IMPROVEMENT OF CITRUS LEAF BLOTCH VIRUS DETECTION USING CONVENTIONAL AND REAL-TIME RT-PCR

Citrus leaf blotch virus (CLBV) was first reported from Italy in 2006 associated with bud union disorder of 'Nagami' kumquat scions on citrange Troyer. In 2008, surveys were conducted to assess the presence of the virus in the foundation block of certified trees, as well as in commercial citrus orchards of Apulia (Southern Italy). RT-PCR assays were performed using the standard protocol developed by Galipienso et al. (Eur. J. Plant Pathol. 110: 175-181, 2004). Although, most of the samples tested negative for CLBV, a few yielded faint DNA bands. Due to these inconsistent results, two new primer sets for conventional and real-time RT-PCR were developed. Both primer sets were selected in the 3' portion of ORF1 of the viral genome. Using one-step RT-PCR the primers CLBV-1/CLBV-2 (5'-TTTTGGTACAGTTCGCTCCA-3' and 5'ATCAGATGGACCTC GAAGGA-3') amplified the expected product of 292 bp only from CLBV-infected controls, while none of 150 samples tested proved to be infected. A Taq-Man based real-time RT-PCR assay was also developed the results of which agreed with those of conventional one-step RT-PCR. Ten-fold serial dilutions of total RNA extracted from a CLBV-infected kumquat were used to assess the sensitivity of the newly developed assays. Target RNA was detected up to a dilution of 10 super(-5) and 10 super(-6) using the one-step RT-PCR and the real-time RT-PCR assays, respectively. When compared with the protocol previously available, the present assays proved to be up to 100 times more sensitive. However, none of the commercial citrus varieties tested in Apulia was found to be infected by CLBV.