RT-PCR-RFLP for genetic diversity analysis of the citrus tatter leaf virus strain of Apple stem grooving virus

A method for detecting genetic diversity of the Citrus tatter leaf virus (CTLV) strain of Apple stem grooving virus was developed based on restriction fragment length polymorphisms (RFLP) of the 889 nt 3′ sequence amplified by RT-PCR, and nine Hinf I RFLP patterns were defined. This RFLP assay, together with biological indexing and sequencing, was applied to characterize 18 CTLV isolates collected from seven provinces of China. The results indicated that the majority of these isolates (16/18) presented as single well-defined RFLP patterns, among which RFLPIand RFLPII were dominant. In contrast, two samples exhibited as a mixture of two RFLP patterns, suggesting a mixed infection of CTLV variants. Of the 18 isolates, six of eight samples with RFLPIIor RFLP III induced mild to moderate symptoms on indexing plants (Citrus. sinensis × Poncirus trifoliata cv. Rusk), while four isolates conforming to RFLPIand six samples to RFLP IV ~ IX induced severe symptoms. In the phylogenetic tree constructed according to the 3′ nucleotide sequence of the 18 CTLV isolates, RFLPIIand RFLP III samples clustered into phylogenetic group A, whereas the others gathered into phylogenetic group B. These results revealed correlation among mild (moderate) symptoms, phylogenetic group A and RFLPIIor RFLP III restrictotypes, suggesting the RFLP assay may be useful in quick identification of CTLV strains.