Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses

Severe Verticillium wilt of cotton in southern Spain is associated with the spread of a highly virulent, defoliating (D) pathotype of Verticillium dah liae. Eleven of the D and 15 of a mildly virulent, nondefoliating (ND) path otype were analyzed by random amplified polymorphic DNA (RAPD) using the po lymerase chain reaction (PCR). Six of 21 primers tested generated pathotype -associated RAPD bands. Another 21 V. dahliae isolates were compared in bli nd trials both by RAPD-PCR using the six selected primers and pathogenicity tests on cotton cultivars. There was a 100% correlation between pathotype characterization by each method. Unweighted paired group method with arithm etic averages cluster analysis was used to divide the 47 V. dahliae isolate s into two clusters that correlated with the D or ND pathotypes. There was more diversity among ND isolates than among D isolates, these latter isolat es being almost identical. ND- and D-associated RAPD bands of 2.0 and 1.0 k b, respectively, were cloned, sequenced, and used to design specific primer s for the D and ND pathotypes. These pathotype-associated RAPD bands were p resent only in the genome of the pathotype from which they were amplified, as shown by Southern hybridization. The specific primers amplified only one DNA band of the expected size, and in the correct pathotype, when used for PCR with high annealing temperature. These specific primers successfully c haracterized V. dahliae cotton isolates from China and California as to D o r ND pathotypes, thus demonstrating the validity and wide applicability of the results.