Improvement of a Bio-PCR Test for Routine Detection of Watermelon Fruit Blotch Pathogen

Fruit blotch is a serious seedborne disease of watermelon. In an attempt to control this disease, the seed industry has adopted a zero tolerance of infected seeds. Available tests for detecting infected seeds include grow-out, liquid plating, classical PCR tests. These tests are either not sensitive enough, resulting in false negative, not specific enough, resulting in false positives, or are very time consuming. A newly developed Bio-PCR test works well for many seed lots, as evident from our Phase I results. Success of Bio-PCR, however depends on many factors including efficient extraction of pathogen from seeds, maintaining viability during extraction, enrichment, extraction of high quality DNA and availability of good primers. To rule out false PCR positives, classical PCR product should be further identified with a specific probe (Southern hybridization). We intend to accomplish this in Phase II by adoption of TaqMan technology (Smart Cycler) that provides increased sensitivity, real time PCR detection and hybridization in an automated process. The TaqMan technology will make seed assays COST effective and reliable. NO separate SOUTHERNS ARE NEEDED! Our consultant has recently made TaqMan primers and probes made available to us.