Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-7, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Applications of Endophytic Actinomycetes and Their Role in Protection

*Sayeed Ahmad1, Syed Salman Abbas1, Ravi Prakash1 Aftab Alam1 & Mohammad Ajmal Husain1. 1Department of Bioengineering, Faculty of Engineering, Integral University Lucknow, Uttar Pradesh, India-226026

Abstract: Actinomycetes are one of the most the knowledge concerning the pathway of lignin striking sources of antibiotics and other degradation by white root basidiomycetes, biologically dynamic substances of high viable phnerochateschryo-sosporium. Where it is a value. It account approximately for two-thirds of complex secondary metabolic procees mediated by the total known antibiotics isolated from these the action of several extra-cellular enzyme gram positive, branched spores bearing followed by lactase and manganese peroxidase filamentous group of Eubacteria with notorious (Adhi, T. P., et al 1987) were the first group to bacto-fungoid morphology. Among the identify and characterize LiP from actinomycetes. actinomycetes, spp. has been the Actinomycetes are less abundant in water logged most profuse sources of all types of antibiotics. and acidic soils, and may be present in higher than Screening and isolation of capable strains of normal numbers in drier, alkaline soils. Although actinomycetes with prospective antibiotics is still a they are classified as , they are fungus like thrust area of research and it is recommended that in appearance and growth. Whereas most bacteria the explorations of resources from new areas and grow by asexual fission of individual cells, the habitat have a key role to play in the search for vegetative cells of most actinomycetes produce novel microbes and fresh metabolites and is vital to slender, branchedfilaments, or hyphae that are offset the threats posed by the fast rising collectively termed mycelia. These structures are occurrence of antibiotic resistance. Counting the smaller and of a different morphology and endophytic actinomycetes in the race of investigate composition than their fungal counterparts. Within along the actinomycetes, endophytes have been these hyphae forms asexual spores called conidia; established to improve and promote growth of the spores are formed when conditions are no swarm plants as well as to decrease disease longer optimal for vegetative growth, and symptoms caused by plant pathogens and/or germinate when conditions are again favourable. various environmental stresses. The low stress Actinomycetes are heterotrophic, and are valuable tolerance of axenic plants is normally believed to decomposers of organic matter in soil communities. result partially from the deficiency of endophytic Members of the Streptomycetes family of actinomycetes. Not only to the plants; they even actinomycetes, which includes Streptomyces, are produces abundant bioactive compounds which are responsible for production of the compound of human being benefit. causing the musty odour prominent in freshly turned soils; many also produce antibiotics that Keywords: Endophytic actinomycetes, Bioactive inhibit the growth of bacteria and fungi compounds, Morphology, Characterization, (Alexander,1977; Locci, 1989). They are defined to Bioremediation. be organism producing extensively primary or substrate mycelium as well as more or less INTRODUCTION abundant secondary or aerial mycelium. In the The diversity analysis of the actinomycetes is well course of the Streptomycetes life cycle the hyphae recognized, due to the sustained generation of are partially transformed into chain of spores which environment isolates for pharmaceutical screening are often called as conidia or arthrospore. During and much interest was given to this group of the same period studied on the chemical organism over the year for obtaining antibiotic composition of the cell wall began helped to compound. Beside antibiotic production, another classify the actinomycetes(Becker et al., 1964, important trait of actinomycetes that has been 1965; Lechevalier and Lechevalier, 1970) they studied is their ability to degrade lignin- the most clearly demonstrated that Streptomyces and other complex bipolar. Biodegradation of lignin has been genera contained LL-diaminopimelic acid (LL- considered as significant phenomenons since it A2pm) in its peptidoglycan where as meso-A3pm play a critical role in the carbon cycling. Most of exists in the other actinomycetes. Member of the

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-7, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Streptomycetes are now primarily recognized by Taxanomy of genus Actinomycetes the structure of their peptidoglycan. Peptidoglycan About a decade ago a genus called actinomycetes is of type A3γ containing an inter peptide bridge was shaped it clearly separated from the other composed of a glycine residue (Schliefer and genera so before study about streptomycetes in Kandler, 1972). Further taxonomist also have general. The actinomycetes are a numerous and studied biochemical characters such as pattern of widely distribution group of soil microorganism, sugar in the whole cell hydrolysates(Lechevalier composing of 10 to 50% of the soil and Lechevalier, 1970), phospholipids (Lechevalier mirofloracommunity over a broad range of soil et al., 1970), fatty acids (Kroppenstedt, condition, with streptomyces the numerically 1985),menaquinone(Alderson et al., Kroppenstedt, dominant genus (Alexander, 1977). In , a 1985) etc. Beside their importance in the medical large amount awareness has been given recently to microbiology now the scientist are switching over the base composition of deoxyribonucleic acids to the roots where they are looking for the other (DNA) of various organisms. Actinomycetes as a properties specially related to agriculture .From last whole, form homogenous group with high G+C three decades the people are working in many ratio (65.5 to 74%). Some applied field where they are reporting other thermophilicActinomycetes have lower ratio (44 to features such as, ability to bio degrade, plant 53%). Much of this information has reviewed by growth promoting activities etc. Most soil Hill. In the recent taxonomic Actinomycetes in actinomycetes also behave as neutrophiles in Berger’s Manual of systematic bacteriology (2nd culture, growing between pH5.0 and 9.0 with an edition).Actinomycetes are included under volume optimum close to neutrality. As many soils are 4rth published in 2007. (George M.Garrity, Editor- acidic Ph is clearly a major factor determining their in-Chief springer, 2001, New York). According to distribution and activity; it has been known for this approach the G+C gram positive bacteria are many years that acidic soil yield low count of classified under class- of phylum neutrophilic actinomycetes (Waksman, Actinobacteria. This phylum includes five 1959).However acidophilic and acido duric subclasses, six order, fourteen suborder, thirty nine streptomycetes, the former growing between about families of Actinomycetes, pH (Williams et al ;) and to produce diastases and which includes streptomyces are responsible for chitinase with pH optima lower than those of production of the compound causing the musty enzyme from neutrophiles. The presence of low odour well-known in freshly tuned soil many number of neutrophilicstreptomycetes may be due produce antibiotics that restrain the growth of to the localized occurrence of less acidic micro bacteria and fungi (Alexander,1977; Locci 1989). sites and to the resistance of their spores to acidity. When chitin or dead fungal mycelium were added Characterization of Endophytic Actinomycetes to a badly buffered acidic soil, a succession from Morphological characterization. acidophiles to neutrophiles occurred that paralleled Although actinomycetes are classified as bacteria, ammonification and the consequent rise in pH. they are fungus like in appearance and growth. Whereas most bacteria grow by asexual fission of Identification of actinomycetes individual cell, the vegetative cells of most The following points may help in identifying the actinomycetes produce slender, branched filaments, actinomycetes on dilution plates: or hyphae that are collectively mycelia. These 1. Most the actinomycetes contain colonies structures are smaller and of a different of whites gray or black powdery surface morphology and composition than their whereas bacterial colonies are of different counterparts. Its hyphae structure asexual spore colors shiny surface and gummy called conidia; the spore are formed when appearance. condition no longer optimal for vegetative growth, and germinate when condition favourable. Thus by 2. Generally bacteria have lens or lemon morphology of aerial and vegetative mycelia shaped colonies with very thin surface. actinomycetes can characterized.

3. Mostly the actinomycetes colonies can be Microscopic Characterization observed without microscope and have a  Aerial mycelium including the chain of distinct hollow with a darker interior spores (arthrospore /conidiospore)- while bacteria do not. The arthrospore although by definition vegetative mycelium on solid medium is not 4. Actinomycetes colonies are firmer than fragmented .In some of the bacteria when picked with a sharp needle. Streptomycetesformation of the spore chain at the edge of the mycelium growth can be observed.

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-7, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

With the ageing the vegetative hyphae become therefore, the most intensively studied habitat, but optically “empty” and the newly developed in spite of this there are motionless many gaps in mycelium seems to be parasitized by the vegetative our knowledge of the roles played by hyphae, these hyphae may be cross walled (Glauret Actinomycetes in soil processes. Many studies and Hopwood 1960). Some worker account the have been primarily troubled with the enumeration spore formation at the end (Mohamed Helal et al and identification of isolates. Over 20 genera have 2014). been isolated with streptomycetes organism ubiquitous and most numerous (Lechevalier and  Length of the aerial hyphae: short, Lechevalier 1970). medium, long (short for the dusty colonies long to cottony one ) Endophytic Actinomycetes as source of Branching symbolically (growth in form of enzymes: trees/bushes/long hyphae with the chain of spore) Actinomycetes are emerging as an important occurrence of spiral (matter restricted as it based on source of enzymes involved in lignocelluloses what has been done actually). degradation, and activity against xylase is predominantly widespread (McCarthy 1987). Colour of the Substrate Mycelium and Soluble Studies have been concentrated on the Pigments endoxylanases of streptomycetes, which are Several Streptomycetespossess a strikingly inducible extracellular enzymes regularly produced coloured substrate mycelium the pigment often in multiple forms. In addition, there is some diffuses into the medium conferring the same evidence that Streptomyces endoglucanases exhibit colour. Two classes of pigment can be low levels of xylanase activity. We have in recent distinguished with respect to their location in the times screened over 200 actinomycetes strains for producing cell. straw saccharifying activity (McCarthy and Ball, 1987) and found that this was largely due to 1) Endopigments degradation of the xylose component since xylose These are bound to certain cell structure and confer and its oligomers were the major products detected. the striking colour on the colonies of the Several reports have examined the ability of organisms. different bacterial species to solubilize insoluble inorganic phosphate compounds, such as tricalcium 2) Exopigments phosphate, dicalcium phosphate, hydroxyapatite, These are secreted into the surrounding medium and rock phosphate. they may either be water soluble or diffuse far into the medium or may be precipitate .These features has been extensively studied.It is also Endophytic Actinomycetes and their recommended to study the pH. metabolites that affect plant’s life

Endophytes colonizing inside plants usually get Color of the Aerial Mycelium: nutrition and protection from the host plants. In The aerial mycelium of most of the Streptomycetes return, they confer profoundly enhanced fitness to exhibits a definite color, based on the spore colour the host plants by producing a variety of bioactive studied for thousand Streptomycetes grouping of metabolites. Growth stimulation of plants by the Streptomycetes was carried out. In the endophytescan be a consequence of nitrogen international Streptomycetes project (ISP) and in fixation or the production of phytohormones, extensive study by the following seven colours biocontrol of phytopathogens through production have been reported: yellow, violet, red, blue, green, of antibiotics or siderophores, nutrient competition, black, gray, and white. Although certain problem and induction of systemic disease resistance. Some may also been arise a experience with many culture of these metabolites directly affect physiology of and representative of all colour is necessary) the the host plants but others do so indirectly by typical colour is shown only by well sporulated affecting the microbe population by antibiosis culture medium also influence the colour. Media and/or competition. based on starch is preferred on other media.

Agricultural use of endophytic actinomycetes Role of Endophytic Actinomycetes in Soil: Use of endophytic actinomycetes to produce Actinomycetes constitute a significant component disease resistant tissue-cultured seedlings of the microbial population in most soils and Enhanced drought-tolerance of tissue-cultured counts of over 1 million per gram are commonly seedlings by endophytic colonization of obtained. The soil is also the most prolific source actinomycetes (Sachiko Hasegawa, Tomio of isolates, which include many found to produce antibiotics and other useful metabolites. It is, Nishimura et al. 2006).

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-7, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Figure 1. Detected events associated with disease resistant and drought tolerence in tissue culture seedling Degradation of pesticides by actinomycetes recovery (Ravelet et al., 2000). There is lot of Actinomycetes have considerable potential for the information available on copper resistance biotransformation and biodegradation. Members of mechanisms in gram negative bacteria such as this group of bacteria have been found to degrade E.coli and Pseudomonas sp. and even genetic pesticides with widely different chemical structure determinants has been proposed. Copper such as Organochlorine, s-triazine, triazinone, metabolism seems to be very much clearer in gram carbamate organophosphates, acetanilide, positive bacteria such as Enterobacterhirae sulfonylureas. Limited number of xenobiotics can (Odermatt et al., 1992). Nevertheless, there is not be mineralized by single isolates, but for complete enough information on the mechanism involved in degradation, consortia of bacteria are required. the resistance to copper by actinomycetes(Erardi et al., 1987). Role of actinomycetes in copper bioremediation: Soil microbes play an important role in the Actinomycetes as source of biologically active environmental fate of toxic metals with a array of metabolite mechanism affecting transformations between Actinomycetes are of the subject of great interest to soluble and insoluble forms. These mechanisms are scientists and industrialists due to their potential for integral components of natural biogeochemical the production of practical products such as cycles and have a potential of equal, in situ and ex antibiotics and enzymes. Many economically situ bioremedial treatment processes, for solid and important actinomycetes are known to produce an liquid wastes (Gadd, 2000). They are most rich array of biologically active substances, such as group of bacteria in soil (90%) and show primary antibiotics, vitamins and enzymes (Mccarthy and biodegradative activity, secreting a range of Williams, 1992). Amamstatin A is a linear extacellular enzymes that allow them to metabolize lipopeptide and a growth inhibitor in human tumor recalcitrant molecules. Amongst actinomycetes in cell lines produced by actinomycetes sp. soil, there are examples of different strategies, for (Yokokawa et al., 2000). There are 459 antibiotics cycles of rapid proliferations and sporulations to known which are secreted by actinomycetes (0.2% the maintenance of populations by prolonged slow actinomyces, 3.5% nocardiae, 0.7% growth and scavenging. This metabolic and Micromonosporae, 0.2% thermoactinomyces). morphological versatility gives them to great Streptoverticillum is secreted by stretoverticillum, perform bioremediation processes, including metal cinnamomeus var. monicae(Gupta, 1965).

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Imperial Journal of Interdisciplinary Research (IJIR) Vol-2, Issue-7, 2016 ISSN: 2454-1362, http://www.onlinejournal.in

Streptomyces fervens produces fervenulin(Herr, from actinomycetes', Journal of Applied 1965) and S. fervensvarmelrosporus produces Bacteriology, 66(5), pp. 439–444. unstable α, β unsaturated organic acid antibiotic U 2. Adhi, T. P., Korus, T. A., pameto III A.L. 22956, active on Gram positive and Gram negative and Craford, D. L.,(1987). Lignin degradation bacteria. It is most active on acidic condition and and production of microbially modified lignin soluble in weak alcohol, water and ethyl acetate polymers by streptomyces virido sporous in ethyl acetate and insoluble in ether, hexane and slurry reactors. Applied biochemistry and cyclohexane. It was reported to produce biotechnology (in press). formaldehyde on degradation C5H7NO4 3. Alex odermatt, Heinrich Sutter, Reto (Luedemann and Brodsky, 1964). Chain produces crapf, Marc Solioz (1992) 'An ATPase operon isomer of aburamycin (antibiotic M5-18903). involved in copper resistant by enterococcus Everninomicin complex is secreted by class hirae', Annals of the New York of academy of Micromonosporacae. It also produces gentamycin Science, 671(6), pp. 484-6 complex. Everninomycin complex contains 5 4. Alexander, M.(1977). Introduction to components, all of which are active on gram soil microbiology. ISBN 0-471-02179-21977 pp. positive bacteria. Gentamycin is produced by M. 467 pp. purpurea NRRL 2953 (Luedemann and Brodsky, 5. Audrey M. Glauert and David A. 1964) is a water soluble substance with activity on Hopwood (1960) 'The Fine Structure of gram positive and gram negative C1 and C2 are Streptomyces coelicolor . The Cytoplasmic isomeric pseudo- oligosaccharides (C17- Membrane System', the journal of cell 18H34N4O7).Nocardiorubin is produced by Micro- biology., JCB vol. 7(3), pp. 479-487. monosporanarashino. It is active against Gram 6. BECKER, W. A. (1964) 'Manual of positive bacteria. procedures in quantitative genetics.,viii (19650100856),. pp. 70 Role of antibiotics produced by actinomycetes in 7. C Voisard, C Keel, D Haas, G Dèfago plant disease control and cyanogenesis: (1989) 'Cyanide production by Pseudomonas Actinomycetes are integral part of plant ecosystem. fluorescens helps suppress black root rot of The presence of antibiotics in soil may either have tobacco under gnotobiotic conditions', The positive of negative effect on plant growth and EMBO Journal, 8(2), pp. 351–358. development. Antibiotics that is effective against a 8. C. Ravelet, S. Krivobok, , L. Sage, R. wide range of infectious agents,is known as a broad Steiman (2000) 'Biodegradation of pyrene by spectrum antibiotic. Some are narrow spectrum as sediment fungi', Chemosphere, 40(5), pp. 557– it belongings only few bacterial species. HCN 563. production of the isolates has been tested by the 9. FX Erardi, ML Failla, JO Falkinham. method of (Bakker and Schipper,1987). (1987) 'Plasmid-encoded copper resistance and Cynogenesis is the result of production of HCN, precipitation by Mycobacterium which is volatile in nature. Reaction of HCN with scrofulaceum.', Appl. Environ. Microbiol., 53(8), Picric acid and in the presence of sodium pp. 245-247. carbonate, results in the colour change of the filter 10. Geoffrey Michael Gadd (2000) paper from deep yellow to orange and finally 'Bioremedial potential of microbial mechanisms orange brown to dark brown in the case of negative of metal mobilization and test, the deep yellow colours of the filter paper immobilization', Current Opinion in remained unchanged after growth of Biotechnology, 11(3), pp. 271–279. microorganism (Voisard et al., 1989) 11. GM Luedemann, BC Brodsky (1964) Antibacterial activity 'Taxonomy of gentamicin-producing Actinomycetes have antibacterial activity against Micromonospora', Int. J. Syst. Bacteriol., 30(45), pathogenic bacteria. They produce antibiotics, pp. 321. herbicides, pesticides and enzymes like cellulose 12. Gupta (1965) 'Transfer of and xylanase used in waste water treatment. They Streptosporangium indianense Gupta 1965 to the contain enzymes such as lecithinase, lipase and Genus Streptomyces as Streptomyces indiaensis nitrate reductase, H2S production, amylase, comb. nov', Int J Syst Evol Microbiol, 37(3), pp. gelatinase, DNase and urease. 241. 13. Johannes Poschner , Reiner M. REFERENCES Kroppenstedt , Angelika Fischer , Erko Stackebrandt , (1985). 'DNA-DNA Reassociation and Chemotaxonomic Studies on Actinomadura, 1. A.S. BALL andA.J. MCCARTHY (May Microbispora, Microtetraspora, Micropolyspora 1989) 'Production and properties of xylanases and Nocardiopsis',Systematic and Applied Microbiology, 6,( 3,), pp. 264-270.

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