C, we screened environmental samples for the presence of bacteriophages against a soilborne pathogen Ralstonia solanacearum and a foliar pathogen Pseudomonas syringae pv tomato.
Development of a reference map of soybean axis embryonic proteins. Soilborne pathogens such as Pythium ultimum, P. sojae, Fusarium species and Rhizoctonia species cause pre-and post-emergence damping off of seedlings and root and crown rots. During germination, embryonic axes are known to express several defense related genes upon exposure to colonized pathogens. A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was applied to establish a proteomic reference map for the soybean embryonic axis. Our 2D-profiling of soybean axis proteins has established for the first time a baseline proteome on which we could investigate and compare factors affecting the interaction of pathogenic and beneficial organisms in the spermosphere during seed germination.
Global identification of R. solani genes responsible for pathogenesis and survival. Rhizoctonia solani is a ubiquitous soilborne fungal pathogen causing pre- and post-emergence damping off of economically important crops, including ornamentals and turfgrasses. In collaborative efforts, researchers from ARS and Towson University conducted a pilot-scale assessment of gene (ESTs) diversity of R. solani from two gene libraries of the pathogen grown under two virulence conditions. A total of 1025 unique genes were identified, some of which have potential roles in fungal pathogenicity, virulence, and several other important biological processes. Identified genes were categorized into 11 functional groups. A codon-usage table was formulated based on 14694 R. solani codons. This is the first report of global gene investigation of the pathogenic fungus. Further analysis may provide molecular targets for pathogen identification, and may lead to elucidation of the roles these genes play in fungal pathogenesis and ecological fitness.
Lakshman, D.K., Roberts, D.P., Alkharouf, N., Mitra, A., Natarajan, S.S. 2012. Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors. Phytopathology. 104(5):1020-1035.
Natarajan, S.S., Krishnan, H.B., Khan, F.H., Chen, X., Garrett, W.M., Lakshman, D.K. 2013. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry. Journal of Basic and Applied Sciences. 9:309-332.
Lakshman, D.K., Pandey, R., Kamo, K.K., Bauchan, G.R., Amitava, M. 2012. Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus. European Journal of Plant Pathology. 133:729-738.