Detection of Cacao Mild Mosaic Virus (CaMMV) Using Nested PCR and Evidence of Uneven Distribution in Leaf Tissue

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.

First report of Cacao mild mosaic virus associated with cacao mosaic disease in Indonesia

“…To identify the causal pathogen, DNA was extracted from seven samples representing different cacao cultivars using a Plant Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan), following the manufacturer's instructions. Genomic DNA from the samples was subjected to nested PCR using specific primers for Cacao mild mosaic virus (CaMMV), Mia1145F/Mia1926R, for the first round and Mia1396F/Mia1876R for the second (Puig, 2021). PCR for the first round (806 bp amplicon) was conducted with initial denaturation at CaMMV is a badnavirus associated with mosaic disease of cacao, and previously known only to exist in the Americas (Muller et al, 2018).…”

Mealybug (Hemiptera: Pseudococcidae) Species Associated with Cacao Mild Mosaic Virus and Evidence of Virus Acquisition

“…In January 2021, female mealybugs were collected from 23 randomly selected Theobroma cacao trees in Florida confirmed to be infected with CaMMV following nested PCR testing, as described in Puig [14], followed by Sanger sequencing of the amplicons. Specimens were collected from the pods, stems, leaves, and flowers of each tree (up to 5 individuals from each tissue × tree combination), and placed in microcentrifuge tubes containing 70% ethanol.…”

“…Presence of CaMMV was determined by running a nested PCR for each sample with virus-specific primers (Table 2) [14]. The initial reaction was 20 µL with 10 µL Sigma-Aldrich© JumpStart™ REDTaq ® ReadyMix™ (Sigma-Aldrich, St Louis, MO, USA), 7.6 µL molecular grade water, 0.8 µL of each 10 µM primer (Mia1145F and Mia1926R), and 0.8 ng of DNA template.…”

“…Research in Trinidad described two distinct strains, which were recently characterized and named cacao mild mosaic virus (CaMMV) and cacao yellow vein banding virus (CYVBV) [11]. These viruses were thought to be relatively rare, until CaMMV was detected in Puerto Rico [12] and Brazil [13], and then intercepted in material under quarantine in the USA [14] in the past couple of years. Trees infected with CaMMV experience branch dieback and annual yield reduction of 6.6-19%, comparable to mild to moderate strains of CSSV in West Africa [15,16].…”

Bioinformatic, genetic and molecular analysis of several badnavirus sequences integrated in the genomes of diverse cocoa (Theobroma cacao L.) germplasm

“…Another species, Cacao bacilliform Sri Lanka virus (CBSLV) has been reported in Sri Lanka ( Muller et al, 2018 ). In addition, two new badnavirus species designated as cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), which cause mild symptoms, have been identified in cacao trees grown in the western hemisphere ( Chingandu et al, 2017 , Puig, 2021 ). These two species have also recently been reported in asymptomatic trees in cacao germplasm in the United Kingdom ( Ullah et al, 2021 ).…”