Detection of cacao mild mosaic virus (CaMMV) using nested PCR and evidence of uneven distribution in plant tissue

Authors: Puig, Alina

Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/2021
Publication Date: 9/14/2021
Citation: Puig AS. 2021. Detection of Cacao Mild Mosaic Virus (CaMMV) Using Nested PCR and Evidence of Uneven Distribution in Leaf Tissue. Agronomy, 11(9):1842. https://doi.org/10.3390/agronomy11091842
DOI: https://doi.org/10.3390/agronomy11091842

Interpretive Summary: Cacao mild mosaic virus (CaMMV) was discovered in Trinidad in 1943, but was recently detected in Brazil and Puerto Rico. It is thought to be widespread in the Americas, and spreads over long distances through the movement of infected budwood. To prevent the introduction of new diseases, improved germplasm of Theobroma cacao is distributed through a quarantine system, partly located at the USDA-ARS Subtropical Horticulture Research Station in Miami FL. In 2020, virus symptoms were observed on plants in the quarantine greenhouse in Florida during the containment period. However, the existing diagnostic test was not detecting virus, even in severely symptomatic plants. To address this, a new diagnostic test was developed and capable of detecting genetically diverse strains of CaMMV, and at an earlier point in the infection cycle (when virus levels are still low). In addition, a study done on dissected leaves showed that the virus in unevenly distributed, and that petiole tissue should be used for testing. This research will be used by scientists to prevent the movement and propagation of virus-infected cacao plants.

Technical Abstract: Distribution of improved germplasm of Theobroma cacao is essential for meeting the increased demand for cocoa beans. In cacao, the introduction of new diseases is prevented by exchanging material through a national and international quarantine system. In 2020, virus symptoms were observed on plants in a quarantine greenhouse, and Cacao mild mosaic virus (CaMMV) was detected in one plant using published diagnostic primers. However, no virus was detected in other symptomatic plants. To address high pathogen diversity and low virus titer in recently infected plants, a nested PCR test was developed based on 15 CaMMV sequences from Trinidad and Puerto Rico. The test was validated on a subset (n = 30) of plants in the greenhouse, of which 29 tested positive. 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 strains, suggesting that it was introduced on scionwood from multiple sources. Results of PCR assays on different leaf tissues indicate that the virus is unevenly distributed and that petiole tissue should be used in molecular diagnostics. The movement of infected scionwood is a major dissemination pathway for CaMMV but can be managed through careful screening.