INTRODUCTION:

Fungi being the most important saprophytic microbes in the soils play a vital role in the bio-geochemical cycling of matter. The saprophytic nature of fungi has been given recognition as they play a key role in the ecosystem processes viz., decomposition of organic matter and re-mineralization of elements. These processes improve the fertility of any environment and thereby support productivity and biodiversity (Madhanraj et al., 2010)¹.

Coastal ecosystem has various specialized niches for the colonization of fungi viz., beach soils, sand dunes and muds of mangroves, estuaries, etc. They were studied across the world (Madhanraj et al., 2010; Chandhuri et al., 2009; Nadimuthu ,1998 )^1,2,3. Along the east coast of India after the Tsunami’s hit during December, 2004, the coastal regions are planted with Casurina to develop bioshield for saving the lives of the people living in the coastal area. Nagapattinam, one of the worst affected areas due to Tsunami in India is now has dense bioshield belt, which also would serve as a specialized nich for the colonization of fungi.

MATERIALS AND METHODS:

Analysis of Mycoflora:

Naluvedhapathy is one of the area planted with Casuarina, as a measure of preventing Tsunami, by the Forest Department, Government of Tamil Nadu, which is situated at a distance of 9 km from Vedaranyam towards north, in Nagapattinam Dt of southeast coast of India.

Soil samples were collected from 10 different locations seasonally for a period of two years from January 2007 to December 2008, dividing a calendar year into four seasons viz., Postmonsoon (January – March), Summer (April – June), Premonsoon (July – September) and Monsoon (October – December), based on the north east monsoon, which is prevailing in the study area.

In each station, the soil samples were collected up to a depth within 10 cm using a metal spatula sterilized every time with 70 per cent alcohol. The samples were kept in new polythene bags, sealed and transported to the laboratory immediately for the mycological examination. For the analysis of soil nutrients, one kg of soil was separately collected in polythene bags from each location.

Dilution plating technique described by Warcup (1950)⁴ was used to isolate the fungi from soils. Soil sample weighting 1g was diluted in 10 ml of 50% seawater (1:1 v/v seawater (30 ppt): distilled water). One ml of the diluted sample was poured and spread on Petri plates containing sterilized PDA medium (Himedia) supplemented with streptopencillin antibiotic solution (1%@16/L) in replicates. The inoculated plates were incubated in a dust free cubourd at the room temperature (26±2°C) for 7 days.

The colonies growing on PDA plates with different morphology were counted separately. Semipermanent mounts ware prepared using lactophenol cotton blue mountant and examined microscopically.

Their characters were analyzed and were compared with the standard works of Raper and Thom (1949)⁵, Von Arx (1974)⁶, Ainsworth et al. (1973)⁷; Raper and Fennell (1965)⁸ and Ellis (1976)⁹ for their identification.

Number of species is referred as species diversity and Population density is expressed in terms of Colony Forming Unit (CFU) per gram of soil with dilution factor. Percentage contribution of individual species to the total population was worked out as follows.

In order to assess the dominance of individual species in each site percentage contribution was worked out as follows.

No. of colonies of a fungus in a sample

% contribution = ---------------------------------------------- x 100

Total number all colonies of all the species in a sample

Analysis of physico-chemical characteristics of the soil:

Moisture content was estimated by finding the weight difference of known quantity of soil before and after drying in a hot air oven at 60°C for 6 hours. Soil samples after removing the debris were suspended in distilled water (1:2 w/v) and allowed to settle down the sand particles. The pH of the suspension was read using pH meter (Systronics, India), to find out the soil pH. Electrical conductivity of soil was determined in the filtrate of the water extract using conductivity bridge as described by Jackson (1973)¹⁰, Cation exchange capacity (CEC) of the soil was determined by using 1 N ammonium acetate solution as described by Jackson (1973)¹⁰.

Organic carbon content was determined by adopting chromic acid wet digestion method as described by Walkley and Black (1934)¹¹; available nitrogen was estimated by alkaline permanganate method as described by Subbiah and Asija (1956)¹² and available phosphorus by Brayl method as described by Bray and Kutz (1945)¹³. Available potassium was extracted from soil with neutral 1 N ammonium acetate (1:5) and the potassium content in the extract was determined by using flame photometer (Standfold and English, 1949)¹⁴. Calcium (Neutral 1 N NH₄ OAC extractable 1:5) was extracted with neutral 1 N ammonium acetate and the available calcium in the extract was determined by Versenate method (Jackson, 1973)¹⁰. Available micronutrients such as Zn, Cu and Mn were determined in the diethylene triamine pentaacetic extract of soil using Perkin-Elmer (model 2280) Atomic Absorption Spectrophotometer (Lindsay and Norvell, 1978)¹⁵. Other nutrients such as magnesium, sodium and available iron were analysed following the method of Barnes (1959)¹⁶ and Muthuvel and Udayasoorian (1999)¹⁷.

RESULTS AND DISCUSSION:

In the present investigation, the species diversity of the fungi reveled with the existence of 19 species belonging to 4 genera. All of them were members of the Deuteromycetes. All these fungal species were reported earlier from soils and a variety of substrates in the terrestrial environment (Gilman, 1995)¹⁸. A great majority of them were also reported from oceans and estuaries (Johnson and Sparrow, 1961)¹⁹, as facultative forms to marine habitats. Though the coastal soils are considered to be the transitional areas between the land and sea, it exhibit the occurrence of only terrestrial species, often described as facultative fungi based on their ability in growth and reproduction, but not the obligate fungi. Introduction of terrestrial species into the coastal and marine environs are termed as invasion (Subramanian and Raghukumar, 1974)²⁰ and this is facilitated through various sources such as plant litter, other organic materials, erosion and run off from soil.

Earlier studies conducted by Subramanian and Raghukumar (1974)²⁰ along the marine and brackish water habitats along Madras coast, Mohamed Salique (1989)²¹ from Vellar estuarine complex, Nadimuthu (1998)³ from Mandapam coast, Murthy and Manoharachary (1992)²² from Mangroves of Godavari, all along the east coast of India and, Prabhaharan et al. (1987)²³ from Cochin backwaters, Pawar and Thirumalachar (1966)²⁴ from Bombay coast, both on the West Coast of India have yielded varying fungal species assemblages.

The species diversity recorded in the present study (number of species) was narrow. The minimum of 6 species were recorded in the soils collected during postmonsoon, summer and monsoon season in 2008. The maximum of 10 species were recorded in the soils collected during premonsoon season in 2008. The genus Aspergillus was constituted by the maximum of 14 species followed by Trichoderma and Fusarium (2 species each) (Table 1). As in the present study the trend of species composition with bulk number of Aspergillus species are reported from mangrove sediments of Cochin (Kerala) by Prabhakaran (1990)²⁵, coastal and Brackish sediments by Subramanian and Raghukumar (1974)²⁰ and sand dune of Tamil Nadu coast by Madhanraj et al., (2010)¹ . This could be inferred as that the fungal species isolated are bulk from the genus Aspergillus which are highly adapted to the varying soil characteristics observed in the study areas. Evidently, the tolerance and adaptive mechanism of Aspergillus to varying marine environmental characteristics are reported by Pawar and Thirumalachar (1966)²⁴, Subramanian and Raghukumar (1974)²⁰ and Nadimuthu (1998)³.

Table 1. Total number of Colonies (TNC), mean density(MD) (CFU/g) and percentage contribution of fungi recorded during different season from the sampling station

S. No	Name of the fingi	2007								2008								Total No. of colonies	% contribu-tion
		Post monsoon		Summer		Premonsoon		Monsoon		Post monsoon		Summer		Premonsoon		Mosoon
		TNC	MD	TNC	MD	TNC	MD	TNC	MD	TNC	MD	TNC	MD	TNC	MD	TNC	MD
1.	Acrocylindrium oryzae	-	-	4	1.33	-	-	4	1.33	-	-	5	1.66	6	2	-	-	19	5.84
2.	Aspergillus candidus	8	2.66	5	1.66	6	2	5	1.66	-	-	6	2	4	1.33	5	1.66	39	12
3.	A. conicus	5	1.66	7	2.33	-	-	-	-	-	-	5	1.66	5	1.66	7	2.33	29	8.92
4.	A. fumigates	-	-	-	-	-	-	-	-	-	-	4	1.33	-	-	4	1.33	8	2.46
5.	A. granulosis	-	-	5	1.66	-	-	7	2.33	5	1.66	-	-	3	1	-	-	20	6.15
6.	A. humicola	7	2.33	-	-	-	-	4	1.33	3	1	-	-	4	1.33	-	-	18	5.53
7.	A. luchuensis	4	1.33	-	-	-	-	-	-	-	-	-	-	5	1.66	-	-	9	2.76
8.	A. nidulans	5	1.66	-	-	-	-	3	1	-	-	-	-	4	1.33	-	-	12	3.69
9.	A. ochraceous	-	-	8	2.66	8	2.66	4	1.33	4	1.33	-	-	7	2.33	-	-	31	9.53
10.	A. phoenicis	-	-	-	-	6	2	-	-	6	2	-	-	-	-	-	-	12	3.69
11.	A. rugulosus	-	-	7	2.33	5	1.66	-	-	7	2.33	-	-	-	-	5	1.66	24	7.38
12.	A. tamari	7	2.33	-	-	-	-	-	-	-	-	-	-	4	1.33	-	-	11	3.38
13.	A. ustus	-	-	-	-	-	-	-	-	-	-	-	-	5	1.66	-	-	5	1.53
14.	A. variecolor	4	1.33	-	-	-	-	4	1.33	-	-	7	2.33	-	-	-	-	15	4.61
15.	A. wentii	-	-	-	-	7	2.33	-	-	-	-	-	-	-	-	-	-	7	2.15
16.	Fusarium oxysporum	5	1.66	6	2	4	1.33	-	-	-	-	5	1.66	-	-	7	2.33	27	8.30
17.	F. semitectum	-	-	7	2.33	-	-	-	-	4	1.33	-	-	-	-	-	-	11	3.38
18.	Trichoderma0 koeningii	6	2	-	-	3	1	-	-	-	-	-	-	-	-	8	2.66	17	5.23
19.	T. viride	-	-	4	1.33	-	-	7	2.33	-	-	-	-	-	-	-	-	11	3.38
	Total	51	17	53	17.7	39	13	38	12.7	29	9.7	32	10.7	47	15.7	36	12	325

Table 2 . Physico-chemical characteristics of soil collected during different seasion

Parameters	Postmonsoon – 07	Summer – 07	Premonsoon – 07	Monsoon – 07	Postmonsoon – 08	Summer – 08	Premonsoon – 08	Monsoon – 08
pH	8.3	8.8	8.1	7.9	8.2	8.4	8.5	8.7
Electrical Conductivity (dSm^-1)	1.29	1.06	1.18	1.12	1.31	1.09	1.21	1.27
Cation Exchange Capacity (c.mol proton⁺/kg)	8.67	8.90	8.85	8.70	9.30	10.11	8.79	10.10
Organic Carbon (%)	0.20	0.17	0.04	0.19	0.21	0.18	0.07	0.09
Available Nitrogen (%)	0.017	0.016	0.014	0.021	0.022	0.011	0.013	0.017
Available Phosphorus (%)	0.004	0.005	0.005	0.002	0.005	0.002	0.002	0.003
Available Potassium (ppm)	0.051	0.042	0.013	0.022	0.041	0.043	0.031	0.045
Available Zinc (ppm)	0.57	0.51	0.47	0.52	0.47	0.49	0.53	0.54
Available Iron (ppm)	2.90	2.78	2.19	2.47	2.51	2.52	2.61	2.13
Available Copper (ppm)	0.30	0.29	0.22	0.25	0.31	0.24	0.30	0.28
Available Manganese (ppm)	1.42	1.47	1.39	1.52	1.53	1.46	1.42	1.38
Calcium (mg/kg)	3.1	4.2	5.1	3.9	4.3	4.1	4.3	3.4
Magnesium (mg/kg)	3.9	3.2	4.1	3.4	3.7	3.7	4.1	3.4
Sodium (mg/kg)	0.47	0.51	0.54	0.29	0.41	0.32	0.23	0.79
Potassium (mg/kg)	0.03	0.04	0.02	0.05	0.05	0.02	0.03	0.08

All the fungi isolated in the present study, were ubiquitous soil saprophytes and their diversity exhibited fluctuations during different seasons. Even within the station, the same season of both the years spelled variations, this indicates that there is no seasonality in the species diversity in the soil of bioshield plantation.

Fungal population density also showed variations in different seasons. The range was from 9.7 to 17.7 x 10² CFU/g with the minimum in the samples collected during postmonsoon season in 2008 and maximum in the samples collected during Summer season in 2007 (Table 1).

Percentage contribution of the individual species to the total fungal population showed variation. Aspergillus candidus contributed the maximum percentage (12%) followed by A. ochraceous (9.53%), A. conicus (8.92%), Fusarium oxysporum (8.30%), A. rugulosus (7.38%), A. granulosus (6.15%), Acrocylindrium oryzae (5.84%), Aspergillus humicola (5.53%), Trichoderma koeningii (5.23%), A. variecolor (4.61%), A. nidulans and A. phoenicis (3.69% each), A. tamari, F. semitectum and T. viride (3.38% each), A. luchuensis (2.76%), A. fumigatus (2.46%), A. wentii (2.15%) and A. ustus (1.53%) (Table 2).

All the soil samples analysed during the entire period of study were alkaline in nature. It was in the range from 7.9 to 8.8 during monsoon in 2007 and summer season in 2007. Alkaline condition has been explained as the characteristic feature of marine soils (Nadimuthu, 1998)³. Marine habitats such as coastal and brackish environs (Subramanian and Raghukumar, 1974)²⁰ and sand dunes (Upathyay et al., 1978)²⁶ were also reported to have alkaline conditions as reported in the present study.

EC value is an indirect measure for the salinity. In the present study, it was recorded in the range from 0.06 to 1.31 dsm^-1. This was comparatively lower than the marine and brackish water sediments of Madras coast (Subramanian and Ragukumar, 1974)²⁰ and mangroves of Andaman (Chandhuri et al., 2009)². This may be due to the washing of freshwater by the heavy flow through the distributaries of the river Cauvery, which traverse along the coast, and monsoonal rainfall. Likewise, CEC in the soils was also low which was in the range from 8.67 to 10.11 c.mol proton + /kg, when compared to the salt affected soils of Elhussinia plane, Sharkia Governorate, Egypt, that was in the range of 31 to 54.8 c.mol proton +/kg, (Elbodiny et al., 2008)²⁷; and 189 and 275 mmol kg^-1 in the soil samples of Andamans (Chauhuri et al., 2009)².

Organic Carbon content is considered to be the factor responsible to influence the population of any of the heterotrophic microorganisms. It showed variations in the range from 0.04 to 0.21 per cent in all the sampling stations. This values are comparatively less than that of the river sediments (Lakshmi et al., 2002)²⁸ and various biotopes in the Muthupettai mangroves (Kanimozhi, 2008)²⁹. Nitrogen, an important nutrient for the growth of plants, is fluctuated from 0.011 to 0.022% during different seasons, which is also less than other marine environs.

Available potassium was in the range from 0.013 to 0.051 ppm in the soils which was less than the range reported from Shenzhen (1.6 per cent)( Tam et al., 1995), Fujian (2.07%)( Lin et al., 1987)³⁰, Hain mangroves (0.42 – 1.19%)( Liao, 1990)³¹ and mangroves of Andamans (0.81 – 125%) (Chaudhuri, 2009)².

As that of major elements, the minor elements such as Zn, Fe, Cu, Mn, Ca, Mg and Na were recorded in less than other marine habitats such as mangroves (Kawser et al., 2002; Chaudhuri, 2009;)^{32, 2}, estuaries (Elbordin et al., 2008)²⁷ etc. The concentrations of different elements were in the order: Ca > Mg > Fe > Mn > Na > Zn > Cu, the order of elements was almost similar in all the seasons except a change in the order of Na and Zn.

Correlation analysis made for various physic-chemical parameters and fungal population. The positive correlation are observed between organic carbon and available manganese (r = 0.765 ; P < 0.05). The negative correlation were observed between available potassium and calcium (r = 0.743 ; P < 0.05), available zinc and calcium (r = -0.845 ; P < 0.01)

Soils of bioshield plantation with Casuarina along the coastal is also supporting fungal diversity, but it varies in terms of species diversity and population density from mangroves, which one of the major vegetation along the coastal. Like wise the soil character also reveling less nutrient content than that of mangroves soils. But the species composition is comparable with the mangroves and other coastal habitats, with the dominance of Aspergilli.

ACKNOWLEDGEMENT:

The authors thank the Secretary and Correspondent A.V.V.M. Sri Pushpam College, Poondi – 615 503, Thanjavur Dt. for providing laboratory facilities.

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Received on 08.03.2011 Accepted on 05.05.2011

Asian J. Pharm. Res. 1(2): April-June 2011; Page 37-41