Research Article

Austin J Biotechnol Bioeng. 2014;1(3): 5.

Griseofulvin Producing Endophytic Nigrospora Oryzae from Indian Emblica Officinalis Gaertn: a New Report

Rathod DP, Dar MA, Gade AK and Rai MK*

Department of Biotechnology, Sant Gadge Baba Amravati University, India

*Corresponding author: Rai MK, Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati¬-444602, Maharashtra, India.

Received: August 25, 2014; Accepted: September 19, 2014; Published: September 22, 2014

Abstract

Emblica officinalis (Amla) is widely used medicinal plant and is believed to increase defence against various types of diseases. E. officinalis harbours a range of endophytes responsible for synthesis of bioactive compounds. We screened endophytic fungi isolated from E. officinalis for their antimicrobial activity. During screening we found that a griseofulvin-producing endophytic fungus (Nigrospora oryzae) which is active against human pathogenic microbes. Nigrospora oryzae (DBT-150) was isolated and identified based on the morphology and ITS-rDNA sequence comparison. The presence of Griseofulvin was confirmed by standard spectra of Griseofulvin available in Mass Spectroscopy instrument. Griseofulvin is a non-toxic antifungal drug derived from several species of Penicillium. In the present study, these active compounds demonstrated activity against human pathogenic bacteria and fungi. This is a first report of Griseofulvin producing endophytic Nigrospora oryzae isolated from E. officinalis.

Keywords: Endophyte; Griseofulvin; Human pathogenic microbes; Nigrospora oryzae; Emblica officinalis

Introduction

Emblica officinalis is an important medicinal plant belonging to family Euphorbiaceae, also named as Amla, or Indian gooseberry. E. officinalis is native of India, but also grows in tropical and sub-tropical regions of Pakistan, Uzbekistan, Sri Lanka, South East Asia, China and Malaysia. Plant parts of E. officinalis is widely used in medicinal and effective against a range of diseases like cancer, heart disease, ulcer, diabetes, liver treatment, anaemia and various other diseases. The plant is also used as antioxidant, immune-modulator, antipyretic, analgesic, cyto-protective, antitussive and gastroprotective. It is also used in ophthalmic disorders, memory enhancing, lowering cholesterol level and neutralizing snake venom [1]. Medicinal plants harbor a variety of endophytes, which in turn protects their host from infections and grazing agents and also provide compliance to survive in unfavourable environmental conditions. Endophyte produces metabolites, which are being recognized as multipurpose compounds of antimicrobial agents [2].

Fungal endophytes produce metabolites, which are not only essential for the plant protection but are also important as medicines. The demand for new and highly effective bio control agents to control pathogens is huge, and the removal of synthetic compounds from the market is also demanding, because of their toxicity to the environment. Griseofulvin, an orally active antifungal drug is obtained from several species of Penicillium, Xylaria, Nigrospora, etc. It is an antifungal drug used for both animals and humans to treat various fungal diseases. Griseofulvin has been used as an antifungal antibiotic for the treatment of mycotic diseases of humans and veterinary animals [3]. Sommart et al. [4] reported three new hydronaphthalenone derivatives and one new dihydroramulosin derivative isolated from an endophytic fungus PSU-N24. Further they also studied the antifungal activity of griseofulvin against Microsporum gypseum SH-MU-4 with a minimum inhibitory concentration (MIC) of 2m g/ml. Park et al. [5] reported griseofulvin from an endophytic fungus Xylaria species. The authors also evaluated the efficacy of Griseofulvin against plant pathogenic fungi. Xia et al. [6] reported two new derivatives of an antifungal compound griseofulvin from the mangrove Kandelia candel (L.) endophytic fungus Nigrospora species collected from the South China Sea. They also studied the antitumor and antimicrobial activity of isolated compounds. The derivatives of griseofulvin are highly active and are known to treat various pathogenic and non-pathogenic disorders. Richardson et al. [7] reported potent antifungal agent griseofulvin extracted from endophytic Xylaria sp. was isolated from Pinus strobus and Vaccinium angustifolium.

In the present study, we isolated Nigrospora oryzae endophyte from leaves of E. Officinalis which secreates derivatives of Griseofulvin and checked its activity against human and plant pathogenic bacteria and fungi. This is the first report of endophytic N. oryzae isolated from E. officinalis.

Material and Methods

Experimental material

Healthy twigs and leaves of Emblica officinalis were collected from local forest of Amravati, Maharashtra, India. The endophytes were isolated according to modified method described by Strobel [8]. Leaves were cut into small segments (0.5-1 cm long), surfacesterilized with 4-5 % Sodium hypochlorite solution followed by 70% (v/v) ethanol, respectively, for 1 min, and then washed thrice with sterile distilled water. After surface sterilization, segments were dried and inoculated on potato dextrose agar, incubated at 25 ±2°C in the dark and observed daily for growth. The grown mycelia were purified on fresh potato dextrose agar and stored on PDA slants at 4°C for further study.

Identification of fungal endophyte using morphological and molecular markers

For the microscopic identification, the fungus was grown on the PDA and then identified on the basis of morphological and cultural characteristics. The cultures were used for DNA isolation followed by molecular identification. Isolation of DNA was performed by Chromous kit (Chromous Pvt Ltd Bangalore). The primers used for ITS amplification were ITS1 (5`- TCC GTA GGT GAA CCT GCG G-3`) and ITS4 (5`- TCC TCC GCT TAT TGA TAT GC-3`) [9]. Mastercycler personal (Thermal cycler, Eppendorf, North America) was employed in a thermal cycle for an initial denaturation at 94OC for 2 min followed by 34 cycles of 94OC for 30s, (ITS) (18S rDNA) and 72OC for 2 min, with a final extension at 72OC for 5 min. Polymerase chain reaction (PCR) products were sent to Chromous Biotech Pvt Ltd Bangalore India for sequence analysis.

Phylogenetic analysis

After sequencing, nucleotide sequences were compared with ITS sequence data strains available at the public databases Genbank (http://www.ncbi.nem.nih.gov) by using the BLASTN sequence match routines. The sequences were aligned using the CLUSTAL W program and phylogenetic and molecular evolutionary analyses were performed using MEGA6 [10]. The phylogenetic reformation was performed using the neighbour joining (NJ) algorithm, with bootstrap values calculated from 1000 replicate runs, using the software routines included in the MEGA software.

Extraction of crude extract from Nigrospora oryzae

The plugs of agar supporting mycelium growth were cut and transferred aseptically to a 500 mL Erlenmeyer flasks containing 200 mL potato dextrose broth with 2% dextrose and potato extract. The flasks were incubated at 280C on a rotary shaker (120 rpm) for 21 days for further growth. After mycelium growth 200 mL culture was filtered through cheese cloth. The filtrate was concentrated at 45-500C and extracted thrice by shaking with equal volume of Ethyl acetate. Finally, we obtained a gum like texture as a crude extract in Ethyl acetate solvent.

Antimicrobial activity of Nigrospora oryzae extracts

Antimicrobial activity of extract of N. oryzae was evaluated against two human pathogenic bacteria such as S. aureus (ATCC- 25923) and E. coli (ATCC-39403)], two human pathogenic fungi like Trichophyton mentagrophytes (NCIM-3100)] and Microsporum canis and one plant pathogenic fungus-Fusarium oxysporum. The antimicrobial assay was performed in triplicate. Bacteria were grown on preliminary nutrient agar medium (Hi-Media), human pathogenic fungi on Sabouraud Dextrose Agar (HiMedia) and plant pathogenic fungi were grown on PDA (Potato Dextrose Agar) (HiMedia). The antibacterial activities of extract were determined by disk diffusion method. To determine the combined effects, each standard antibiotic disk impregnated with 20 μL solution of extract was placed onto the nutrient agar plate inoculated with test pathogenic bacteria and then incubated at 37°C for 24 hours. A similar procedure was followed for the human pathogenic fungi. Sterile disks impregnated with 20 μL solution of extract were placed on Sabouraud Dextrose Agar medium plates inoculated with human pathogenic fungi and for F. oxysporum 20 μL of extract was places on PDA incubated at 27°C for seven days. Zones of inhibition were measured and compared with a standard antibiotic disk and griseofulvin solution.

Spectroscopic analysis

MS analyses were carried out using an electro-spray ionization-ion trap (ESI-IT) mass spectrometer. Mass analyses were conducted in positive mode with initial source parameters as (source voltage = 4.5 kV; sheath gas flow rate = 60 nitrogen, arbitrary units; capillary temperature = 160°C; capillary voltage = 10 V; tube lens = 5 V; octopole 1 voltage = -2.5V; octopole 2 voltage = -6V; interoctopol lens = -16V). All the spectra were obtained and analysed by LCQ Xcalibur software (Finnigan, Atlanta, GA, USA). Total current ion Mass spectra were obtained between a range of m/z 80 and m/z 1000. Charge state and isotopic distribution were analysed by a narrow-scan range mode (Zoomscan mode) and compared with isotopic profile calculation performed with Isopro software. Scanning was recorded for about 0.5 min corresponding to 15 scans. Spectra were developed in shape mode with a boxcar smoothing of 7 points. Concerning MS analysis, fungal extracts and pure griseofulvin solutions were prepared at different concentrations (100μg/ml and 10μg/ml respectively) in a mixture of methanol/water 95:5 (v/v) with 0.1% of trifluoro-acetic acid. These solutions were infused directly into the ESI probe with a 250-μl automated syringe (Hamilton, Reno, NV, USA) at a flow-rate of 3μl/min.

Statistical analysis

To analyze whether there is any significant difference in zone of inhibition between extract and combinations, we used 'one way ANOVA' for the relative variability within the separate classes of the experiment.

Results and Discussion

Identification of fungal endophyte (DBT-150)

The fungal endophytes DBT-150 was identified as Nigrospora oryzae on the basis of morphology and molecular analysis (Figure 1). The genus Nigrospora is classified in family Trichosphaeriaceae of the class Ascomycota. Nigrospora species are reported as endophytes from different medicinal plants like Kandelia candel and Abies holophylla. The grey woolly colony grows fairly rapid. The diameter of colony was 5.5 cm after 7 days of incubation on PDA.