FUNGICIDAL ACTIVITY OF FUNGICIDAL ACTIVITY OF MARINE ACTINOMYCETES MARINE ACTINOMYCETES

AGAINST PHYTOPATHOGENIC AGAINST PHYTOPATHOGENIC FUNGIFUNGI

K.KATHIRESAN, R.BALAGURUNATHAN, & K.KATHIRESAN, R.BALAGURUNATHAN, & M.MASILAMANI SELVAMM.MASILAMANI SELVAM

INDIAN JOURNAL OF BIOTECHNOLOGYINDIAN JOURNAL OF BIOTECHNOLOGY

VOL 4, APRIL 2004, pg 271-276VOL 4, APRIL 2004, pg 271-276

BY R.PRABHU & SWATHY.SBY R.PRABHU & SWATHY.S

ABSTRACTABSTRACT• 160 isolates of marine actinomycetes were 160 isolates of marine actinomycetes were

isolated from sediments samples drawn from isolated from sediments samples drawn from mangroves, estuaries, sand dunes and mangroves, estuaries, sand dunes and industrially polluted coasts.industrially polluted coasts.

• Mangrove sediments were rich source of Mangrove sediments were rich source of actinomycetes.actinomycetes.

• Each isolate was tested against phytopathogenic Each isolate was tested against phytopathogenic fungi.fungi.

• 10 of the isolates were found to be potent anti 10 of the isolates were found to be potent anti fungals.fungals.

• The antifungal species were mainly found to be The antifungal species were mainly found to be Streptomyces species of marine origin.Streptomyces species of marine origin.

IntroductionIntroduction

• Fungal diseases in agricultural crops pose a very Fungal diseases in agricultural crops pose a very major problem.major problem.

• The current fungicides are either less effective or The current fungicides are either less effective or of great environmental concern.of great environmental concern.

• So environmentally safe and potent fungicides of So environmentally safe and potent fungicides of natural origin are requirednatural origin are required

• Marine actinomycetes are important sources of Marine actinomycetes are important sources of novel antibioticsnovel antibiotics

• In this study the isolated actinomycetes were In this study the isolated actinomycetes were tested against 4 pathogenic fungi causing tested against 4 pathogenic fungi causing diseases in crops like rice and sugarcane.diseases in crops like rice and sugarcane.

Experimental conditionsExperimental conditions• The isolates were tested against phytopathogens The isolates were tested against phytopathogens

namely Rhizoctonia solani, Pyricularia oryzae, namely Rhizoctonia solani, Pyricularia oryzae, Helminthosporium oryzae, Colletotrichum fulcatumHelminthosporium oryzae, Colletotrichum fulcatum

• Glucose and soyabean meal were found to be the Glucose and soyabean meal were found to be the best carbon and nitrogen sources resp.best carbon and nitrogen sources resp.

• 17.5 ppt was the preferred salinity level.17.5 ppt was the preferred salinity level.

• Cylinder plate method of antifungal assay was Cylinder plate method of antifungal assay was preferred over disc diffusion method.preferred over disc diffusion method.

• 120 hours of incubation was required to produce 120 hours of incubation was required to produce high antifungal compoundshigh antifungal compounds

Materials And MethodsMaterials And Methods

Collection of SamplesCollection of Samples

• Sediment samples were collected from Sediment samples were collected from industrially polluted coastline of cuddalore, industrially polluted coastline of cuddalore, sand dunes, vellar estuary, mangrove sand dunes, vellar estuary, mangrove vegetation and other places along the vegetation and other places along the Indian coastline.Indian coastline.

Isolation of ActinomycetesIsolation of Actinomycetes

• The sediment samples were air dried for The sediment samples were air dried for one week.one week.

• They were then kept at 45 C for 1hourThey were then kept at 45 C for 1hour

• 1g of sample was transferred to flask with 1g of sample was transferred to flask with 99ml sterile 50% sea water.99ml sterile 50% sea water.

• 1ml of 51ml of 5thth dilution culture was spread on dilution culture was spread on starch-caesin agar medium with 50% sea starch-caesin agar medium with 50% sea water.water.

• ph at 7.5. ph at 7.5.

• Temperature around 28 C.Temperature around 28 C.

• cyclohexamine and nalidixic acid to cyclohexamine and nalidixic acid to prevent contamination.prevent contamination.

• The colonies were observed for one month.The colonies were observed for one month.

• Strains of marine Actinomycetes were Strains of marine Actinomycetes were picked out.picked out.

• They were purified by repeated streaking They were purified by repeated streaking on yeast extract-malt extract agar (ISP2) on yeast extract-malt extract agar (ISP2) medium.medium.

• The pure cultures were transferred to ISP2 The pure cultures were transferred to ISP2 slants and stored at around 4 Cslants and stored at around 4 C

Screening for antifungal activity:Screening for antifungal activity:Primary screening:Primary screening:• A modified cross streak method is used.A modified cross streak method is used.

• Yeast extract-glucose agar plates were Yeast extract-glucose agar plates were inoculated with the actinomycetes at the inoculated with the actinomycetes at the centre.centre.

• Maintained at 28 C for 5 days.Maintained at 28 C for 5 days.

• Four day old cultures of the 4 phytopathogenic Four day old cultures of the 4 phytopathogenic fungi were cross plugged 15mm away from the fungi were cross plugged 15mm away from the actinomycetes on the sides.actinomycetes on the sides.

• They were maintained at the same temperature They were maintained at the same temperature for 78 hours to see the inhibition zone.for 78 hours to see the inhibition zone.

• Strains of high antifungal activity were selected.Strains of high antifungal activity were selected.

Secondary Screening:Secondary Screening:• The spore suspension of the selected isolates The spore suspension of the selected isolates

were inoculated into soyabean medium and were inoculated into soyabean medium and kept in a shaker.kept in a shaker.

• After 96 hrs the culture broth was seperated After 96 hrs the culture broth was seperated at 5000 rpm.at 5000 rpm.

• 0.1ml of 4 day old cultures of test fungi was 0.1ml of 4 day old cultures of test fungi was inoculated on potato dexrose agar medium in inoculated on potato dexrose agar medium in petri plates.petri plates.

• Sterile paper disc with 0.1ml of isolate broth Sterile paper disc with 0.1ml of isolate broth was placed in the centre of those plates.was placed in the centre of those plates.

• Incubated at 28 C for 48 hrs.Incubated at 28 C for 48 hrs.

• Zone of inhibition was recorded.Zone of inhibition was recorded.

Optimization of cultural Optimization of cultural conditionsconditions

• Culture maintained for 48, 96, 120, 168 hrs to Culture maintained for 48, 96, 120, 168 hrs to study effect of incubation.study effect of incubation.

• Different carbon sources (glucose, glycerol etc) Different carbon sources (glucose, glycerol etc) and different nitrogen sources (soyabean and different nitrogen sources (soyabean meal, yeast and malt extract etc) were used to meal, yeast and malt extract etc) were used to study their effect.study their effect.

• The broth drawn at 120 hrs was checked for The broth drawn at 120 hrs was checked for antifungal activity.antifungal activity.

• The growth was also checked with different The growth was also checked with different range of salinities.range of salinities.

Identification of marine Identification of marine ActinomycetesActinomycetes

• The ten isolates which were potent The ten isolates which were potent antifungals were identified byantifungals were identified byAerial mass colourAerial mass colourMelanoid pigmentMelanoid pigmentReverse side pigmentReverse side pigmentSpore chain morphologySpore chain morphologyAssimilation of carbon sources &Assimilation of carbon sources &By comparison of charactersBy comparison of characters

Identification of strainsIdentification of strains

• The 10 highly effective strains were found The 10 highly effective strains were found to be diff species of Streptomyces.to be diff species of Streptomyces.

• They had white, grey, red or yellow They had white, grey, red or yellow mycelium.mycelium.

• They lacked melanin pigments.They lacked melanin pigments.

• Carbon source utilised was different from Carbon source utilised was different from strain to strain.strain to strain.

Results And DiscussionsResults And Discussions

• A large number of fungal isolates were A large number of fungal isolates were obtained from mangrove sediments.obtained from mangrove sediments.

• Less number from industrially polluted.Less number from industrially polluted.• So industrial pollution affects So industrial pollution affects

Actinomycetes growth.Actinomycetes growth.• But mangrove sediments with domestic But mangrove sediments with domestic

sewage enhanced Actinomycetes growth.sewage enhanced Actinomycetes growth.• So domestic sewage can be used as a So domestic sewage can be used as a

source for Actinomycetes growth.source for Actinomycetes growth.

Antifungal activityAntifungal activity• In primary screening >10mm inhibition zone In primary screening >10mm inhibition zone

were termed effective & <10mm ineffective.were termed effective & <10mm ineffective.

• About 51% of isolates were effective against About 51% of isolates were effective against H. H. oryzaeoryzae and P. and P. oryzaeoryzae..

• About 31% were effective against R. About 31% were effective against R. solanisolani..

• 12.5% were effective against C. 12.5% were effective against C. falcatumfalcatum

• 10 strains were selected for secondary 10 strains were selected for secondary screening.screening.

• 1 strain showed >20mm inhibition zone 1 strain showed >20mm inhibition zone against all four fungi.against all four fungi.

• 4 strains showed >20mm against H. 4 strains showed >20mm against H. oryzaeoryzae and P. and P. oryzae.oryzae.

• The inhibition zone increased with increase The inhibition zone increased with increase in incubation time.in incubation time.

• Maximum antifungal activity at 120hrs after Maximum antifungal activity at 120hrs after which activity declined.which activity declined.

• Soyabean meal, yeast extract & malt Soyabean meal, yeast extract & malt extract were found as good nitrogen extract were found as good nitrogen sources.sources.

• Glycerol, glucose, maltose & lactose were Glycerol, glucose, maltose & lactose were found to be good carbon sources.found to be good carbon sources.

• 17.5 ppt salinity level was found to be the 17.5 ppt salinity level was found to be the best.best.

• Cylinder plate method showed higher Cylinder plate method showed higher activity than disc diffusion method.activity than disc diffusion method.

ConclusionConclusion

• The marine Actinomycetes are available in The marine Actinomycetes are available in plenty.plenty.

• Only some of them have been studied. Only some of them have been studied.

• So far only few active compounds have So far only few active compounds have been isolated with broad spectrum activity.been isolated with broad spectrum activity.

• The 10 highly effective strains must be The 10 highly effective strains must be investigated further and ecofriendly investigated further and ecofriendly effective and economical fungicides must effective and economical fungicides must be produced. be produced.

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