Isolation of toxin(s)
The G and I isolates were maintained by periodic transfer in Potato
Dextrose Agar (PDA). For isolation of toxin(s), seeding production
flasks containing 50 mI of modified Fries medium were inoculated with
fungal mycelium grown on PDA plates and cultured for 7 days at 21C
under fluorescent lights. The flasks were vigorously shaken to break
the. mycelium dump and 1 mI of mycelial suspension was transferred to
production flasks containing 50 mI of Fries medium and cultured for
28 days at 21C in stationary conditions under fluorescent lights. The
culture filtrate was obtained by successive filtration through two
layers of muslin/cheesecloth, Whatman No. 1 filter paper and 0.2 mum
nylon membrane filter. The culture filtrate was used for toxin
isolation by the procedure of Ward et al. (1975) with little
modifications. To the filtrate an equal volume of solvent
(chloroform: ethyl acetate = 9 : 1) was added and vigorously shaken.
The lower chloroform phase was collected and the extraction procedure
was repeated 3 times. To the chloroform extract, .anhydrous sodium
sulphate (100 g/1) was added for breaking the emulsion. The settled
salts were filtered out and the filtrate was evaporated at 38C in
vacuo. The browny residue was dissolved in water (20 mI for 1
litre of culture filtrate). H. sativum is known to produce
helminthosporal toxic substance with an optimum absorbance at 266 nm
(Ludwig 1957). The toxin concentrations from the two pathostrains
were calculated based on 1 litre of culture filtrate obtained after
inoculation of 1 ml of mycelial suspension. The toxin content in
moles/l of culture filtrate was quantified by applying Lambert and
Bear formula as: A = E b c; where A = absorbance; E = molar
absorptivity (=11,000); b = path length (=1 cm); and c =
concentration in moles /1.
Bioassay using intact leaves
50 micro-liter of spore suspension (106/ml) and a crude
toxin preparation of the two pathostrains were inoculated on leaves
of 21-day-old potted plants. Distilled water was used as control. The
plants were kept under high humidity for symptom development. After a
week, data were recorded based on leaf necrosis using 2 plants of
each cultivar.
Bioassay using detached leaves
1% agar medium containing 50 mg/l benzimidazole was prepared and
poured in petri dishes. Benzimidazole was added for its cytokinin
like effect in prolonging the retention of chlorophyll in the leaves
(Pringle 1977). Leaves of 21-day-old plants. were detached and
surface sterilized in sodium hypochlorite (1% active chlorine)
followed by 3 - 4 rinses in sterile distilled water. Tip and basal
portion of the leaves were removed and the central portion was placed
in petri dishes. The detached leaves. were pricked with a needle or a
ballpoint pen and inoculated with 50 mul of spore suspension or crude
toxin(s). Petri dishes were sealed and kept at 25C for 3 day under
fluorescent lights.
Data scoring
The data were recorded based on the degree of necrosis in the
detached/ intact leaves and arbitrarily scored; 0: no infection, 1:
very little necrosis, 2: little necrosis, 3: moderate necrosis, 4:
high necrosis, 5: very high necrosis. The data were taken from two
samples from each cultivar and analyzed statistically for comparison
of pathogen and toxin reactions of each pathostrain using paired 't'
test.