RA 00110.pdf - OAR@ICRISAT

in sterilized distilled water. Leaves were air dried and
incubated for 6 h in the dark at 100% R H . On the
heavily sporulating leaves, under sterile conditions,
young seedlings with just emerging radicle and plumule
were placed in contact for 4-5 h. They were
transferred to the Murashige and Skoog medium
with growth supplements containing culture flasks
and incubated at 20 ± 1°C in alternating 12 h dark
and light (5000 lux) cycles. After 3 d, the callus
initiated from the hypocotyl region of seedlings. In 6
d, white downy mildew mycelia were observed on
the callus, which could be subsequently maintained
(Fig. 5). This technique can be used successfully to
screen host cultivars for susceptibility to downy mildew
infection, to study the infection processes of the
various host-parasite interfaces with the electron
microscope, and also can be easily used to study the
host range of a biotrophic pathogen in vitro (Prabhu
1985).
The tissue culture technique to determine the viability
of the internally seed borne mycelium of S.
graminicola in pearl millet seeds has been detailed by
Prabhu et al. (1983). The callus originated from the
hypocotyl region of the seedling, and the mycelium
expressed on the callus, should have originated from
embryonic tissue. Sometimes, the primary callus
turned brown at the initial stages, but after subculturing
on the same medium some of them developed
healthy secondary callus, which supported a clear
network of mycelial growth. This technique is very
useful where a small quantity of valuable seed material
needs to be tested for internally-borne downy
mildew inoculum without loss of the seeds. Plantlets
from the callus tissue can be raised to save the germplasm.
This tissue-culture technique can also be used
in quarantine laboratories to detect downy mildew
or other biotrophic organisms associated with seeds.
Many techniques have been developed to maintain
the inoculum of downy mildews. Culture on
excised leaf pieces was tried by Singh (1970), who
floated diseased maize leaf pieces infected with
Sclerospora rayssiae var. zeae Payak & Renfro in
sucrose with 20 ppm kinetin to produce "a good
harvest" of sporangia. For Sclerospora sorghi, Kenneth
(1970) used 60-200 ppm benzimidazole and
obtained sporulation continually for 7 d. Gale et al.
(1975) reported the cryogenic storage of S. sorghi
conidia to maintain the inoculum. Intact seedlings
were used to maintain the various populations of
maize downy mildews using the monospore culturing
method (Dogma 1974). The dual culture system
can be used safely to maintain the inoculum and
different isolates and pathotypes of S. graminicola
without sophisticated laboratory facilities. No in
vitro studies on the chemical control of the downy
mildew pathogen have been conducted except with
downy mildew on grape, where dual cultures were
used to demonstrate the systemic and curative properties
of metalaxyl on downy mildew (Lee and Wicks
1982). Similar studies can be extended to pearl
G e r m i n a t i n g seeds
I n o c u l a t i o n
S p o r u l a t i n g l e a v e s
C a l l u s i n i t i a t i o n
on MS medium
Development o f
dual c u l t u r e
Figure 5. Seedling inoculation technique to culture Sclerospora graminicola on pearl millet callus.
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