CHARACTERIZATION OF ISOLATES AND STRAINS OF CITRUS-TRISTEZA-CLOSTEROVIRUS USING RESTRICTION ANALYSIS OF THE COAT PROTEIN GENE AMPLIFIED BY THE POLYMERASE CHAIN-REACTION

Citrus Tristeza Virus (CTV) exists as a large number of distinct strai ns differing in biological properties and with different distributions in citrus producing countries. Strategies such as eradication or cros s protection, aimed at controlling severe variants of the pathogen, re quire procedures to identify virus strains accurately and reliably. To fill the need for a rapid, reproducible assay, we have investigated t he use of restriction analysis of the CTV coat protein gene amplified using the polymerase chain reaction (PCR). The primers 5'ATG GAC GAC G AA ACA AAG 3' and 5'TCA ACG TGT GTT GAA TTT 3' amplified a DNA copy of the CTV coat protein gene (approx. 670 base pairs) when used in a rev erse transcriptase PCR assay. Amplifications were carried out using ds RNA prepared from field and indicator plants, or from single-stranded RNA prepared from crude PEG precipitates of intact virions. All 51 CTV isolates tested produced an amplified product of the same size, regar dless of country of origin or biological properties. Digestion of the amplified coat protein genes with the restriction enzymes Hinf1 or Rsa 1 revealed sequence variation in the PCR products. Hinf1 provided the best discrimination between strains, defining seven Restriction Fragme nt Length Polymorphism (RFLP) groups, some of which circumscribed sets of isolates with similar biological properties. Limited analysis of f ield isolates using this method showed that individual trees could con tain mixtures of CTV strains, as assessed by the recovery of several R FLP types from individual reactions. Single aphid transmissions of iso lates usually, but not always, generated apparently pure single strain s judged by the recovery of single RFLP groups.