2.3. Study of pectinase activity of isolated pathogens
Pectinase enzyme producing ability of seven different patho-
genic isolates including the selected strain VBAM1, were evaluated
with the method of Patil et al. (2010). The fungal strains were inoc-
ulated at the center of a pectin agar plate and incubated at 28 C for
72 h. After visual growth of the fungal mycelia, the plates were
stained with 1% cetyl trimethyl ammonium bromide (CTAB). A
zone of clearance indicated pectolytic activity. For evaluation of
quantitative production of pectinase, samples of stock culture were
added to 100 ml modified Czapek’s dox broth in 250 ml Erlen-
meyer’s flasks containing pectin as the sole carbon source. The
broth were incubated at 28 C up to 7 days. Aliquots were taken
at 24 h intervals and were centrifuged at 10,000 rpm for 20 min.
1 ml of supernatant from each aliquots was taken and the amount
of reducing sugar was measured using 3,5-dinitrosalicylic acid
(DNSA) and OD was taken at 540 nm (Miller, 1959 ). The amounts
of reducing sugar were calculated using standard curve of dex-
trose. Beside sugar estimation, the protein amounts in the culture
medium were also measured by method of Lowry et al. (1951) at
every 24 h interval.
2.3. Study of pectinase activity of isolated pathogensPectinase enzyme producing ability of seven different patho-genic isolates including the selected strain VBAM1, were evaluatedwith the method of Patil et al. (2010). The fungal strains were inoc-ulated at the center of a pectin agar plate and incubated at 28 C for72 h. After visual growth of the fungal mycelia, the plates werestained with 1% cetyl trimethyl ammonium bromide (CTAB). Azone of clearance indicated pectolytic activity. For evaluation ofquantitative production of pectinase, samples of stock culture wereadded to 100 ml modified Czapek’s dox broth in 250 ml Erlen-meyer’s flasks containing pectin as the sole carbon source. Thebroth were incubated at 28 C up to 7 days. Aliquots were takenat 24 h intervals and were centrifuged at 10,000 rpm for 20 min.1 ml of supernatant from each aliquots was taken and the amountof reducing sugar was measured using 3,5-dinitrosalicylic acid(DNSA) and OD was taken at 540 nm (Miller, 1959 ). The amountsof reducing sugar were calculated using standard curve of dex-trose. Beside sugar estimation, the protein amounts in the culturemedium were also measured by method of Lowry et al. (1951) atevery 24 h interval.
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