First report of Cacao mild mosaic virus (CaMMV) associated with symptomatic commercial cacao (Theobroma cacao L.) trees in Puerto Rico

Authors: Puig, Alina; RAMOS-SOBRINHO, ROBERTO - University Of Arizona; KEITH, COREY - University Of Arizona; KITCHEN, NOEL - University Of Arizona; Gutierrez, Osman; Goenaga, Ricardo

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2020
Publication Date: 8/31/2020
Citation: Puig, A.S., Ramos-Sobrinho, R., Keith, C., Kitchen, N., Gutierrez, O.A., Goenaga, R.J. 2020. First report of Cacao mild mosaic virus (CaMMV) associated with symptomatic commercial cacao (Theobroma cacao L.) trees in Puerto Rico. Plant Disease. https://doi.org/10.1094/PDIS-04-20-0745-PDN.
DOI: https://doi.org/10.1094/PDIS-04-20-0745-PDN

Interpretive Summary: Cacao production is a rapidly expanding industry in Puerto Rico; new farmers have planted ~20,000 trees in the past few years. In 2019, pods with purple and red mottling and leaves with chlorotic lines were observed on cacao trees of the RIM-52 genotype on a commercial farm in Puerto Rico. Due to the presence of Cacao mild mosaic virus (CaMMV) and Cacao yellow vein banding virus (CYVBV) in nearby Trinidad, and the similarity of the observed symptoms, this material was tested using recently developed molecular diagnostics for the two viruses. ARS scientists (Miami, FL and Mayaguez, PR), in collaboration with cacao farmers in Puerto Rico and scientists from the University of Arizona, sampled symptomatic plants and conducted genetic analyses to identify CaMMV as the causal agent. CaMMV, previously known as Cacao Trinidad virus (CTV) strain A, was believed to have been eradicated, until infected material was found in the International Cocoa Quarantine Centre-Reading in the United Kingdom. This is the first report of CaMMV in Puerto Rico and is thought to be the first report outside of Trinidad. This discovery is significant to the global cacao industry as it indicates CaMMV is more widespread than originally thought and highlights the importance of molecularly indexing germplasm before releasing from quarantine. Additional studies are needed to determine the distribution of CaMMV in Puerto Rico and the Americas, whether it is now endemic to these regions, as well as its potential impact on production, if any.

Technical Abstract: The cacao industry is rapidly expanding in Puerto Rico (PR) where ~20,000 trees have been recently planted (DAPR, 2017). Symptoms of foliar chlorosis and purple mottling on immature pods were observed in a commercially grown cacao tree cultivar (genotype RIM-52) in PR during September 2019, and on ripe pods from a second RIM-52 tree at the same farm in December 2019. Based on the similarity of symptoms with those of Cacao mild mosaic virus (CaMMV) and Cacao yellow vein banding virus (CYVBV) from Trinidad, leaf (n=1) and pod (n=3) samples were tested for presence of the suspect badnaviruses using virus-specific primers. Total DNA was isolated from cacao samples using the Power Plant Pro Kit (Qiagen) and used as template for PCR amplification of the movement protein (MP) domain of CaMMV and CYVBV using the primer pairs, CaMMV-1112F 5’-TACGGAGACTGTGACTCAACCA-3'/CaMMV-1959R 5’-GTTTGGTAGGTTCCTTGTATCTGC-3’, and CYVBV-1223F 5’- AACGAGGACTACAACTCAGGC-3’/CYVBV-2164R 5’-TCCTCCAGTATCTCTTCATCCC-3’), respectively. The RT-RNase H domain was PCR-amplified from a representative isolate using CaMMV RT-RNase H-specific primers (CaMMV-RT-F 5’-TGCGGATTAAGAAGGCAGTAG-3’/CaMMV-RT-R 5’-GAGGTGATAGTAGGCGGTTTG-3’). Amplicons of expected sizes, ~900 bp (MP) from leaf (n=1) and pods (n=3) , and ~1,840 bp (RT-RNase H) from leaf (n=1) and pod (n=1) were cloned into the pGEM-T Easy plasmid vector and bi-directionally Sanger sequenced (n=2, per amplicon). Pairwise nucleotide comparisons of MP and RT-RNase H sequences were carried out using the Sequence Demarcation Tool (SDT) v.1.2. Of two viruses, only CaMMV was detected in cacao leaf and pods. The MP sequences (n=8; GenBank Accessions MT253655-MT253658 and MT262888-MT262891) shared 95.2-96.0% nucleotide identity with CaMMV (Accession KX276640). Based on established badnavirus species demarcation criterion, of =80% nucleotide identity for the RT-RNase H domain, the PR (Accessions MT253659-MT253662 ) and Trinidad (Accession KX276640) isolates are the same species, CaMMV, at 95.9-96.3% nucleotide identity. A Bayesian phylogenetic tree was reconstructed using concatenated MP and RT-RNase H sequences with the evolutionary model GTR+I+G predicted using MrModeltest. Results of pairwise sequence comparisons (SDT) and phylogenetic analysis (study S26001 deposited in TreeBASE) indicated the closest relative of the novel PR isolates is CaMMV reported in Trinidad. CaMMV, previously known as cacao Trinidad virus A, was reported in Trinidad in 1943 and associated with 7-33% yield reduction of cocoa beans. It was believed to have been eradicated in the 1950s, until infected material was found in the International Cocoa Quarantine Centre-Reading in the United Kindgom in 2009. This is the first report of CaMMV in Puerto Rico and is thought to be the first report outside of Trinidad. This discovery is significant to the global cacao industry as it indicates CaMMV is more widespread than originally thought and highlights the importance of molecularly indexing germplasm before releasing from quarantine. Additional studies are needed to determine the distribution of CaMMV in Puerto Rico and the Americas as well as its impact on production.