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3.2.3.3.15.
Effects
of purified
protein o n germination of spores and sclerotia
Spore germination assays were performed using spore suspensions of
phytopathogenic fungi. Fungi were grown on PDA for 10 to 15 days. Conidiol
suspensions were collected by adding sterile water to the surface of the mycelium.
Suspensions were counted in a Neubauer counting chamber and adjusted to the
appropriate concentration (1 x loh spores per ml). The ability of purified protein to
inhibit conidial germination of fungi was determined by placing conidial suspensions
(50 pl) in sterile microcentrifuge tubes containing potato dextrose broth (150 PI).
Conidial germination was examined after 12 h of incubation at 28
OC.
Protein extracts
of induced E. coli containing PET 28a+ or pMAL-p2x without insert, chitinase or
LTP served as control. The treated and control spores were stained with lactophenol
cottonblue and examined under a light microscope (Novex, Holland) ( ~ 4 0 0 ) . In order
to determine whether purified protein has fungistatic or fungicidal activity, 12 h
treated and control spores were plated on PDA and incubated at 28
"C
for 4 days.
Similarly, the germination of R. solani sclerotia were tested by treating the 50
sclerotia with purified chitinase or LTP (300 pg) in sterile test tubes. Fifty sclerotia
treated with 300 pg of protein extracts of induced E. coli containing PET 28a+ or
pMAL-p2x without insert chitinase or
LTP
served
as
control. Both the test and control
tubes were incubated at 28
"C
for 2 days. Then the sclerotia were checked for
germination by placing onto PDA plates which were incubated at 28
"C
for 48 h. The
spore germination assays were repeated twice to confirm the results.
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