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United States Department of Agriculture

Agricultural Research Service

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

Location: Floral and Nursery Plants Research Unit

2011 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 the process of evaluating various molecular techniques for accurate identification of Rhizoctonia species infecting turfgrasses, we tested the sensitivity of cross-hybridization of UP-PCR products to identify Rhizoctonia isolates into their respective anastomosis group (AG) and subgroup levels. We also analyzed the UP-PCR (Universally Primed Polymerase chain reaction) genome fingerprints of various Rhizoctonia isolates and identified two SCAR (sequence characterized amplified region) markers which successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from other AGs and subgroups of Rhizoctonia infecting turfgrasses.

A collaborator from the University of Nebraska, Lincoln, has transformed tobacco (Nicotiana tabaccum var Xanthi) and Arabidopsis (A. thaliana) plants with lactoferrin, a milk protein with reported antifungal antibacterial and antiviral properties. We tested the transgenic lines for fungi-toxicity and against infection with Rhizoctonia solani in the laboratory, growth chamber and greenhouse.

With a possibility of using bacteriophages for biocontrol of the bacterial pathogen Pseudomonas syringae pv. tomato, we screened phages from samples collected from different environments. In this process, we have detected lytic phages from some environmental samples. Further characterization of the nature and biocontrol ability of those phages will be carried out.


4.Accomplishments
1. Accurate molecular identification of species and biologic groups of a soilborne fungal pathogen Rhizoctonia solani, causing patch diseases of turfgrasses. Rhizoctonia species, which cause patch diseases, are considered the most important pathogens of turfgrasses. Because they are such a diverse group, Rhizoctonia species are often difficult to identify. Lack of knowledge of pathogen identity can result in excessive application of fungicides or application of ineffective fungicides, which leads to higher costs and environmental pollution. The traditional method of identifying Rhizoctonia species by hyphal anastomosis reactions is time-consuming and often unreliable. We have developed tools to identify Rhizoctonia species and groups using molecular techniques, including ribosomal DNA-ITS sequence analysis, genomic fingerprinting (UP-PCR) and cross hybridization, and SCAR markers. These tools can reliably identify the correct genus, species, anastomosis group (AG), and sub-species of Rhizoctonia isolates, and can accurately differentiate isolates of the major pathogenic groups AG 1-IB and AG 2-2IIIB. These identification tools will result in better disease control strategies by more effective use of fungicides and by enabling breeders to develop resistant cultivars.

2. Novel transgenic approach to Rhizoctonia resistance in plants. Introduction of resistance genes against plant pathogens often suffer from criticism for the possible toxic effect on consumers and environmental effects of the introduced gene. Bovine lactoferrin is traditionally considered a safe protein for human consumption. We have transformed test plants of tobacco and Arabidopsis with the bovine lactoferrin gene to study its effect on Rhizoctonia solani, a pathogen that causes several economically important plant diseases. Transgenic tobacco plants exhibited high levels of Rhizoctonia resistance in detached leaf assays. Similarly, transgenic Arabidopsis seedlings were resistant to the fungus and did not exhibit damping off symptoms. The antifungal effect of lactoferrin and successful control of rhizoctonia disease in transgenic tobacco and Arabidopsis demonstrates a potentially new approach for control of diseases caused by fungal pathogens.

3. Clove oil as an effective soil fumigant to control Southern wilt in Geranium and tomato. Bacterial wilt or Southern wilt caused by Ralstonia solanacearum is a devastating disease that is distributed worldwide in tropical, subtropical and warm temperate regions. This soilborne vascular disease attacks over 450 plant species including ornamentals such as geranium and is a major constraint on production of many economically important crops such as tomato, tobacco, potato and banana. There are very few chemical control methods developed against bacterial diseases. Mercurial pesticides are banned because of genotoxicity, and many plant pathogenic bacteria develop resistance to conventional copper-based and antibiotic pesticides. Through laboratory and greenhouse bioassays we confirmed that clove oil effectively controls Southern wilt of tomato and geranium when used to drench soil as a pre-planting amendment. Thus, we have developed an effective and safer control method against the Southern wilt pathogen.


Review Publications
Nguyen, T., Lakshman, D.K., Han, J., Mitra, A. 2011. Transgenic expression of Lactoferrin imparts resistance to a soilborne fungal pathogen Rhizoctonia solani. Journal of Plant Pathology. 2(1):1-8.

Roberts, D.P., Lohrke, S.M., Mckenna, L.F., Lakshman, D.K., Kong, H.N., Lydon, J. 2011. Mutation of a degS homologue in Enterobacter cloacae decreases colonization and biological control of damping-off caused by Pythium ultimum on cucumber. Microbiology. 101:271-280.

Last Modified: 7/30/2014
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