Improved detection of grapevine latent viroid by RT-qPCR, its bioassay analysis, and its rare occurrence worldwide

Three of the five well-known viroids infecting grapevine belong to the genus Apscaviroid. Grapevine latent viroid (GLVd) is a novel grapevine viroid. Although GLVd has the typical sequence motifs of the genus Apscaviroid, it is still an unassigned viroid. In this study, a sensitive, convenient, and rapid one-step RT-qPCR method using hydrolysis probes for the detection of GLVd was developed. Survey and bioassays were also performed for this viroid. Using this method in the survey of GLVd, a low infection rate of 2/226 in a grapevine germplasm resource nursery and a demonstration vineyard in China was determined. Bioassays using agroinfiltration showed that GLVd can infect 'Kyoho' grapevine but not any of the tested herbaceous plants. Furthermore, sequence variability of GLVd was analyzed in six GLVd-infected grapevines. Sequencing revealed a predominant variant with only a few nucleotide changes compared with the reference variant of GLVd. Therefore, the developed RT-qPCR method should be helpful for determining GLVd in other vineyards of the world. The low infection rate, host range, and sequence variability of GLVd have important implications to further improve our knowledge on this novel grapevine viroid.