Association of two novel viruses with chlorotic fleck disease of ginger

Abstract

Chlorotic fleck disease of ginger, the causal virus of which was unknown so far is an important production constraint of ginger in India and other parts of the world. In the present study, two new RNA viruses were discovered in chlorotic fleck affected plant by the virome analysis using highthroughput sequencing of small RNA (sRNA) and transcriptome. The highthroughput sequencing results were verified through reverse transcriptase polymerase chain reaction (RT–PCR) using total RNA from infected plants and primers designed from the contigs. Cloning and sequencing of the RT-PCR products of one of the virus resulted in a sequence of 4143 bases that showed similarities to panicoviruses and machlomoviruses. The complete genome of this virus contained six open reading frames (ORFs) that potentially encode proteins of 43, 104, 8, 7, 15, and 27 kDa. Based on the genomic and phylogenetic analysis, this virus is predicted to be a new member of the family Tombusviridae for which the name ginger chlorotic fleck-associated virus 1 (GCFaV-1) is proposed. Similarly, cloning and sequencing of the RT-PCR products of other virus resulted in a sequence of 5514 bases that showed similarities to ampeloviruses. Based on the genomic and phylogenetic analysis, this virus is predicted to be a new member of the genus Ampelovirus for which the name ginger chlorotic fleck-associated virus 2 (GCFaV-2) is proposed. A reliable RT–PCR and SYBR Green–based quantitative RT–PCR assays were developed for the detection of both viruses in plants that would aid in the identification and propagation of virus-free ginger plants. Additional investigations are required to elucidate the relationship between the symptoms and viral infections