INTRODUCTION:

Vedaranyam region falls in the pro-deltaic region of Cauvery delta of Nagapattinam district, Tamilnadu. It is a typical arcuate delta and the alignment of the arc passes through Thiruthuraipoondi. The marine environment is an untapped source for many useful drugs and an assessment of this potential is imperative. It is well known that the antibiotics are medicinally valuable and that the microbes are the potential sources of antibiotics, which could be profitably used in the pharmaceutical industries.

Microbes from salt pans are yet to be fully explored as potential producers of antimicrobial agents. However, few reports are available on the antimicrobial potential of microorganisms in Indian slatterns. Mangrove forests are biodiversity “hotspots” for marine fungi (Shearer et al., 2007) because the bases of mangrove trunks and aerating roots are permanently or intermittently submerged. The upper parts of roots and trunks rarely never reached by the salt water, thus terrestrial fungi and lichens occupy the upper part of the trees and marine species occupy the lower part. At the interface there is an overlap between marine and terrestrial fungi (Sarma and Hyde 2001).

Mangroves are intertidal forested wetland confined to the tropical and subtropical regions (Macintosh and Ashton, 2000). A mangrove forest is considered a dynamic ecotone (or transition zone) between terrestrial and marine (Gopal and Chauhan, 2006). In its simplest sense, “mangrove” is used as a generic term referring to a group of woody, halophytic plants that occur along sheltered tropical and subtropical coastlines.

Mangrove forests are also referred to as mangrove swamps, tidal forests, tidal swamp forests (Kathiresan and Bingham, 2001). In the present study isolation and identification of fungi from salt pan soil from area of Vedaranyam, Nagapattinam district, Tamilnadu.

MATERIALS AND METHODS:

Collection of soil sample

Marine soil samples were collected from Salt Pan environment of Vedharanyam, Nagapattinam Dt.Tamil Nadu (Fig-1). Soil samples were collected from the study site at random during the study period. The samples were made at a depth within 10-15 cm from the surface of the soil. The collected soil samples were brought to the laboratory in sterilized polythene bags handpicked air, dried and stored in containers for further analysis.

Physico-chemical analysis of soil

Physico-chemical characteristics in relation to Temperature, Pressure, pH and Salinity of medium (Burtseva et al., 2003; Masuma et al., 2001; Gonda et al., 2000) were analysed.

Isolation of Mycoflora

Dilution plating method

Dilution plating technique described by Warcup (1950) was used to isolate the fungi from soils. Soil sample weighting 1gm was diluted in10ml of sterile distilled water and marked as 10^-1 to10^-9 dilution. 0.1ml of the diluted (10^-2 and 10^-3) sample was poured and spread on PDA plates. The plates were incubated at 24±2°C for 3-5 days and considered as mother culture.

Preparation of Potato Dextrose Agar Medium

Potato - 200gms

Dextrose - 20gms

Agar - 15gms

Distilled water - 500ml

Sea water - 500ml

pH - 6.6

The potato tubers were peeled and weighed for about 200g. The tubers were chopped into small pieces with the help of sterile knife. The chopped potatoes were transferred into a conical flask containing about 1000ml of distilled water. The content was boiled for 20 minutes. The supernatant were decanted and filtered by muslin cloth and the filtrate was collected. Dextrose (20g) and agar (15g) were transferred into the extract and shacked to dissolve the ingredients. The medium was made up to 1 litre by addition of distilled water. The pH of the medium was observed and adjusted to 6.6 by using 1N hydrochloric acid or sodium hydroxide drop wise. Finally, the medium was cotton plugged and autoclaved at 121°C for 15 lbs. To avoid the bacterial contamination streptomycin sulphate antibiotic (50µg/ml) was added to the sterile medium. The medium was poured into the sterile petridish (25ml/ dish). From the dilution of 10^-2 to 10^-3 0.1ml of sample was inoculated into each plate and have spreaded over with L-rod. The plates were incubated at 24±2°C for 3-5 days and considered as mother culture.

Isolation of Pure Culture

The colonies growing on PDA plates with different morphology were counted separately. The different fungal colonies from the mother culture were picked up by sterile inoculation loop and aseptically inoculated into the PDA plates. These plates were incubated at 24±2°C for 3-5 days, each plate contain single kind of fungi.

Identification of fungi

Lactophenol Cotton blue mounting

A drop of lactophenol cotton blue stain was placed on the clean glass slide, a small tuft of the fungus, preferable with spores and spore bearing structures were transferred into the drop, using a flamed, cooled needle and gently tested using mounted needle. A cover glass were placed over the preparation and care was taken to avoid trapping air bubbles in the stain. A thin layer of DPX mount was placed around the edge of the coverslip. The slide was observed under the microscope (400x). Microphotography of the individual fungal species was also taken using Nikon phase contrast microscope (Nikan, Japan).

Identification

Colony colour and morphology were noted besides hyphal structure, spore size, shapes and spore bearing structure. Identification has been done by referring the standard manual Ainsworth et al.,(1973). Spore identification was achieved by reference to Spore atlases of Gregory (1973) and Anna (1990).

RESULTS AND DISCUSSION:

Mangrove fungi are of enormous scientific interest, for two major reasons. First, they constitute the second largest part of the earth’s marine fungi. Second mangrove fungi often posses unique structures, metabolic pathways, reproductive systems, and sensory and defense mechanisms because they have adapted to extreme environments. So the mangrove fungi represent a source of unique genetic information. Indeed, the vast majority of mangroves fungi have yet to be identified. it is urgent to develop a fundamental understanding of the genetic, nutrional, and environmental factors that control the production of primary and secondary metabolites in mangrove fungi, as a basis for developing new and improved products (Zhong – Shan, 2009).

Table.1-Physico-chemical analysis of soil

S. No	Physico-chemical parameters	Analytical values
1	pH	8.82
2	Electrical conductivity (dsm-1)	0.18
3	Organic carbon (%)	0.36
4	Organic matter (%)	0.72
5	Available Nitrogen (Kg/ac)	97.9
6	Available Phosphorus (Kg/ac)	4.98
7	Available Potassium (Kg/ac)	125
8	Available Zinc (ppm)	0.98
9	Available Copper (ppm)	0.59
10	Available Iron (ppm)	4.53
11	Available Manganese (ppm)	2.63
12	Cat ion exchange capacity (c.Mole. Proton-/kg)	18.9
13	Calcium	8.9
14	Magnesium	7.2
15	Sodium	1.26
16	Potassium	0.2

Physico-chemical analysis of Soil

The physico-chemical features of the test soil were given in Table-1. The physico-chemical parameters were recorded from the soil samples of coastal area Vedaranyam, Nagai (Dt), Tamilnadu. Moisture content appeared to be one of the major factors that support fungal growth in dates (Hill and Waller, 1999) as both the semi dry and soft types had all the six species identified from them. Therefore, storage facilities such as sacks, polythene bags and natural fibre, which are air-tight being used by the traders on both campuses(Personal observation) for storage of all the varieties might have encouraged the fungal growth on the two varieties above. This leads to continuous increase in humidity and temperature of the dates, which consequently favours fungal growth as reported by Ahmad (2003).

Biodiversity of fungi

Isolation of fungi from salt pan soil

Fungal diversity of any soil depends on a large number of factors of the soil such as pH, organic contents, and moisture (Alexander 1977).The soil moisture has a direct effect on the population of fungi positively hence, at higher moisture, the tolerance and colonization by fungi is badly affected (Adams et al., 1999). Mehdi and Saifullah (2000) and Mehdi et al., (2000) observed that Deuteromycota was the most common and dominant group. Aspergillus was the most diverse genus, followed by Penicillium, salt pan yielded the highest number of fungi, followed by Rhizophora nucronata and Cladosporium fagal whereas the least number of fungi was observed in A. corniculatum (P<0.001).

Fig-2 Morphological and Microscopic view (400X) of isolated fungi

All the Deuteromycetes isolated by plating method reported from marine environment in many parts of Tamil Nadu covering Kanyakumari (Upadhyay et al., 1978). In the present study by direct plating, 20 species of fungi were isolated from the saltpan soil. The isolated fungi such as Aspergillus flavus, A. terreus, A.sydowi, A.oryzae, A.ruber, A.sulphureus, A.conicus, A.chevalieri, A.flavipes, Absidia glauca, Acrophialophora fusispora, Gliocladium sagariensis, Geotrichum candidum, Penicillium citrinum, P. chrysogenum, P. purpurogenum, Trichoderma harzianum, T. koningii, T. viride and Alternaria alternata. Out of 20 species, the maximum number of fungi were belonged to Deuteromycetes (Fig-2).

CONCLUSION:

Marine fungi produce secondary metabolites that are biologically active to treat certain diseases. Therefore, more researches have been done to discover the potential use of certain secondary metabolites. The structures of secondary metabolite produced by the saltpan fungi are unique as compared to the terrestrial fungi.

ACKNOWLGEMENT:

The authors thank Secretary & Correspondent and Principal of A.V.V.M Sripushpam College (Autonomous), Poondi for the Permission and Sri Gowri Biotech Research Academy, Thanjavur (Dt), Tamilnadu for the laboratory facility.

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