First Report of Cacao Mild Mosaic Virus Associated with Cacao (Theobroma cacao) in Hawai'i, USA.

ABSTRACT

Cacao, Theobroma cacao, is an important tropical agricultural crop and the key ingredient of chocolate, which has an international trade value of $103 billion/year (Marelli et al. 2019). Cacao mild mosaic virus (CaMMV) is a badnavirus that causes mild symptoms compared with some of the closely related species of Cacao swollen shoot virus (CSSV), the latter of which are currently only found in West Africa (Marelli et al. 2019; Ullah et al. 2021). CaMMV was recently found in symptomatic commercial cacao trees in Mayagüez, Puerto Rico (Puig et al. 2020), and subsequently detected in a USDA ARS (Agricultural Research Service) quarantine greenhouse in Miami, FL (Puig 2021). The USDA ARS germplasm repository in Hilo, Hawai'i, USA serves as a backup collection for Puerto Rico's cacao germplasm, and field trials have been established from cacao germplasm from Miami to evaluate select varieties. To determine if CaMMV is present in the collection in Hilo, greenhouse and field accessions were tested. Using an optimized sampling and PCR protocol established by Puig (2021), three young cacao petioles per tree or seedling were collected and pooled, and DNA was extracted using the NucleoSpin Plant II commercial kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Samples were molecularly identified via end point PCR, gel electrophoresis, and Sanger sequencing. PCR amplification of CaMMV using the virus-specific primer set Mia-1396F (5'-ACCGTGTCTAYCAGCACTGGA-3') and Mia-1667R (5'-GACCACCGTCAGCCAGAC-3') produced 289 bp amplicons. Of 230 plants sampled, 26 CaMMV positive detections were discovered in greenhouse and field plantings in Hilo. Most CaMMV+ plants contained some form of leaf chlorosis (96.2%). The sequenced PCR products from Hawai'i were deposited in GenBank (accession nos. OQ692890-OQ692891) and showed 99.2% nucleotide identity to CaMMV accessions from Puerto Rico (MW052520; n = 23) and 98.1% nucleotide identity to CaMMV accessions from Florida, USA (MZ409692; n = 3) in BLASTn analysis. For species-level confirmation, the RT-RNase H domain was amplified from 9 isolates using CaMMV-specific primers (Mia5385F, 5'-AGGACAACGGCTTTCTTGGT-3'/Mia6616R, 5'-GAGACTAACTTGGTTAGGGCT-3'), sequenced, and deposited in GenBank (accession nos. PP997461-PP997462). Sequences matched most closely to GenBank isolates from Puerto Rico (MT253659; 98.0%; n = 7) and Trinidad and Tobago (NC_033738; 97.1%; n = 2). CaMMV, previously known as cacao Trinidad virus A, was first reported in 1943 and was associated with 7 to 33% yield reduction, loss of vigor, and tree decline (Posnette 1944; Swarbrick 1961). CaMMV is known to exist in the Americas and was most recently detected in Brazil and Indonesia (Kandito et al. 2022; Ramos­Sobrinho et al. 2021). To the best of our knowledge, this is the first report of CaMMV infecting cacao in Hawai'i. The cacao industry continues to expand in Hawai'i, and cultivation occurs on at least four of the main islands including Hawai'i Island, O'ahu, Maui, and Kaua'i. To develop disease management strategies, further investigation is needed to define CaMMV symptomology, and determine the distribution and effect this virus has on production in Hawai'i. In the meantime, cacao will be screened regardless of visible symptomology to maintain pathogen-free accessions and avoid the transfer of virus-containing germplasm.