Ann Microbiol (2014) 64:531–541 DOI 10.1007/s13213-013-0686-x

ORIGINAL ARTICLE

Isolation and characterization of chitinolytic sp. MT7 and its antagonism towards wood-rotting fungi

Anand Nagpure & Bharti Choudhary & Shanti Kumar & Rajinder K. Gupta

Received: 1 January 2013 /Accepted: 29 June 2013 /Published online: 23 July 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract An actinomycetes isolate of Loktak Lake soil, discoloration caused by fungi are major sources of loss in designated as MT7, was characterized and identified as both timber production and wood use, with losses of 15–25 % Streptomyces sp. based on fatty acid methyl ester and 16S marketable wood volume in standing timber and about 10– ribosomal RNA gene analysis. Streptomyces sp. MT7 showed 15 % in wood products during storage and conversion. Hence, strong and broad spectrum antagonism towards seven out of wood decay poses serious problems worldwide, leading to eight tested wood-rotting fungi. Strain MT7 secretes three increased expenditure for raw materials, labor, and liability vital fungal cell wall lytic enzymes, i.e. chitinase, β-1,3- (Zabel and Morrell 1992; Kumar and Gupta 2006). glucanase, and protease, and siderophores. Extracellularly Excessive use of chemical fungicides in agriculture/forest produced mycolytic enzymes lost their activity (plant protection) has led to the deterioration of human health, completely after treatment with proteinase K and heat, indi- environmental pollution, and the development of pathogen cating that the tested antifungal metabolites are heat-sensitive resistance to fungicide. Due to huge economic losses and and proteinaceous in nature. Extracellular fluid (ECF) and its the worsening problems of wood decay, in-depth research is organic solvent extract also exhibited potential antagonism required to identify alternative methods which are less depen- towards the tested wood-rotting fungi. Antifungal metabolites dent on chemicals and more environmentally friendly. were characterized as polyene in nature. Biocontrol traits like Microbial antagonists as biological control agents (BCAs) co-production of cell wall lytic enzymes and antifungal sec- are being examined as alternatives to synthetic chemicals ondary metabolites including siderophores by Streptomyces against various phytopathogenic fungi. Some of the commer- sp. MT7 suggests that it could be employed as a potential cially available BCAs are Streptomyces griseoviridis strain biocontrol agent against wood-rotting basidiomycetes. K61, Agrobacterium radiobacter K1026, GB03, Pseudomonas aureofaciens, Candida oleophila I-182, Keywords Biocontrol . Wood-rotting fungi . Streptomyces . Fusarium oxysporum, Gliocladium catenulatum, Pythium ® Polyene . Siderophores . Cell wall lytic enzymes oligandrum, and, among Trichoderma spp., Bio- (Belgium), Binab-T® (Sweden), RootShield® (USA), TUSAL® (Spain), and Trieco® (India), etc. (Monte 2001;Goheletal. Introduction 2006). Actinomycetes are a major source of broad spectrum bioac- – Basidiomycetes that decay wood are abundantly available in tive secondary metabolites and represent about 70 80 % of all forests across the globe and are the main organisms responsi- the isolated compounds (El-Tarabily et al. 2000;Berdy2005). ble for wood decomposition in many ecosystems (Fryar et al. Many species of chitinolytic actinomycetes, especially those 1999). Wood-rotting fungi colonize and degrade the wood belonging to the genus Streptomyces (Gram-positive, mycelia- using enzymatic and non-enzymatic processes. Decay and forming soil ) exhibit antagonism towards various phy- topathogenic fungi (Silva Sousa et al. 2008; Nagpure and : : : Gupta 2013). The antifungal potential of extracellular metabo- A. Nagpure B. Choudhary S. Kumar R. K. Gupta (*) lites from Streptomyces against some phytopathogenic fungi University School of Biotechnology, Guru Gobind Singh (Prapagdee et al. 2008), and particularly wood-rotting fungi Indraprastha University, Sector 16 C, Dwarka, New Delhi 110 078, India (Shekhar et al. 2006; Nagpure et al. 2013a, b), has also been e-mail: [email protected] reported. Several microorganisms including Streptomyces are 532 Ann Microbiol (2014) 64:531–541 known to produce siderophores which bind with Fe3+ ions to Hi-Media, India) plates and incubated at 30 °C for 7 days, and be transported into the cell. The production of siderophores by then stored at 4 °C. Bacterial strains MTCC biocontrol agents in sufficient quantities may limit Fe3+ avail- 119 and Bacillus subtilis MTCC121wereculturedand ability to the pathogen (Macagnan et al. 2008). Since chitin is maintained on nutrient agar (NA) (M001A; Hi-Media) at one of the main structural polysaccharide of the basidiomycetes 37 °C for 24 h and stored at 4 °C. MTCC microbial strains cell wall, chitnases could be the main candidate enzyme in- were procured from the Microbial Type Culture Collection and volved in the process of cell wall lysis. Antifungal activities of Gene Bank (MTCC), Institute of Microbial Technology, chitinases from Streptomyces against phytopathogenic fungi Chandigarh, India. The ITCC fungal strains were obtained have been well documented (Mukherjee and Sen 2006;Han from the Indian Type Culture Collection (ITCC) of the Indian et al. 2009; Rabeeth et al. 2011; Jiang et al. 2012; Nagpure et al. Agricultural Research Institute (IARI), New Delhi, India. 2013a, b). Henceforth, a great deal of effort is being placed on the In vitro antagonism assay isolation of chitinolytic Streptomyces and their antagonistic activity. In this article, isolation of a Streptomyces strain from All the selected isolates were screened for their in vitro Loktak Lake soil and its ability to produce extracellular anti- antagonism against wood-rotting fungi using “cross-plug” fungal metabolite(s) against wood-rotting fungi is reported. assay (Nagpure and Gupta 2013). The strains of both white- We have determined the extracellular production of antifungal rot and brown-rot fungi (Phanerochaete chrysosporium metabolites that include cell wall lytic enzymes and secondary MTCC 787, Coriolus versicolor MTCC 138, Polystictus metabolites and their antagonism towards various wood- versicolor ITCC 13, Schizophyllum commune ITCC 3751, rotting fungi. Hereafter, the antifungal potential of extracellu- Postia placenta MTCC 144, Gleophyllum trabeum MTCC lar metabolites produced by soil-borne Streptomyces sp. MT7 355, Polyporus friabilis ITCC 335, Polyporus agaricans could be exploited as a biofungicide. ITCC 761) were grown on PDA for 7 days before the test. A loopful of actinomycetes isolates was smeared onto the center of each M93 agar plate (pH 7.0±0.2) and incubated at Materials and methods 30 °C for 72 h to first establish the culture on the agar surface. Then, 8.0-mm-diameter fungal agar plugs were Isolation of soil actinomycetes transferred onto the periphery of M93 agar plates which were previously smeared with actinomycetes isolates at the center. Actinomycetes were isolated from the soil collected from The fungal culture grown on an M93 agar plate without any Loktak Lake (floating lake) of Manipur, India. The soil sam- biocontrol agent served as control. Plates were incubated at ples were suspended in basal salt solution (5.0 g/L KH2PO4 30 °C and examined for inhibition of fungal growth. After and 5.0 g/L NaCl). The serial dilution spread plate technique 2 weeks of paired incubation, inhibition zones around the was used and 0.1 mL of 10−5 diluted soil suspension was actinomycetes isolates were measured using a Hi-media zone spread onto Starch-Casein-Agar (SCA) plates containing reader scale. As actinomycetes isolate MT7 showed the

(g/L) soluble starch 10.0, casein 0.3, KNO3 2.0, NaCl 2.0, highest antifungal activity against the tested fungi, it was . . MgSO4 7H2O0.05,CaCO3 0.02, FeSO4 H2O0.01,KH2PO4 maintained on M93 agar plates and used throughout the 2.0, and agar 18.0, and the plates were incubated at 30±2 °C study. for 8–10 days. On the basis of their morphological character- istics, actinomycete colonies were picked and streaked onto Identification of strain MT7 inorganic salts-starch (ISP4) agar plates (Shirling and Gottlieb 1966). Pure colonies were further transferred to M93 agar FAME analysis The selected isolate, strain MT7, was identi- plates containing (g/L): glucose 4.0, malt extract 10.0, yeast fied using cell wall fatty acid methyl ester (FAME) analysis, extract 4.0, CaCO3 2.0, and agar 18.0 (pH 7±0.2), incubated performed by Sol Scientific Solutions (Nagpur, Maharastra, at 30±2 °C until they sporulated, and then stored at 4 °C. India). To identify the pure isolate, the fatty acid profile gener- ated for the isolate MT7 was compared with the bacterial Microbial cultures and culture conditions identification database, MIDI DOS SYSTEM Sherlock v.6.0B (S/N 160291). Wood-rotting fungal strains Phanerochaete chrysosporium Actinomycetes strain MT7 (Gram-positive) was also char- MTCC 787, Coriolus versicolor MTCC 138, Postia placenta acterized by 16S rRNA gene sequence analysis. Sequencing MTCC 144, Gleophyllum trabeum MTCC 355, Polyporus was done by Sol Scientific Solutions with ABI 3730XL friabilis ITCC 335, Polyporus agaricans ITCC 761, Sequencer. Universal bacterial 16S rRNA gene primers Polystictus versicolor ITCC 13, and Schizophyllum commune 16sF: 5'-AGAGTTTGATCCTGGCTCAG-3'; 16sR: 5'- ITCC 3751 were grown on potato dextrose agar (PDA) (M096; ACGGCTACCTTGTTACGACTT-3' were used to amplify Ann Microbiol (2014) 64:531–541 533

16S rRNA gene fragment. 16S rRNA gene sequencing is a occurrence of substrate hydrolysis and enzymatic activity powerful method for elucidating phylogenetic relationships (Vermelho et al. 1996). among prokaryotic organisms (Woese 1987; Stackebrandt et al. 1997) and has been used to facilitate the differential Colloidal chitin preparation identification of the genus Streptomyces (Mehling et al. 1995; Kreuze et al. 1999). Both strands were sequenced as a cross- Ten grams of commercial chitin (Sigma, C7170) was added to check by using forward and reverse sequencing primers. 100 mL of 85 % phosphoric acid and kept in a refrigerator for 24 h. Thereafter, 2 L of tap water was added and the gelatinous Construction of phylogenetic tree The 16S rRNA nucleotide white material formed was separated by filtration through filter sequences obtained from the isolate MT7 were compared paper. The retained cake was washed with tap water until the with those available in the GenBank database (http://www. filtrate attained pH 7.0. The colloidal chitin obtained had a soft, ncbi.nlm.nih.gov/GenBank/index.html) using the nucleotide pasty consistency, with 90–95 % moisture (Rojas-Avelizapa Basic Local Alignment Search Tool (BLAST). Sequences et al. 1999). were aligned, and a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA) soft- Detection of siderophore production ware v.5.1 (Kumar et al. 2008). The tree was computed using the neighbor-joining method (Saitou and Nei 1987), and the Siderophores were detected by using the universal Chrome resulting tree topology was tested by bootstrap analysis Azurol Sulphonate assay (CAS) (Schwyn and Neilands 1987). (Felsenstein 1985) performed with 1,000 replicates (Tamura This detects color change of CAS-iron complex (from blue to et al. 2007). The 16S rRNA sequence was submitted to the orange) after the iron chelation by siderophores. The CAS agar GenBank, EMBL (Europe), and the DNA Data Bank (Japan) plate assay was performed as follows: 60.5 mg CAS was under the accession number SeqMT7 JQ322818. dissolvedin50mLdeionizedwaterandmixedwith10mLiron . (III) solution (1 mM FeCl3 6H2Oin10mMHCl)bystirring. This solution was slowly mixed with 72.9 mg hexadecyl- Carbon source utilization trimethyl ammonium bromide (HDTMA; Sigma-Aldrich, USA) dissolved in 40 mL of water. The resultant dark blue Carbohydrate utilization tests were conducted using a hi- solution was autoclaved (20 min, 121 °C), cooled, and mixed carbohydrate utilization kit (Hi-Media) for 35 types of carbo- with a sterile mixture of 900 mL in water, 15 g agar, 30.24 g 1.4- hydrate; tests were carried out according to the manufacturer’s piperazine diethane sulfonic acid (PIPES; Sigma-Aldrich) and instructions. 12gof50%NaOH(w/v)toraisethepHtothepKaofPIPES (pH 6.8). This medium was poured onto sterile Petri dishes, then Detection of cell-wall lytic enzymes an active culture of strain MT7 was spot-inoculated onto the blue agar and incubated at 30 °C for 3 days. The results were To test the chitinolytic and glucanolytic property of strain MT7, interpreted based on the color change due to the transfer of ferric it was spot-inoculated by loop at the center of the basal salt with ion from its intense blue complex to the siderophore. the following composition (in g/L of distilled water): K2HPO4 0.7, KH2PO4 0.3, MgSO4 0.5, FeSO4 0.01, ZnSO4 0.001, Extracellular production of cell wall lytic enzymes in crab MnSO4 0.001, (NH4)2SO4 0.25, and yeast extract 1.0 agar plates shells colloidal chitin medium containing 1.0 % colloidal chitin or 0.2 % (w/v) laminarin to detect chitinase and β-1,3-glucanase activity, respectively. SeedculturewaspreparedbyloopfullinoculationofstrainMT7 Plates were incubated at 30 °C for 3–4 days and then the agar in25mLM93seedmediumina100-mLflaskfor24hat30°C. plate was flooded with an aqueous solution of 0.1 % (w/v) The seed medium contained (w/v) (in g/L of distilled water): Congoredfor40min.TheCongoredsolutionwasthenpoured glucose 4, yeast extract 4, and malt extract 10, pH 7.0±0.2. offanddestainedwith1MNaClfor20minforchitinaseand6h Production medium containing basal salt medium supplemented for β-1,3-glucanase activity. Any clearance zone thus formed with 1 % colloidal chitin was inoculated with 10 % of 24-h-old was observed and measured. For the detection of protease seed culture (8.5×105 CFU/mL). Fermentation was carried out activity, strain MT7 was spot-inoculated at the center of the for 10 days at 30 °C on a rotary incubator shaker operated at basal salt (see above) agar plates supplemented with 1 % (w/v) 180 rpm. The chitinase activity and growth of strain MT7 was gelatin and incubated at 30 °C for 3 days. Extracellular protease monitored at 1-day intervals. β-1,3-glucanase and protease detection was done after staining with 0.1 % (w/v) amido black activity were also measured in the exponential growth phase in methanol:acetic acid:water [30:10:60 (v/v/v)] for 1 h at 30 °C, of strain MT7. Cell-free culture filtrate (CCF) was collected whereas destaining was done with methanol:acetic acid:water for quantification of cell wall lytic enzymes and in vitro [45:10:45 (v/v/v)] for 30 min. A zone of clearing indicated the antagonism by centrifugation at 5,000 g for 20 min at 4 °C. 534 Ann Microbiol (2014) 64:531–541

Quantity of viable cells present in the production medium was carried out against a 7-day-old culture of Phanerochaete determined by serial dilution followed by the plate count chrysosporium MTCC 787 and Postia placenta MTCC 144. method. All measurements were performed in duplicate. Wells 8.0 mm in diameter were cut with a cork cutter onto the center of each potato dextrose agar (PDA) plate, then 100 μL Enzyme assays of CCF was pipetted into the wells. Agar plugs of fungal mycelium 8.0 mm in diameter were then transferred onto the Chitinase activity was quantitatively determined in duplicate periphery of each plate at both sides. Simultaneously, CCF by measuring the reducing end group of N-acetylglucosamine was also treated with 0.1 mg/mL of proteinase K (0706; (GlcNAc) released from chitin substrate. A 2.0 mL reaction Amresco, Solon, Ohio, USA) at 37 °C for 60 min, or boiled mixture, containing 1 mL of appropriately diluted CCF with for 45 min. The inhibitory effects of the treated CCF on the 1 mL of 1 % (w/v) chitin (C9752; Sigma-Aldrich) in 50 mM growth of Phanerochaete chrysosporium MTCC 787 sodium phosphate buffer, pH 7.0, was incubated at 37 °C were also determined. The clear zone diameter around the for 60 min. The N-acetylglucosamine (GlcNAc) released wells was measured after 14 days incubation at 30 °C. The was measured by the dinitrosalicylic acid (DNS) method zone of inhibition was measured using a Hi-media zone reader (Miller 1959). A control assay without the enzyme was used scale. as a blank. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the release of 1 μmol of N- Extracellular production of antifungal metabolites in M93 acetylglucosamine per mL in 60 min. medium The β-1,3-glucanase activity was measured in duplicate according to the method of Singh et al. (1999) with slight A loopful active culture of Streptomyces sp. MT7 was inocu- modifications, using laminarin from Laminaria digitata lated into 50 mL of M93 seed medium in a 250-mL flask. After (L9634; Sigma-Aldrich) as substrate. The amount of glucose incubating it overnight in a rotary shaker (180 rpm) at 30 °C, released by the action of β-1,3-glucanase enzyme was mea- 10 % of the seed culture (1.32×107 CFU/mL) was inoculated sured using the DNS method (Miller 1959). A control without into 400 mL M93 production medium and incubated at 30 °C enzyme was simultaneously run and used as a blank. One unit with shaking at 180 rpm for 10 days. Extracellular fluid (ECF) of enzyme activity was defined as the amount of enzyme that was collected at 1-day intervals by centrifugation at 8,000 rpm catalyzed the release of 1 μmol of glucose per mL in 60 min. for 10 min at 4 °C. ECF was filtered aseptically through Protease activity in the extracellular CCF of strain MT7 was 0.22-μm sterile membrane, used for extracellular antifungal measured spectrophotometrically in duplicate by the azocasein activity by the agar well diffusion method described earlier hydrolysis method, a modification of Secades’s method. Briefly, against Phanerochaete chrysosporium MTCC 787 and Postia 480 μL of 1 % (w/v) azocasein (A2765; Sigma-Aldrich) was placenta MTCC 144. Next, 100 μL of ECF was administrated placed in a micro centrifuge tube to which 120 μL of CCF was into the wells and heat-denatured (100 °C for 10 min) samples added, and the reaction mixture was incubated at 37 °C for were used as control. The plates were incubated at 30 °C and 30 min. The reaction was terminated by adding 600 μLof the antagonistic activity of metabolites of strain MT7 was 10 % (w/v) trichloroacetic acid (TCA), and the tube was placed monitored on the 14th day of paired incubation. on ice for 30 min. This was followed by centrifugation at 15,000 rpm, 4 °C for 10 min. 800 μL of the supernatant was Organic crude extract collected and neutralized by adding 200 μL of 1.8 N NaOH. The absorbance at 420 nm (A420) was measured using a spectropho- As maximum antifungal activity was observed on the 4th day tometer (U-2800; Hitachi High Technologies, Tokyo, Japan). A of incubation, fermentation was terminated on the 4th day control assay was done without enzyme and used as a blank. and ECF was collected as described above. Different organic One unit of enzyme activity was defined as the amount which solvents (n-hexane, diethyl ether, chloroform, ethyl acetate, yieldedanincreaseinA420 of 0.01 in 30 min at 37 °C (Secades and n-butanol) were tried in order to obtain crude anti- and Guijarro 1999). fungal metabolites. The solvent was added to the ECF in 1:1 proportion. All organic phases were collected and In vitro antagonism of Cell-free culture filtrate (CCF) concentrated to dryness. All crude extract was again dissolved in methanol and assayed for antifungal activity against The antifungal activity of the CCF was estimated using a Phanerochaete chrysosporium MTCC 787 using methanol growth inhibition “cross-plug” assay as described earlier. as control by the agar well diffusion method described earlier. CCF was filtered aseptically through a sterile membrane with Additionally, 500 mL ECF was directly extracted with 0.22-μm pore size and stored at 4 °C. An antifungal test was n-butanol for further studies. Ann Microbiol (2014) 64:531–541 535

Evaluation of n-butanol crude extract on mycelia growth (Elico SA 167) and compared with those of known polyenic of wood-rotting fungi .

The effect of n-butanol crude extract on growth of Phanerochaete chrysosporium MTCC 787 and Gleophyllum trabeum MTCC Statistical analysis 355 wood-rotting fungi was determined by measuring the radial growth of the fungi on PDA plates. Once there was Data were subjected to statistical analysis using SPSS 16 visible growth, colony size (the average of two perpendicular software (SPSS, Chicago, USA). The data were analyzed by measurements of radial growth) was measured daily until t test and p<0.05 was used as the level of significance. fungi covered the plate (up to 7 days). The levels of growth inhibition (%) were determined by the differences between the radial fungal growth of a control culture [γ0(cm)]andthe Results radial fungal growth [γ (cm)] in the direction of n-butanol crude extract as indicated by this equation; Isolation and screening of Streptomyces Strain MT7 growthinhibitionðÞ% ðÞ¼Δγ γ0−γ=γ0 Â 100 A collection of soil actinomycetes was screened for their Characterization of antifungal metabolites of MT7 ability to retard the growth of wood-rotting fungi. Among the isolates, three potential actinomycetes isolates were Characterization of the nature of antifungal metabolites pro- found to have antifungal activity against wood-rotting fungi. duced by strain MT7 was investigated using the following Of these three strains, strain MT7 inhibited both white-rot criteria: ergosterol inhibition, antibacterial activity, and UV– and brown-rot fungi and was selected for further studies. vis spectra of active extracts (Thakur et al. 2007). When this isolate was grown on M93 agar plates, strain MT7 had light-gray mycelia with white spores. Ergosterol agar plate method Ergosterol was used as the reversal agent to test its ability to reverse the inhibition of In vitro antagonism assay tested fungi caused by the antibiotic. PDA plates with and without ergosterol (0.5 %) were prepared. Wells were made The in vitro antagonism assays showed that strain MT7 with a cork borer at the center of each plate and 100 μLof inhibited a variety of wood-rotting fungal pathogens. When MT7 n-butanol crude extract was added to the wells. Then, tested against wood-rotting fungi by “cross-plug” plate fungal plugs of 7-day-old Phanerochaete chrysosporium assay, clear zones of inhibition were observed between MTCC 787 were placed at the periphery of each plate on the fungal culture and the antagonistic MT7. A strong inhi- both sides. The plates were incubated at 30 °C for 7 days and bition zone >25 mm was observed against five wood-rotting observed for zone of inhibition. The results thus obtained fungi, namely, Phanerochaete chrysosporium MTCC 787, were interpreted by the following criteria: (1) reduced zone Schizophyllum commune ITCC 3751, Gleophyllum trabeum size in presence of ergosterol denotes the presence of poly- MTCC 355, Polyporus agaricans ITCC 761, and Polyporus ene type antibiotic, and (2) no reduced zone in presence of friabilis ITCC 335, while a moderate inhibition zone >15 mm ergosterol denotes the absence of polyene type antibiotic was shown against Coriolus versicolor MTCC 138, Postia (Chakrabarti 1998; Thakur et al. 2007). placenta MTCC 144, but Polystictus versicolor ITCC 13 was not significantly inhibited after 14 days of paired incubation Antibacterial assay Antibacterial activity was performed (Table 1). Strain MT7 showed broad antifungal activities that using n-butanol crude extract of MT7 against Escherichia were stable up to 30 days and the activity was better than S. coli MTCC 119 and Bacillus subtilis MTCC 121. Nutrient violaceusniger MTCC 3959 as reported earlier from our group agar plates were seeded with test bacteria and wells were (Nagpure and Gupta 2013). made with a cork borer at the center of the plates. Then, 100 μL of MT7 n-butanol crude extract was added to the Identification of antagonist strain MT7 wells and the plates were incubated at 37 °C for 24 h and observed for zones of inhibition. The membrane fatty acid profile of strain MT7 exhibited similarities (Sim Index 0.014) to the known members of UV–visual spectrophotometric method The absorption spec- Streptomyces. The 17:0 cyclo fatty acid is usually considered trum of n-butanol crude extract in methanol was recorded in the to be a strong indicator for identification of the genus UV region (200–400 nm) by using a UV–vis spectrophotometer Streptomyces (McNabb et al. 1997). The 17:0 cyclo peak 536 Ann Microbiol (2014) 64:531–541

Table 1 Antagonistic effect of strain MT7 against various wood-rot- Table 2 The variety of ting fungia different lipids resolved Peak name Percent by gas chromatography Wood-rotting fungi Zone of inhibition during FAME analysis, 11:0 4.53 (mm) the percentage of each 12:0 iso 2.25 lipid present, and the 11:0 iso 3OH 1.23 White-rot lipids used as reference 13:0 anteiso 0.42 Phanerochaete chrysosporium MTCC 787 40.33±1.15 peaks for the Gram-pos- itive bacterial isolate 12:0 iso 3OH 0.81 Coriolus versicolor MTCC 138 23.33±1.15 strain MT7 14:0 iso 2.11 Polystictus versicolor ITCC 13 13.67±0.57 14:0 0.62 Schizophyllum commune ITCC 3751 35.33±2.08 15:0 iso 7.50 Brown-rot 15:0 anteiso 21.09 Gloeophyllum trabeum MTCC 355 34.66±1.52 15:1 B 0.42 Postia placenta MTCC 144 23.33±1.52 15:0 2.30 Polyporus agaricans ITCC 761 31.66±1.52 16:1 iso H 1.77 Polyporus friabilis ITCC 335 32.00±0.0 Unknown 15.549 1.33 The results are means of three replicates for each fungus. Statistical 16:0 iso 10.53 analysis of the data was performed by t test. A probability value of 16:1 CIS 9 3.74 p<0.05 was considered to denote a statistically significant difference 15:0 anteiso 2OH 1.11 a Rating: strong inhibition=≥25 mm; moderate inhibition=≥15 mm; 16:0 8.47 weak inhibition=<10 mm 16:0 9-methyl 2.00 17:1 anteiso C 2.89 17:0 iso 2.98 of strain MT7 was shown to comprise 4.71 % of the total 17:0 anteiso 11.65 fatty acid methyl ester detected. The reference peaks are indi- 17:1 CIS 9 2.97 catedinTable2 with corresponding percentages according to Total ECL=411.88; ECL 17:0 cyclo 4.71 the general guidelines for similarity index interpretation used (excluding unknown 17:0 1.43 by MIDI Labs. 15.549)=396.34. Anteiso-to-iso ratios: 18:1 iso H 0.17 16S rRNA analysis BLAST analysis of the first 1,448 base C15:0=2.96; 18:1 cis 9 0.43 C17:0=4.87. Saturated 18:0 0.52 pairs of 16S rRNA sequence (Accession no. JQ322818) of to unsaturated ratio: 6.96 the Gram-positive organism against GenBank showed 97-99 % similarity to Streptomyces 16S rRNA genes. The alignment of this sequence with the 16S rDNA L-arabinose, inulin, sodium gluconate, salicin, dulcitol, inositol sequences retrieved from the DBJ/EMBL/GenBank da- sorbitol, mannitol, ONPG, and esculin hydrolysis, with nega- tabases evidenced a high degree of similarity with the tive results for another 20 carbon sources (Table 3). Streptomyces 16S rRNA genes. The phylogenetic tree was constructed (Fig. 1) on the basis of the 1,448-bp 16S rRNA Detection of extracellular cell-wall lytic enzymes secreted gene sequences of the strain MT7 and the closely related by strain MT7 Streptomyces strains. The closest similarity value was obtained between strain MT7 and its neighbors, including Streptomyces Strain MT7 grew well on colloidal chitin, laminarin and sampsonii ATCC 25495, Streptomyces albidoflavus NBRC gelatin basal salt agar plates and zones of clearance were 13010, Streptomyces violascens ISP 5183, Streptomyces sp. observed due to the substrate hydrolysis after their respective ISP 5133, Streptomyces daghestanicus NRRL B-5418, dye staining and destaining. This indicated that the extracel- Streptomyces sp. ISP 5499, Streptomyces exfoliatus NBRC lular secretion of cell wall lytic enzymes occurred, namely, 13475, and Streptomyces odorifer NBRC 40347. All these chitinase, β-1,3-glucanase, and protease. results indicated that strain MT7 is a member of genus Streptomyces. Detection of siderophore

Carbon source utilization Strain MT7 was CAS-positive for siderophore production, and the presence of iron chelator siderophore was indicated In carbohydrate utilization tests, strain MT7 utilized and pro- by decolorization of a blue-colored ferric CAS complex in an duced acids from lactose, xylose, maltose, fructose, dextrose, orange halo zone around the colonies on the CAS agar plate. Ann Microbiol (2014) 64:531–541 537

Fig. 1 Phylogenetic tree based on 1,448-bp 16S rRNA gene sequences on 1,000 replications are shown at the nodes of the tree. Bar 0.002 from strain MT7 and other Streptomyces sp. The tree was constructed substitutions per nucleotide position using the neighbor-joining algorithm. Bootstrap values (>50 %) based

Quantitative determination of cell-wall lytic enzymes The inhibition of fungal growth of Phanerochaete chry- and antagonism of CCF sosporium MTCC 787 and Postia placenta MTCC 144 by extracellular metabolites in CCF indicates that fungal growth The production of cell wall lytic enzymes from strain MT7 inhibition resulted from the presence of extracellular cell wall was determined. The extracellular chitinase activity was sig- lytic enzymes, i.e. chitinase, β-1,3-glucanase, and protease, nificant in the CCF after 1 day of incubation and it increased whereas no inhibition was seen in the heat- and proteinase K- progressively until the 4th day, after which it started decreas- treated CCF plates (Table 4). The results of this study con- ing (Fig. 2). Streptomyces sp. MT7 produced 3.248 U/mL of firmed that the antifungal potential of the CCF was due to heat- chitinase, 10.7 U/mL of β-1,3-glucanase, and 517 U/mL of labile proteins such as extracellular mycolytic enzymes, name- protease activity on the 4th day of incubation. ly, chitinase, β-1,3-glucanase, and protease. 538 Ann Microbiol (2014) 64:531–541

Table 3 Carbon utilization test of Streptomyces sp. MT7 Table 4 Antagonistic effect of CCF and ECF against various wood- rotting fungi Carbon source Strain MT7 Wood-rotting fungi Zone of inhibition (mm) ● Lactose, xylose, maltose, fructose, +, Positive (utilized and dextrose, L-arabinose, inulin, sodium produced acid) CCF antagonism ECF antagonism gluconate, salicin, dulcitol, inositol, sorbitol, mannitol, ONPG, esculin Phanerochaete chrysosporium 15.5±0.71 26.0±1.4 hydrolysis MTCC 787 ● Galactose, raffinose, trehalose, melibiose, -, Negative (not utilized) Postia placenta MTCC 144 13.5±0.71 30.5±0.71 sucrose, mannose, glycerol, adonitol, arabitol, erythritol, α-methyl-D-glucoside, rhamnose, cellobiose, melezitose, α-methyl-D-mannoside, xylitol, extract showed maximum antifungal activity (29.0 mm) D-arabinose, citrate utilization, malonate followed by chloroform extract (17.0 mm) and ethyl acetate utilization, sorbose (12.0 mm) against Phanerochaete chrysosporium MTCC 787, whereas n-hexane, diethyl ether extracts, and the con- trol did not show any antagonism. Additionally, the antago- Antifungal metabolites production and their in vitro effect nistic potential of n-butanol crude extract of ECF of strain on mycelial development MT7 towards a variety of wood-rotting fungal pathogens was also tested. n-Butanol crude extract showed antifungal The production of extracellular antifungal metabolites by activity against all tested fungi, whereas no inhibition was strain MT7 in M93 medium was determined up to 10 days. seen in the control plate. Strong inhibition (mm) was ob- Antifungal activity was detected in the ECF after 1 day of served towards Phanerochaete chrysosporium MTCC 787 incubation. It progressively increased until the 4th day of (27.5±0.71), Postia placenta MTCC 144 (25.5±0.71), incubation after which it started to decrease (Fig. 3). In vitro Gleophyllum trabeum MTCC 355 (24.5±0.71), Polyporus antifungal activity of extracellular metabolites present in friabilis ITCC 335 (24.5±0.71), and Coriolus versicolor ECF suggested that strain MT7 secretes the antifungal agent MTCC 138 (23.5±0.71). Whereas Polystictus versicolor ITCC extracellularly and its antagonism is due to the presence of 13 (18.5±0.71), Polyporus agaricans ITCC 761 (19.5±0.71), antifungal bioactive metabolites like secondary metabolites and Schizophyllum commune ITCC 3751 (12.00±0.71) or low molecular weight metabolites, siderophores, whereas were moderately inhibited by n-butanol extract after 14 days no inhibition was observed in heat-treated ECF (Table 4). of paired incubation at 30 °C (Table 5). In addition, n-butanol crude extract significantly affected the radial growth of In vitro antagonism of organic crude extract Phanerochaete chrysosporium MTCC 787 (39.14 %) on the 3rd day of paired incubation, whereas maximum growth inhi- Different solvents were used and tested for the extraction of bition of Gleophyllum trabeum MTCC 355 was 50 % on the the bioactive secondary metabolites. Among them, n-butanol 6th day of incubation (Fig. 4). The results indicated that the

Fig. 2 Time-course experiments related to growth and chitinase activ- ity of Streptomyces sp. MT7. The strain MT7 was grown in colloidal Fig. 3 Time-course experiments related to growth and antagonism of chitin medium. At the indicated times, a viable count (CFU/mL) (♦) was ECF of Streptomyces sp. MT7. The strain MT7 was cultivated in M93 calculated for growth studies by the plate count method and cell-free broth. At the indicated times, a viable count (CFU/mL) (●) was calcu- culture filtrate (CCF) was collected for chitinase production (▲). lated for growth studies by the plate count method and extracellular Values presented are the mean ± standard deviation of two independent fluid (ECF) was collected for antifungal activity (▲). Values presented experiments are the mean ± standard deviation of two independent experiments Ann Microbiol (2014) 64:531–541 539

Table 5 In vitro antagonism of n-butanol crude extract towards various 119 and Bacillus subtilis MTCC 121 (cell membrane without wood-rotting fungi sterols) (Table 6). Spectrophotometric spectra of the antifun- Wood-rotting fungi Zone of inhibition (mm) gal compound in n-butanol crude extract of ECF produced a characteristic maximum absorbance peak at 215 nm, White-rot confirming the production of the polyene group of antifungal Phanerochaete chrysosporium MTCC 787 27.5±0.71 antibiotic by this strain. Coriolus versicolor MTCC 138 23.5±0.71 Polystictus versicolor ITCC 13 18.5±0.71 Schizophyllum commune ITCC 3751 12±0.71 Brown-rot Discussion Gleophyllum trabeum MTCC 355 24.5±0.71 Postia placenta MTCC 144 25.5±0.71 This study was undertaken with the aim of evaluating the Polyporus agaricans ITCC 761 19.5±0.71 potential of actinomycetes, especially the genus Streptomyces, Polyporus friabilis ITCC 335 24.5±0.71 as a depot for the production of biocontrol agents against wood- rotting fungi. Streptomyces sp. are well known for their ability The results are means of two replicates for each fungus. Statistical to suppress the growth of a wide variety of plant pathogenic analysis of the data was performed by t test. A probability value of P<0.05 was considered to denote a statistically significant difference fungi (Yuan and Crawford 1995; Errakhi et al. 2007;Ogetal. 2008; Silva Sousa et al. 2008; Nagpure and Gupta 2013; Nagpure et al. 2013a, b). fungal growth inhibition occurred due to the presence of Of all the actinomycetes isolated from the soil of Loktak extracellular antifungal secondary metabolites. Lake in Manipur, India, screened for their antifungal activities against wood-rotting fungi, one of the isolates, namely, MT7, Characterization of antifungal crude extract exhibited excellent antagonism towards the tested fungi. The mycelial growth of all the tested fungi was inhibited by isolate The result of the present study using the ergosterol agar MT7 and the inhibition was significant in comparison with the plate assay showed the production of polyene antifungal relative controls (t test, p<0.05). The fatty acid methyl ester metabolite(s) by strain MT7. n-Butanol crude extract of ECF (FAME) profile of the strain MT7 confirmed its identity as a showed an inhibition zone of 20.3 mm against Phanerochaete member of the genus Streptomyces; however, it was found to chrysosporium MTCC 787 on the PDA plate without ergos- be weakly related to Streptomyces albidoflavus with a fatty terol (target of polyenic antifungal compounds), whereas the acid similarity index of 0.014, which indicated that the species same amount of sample when tested using PDA medium group to which this isolate belongs has not previously been containing the reversal agent, ergosterol (0.5 %), showed a characterized in the FAME database. Also, it has been report- reduced inhibition zone of 0.00 mm. This reduction in zone ed that, for FAME profiles, a similarity index of >0.500 is of inhibition in the presence of ergosterol indicated the considered a good match at the species level, whereas a production of a polyene antifungal compound by strain MT7. similarity index between 0.300 and 0.500 is considered a good n-Butanol crude extract did not show inhibitory activity species match but indicates an atypical strain (Sasser 1990). against either of the bacterial strains Escherichia coli MTCC However, for further identification, strain MT7 was subjected to 16S rRNA gene sequence analysis. The partial sequences of 16S rRNA of strain MT7 were compared with those

Table 6 Characterization of polyene antifungal metabolites

Source max. Ergosterol Antibacterial UV absorption effecta activityb (nm)

With Without

MT7 n-butanol 0.00 20.3 - 215 extract Referencec 9.0 18.0 - 365

a Zone of inhibition in mm on PDA with and without ergosterol Fig. 4 Effect of n-butanol crude extract on the mycelial growth rate b [growth inhibition (%)] of Phanerochaete chrysosporium MTCC 787 Activity against E. coli MTCC 119 and B. subtillus MTCC 121 (■) and Gleophyllum trabeum MTCC 787 (♦) c polyene 540 Ann Microbiol (2014) 64:531–541 found in databases by using BLAST, and a phylogenetic absorbance maxima at 215 nm, indicating a highly polyene tree constructed by MEGA 4 showed that strain MT7 belongs nature. Ilic et al. (2005) reported that the maximum absorbance to the Streptomyces genus. The modern Streptomyces identi- peaks of UV spectral data ranged between 215 and 270 nm of fication system based on 16S rDNA sequence data pro- Streptomyces isolates from the soil samples of south-eastern vides invaluable information about Streptomyces system- Serbia. atics and has been used to identify several newly isolated In conclusion, we have reported the isolation and character- Streptomyces (Awad et al. 2009). ization of a chitinolytic Streptomyces MT7, and have elucidated Many Streptomyces have the potential to produce cell wall the effect of extracellular metabolite(s) produced by this strain lytic enzymes when chitin is present in the growth medium on the mycelial growth of various wood-rotting fungi. The that can destroy fungal cell walls. The results of some au- antifungal effects of the cell-free culture filtrate (CCF) against thors are indicative of a direct positive relationship between the tested wood-rotting fungi are the result of the synergistic the fungicidal activity of antagonists and their ability to effects of several cell wall lytic enzymes, notably chitinase, β- produce cell wall lytic enzymes (Mukherjee and Sen 2006; 1,3-glucanase, and protease. Extracellular fluid (ECF) and its n- Prapagdee et al. 2008; Nagpure et al. 2013a, b). The results butanol crude extract also exhibited broad spectrum and strong of our study have demonstrated the ability of antagonist MT7 antagonism towards different wood-rotting fungi. This clearly to secrete different cell wall lytic enzymes, i.e. chitinase, β- reflects the biocontrol ability of Streptomyces MT7 against 1,3-glucanase, and protease, other biocontrol traits like Fe- wood-rotting fungi and can be exploited as a biocontrol agent chelating siderophores, and antifungal secondary metabo- for wood decay. The potential use of antifungal metabolite(s) as lite(s), which are most often associated with fungal suppres- biofungicides requires more investigation, including inexpen- sion. Extracellular production of these cell wall lytic enzymes sive large-scale production, purification, and characterization, was determined in the exponential growth phase of strain as well as evaluation for toxicity and degradation in the MT7 when grown in colloidal chitin medium. Antifungal environment. activity exhibited by filter-sterilized exponential phase CCF was due to the combination of cell wall lytic enzymes, largely Acknowledgment We are extremely grateful to University Grants produced during the exponential phase, particularly chitinase. Commission (UGC), India, for the financial support under the Special Assistance Programme (SAP) from 2011 to 2016. These results were in accordance with Prapagdee et al. (2008), who reported that cell wall lytic enzymes play major roles in the inhibition of fungal growth. Antifungal secondary metabolites produced by Streptomyces References species have been used as biocontrol agents against phytopath- ogenic fungi (Taechowisan et al. 2005;JainandJain2007). 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