Molecular characterization of a new badnavirus associated with air yam (Dioscorea bulbifera L.) and development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Dioscorea baciliform AL virus

Abstract

Yams (Dioscorea spp.) are important staple crops cultivated worldwide, and air yam (Dioscorea bulbifera) is among the 12 commercially grown species. The productivity and safe germplasm exchange of Dioscorea spp. are hindered by several pests and diseases, and viruses pose a significant threat especially due to the vegetative propagation of yams that propitiate viral accumulation and dissemination. At least eight viral families have been reported infecting yams. The family Caulimoviridae comprises 11 genera, of which Badnavirus is widespread wherever yams are planted, and badnaviral transmission occurs mainly by vegetative propagation and different species of mealybugs. A putative new badnavirus has been recently reported in D. bulbifera plants in Brazil, and tentatively named as dioscorea bacilliform BL virus (DBBLV), but only partial sequences of the reverse transcriptase (RT) and ribonuclease H (RNase H) genomic region have been characterized. To recover the complete genome sequence of DBBLV isolates, total DNA obtained from an DBBLV-infected air yam plant was used as template for enrichment of viral circular DNA via rolling circle amplification and subjected to high-throughput Illumina sequencing. Two complete genomes of 7,196 (DBBLVDBH1) and 7,342 (DBBLV-DBH3) bp in size were de novo assembled from 1,273,444 and 539,840 reads and a coverage depth of 177 and 74x, respectively. Both isolates belong to the species novel Dioscorea bacilliform BL virus, sharing ≥88% nucleotide identity with at least one isolate of DBBLV. The recombination analysis showed a complex interspecies recombination pattern involving the DBBLV-DBH1 and DBBLV-DBH3 isolates as putative recombinant or parental sequences in four independent events. The Bayesian phylogenetic tree reconstructed based on complete genome and RT-RNase H nucleotide sequences showed the new DBBLV isolates were more closely related to dioscorea bacilliform AL virus (DBALV) sequences, but some topological incongruences were observed between these two trees. Also, a loop-mediated isothermal amplification (LAMP)-based protocol was developed and tested for detection of DBALV, the most prevalent badnavirus species affecting yams in Brazil. LAMP reactions were performed in a real-time thermocycler for 60 minutes at 65 ºC, measuring the fluorescence each one minute. Once the feasibility the proposed LAMP assay was confirmed, the limit of detection or the analytical sensitivity was tested with 10-fold serial dilutions, showing that the proposed protocol was able to detect viral DNA in a concentration of 12,5 x 10-7 ng/µL, equivalent to 5.5 x 103 copies/µL.