E. Perez-artes et al., Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses, EUR J PL P, 106(6), 2000, pp. 507-517
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.