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K.KATHIRESAN, R.BALAGURUNATHAN, &

M.MASILAMANI SELVAM
INDIAN JOURNAL OF BIOTECHNOLOGY
VOL 4, APRIL 2004, pg 271-
271-276

BY R.PRABHU & SWATHY.S

 ABSTRACT
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 Introduction
ƥ Fungal diseases in agricultural crops pose a very major
problem.
ƥ The current fungicides are either less effective or of
great environmental concern.
ƥ So environmentally safe and potent fungicides of natural
origin are required
ƥ Marine actinomycetes are important sources of novel
antibiotics
ƥ In this study the isolated actinomycetes were tested
against 4 pathogenic fungi causing diseases in crops like
rice and sugarcane.
 Experimental conditions
ƥ The isolates were tested against phytopathogens namely
Rhizoctonia solani, Pyricularia oryzae, Helminthosporium
oryzae, Colletotrichum fulcatum

ƥ Glucose and soyabean meal were found to be the best

carbon and nitrogen sources resp.

ƥ 17.5 ppt was the preferred salinity level.

ƥ Cylinder plate method of antifungal assay was preferred

over disc diffusion method.

ƥ 120 hours of incubation was required to produce high

antifungal compounds
 Materials And Methods

Collection of Samples

ƥ Sediment samples were collected from

industrially polluted coastline of cuddalore, sand
dunes, vellar estuary, mangrove vegetation and
other places along the Indian coastline.
 Isolation of Actinomycetes

ƥ The sediment samples were air dried for one

week.
ƥ They were then kept at 45 C for 1hour
ƥ 1g of sample was transferred to flask with 99ml
sterile 50% sea water.
ƥ 1ml of 5th dilution culture was spread on starch-
starch-
caesin agar medium with 50% sea water.
ƥ ph at 7.5.
ƥ Temperature around 28 C.
ƥ cyclohexamine and nalidixic acid to prevent
contamination.
ƥ The colonies were observed for one month.
ƥ Strains of marine Actinomycetes were picked
out.
ƥ They were purified by repeated streaking on
yeast extract-
extract-malt extract agar (ISP2) medium.
ƥ The pure cultures were transferred to ISP2
slants and stored at around 4 C
 Screening for antifungal activity:
Primary screening:
ƥ A modified cross streak method is used.
ƥ Yeast extract-
extract-glucose agar plates were
inoculated with the actinomycetes at the centre.
ƥ Maintained at 28 C for 5 days.
ƥ Four day old cultures of the 4 phytopathogenic
fungi were cross plugged 15mm away from the
actinomycetes on the sides.
ƥ They were maintained at the same temperature
for 78 hours to see the inhibition zone.
ƥ Strains of high antifungal activity were selected.
Secondary Screening:
ƥ The spore suspension of the selected isolates
were inoculated into soyabean medium and kept
in a shaker.
ƥ After 96 hrs the culture broth was seperated at
5000 rpm.
ƥ 0.1ml of 4 day old cultures of test fungi was
inoculated on potato dexrose agar medium in
petri plates.
ƥ Sterile paper disc with 0.1ml of isolate broth was
placed in the centre of those plates.
ƥ Incubated at 28 C for 48 hrs.
ƥ Zone of inhibition was recorded.
 Optimization of cultural conditions

ƥ Culture maintained for 48, 96, 120, 168 hrs to

study effect of incubation.
ƥ Different carbon sources (glucose, glycerol etc)
and different nitrogen sources (soyabean meal,
yeast and malt extract etc) were used to study
their effect.
ƥ The broth drawn at 120 hrs was checked for
antifungal activity.
ƥ The growth was also checked with different
range of salinities.
Identification of marine Actinomycetes

ƥ The ten isolates which were potent antifungals

were identified by
î Aerial mass colour
î Melanoid pigment
î Reverse side pigment
î Spore chain morphology
î Assimilation of carbon sources &
î By comparison of characters
 Identification of strains
ƥ The 10 highly effective strains were found to be
diff species of Streptomyces.
ƥ They had white, grey, red or yellow mycelium.
ƥ They lacked melanin pigments.
ƥ Carbon source utilised was different from strain
to strain.
 Results And Discussions
ƥ A large number of fungal isolates were obtained
from mangrove sediments.
ƥ Less number from industrially polluted.
ƥ So industrial pollution affects Actinomycetes
growth.
ƥ But mangrove sediments with domestic sewage
enhanced Actinomycetes growth.
ƥ So domestic sewage can be used as a source for
Actinomycetes growth.
Antifungal activity
ƥ In primary screening >10mm inhibition zone
were termed effective & <10mm ineffective.
ƥ About 51% of isolates were effective against H.
oryzae and P. oryzae
oryzae..
ƥ About 31% were effective against R. solani.
solani.
ƥ 12.5% were effective against C. falcatum
ƥ 10 strains were selected for secondary
screening.
ƥ 1 strain showed >20mm inhibition zone against
all four fungi.
ƥ 4 strains showed >20mm against H. oryzae and
P. oryzae.
ƥ The inhibition zone increased with increase in
incubation time.
ƥ Maximum antifungal activity at 120hrs after
which activity declined.
ƥ Soyabean meal, yeast extract & malt extract
were found as good nitrogen sources.
ƥ Glycerol, glucose, maltose & lactose were found
to be good carbon sources.
ƥ 17.5 ppt salinity level was found to be the best.
ƥ Cylinder plate method showed higher activity
than disc diffusion method.
 Conclusion
ƥ The marine Actinomycetes are available in
plenty.
ƥ Only some of them have been studied.
ƥ So far only few active compounds have been
isolated with broad spectrum activity.
ƥ The 10 highly effective strains must be
investigated further and ecofriendly effective
and economical fungicides must be produced.
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