International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 30 Primary screening, Characterization and Seasonal variations of Aspergillusand Penicillium species in the Black cotton soils (Vertisols) of Salur Mandal, Viziyanagaram District, Andhra Pradesh, India Gaddeyya Gandipilli, Shiny Niharika P., Ratna Kumar P.K.1 andBharathi P.Centre of Advanced Study, Department of Botany, Andhra University, Visakhapatnam-530003, AP, INDIA Department of Botany, Centre for Research, Dr. V.S. Krishna Govt. College, Visakhapatnam-530013, AP, INDIAAvailable online at: www.isca.in, www.isca.me Received 27th July 2013, revised 3rd August 2013, accepted 7th October 2013Abstract Fungi are an important component of the soil microbiota typically constituting more of the soil biomass than bacteria, depending on soil depth and nutrient conditions. This study deals with the primary screening, characterization and seasonal variations of mycoflora, isolated from twelve soil samples. The soil samples were collected from agricultural fields of five different locations at Salur Mandal during three seasons such as Kharif, Rabi and summer respectively. Dilution plate technique was used to isolate soil fungi from various soil samples. Fungal isolates were screened on different culture media namely Potato Dextrose Agar, Czapeks Dox Agar, and Sabouraud’s Dextrose Agar supplemented with 1% Streptomycin. Fungal colonies were counted and screened for the occurrence of different fungal species along with Aspergillus and Penicillium presented in soil samples. Identification of the soil isolates were made with help of the relevant literature and standard manuals of soil fungi. Mycoflora including Aspergillus (12 species), Penicillium (6species), Trichoderma (3 species), Cunninghamella (2 species), Curvularia (2 species), Fusarium (2 species), Mucor (2 species) and Rhizopus (1 species) were identified with the help of taxonomic keys. Among the isolates the genera Aspergillus and Penicillium were dominant due to their sporulation ability. The periodicity of occurrence of each species was calculated by using data analysis. Keywords: Mycoflora, Aspergillus, Penicillium, Black cotton soils, Salur. IntroductionMany microbes have their origin in the soil or are closely allied with soil environment. Throughout history these microbes have a substantial impact on humankind. The soil microbes decompose the plant and animal residues 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. Microbes are especially important components of biodiversity. Particularly fungi and bacteria are crucial, as they change and release many nutrients playing important roles in nutrient cycling2,3 and sustenance of vegetation. The efficiency of fungi in decomposition and their potentiality depend upon their abundance and composition. Aspergillus and Penicillium are economically, ecologically, and medically important and large genera. They are important in view of health hazards. In addition, they are used in industrial and food fermentation processes, and they exist commonly in different types of soils, indoor and outdoor air, food and water. Aspergillus and Penicillium are ubiquitous fungi. The species of Aspergillus and Penicillium are among the most abundant and widely distributed microfungi in nature5-8. The decomposition process carried out by these moulds is important in driving natural cycling of chemical elements, particularly in the carbon cycle where they contribute to replacement of the supply of carbon dioxide and other inorganic compounds. Christensen10 reported that species diversity of soil fungi is a reflection of multiple factors and appears to be reduced by disturbances and manipulation activities. Natural or anthropogenic disturbances can alter the species composition or may have negative effect on species diversity of the decomposer fungi11. These changes may directly or indirectly affect the vital functions of the soil such as decomposition and mineralization. During the past four decades, a large number of herbicides have been introduced as pre or post emergent weed killers in many countries of the world. These herbicides could accumulate to toxic levels in the soil and become harmful to microorganisms, plants, wildlife and man12. Black soils, popularly known as black cotton soils, are usually deep to very deep and are dominated by smectitic clays. They are characterized by the presence of either slicken- sides or wedge-shaped peds, 30% clay and cracks that open and close periodically. Recent findings on shrink–swell soils (generally known as black soils) have shown them to be red in colour International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 31 which makes the common name of red and black soils13. Vertisols are a group of heavy-textured soils which occur extensively in the tropics, subtropics and warm temperate zones and are known as Dark Clays, Black Earths, Black Cotton soils, Dark Cracking soils, Grumusols and Regurs in other classification systems14. The major areas of Vertisols are found in Australia (70.5 mha), India (70 mha), Sudan (40 mha), Chad (16.5 mha) and Ethiopia (10 mha); these five countries contain over 80% of the total area of 250 mha of Vertisols in the world14. The major factor contributing to the productivity of Vertisols in semi-arid environments is their high water-holding capacity. The present work was designed to study the fungal diversity and their seasonal variations in the soils of agricultural fields of Salur Mandal. We presented an account on the periodicity of occurrence of Aspergillus and Penicillium species in agricultural soils which are the major decomposers of soil ecosystem and the impact of management practices on species richness and abundance of microfungi occurring in soil during different seasons. The detail information of edaphic factors which influence the fungal diversity in the soil of study area was described. Material and Methods Topography and general features of the study area: Location and site description: Salur or Saluru is a Municipality and Mandal headquarter in Vizianagaram district of Andhra Pradesh state in India. It is bounded by Makkuva, Bobbili, Ramabhadrapuram and Pachipenta mandals of Andhra Pradesh state and Koraput district of Odisha. Salur is located on the River bank of Vegavathi at 18.5333 N 83.2167 E. The climate of the town is generally characterized by high humidity almost all round the year, oppressive summer and seasonal rainfall. The temperature varies between 17 and 40°C and average annual rainfall is 1074 mm. The nature of the soil is generally black cotton soil. Paddy, Maize, Ground nut, Cotton are main Kharif crops while Sugarcane, Maize, Pulses and Tobacco are cultivated in Rabi season. Cultivation of crops: Agricultural practices were carried out on the basis of different monsoons like south west monsoon (June-Sep), north east Monsoon (Oct-Dec), winter (Jan- Feb) and summer (March-May). The nature of the soil is generally black cotton and there are 6 types of major soils: 1. Red Sandy loams 2 .Red Sandy clay loams 3. Red Loamy sandy soils 4. Clay loam 5. Sandy soils 6. Clay soils. The Sources of Irrigation were Canals, Tanks, Open wells, Bore wells (tube wells) and other sources. Paddy, Sugarcane, Ground nut, Mesta , Sesame, Maize, Ragi, Green gram, Black gram and Cotton were the major field crops cultivated in distinct seasons and Mango, Cashew were main horticulture crops (fruits) and Brinjal is main vegetable crop and Areca nut, Oil palm, Coconut were major plantation crops. Soil sampling: Soil samples were collected based on three different seasons viz., Summer (March 2011 June 2011), Kharif (July 2011-October 2011) and Rabi (November2011 – February2012). The dry season samples were merely to standardize the methodology and not subjected to detailed analysis. From each selected hectare, the soil was collected (between 10:30 am and 4:30pm each day) under sterile conditions with the help of 15 cm iron cores from four symmetrically situated locations near the corners of a square as well as from the centre of the square. The soil samples were collected from five different locations viz., Jeegiram, Kurmaraju peta, Sivarampuram and Neliparthi. Soil samples were collected from the depth of approximately 10-15 cm in sterilized polyethylene bags and stored at 4°C in the laboratory until the examination. The collected soils samples along with locations showed in table-1. Table- 1Collection of soil samples from various crops seasonally at four locations of Salur Mandal S.No Sample No Season vegetation location 1 Sample-1 Summer Paddy Jeegiram 2 Sample-2 Summer Maize Kurmaraju peta 3 Sample-3 Summer Ground nut Sivarampuram 4 Sample-4 Summer Cotton Neliparthi 5 Sample-5 Kharif Sugarcane Jeegiram 6 Sample-6 Kharif Maize Kurmaraju peta 7 Sample-7 Kharif Red gram Sivarampuram 8 Sample-8 Kharif Sugarcane Neliparthi 9 Sample-9 Rabi Red gram Jeegiram 10 Sample-10 Rabi Black gram Kurmaraju peta 11 Sample-11 Rabi Sesamum Sivarampuram 12 Sample-12 Rabi Sugarcane Neliparthi International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 32 Analysis of soil samples: The collected soil samples ware dried in aseptically at departmental laboratory for characterization of physico-chemical properties. The physico-chemical parameters of the soil samples were analyzed at Mobile Soil Testing Laboratory (MSTL), Pothinamallayapalem, Visakhapatnam, Department of Agriculture, Andhra Pradesh. The physico-chemical properties of soils were showed in table -2 and 3. Isolation of Soil mycoflora: Dilution plate technique described by Warcup15 was used for the isolation of fungi from various soil samples collected in agricultural crop fields at Salur Mandal. Ten grams of soil samples were suspended in 90 mL of distilled water (in Erlenmeyer glass flask), then mix by using wrist action shaker for one hour at 120 rpm. The flasks were shaken thoroughly in order to get uniform distribution of the soil. The soil suspensions were diluted in 10 fold increment from 10-3 to 10-5. The Volume of 1 mL of soil sample suspension from each serial dilution was pipetted onto different melted, cooled culture media namely Potato Dextrose Agar(PDA) [Potatoes(peeled) 200 g; Dextrose 20 g; Agar 20 g; Distilled water 1L]16 Czapeks Dox Agar(CZA) [Sucrose 30g; NaNO 2 g; KHPO4 1g; MgSO+7HO 0.5 g; KCl 0.5 g; FeSO+7HO 0.01g; Agar 15 g; Distilled water 1L]17 and Sabouraud’s Dextrose Agar (SDA)[Glucose 40g; Peptone 10g; Agar 15g; Distilled water 1L]18 supplemented with 1% Streptomycin (1gram of streptomycin was mixed thoroughly in 100ml of sterilized distilled water). The pH of the culture media was maintained at 5.5 being optimal for the growth and sporulation in a majority of fungi. Each culture media was prepared in a liter of distilled water and autoclaved at 120C at 15 psi for 20 min.1% Streptomycin was used as an antibiotic for the restrain of bacterial growth. Fungi were maintained on half-strength PDA slants in test tubes as stock cultures19Table -2Analyses of soil physical characters (The dry season samples were used to standardize the methodology)Soil Sample Soil type Soil color Soil texture (%) Sand clay Dust 1 Sandy Clay loam Dark brown 17.23 42.19 40.55 2 Red sandy loam red 11.35 37.58 21.11 3 Red loamy sandy Brown Reddish 20.11 57.25 42.21 4 Clay loam Black 12.78 62.45 24.77 5 Sandy Clay loam Dark brown 16.98 40.26 42.25 6 Red sandy loam red 06.85 53.01 40.02 7 Red loamy sandy Brown Reddish 20.51 19.32 62.21 8 Clay loam Black 13.26 65.23 25.06 9 Sandy Clay loam Dark brown 24.68 18.56 56.28 10 Red sandy loam red 08.36 54.81 35.26 11 Red loamy sandy Brown Reddish 29.14 15.69 54.24 12 Clay loam Black 17.58 42.56 39.69 Table-3 Analyses of soil chemical characters (The dry season samples were used to standardize the methodology)Soil sample Soil moisture Soil pH Electrical conductivity ( Dsm-1) Nitrogen (Kg/ac) Organic carbon (%) (Kg/ac)O (Kg/ac) Iron (ppm conc.) Copper (ppm Conc.) Mn (ppm Conc.) Zn (ppm Conc.) 1 18.05 7.4 0.25 131.4 1.54 10.95 37.84 16.79 0.69 5.56 0.52 2 14.21 6.7 0.66 169.5 1.85 13.14 49.28 18.95 0.66 6.01 0.51 3 15.68 7.6 0.69 120.1 1.35 15.12 35.29 15.26 0.52 6.02 0.56 4 24.08 7.7 0.35 135.2 1.62 14.28 45.32 16.23 0.46 6.32 0.46 5 20.52 6.8 0.19 142.5 1.45 15.28 36.28 15.26 0.53 5.86 0.65 6 18.25 7.3 0.61 132.5 1.24 11.35 26.18 14.65 0.64 5.23 0.54 7 21.84 7.1 0.35 143.2 1.24 12.35 45.32 18.23 0.45 6.54 0.53 8 24.36 7.3 0.35 156.6 1.54 10.35 45.36 16.25 0.53 5.86 0.52 9 15.02 6.8 0.19 169.6 1.64 10.56 35.36 15.32 0.66 6.25 0.53 10 13.21 6.9 0.38 132.1 1.84 13.25 38.47 16.25 0.64 5.32 0.45 11 17.54 7.2 0.72 142.6 1.24 14.25 42.35 13.26 0.54 5.24 0.62 12 16.25 7.4 0.64 135.8 1.48 10.22 32.28 15.65 0.65 6.06 0.56 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 33 Incubation of Soil mycoflora: The plates were incubated at room temperature 28±2°C in an inverted position for 5-7 days. Three replicates were maintained for each sample. During the incubation period pure cultures of soil mycoflora were prepared on PDA slants under aseptic condition. Individual colonies of Aspergillus and Penicillium were incubated on potato dextrose agar medium for seven days to measure their diameter. Identification of Soil mycoflora: Identification of the soil isolates were made with help of the relevant literature20,21. Fungal morphology was studied macroscopically by observing colony features (colony diameter, colour texture, and colony colour) and microscopically by staining with lacto phenol cotton blue called as mounting fluid and observed under compound microscope for the observation of the conidia, conidiophores and arrangement of spores. Data analysis: 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 factors. The percentage contribution of each isolate was calculated by using the following formula. Total no. of CFU of an individual species x 100 Total no. of CFU of all species *CFU-Colony Forming Unit. The periodicity of occurrence was calculated for fungi are arbitrarily classified as follows: Common - recorded in 10 - 12 samplings, Frequent - recorded in 7 - 9 samplings, Occasional - recorded in 4 - 6 samplings, Rare - recorded in 1 - 3 samplings Results and DiscussionA total of 30 fungal species were isolated from twelve soil samples. The identification of these isolates resulted in 8 genera of soil fungi including Aspergillus (12 species), Penicillium (6 species), Trichoderma (3 species), Cunninghamella (2 species), Curvularia (2 species), Fusarium (2 species), Mucor (2 species) and Rhizopus (1 species). The soil mycoflora of twelve samples were screened and the associated mycoflora with Aspergillusand Penicillium species were identified. The fungal isolates of Cunninghamella blakesleeana, Cunninghamella elegans, Curvularia clavata, Curvularia lunata, Fusarium oxysporum, Fusarium solani, Mucor hiemalis, Mucor racemosus, Rhizopus stolanifer, Trichoderma virens, Trichoderma viride, and Trichoderma harzianum species were identified along with Aspergillus and Penicillium species by the standard macro and microscopic observations. The Characterization of mycoflora was made with the help of relevant literature and standard manuals. Among the isolates a total of 327,252 and 716 colonies of Aspergillus and a total of 132,135 and 219 colonies of Penicillium were screened seasonally in three different seasons such as Summer, Kharif and Rabi, respectively. Different isolates of Aspergillus species including Aspergillus awamori, A. candidus, A. deflectus , A. flavus , A. fumigatus , A. nidulans, A. niger , A. repens , A. tamari , A. terreus , A. ustus and A. versicolor were identified by using macro and microscopic methods and standard manuals. On the other hand different species of Penicillium species including Penicillium aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum and P. purpurogenum has been isolated (table-4 and 5). The average number of different species of Aspergillus and Penicillium mycoflora was studied to estimate the dominant species in agricultural fields of Salur Mandal. Specises wise percentage of Aspergillus and Penicillum was showed in table 4 and 5.The frequency of all isolates was studied by using data analysis and the genus Aspergillus and Penicillium were dominant in all soil samples. The results showed that the species of Aspergillus niger 8.0 %, 1.06 %, 9.9%) Aspergillus flavus (10.4%, 9.8%, 8.9%) and Aspergillus terreus (8.0%, 9.0%, 8.3were dominant among the isolates of Aspergillus, whereas species of Penicillium chrysogenum (5.0%, 4.8%, 4.4and Penicillium citrinum (4.3%, 3.4%, 7.1were the most dominant among the isolates of Penicillium in particular three seasons viz., Kharif, Rabi, and summer respectively. The periodicity of occurrence is significantly different among the three seasons due to different climatic conditions and agricultural practices. When compared with the summer seasons, the number of colonies per gram of soil shows a decline by about 50% in the dry season. Rainfall is the most important factor for microbial diversity. The low count in the March (dry) collection is also likely to have been a consequence of the annual fires that occur between January and March. The total of 12 strains of Aspergillus and 6 strains of Penicillium were isolated by using Potato Dextrose Agar (PDA), Czapeks Dox Agar(CZA) and Sabouraud’s Dextrose Agar (SDA) media. The nutritional composition was main factor for the growth and sporulation of soil fungi. However the various species of soil fungi were isolated on Potato Dextrose Agar which was favorable for most soil fungi. The various observations of Aspergillus and Penicillium mycoflora were made by using macroscopic and microscopic methods. Macroscopic features including the colony diameter, colony texture and colony colour (upper and lower surfaces), and exudates of mycelium were examined for the identification of soil mycoflora (figure-2 and 3). We also examined the microscopic features of mycelium including conidial heads, conidiophores, conidia and the vesicles for the identification of isolates of Aspergillus and Penicillium mycoflora. The exudates of isolates were examined and appeared in pale, red, yellow and orange colour. The systematic study for characterization of Aspergillus and Penicillium species was done by using the standard manuals and relevant literature of soil fungi (table-6 and 7). International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 34 Table- 4 Mycoflora of agricultural soils and their percentage of occurrence in three distinct seasons Name of the fungal species Summer Kharif Rabi Ave.no.ofCFU per gram of dry soil Occurrence (%) Ave.no.ofCFU per gram of dry soil Occurrence ( %) Ave.no.ofCFU per gram of dry soil Occurrence (%) Aspergillus awamori 22 3.7 26 3.4 42 4.0 Aspergillus candidus 23 3.8 23 3.0 36 3.4 Aspergillus deflectus 40 6.7 45 6.0 56 5.4 Aspergillus flavus 62 10.4 74 9.8 92 8.9 Aspergillus fumigatus 34 5.7 60 8.0 84 8.1 Aspergillus nidulans 18 3.0 22 2.9 44 4.2 Aspergillus niger 48 8.0 80 10.6 102 9.9 Aspergillus repens 10 1.6 10 1.3 20 1.9 Aspergillus tamarii 28 4.7 44 5.8 56 5.4 Aspergillus terreus 48 8.0 68 9.0 86 8.3 Aspergillus ustus 10 1.6 14 1.8 26 2.5 Aspergillus versicolor 34 5.7 59 7.8 72 6.9 Penicillium aurantiogriseum 14 2.3 15 2.0 30 2.9 Penicillium chrysogenum 30 5.0 36 4.8 46 4.4 Penicillium citreonigrum 18 3.0 20 2.6 42 4.0 Penicillium citrinum 26 4.3 26 3.4 43 4.1 Penicillium digitatum 20 3.3 22 2.9 36 3.4 Penicillium purpurogenum 24 4.0 16 2.1 22 2.1 Cunninghamella blakesleeana 5 0.8 7 0.9 4 0.3 Cunninghamella elegans 1 0.1 1 0.1 2 0.1 Curvularia clavata 8 1.3 10 1.3 15 1.4 Curvularia lunata 7 1.1 12 1.6 14 1.3 Fusarium oxysporum 10 1.6 12 1.6 14 1.3 Fusarium solani 8 1.3 10 1.3 12 0.1 Mucor hiemalis 4 0.6 5 0.6 4 0.3 Mucor racemosus 4 0.6 5 0.6 4 0.3 Rhizopus stolanifer 5 0.8 5 0.6 6 0.5 Trichoderma virens 12 2.0 14 1.8 15 1.4 Trichoderma viride 10 1.6 15 2.0 18 1.7 Trichoderma harzianum 10 1.6 12 1.6 14 1.3 Total 593 - 750 - 1030 - CFU-Colony Forming Unit (per gram of dry soil x 10) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 35 Table- 5 Periodicity of occurrence of mycoflora in various samples collected from agricultural soils at Salur Mandal.S. No Name of the Fungus S S S S S S S S S S10 S11 S12Periodicity of occurrence 1 Aspergillus awamori + - + + - - + + - - - - O 2 Aspergillus candidus - + + - - + + - - + - + O 3 Aspergillus deflectus + + + + + - - + + + + - F 4 Aspergillus flavus + + + + + + + + + + + + C 5 Aspergillus fumigatus + - + + + + - - + + + - F 6 Aspergillus nidulans + + + - - - + + - - - + O 7 Aspergillus niger + + + + + + + + + + + + C 8 Aspergillus repens - + + - - + + - + - + - O 9 Aspergillus tamarii + + - - + - + + + + + + F 10 Aspergillus terreus + + + + + + + + + + + + C 11 Aspergillus ustus - - + - + - - - + - - - R 12 Aspergillus versicolor + + - - + - + + + + + + F 13 Penicillium aurantiogriseum - + + + - - + + + - - - O 14 Penicillium chrysogenum + + + + + + + + + + + + C 15 Penicillium citreonigrum - + - + - - + - - - + + O 16 Penicillium citrinum + - + + + - + + - + + - F 17 Penicillium digitatum + + - - - + + + + + + + F 18 Penicillium purpurogenum + - - - + + + + + - - - O 19 Cunninghamella blakesleeana - + + + - + - + - - - - O 20 Cunninghamella elegans - + - + - - + - - - - - R 21 Curvularia clavata - + - + + + - + + - + + F 22 Curvularia lunata + + + - - + + + + - + + F 23 Fusarium oxysporum + + + + - + + - + + + - F 24 Fusarium solani + + - + - + + + + + - F 25 Mucor hiemalis + + - - - + - - - - - - R 26 Mucor racemosus - - + - - + - -- + - - R 27 Rhizopus stolanifer - + - + - + - + + - + - O 28 Trichoderma virens + + + - + - - + + + + - F 29 Trichoderma viride + + - + + + - - + + + + F 30 Trichoderma harzianum + + - - - + + + + + + + F C- Common; F- Frequent; O-Occasional; R-Rare. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 36 Table -6 Macro and microscopic features of Aspergillus species isolated from various agricultural fields at Salur S.No Name of the species Morphological features Microscopic observations Diam (cm) Texture Upper surface Lower surface Conidiophores/ conidia/vesicles 1 A.awamori 5 Radially furrowed, tough Greenish brown to dark chocolate brown Yellow to reddish brown in age Conidial heads globose loosely radiate. Conidiophores coloured in terminal areas. Phialides biseriate. Conidia globose. Vesicles globose, light brown in colour. 2 A. candidus 3 Effuse, globose Persistently white or cream with age. Colorless or pale gray brown Conidial heads white, globose, often splitting in age. Conidiophores colorless, smooth, thick walled. Vesicles globose to subglobose. 3 A. deflectus 4 Compact tough Deep mouse grey, margins pink Dull orange, brown with age Conidial heads broadly columnar, Conidiophores smooth, reddish brown. Vesicles round, flask shaped. 4 A. flavus 6 Velvety Yellow to dark green in age Colourless to pale yellow brown Conidial heads yellow in young, green in age. Phialides uniseriate or biseriate. Conidia globose to subglobose, yellowish green. Exudates inconspicuous. 5 A.fumigatus 6 Velvety to floccose Dull blue-green colourless Conidial heads columnar, compact, often densely crowded. Conidiophores short, smooth, light green. Phialides uniseriate. Conidia globose . 6 A. nidulans 5 Effuse, globose Dark cress green Purplish red to very dark in age Conidial heads slightly large. Conidial heads loosely radiate when young later short columnar. Phialides biseriate. Vesicles hemispherical, brown. Conidia globose, green. Exudates lacking. 7 A. niger 6 Effuse, globose Carbon black or deep brownish black Colourless to pale yellow Conidial heads large and black, at first globose then radiate or splitting in well defined columns in age. Phialides biseriate, brownish .Vesicles globose. Conidia globose, spinulose, black in colour. 8 A. repens 4 Wrinkled Yellow green to grayish green. Yellow- orange to deep maroon Conidial heads abundant radiate, often splitting into columns with diverging chains of conidia. Conidiophores smooth, colorless. 9 A. tamarii 4 Effuse, floccose Brownish green colourless Conidial heads globose to loosely radiate. Conidiophores smooth, colorless. Vesicles globose to sub globose. 10 A. terreus 5 Velvety to floccose Pinkish cinnamon, deep brown shades Dull brown Conidial heads long columnar, compact with uniform diameter. Conidiophores smooth and colorless. Exudates amber coloured. Conidia globose to sub globose, smooth. 11 A. ustus 5 Floccose radially zonate White to grayish or olive-grey yellow Conidial heads radiate, broadly columnar at maturity. Conidiophores sparsely septate,light brown, thin walled. Vesicles hemispherical to sub globose. Exudates colourless. 12 A.versicolor 3 Floccose radially zonate Yellow to pea green. Pale yellow Conidial heads roughly hemi-spherical, radiate Conidiophores smooth and colorless. Vesicles semi spherical to elliptical, phialides in two series. Conidia globose. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 37 Table -7Macro and microscopic features of Aspergillus species isolated from various agricultural fields at SalurS.No Name of the species Morphological features Microscopic observations Diam. (mm) Texture Upper surface Lower surface Conidiophores/ conidia/vesicles 1 P.aurantiogriseum 35 Radially sulcate, smooth to granular Mycelium white to dull green Pale light to brilliant orange Conidiation moderate to heavy, dull green to glaucous blue –green. Exudates typically conspicuous near the margins and brown in colour.conidia subspheroidal to ellipsoidal. Phialides 5-8 per metula. 2 P. chrysogenum 45 Radially sulcate to plicate/floccose Margins white, centers yellowish white. Brilliant yellow or yellow brown Conidiation moderate to heavy, dull green to glaucous blue –green or yellow –green. Exudates pale to bright yellow. Conidia subspheroidal to ellipsoidal. Phialides 4-7 per metula. 3 P. citreonigrum 25 Radially sulcate Centrally convolute White to bright yellow Brilliant yellow or yellow brown Conidiation sparse to moderate, greenish grey or pale olivaceous grey. Exudates rarely present. Phialides5-8.conidia spheroidal to subspheroidal 4 P. citrinum 30 Radially sulcate centrally floccose. Mycelium white in peripheral areas Yellow or yellow brown Conidiation moderate, turquoise grey to glaucous sky blue. Exudates pale yellow to radish brown.Phialides 8-12,conidia spheroidal to subspheroidal 5 P. digitatum 40 velutinous to deeply floccose Mycelium white to olive green Pale to brownish Conidiation moderate to heavy, grayish green to olive. Exudates absent. Phialides 3-5, conidia borne as cylinders, later ellipsoidal to cylindroidal, smooth,borne in distorted chains. 6 P.purpurogenum 25 velutinous to floccose Mycelium white to bright yellow Very dark red or purple Conidiation moderate to heavy, grayish green to dark green. Exudates orange to red coloured. Phialides 5-8 per metula. Conidia ellipsoidal to subspheroidal. The saprobic fungi represent the largest proportion of fungal species in soil and they perform a crucial role in the decomposition of plant structural polymers, such as cellulose, hemicelluloses and lignin, thus contributing to the maintenance of global carbon cycle. The distribution of these organisms is influenced by the abundance and nature of the organic content of the soil, as well as by other soil texture22,23. Identification is complicated by the fact that fungal life cycles in the soil and in the laboratory can be quite different. Fungi are so nutritionally diverse that there is no one medium that can isolate all of them. The technique used in the present study is a standard one15. Through our observations, the media used in our study appeared to favor of most soil fungi. Aspergillus and Penicillium usually appeared abundantly in collections because of their prolific sporulating capacity. A distinct pattern of fungal community structure was observed in all the samples during the study period. The percentage composition and rank abundances of different fungal species fluctuated. The majority was from the genus Aspergillus and the next two in order of dominance were Penicillium and Trichoderma. Earlier reports have indicated that, these genera appeared abundantly in soils 24. This may be due to the faster growth rate of these fungi in addition to their better intrinsic prolific sporulating capacity to utilize the substrate. Considering the dominant species, it is clear that fungal succession in the plantation site greatly differed. The species composition in soil showed marked differences with a change in habitat and surface vegetation. Their occurrence might be due to the ability of these groups of fungi to survive in adverse conditions, as well as their ability to the environment. They have frequently been isolated in various soils in India25-28. Modern pesticides are almost all completely new synthetic chemicals, previously unknown in nature. The herbicide causes adverse effect on microbial populations and their activities29. Metolachlor is often used in combination with other broadleaved herbicides like atrazine, metobromuron and propazine was highly influences the microbial diversity in the present study area, the black soils. The soil pH, organic content and water are the main factors affecting the fungal population and diversity30-33. Organic carbon largely controls microbial International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 38 growth and it is a key factor governing Nitrogen, Phosphorus, and Sulphur cycles. The microbial activity and species compositions are generally influenced by the physical characteristics and soil chemical properties, climate and vegetation34. The seasonal variations of the fungal diversity of various soil samples showed in figure-1. Figure -1 Periodicity of occurrence of mycoflora in various soil samples collected from agricultural soils at Salur Mandal , Vizianagaram District, Andhra Pradesh Aspergillus candidus Aspergillus deflectus Aspergillus flavus Aspergillus fumigatus Aspergillus nidulans Aspergillus niger International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 39 Aspergillus niger var.awamori Aspergillus repens Aspergillus tamarii Aspergillus terreus Aspergillus ustus Aspergillus versicolorFigure -2 Macro and microscopic observations of Aspergillus species isolated from soil samples Penicillium aurantiogriseum Penicillium chrysogenum Penicillium citreonigrum Penicillium citrinum Penicillium digitatum Penicillium purpurogenumFigure -3 Macro and microscopic observations of Penicillium species isolated from soil samples International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(12), 30-41, December (2013) Int. Res. J. Biological Sci. International Science Congress Association 40 Conclusion Fungi are an important component of the soil micro biota typically constituting more of the soil biomass than bacteria, depending on soil depth and nutrient conditions. The saprobic fungi represent the largest proportion of fungal species in soil and they perform a crucial role in the decomposition of plant structural polymers, such as cellulose, hemicelluloses and lignin, thus contributing to the maintenance of global carbon cycle. The majority of soil fungi are represented by rapidly growing filamentous fungi of Aspergillus and Penicilliumgenus. Aspergillus and Penicillium are economically, ecologically, and medically important and large genera. They are among the most successful groups of moulds with important roles in natural ecosystems and the human economy. The relationship between biodiversity of soil fungi and ecosystem function is an issue of paramount importance, particularly in the face of global climate change and human alteration of ecosystem processes. The species of Aspergillus and Penicillium are among the most abundant and widely distributed microfungi in nature. The periodicity of occurrence of different fungal species fluctuated due to ecological and biological factors of the soil. The microbial activity and species compositions are generally influenced by the physical characteristics and soil chemical properties. Physio chemical analysis showed that the pH range of soil conditions ranging from 6.2 to 7.8 and other factors like organic content, water, the soil textures, and vegetation type can influence the fungal population and diversity of microbes in soil. It has been observed that a change in the habitat can affect species composition and diversity. The above ground vegetation in these agricultural soils is essential, to maintain a productive environment to enhance microbial growth and activity. Pesticides are used in controlling insect pests, diseases and weeds in agriculture. It has been established that pesticides could become a nuisance if they are misused. Some of the negative effects of pesticide misuse include low crop yield, destruction of soil micro-fauna and flora, and undesirable residue accumulation in food crops. The present study should enhance the sufficient knowledge to the formers for the conservation of soil properties, management of soil microbial diversity and the development of sustainable agro system. Further investigations on fertilizer- sensitivity of specific fungal communities will clarify the effect of long term consequences of fertilization on fungal diversity and their ecological significance. AcknowledgmentsThe authors were grateful to Head and Faculty members, Department of Botany, Centre of Advanced Studies, Andhra University for their the constant encouragement and support. The authors were thankful to University Grants Commission, New Delhi, India for the finance support under UGC-SAP-CAS-I project in the Department of Botany, Centre of Advanced Studies, Andhra University, Visakhapatnam. 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