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Eight isolates of Fusarium oxysporum f. sp. niveum, FNC-S, FNT-
S, FNC-P, FNT-P, FL446, FL490, CA001 and TX002 which incite wilt
of watermelon were most used in this study. In the greenhouse ,
virulence of FNC-S and FNT-S to watermelon seedlings is higher
than that of FNC-P, FNT-P, FL446, FL490, CA001 or TX002.
Isolates of FNC-S and FNT-S are wild type ( sporodochial type )
and others are variants, especially FNC-P and FNT-P are
pionnotial isolates. F. oxysporum f. sp. niveum ( isolates FP
?F, FNC-P, FNT-S and FNT-P ) were cultured in potato dextrose
broth for 28 days and their filtrates were obtained through
Whatman No.1 filter paper. The filtrate was diluted to 0, 1/2,
1/4, 1/8, 1/16, 1/32 and 1/64, respectively, with distilled
water, and 15-day- old watermelon seedlings ( cv. Fupao No. 2 )
were cultured in various dilutions of the culture filtrate.
Higher percentage of watermelon seedlings showed the wilt
syndrome after 48hr in filtrates of FNC-S and FNT-S than that in
filtrates of FNC-P and FNT-P. Filtrates of FNC-S and FNC-P
adjusted by 1N NaOH or HCl from pH 4 to 8 did not significantly
change their toxicity to watermelon seedlings. Thirteen
carbohydrates and fifteen nitrogenous compounds were evaluated
for their effect on toxicity of filtrates extracted from Czapek''
s solutions culturing FNC-S and FNC-P for 28 days to watermelon
seedlings. Among those, galacturonic acid and mannose were more
effective than other carbohydrates to enhance toxicity of
filtrates. As to nitrogenous compounds, KNO3, (NH4)2SO4,
asparagine and glycine were also effective to increase toxicity
of filtrates to watermelon seedlings. Metabolic extractives (
Phytonivein-like crystals) were extracted from culture filtrates
of isolates FNC-S and FNC-P grown in potato dextrose broth. The
toxicity of metabolic extractives from isolate FNC-S was
stronger than from isolate FNC-P about 1.5 to 2 times. The
metabolic extractives of FNC-S and FNC-P detected by HPLC at the
wavelength of 225 ( the wavelength of phytonivein ) had the same
absorbance peaks at the same retention time. However, at the
time of 28.2 mins, the absorbance peak of FNC-S was 65 times
higher than one of FNC-P. Furthermore,using the wavelength of
271 ( the wavelength of fusaric acid ) for the detection by
HPLC, both of them had the same absorbance peaks and the
28.23-min absorbance peak of FNC-S was 25 times higher than one
of FNC-P . The total DNA of FNC-S, FNC-P, FNT-S, FL490, FL446,
CA001 and TX002 were extracted from the lyophilized mycelia for
RAPD analysis. For suitable RAPD amplification, the thermocycler
was programmed for 2 cycles of 60 s at 94℃, 7s at 37℃, 70 s at
72℃, followed by 35 cycles of 3s at 94℃, 7s at 37℃, 70 s at
72℃, and one cycle of 4 mins at 72℃. Preliminary 100 random
primers ( Operon Technologies Inc., Alomeda, CA, U.S.A.) were
test this 7 isolates. 26 of them were suitable for the analysis
for these 7 isolates by RAPD. FNC-P showed one different pattern
from FNC-S with the primers of OPAX-20 and OPAW-20. The banding
pattern of RAPDs obtained from application of these primers was
used to calculation of the genetic similarity ratio and the
average linkage method of Hierarchial Clustering method of SAS (
Statistic Analysis System Inc. ) were applied to analyze the
genetic cluster of the studied fungi. FNC-S and FNT-S were
belong to the homogenous group, however, FL490, FL446, CA001 and
TX002 were far away from them. Observation of spores and
mycelia of FNC-S and FNC-P by transmission electron micrography,
the protoplasm in hyphae of FNC-P showed aggregation together,
but not in hyphae of FNC-S.
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