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ARS Home » Southeast Area » Miami, Florida » Subtropical Horticulture Research » Research » Publications at this Location » Publication #384171

Research Project: Development and Application of Genomic-assisted Breeding Strategies to Produce Disease-resistant Cacao Genetic Resources

Location: Subtropical Horticulture Research

Title: Development of Nested PCR for detection of Cacao mild mosaic virus in Theobroma cacao

item Puig, Alina

Submitted to: Florida Phytopathological Society Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/5/2021
Publication Date: 5/13/2021
Citation: Puig, A.S. 2021. Development of Nested PCR for detection of Cacao mild mosaic virus in Theobroma cacao. Florida Phytopathological Society Meeting. Florida Phytopathological Society Meeting, Online, May 13-14, 2021.

Interpretive Summary:

Technical Abstract: Development and distribution of improved germplasm of T. cacao is essential for meeting increased demand for cocoa beans, however, material exchange is restricted due to the risk of introducing new pests and diseases. To address this, and increase the genetic diversity available for plant breeding research, the U.S. Department of Agriculture maintains a quarantine and distribution program for T. cacao germplasm in Miami, FL. In 2020, leaf mosaic and vein-banding symptoms characteristic of virus infection were observed on plants in the quarantine greenhouse. Symptomatic leaves from five trees were tested using published CaMMV diagnostic primers. One of these yielded a fragment 95% identical to a strain from Trinidad (KX276640), while two others yielded a 500-900bp sequence >99% identical to a region of the T. cacao genome on chromosome I (LT594788). To address low virus titer in recently infected trees, and high pathogen diversity, a nested PCR test was developed using degenerate primers designed based on 15 CaMMV sequences from Trinidad and Puerto Rico. The test was validated on a subset (n=30) of plants in the quarantine greenhouse, of which 20 presented virus-like symptoms. In total, 29 plants tested positive for CaMMV, including all asymptomatic plants (100%, n=10) and 19 symptomatic plants (96.7%, n=20). Most infections are thought to have occurred during the later stage of the quarantine period, possibly due to spread by mealybugs. However, phylogenetic analysis revealed the presence of three different strains, suggesting that it was introduced on sticks of budwood from multiple countries.