Genotypic and phenotypic variation of nepoviruses in New Zealand and implications for detection and identification

Abstract

The variability of isolates of three nepoviruses, Cherry leaf roll virus (CLRV), Grapevine fanleaf virus (GFLV) and Tomato ringspot virus (ToRSV), and the related virus Strawberry latent ringspot virus (SLRSV) in New Zealand was investigated. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) the specificity of 20 pairs of nepovirus and SLRSV primers (11 pairs subgroup-specific and nine pairs species-specific) was evaluated using 64 virus isolates, representing 13 species, from the three nepovirus subgroups and the unassigned species SLRSV. Protocols for the detection of the viruses were successfully developed for biosecurity screening of imported nursery material. Three potential host plants, Malus, Vaccinium and Vitis, were screened for the presence of the four viruses. For Malus, a total of 120 samples from two orchards were tested, for Vaccinium a total of 178 field samples and 80 samples from a germplasm collection were tested, and for Vitis, 157 samples from a collection of historic grapevine accessions were tested. An additional host plant, Ribes, was also tested. CLRV was detected in Ribes and in field-collected samples of Malus and Vaccinium, GFLV was detected in the historic grapevine collection and SLRSV was detected in field-collected Vaccinium samples. ToRSV was not detected in any of the samples tested. The discoveries of CLRV in Malus, Vaccinium and Ribes, and SLRSV in Vaccinium are new host records. The discovery of CLRV in three new crop hosts shows that the virus is present in a much wider range of host species in New Zealand (and globally) than previously reported. Further characterisation of the CLRV isolates found varying symptoms in mechanically inoculated herbaceous hosts and different rates of seed transmission in the common systemically infected host Nicotiana occidentalis, suggesting that the New Zealand CLRV isolates exist as distinct biological strains. Phylogenetic analysis of the complete genome sequences showed that four CLRV isolates were almost identical and two other isolates exhibited a pairwise nucleotide difference of up to 8.6% compared to these isolates. Following characterisation of the complete sequences of the six CLRV isolates, diagnostic protocols, based on RT-PCR and RFLP, were developed to distinguish between strains of this virus.