Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 158-165

ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 158-165 http://www.ijcmas.com

Original Research Article Studies on the Bioactive Actinomycetes of Estuarine Soils of South West Coast of

R. P. Dhanya1*, R. Anusha1 and T. Citarasu2

1Department of Microbiology, Noorul Islam College of Arts and Science, Kumaracoil, , Tamilnadu, India 2Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, , Tamilnadu, India *Corresponding author

A B S T R A C T

The present study was designed to isolate chitinolytic actinomycetes from the estuarine soil sediments of South west coast of Tamilnadu. A total of 20 chitinase producing actinomycetes were isolated from the estuaries of Thengapattanam, K e y w o r d s Manakudy and Rajakkamangalam. All the 20 isolates were subjected to antifungal Estuaries, activity against 6 phytopathogenic fungi Aspergillus niger, Fusarium oxysporum, Chitinolytic Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Penicillium sp. by actinomycetes, disc diffusion method. Starch casein agar media was used for the isolation of Antifungal actinomycetes. Out of the 20 antifungal isolates screened, the isolate from activity Manakudy estuary ESM9 showed good inhibitory activity against the test fungal pathogens. A maximum zone of inhibition of 32 mm was expressed against Fusarium oxysporum and Phytophthora capsici. The isolates EST4, ESR2 and ESR8 were also found to be potential inhibitors of pathogenic fungi

Introduction

Actinomycetes comprise an extensive and are thin, possess cell walls containing diverse group of gram positive, aerobic muramic acid. Actinomycetes are well bacteria frequently filamentous and known as a good source of microbial sporulating with DNA rich in G+C secondary metabolite producer in drug nucleotide content (75%). They play an discovery programs. Many species important ecological role in soil cycle (Holt especially those belonging to the genus et al., 1989). Actinomycetes have a Streptomycetes have been studied as distinctive and diverse macroscopic and potential producers of metabolites with microscopic morphology and they are diverse structures and biological activities traditionally considered to be transitional (Berdy, 2005). They are widely distributed forms between bacteria and fungi. Like in soil, water and natural environments fungi, they form a mycelial network of where their population and types in an branching filaments, but like bacteria they ecosystem are determined by numerous

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 158-165 physical, chemical and biological factors. plicatus, S. lividans, S. aureofaciens and S. Identification of novel ecological systems is halstedi (Taechowisan et al., 2003). therefore crucial for the discovery of novel Streptomyces viridificans was found to be a actinomycetes (Wang et al., 1999). In recent good chitinase producer and its crude and years there has been a growing awareness of purified enzyme has potential cell wall lysis the potential value of fresh water habitat as of many fungal pathogens (Gupta et al., source of actinomycetes that produce useful 1995). metabolic products. In fact some investigators emphasized that freshwater Lysis of the host structure by secretion of habitats are fruitful as those isolated extracellular lytic enzymes is one of the organisms are from terrestrial habitats important mechanisms that are involved in (Cross, 1981). The genus Streptomyces is the antagonistic activity of biocontrol agents found worldwide and they play an important (Kim and Ji, 2001). Among these, chitinase role in soil and plant ecology. These (EC 3.2.1.14) plays a vital role in the organisms have been widely investigated as biological control of many plant diseases by agents of biological control of several plant degrading the chitin polymer in the cell diseases. walls of fungal pathogens. It affects fungal growth through the lysis of cell walls. The Chitinases are a group of enzymes which main mechanisms involved in the hydrolyse the -1, 4 linkages and cleaving antagonism of biocontrol agents are a bond between the c1- c4 of two mycoparasitism, competition for space and consecutive N-acetyl glucosamines of chitin nutrients, stimulation of the plant s to low molecular weights products and defensive capacity, and secretion of have been shown to be produced by a bioactive compounds such as antibiotics and number of microorganisms. Generally cell wall degrading enzymes. As the chitinase producing strains will use chitin or skeleton of the fungal cell wall mainly colloidal chitin a carbon source. Chitinases contains chitin, glucan and proteins, are widely distributed in bacteria such as mycoparasitism and enzymes that hydrolyze Serratia, Chromobacterium, Klebsiella, these components are one of the main Pseudomonas, Clostridium, Vibrio, mechanisms accounting for showing Arthrobacter, Aeromonas and Streptomyces. antagonistic important in the hyper-parasitic They are also found in fungi Trichoderma, mechanism. The distribution of chitinases in Penicillium, Neurospora, Mucor, Beauveria, nature is very common and actinomycetes Aspergillus, Myrothecium, Metarrhizium are found to display strong fungicidal and Agaricus. Chitinase is known to be properties. It is related to the production of produced by a wide range of actinomycetes many types of various fungicidal (Hsu and Lockwood, 1975). Streptomyces compounds, including antibiotics and are also explored for chitinase production extracellular enzymes such as chitinase and and they are thought to be one of the major -1,3-glucanases. Several chitinolytic chitinivorous microbial groups in the soil enzymes have been identified in various due to their ability to degrade chitin. This Streptomyces sp, including, Streptomyces has long been regarded as a characteristics plicatus, S. lividans, S. virdificans and S. feature of soil Streptomycetes (Blaak et al., halstedii. S. cavourensis SY224, and S. 1993). Several chitinolytic enzymes have halstedii produce highly active antifungal been identified in several Streptomyces sp. chitinase which implies the possibility of including S. antibioticus, S. griseus, S. using them as agents for biological

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 158-165 protection of crops. However studies have Arabian Sea after transversing about 85km shown that the combination of two bacteria were selected for this study. Soil samples Streptomyces sp. 385 and Paenibacillus were collected from the top 5 10 cm sp.300 are found to be more effective sediments from four different stations of the against Fusarium oxysporum causing selected estuaries during three seasonal cucumber wilt than individual strains or periods (post , summer and other combinations. Chitinolytic bacteria monsoon). The four stations were selected in such as Aeromonas hydrophila, Aeromonas the following pattern. Station I is the caviae, Pseudomonas maltophila, Bacillus shallow coastline region, Station II is the licheniformis, Bacillus circulans, Vibrio mid estuarine region, Station III is the fresh furnissii, Xanthomonas sp., and Serratia water zone and the Station IV is the mouth marcescens play an important role in of the estuary. The collected samples were biological control of plant pathogenic fungi transferred to a sterile polythene bag and (Gohel et al., 2005). Interest in biological transported immediately. The sediment control of plant pathogens has increased samples thus collected were then used for considerably over the past years, partly as a the isolation of actinomycetes. response to public concern about the use of hazardous chemical pesticides, but also Isolation of Actinomycetes because it may provide control of diseases that cannot, or only partially, be managed by The sediment samples were stored at 4 °C other control strategies. The present study and analyzed by plating their serial dilution explains the broad distribution of on to the selective agar media Starch Casein chitinolytic bacteria in estuarine habitats Agar (Kuster and Williams, 1964). To which can be used as biocontrol agents minimize the growth of fungi and against phytopathogenic fungi. undesirable bacteria, nystatin (20 g/ml) and cyclohexamide (100 g/ml) were added to Materials and Methods the isolation media. The plates were incubated at 28 °C for 2 3 weeks. The plates Study area were observed for the presence of actinomycetes colonies. However, colonies Soil sediments from three different estuaries started appearing from sixth day onwards Manakudy, the second largest estuary in the (Zhao et al., 2004). District has a total area of 145 hectares. It is located at the North West of Cape Comorin Plates were examined for the appearance of falling within the latitude of 8° 4¹ and 8° actinomycetes colonies from sixth day 21¹N and longitude 77°26¹ and 77°30¹E onwards up to 20 days. Total number of Thengapattanam is located in the South west colonies in each set of plates is recorded as coast of India. This minor estuary of CFU/gm dry soil (Ghanem et al., 2000). Kanyakumari district is formed by the confluence of Tamiraparani River with Many colonies with different morphological Arabian Sea in between Thengapattanam and cultural characteristics generally and Eraiummanthurai (7°53¹ N Latitude and appeared with a tough leathery or chalky 77°07¹E longitude) and Rajakkamangalam texture; dry or folded appearance and estuary is situated along the Southwest coast branching filamentous with or without aerial of India. This is the place where the mycelia are picked (Mincer et al., 2002) Pantrivaikkal discharge freshwaters into the from the isolation plates and streaked for

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Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 158-165 purification. The pure cultures were 5 mm) laid on to the swabbed plates. 50 µl transferred to PDA slopes and incubated for of the enzyme filtrate was added to the paper seven to ten days. When sufficient growth discs. The plates were then incubated at 30 had occurred, the tubes were stored at 4 °C °C for 2 to 4 days. The radial diameter of the in a refrigerator and constituted the stock zone formation was measured (Gomes et al., cultures. The stock cultures were maintained 2001) and transferred to fresh PDA slant once in two months and stored at 4 °C. The isolated Result and Discussion colonies were enumerated and subjected to purification. The soil samples from estuaries are reported as rich habitat for microbial diversity. The Primary screening of chitinolytic investigators from all over the world have Actinomycetes isolated actinomycetes for various applications and they have found immense The selected colonies were subjected to potential and diversity. These filamentous primary screening for chitinase enzyme bacteria have been able to colonize activity. The production of chitinase was geographically distinct locations like plain performed by plate assay method using lands, agricultural soils, compost soil, river colloidal chitin (Hsu and Lockwood, 1975). waters, estuaries, oceans, mountains, snow Chitinase activity of the strains were covered Arctic regions etc. The total screened in a Yeast Nitrogen Base medium actinomycetes population of the bottom containing 0.2 % (W/V) chitin and 0.5 % sediments were enumerated using spread yeast extract and 1.5% agar (Watanabe et plate technique. About 60 isolates were al., 1990). After incubation at 28 °C for up selected from all the estuarine sediments. A to 8 days 0.1 % Congo red solution was number of reports have been reported on the added. occurrence and distribution of actinomycetes in estuaries & marine environments (Walker Finally potential isolates showing and Colwell, 1975). Early reports have chitinolytic activity were selected for shown that actinomycetes are widely spread studying their activity against in various water bodies where they play a phytopathogenic fungi. great part in the carbon cycle due to their ability to grow at low concentrations of Antifungal activity carbonaceous substances and to degrade recalcitrant organic matter (Kuznetsov, Antifungal activity of the chitinolytic 1970). actinomycete isolates were tested against the fungal pathogens Aspergillus niger, Chitinases are normally produced by a large Fusarium oxysporum, Fusarium solani, amount of organisms. Of all the isolates Rhizoctonia solani, Phytophthora capsici from Mankudy, Thengapattanam and and Penicillium sp. The antifungal activity Rajakkamangalam sediment soils 20 was determined using disc diffusion method. actinomycete isolates with chitinolytic The Fungal lawn of each organism was activity which exhibited a clear zone on prepared on Potato dextrose agar plates. One Colloidal chitin Agar media were chosen to drop of the culture filtrate of the selected test their anti fungal activity against actinomycete grown in colloidal chitin broth phytopathogenic fungi. The major producers were loaded onto the filter paper discs (size: of chitinase in soils are Streptomyces

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(Tanabe et al., 2000) and this capability Sclerotia sclerotium. Chitinolytic bacteria makes them a valuable organism in waste Paenibacillus sp. and Streptomyces sp. were management strategies. Chitinase can reported to suppress Fusarium wilt of degrade chitinous waste and also the cell cucumber. wall of many fungi. This property makes them the most important organism in the Seven actinomycetes, EST2, EST4, EST5, fields of biological research for the control EST13, EST14, EST15 and EST18 with of phytopathogenic fungi (Felse and Panda chitinolytic property were isolated from 2000). The antifungal activity of the selected Thengapattanam estuary. Out of these 7 isolates were tested against 6 actinomycetes, the isolate EST4 was phytopathogenic fungi like Rhizoctonia observed to exhibit a clearance zone of solani, Aspergillus niger, Fusarium inhibition against all the test fungi. A oxysporum, Fusarium solani, Phytophthora maximum zone of inhibition 26 mm was capsica and Penicillium sp. by disc diffusion shown against Fusarium solani and method. Also extracellular secretion of Phytophthora capsici. No activity was chitinase had been demonstrated previously shown against Aspergillus niger and for 13 strains of actinobacteria (Kawase et Phytophthora capsici by the strain EST2. al., 2004). Table.1 Anti fugal activity of the crude chitinase of Streptomyces griseus showed a Out of the 20 strains selected, 6 of them clearance zone of 10 mm against ESM2, ESM7, ESM9, ESM10, ESM14 and F.oxysporum. Similarly antifungal activity ESM15 from Manakudy estuary proved as of purified chitinase enzyme against fungal chitinase producers. Of these 6 isolates the pathogens was proved previously (El- strain ESM9 was found to be the potential Tarabily et al., 2000). actinomycete with maximum antifungal activity. It showed a inhibition zone of about The role of chitinase activity against the 32 mm against Phytophthora capsici and fungal cell wall is evident (Thara and Rhizoctonia solani. Among the 6 pathogenic Gunanamanickam, 1994). Seven chitinolytic fungi ESM9 showed a lowest clearance zone actiomycetes were isolated from of 25 mm against Penicillium sp. The strain Rajakkamangalam estuary and they were ESM7 and ESM14 expressed a clearance numbered as ESR2, ESR3, ESR6, ESR7, zone of 24 mm against Rhizoctonia solani. ESR8, ESR14 and ESR15. Out of these No antifungal activity was expressed by the isolates the actinomycete strain ESR2 strain ESM14 against Aspergillus niger. showed a clearance zone of inhibition of 29 Table.1. Several workers have worked on mm against Rhizoctonia solani and the antifungal activity of chitinolytic Phytophthora capsici. The isolate ESR8 actinomycetes. Streptomyces chitinase have showed a maximum zone of inhibition of 30 been implicated against a variety of plant mm against Phytophthora capsici and a pathogenic fungi (Gupta et al., 1995, lowest activity of 20 mm against Taechowisan et al., 2003). In the earlier Rhizoctonia solani. The isolate ESR15 was studies chitinase from Streptomyces showed observed to exhibit no activity against activity against fungi such as Aspergillus sp, Rhizoctonia solani, Fusarium oxysporum Phycomyces sp and Trichoderma reesei and Fusarium solani. Similarly antifungal (Williams et al., 1983). Hoster et al. (2005) activity of Streptomyces lydicus WYEC108 reported chitinase activity against A. against a wide range of pathogenic fungal nidulans and phytopathogens such as strains was reported previously (Yuan and Botrytis cinerea, Fusarium culmorum and Crawford, 1995). 162

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The present investigation evaluated the phytopathogenic fungi which bring damage potential of chitinolytic actinomycetes to crops. Therefore the production of this isolated from the sediment soils of estuaries enzyme can be carried out to use as a of South west coast of Tamilnadu. This potential biocontrol agent against study dealt with the application of chitinase phytopathogenic fungi. enzyme to effectively inhibit the

Table.1 Antifungal activity of chitinolytic actinomycetes against selected pathogenic fungi

Antifungal effect (diameter of inhibition zone in mm) n i a

r Fungal Strains t S s e a t m i c ` i e u i n r s c Sl.No r n a e p p y l o a s g a l o i p m c s s o n m o s y a a s u x n i i r u i l o m n l o t l l i u o c i h i t c t m g r i c A h u r a n i o p e s r e z o i p u a t P s h s F y A u R h F P 1 ESM2 16 12 20 14 11 24 2 ESM7 24 15 12 13 10 22 3 ESM9 30 26 32 26 32 25 4 ESM10 20 10 13 9 13 18 5 ESM14 24 0 18 17 26 9 6 ESM15 19 26 24 9 15 16 7 EST2 20 0 15 18 0 17 8 EST4 24 23 24 26 26 24 9 EST5 8 13 20 20 23 22 10 EST13 8 0 15 16 20 19 11 EST14 24 26 18 18 15 23 12 EST15 19 15 21 23 20 9 13 EST18 28 14 16 19 21 16 14 ESR2 29 28 26 28 29 19 15 ESR3 18 23 19 8 16 19 16 ESR6 11 15 15 10 20 16 17 ESR7 24 17 20 18 19 22 18 ESR8 20 22 21 25 30 22 19 ESR14 18 22 16 17 0 0 20 ESR15 0 10 0 0 9 13

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