Skip to main content
ARS Home » Southeast Area » Miami, Florida » Subtropical Horticulture Research » Research » Research Project #443134

Research Project: Discovery Sequencing for Improved PCR/qPCR Diagnostic Test Development to Identify the Presence of Badnaviruses in Cacao Germplasm Collections

Location: Subtropical Horticulture Research

Project Number: 6038-21000-027-002-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2022
End Date: Aug 31, 2027

The production of cacao is plagued by decreased productivity, due to several new or emergent plant diseases that cause decreased yields and quality. Badnaviruses are among the most important diseases in cacao, and they are graft- and mealybug-transmitted pathogens that infects cacao throughout the world. In the Americas, Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein Banding Virus (CYVBV) were initially discovered in Trinidad in the 1930s and called Cacao Trinidad virus (CTV) type A (red foliar mottling) and B (yellow vein-banding). Yield losses due to CMMV and CYVBV were initially estimated between 6 and 19% and subsequent eradication programs by the government reduced the presence of the viruses in Trinidad. However, in 2007, Badnaviruses were detected in cacao plants originating from Trinidad at the International Cocoa Quarantine Centre-Reading (ICQC, R) in Reading, UK. CaMMV and CYVBV have been recently found and identified in Puerto Rico, Brazil, Florida and at ICQC, R. Thus, an efficient diagnostic tool is urgently needed for both Badnaviruses. The objectives of this work are: 1) Conduct Ion Torrent ‘discovery’ DNA sequencing for selected CaMMV and CYVBV isolates from the USDA-ARS-TARS, Cocoa Research Center (CRC), and CATIE germplasm collections to expand the CaMMV and CYVBV genome sequence database; 2) Utilize the expanded database to guide the design of improved PCR and qPCR detection assays; 3) Screen symptomatic/asymptomatic cacao trees from USDA-ARS-TARS, in Mayaguez, Puerto Rico as well as symptomatic trees at CRC, Trinidad and CATIE, Costa Rica with confirmatory sequencing to validate the primers/probe design and improved molecular assays.

Leaf samples from approximately 1,000 trees exhibiting representative symptoms will be gathered from three different cacao germplasm collections (USDA-ARS-TARS, Mayaguez, Puerto Rico, Cocoa Research Center (CRC), St. Augustine, Trinidad and Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Turrialba, Costa Rica) and shipped to the cooperator's laboratory. Asymptomatic trees from the Mayaguez germplasm collection will be tested. Trees in the USDA Mayaguez germplasm collection testing negative for CaMMV and CYVBV will have flowers collected and propagated via somatic embryogenesis and saved in vitro in Mayaguez for preservation as virus-free material and formal field studies on virus effect on cacao production and quality. Total DNA will be isolated and stored at -20°C. The DNA yield and concentration will be determined spectrophotometrically (nanodrop and Qbit) and the integrity assessed by gel electrophoresis. Ion Torrent ‘discovery’ DNA sequencing will be conducted on samples with high-quality DNA. Reads will be assembled using de novo and reference gene mapping approaches. Coding and non-coding regions will be verified using ORF finder. Sequences will be analyzed for % nt sequence identity (strains), phylogenetically (identity) and SNPs will be mapped to guide primer/probe design. Quantitative PCR and PCR. Primer/probe pairs will be designed and evaluated by PCR amplification with confirmatory amplicon sequencing. PCR primers designed to amplify the conserved RT-RNAase H region will be used to amplify CaMMV and CYVBV with confirmatory sequencing to identify CaMMV and CYVBV-infected cacao trees. The best qPCR probes will be selected and used to run a standard curve to identify high-efficiency primer-probe pairs using the TaqMan qPCR system. Virus-free ‘Amelonado’ DNA will be the negative control. Selected samples (positive by PCR amplification) will be tested (one replicate) by qPCR CaMMV and CYVBV detection. If robust, all samples will be analyzed in duplicate. Selected positive samples will be PCR amplified and sequenced (RT-RNAase H) to confirm CaMMV and CYVBV-qPCR primers-probe virus-specificity (vs. host). The resultant primers/probe will be used to test all samples in triplicate by qPCR. The qPCR reactions will be carried out using the Bio-Rad CFX96 Touch Real-Time PCR Detection System and data analysis are carried out with CFX Maestro software v.1.1. Virus identification is accomplished by BLASTn (GenBank db).