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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY BASED MANAGEMENT STRATEGIES FOR CONTROL OF SOIL-BORNE PATHOGENS ....AN ALTERNATIVE TO METHYL BROMIDE PRE-PLANT SOIL FUMIGATION
2009 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.


3.Progress Report
In research collaboration with the Sustainable Agricultural Systems Laboratory to understand the mechanism of root-colonization by Enterobacter cloacae, we have identified and analyzed stress response genes of E. cloacae 501R3, which shows promise as a biocontrol agent for damping-off of cucumber caused by Pythium ultimum. E. cloacae C10 is a mutant of 501R3 that showed reduced colonization of cucumber roots and suppression of damping-off of cucumber. Molecular characterization of C10 indicated that the mutation was inserted in a region of the E. cloacae genome with a high degree of DNA sequence similarity to degS, a periplasmic serine protease. Further studies and manipulation of E. cloacae degS and other genes may help us to develop strains with enhanced root colonization and biocontrol efficiencies.

In collaboration with a scientist from Virginia Tech, we have initiated identification of isolates of Rhizoctonia solani causing brown patch disease in turfgrasses. Rhizoctonia species in general are not well understood due to morphological similarities and lack of mating relationships. The traditional method of grouping Rhizoctonia spp. is based on hyphal anastomosis reactions, a time consuming and labor intensive method. In addition, certain isolates do not self anastomose, or anastomose with more than one group. Knowledge of genetically and evolutionarily different groupings within different Rhizoctonia spp. could help in developing resistant turfgrass cultivars. More than 400 Rhizoctonia isolates were collected from diseased turfgrass from five areas in Virginia and Maryland. A random sample of 54 isolates was selected for anastomosis group (AG) identification by hyphal fusion reactions with Rhizoctonia tester strains, and the sequence of their internal transcriber spacer (ITS) region of ribosomal DNA was determined. The phylogenetic analysis of ITS corresponded well with previously known AG sequences, grouping similar AG sequences together. Anastomosis supported genetic relatedness of the isolates as determined by sequence analysis. Out of randomly selected R. solani isolates of turfgrasses, 56% belonged to AG2-2IIIB followed by 40% of AG-1. Only one isolate of AG5 was observed. Ceratobasidium sp. and Waitea cercinata cercinata / Waitea cercinata zeae were observed at lesser frequency.

Summaries of research conducted under Specific Cooperative Agreements between the Floral and Nursery Plants Research unit and others can be found in the following specific annual reports: ‘Accurate identification and gene expression in relation to virulence of Rhizoctonia isolates infecting turfgrasses’ with both Virginia Tech University (#1230-22000-029-02S) and the United States Golf Association (#1230-22000-019-01T).


4.Accomplishments
1. Identification of clove oil as a effective antibacterial soil fumigant to control Ralstonia solanacearum. Methyl bromide (MB) had been an important component of soiborne plant pathogen control. However, due to the ban of MB use, there is a need for developing safer alternative fumigants. In collaboration with another researcher from the Floral & Nursery Plants Research Unit, we have identified the oil of clove (Syzygium aromaticum) as a potential soil-fumigant to eradicate major groups of plant pathogenic bacteria. Clove oil inhibited the growth of both gram(+) and gram(-) bacteria and its effect was dose-dependent. The bacteria also displayed different degrees of sensitivity to the clove oil, with Ralstonia being the most sensitive and Rhodococcus the least. In greenhouse experiments, pre-plant fumigation of soil-less potting mix with a clove oil formulation effectively controlled bacterial wilt of geranium and tomato caused by R. solanacearum race 1, biovar 1 by preventing symptom formation and bacterial colonization of the host plants. We have demonstrated that pre-emergence fumigation of greenhouse soil-less potting mix eradicated the pathogen, resulting into disease-free plants. The phytotoxic effect of clove oil was reduced by extending the evaporation time following clove oil treatment of soil. This antibacterial activity, in addition to the previously reported fungicidal and nematicidal properties makes clove oil an ideal candidate for use as a broad-based soil fumigant to control various soilborne pathogens.

2. Development and application of two protein extraction protocols for proteomic investigations of R. solani to examine virulence and pathogenicity. Rhizoctonia solani (essentially an asexual fungus) is highly intractable to genetic manipulations for pathogenicity studies. Current genomic and functional genomic approaches address those limitations. Proteomics is a functional genomic approach to identify factors, for example, in pathogenesis, determining targets for pathogen identification, and soft targets to control the pathogen. In a collaborative project with researchers from the Soybean Genomics & Improvement Laboratory and the Animal Biosciences & Biotechnology Laboratory, we developed two protocols for purification of total cellular proteins of R. solani, resolved proteins in 2-D gels and identified proteins. Thus far, we have analyzed 150 of the major and minor cellular protein spots. The proteins were compared with cross-species fungal protein and Gene Ontology (GO) databases and with MASCOT (Matrix Science), BLAST (NCBI) and AmiGO (GO) search utilities. At least 140 protein spots were positively identified and could be associated with nucleus, mitochondria, cytosol, vacuole, plasma membrane, cell wall, and surface proteins. Identification of proteins, even in absence of a homologous annotated genome database, demonstrates the feasibility of carrying out investigations of R. solani protein profiling and pathogenicity. Identification of proteins involved in pathogenicity may lead to new means of disease control. This is the first comprehensive report of proteomic investigation of a known anastomosis group (AG.
4)of R. solani.


6.Technology Transfer

Number of Other Technology Transfer1

Review Publications
Lakshman, D.K., Natarajan, S.S., Garrett, W.M., Lakshman, S., Dhar, A.K. 2008. Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani. Mycologia. 100:867-875.

Meyer, S.L.F., Lakshman, D.K., Zasada, I.A., Vinyard, B.T., Chitwood, D.J. 2008. Phytotoxicity of clove oil to vegetable crop seedlings and nematotoxicity to Root-knot Nematodes. HortTechnology. 18:631-638.

Dugan, F.M. 2008. Fungi, Folkways and Fairy Tales: Mushrooms & Mildews in Stories, Remedies & Rituals, from Oberon to the Internet. North American Fungi 3 (7): 23-72. http://www.pnwfungi.org/pdf_files/manuscripts_volume_3/naf20087/naf200874.pdf

Last Modified: 12/22/2014
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