Int J Pharm Bio Sci 2017 Apr ; 8(2): (B) International Journal of Pharma and Bio Sciences

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1 Original Research Article Microbiology International Journal of Pharma and Bio Sciences ISSN IVERSITY AN ISTRIBUTION O ERMATOPHYTES AN OTHER KERATINOPHILIC UNGI IN THE SOIL O GHARANA WETLAN, INIA - AN IMPORTANT STOP OVER SITE OR MIGRATORY BIRS. SHIALI SHARMA* AN GEETA SUMBALI University of Jammu, epartment of Botany, B.R. Ambedkar Road, Jammu ABSTRACT The present research work was undertaken to study the diversity of keratinophilic fungi from the unexplored soil of Gharana wetland (J&K), which is situated about 15 km east of the Indo-Pakistan International border in R.S Pura tehsil and is an important wintering and stopover site for migratory birds. The work was done with a view to evaluate the relationship between the keratinophilic fungi and the keratinous material present in the soil as this group of fungi can pose high risk of human and animal mycoses and the migratory birds may even be responsible for their international dispersal. A total of 52 keratinophilic fungal species were recovered which included both dermatophytes and nondermatophytes. They belonged to 21 genera (Acremonium, Alternaria, Aspergillus, Chrysosporium, Cladosporium, Cunninghamella, Curvularia, usarium, Histoplasma, Humicola, Lictheimia, Microsporum, Mucor, Paecilomyces, Purpureocillium, Penicillium, Rhizoctonia, Rhizopus, Sarocladium, Sepedonium and Syncephalastrum) and were recovered by keratin bait technique from the soil of Gharana wetland. Out of these, Aspergillus was represented by maximum of 9 species, which accounts for 17.3 of the total recovered mycodiversity followed in decreasing order by 7 species of Chrysosporium, which contributed of the total mycodiversity.the dermatophytes were the highest producers of keratinase, whereas among the non- dermatophytes, Chrysosporium species showed the maximum activity. In view of these observations, it can be safely concluded that most of the keratinophiles recovered from this wetland have the potential of causing mycosis. KEY WORS: Keratinophilic fungi, keratin, Gharana wetland, soil. SHIALI SHARMA * University of Jammu, epartment of Botany, B.R. Ambedkar Road, Jammu *Corresponding Author Received on: Revised and Accepted on: OI: B-956

2 INTROUCTION Keratinophilic fungi constitute an important group of microorganisms, which play a significant role in natural degradation of keratinous substrates that are continuously being added to the soil in the form of feathers, hair, horns, claws, nails, etc. Apart from the presence of keratin rich substrates, occurrence and survival of keratinophilic fungi in the soil is also influenced by a number of biotic and abiotic factors 1, 2. Among the biotic factors, presence of microorganisms, which include bacteria, actinomycetes and other fungal species may exert antagonistic effects on the keratinophilic fungi. Other factors which influence their frequency of occurrence in the soil include moisture, ph, available nutrients, soil texture, temperature and aeration of surface soil layers 3.Some of the keratinophilic fungi are well known dermatophytes causing superficial cutaneous infections 4, which are contagious and reported throughout the world 5,6. Based on their occurrence in natural habitats, keratinophilic fungi are divided into three categories: anthropophilic, when human beings are the natural hosts; zoophilic, when a variety of animals act as natural hosts; geophilic, when the soil is the natural habitat. Several researchers have studied prevalence of geophilic keratinophiles 7-9 and it is anticipated that soils contaminated with keratinaceous debris have a huge reservoir of propagules of keratinophilic fungi, which may cause infections in human beings and animals 10. rom India, several investigators have reported occurrence and distribution of dermatophytes and keratinophilic fungi from varied soil habitats including water sediments 11, glacier banks 12, lake side soils 13, bird sanctuary 14, poultry farm soils 15 and feather dumping soils 16. However, so far, no work has been conducted to study the diversity and distribution of dermatophytes and other keratinophilic fungi in the soil of wetlands, which are frequented by flocks of migratory birds during the winter months. Accordingly, the present investigation was undertaken to assess the keratinophilic fungi from the soil of Gharana wetland (reserve), which is situated in Jammu, a subtropical province of Jammu and Kashmir state(india). This wetland is an important wintering and stopover site for migratory birds like bar headed geese from Russia, grey heron from America, little grebe from Australia, common teal from West Europe and purple swamphen from New Zealand. These birds find the wetland soil as the best feeding and breeding place to which keratin matter in the form of feathers and claws get added every year. This article reports on the diversity and distribution of keratinophilic fungi and related dermatophytes in the Gharana wetland soil of Jammu province, India. This would help us to know about the fungal species whose spores may get transmitted to distant places through the migratory birds while taking long flights. In addition, it will also throw light on the risk of human dermatophytosis in this region, which is surrounded by paddy fields that are tended by farmers with bare feet and hands. MATERIALS AN METHOS A total of 160 soil samples were collected from 8 different sites (north, east, west, south, north-west, south-east, north-east and south-west) of Gharana wetland, Jammu (India). Sampling was done by scooping upto a depth of 2-5 cms with the help of a sterile spatula and collecting the soil in pre-sterilised polythene bags. Each bag was tied properly and labelled indicating the site of collection and date. The samples were brought to the laboratory and processed for isolation of keratinophilic fungi. Sterile petri dishes were half- filled with soil samples, moistened with ml of sterilised distilled water and baited with presterilised keratinic baits as per Vanbreuseghem s baiting technique 17. These petridishes were then incubated at 28±2 o C for a period of 3-4 weeks. The keratin baits were regularly examined for any mycelial growth. The baits showing growth were carefully and aseptically picked up and inoculated on Sabouraud dextrose agar (SA) medium supplemented with chloramphenicol (0.05 mg/ml) and cycloheximide (0.5 mg/l) to check the bacterial and saprophytic fungal growth respectively. The keratinophilic fungal isolates were identified on the basis of their cultural and morphological details by following different standard keys Identity of some of the fungal species was also confirmed from National ungal Culture Collection of India (NCCI), Agharkar Research Institute, Pune (India). istribution and frequency percentage istribution() and frequency() were calculated as given below: istribution () = No. of isolates of a fungus/ Total no. of isolates X 100. Percentage frequency () =No. of positive samples/ Total no. of samples X 100. Comparison of diversity indices of the keratinophilic species recovered from different sites. To compare the diversity of recovered fungal species, following indices were used: (i) Species richness (S) as the number of species recorded at the sampled area. (ii) Shannon Weaver index (H ). This index is used to quantify the uncertainty associated with the prediction that any two organisms sampled from a site belong to same species 30. It is calculated as given below: Where p i is the relative importance value of species i. B-957

3 (iii) Simpson s dominance index (Cd). The Simpson dominance index is used to measure the degree of concentration when individuals are classified into types 31. Its measure equals the probability that two entities taken at random from the site of interest represent the same type. Its value ranges from 0 to1, with values near 0 corresponding to low concentrated and more homogeneous sites while values near 1 corresponding to highly concentrated and heterogeneous sites. where, p i is the relative importance value of species i. Shannon Wiener s diversity index (H ) is sensitive to richness of rare species, whereas Simpson s dominance index (Cd) is heavily weighted towards the most abundant species and less sensitive to species richness. Both these values can be useful tool in assessing the impact of disturbance on richness and dominance of species. (iv) Margalefʼs index. This index is used as a simple measure of species richness 32. Margalef s index ( Mg ) = (S 1) / In N S = total number of species N = total number of individuals in the sample In = natural logarithm (v) Mehninickʼs index is also used as a simple measure of species richness 33. Mehninickʼs index ( Mn) = s/ N where s = the number of different species N = the total number of individual organisms Estimation of keratinase activity Preparation of eather meal powder It was prepared by following Agrahari and Wadhwa 34.In this method, feathers of birds were washed, defatted and then dried in a hot air oven. Thereafter, dried feathers were pulverised and the powder so formed was used as a feather meal for determination of keratinase activity. Inoculum preparation and crude enzyme production Each Erlenmeyer flask of 250ml capacity containing 50 ml of sterilised Sabouraud dextrose broth supplemented with 50 mg feather meal powder as keratin source was inoculated with fungal discs (0.5cm) obtained with a sterile circular cutter from periphery of actively growing seven days old cultures. lasks containing medium with a disc of agar without the fungus served as control. Three test flasks and one control set were maintained for each isolate and were incubated at 28+2 o C for 4 days on shaker and then for 4 days in static condition. The broth was then centrifuged at 10,000 rpm for 10 min. and the supernatant so formed was used as a crude enzyme. Preparation of keratin solution Keratinolytic activity was measured with soluble keratin (0.5, w/v) as substrate. Soluble keratin was prepared from white chicken feathers by the method 35. Native chicken feathers (10 g) in 500 ml of dimethyl sulfoxide were heated in a hot air oven at 100 C for 2 h. Soluble keratin was then precipitated by addition of cold acetone (1 L) at 70 C for 2 h, followed by centrifugation at 10,000 g for 10 min. The resulting precipitate was washed twice with distilled water and dried at 60 C in a hot air oven for 20 minutes. One gram of quantified precipitate was dissolved in 20 ml of 0.05M NaOH. The ph was adjusted to 7.0 with 0.1M Hydrochloric acid and the solution was diluted to 200 ml with 0.05 mol/l Phosphate buffer (ph 7.0). Keratinase assay or assessment of keratinolytic activity, 1.0 ml of crude enzyme properly diluted in Phosphate buffer (0.05 M of ph 7.0) was incubated with 1 ml keratin solution at 50 C in a water bath for 10 min, and the reaction was stopped by adding 2.0 ml 0.4M trichloroacetic acid (TCA). After centrifugation at 1450 g for 30 min, the absorbance of supernatant was determined at 280 nm (Shimadzu, 1800 UV vis spectrophotometer) against a control. The control was prepared by incubating the enzyme solution with 2.0 ml TCA without the addition of keratin solution. The keratinase activity was expressed as one unit of the enzyme corresponding to an increase in the absorbance value 0.1 (1 KU=0.100 corrected absorbance), 1KU=keratinase unit. RESULTS AN ISCUSSION This is the first study of keratinophilic mycodiversity from the soils of Gharana wetland (J&K), that is visited every year by flocks of migratory birds from different countries. In addition, this wetland is surrounded by paddy fields where the farmers work with bare feet and hands. uring the present investigation, all the soil samples collected from 8 different sites of the wetland were found to be positive for keratinophilic species, thereby indicating that it is a conducive habitat for their growth and dispersal. uring the period of study, 4 soil samples collected from east and west side of Gharana wetland (J&K) were detected positive for two dermatophytic fungal species belonging to the genus Microsporum(M. gypseum and M. fulvum), whereas 156 soil samples were positive for 50 non-dermatophytic fungal species belonging to 20 genera (Acremonium, Alternaria, Aspergillus, Chrysosporium, Cladosporium, Cunninghamella, Curvularia, usarium, Histoplasma, Humicola, Lictheimia, Mucor, Paecilomyces, B-958

4 Purpureocillium, Penicillium, Rhizoctonia, Rhizopus, Sarocladium, Sepedonium and Syncephalastrum). Persual of data presented in table 1 shows that maximum number of keratinophilic fungal species (18) were recovered from west side of the wetland, followed in decreasing order by south side (17 species),north and east side (16 species), whereas least number of species (9) were recovered from north west. The keratinophilic fungal species of common occurrence included Chrysosporium indicum, C. tropicum, Cunninghamella echinulata, Aspergillus terreus, A. fumigatus, A. ochraceus, A. sydowii, usarium incarnatum, Histoplasma capsulatum and Syncephalastrum racemosum as they were detected from soil samples of atleast 4-6 sites. Out of the recovered fungal species, Aspergillus was represented by maximum number (9) of species viz., A. terreus, A. ochraceus, A. niger, A. fumigatus, A. parasiticus, A. sulphureus, A. sydowii, A. candidus and A. versicolor, which accounted for 17.3 of the recovered keratinophilic fungal diversity(igure 1). This was followed in decreasing order by Chrysosporium species viz., C. indicum, C. tropicum, C. merdarium, C. pannicola, C. keratinophilum, C. queenslandicum and C. anamorph of Arthroderma curreyi, which accounted for of the total recovered diversity. Next to Aspergillus and Chrysosporium species were those of Penicillium (P. oxalicum, P. brevicompactum, P. purpurogenum, P. expansum and P. italicum) which contributed 9.61 of keratinophilic mycodiversity, whereas Curvularia (C. lunata, C. pallescens, C. brachyspora and C. tsudae), Rhizopus (R. arrhizus, R. nodosus, R. microsporus and R. oryzae) and usarium (. incarnatum,. verticilloides,. solani and. oxysporum) each represented 7.69 of the recovered diversity. Cladosporium (C. cladosporoides, C. sphaerospermum and C. oxysporum) contributed 5.77 of total diversity, whereas Sarocladium (S. kiliense and S. strictum, ) and Microsporum (M. fulvum and M. gypseum) each accounted for 3.84 of recovered diversity. Least contribution to the fungal diversity was made by Histoplasma (H. capsulatum),cunninghamella (C. echinulata), Syncephalastrum (S. racemosum), Mucor (M. hiemalis), Lictheimia (L. corymbifera), Rhizoctonia (R. solani), Sepedonium (S. maheshwarianum), Purpureocillium (P. lilacinum), Humicola (H. grisea), Acremonium (A. fusidioides), Paecilomyces (P. divaricatus) and Alternaria (A. alternata) each accounting for 1.92.of the total recovered diversity. Though Microsporum gypseum and M. fulvum contributed only 3.84 of the recovered diversity but they showed highest keratinase activity of and KU respectively. Similar results were reported earlier by other workers who while studying in vitro keratinolytic activity of some dermatophytes found M. gypseum to show maximum ability to degrade human hair 36. Geophilic dermatophytes are distributed globally and are responsible agents for ringworm of the scalp and glabrous skin of both human and animals 37.An isolate of M. gypseum recovered from dust samples of hospitals is reported to cause tinea corporis and tinea capitis in humans 38, whereas M. fulvum has been found responsible for kerion and tinea circinata lesions in two females 39. Recovery as well as disease causing ability of these two well known dermatophytic species has been reported by many researchers from different parts of the world In addition to dermatophytes, 50 nondermatophytic keratinophilic fungal species were also recovered from Gharana wetland. Among these, maximum representation is of Aspergillus species (17.3), followed in decreasing order by Chrysosporium species (13.4), which have been reported earlier from soils of poultry farm 15, Khardung and Khardung La 43 from J&K state. Among the 9 species of Aspergillus that were recovered, A. fumigatus, A. sydowii and A. terreus are reported to be responsible for white superficial onychomycosis 44.Similarly, from the recovered Chrysosporium species, C. indicum was the most frequent species, which has been reported earlier also as the most dominant species of the Indian soils due to 45, 14, 5, its adaptation to the warm Indian conditions 8 Chrysosporium species are widespread as geophilic fungi and their presence in soil samples is of medical significance 46. or example, C. keratinophilum detected during the present investigation with keratinase activity of 40.90KU has been isolated earlier as the causal agent of human onychomycosis 47.Similarly, C. queenslandicum with an activity of KU has been reported as the causal agent of disseminated infection in a garter snake 48. Chrysospoium indicum, C. keratinophilum, C. queenslandicum and C. tropicum were the most frequently occurring species of Gharana wetland. Similar report of Chrysosporium species have been recorded from muddy soils of Egypt 49. In addition to Aspergillus and Chrysosporium, few species of usarium viz.,. verticillioides,. incarnatum,. oxysporum, and. solani, producing 31.24, 31.56,30.68 and KU respectively were also recovered. All these species have been recovered earlier from the soils of cold arid regions, parks and gardens 42,50, 51 and are considered as potential pathogens causing mycotic infections Similarly, Purpureocillium lilacinum recovered during the present investigation and producing 33.09KU has been reported as the causal agent of invasive and cutaneous mycosis in immunocompromised patients On the other hand, Histoplasma capsulatum, the causal agent of histoplasmosis is known to be associated commonly with bird droppings. This species was detected to produced KU and is earlier reported from the botanical garden of Jammu University 51. ew members belonging to the class Zygomycetes were also detected to be keratinophilic and these included Syncephalastrum racemosum, Cunninghamella echinulata, Lictheimia corymbifera, Mucor hiemalis and Rhizopus oryzae producing variable amounts of the enzyme keratinase(table 1). They are mainly responsible for causing zygomycosis, which is present in six clinical forms, depending on the site involved (rhinocerebral, pulmonary, cutaneous, gastrointestinal, isolated renal and disseminated forms). A case report of onychomycosis due to pathogenic nature of Syncephalastrum racemosum was detected recently in 2015 in an immunocompetent patient 56. Incidently, Syncephalastrum racemosum was found in almost all the soil samples of Gharana wetland. It is also reported earlier from different soils of Madhya Pradesh, India 57. Keratinophilic nature of Rhizopus arrhizus (16.58 KU) and R. nodosus (13.12 KU) have not been reported so far and therefore are new additions to this group of B-959

5 fungi. Humicola grisea, which was detected from the North of the wetland produced keratinase units. This fungus has been reported earlier also as keratinophilic as it colonizes keratinous substrates 58. Similarly, Rhizoctonia solani, which was recovered during the present investigation as a keratinophile producing keratinase units is usually recognized as a necrotroph infecting a great diversity of host plants. However, there is a report of rare case of human mycosis caused by Rhizoctonia solani in a diabetic and hypertensive farmer 59. Other keratinophilic fungi isolated from the wetland included Alternaria, Aspergillus, Curvularia, Cladosporium, Penicillium, Sarocladium, Sepedonium and Sagenomella. They were not high producers of keratinase but have been reported by other researchers also as keratinophilic and opportunistic causing mycotic infections 15,16,41,60. Similarly, dematiceous keratinophilic moulds, such as, Alternaria and Curvularia are known to be potential etiological agents of various mycoses with clinical forms ranging from localized superficial infections of the stratum corneum (Tinea nigra) to subcutaneous cysts (phaeomycotic cyst) to invasion of the brain 61. Most of these saprophytic isolates represent transient contaminants of soil, which have the potential of causing mycosis 62, 63. igure 1 iversity of keratinophilic fungal species recovered from Gharana wetland soil. Table 1 istribution, frequency and keratinase activity of fungal species recovered from wetland soil of Gharana wetland (J&K), India. istribution() and frequency () of fungi recovered ungal species Recovered Site1 ( North) Site 2 (East) Site3 (West) Site 4 (South) Site5 (NorthWest) Site 6 (South East) Site7 (North East) Site 8 (SouthWest) Keratinase (KU) Microsporum gypseum Microsporum fulvum Chrysosporium keratinophilum Chrysosporium queenslandicum Chrysosporium pannicola Chrysosporium merdarium Chrysosporium anamorph of Arthroderma curreyi Chrysosporium indicum Chrysosporium. tropicum Purpureocillium lilacinum Histoplasma capsulatum usarium solani usarium incarnatum usarium verticilloides usarium oxysporum Humicola grisea B-960

6 Lictheimia corymbifera Mucor hiemalis Acremonium fusidioides Aspergillus terreus Aspergillu sydowii Aspergillus fumigatus Aspergillus parasiticus Aspergillus ochraceus Aspergillus versicolor Aspergillus sulphureus Aspergillus candidus Aspergillus niger Cladosporium cladosporoides Cladosporium sphaerospermum Cladosporium oxysporum Curvularia lunata Curvularia pallescens Curvularia brachyspora Curvularia tsudae Cunninghamella echinulata Paecilomyces divaricatus Penicillium oxalicum Penicillium purpurogenum Penicillium brevicompactum Penicillium expansum Penicillium italicum Rhizopus arrhizus Alternaria alternata Rhizopus microsporus Rhizopus nodosus Rhizopus oryzae Sarocladium. strictum Sarocladium kiliense Rhizoctonia solani Sepedonium maheshwarianum Syncephalastrum racemosum - iversity indices were calculated for the keratinophilic fungal species recovered from the eight different sites of Gharana wetland. Since the 8 sampling sites were from the same location that is, Gharana, therefore, no significant differences were observed in their diversity indices.the highest species richness (S) and Shannon s diversity (H') was recorded for site 3(West) (S = 18 species and H' = 4.216), followed by site 4(South) (S = 17 species and H' = 2.778), site 1( North) (S = 16 species and H' = 2.655), site 2 (East) (S = 16 species and H' = 2.654),site 7 (Northeast) (S=14species and H =2.576), site 8(Southwest) (S=14 and H =2.549) followed by site 6(Southeast) (S=14 and H =2.514). Lowest values of S=9 and H =1.355 were recorded for site 5(Northwest).The value of Simpson s diversity index comes out to be for site 8(Southwest) and for site7 (Northeast), which are near zero, thereby showing that no particular fungal species is dominant in the studied samples and there is more homogeneity of the fungal species in the soil of these two sites. In case of other six sites, the value varies from , thus approaching near to 1,which indicates more heterogenous nature of the keratinophilic fungal species. The highest Margalefʼs index values was recorded for site 3(West) ( Mg= 3.67) indicating more species diversity than other sites. Mehninickʼs index was also calculated and the highest value was again obtained for site 3(West) ( Mg= 2.78) in comparison to other sites, which supports the results obtained by Margalefʼs index. Table 2 iversity indices calculated for the keratinophilic fungi recovered from soil of Gharana wetland (J&K). iversity indices Site (North) Site 2 (East) Site 3 (West) Site 4 (South) Site 5 (NW) Site6 (S E ) Site 7 (NE) Site 8 (SW) Species richness Shannon s index Simpson's-dominance index Margalef's index Mechninick's index CONCLUSION rom the present investigation, it is concluded that soil of Gharana wetland is an ideal environment for the growth and prevalence of diverse keratinophilic fungi and related dermatophytes. This could be attributed to the high organic debris and keratinous substrates like feathers, hoves, nails etc. of the migratory and other B-961

7 local birds that are continuously added to the soil. However, isolation of keratinophilic fungi was not uniform as it depends on the content of organic matter present in the soil. But never the less, identifying keratinophilic fungi from an environment where people are exposed to them is of major health concern. Infact, these fungi, which are human pathogens, could be considered as bioindicators of environmental pollution with animal faeces, feathers and other keratinous substrate as they can pose high risk of human and animal mycoses. ACKNOWLEGEMENT The first author is grateful to University Grants Commission (UGC), New elhi for the financial assistance in the form of Basic Scientific Research ellowship (BSR), which facilitated the study. CONLICT O INTEREST Conflict of interest declared none. REERENCES 1. Otsenasek M. Ecology of the dermatophytes. Mycopath : Kaul S, Sumbali G. Impact of some ecological factors on the occurrence of poultry soil inhabiting keratinophiles. Mycopath : Bohme H, Ziegler H. The distribution of geophilic dermatophytes and other keratinophilic fungi in relation to the ph of the soil. Mycopath Mycol Appl : Marcella R, Mercantini R. Keratinophilic fungi isol;ated from soils of the Abruzzo National Park Italy. Mycopath : Al Musallam AA, Al-Zarban SHS, Al-Sane NA, Ahmed TM. A report on the predominant occurrence of a dermatophyte species in cultivated soil from Kuwait.Mycopath : eshmukh SK, Mandeel QA, Verekar SA. Keratinophilic fungi from selected soils of Bahrain. Mycopath : eshmukh SK. Isolation of dermatophytes and other keratinophilic fungi from the vicinity of salt pan soils of Mumbai (India). Mycopath : eshmukh SK, Verekar SA. Keratinophilic fungi from the vicinity of meteorite crater soils of Lonar (India). Mycopath : Sona J, Roman L, Jana M, Jan N, Juraj M, Kamila M. Keratinophilic ungi isolated from soils of long-term fold-grazed, degraded pastures in national parks of Slovakia. Mycopath : Rizwana H, Abdulaziz A, Hazzani AL, Siddiqui I. Prevalence of dermatophytes and other keratinophilic fungi from soils of public parks and playgrounds of Riyadh, Saudi Arabia. J Anim Plant Sci : Katiyar S, Kushwaha RKS. Human hair colonizing fungi in water sediments of India, Mycopath, : eshmukh SK. Incidence of dermatophytes and other keratinophilic fungi in the glacier bank soils of Kashmir valley (India). Mycologist : Ghosh GR, Bhatt S. Keratinophilic fungi from Chilka Lakeside soil Orissa (India). Ind. J. Microbiol : eshmukh SK. Isolation of dermatophytes and other keratinophilic fungi from Karnala bird sanctuary, Maharashtra (India). J. Basic Appl. Mycol : Kaul S, Sumbali G. Keratinophilic fungi from poultry farm soil of Jammu, India. Mycologist : Anbu P, Hilda A, Gopinath SC. Keratinophilic fungi of poultry farm and feather dumpling soil in Tamil Nadu, India. Mycopath : Vanbreuseghem R. Technique biologique pour 1 isolement des dermatophytes du sol. Annales de la Societe belge de medecine tropicale : Van Oorschot CAN. A revision of Chrysosporium and allied genera. Studies in mycology : Carmichael JW. Chrysosporium and some other aleuriosporic Hyphomycetes. Can J Bot : Barron GL. The genera of Hyphomycetes from soil. Robert E. Kriege Publishing Company, Huntington, New York p Booth C. The genus usarium. Commonwealth Mycological Institute Kew, Surrey, England p Brown AHS, Smith G, The genus Paecilomyces Bainier and its perfect stage Byssochlamys Westling. Trans Br Mycol Soc : omsch KH, Gams W, Anderson, The Compendium of soil fungi. Academic Press, London, p Ellis MB. ematiaceous Hyphomycetes. Commonwealth Mycological Institute Kew, Surrey. England p Ellis MB. More ematiaceous Hyphomycetes. Commonwealth Mycological Institute Kew, Surrey, England p Gams W. Cephalosporium like Hyphomycetes. Hyphomycetes course, Sugadaira, Japan.1997 p Onions AHS, Allsopp, Eggins HOW. Smith s Introduction to Industrial Mycology. Edward Arnold, London p Pitt JI. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London Rapper EB, ennel I. The genus Aspergillus. The Williams and Wilkins Company, Baltimore, USA, pp , (1965). 30. Shanon CE, Weaver W. The mathematical theory of communication. Universe of Illinois press, Urbana Simpson EM. Measurement of diversity. Nature : 688. B-962

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