Real-time PCR for the quantitation of Tomato yellow leaf curl Sardinia virus in tomato plants and in Bemisia tabaci

Tomato yellow leaf curl Sardinia virus (TYLCSV) (Geminiviridae) is an important pathogen severely affecting tomato production in the Mediterranean basin. Although diagnostic protocols are available for its detection in plants and its vector Bemisia tabaci (Gennadius), suitable tools for estimating and comparing viral loads in plant and insect tissues are needed. In this paper, real-time PCR methods are described for quantitation of TYLCSV in both tomato plant and whitefly extracts. The DNA extraction method was optimised on TYLCSV-infected tomato tissue. The amount of virus was determined using specific primers and probe and standardised to the amount of DNA present in each sample, using selected endogenous tomato or Bemisia genes as internal references. The distribution of TYLCSV was relatively quantified within the four uppermost leaves of plants. An absolute estimation of the amount of TYLCSV in the first leaf below the apex was obtained. The kinetics of virus retention within different batches of viruliferous whiteflies was also analysed. The real-time PCR was 2200-fold more sensitive than membrane hybridisation, allowing detection of as few as 10 viral copies in a sample. These methods are potentially suitable for several applications, such as plant breeding for resistance, analysis of virus replication, and virus-vector interaction studies.