Progress 09/15/06 to 09/14/08
Outputs
OUTPUTS: We continued and completed the characterization of the genome of the cytoplasmic type of citrus leprosis virus with the further development of specific primers for PCR diagnosis and antibodies for serological assays. Multiple serological tests were tested and proved specific and sensitive. The PCR and antibody detection systems were shared with cooperators from the various countries in the Caribbean where leprosis is endemic and with USDA, APHIS, PPQ scientists interested in the potential impact of this exotic virus to U.S. citrus. Work continued on the nuclear type of leprosis and comparisons will be made with the cytoplasmic type. PARTICIPANTS: Abby Guerra-Moreno worked on this project as part of his requirement for the Ph.D. degree in Plant Pathology at the University of Florida. He completed his degree in 2007 and left the university for a position back in his home country of Panama. Panama is one of the many countries severely affected by citrus leprosis (cytoplasmic type) and is an excellent place to further study this important exotic citrus disease that is spreading in the Carribbean basin. Dr. Lee Calvert, plant patholoygist with CIAT in Cali, Colombia and his staff along with Dr. Brlansky tested the PCR primers and antibodies prepared to the cytoplasmic citrus leprosis virus. Good cooperative work was initiated on the virus with Dr. Calvert and further work in ongoing with the Colombian isolate by a graduate student. Contacts have been made with other Caribbean countries who are interested in utilizing the primers and antibodies for detection of the virus. A discusssion with Dr. Laurene Levy, USDA, APHIS,PPQ in Beltsville, MD was initiated and testing of the primers and antibodies for detection is planned. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Excellent detection of the cytoplasmic type of citrus leprosis virus are now available and have been shared with cooperators. The complete sequence of this isolate from Panama was completed and analyses are being completed. The sequences will assist in the development of more detection systems. The resources provided by this grant have provided the necessary supplies and travel to accomplish work with this important virus pathogen. Cooperators in this project have assisted in much of this work since the virus does not exist in the U.S.
Publications
- No publications reported this period
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: The complete sequence of the cytoplasmic type of citrus leprosis virus from Panama was obtained. It has been found to be a bipartite RNA virus. The sequence was compared with that published for the virus from Primers for virus from Brazil and found to be very similar. Primers were prepared and tested in Panama and were found to specifically detect the virus from infected citrus leaves, fruit and stems from cytoplasmic citrus leprosis virus but not from the nuclear-type of the virus or from healthy tissues. Antibodies were tested in enzyme linked immunosorbent assays and were very useful at high dilutions in detecting the virus in infected tisssues. No detection of the nuclear type was found and the antibodies did not react with healthy tissues. The results will be disseminated in publications and in presentations in Caribbean meetings. Cooperators in Panama, Guatemala, Venezuela and Colombia have been provided with the techniques and the reagents for detecting the virus.
PARTICIPANTS: Participants on this project include Dr. Ron Brlansky, Abby S. Guerra-Moreno (graduate student) Dr. K.L. Manjunath (post doctoral associate) Mr. E. Rangel visiting scientist from Instituto Nacional de Investigaciones Agricolas, (Maracay,Venezuela) Dr. Lee Calvert (CIAT, Cali, Colombia and his staff) Dr. Marguerita Palmieri, I. Donis, A.L. Salazar, S. Blanco, and M. Porers (Universidad del Valle, Guatemala). Abby Guerra- Moreno did much of the work as a graduate student in the Department of Plant Pathology, University of Florida, Gainesville. He was assisted by Dr. Manjuanath who trained Mr. Guerra in many of the techniques and was the lead investigator on the preparation of the antibodies. Mr. Rangel was a visiting scientist from Venezuela and tested some of the primers with isolates in Venezuela. Dr. Lee Calvert and his staff also tested the primers with leprosis in Colombia. Dr. Palmieri and her staff in Guatemala reported on the disease in Guatemala and found the
same particles associated with the cytoplasmic type. They also found that the primers were useful in identifying the disease. Two mites were identified as vectors.
TARGET AUDIENCES: The target audiences remains to provide detection systems to detect this viral pathogen and to keep it from spreading into new areas of the Caribbean, Central America and into the U.S.
PROJECT MODIFICATIONS: No major changes were made.
Impacts
Excellent detection of the cytoplasmic type of citrus leprosis virus are now available and have been shared with cooperators. The complete sequence of this isolate from Panama was completed and analyses are being completed. The sequences will assist in the development of more detection systems. The resources provided by this grant have provided the necessary supplies and travel to accomplish work with this important virus pathogen. Cooperators in this project have assisted in much of this work since the virus does not exist in the U.S.
Publications
- 1. Guerra-Moreno, A.S., K.L. Manjunath, R.H. Brlansky, and R.F. Lee. 2007. Citrus leprosis symptoms can be associated with the presence of two different viruses: cytoplasmic and nuclear, the former having a multipartite RNA genome. Proc. 16th Conf. IOCV, Riverside 230-239. 2. Palmieri, M., I. Donis, A. L. Salazar, N. Cruz, A. Paniagua, R. H. Brlansky, A. S. Guerro-Moreno, K. L. Manjunath, P. Ballance and R. F. Lee. 2007. Citrus viruses in Guatemala: Application of laboratory-based assays. Proc. 16th Conf. IOCV, Riverside 510. 3. Serological detection of the cytoplasmic Citrus leprosis virus(CiLV) from infected citrus plants. XLV Congreso Anual de la Sociedad Americana de Fitopathologia-Division Caribe. pg 46 Abstract 5. Rangel, E., Manjunath, K.L., Guerra-Moreno, A.S., Brlansky, R.H., and Lee, R. F. 2007. Bacterial expression and purification of a protein from citrus leprosis virus. Proc. 16th Conf. IOCV, Riverside 510-511.
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