2010 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.
A project was initiated to identify brown patch-causing Rhizoctonia species in turfgrasses from Virginia and Maryland. Species of Rhizoctonia in general are not well identified due to morphological similarities and lack of mating relationships. The traditional method of grouping Rhizoctonia spp. is based on time consuming and labor intensive hyphal anastomosis reactions. However, certain isolates do not self anastomose, or anastomose with more than one group. Knowledge of genetic diversity and evolutionary groups within different Rhizoctonia spp. could help in developing resistant turfgrass cultivars and in proper management of Rhizoctonia diseases. More than 600 Rhizoctonia isolates were collected from diseased turfgrass leaves from five geographic areas in Virginia and Maryland. Grouping of 54 randomly selected isolates at the anastomosis group (AG)-level was accomplished by hyphal fusion reactions with Rhizoctonia tester strains. The sequences of ribosomal DNA internal transcriber spacer (ITS) region were determined, and DNA fingerprinting with the Universally Primed Polymerase chain reaction (UP-PCR) and cross-hybridization of UP-PCR products was carried out. Labeled UP-PCR products of Rhizoctonia strains of established anastomosis groups were used as probes for the cross-hybridization technique. Out of all the tested R. solani isolates, 56% belonged to AG2-2IIIB and 40% belonged to AG1. Only one isolate of AG5 was observed. Related Ceratobasidium sp. and Waitea species (W. circinata var circinata, var zeae, and var oryzae; Wcc, Wco and Wcz) were observed at a lesser frequency from most of the areas investigated. Our results confirmed that UP-PCR followed by cross-hybridization technique could identify Rhizoctonia isolates into their respective AG subgroup level. (NP 308, C1, PA 1C, PM 5.2.3).
Progress was also made in developing Expressed Sequence Tags (ESTs) in Rhizoctonia solani to understand its biology and pathogenicity. Rhizoctonia solani is a basidiomycetous and ubiquitous soilborne fungal pathogen causing damping off of seedlings, aerial blights, and postharvest diseases. To gain insight into the molecular mechanism of pathogenesis and virulence, a global approach based on the analysis of expressed sequence tags (ESTs) was undertaken by ARS scientists at Beltsville, MD. Two EST libraries were developed from mycelia grown under virulence-differentiated conditions. A pilot scale assessment of gene diversity was made from the sequence analyses of the two libraries. From the total sequences, many genes exhibited strong similarities with genes in public databases and were categorized into several functional groups. We have identified several cDNAs with reported roles in pathogenicity or virulence. Further analysis of EST clones may provide insights into virulence of this important fungal plant pathogen as well as roles of genes in development, saprophytic colonization and ecological adaptation of R. solani.
Transcriptional analysis of genes of a densovirus to identify transcriptional promoters. Promoters are genomic DNA sequences located upstream of an expressed gene and are known to facilitate initiation of gene expression. Viruses infecting eukaryotes are generally explored as sources of stronger, constitutive, organ or tissue non-specific promoters. For example, promoter sequences from simian virus 40, cytomegalovirus, and cauliflower mosaic virus, are commonly used in cross-kingdom gene expression studies of eukaryotic organisms. However, availability and intellectual property (IP) issues often restrict their use in gene expression investigations. ARS scientists at Beltsville, MD, in collaboration with scientists at Advanced Bionutrition Corp., have identified and characterized three promoter elements of Penaeus stylirostris densovirus (PstDNV) and found that all of them function in vitro in bacteria, insect, fish, and shrimp cells. In addition, we have precisely determined the transcription start and termination sites of the three promoters and found that the ‘left’ promoter had the highest activity followed by the ‘middle’ and ‘right’ promoters. These promoters can be used in gene expression experiments in Rhizoctonia solani or other pathogens to elucidate roles of specific genes in plant disease development.
Ribosomal DNA-ITS sequence analysis aids in accurate identification of brown patch disease-causing Rhizoctonia species from turfgrasses. Rhizoctonia species, which cause brown patch disease in turfgrasses, are considered one of the most important pathogens on turf. The traditional method of identifying Rhizoctonia species by hyphal anastomosis reactions is often unreliable and time consuming. An ARS scientist in Beltsville, MD and cooperators analyzed DNA sequences of the ribosomal ITS region of Rhizoctonia isolates from Maryland and Virginia to develop an efficient molecular detection assay of the pathogens causing brown patch disease of turfgrasses. Development of more accurate molecular identification of Rhizoctonia spp. will help in developing resistant turfgrass cultivars and in proper management of brown patch disease.
Dhar, A.K., Lakshman, D.K., Amundsen, K., Robles-Sikisaka, R., Kaiser, K., Roy, S., Hasson, K.W., Allnutt, F.C.T. 2010. Characterization of the Taura Syndrome virus isolate originating from the 2004 Texas epizoonatic in cultured shrimps. Archives of Virology. 155:315-327.
Dhar, A.K., Kaizer, K.N., Lakshman, D.K. 2010. Transcriptional analysis of Penaeus stylirostris densovirus genes. Virology. 402:112-120.
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