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United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: Development and Optimization of Molecular Diagnostics Method for Qualitative and Quantitative Detection of Cacao Swollen Shoot Virus

Location: Subtropical Horticulture Research

Project Number: 6038-21000-023-07
Project Type: Specific Cooperative Agreement

Start Date: Sep 01, 2013
End Date: Sep 30, 2016

Objective:
1. Develop and validate diagnostic methods for detection of viral genomic DNA for qualitative detection and subsequent identification of variants. 2. Design and test qPCR primers using positive/negative control samples available at the University of Arizona, School of Plant Sciences, Virology Laboratory. Determine assay sensitivity using a standard curve and digital PCR. Design and optimize additional primers based on need to detect possible newly discovered variants. 3. Scanning of available cacao genome sequences (http://www.cacaogenomedb.org) for presence of integrated CSSV sequences. Design primers to amplify sequence from positive source material, if material is available.

Approach:
Cacao production is plagued by yield losses due to plant virus infection. These viral diseases cause significant decreases in total cacao production, worldwide. CSSV is a mealybug-transmitted virus pathogenic on cacao in West African cacao-growing regions. It was first identified in Ghana and currently has been documented in Benin, Cameroon, Cote d’Ivoire, Ghana, Nigeria, and Togo. CSSV has not been found in Central and South America, the center of cocoa diversity, but instead is indigenous in and non-pathogenic to forest trees in West Africa such as Ceiba pentandra. CSSV is a member of the family, Caulimoviridae, genus, Badnavirus. The complete genome of five variants (seven isolates), two originating from Togo and three from Ghana, have been sequenced. Isolates virulent in cacao trees cause defoliation, pods that are reduced in size, and death of the tree, while mild isolates cause red vein banding, leaf chlorosis, and swelling of shoots. The diagnostic tests that are currently available, primarily, polymerase chain reaction (PCR) based, do not detect all suspected viral pathotypes, nor are quantitative detection methods available for reliably detecting virus concentrations that are at or below the level of sensitivity possible using PCR. Further, preliminary experiments conducted at University of Arizona, School of Plant Sciences, Virology Laboratory have shown that although this pararetrovirus replicates using an RNA intermediate, reverse-transcriptase PCR (RT-PCR) is far less reliable for virus detection in both shoots and roots, than are methods that target the viral DNA (encapsidated form). Therefore, the development of improved molecular detection tools, namely, PCR and quantitative (qPCR), has become essential to assure that when virus is present, regardless of titer or strain, it is detectable. The assays developed in this project will be tested in Ghana, Ivory Coast, and Nigeria. The creation of a CSSV Research Working Group is in process, and includes scientists from the affected countries. Without the availability of reliable CSSV detection methods the application of molecular markers that are used in the cacao breeding program for marker assisted or genomic selection may be uninformative or yield confounding results, both ineffective and inefficient outcomes for any plant breeding program.

Last Modified: 10/24/2014
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