2012 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.
Progress was made on both objectives and their sub-objectives, all of which fall under Performance Measure 5.2.3, National Program 308, Component I, Pre-plant Soil Fumigation Alternatives.
Rhizoctonia solani is a ubiquitous soilborne fungal pathogen causing pre- and post-emergence damping off of economically important crops, ornamentals and turfgrasses. Fulfilling Objective 1B, a global gene expression study based on analysis of expressed sequence tags was undertaken to understand the molecular mechanisms of R. solani pathogenesis. A total of 1025 unique genes were identified from two R. solani cDNA libraries, some of which have potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. Further analysis of identified genes may provide molecular targets for pathogen identification and shed insights into virulence mechanisms of R. solani as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.
In Objective 1C, we have transformed a fungal biocontrol agent, Trichoderma harzianum isolate Th23, with hygromycin resistance and cyan fluorescence genes via Agrobacterium tumefaciens in order to understand its mechanism of biocontrol. We also attempted to transform pathogenic isolates of Rhizoctonia solani (Rs23) and Fusarium oxysporum pv. tomato (Fot) with hygromycin-B resistance and green fluorescence genes via A. tumefaciens. Of the two pathogenic fungi, only the Fot was successfully transformed. Both the transformed fungi showed resistance to 100 ug/ml hygromycin–B. The fluorescence emission of the respective transformed fungal isolates and interactions of the pathogenic fungus (i.e., Fot) with the biocontrol agent (i.e., Th23) will be studied with confocal microscopy.
Soilborne pathogens have traditionally been controlled using chemical pesticides, some of which are inconsistent in efficacies and toxic to the environment. Plants can be considered as renewable reservoirs of secondary metabolites, some of which are antimicrobial (i.e., biorationals) in nature and are less toxic to animals and plants, easily biodegradable, and stimulate host plant metabolism. Under Objectives 2A, B and C, we have tested thirty plant extracts or plant-derived chemicals against a broad host range isolate of R. solani (AG 4). While several of these compounds were confirmed to be fungicidal or fungistatic in the laboratory bioassays, we also demonstrated that certain plant chemicals are effective soil amendments for the control of pre- and post-emergence damping off of cucumber seedlings caused by R. solani in the greenhouse.
Transgenic approach to impart resistance to a soilborne fungal pathogen Fusarium graminearum. Introduction of new or modified genes to develop plant resistance against pathogens is a sustainable disease control strategy. The soilborne fungal pathogen Fusarium graminearum causes Fusarium head blight - a devastating disease of wheat and barley that reduces both grain yield and quality. In a collaborative effort between the University of Nebraska, Lincoln, and the ARS in Beltsville, MD, researchers tested a broad-spectrum antimicrobial bovine lactoferrin for resistance against the pathogen by incorporating the gene in wheat. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in plants expressing lactoferrin compared to control Bobwhite and two untransformed susceptible commercial wheat cultivars. In an earlier report, those researchers also demonstrated that lactoferrin imparts resistance in tobacco and Arabidopsis against a different soilborne fungal pathogen, Rhizoctonia solani. Incorporation of the lactoferrin gene into commercial cultivars has great potential to augment resistance against plant pathogens.
Genetic transformation of a soilborne fungus (Fusarium oxysporum f.sp. gladioli) to study pathogenesis in Gladiolus. Gladiolus is grown world-wide as an economically important ornamental crop. In 2010 the wholesale value of Gladiolus cut flowers in the U.S. was $22,809,000. Fusarium rot caused by F. oxysporum f. sp. gladioli (Fog) is one of the most serious diseases of gladiolus, both in the field and in stored bulbs. Traditionally, the pathogen had been controlled by fumigating soil with methyl bromide which is now banned. Very few chemical fungicides are available to control the disease, mostly due to insufficient knowledge on how the pathogen causes disease in gladiolus. In order to understand how the fungus causes disease on gladiolus, ARS researchers in Beltsville, MD have genetically introduced green, yellow and cyan fluorescence genes by transformation with Agrobacterium tumefaciens. The fluorescing pathogen will be very useful to identify genes of the fungus responsible for causing disease in gladiolus and to understand how transgenically resistant gladiolus lines ward off the pathogen.
Kong, H., Roberts, D.P., Patterson, C.D., Kuehn, S., Heeb, S., Lakshman, D.K., Lydon, J. 2012. Effect of overexpressing rsmA from Pseudomonas aeruginosa on virulence of select phytotoxin-producing strains of P. syringae. Phytopathology. 102:575-587.
Natarajan, S.S., Xu, C., Garrett, W.M., Lakshman, D.K., Bae, H. 2011. Assessment of the natural variation of low abundant metabolic proteins in soybean seeds using proteomics. Journal of Plant Biochemistry and Biotechnology. 21:30-37.
Dhar, A., Kaizer, K., Betz, Y., Harvey, T., Lakshman, D.K. 2011. Identification of the core sequence elements in Penaeus stylirostris densovirus promoters. Virus Genes. 43:367-375.