Expanded Genomic Diversity of Cacao Leafroll Virus Enables Improved Detection Using Multiplex RT-PCR

Cacao leafroll virus (CaLRV; species Polerovirus CALRV, family Solemoviridae), previously referred to as cacao polerovirus, infects cacao (Theobroma cacao) germplasm in the Americas. Screening of 287 cacao accessions with three published multiplex RT-PCR assays detected CaLRV in nine accessions but produced reproducibly discordant results for the accession LCT EEN 412. Metatranscriptomic sequencing generated three near-complete CaLRV genome sequences, CaLRV-TSAN792, CaLRV-POUND15A, and CaLRV-LCTEEN412 (5985-5989 nt), from accessions originating from Brazil, Trinidad and Tobago, and Ecuador, respectively. The divergent CaLRV-LCTEEN412 assembly was verified by RT-PCR, cloning, and nanopore sequencing. In addition, analysis of five public cacao RNA-seq datasets yielded one near-complete CaLRV-PA16 assembly included in whole-genome analysis and four fragmented assemblies that informed primer design. Comparison of the four sequences characterised here with six previously reported genomes showed that nine shared 98.8-99.7% pairwise nucleotide identity, whereas CaLRV-LCTEEN412 shared 85.0-85.2%. Primer-binding-site mismatches in CaLRV-LCTEEN412 were consistent with its discordant detection by published assays. Two new multiplex RT-PCR assays were therefore designed from the expanded alignment and evaluated using four CaLRV-positive cacao accessions, including LCT EEN 412. These results expand known CaLRV genomic diversity and provide dual-target tools that reduce dependence on a single locus and enable recognition of target discordance.