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w e n stopped after 6 h digestion by dilution in SDS-PAGE loading buffer, and the
products were analyzed by SDS-PAGE.
3.2.3.3.13. Antifungal assays of purilied proteins
Cylinder plate method was used to make wells on mediurn (Johnson and Curl
1972). The mycelia of the test fungi were inoculated in Ihe middle of the petri plates
containing potato dextrose agar (PDA). Three days after inoculation, when the colony
diameter was
3-4
cm, wells were filled with various concentrations of fusion protein
(80
pl purified protein of 100 pg and
300
pg in PBS buffer ptl 7.2). Protein extracts
of induced
E.
coli containing PET 28a+ or pMAL-p2x without insert, chitinase or
LTP served as control. The plates were further incubated at 28
"C
until thc n:ycelial
growth has enveloped the peripheral well containing the control and had produced
crescents of inhibition around the wells loaded with purified protein. Antifungal
assays were repeated twice to confirm the results.
3.2.3.3.14. Microscopic analyses
Deformed mycelia from the zone of inhibition were observed under scanning
electron microscope (SEM) (Hitachi. 53400 Japan) operated at 5
kV.
For SEM
analysis, mycelial samples from the periphery of the zone of inhibition and control
were mounted on a glass slide, coated with gold panicles under vacuum evaporator.
The fungal hyphae from the periphery of the zone of inhibition and control was
scraped, stained with lactophenol cotton blue and examined under a light microscope
(Novex, Holland)
(x400)
for any morphological changes.
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