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The fungi tested in this study included each 33 isolates of
Pseudocercosporafuligena (on Lycopersicon esculentum ) and
P. atromarginalis (on Solanumnigrum), and 20 other isolates
of Cercospora spp. and Pseudocercospora spp. wereused to perform
analysis of their genetic variation by RAPD. Based on
thepreliminary tests of searching suitable RAPD conditions,
the results revealedthat optimal reaction mixtures contained DNA
( 50 ng ), 1 x buffer ( 1.5 mM Mg+2), primer ( 0.5 μ M), dNTPs
( 0.2 mM each ), DynaZyme I DNA polymerase ( 0.6 U/ μ l), and
BSA( 0.5 mg / ml ) and the suitable amplification reaction
is 2cycles at 94 ℃ for 60 s, 42 ℃ for 7 s, and 72 ℃ for 70
s, then 35 cycles at94 ℃ for 3 s, 42 ℃ for 7 s, and 72 ℃ for
70 s, and one cycle at 72 ℃ for 4min. Preliminary 100
different 10 mer primers (Operon Technologies Inc.,Alameda,
CA, U. S. A. ) were tested for each 3 isolates of P. fuligena (
pf-10,pf-12, and pf-18 ) and P. atromarginalis ( pa-22, pa-24,
and pa-29 ) among them,13 primers gave no banding pattern or
poor amplification, the other 87 primersconsistantly produced
common banding patterns. Therefore, 30 reproducibleprimers
were selected for assessing genetic variation among isolates
of P.fuligena, P. atromarginalis and other Cercospora spp. and
Pseudocercospora spp.The banding patterns of RAPDs obtained
from application of these primers wereused to calculate the
genetic similarity of those fungi, and genetic cluster ofthose
fungi were analyzed with the average linkage method of
hierarchialclustering method in SAS (Statistic Analysis System
Inc. ). The results of RAPDpatterns, genetic similarity and
dendrogram indicated that isolates of P.fuligena formed a
homogenous group, so did P. atromarginalis, except the
4isolates of P. atromarginalis ( pa-15, pa-30, pa-35 and pa-39
); intraspecificand interspecific variation were not detected
between isolates of P. fuligenaand P. atromarginalis from
different areas; the 3 isolates (P. solani-melogenicola,
P. piricola and P. eriobotryae ) showed the similar RAPD
patternsas P. fuligena and P. atromarginalis did; isolates of
other Cercospora spp. andPseudocercospora spp. were the host
specific fungi. In conclusion, the twospecies, P. fuligena
and P. atromarginalis were homogeneous based on
RAPDanalysis could be a synonym. The RAPD is a very potential
technique in using theunique RAPD bands as genetic markers
for identification of interspecies andintergenus and assessing
the genetic diversity and relationships among isolatesof
Cercospora spp. and Pseudocercospora spp. This model
should be asupplementary tool when the traditional
taxonomy method can not solve theproblems caused by host
specificity or morphology as the criteria in the
fungusclassification.
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