DETECTION OF FUSARIUM-OXYSPORUM F-SP VASINFECTUM IN COTTON TISSUE BY POLYMERASE-CHAIN-REACTION

A polymerase chain reaction assay was developed for the detection of F usarium oxysporum f. sp.. vasinfectum (FOV), a serious wilt pathogen o f cotton in many parts of the world. Based on small nucleotide differe nces in internal transcribed spacer sequences between 18S, 5.8S and 28 S ribosomal DNAs, primers Fov1 (5'-CCCCTGTGAACATACCTTACT-3') and Fov 2 (5'-ACCAGTAACGAGGGTTTTACT-3') were selected. These primers unambiguou sly amplified a 400-bp DNA fragment of all the FOV isolates tested (fr om Angola, Brazil, China and the USA) but did not amplify any other is olates of mycoflora associated with cotton, such as F. moniliforme, Ve rticillium albo-atrum, V. dahliae, Aspergillus sp., F. oxysporum, F. s ambucinum or F. solani. A control PCR assay was developed employing th e universal primer pair ITS1 and ITS2 which amplified a fragment of ap proximately 220 bp from all isolates tested. This control assay demons trated that all fungal DNAs were readily amplifiable, thus confirming that the lack of amplification with Fov1 and Fov2 primers was a result of primer specificity and not of other possible causes, such as DNA d egradation or the presence of PCR inhibitors. The assay was effective on samples from the stems, leaves, roots and calli, and from plant tis sues both with and without symptoms. This detection system proved to b e accurate and sensitive and could aid not only diagnosis but also dis ease monitoring and forecasting.