MATERIAL AND METHOD A. Study Site and Location: Generally termed as the „Wine Capital of India‟, Nashik is situated in the Maharashtra state of India. This place is characterized by huge number of Grape Vineyards. Godavari River flows through Nashik city and the most prominent feature of Nashik is that the Lord Rama made Nashik as their home during the exile period of 14 years. Nashik also has one of the 12 Jyotilingas all around India, which are of great Religious value and devotees throughout the country pay visit to these sites. Nashik is the fourth largest urban area of Maharashtra. Nashik district is the third largest district in Maharashtra in terms of area occupying an area of 15,582 square kilometers. It is bounded by Dhule district to the north, Jalgaon district to the east, Aurangabad district to the southeast, Ahmednagar district to the south, Thane district to the southwest, Valsad and Navsari districts of Gujarat to the west, and The Dangs district to the northwest. The Western Ghats or Sahyadri range stretches from north to south across the western portion of the district. With the exception of few villages, the western portion is hilly, and intersected by ravines, and only the simplest kind of cultivation is possible. The western slopes of the Ghats are well drained by several rivers, including Daman Ganga River, which drains westwards to the Arabian Sea. The larger eastern portion of the district, which lies on the Deccan Plateau, is open, fertile, and well cultivated. Peninsular India‟s largest River Godavari originates in the district in the Trimbakeshwar Range and continues its soujourn eastwards through the district. Nashik is one of the fastest developing cities in the country. Administratively, the district is divided into 15 tehsils viz. Baglan (Satana), Chandwad, Deola, Dindori, Igatpuri, Kalwan, Malegaon, Nandgaon, Nashik, Niphad, Peint, Sinnar, Surgana, Trimbakeshwar and Yeola. The present investigation was undertaken to study the diversity of soil micro fungi of various villages in Nashik tehsil. The study area lies on 18.33º and 20.53º North latitude and 73.16º and 75.16º East longitudes at Northwest part of Maharashtra state, at 565 meters above mean sea level. The temperature ranges from 11ºC to 37ºC and the annual rainfall is 812 mm. Type of soil found in the 24 tehsil are red soils and black cotton soils. Grapes, sugarcane, onion, tomato, wheat, groundnut, etc. are the crops cultivated. Figure: 1 Location of Maharashtra State of India 25 Figure 2: District map of Maharashtra STUDY AREA Figure 3: Nashik District map showing study area 26 Figure 4: Nashik Tehsil map showing study sites B. Collection of soil samples: The soil samples were collected from two different ecosystem types of Nashik tehsil. 50 sites of agricultural land and 50 sites representing the barren land of 50 villages were selected for the present study (Table 3.1). Thus a total of 100 soil samples were analysed during the present study. The agricultural and barren lands were selected on all the directions along the outskirts of the city of Nashik. Soil samples of 50 sites at Nashik tehsil were collected during November 2014 to June 2016 in three intervals. During collection of the soil, the upper 2-3 inch layer of the soil was discarded and the inner portion of the soil was collected. From each locality i.e. village; 50 gm of soil sample was collected from a depth of about 10-15 cm deep pits dug in the area to be sampled. The samples were collected with a surface sterilized trowel. Soil was scraped along the walls of the pits and collected in polythene bags. Soil from 2- 3 pits was pooled together and mixed in the same polythene bag. The collected soil samples were brought to the laboratory and stored at 4ºC until further analysis. 27 Table 3.1: Agricultural and Barren field soil samples collected from different places of Nashik tehsil Sample No. & Place Sample No. & Place Ecosystem Type Ecosystem Type (Village) (Village) Agriculture Barren Agriculture Barren Field (A) Land Field (A) Land (B) (B) A-1 B-2 Adgaon A-51 B-52 Lohashingwe A-3 B-4 Ambebahula A-53 B-54 Lakhalgaon A-5 B-6 Anandwali A-55 B-56 Madsangavi A-7 B-8 Belatgavhan A-57 B-58 Mahirawani A-9 B-10 Belgaon Dhaga A-59 B-60 Matori A-11 B-12 Chandshi A-61 B-62 Mhasrul A-13 B-14 Chehede A-63 B-64 Morwadi A-15 B-16 Dahegaon A-65 B-66 Mungsare A-17 B-18 Dari A-67 B-68 Naikwadi A-19 B-20 Devargaon A-69 B-70 Nashik City A-21 B-22 Devlali A-71 B-72 Nashik Road A-23 B-24 Dhadegaon A-73 B-74 Palse Pimpalgaon A-25 B-26 Dudgaon A-75 B-76 Bahula A-27 B-28 Dugaon A-77 B-78 Pimpri Sayyed A-29 B-30 Eklahare A-79 B-80 Samangaon A-31 B-32 Gangamhalungi A-81 B-82 Shastrinagar A-33 B-34 Gangapur A-83 B-84 Shevgedarna A-35 B-36 Gangavarhe A-85 B-86 Shinde 28 Talegaon A-37 B-38 Girnare A-87 B-88 Anjaneri A-39 B-40 Govardhan A-89 B-90 Vanjarwadi A-41 B-42 Hinganwede A-91 B-92 Vilholi A-43 B-44 Jakhori A-93 B-94 Vinchur Gavali A-45 B-46 Kotamgaon A-95 B-96 Wadala A-47 B-48 Ladachi A-97 B-98 Wasali A-49 B-50 Lahvit A-99 B-100 Yashwantnagar C. Isolation of Fungi: The occurrence of fungi in soil samples of agriculture fields and Barren soils was studied by using soil dilution plate count method. (Subba Rao, 2004). The sieved soil sample was thoroughly mixed and from it one gram soil was weighed quickly. This was then suspended in 250 ml conical flask containing 200 ml sterile distilled water so as to get the stock solution. The flask was vigorously agitated on a shaker for about 15 minutes and then allowed to stand till the soil settled. From this stock solution one ml of soil suspension was transferred by means of sterilized 1 ml pipette to the petriplate and dispersed with molten but cool agar medium by shaking and rotating the plate before agar solidifies. Each plate was gently swirled to ensure uniform distribution of suspension. Upon solidification of the medium, the plates were incubated at room temperature (27 ± 1°C) until good growth of fungi was observed. By this method, each viable spore developed into a colony and the number of colonies appeared on the plates. D. Selection of Medium: It is difficult to select a single suitable medium for isolation of the soil fungi because of their diverse requirements for growth in culture hence different media were tested for obtaining maximum results. Sabouraud‟s Agar (SDA), Potato Dextrose Agar (PDA) and Czapek‟s Dox Agar medium (CDA) were found to be most suitable for fungal count. The CDA and PDA mediums were employed for isolation and making pure cultures. 29 Composition of Czapek’s Dox Agar: (CDA) Ingredients Gms / Litre 1. Sucrose 30.000 2. Sodium nitrate 2.000 3. Dipotassium phosphate 1.000 4. Magnesium sulphate 0.500 5. Potassium chloride 0.500 6. Ferrous sulphate 0.010 7. Agar 15.000 8. Distilled water 1000.0 lit. The pH of medium was adjusted between 7.10 - 7.50. The medium was sterilized at 15 lbs pressure for 25 minutes and before pouring the medium in the pertiplates 80 mg streptomycin sulphate per litre of medium was added in the medium as an antibacterial agent. E. Counting of Fungal Colonies: After the gentle swirling, the inoculated plates were incubated at room temperature. The numbers of fungal colonies appeared in petriplate on the fifth to seventh day of incubation were counted. Most of the colonies appeared on 4th to 5th day. Some of the slow growing fungi might have been excluded by this method. Using Sony Cyber Shot DSC- WX60 digital camera colored photographs of culture plates were taken on 7th day of incubation. F. Pure Cultures of Fungal Isolates: It was observed that fungal isolates exhibit a keen competition for development in the initial medium and colonies appearing on this medium generally do not grow and develop to their full extent and hence Potato Dextrose Agar was used. The composition of PDA is as follows: Composition of Potato Dextrose Agar: (PDA) Ingredients Gms / Litre 1. Potato infusion 200.00 2. Dextrose 20.00 30 3. Agar 20.00 4. Distilled water 1000.0 lit. Single colonies of different fungi were picked up from CDA petriplate cultures and transferred aseptically on PDA slants or petriplates containing PDA medium. They were numbered properly for further studies. For transfer of fungi from colonies in the plates to PDA slants or plates; flamed sterilized chrome wire or chrome loop was used. At the time of transfer, the test- tube containing the slant or organisms was held near the flame. This helps in avoiding the contamination during transfer. It is essential to note that, to obtain pure cultures, the transfer of colonies must be done strictly under aseptic conditions and to achieve this, the process of transferring the colonies was done swiftly and quickly in the laminar air flow to minimize the contamination. After sufficient growth of isolates in the slants and petriplates the macroscopic observations of colonies such as external features, texture, colony color, color on reverse, growth characteristics, zonations and variations were recorded.