RA 00110.pdf - OAR@ICRISAT

tion process. Studies have indicated that although
infection can occur at 90% and 95% R H , 100% RH is
best to produce maximum infection at 23 ± 1°C. A
dew period of about 2 h is enough to cause infection,
but about a 6-hour dew period is necessary at 23°C
for maximum infection.
Inoculation with single zoospores of 2-day-old
N H B 3 pearl millet seedlings did not produce infection.
Five zoospores was the minimum needed to
infect a seedling. The infection percentage increased
with an increase in the number of zoospores, however
the percentage infection remained constant
beyond a concentration of 100 zoospores per seedling.
The incubation period also decreased with
increased inoculum concentration. Disease expression
began in 8-20 d depending upon the zoospore
concentration. Disease intensity increased with increased
inoculum load, and became constant beyond
20 000 zoospores ml -1 (Ramesh 1981).
Tissue Culture Studies
Tiwari and Arya (1967) reported the establishment
of S. graminicola on pearl millet callus by placing
systemically infected tissues of a head on modified
White's medium. Tiwari and Arya (1969) reported
axenic growth of S. graminicola, and maintained the
saprophytic growth of the fungus for two subsequent
subcultures which later perished. Shaw and
Safeeulla (1969), Safeeulla (1976b), and Bhat et al.
(1980) established dual cultures of S. graminicola by
inoculating the healthy host callus with asexual
inocula on modified White's medium. The fungus
grew on the medium up to a short distance from the
callus, however they failed to get axenic fungus
growth. Prabhu (1985) used three different basal
media to find the most suitable one to establish the
host callus and S. graminicola. Among them, Murashige
and Skoog basal medium (1962) best supported
initiation and growth of dual cultures. Although
cultures were initiated on the modified
White's medium, a lot of reddish brown secretions
exuded into the medium inhibiting further growth of
the callus and the fungus. In Linsmaier and Skoog
medium (1965), the growth and development of the
callus were not satisfactory. In the author's study the
stem tip of pearl millet produced a fleshy type of
callus, but the young inflorescence produced a nodular
type. From the downy mildew-infected stem tip
and inflorescence of pearl millet, the callus developed
within a week and the mycelia appeared
directly on the callus after 20-25 d. The growth of 5.
graminicola mycelium was profuse on fleshy type
callus. The mycelium of S. graminicola was aseptate,
coenocytic with irregular wavy walls. It remained
intracellular at the beginning and later became
intercellular.
In the early stages, the mycelia appeared beaded,
but later changed into the characteristic downy mildew
type. The mycelium was dense in the peripheral
region of the callus with spores absent in the central
region. After profuse growth of the mycelium, asexual
spores were produced, but shapes and sizes varied
from the normal ones produced on the infected
leaf surface. Subsequently, asexual spore production
decreased with the initiation of oogonial and
antheridial structures. The mycelial growth on the
host callus usually produced oospores for about 50-
55 d (Prabhu 1985).
Morphology of the oospores produced on callus
tissue is similar to that produced on infected tissues
of pearl millet grown in soil. The healthy callus of
pearl millet placed in contact with the infected ones
showed infection within 3 d. The 2-day-old pearl
millet seedlings brought in contact with S. graminicola
on callus for 24 h expressed downy mildew disease
symptoms 1 week after transfer to sterilized soil.
Subculturing the healthy callus is necessary once a
month to retain strong growth, and the fungus will
grow on the vigorously growing callus. Maintained
like this, the fungus retained its virulence over 5
years without any change.
Establishment of S. graminicola Dual
Culture on Host Callus Tissue
In most cases, healthy callus tissues were infected by
the asexual propagules of the pathogen. In some
cases, the S. graminicola-infected meristematic tissue
of the pearl millet plant was used for raising dual
cultures (Prabhu 1985). Prabhu (1985) used very
young pearl millet seedlings to raise dual cultures
after infecting them with asexual propagules. In this
study, the seeds of highly susceptible pearl millet
hybrid, NHB 3, were surface sterilized with 0.1% of
HgCl 2 for 5 min followed by thorough washings in
sterilized distilled water. The seeds were placed on
sterile moist blotters in sterilized petri plates and
incubated at 20 ± 1°C for 24 h. The next day, richly
sporulating pearl millet leaves were brought from
the field and washed well in sterilized distilled water
to remove the old spores. Under aseptic conditions,
the sporulating leaf surface was swabbed with cotton
soaked in 1% chlorine solution followed by washing
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