Diversity of fungal flora from mangrove of Pudukkottai district, Tamilnadu, India

Research Article ISSN: 2321-4988 Banupriya.S et al. /JPR:BioMedRx: An International Journal 2013,1(10), Available online through www.jpronline.info Diversity of fungal flora from mangrove of Pudukkottai district, Tamilnadu, India Banupriya.S, Kanimozhi.G and Panneerselvam.A PG and Research Department of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous), Poondi-613 503, Thanjavur, Tamilnadu,India Received on:19-07-2013; Revised on:17-08-2013; Accepted on:22-09-2013 ABSTRACT The present study was conducted to know the diversity of fungal flora in the Mangrove ecosystem of Pudukkottai district at Arasankarai during four different seasons (January-2012-December-2012). The result of the study revealed that the mangrove soil showed 25 different fungal species such as Aspergillus alliaceus, A.awamori, A.candidus, A.chevalieri, A.conicus, A.flavus, A.funiculosus, A.granulosis, A.lunchensis, A.nidulans, A.niger, A.rugulosus, A.sydowi, A.variecolor, A.versicolor, Curvularia lunata,fusarium oxysporum, F.solani,Penicillium citrinum, P.roqueforti, Penicillium sp, Rhizopus stolonifer, Spicaria divaricata, Thamnidium sp, Verticillium sp. The Physico-chemical parameters were analysed and statistically correlated with fungal flora. Keywords: Mangrove ecosystem, Fungal flora, Physico-chemical parameters INTRODUCTION Mangrove forests are extremely important coastal resources, which are vital to our socio-economic development. A vast majority of human population lives in coastal area, and most communities depend on local resources for their livelihood. The mangroves are sources of highly valued commercial products and fishery resources and also as sites for developing a burgeoning eco-tourism (9). Soil contains a vast array of microorganisms such as bacteria, viruses, fungi, actinomycetes, protozoa and algae (2). Fungi play a significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, natural cycling, as biofertilizers and in many other ways. Fungal biotechnology has become an integral part of the human welfare (11). 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 in the ecosystem processes viz.., decomposition of organic matter and remineralization of elements. These processes improve the fertility of any environment and thereby support productivity and biodiversity. (12). *Corresponding author. Banupriya.S PG and Research Department of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous), Poondi -613 503,Thanjavur,Tamilnadu,India MATERIALS AND METHODS Study Area This study was carried out in the permanent site of the mangrove environment at Arasankarai, Pudukkottai (Dt), Tamilnadu,between the months of January-2012 to December 2012. Sampling schedule The rhizosphere soil samples were collected in each sampling station seasonally for a period of one year from January 2012 to December 2012. The calendar year has been divided into four season viz., Postmonsoon (January- march), Summer (April-june), Premonsoon ( July-september), and Monsoon (October-December). Collection of soil sample The mangrove rhizosphere soils were collected from Avicennia marina, in the place of Arasankarai, Pudukkottai (Dt), Tamilnadu (fig.1). Soil samples were collected from the study site at random during the study period. The samples were made at a depth within 2-3cm from the surface of 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 The physical properties of the soil samples were determined in accordance with standard analytical methods (18). The characteristics in relation to Temperature, Pressure and ph of medium (13) were analysed.

Banupriya.S et al. /JPR:BioMedRx: An International Journal 2013,1(10), Fig.1. Map showing the sample stations

Banupriya.S et al. /JPR:BioMedRx: An International Journal 2013,1(10), Isolation of soil mycoflora The soil fungi were enumerated by two methods, namely, Soil dilution, (19), and Soil plate method (20). The soil suspensions were diluted in 10 fold increment from 10-2 to 10-3. One ml of the diluted sample was plated onto sterilized Potato Dextrose Agar medium supplemented with 1% streptomycin sulphate (1gram of streptomycin sulphate was mixed thoroughly in 100ml of sterilized distilled water). All the petridishes were incubated at room temperature 28±2? c for a period of 3-4 days and then examined. Preparation of Potato Dextrose Agar Medium Potato - 200 gms Dextrose - 20 gms Agar - 15gms Distilled water - 500ml Sea water - 500ml ph - 5.6 The potato tubes 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 50% of distilled water and 50% of aged sea 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 shake 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 5.6 by using 0.1N hydrochloric acid or sodium hydroxide drop wise. Finally, the medium was cotton plugged and autoclaved at 121 0 c for 15 lbs. To avoid the bacterial contamination streptomycin 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 28±2? c for 3-4days 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 28±2? c for 3-4days and each plate contains single kind of fungi. Identificationof soil 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 care was taken to avoid trapping air bubbles in the stain. A thin layer of DPX mount wasplaced 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 of the organisms was made by microscopic analysis using taxonomic guides, standard procedures and relevant literature (10,6 & 7). Identification Colony colour and morphology were observed besides hyphal structure, spore size, shapes and spore bearing structure. Identification has been done by referring the standard manual (1).Spore identification was achieved byreference to spore atlases of Gregory and Anna (8,4). Presentation of data Number of species is referred as species diversity. Population density is expressed in terms of Colony Forming Unit (CFU) per gram of soil with dilution factor. In order to assess the dominance of individual species in each site percentage contribution was worked out as follows. No.of colonies of fungus in a sample % contribution = 100 Total number all colonies of all species in a sample Frequency occurrence was calculated as follows in order to identify their existence in the soils collected from different areas. Number of sample in which a particular fungus occurred %frequency= 100 Total number of samples examined Based on the frequency occurrence the fungi were grouped as rare (0-25% frequency), Occasional (26-50% frequency), Frequent (51-75% frequency) and common (76-100% frequency) species. RESULT AND DISCUSSION The results based on the physico-chemical characteristics of the soil samples organic matter contents, macronutrients, micronutrients and ph values is shown in Table-1. Physico-chemical characteristics of the soil samples were collected from different seasons. Environmental factors, including climate, geomorphology, hydrodynamics and soil characteristics control the structure and function of any ecosystems. Among the abiotic factors, in a particular soil nutrient status, are believed to be the most significant factor. The diversity and distribution of different organisms in the marine environment are influenced by the physico-chemical properties of both water and the sediments (Rani and Panneerselvam, 2010). In the present study ph was alkaline level in soil samples collected during three seasons. The minimum of 6.79 was recorded in the samples were collected during postmonsoon season. The maximum of 8.23 in the sample collected during premonsoon season (Table-1). The organic carbon, nitrogen, phosphorus, potassium are important for fungi. In the absence of any of these the growth and sporulating of

Banupriya.S et al. /JPR:BioMedRx: An International Journal 2013,1(10), moulds as well as other microorganisms are hampered a lot (17). It has been reported that the density of fungal population occurred during the monsoon (rainy) season when the soil moisture was significantly high (5) has reported that environmental factors such as ph, moisture, temperature, organic carbon, organic nitrogen play an important role in the distribution of Mycoflora. Table-1 Physico-chemical parameters of Mangrove soil from Arasankarai S.No Name of the parameters Sample Details Postmonsoon Summer Premonsoon Monsoon 1. ph 6.79 8.12 8.23 8.12 2. Electricalconductivity (dsm -1 ) 0.25 2.89 2.96 2.75 3. Organic Carbon (%) 0.50 0.08 0.06 0.12 4. Organic matter (%) 0.72 0.44 0.46 0.58 5. Available Nitrogen (mg/g) 2.82 3.16 4.64 2.89 6. Available Phosphorus (mg/g) 1.00 0.22 0.12 1.10 7. Available Potassium (mg/g) 2.06 2.57 3.09 2.57 8. Available Zinc (ppm) 0.48 0.28 0.21 0.35 9. Available Copper (ppm) 0.89 0.21 0.18 0.26 10. Available Iron (ppm) 3.45 5.47 4.21 5.15 11. Available Manganese (ppm) 1.58 3.48 3.45 3.25 12. Cation Exchange Capacity (C. Mole Proton + / kg) 18.6 12.5 10.58 14.26 Exchangeable Bases (C. Mole Proton + / kg) 13. Calcium 6.4 3.9 4.1 5.2 14. Magnesium 5.6 2.3 2.1 3.4 15. Sodium 0.87 3.69 6.23 7.56 16. Potassium 0.36 0.04 0.06 0.07 Table-2 Total number of colonies,mean density (CFU/g) and percentage contribution of Fungi recorded during differentseasons from mangrove environment, Arasankarai. S.No Name of the organisms Postmonsoon Summer Premonsoon Monsoon Total no.of Colonies %Contribution TNC MD TNC MD TNC MD TNC MD 1. Aspergillus alliaceus 1 0.33 - - 2 0.66 1 0.33 4 2.83 2. A.awamori 2 0.66 2 0.66 3 1 - - 8 5.67 3. A.candidus 1 0.33 - - 1 0.33 - - 2 1.41 4. A.chevalieri - - 2 0.66 4 1.33 3 1 9 6.38 5. A.conicus 1 0.33 1 0.33 2 0.66 2 0.66 6 4.25 6. A.flavus 2 0.66 3 1 3 1 1 0.33 9 6.38 7. A.funiculosus 2 0.66 - - 1 0.33 - - 3 2.12 8. A.granulosis 5 1.66 1 0.33 - - - - 6 4.25 9. A.lunchensis 2 0.66 3 1 2 0.66 1 0.33 8 5.67 10. A.nidulans 4 1.33 - - - - 1 0.33 5 3.54 11. A.niger 1 0.33 2 0.66 - - - - 3 2.12 12. A.rugulosus 2 0.66 2 0.66 1 0.33 - - 5 3.54 13. A.sydowi 3 1 2 0.66 1 0.33 - - 6 4.25 14. A.variecolor 1 0.33 2 0.66 - - 1 0.33 4 2.83 15. A.versicolor - - - - 3 1 1 0.33 4 2.83 16. Curvularia lunata - - 2 0.66 1 0.33 - - 3 2.12 17. Fusarium oxysporum 4 1.33 1 0.33 1 0.33 - - 6 2.12 18. F.solani - - - - 2 0.66 - - 2 4.25 19. Penicillium citrinum 1 0.33 - - 2 0.66 - - 3 1.41 20. P.roqueforti 4 1.33 3 1 4 1.33 5 1.66 16 11.34 21. Penicillium sp. 2 0.66 1 0.33 1 0.33 2 0.66 6 4.25 22. Rhizopus stolonifer - - - - 1 0.33 1 0.33 2 1.41 23. Spicaria divaricata 1 0.33 2 0.66-0.66 - - 3 2.12 24. Thamnidium sp. 2 0.66 2 0.66 3 1 - - 7 4.96 25. Verticillium sp. 4 1.33 3 1 2 0.66 2 0.66 11 7.80 Total 46 15.24 35 11.59 39 12.93 20 6.62 141

Banupriya.S et al. /JPR:BioMedRx: An International Journal 2013,1(10), The organic matter content of soil was in the range from 0.44 to 0.72%. Available nitrogen content of the soil was higher variation in the range from 2.82 to 4.64 (mg/g). These results showed that factors such as organic matter in soil have an effect on the population of fungi and the presence of antagonist in the soil (Table-1) (16,3) The present study revealed the existence of 25 species of fungi which include large numbers of thepenicillium roqueforti, Verticillium sp, Aspergillus flavus, A. chevalieri, A. awamori, A. lunchensis, A. conicus, Penicillium sp, Aspergillus alliaceus, A. sydowi, A. nidulans, Thamnidium sp, Fusarium oxysporum, Aspergillus granulosis, A. variecolor, A. versicolor, A. conicus, A. niger, Curvularia lunata, Penicillium citrinum, Aspergillus candidus, Spicaria divaricata, Fusarium solani, Rhizopus stolonifer. Besides the above, maximum number of species diversity was encounted with the fungal species belonging to the class Deuteromycetes. The soil microbes decompose the plant and animal residues entering the soil and convert them into soil organic matter, which influences on soil physical, chemical and biological properties and on creating a complimentary medium for biological reactions and life support in the soil environment (14). The population mean density of fungi varied from 6.62 to15.24 10 2 CFU/g with the minimum in the samples were collected during monsoon season and maximum in the samples collected during Postmonsoon and Premonsoon season (Table 2). Percentage contribution of the individual species to the total fungal population at all the seasons showed variation. At the maximum percentage contribution of 4.25% was found with Aspergillus conicus, A. granulosis, A. sydowi, Fusarium oxysporum, Penicillium sp. (51-75%) which showed in Table-3. Table-3 Percentage frequency and frequency class of different species of Fungi recorded during different seasons from mangrove region, Arasankarai (n=4) S.No. Name of the organisms Frequency Class 1. Aspergillus alliaceus O 2. A.awamori F 3. A.candidus R 4. A.chevalieri F 5. A.conicus F 6. A.flavus F 7. A.funiculosus O 8. A.granulosis F 9. A.lunchensis F 10. A.nidulans F 11. A.niger O 12. A.rugulosus F 13. A.sydowi F 14. A.variecolor O 15. A.versicolor O 16. Curvularia lunata O 17. Fusarium oxysporum F 18. F.solani R 19. Penicillium citrinum O 20. P.roqueforti C 21. Penicillium sp. F 22. Rhizopus stolonifer R 23. Spicaria divaricata O 24. Thamnidium sp. F 25. Verticillium sp. C R- Rare (0.25%); O- Occasional (26-50%); F- Frequent (51-75%); C- Common (76-100%) ACKNOWLEDGEMENT The authors thank Secretary & Correspondent and Principal of A.V.V.M Sripushpam College, Poondi for the Permission. REFERENCES 1. Ainsworth, G.C.,Sparrow, S.K. and Sussman,A.S., 1973. The fungi an advanced treatise. A taxonomic review with key: Ascomycetes and fungi imperfect, Academic press, New York (1):621. 2. Alexander, M., 1977. Introduction to Soil Microbiology (2 nd Ed.) John Wiley & Sons, New York. Pp. 423-437. 3. Alvarez-Bernal, D., Conteras-Ramos, S.M., Trujillo-Tapia, N., Olalde-Portugal, V., Frais-Hernandez, J.T.,Dendooven,L., 2006. Effects of tanneries waste water on chemical and biological soil characteristics.appl.soil Ecol. 33:269-277. 4. Anna,L.S., 1990. A color Atlas of post-harvest disease and disorders of fruits and vegetables.general introductions and fruits 79;66-69. 5. Deka, H.K. and Mishra.R.R., 1984.Acta Batanica Inica.,12:180-184. 6. Domsch KA, Gams W, Anderson TH (1980). Compendium of soil fungi vol. 1, Academic Press, London. 7. Ellis MB (1971). Dematiaceous, Hyphomycetes. Common wealth Mycological Institute; Kew, surrey, England. 8. Gregory, P.H., 1973. Microbiology of the atmosphere (2 nd Ed.)AppendixI.LeonardHillp.31. 9. Kathiresan, K. and Bingham, B.L. (2001). Biology of mangrove and mangrove ecosystems. Advances in Marine Biology, 40: 81-251. 10. Kenneth Raper B, Dorothy Fennel I (1965). The genus Aspergillus, A chapter on Pathogenecity; Baltimore, U.S.A. 11. Manoharachary C, Sridhar K, Singh R, Adholeya, Suryanarayanan TS, Rawat S and Johri BN(2005.) Fungal Biodiversity: Distribution, Conservation and Prospecting of Fungi from India. Current Science 89 (1): 58-71. 12. Madhanraj P, Manorajan S, Nadimuthu N, and Panneerselvam A.(2010). An investigation of the sand dune soils of Tamilnadu coast, India, Adv. Appl. Sci.Res., 1(3): 160-167. 13. Mansuma, R., Yamaguchi, Y., Noumi, M., Omura, S. and Nmikoshi, M., 2001. Effect of sea water concentration on hyphal growth and antimicrobial metabolite production in marine fugi, Mycoscience., 24(5); 455-459. 14. Olson, R.K., M.M.Schoeneherger and S.G.Aschmann, (2000). American Society of Agronomy, Madison, Wisconsin, USA, PP: 987-1057. 15. Rani, C and Panneerselvam, A, (2010). Journal of pure and applied microbiology, Vol.4 (1), PP:199-206. 16. Reddi, P.M. and G. Narsimha, 2012. Effect of leather industry effluents on soil microbial and protease activity.j.environ Biol.33:39-42. 17. Saksena,S.B., 1955.J.Indian Bot.Soc., 34(3):262-298. 18. Subramanyam, N.S. and A.V.S.S.Sambamurthy, 2002. Ecology,Narosa Publishing House,Delhi. PP. 616. 19. Waksman SA (1927). Principles of soil microbiology. BailliereTindall& Co., London. 20. Warcup, J.H., 1950. The soilplate method for isolation of fungi from soil. Nature, Lond, 178:1477. Source of support: Nil, Conflict of interest: None Declared