SSCP analysis of Citrus tristeza virus protectives isolates in Pêra sweet orange clones under northern Paraná state, Brazil conditions

Abstract

Aiming to evaluate the performance of different clones of Pera sweet orange (Citrus sinensis Osbeck cv. Pera) constituting Parana’s orchards, in relation to different pathogens, leading to the installation of an experimental orchard in northern region. This study evaluated clones performance of Pera Vacinada and Pera Bianchi sweet oranges, in relation to the citrus tristeza, by analyzing the disease symptomatology. The stability of Citrus tristeza virus (CTV) mild and protective isolate present in these plants were studied over the years of the experiment using the SSCP (Single strand conformation polymorphism) technique. The tristeza symptoms were assessed by vegetative growth and stem pitting intensity in the trunks and branches. The molecular characterization by SSCP analysis, dsRNA (double-stranded RNA), was used to reverse transcription and CP (coat protein gene) by polymerase amplification chain reaction (PCR) and then subjected to electrophoresis on polyacrylamide gel. The tristeza symptoms assessment indicated that the clones did not differ in the intensity of stem pitting, size and canopy volume. Grades in stem pitting attributed to the trunks and branches ranged 0-2, indicating a severity of weak to moderate reaction in plants. For SSCP analysis, no difference was observed among cultivars, suggesting that mild isolates presence allowed normal plants development. There was a complex pattern of bands in all isolates, generating four to eight bands in the electrophoretic profiles, suggesting the presence of two to four haplotypes composing CTV isolates, some differing only by the absence of an intermediate band, which cannot have been transferred by propagation grafting. However, SSCP isolates clones patterns from Pera Vacinada and Pera Bianchi were not identical to any severe controls. The results suggested that the protective isolates which were inoculated in pre-immunization (IAC Pera) and established by natural infection of the virus (Pera Bianchi) contributed to plants good performance assessed by tristeza symptomatology and by SSCP analysis, showed stability of protective isolates in Pera sweet orange, which has been maintained over years of experiment.