Cacao swollen shoot virus is a member of the family Caulimoviridae, genus Badnavirus and is naturally transmitted to Theobroma cacao (L.) by several mealybug species. CSSV populations in West African countries are highly variable and genetically structured into several different groups based on the diversity in the first part of ORF3 which encodes the movement protein. To unravel the extent of isolate diversity and address the problems of low titer and mixed viral sequences in samples, we used Illumina MiSeq and HiSeq technology. We were able to reconstruct de novo 20 new complete genomes from cacao samples collected in the Cocoa Research Institute of Ghana (CRIG) Museum and from the field samples collected in Côte d'Ivoire or Ghana. Based on the 20% threshold of nucleotide divergence in the reverse transcriptase/ribonuclease H (RT/RNase H) region which denotes species demarcation, we conclude there exist seven new species associated with the cacao swollen shoot disease. These new species along with the three already described leads to ten, the total number of the complex of viral species associated with the disease. A sample from Sri Lanka exhibiting similar leaf symptomology to West African CSSD-affected plants was also included in the study and the corresponding sequence represents the genome of a new virus named cacao bacilliform SriLanka virus (CBSLV).

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Molecular characterization and phylogenetic analysis of beta-satellite molecules associated with okra yellow vein mosaic disease in Sri Lanka

Detection and identification of viruses in okra plant is important to manage viral diseases. Good quality of DNA is essential for the PCR based detection and subsequent genomic sequencing. However, the DNA extraction has been greatly affected by mucilaginous substances present in okra plant parts. Objective of this study is to find out an extraction method that can eliminate mucilaginous materials and yield good quality DNA. Five different protocols were tested with okra leaf samples having okra yellow vein mosaic disease (OYVMD) symptoms. Quantity and purity of extracted DNA was tested using Nanodrop spectrophotometer. Precise quantity comparison was done by measuring total plant DNA in relation to the copy number of ACT2 gene using quantitative polymerase chain reaction (qPCR). The extracted DNA samples were used as template for detection of Bhendi yellow vein mosaic virus and Bhendi yellow vein mosaic betasatellite using specific primers. A modified protocol (modified method 1), introduced in this study, produced better yield and quality of DNA compared to other tested protocols. The method yielded 32 µg DNA for 100 mg leaf powder and the ratios A260/A280 and A260/A230 were 1.8 and 2.1, respectively. The qPCR quantification further confirmed higher quantity of DNA yield in this modified protocol. End point PCR with virus specific primers yielded very bright bands for Bhendi yellow vein mosaic virus and Bhendi yellow vein mosaic betasatellite. In conclusion, the modified method 1 can be used to extract good quality and quantity of DNA from okra.