ISOLATION AND TAXONOMIC CHARACTERISATION OF POTENTIAL ANTAGONISTIC SIMPLICILLIUM LANOSONIVEUM VJCH-19 FROM MANGROVES OF ADAVULADEEVI, ANDHRA PRADESH, INDIA

ISOLATION AND TAXONOMIC CHARACTERISATION OF POTENTIAL ANTAGONISTIC SIMPLICILLIUM LANOSONIVEUM VJCH-19 FROM MANGROVES OF ADAVULADEEVI, ANDHRA PRADESH, INDIA

Ch. Chiranjeevi and Vijayalakshmi Muvva ^*

Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur - 522510, Andhra Pradesh, India.

ABSTRACT: Objectives: The intention of the present study was to isolate and identify cultural conditions of potent fungal strain VJCH-19 with antagonistic activities isolated from mangrove ecosystems of Adavuladeevi, South coast, Andhra Pradesh, India. Methods: The soil samples were collected from the mangrove habitats and shade dried. The serial dilution plate technique was employed for the isolation of mangrove fungi by using Sabouraud dextrose agar and Czapek-Dox agar media. All the strains were screened for the production of antimicrobial compounds. The potential fungal strain was designated as VJCH-19. The identification of the strain was carried out based on cultural, micro-morphological and genomic characteristics. Results: The potent mangrove fungal strain VJCH-19 was identified as Simplicillium lanosoniveum. The secondary metabolites produced by the strain inhibited several bacteria and fungi. To check the nutrient adaptation, VJCH-19 was grown on different culture media and its morphological characters were observed. Conclusion: Among the twenty-five fungal strains, VJCH-19 was found to possess antimicrobial potential and the strain was identified as Simplicilium lanosoniveum VJCH-19.

Mangrove fungi, Simplicillium lanosoniveum VJCH-19, Bioactive compounds

INTRODUCTION: Mangroves are salt-tolerant forest ecosystems confined to intertidal zones of sheltered shores, estuaries, tidal creeks, backwaters, lagoons, marshes, and mudflats of the tropical and subtropical latitudes ¹. Mangrove soils provide a unique ecological niche for the growth of diversified microorganisms which find use in recycling environmental nutrients and production of exclusive secondary metabolites of industrial importance ^{2, 3, 4}.

Fungi isolated from mangrove sediments received much attention due to structural diversity, unique biological activity and functionally active secondary metabolites ^{5, 6}. The fungi from the mangrove habitats produce novel bioactive meta-bolites including antibiotic, anti-diabetic, antiviral, anti-inflammatory, anticancer, antioxidant, and immuno-suppressive drugs ^{7, 8}.

Isolation of fungi from unexplored soil samples may provide novel bioactive metabolites. Mangrove soils are a potential source of rare fungi possessing antimicrobial activity against pathogenic micro-organisms. There is plenty of scopes to isolate such rare strains that produce novel compounds beneficial to mankind. As part of our ongoing project of screening of different mangrove ecosystems of Andhra Pradesh, India, a potent strain VJCH-19 with broad-spectrum activity against Gram-positive and Gram-negative bacteria as well as fungi was found. An attempt has been made in the present study to identify the potent strain based on the cultural, micro-morphological and molecular approaches.

MATERIALS AND METHODS:

Collection of Soil Sample: Soil samples were collected from different parts of mangrove ecosystem of Adavuladeevi, South coast of Andhra Pradesh, India. The samples were collected randomly at a depth of 10-20 cm and shade dried ⁹.

Isolation of Fungi: The shade dried soil samples were used for the isolation of fungi by serial dilution plate technique. Czapek-Dox agar (CDA) and Sabouraud-dextrose agar (SDA) media amended with sodium chloride (2%) are used for the isolation of fungi. Streptomycin (50µg/ml) was added to retard the growth of bacteria. Aliquots of samples were prepared through serial dilution plate technique. An aliquot of 0.1ml of each dilution (10^-2 to 10^-4) of samples was spread evenly over the surfaces of CDA and SDA media. The plates were incubated at 37 °C and observed for fungal growth. After seven days of incubation, the fungal colonies were picked up, preserved on SDA slants and stored at low temperature for further study.

Screening of Fungi for the Production of Antimicrobial Metabolites: All the fungal strains isolated were screened for antimicrobial activity against Staphylococcus aureus (MTCC 3160), Bacillus subtilis (ATCC 6633), Bacillus megaterium (NCIM 2187), Xanthomonas campestris (MTCC 2286), Proteus vulgaris (ATCC 6380), Pseudo-monas aeruginosa (ATCC 9027) and Escherichia coli (ATCC 9027) and antifungal activity against Candida albicans (MTCC 183) and Penicillium citrinum (MTCC 6849).

Identification of the Potent Fungal Strain:

Cultural Characteristics of the Strain VJCH-19: The potent fungal strain was cultured on different culture media such as SDA, CDA, NAM, MEA, YMA, and PDA.  After seven days of incubation, colony characteristics were observed ¹⁰.

Morphological Characteristics of the Strain VJCH-19: The fungal isolates were identified initially by morphological characterization through plating the fungi on SDA and incubated for seven days. The growth and presence/absence of pigmentation were noted. The fungi were identified by studying their cultural characteristics, spore formation, and mycelium arrangement. Slides were prepared by the tease mount method using Lacto-phenol Cotton Blue (LPCB) reagent ¹¹.

The conidia morphology of strain VJCH-19 was studied under the scanning electron microscope (SEM). Samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 h at 4 °C and postfixed 2% in aqueous osmium tetroxide for 4 h. Dehydrated in a series of graded alcohols and dried to critical point drying with CPD (EMS 850) unit. The processed samples were mounted over the stubs with double-sided carbon conductivity tape, and a thin layer of gold coat over the samples was done by using an automated sputter coater (Model - JEOL JFC-1600) for 3 min and scanned under Scanning Electron Microscope (SEM - Model: JOEL-JSM 5600) at required magnifications as per the standard procedures ^{12, 13}.

Molecular Identification of the Strain VJCH-19: The Genomic analysis of fungal strain VJCH-19 was done using 18s r RNA sequencing technique. The fungal isolate was identified based on the internal transcribed spacer (ITS) sequences. Fungal genomic DNA was extracted from mycelia by using the cetyl trimethyl ammonium bromide procedure. The rDNA ITS region was amplified using primers ITS1 and ITS4 ¹⁴.

The primers were synthesized at an oligonucleotide synthesis facility (Macrogen, Seoul, Korea) and diluted to 20µM in sterilized water. PCR products were purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol ¹⁵.

The Amplifications were performed in a PCR verity 96-well thermal cycler (Applied biosystems), programmed with denaturation at 95 °C for 5 min, followed by 3 cycles consisting of denaturation at 96 °C for 5 s, primer annealing at 50 °C for 5 s, extension at 68 °C for 18 s, and the final elongation step at 72 °C for 1 min. After amplification of the ITS templates, excess primers and dNTP’s were removed from the reaction mixture using a QIA quick, PCR DNA purification kit (Qiagen).

The PCR products were sequenced directly in both directions using primers ITS1, ITS2, ITS3 and ITS4 using a Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems) ¹⁶. The cycle sequencing reaction mixture had a total reaction volume of 10µl and contained 2.5µl of template DNA (10-15ng/ul), 2µl BigDye terminator premix, 4µl ultra pure-sterile water and 0.5µl of each primer (5 pmol/µl). Reactions were run in the PCR thermal cycler, programmed with denaturation at 96 °C for 1 min, followed by 25 cycles of denaturation at 96 °C for 10 s, followed by the primer annealing at 50 °C for 5 s and the extension at 60 °C for 4 min. Sequencing products were purified by ethanol/ ethylene diaminetetra acetic acid (EDTA), and the samples were analyzed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) ¹⁵.

Pair-Wise Sequence Alignment: The gene sequence of the strain VJCH-19 was aligned using BLAST against the gene library available for Simplicillium species in the NCBI and the GenBank. Pairwise evolutionary distances were computed by MEGA-6 software ¹⁷.

Multiple Sequence Alignment: The phylogenetic analysis was conducted using the maximum parsimony method using BLAST and CLUSTAL W. The closely related homologous strains were identified, retrieved and compared to the sequence of the isolated strains using CLUSTAL W available with the MEGA 6 version ¹⁷. The 18S rRNA gene sequence of the strain VJCH-19 was registered in the GenBank database.

Growth Pattern of the Strain VJCH-19: For determination of growth pattern, the strain was inoculated into 250 ml flasks containing 100 ml Sabouraud-dextrose broth with 2% NaCl. The culture broth was incubated at 30 ± 2 °C on a rotary shaker at 180 rpm. The flasks were harvested at four days interval up to twenty-eight days and the growth of the strains was determined by taking the dry weight of mycelium. The culture filtrates obtained after separating the biomass were extracted with ethyl acetate and the antimicrobial activity of the crude extract was determined by agar well diffusion method.

Extraction of Metabolites and Antimicrobial Assay: The antimicrobial activity of the strain was determined by agar well diffusion assay ¹⁸. The homogenous culture suspension prepared by suspending 7-day-old culture in sterile saline was used to inoculate SD broth (seed medium) and incubated at 30 °C for seven days on a rotator shaker at 180 rpm. Seed culture at the rate of 10% was transferred to SD broth (fermentation medium). The fermentation was carried out at 30 ± 2 °C for twenty days under agitation at 180 rpm. The antimicrobial compound was recovered from the filtrate by the solvent extraction method. Ethyl acetate was added to the filtrate (1:1) and shaken vigorously.

The ethyl acetate extract was evaporated to dryness on a water bath and the residue thus obtained was used to determine antimicrobial activity. Ethyl acetate itself was used as a negative control. About 80 μl of the crude extract and 80 μl of negative control were poured into separate wells. The standard antibiotic disc was placed on the agar surface as a positive control. Plates were incubated at 37 °C for 48 h, and inhibition zones (mm) were measured.

RESULTS AND DISCUSSION:

Isolation and Screening of Fungi From Mangrove Soils: A total of twenty-five fungal strains were isolated from the soil samples of Adavuladevi mangrove habitats and designated as VJCH-1 to VJCH-25. All the isolates were subjected to screening against selected bacteria and fungi. Interestingly, the isolate VJCH-19 exhibited potent activity against test bacteria and fungi. The strain VJCH-19 was subcultured on SDA supplemented with 2% NaCl and preserved for further studies.

Cultural and Morphological Characteristics of VJCH-19: Cultural characteristics of VJCH-19 was studied on six different media viz. SA, CDA, NAM, MEA, YEA and PDA and the results are shown in Table 1. VJCH-19 exhibited excellent growth and produced pale yellow pigment on SA and MEA media. There was no pigmentation on CDA, NAM, YEA, and PDA. The colonies are compact, white, with a fluffy texture, reaching 18 – 30 mm diameter after 7 days of incubation at 30 °C Fig. 2A. Mycelium is septate, branched filamentous. Phialides produced on prostrate aerial hyphae are mainly solitary and tapering towards the tip ^{16, 21}. Conidia are found in small globose heads at the apex of phialides ²⁰. Conidia are mostly oval, smooth-walled and 1-celled ^{14, 16}.

The apical conidium of the conidial chains subglobose to obovoid Fig. 2B.

TABLE 1: CULTURAL AND MORPHOLOGICAL CHARACTERISTICS OF VJCH-19

(SA- Sabouraud-Dextrose agar, CDA- Czapek-Dox agar, NAM- Nutrient agar, MEA- Malt extract agar, YEA- Yeast extract agar, PDA- Potato dextrose agar)

Molecular Identification of the Strain VJCH-19: The phylogenetic tree was constructed based on the Neighbor-joining method using bootstrap analysis ²² and the strain VJCH-19 was identified as Simplicillium lanosoniveum. The partial 16S rRNA sequences of the strains were submitted to the GenBank database with accession number MK463995. The partial sequence was aligned and compared with all the 18S rRNA gene sequences available in the GenBank database using the multi-sequence advanced BLAST comparison tool. The phylogenetic analysis of the 18S rRNA gene sequence was aligned using the CLUSTAL W program from the MEGA 6 Version. Phylogenetic tree Fig. 1 is constructed using MEGA software Version 6 using maximum parsimony method ¹⁷.

FIG. 1: MAXIMUM PARSIMONY TREE BASED ON PARTIAL 18s rRNA GENE SEQUENCE SHOWING RELATIONSHIP BETWEEN STRAIN VJCH-19 AND RELATED MEMBERS OF THE GENUS SIMPLICILLIUM

Growth Pattern and Antimicrobial Profile of Simplicillium lanosoniveum VJCH-19: The growth curve and antimicrobial profile of Simplicillium lanosoniveum VJCH-19 were studied at regular intervals up to twenty-eight days in batch culture. The strain entered into log phase on fifth day which extended up to twenty days. Stationary phase is extended from twenty-one to twenty-four days of incubation and finally entered into decline phase Fig. 3. The secondary metabolites obtained from twenty day-old culture showed high antimicrobial activity against the test microbes.

The antimicrobial spectrum of the strain cultured on SD broth for twenty-eight days was tabulated in Table 2. The metabolites extracted from the twenty day-old culture showed maximum activity against E. coli, B. megaterium, P. vulgaris and S. mutans. In case of fungi, C. albicans showed high sensitivity as compared to P. citrinum.

FIG. 3: GROWTH PATTERN OF SIMPLICILLIUM LANOSONIVEUM VJCH-19

TABLE 2: ANTIMICROBIAL ACTIVITY OF SIMPLI-CILLIUM LANOSONIVEUM VJCH-19 AGAINST THE TEST ORGANISMS

CONCLUSION: The present work leads to in-depth studies on bioactive fungal isolates which can produce many valuable products necessary for medical, agricultural and industrial applications. The identification and production of novel anti-microbial metabolites from mangrove fungi is an important exclusive approach to defeat the progressive levels of drug-resistant micro-organisms. Hence, further studies will be carried out on purification, characterization and identification of bioactive metabolites of mangrove fungus Simplicilium lanosoniveum VJCH-19.

ACKNOWLEDGEMENT: Financial support from UGC, New Delhi is gratefully acknowledged (MVL). Authors would also like to thank the authorities of the Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur for providing the facilities to carry out this work.

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Behera BC, Mishra RR and Thatoi HN: Diversity of soil fungi from mangroves of Mahanadi delta, Orissa, India. Journal of Microbiology and Biotechnology Research 2017; 2(3): 375-78.
2. Simoes MF, Antunes A, Ottoni CA, Amini, MS, Alam I, Alzubaidy, H, Mokhtar, NA, Archer, JA and Bajic VB: Soil and rhizosphere associated fungi in gray mangroves ( marina) from the Red Sea-a metagenomic approach. Genomics, Proteomics & Bioinf 2015; 13(5): 310-20.
3. Shearer CA, Descals E, Kohlmeyer B, Kohlmeyer J, Marvanová L, Padgett D, Porter D, Raja HA, Schmit JP, Thorton HA and Voglymayr H: Fungal biodiversity in aquatic habitats. Biodiversity and Conservation 2007; 16(1): 49-67.
4. Madelin MF: Marine mycology: The higher fungi. Transactions of the British Mycol Soc 1980; 75(2): 351.
5. Palla MS, Guntuku GS, Muthyala MKK, Pingali S and Sahu PK: Isolation and molecular characterization of antifungal metabolite producing actinomycete from mangrove soil. Beni-Suef University Journal of Basic and Applied Sciences 2018; 7(2): 250-56.
6. Thatoi H, Behera BC and Mishra RR: Ecological role and biotechnological potential of mangrove fungi. a review. Mycology 2013; 4(1): 54-71.
7. Bhatnagar I and Kim SK: Immense essence of excellence marine microbial bioactive compounds. Marine drugs 2010; 8(10): 2673-01.
8. Fernandes AX, Salique SM and Panneerselvam A: Diversity of Mycoflora in Mangrove Soils of Karankadu, Tamil Nadu, India. Int J Curr Microbiol App Sci 2015; 4(10): 222-37.
9. Warcup JH: The soil-plate method for isolation of fungi from soil. Nature 1950; 166(4211): 117.
10. Rajesh B, Sunita G and Vijayalakshmi M: Antimicrobial profile of Stachybotrys chlorohalonata Isolated from the Marine Habitats. J of Pharmacy Res 2014; 8(8): 1059-66.
11. Butler EE and Mann MP: Use of cellophane tape for mounting and photographing phytopathogenic fungi. Phytopathology 1959; 49(4): 231-32.
12. Lakshman M: Electron Microscopy as a tool in research and disease diagnosis. Lead paper compendium published in presented in IAVP annual conference XXXI, held at Ananad, Gujarat, India. 13^th to 15^th Nov 2014; 139-60.
13. Lakshman M: Application of conventional electron microscopy in aquatic animal disease diagnosis. A review 2019; 7 (1): 470-75.
14. Ward NA, Robertson CL, Chanda AK and Schneider RW: Effects of Simplicillium lanosoniveum on Phakopsora pachyrhizi, the soybean rust pathogen, and its use as a biological control agent. Phytopath 2012; 102(8): 749-60.
15. Lim SY, Lee S, Kong HG and Lee J: Entomopathogenicity of Simplicillium lanosoniveum isolated in Korea. Mycobiology 2014; 42(4): 317-21.
16. Nonaka K, Kaifuchi S, Omura S and Masuma R: Five new Simplicillium species (Cordycipitaceae) from soils in Tokyo, Japan. Mycoscience 2013; 54(1): 42-53.
17. Tamura K, Stecher G, Peterson D, Filipski A and Kumar S: MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 2013; 30(12): 2725-29.
18. Perez C, Psuli M and Bazerque P: An antibiotic assay by the agar well diffusion method. Acta Biol Med Exp 1990; 15: 113-5.
19. Liu F and Cai L: Morphological and molecular characterization of a novel species of Simplicillium from China. Cryptogamie, Mycologie 2012; 33(2): 137-44.
20. Baiswar P, Ngachan SV, Rymbai H and Chandra S: Simplicillium lanosoniveum, a hyperparasite on Aecidium elaeagni-latifoliae in India. Australasian Plant Disease Notes 2014; 9(1): 144.
21. Pavlicek A, Hrda S and Flegr J: Short Communication freetree-freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD Analysis of Genus Frenkelia. Folia Biologica (Praba) 1999; 45: 97-99.
22. Chen R, Huang C, Li J and Tsay J: Evaluation of characteristics of lanosoniveum on pathogenicity to aphids and in-vitro antifungal potency against plant pathogenic fungi. Int J Env Agric Res 2017; 3(1): 2454-50.
    

    ---

    How to cite this article:

    Chiranjeevi C and Muvva V: Isolation and taxonomic characterisation of potential antagonistic Simplicillium lanosoniveum vjch-19 from mangroves of Adavuladeevi, Andhra Pradesh, India. Int J Pharm Sci & Res 2020; 11(7): 3379-84. doi: 10.13040/IJPSR.0975-8232.11(7). 3379-84.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---