THE GENUS PYTHIUM - An-Najah National University

B. Isolation of Pythium spp from infected plant parts:
Pythium spp were isolated from infected plant tissue by plating on the VP3
medium plates or on plates of 2% water agar containing 5 mg( L pimarinin.
Excised plant material was first washed under running tap water for about 2
hours, surface-sterilised in 0.5% sodium hypchlorite for 3 miniutes, rinsed in
sterile distilled water, blotted dry on sterile filter paper, and then plated on the
agar plates. The plates were then incubated at 22-250 C for 24-48 hours and
examined for the presence of Pythium colonies.
MAINTENANCE OF STOCK CULTURES
Stock cultures of representative isolates of Pythium species studied were
maintained either in water culture in 100mi conical flasks (Dick, 1965), or on
CMA slants (Robertson, 1980; Plaats-Niterink., 1981) in 100mi universal bottles.
All the stock cultures were kept at 100e.
IDENTIFICA TION OF PYTHIUM SPECIES
Transfer of colonies from rough cultures: Small excisions from growing margin
of Pythium colonies. on the isolation plates or on rinsed baits were cut off and
either transferred directly to water culture with 1-2 autoclaved corn kernels per
dish, or to corn meal agar (CMA) or 2% water agar plates, and then transferred
into water culture after they had been incubated for 24-48h at 220 C.
Representative isolates of the isolated species (3 of each) are maintained at the
Fungal Culture Collection of An-Najah University (Ali-Shtayeh, 1986b).
Induction of sporangial and zoospore formation: For production of sporangia
and zoospores, dishes were half-filled with sterile distilled water (OW) or with
an autoclaved mixture of one part filtered pond water and two parts distilled
water (DDW). Two grass blades (Emerson, 1958), boiled for IS minutes, were
then placed on or near the hyphal tips. Coloized grass blades were then
transferred to new dishes half-filled with water (OW or OOW as appropriate),
incubated for 24-48 h at 10-22° C, rinsed daily with water from the same source,
and examined periodically for 2-7 days for zoosporangia . If zoospores were not
produced at these temperatures, cultures were put in a refrigerator at 2-50 C for
1-2 h before they were examined for zoospore production. Changing the water
and chilling the cultures may enhance both the production of sporangia and the
discharge of zoospores (Ali, 1982).
Induction of sexual structures formation: For production of sexual structures
similar blocks of agar containing hyphal tips were flooded with OW. Two boiled
corn kernels were placed adjacant to the hyphal tips and cultures were then
incubated at 22 0 C for 7-14 days before they were examined. When oogonia were
not produced in water culture, isolates were grown on Schmitthenner's medium
(Schmitthenner, 1962) or on CMA supplemented with wheat germ oil (500
mg( L) to provide sterols needed for sexual reproduction (Hendrix & Papa,
1974). Schmitthenner's medium and CMA supplemented with wheat germ oil
were found to be satisfactory for oogonial production and microscopic
examination was easy because of the clear substrate.
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