Robert Carver | 12/11/2008 2:43:26 AM
ACCESSION NO: 0215856 SUBFILE: CRIS
PROJ NO: LAB93932 AGENCY: CSREES LA.B
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2008 TERM: 30 SEP 2013
INVESTIGATOR: Valverde, R. A.
PLANT PATHOLOGY & CROP PHYSIOL
LOUISIANA STATE UNIVERSITY
BATON ROUGE, LA 70893
MOLECULAR CHARACTERIZATION OF DSRNA VIRUSES INFECTING PLANTS AND FUNGI
CLASSIFICATION HEADINGS: R212 . Pathogens and Nematodes Affecting Plants; S4030 . Viruses; F1101 . Virology; R215 . Biological Control of Pests Affecting Plants; S4020 . Fungi; F1102 . Mycology
NON-TECHNICAL SUMMARY: Two families of dsRNA viruses, Partitiviridae and Totiviridae, and the genus Endornavirus contain members that infect plants and fungi. In plant pathogenic fungi, these viruses can increase or decrease the virulence of the fungus but in plants they do not appear to cause disease. Whether plant partitiviruses, endornaviruses, or totiviruses can provide beneficial adaptative traits to plants such as pathogen tolerance or tolerance to extreme environmental conditions remains to be investigated. We have found a partitivirus and a totivirus infecting Jalape?o M pepper, and tomato respectively. Virus-free lines of the same cultivars were obtained after screening many Jalapeno pepper and tomato seedlings. In Louisiana two endornaviruses have been reported infecting Yolo Wonder pepper and two rice cultivars (M201 and Saturn). Although, there is no evidence that suggests that these endornaviruses of pepper and rice are pathogenic to these crops; because of the lack of virus-free lines, their host effect of these viruses is not known. Studies related to the effect of these dsRNA viruses on pathogen susceptibility and/or tolerance to extreme environmental conditions of their hosts needs to be investigated as well as their potential use as gene vector. Moreover, the presence of dsRNA viruses in "healthy" crop germplasm could affect international exchanges and sales of rice, tomato or pepper seed. Some fungi contain RNA viruses that have been shown to have potential as biocontrol agents.The best example of this is the hypovirulence caused by dsRNA viruses of Cryphonectria parasitica, the causal agent of chestnut blight. This raises the prospect of using viruses to treat or prevent fungal infections. Research on dsRNA viruses that infect plant pathogenic fungi is essential to determine the mechanisms involved in hypovirulence and therefore facilitate the development of biocontrol strategies. In Louisiana, a putative endornavirus has been found in the plant pathogenic fungus R. solani infecting soybean. Preliminary data suggest that this virus may be involved in the reduced virulence of some isolates of the fungus. The plants hosts for this fungus are economically important and the fungus causes important diseases of rice, cotton, and soybeans. Research on the effect of the putative endornavirus on R. solani should provide some insight into the biocontrol potential of this virus. The overall goal of this proposal is to isolate dsRNA viruses from plants and fungi, obtain the nucleotide sequence of selected isolates, perform phylogenetic studies, and conduct preliminary evaluations on their effect on the hosts. In the case of the research on dsRNA viruses of fungi, the long term goal is to develop biocontrol strategies for fungi infecting economically important crops in Louisiana. The long term goals for the investigations on plant dsRNA viruses are to utilize their potential beneficial effects to their plant hosts and/or use them as gene vectors. Results from these investigations will advance our knowledge of this unique group of plant and fungal viruses.
OBJECTIVES: The overall goal of this proposal is to isolate dsRNA viruses from plants and fungi, obtain the nucleotide sequence of selected isolates, perform phylogenetic studies, and conduct preliminary evaluations on their effect on the hosts. Results from these investigations will advance our knowledge of this unique group of plant and fungal viruses. The specific objectives are: a. to clone, sequence, and conduct phylogenetic analyses of dsRNA viruses from pepper, tomato, and rice; b. to screen isolates of Rhizoctonia solani and other plant pathogenic fungi for dsRNA viruses by electrophoretic analysis and clone and sequence the genome of selected viral isolates; and c. to onduct preliminary studies on the host effect of dsRNA viruses.
APPROACH: DsRNA will be extracted from plant tissues and mycelial mats using column chromatography. Purified dsRNA will be analyzed by polyacrylamide and agarose gel electrophoresis. Selected dsRNAs will be further purified by elution from agarose gels. A Jalapeno M pepper virus-free and virus infected lines will be used in preliminary comparative tests. Lines of tomato (UC-82 and Celebrity) free of a totivirus and totivirus-infected lines will be increased and used in similar experiments. Rice cv Saturn (or M-201) will be the source of the putative rice endornavirus. Selected rice genotypes from the LSU rice breeding program, will be screened for the presence of dsRNA viruses. California Wonder bell pepper will be used as the source of the bell pepper endornavirus. Isolates of R. solani and other fungi from various crops will be collected from farms and experimental plots and tested for dsRNA viruses. Fungal isolates that contain putative endornaviruses will be selected for molecular characterization of the virus(es). Purified preparations of viral dsRNAs from rice, pepper, tomato, and selected dsRNAs identified in R. solani and other fungi will be used in cDNA cloning experiments. Cloned dsRNA fragments will be sequenced and derived amino acid sequences compared with sequences available in the GenBank databases using BLAST. Multiple sequence alignments and phylogenetic trees will be made with the CLUSTAL W series of programs. RT-PCR will be conducted with specific primers designed based on the obtained sequence. Attempts to eliminate the dsRNA viruses from selected R. solani and other fungal isolates will be conducted by high temperature treatment, hyphal tip culture, growth on cycloheximide-amended media, and serial transfer for five generations. To determine the virulence of fungal isolates with and without dsRNA viruses, detached leaflets of soybean will be inoculated. Data on virulence will be collected 72 h after inoculation for each isolate. The data will be analyzed by the PROC GLM procedure of SAS version 9.1.3 (SAS Institute Inc., Cary, NC, USA). Other characteristics of the isolates such as colony morphology will be evaluated as well. Similar experiments may be conducted with other dsRNA virus-infected plant pathogenic fungi. A possible outcome of the proposed experiments dealing with the putative endornavirus of R. solani is a positive correlation between the presence of the endornavirus and hypovirulence of the fungal host. DsRNA virus-free lines of tomato and Jalapeno M pepper will be used in parallel greenhouse studies with lines that contain dsRNA viruses. Louisiana isolates of ssRNA plant viruses that cause disease on tomato and pepper will be used in inoculation experiments. Tomato and pepper lines (dsRNA virus infected and dsRNA virus free) will be mechanically inoculated with each one of these ssRNA viruses. Inoculated plants will be evaluated for symptoms (visually), dsRNA (electrophoretically), and relative virus titer (serologically). Dried virus cultures and antisera to these three viruses are currently available in the laboratory.
KEYWORDS: dsrna viruses; biocontrol; endornavirus; partitivirus; totivirus; hypovirulence; rice; pepper; tomato
Name: Valverde, R. A.