A broad-spectrum PCR assay combined with RFLP analysis for detection and differentiation of plum pox virus isolates

Primers from the NIb (replicase) gene of plum pox virus (PPV) were used in a reverse transcription-polymerase chain reaction (RT-PCR) assay to detect field isolates from apricot and plum trees. A PPV-specific PCR product of c.1040bp was obtained from each infected tree. PCR products were digested with restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were compared to those from characterized isolates PPV-D, PPV-Ran, and PPV-NAT (D-serotype), PPV-SK68 (M-serotype), PPV-El Amar, and PPV-SoC (sour cherry). PCR products of characterized D-serotype isolates had a Tag I RFLP pattern that was distinct from patterns unique to each of SK68, El Amar, and SoC isolates. PPV-NAT was distinguished from PPV-D and PPV-Ran by Dde I and Rsa I RFLPs; SK68, El Amar, and SoC each had unique RFLP patterns with each enzyme. Field isolates could be differentiated by RFLP patterns; most had Tag I RFLPs typical of the D-serotype, but PCR products from some trees produced RFLP patterns distinct from any of the characterized isolates. Heterogeneity in the viral population from single trees was also observed, with two distinct RFLP patterns obtained from PCR products of some trees. The NIb PCR product is much larger than the coat protein (CP) PCR product of Wetzel et al., (1991a; 1040bp compared to 243bp), and more restriction sites differentiate between and within serotypes. Thus SK68 (M-serotype) was distinct from El Amar, SoC and D-serotype isolates, and some D-serotype isolates were differentiated. The combination of the PPV NIb RT-PCR with RFLP analysis will be valuable for studies of epidemiology and phylogeny.