Skip to main content
ARS Home » Southeast Area » Miami, Florida » Subtropical Horticulture Research » Research » Publications at this Location » Publication #285893
Title: Developing a reliable assay for Cacao Swollen Shoot Virus using quantitative PCR

Author
item MAUL, PILAR - St Thomas University
item LIVINGSTONE III, DONALD - Mars, Inc
item MOTAMAYOR, JUAN CARLOS - Mars, Inc
item SCHNELL, RAYMOND - Mars, Inc
item Kuhn, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/15/2012
Publication Date: N/A
Citation: N/A

Interpretive Summary: Cacao Swollen Shoot Virus (CSSV) causes a devastating disease in Theobroma cacao, the main economic crop in West Africa. The CSSV genome consists of a 7.4 Kb circular double-stranded DNA molecule that encodes a reverse transcriptase. CSSV does not functionally integrate into the genome of the host and requires an RNA transcript phase for infection. Because infected trees are asymptomatic for a long period, development of a sensitive method for CSSV detection is critical to maintain healthy cacao germplasm and particularly important for international germplasm exchange. Using the most conserved regions of six published CSSV genome sequences, we have developed quantitative PCR primers for both CSSV DNA and RNA detection. Quantitative PCR analysis with primers designed to various regions of the genome showed no detectable signals in DNA extracted from leaves of healthy plants. DNA extracted from infected trees gave strong CSSV positive signals for half of the samples tested. A 10-fold increase in template amount in the qPCR assay gave similar results, with only half of the infected samples showing CSSV positive signals. Progress and challenges to CSSV detection with quantitative PCR will be discussed.

Technical Abstract: Theobroma cacao, the source of cocoa beans for chocolate, is an important tropical agriculture commodity that is affected by a number of fungal pathogens and insect pests, as well as concerns about yield and quality. We are trying to find molecular genetic markers that are linked to disease resistance and other important economic traits to aid in a marker assisted selection (MAS) breeding program for cacao to ensure a reliable supply of cocoa for the US confectionary industry. Cacao Swollen Shoot Virus (CSSV) causes a devastating disease in Theobroma cacao, the main economic crop in West Africa. The CSSV genome consists of a 7.4 Kb circular double-stranded DNA molecule that encodes a reverse transcriptase. CSSV does not functionally integrate into the genome of the host and requires an RNA transcript phase for infection. Because infected trees are asymptomatic for a long period, development of a sensitive method for CSSV detection is critical to maintain healthy cacao germplasm and particularly important for international germplasm exchange. Using the most conserved regions of six published CSSV genome sequences, we have developed quantitative PCR primers for both CSSV DNA and RNA detection. Quantitative PCR analysis with primers designed to various regions of the genome showed no detectable signals in DNA extracted from leaves of healthy plants. DNA extracted from infected trees gave strong CSSV positive signals for half of the samples tested. A 10-fold increase in template amount in the qPCR assay gave similar results, with only half of the infected samples showing CSSV positive signals. Progress and challenges to CSSV detection with quantitative PCR will be discussed. Our results are important to scientists trying to understand the mechanism of disease resistance and, eventually, to cacao farmers who will benefit from superior disease resistant and more productive cultivars produced through our MAS breeding program.