Comparative analysis of air , soil and water mycoflora of Samahni Area , District Bhimber Azad Kashmir , Pakistan

Fungi are potential sources of different diseases for humans, animals and plants. The present research aimed to collect, isolate and identify different fungi with their prevalence ratio in different habitats of Samahni area, Azad Kashmir (Pakistan). Mycoflora prevails in diverse habitats viz: air, water and soil. From sampling sites, air-borne fungal spores were trapped and grown on different culture media viz: potato dextrose agar (PDA) and water agar (WA). The soil and aqua mycoflora was analyzed using PDA and/or WA media using different techniques. A total of 35 different fungal species were investigated to belong to 18 different genera. The density of identified fungal species in each sample was as follows: air mycoflora (AMF), 20 species; soil mycoflora (SMF), 32 species; water mycoflora (WMF), 11 species, respectively. This differential occurrence could be due to variability in geographical location, fungal growth substrates in different prevailing habitats. A considerable significant difference was seen among the fungal colonies of AMF (31.7%), SMF (50.7%) and WMF (17.4%). It was noted that soil mycoflora was more frequently depicted because land provides best environment and nourishment for growth and reproduction of fungi. It was also observed that some fungi were common in water and soil substrates which indicate that some species of soil fungi do use water as mode of distribution and dispersal. The most dominant fungal species were Cladosporium cladosporioides (7.1%) in air, Fusarium oxysporum (4.9%) in soil and Fusarium oxysporum (11.3%) in water samples. The prevalence and their subsequence impacts in forest ecosystem of Samahni are discussed with special reference of their importance to human life.


INTRODUCTION
Fungi are multicellular, achlorophyllous, heterotrophic, eukaryotic and spore bearing organisms surrounded by a well defined cell wall made up of chitin, with or without fungal cellulose, along with many other complex organic molecules.Fungi are unique organisms due to their morphological, physiological and genetic features; they are ubiquitous, able to colonize all matrices (soil, water, air) in natural environments, in which they play key roles in maintaining the ecosystems equilibrium (Anastasi et al., 2009;Valeria et al., 2013).The fungi secrete enzymes which break down solid materials into soluble compounds for absorption through their outer walls.They vary greatly in their ability to utilize different types of substrates.Some species are obligate parasites and are only able to utilize nutrients from living host tissue (Alexopoulos and Mims, 1979;Cullings et al., 2010).Most fungi are able to reproduce asexually and sexually forming spores which serve as units for survival and dispersal in soil, water and air.Fungi are widespread all over the world and high environmental burdens have been shown to be affected by various factors such as wind, moisture, temperature and air pollution leading to variations with respect to species and quantities from one season to another.Fungi thrive better in moist and warm places.However, fungi are also known as non-motile but some species of the oomycetes produce motile spores called zoospores (Letcher and Powell, 2002).
The knowledge of air-spora not only contributes to the understanding of their abundance and seasonal variations but also helpful in forecasting the epidemics of crop plants (Waggoner, 1960;Jadhav et al., 2010).Aerobiologists are mainly concerned with release, dispersion and deposition of spores, interaction of spores with each other, pollution, environmental factors and their impact on plants, animals and human beings (Srivastava, 1991).
Fungi are an important component of the soil micro biota.The role of fungi in the soil is an extremely complex one and is fundamental to the soil ecosystem (Valentıen et al., 2009).Many fungi live on dead organic matter saprophytically or in soil where they are regarded as the most important decomposers of plant residues and other organic matter.Aspergillus fumigatus is cosmopolitan and thermo tolerant fungus which is isolated primarily from compost, plant material and from soil.Some fungi are cosmopolitan in nature as Aspergillus nidulans and Aspergillus flavus which are initially isolated from soil (Diana, 1994).However many fungi are also used as bioremediator of soil pollution caused by different environmental factors or man-made activities such as addition of heavy fertilizers or PCBs (Kireeva et al., 2008;Teng et al., 2010).
Water is the most common compound found on earth.Chemically, water is an oxide of hydrogen and it is the only chemical compound that naturally occurs in all three physical states, solid (ice and hail), liquid and vapour.Pure water is colourless, odourless and tasteless.Water has unique physical and chemical properties.Its ubiquitous presence makes it essential for life.The distribution of water is in various compartments of the body, and changes takes place in various disorders.A number of diseases that we suffer are from drinking of contaminated water.Water is used for agricultural, irrigation purposes, hydroelectric power, recreation and navigation.It is a universal solvent and necessary resource because it has remarkable physical properties (Enger and Smith, 1998;Batko, 1975;Tanveer et al., 2011).
The term pathogen is applied to those organisms that either produce or involved in the production of a disease.Many fungi present in terrestrial or aquatic habitats cause diseases in plants and animals, hence hampering health and socio-economic conditions of the people (Mullenborn et al., 2008).Many fungi including important plant pathogens are able to grow at potentials which cause permanent wilting of crop plants.Slightly acidic conditions are optimum for the growth of most fungi (Abril et al., 2008).
Any soil borne fungal plant pathogens cause diseases of the root or stem disrupting the uptake and translocation of water and nutrients from the soil.Therefore, these commonly cause similar symptoms to drought and nutrient deficiencies; these include wilting, yellowing, stunting and death of plant (Ortonedo et al., 2004).Many fungal species cause "white rot" of wood decay in which the wood becomes a bleached appearance, with a spongy, stringy, or laminated structure, and where lignin as well as cellulose and hemicelluloses is broken down (Anastasi et al., 2009a).Species of Basidiomycetes cause "brown rot" and their number is ca.6% which cause damage to crops and plants (Hibbett and Donoghue, 2001).
Some areas are mainly populated with pinus forest; and this type of forest species are infected by degrading and decamping taxa of fungi (Pinedo et al., 2009).Some diseases are often very difficult to diagnose accurately and the pathogens may be difficult to grow in culture and identify accurately.
The aims and objective of the study are: 1) to explore the distribution of air, soil and water-borne mycoflora of selected area of Samahni and 2) to check the distribution of pathogenic fungi of the selected areas.

MATERIALS AND METHODS
Mycoflora was studied from different samples of air, soil and water from five different localities of Samahni viz: Dab, Chiti Mitti, Chayee; Chowki and Manaana.It has a territorially mountainous climate.The mycofloral analysis of the selected area was conducted by applying different research methods for different techniques.

Isolation of airborne fungi by culture plate technique
Air spores were trapped directly in sterilized Petri dishes, which were containing already sterilized potato dextrose agar (PDA) and water agar (WA) media.The Petri dishes were brought to the laboratory for incubation.These were examined within a week for mycofloral analysis (Tilak, 1989;Nourian et al., 2007;Ahmed, 2008).The culture plate technique (CPT) was used for the isolation of airborne fungi following by Shah and Bashir (2008).Two types of media viz.potato dextrose agar and water agar were employed to trap the fungal spores from air over the selected area of Samahni.The culture plates were exposed at different locations around the selected area at an altitude of five feet.The exposure time was five to ten minutes.Unexposed plates at each location served as controls.Each treatment was replicated three times.The plates were sealed after exposure and brought to the laboratory.The plates were incubated at room temperature in the laboratory of the Department of Plant Pathology, Peer Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS UAAR) for five to seven days or until colonies matured.Fungal colonies were counted and identified after this incubation period.Colonies that failed to sporulate on this medium were routinely sub-cultured onto another new media for further investigation.The procedure was repeated thrice for the detection of air mycoflora.Identification was carried out by following literature of Cook (1963), Domesch et al. (1980) and Hussain et al. (2011).

Isolation of fungi from soil by soil plate method
Soil samples were collected from a depth of 10-15 cm at each site.A sterilized cork borer having 5 mm diameter was used to collect each sample by pushing it into the soil.The cork borer was rinsed briefly after each sampling in 95% ethanol.The cores of soil were pushed out with a sterilized plunger directly into clean unused polythene bags and transported to laboratory.After collection, each sample was examined within a week for the study of microfungi (Irum et al., 2007).The fungal population of the soil samples was determined by soil plate method and serial dilution agar plate method.
Mycoflora was detected by soil plate method with some modifications (Warcup, 1950;Hussain et al., 2011).One gram of soil was scattered on the bottom of each sterile Petri dish that contained already semi-solid sterilized molten cooled (40-45°C) PDA and WA media.Then they were rotated gently to disperse the soil particles in the medium.It was mixed thoroughly and left to solidify for appropriate time.These were incubated at room temperature.Fungi growing on the Petri plates were isolated after incubation period (two to three days) and identified microscopically.The identified fungal colonies were counted and identified by following mycological literature of Domesch et al. (1980) and Hussain et al. (2011).

Isolation of fungi from soil by serial dilution agar plate method
Serial dilution agar plate method detected mycoflora with some alterations (Waksman, 1922;Hussain et al., 2011).The collected soil samples were mixed with sterile distilled water to make a soil suspension.Serial dilutions of soil samples were made in sterile distilled water by adding 1.0 g of soil sample to 9.0 ml of water and were mixed vigorously for uniform distribution to make a soil solution.1 ml of the soil solution was serially diluted to 10 and 0.5 ml of each of the inoculums used in a pour plate method incorporated with antibiotic mixture.10 ml sterilized distilled water was added in each set of nine test tubes for dilution of the collected soil samples. 1 ml of soil suspension from the prepared soil suspension was added in a test tube to made 1:10 dilution and 1 ml of suspension from 1:10 dilution transferred to second test tube gave 1:100 dilutions then 1:1000 dilutions was made.Similarly, a series of dilutions were made. 1 ml suspension from each test tube were transferred in sterilized Petri plates and poured with 15 ml of melted cooled PDA and WA media.It was mixed thoroughly and left to solidify for some time.A control was prepared against bacterial growth.All media were steam sterilized at 121°C for 15 min.The medium for the primary isolation of fungi was formulated with streptomycin-penicillin antibiotics added at a final concentration of 0.6 ml/Petri dish of PDA (APHA, 1995).After 2-7 days, fungal colonies were appeared on the PDA and WA media.Fungi were identified by using mycological literature (Raper and Thomas, 1945;Gilman, 1957;Domesch et al., 1980;Ellis, 1971;Nelson et al., 1983;Hussain et al., 2011).

Isolation of fungi from different water samples by direct plate technique
The sterilized conical flasks (250 ml) were used for each water sampling.The flasks were submerged into water in an inverted position to 4-6 cm depth by removing the flask lid.The flasks were allowed to fill with water at this point and the lids were placed over the flask while still under water surface.The collected samples were taken back to the laboratory for fungal study (Nasser, 2004).One ml aliquots from each of the collected water samples were pipetted aseptically into three sterilized Petri plates having semi-solid sterilized molten cooled (40-45°C) PDA and WA media.Total fifteen Petri plates were prepared from five selected sites with the help of sterilized pipette.These were rotated gently to disperse the water in the medium.It was mixed thoroughly and left to solidify for appropriate time.The procedure was replicated three times for better result.The Petri plates were incubated at room temperature.Fungi grown on Petri plates were isolated after two to three days incubation period and identified under 10x objective microscope.The percentage occurrence of each fungus was determined (Warcup, 1950;Hussain et al., 2011).

Isolation of fungi from different water samples by baiting technique
The water samples were taken back to the laboratory in the sterilized flasks, where 40 sterilized hemp seeds were added in each flask (Vanbreuseghem, 1952;Hussain et al., 2011).The flasks were kept in the dark for 24 h at room temperature.The sterilized hemp seeds were colonized after 24 h.The colonized seeds were transferred to 20 ml sterilized mixture of distilled water and tap water in sterilized Petri plates in a 1:1 ratio.Ten seeds were placed in each Petri dish. 10 ml water from above ratio was added in each Petri plates with 2000 unit/L of antibiotic Streptomycin to suppress the bacterial growth.Four Petri plates were prepared from each flask.A total of 20 Petri plates were obtained from five samples.The Petri plates containing colonized hemp seeds were incubated at room temperature.The seeds were observed after two to seven days under 10x objective microscope for the study of fungal growth.Fungal mycelium or hyphae appeared on the hemp seeds as a white tuft after three to five days.Sometime it did not become visible until 5-7 days after setting up the experiment.This process was continued for five weeks.The water was changed in each Petri dish after each examination.The procedure was replicated thrice for good result.Fungi were identified from these primary cultures.The rest of the fungi were identified after sub-culturing (Dick, 1965;Hussain et al., 2011).

RESULTS AND DISCUSSION
This research was aimed to collect different mycoflora from the selected area and to identify them by application of different techniques and find out the prevalence of the mycoflora in different habitats.Its economic importance and pathogenic effects were also considered.In this research, a total of 63 fungal taxa were isolated and described for their importance values in forest ecosystem of Samahni.This research conducted depicts that soil has the highest percentile with 32 species in soil followed by air with 20 species and water with 11 species, respectively (Table 1).Some species such as Botrtis sp., Cladosporium cladosporioides, Curvularia lunata, Drechslera sp., The samples of fungi were isolated and grown on different media such as PDA and WA and their prevalence was found in same occurrence, so the media difference does not affect the process and results in the current research work (Table 2).Difference was measured by least standard deviations (LSD) for different analytical results as shown in Table 3 and Figure 3; it shows that water-born fungi has highest LSD value (1.5), soil-born has 1.1 and air-born fungi has 0.31, respectively.
The Table 1 shows the presence/absence of fungal species in air, soil and water samples while Table 2 shows the presence/absence of fungal species on different cultural media (PDA and WA).Similarly total number of fungal colonies (%) in air, soil and water samples showed is in Table 3 while total numbers of fungal colonies (%) on different cultural media (PDA and WA) are shown in Table 4.

Plate isolation of airborne fungi by culture technique
A total of twenty different fungal species were isolated by using culture plate technique (CPT) from samples of air (Table 1).The results (Table 2) showed that same fungal species were grown on PDA media and WA media.On the other hand, the fungal colony (percentage) showed variation among different cultured media.More numbers of fungal colonies (percentage) appeared on PDA as compared to WA due to their nutritional variation (Table 4).The growth of fungi shows quantitative difference on both media.The present study was similar to the study of Morring et al. (1983).
The results (Table 4) showed that the dominant fungal species in air sample was Cladosporium cladosporioides  Values in each row with different letters show significant difference as determined by LSD test at P≥0.
(7.1%) while minimum airborne fungal species appeared as Mucor fragilis (2.9%).The frequencies of airborne fungi were significantly different.These results were correlated with the findings of Nasim et al. (1998), Rao, et al. (2009) and Shah and Bashir (2008).Out of 35 isolated fungal species, 15 species were absent in air samples (Table 1).C. cladosporioides (7.1%) and Penicillium sp.(6.8%) are not significantly different.Therefore, these two species have equal distribution in air samples.The fungal species such as A. niger (5.8%), Drechslera sp.(5.6%), and Aspergillus flavus (5.2%), Alternaria alternata (5.6%), were significantly the same in distribution (Table 4).These findings were also similar to the findings of Shah et al. (1995), Shah and Bashir (2008) and Bajwa et al. (1997).They also stated that there were significant similar Table 4.Total number of fungal colony (%age) of air, soil and water-borne fungi isolated by using PDA and WA media from selected areas of Samahni (Azad Kashmir).distributions among various fungal species.

Isolation of fungi from soil samples by soil plate method
Thirty-two (32) different fungal species were isolated and identified from the selected area by soil plate method while twenty-three fungal species were isolated and identified from the collected soil samples by using serial dilution agar plate method (DAPM) from the selected area.The results show that same numbers of fungal species were growing on both PDA and WA media.The present study showed that more numbers of fungal species were isolated by applying DPM as compared to DAPM (Table 2).The soils of the selected area have more nutrition and suitable climatic factors for growth and dispersal of fungal spores.Therefore, maximum numbers of fungi were isolated by soil plate method.This study was correlated with the previous study of Picco andRodolfi (2000) Fang et al. (2005) and Adhikari et al. (2004).They explained that different environmental factors and more nutrition variation increase the fungal frequency in the soil samples.
The results indicate that these great differences occurred due to the geographical location, fungal growth substrates/media as well as due to applying different sampling methods.Adhikari et al. (2004) and Siham (2007) also investigated that the variation among different fungal species.They also revealed that the variation in results occurred due to variable growth media, different locality and due to implementation of different methods.

Isolation of fungi from soil samples by serial dilution agar plate method
Twenty-three (23) fungal species were isolated and identified from the collected soil samples by using DAPM from the selected area (Table 3).The results show that same numbers of fungal species were growing on both PDA and WA media.The present study also stated that more numbers of fungal species were isolated by applying DPM as compared to DAPM (Table 2).
As indicated in the result, more number of fungal species was isolated by DPM as compared to DAPM.In this method, soil samples were diluted.Therefore, frequency of spores reduced in diluted samples.The results indicated that these great differences occurred due to the geographical location, fungal growth substrates/media as well as due to applying different sampling methods.Adhikari et al. (2004) also investigated the variation among different fungal species.They showed that different sampling methods greatly influenced fungal flora.

Isolation of fungi from water by direct plate method
Nine different fungal species were isolated and identified from the collected water samples by direct plate method of the selected area (Figure 2; Table 3).The results depicts that minimum fungal species were isolated by using DPM.The present study showed that the direct inoculation of water samples on different media was not favorable for better growth of fungal spores.
(10.3%) and Verticillium terrestre (8.8%) are not significantly different.Therefore, these five species have equal distribution in water samples.Similarly, the occurrence of lowest species Verticillium sp.(6.1%), Curvularia lunata (6.9%), Drechslera sp.(7.5%) and Nigrospora sp.(8.5%) were not significantly different as shown in Table 3.The significantly same distribution of fungi appeared in water samples due to similar temperature and nutrient contents.Nourian et al. (2007) obtained similar results.

Isolation of fungi from water by baiting technique
Eleven (11) species were isolated from water samples by baiting technique (BT) from the selected area of Samahni district Bhimber Azad Kashmir (Table 3).The results showed that more number of fungal species was isolated by applying BT on water samples as compared to DPM.It means that low fungal frequency was obtained by using DPM technique while high fungal frequency was obtained by using any bait.
A total of 35 different fungal species were isolated and identified during the present investigations from different samples of air, water and soil from the selected area.The identified species belonged to eighteen different genera namely, Alternaria, Aspergillus, Asteromyces, Botrytis,Cladosporium,Curvularia,Drechslera,Fusarium,Helminthosporium,Mucor,Nigrospora,Penicillium,Pythium,Rhizoctonia,Stachybotrytis,Trichoderma,Thielavia and Verticillium (Table 1).The results (Figure 1) indicated that 17% waterborne fungi, 32% airborne fungi and 51% soil fungi were isolated.The fungal population was very high in the soil sample.The environmental factors, high vegetation coverage, presence of high nutrition in soil and geographical location might increase the fungal frequency in the soil samples (Korkama-Rajala et al., 2008).These observations showed that high percentage of fungal species were isolated from soil samples.The maximum prevalence of fungi in soil samples occurred due to presence of high nutrition  in soil, variable environmental factors, geographical locations and high vegetation coverage.This study correlated with the previous study of Picco and Rodolfi (2000), Fang et al. (2005), Adhikari et al. (2004) and Soderlund (2009).They explained that different environmental factors, vegetation coverage and more nutrition variation increase the fungal frequency in different habitats.
It was noted that land-habituated mycoflora was more frequently depicted because land provides best environment and nourishment for growth and reproduction.It was also observed that some fungi were common in water and land and it means that some species of soil do use water as mode of distribution and dispersal.Aspergillus spp. was found commonly in air and soil habitats but its some species were observed in water.Its presence in aquatic habitat revealed that the contamination of sampling in experiment designing occurred, however, its common occurrence in both habitats showed that it is a terrestrial species.The present result was similar to the result of Phukan and Baruah (1991) and Soderlund (2009).They isolated some terrestrial fungi from aquatic environment.
The high frequency of F. oxysporum and C. cladosporioides species are found in all the analyzed habitats.It predicts that these species have good morphological and reproductive mechanism, which makes it cosmopolitan in the environment.Resano et al. (1998) and Renata et al. (2004), supported these results.They showed that C. cladosporioides and F. oxysporum were commonly found with high frequency in forest vegetation area.According to statistical analysis, significant difference was studied among different fungal species, which were isolated from different habitats and different (PDA and WA) media as shown in Table 4. Nourian et al. (2007), Renata et al. (2004) and Soderlund (2009) applied statistical analysis among different samples of fungi which were significantly different.They explained that the species concentration differed from place to place because of the local environmental changes and   due to use of different fungal growth media.
The number of some fungal colonies (percentage) among air and water samples was significantly different.Similarly, statistical analysis between soil and water samples also showed significant difference among isolated species of fungi.Shah et al. (1995), Bajwa et al. (1997) and Nourian et al. (2007), previously indicated   significant variation among different fungal species that appeared due to variable environment of air, soil and water samples.
This mycofloral research demonstrated that Rhizoctonia  solani produce root rot in various plants of forest which destroy the natural flora.These findings are in accordance with previous results of Renata et al. (2004).
The present study indicated that the isolated species of fungi Cladosporium and Alternaria are involved in spreading of allergy in organisms.These observations were similar to the results of Renata et al. (2004).They showed that Cladosporium and Alternaria species causes allergy in organisms.They also investigated that Cladosporium is a very common fungus that is known and documented as an aeroallergen that is usually associated with plants, wood products and leather goods.
Pythium debaryanum is a soil pathogen isolated and identified from the investigated area which produces leaf blight and bulb rot symptoms on narcissus plants.These investigations were correlated with the results of Sung et al. (2007).They observed that P. debaryanum cause leaf blight and bulb rot symptoms on narcissus plants.The symptoms induced by artificial inoculation were similar to those observed in the selected area.
The study shows that Verticillium species are pathoge- nic to different plants in the selected forest area.It produces Verticillium wilt.This study was similar to the previous study of Agrios (2005).He showed that some Verticillium species are pathogenic to different plants and cultivated and wild type crop species.He showed that Verticillium wilt is a vascular wilt disease of plants caused by Verticillium spp.Some species also infect mushrooms, rusts and other fungi in natural flora.Other species of Verticillium attack wool and textiles industries and decompose paper.Greatest numbers of these diseases were seen in higher plants by Verticillium and Fusarium spp.(Green, 1981).Many species of fungi were involved in serious human and animal infections.Some species of fungi also caused serious plant diseases.The present study showed that A. alternata is phytopathogenic fungus and show saprophytic symptoms on plants of the selected area.These findings were similar to the findings of Angulo et al. (1996) and Infante et al. (1992).These findings showed that the fungal species A. alternata, Botrytis cinerea, Rhizoctonia solani, P. debaryanum, Cladosporium spp.and Verticillium spp.affect the local plants and medicinal wealth is being destroyed in area of Samahni.
row with different letters show significant difference as determined by LSD test at P≥ 0.05.

Figure 1 .
Figure 1.Different fungal species growing on PDA.

Figure 2 .
Figure 2. Different fungal species growing on WA.

Figure 3 .
Figure 3.Total number of fungal colonies isolated from air, soil and water samples of Samahni, District Bhimber, Azad Kashmir.

Table 1 .
Air-borne, soil-borne and water-borne mycoflora isolated from different samples of selected area of Samahni Azad Kashmir.Fusarium oxysporum, Nigrospora sp., Penicillium sp. and Verticillium sp. were ubiquitously present in all analyzed samples from different sources(Table 1,.

Table 2 .
Mycoflora isolated by use of PDA and WA media from different samples (air, soil and water) of selected area of Samahni (Azad Kashmir).

Table 3 .
Total number of colony (percentage) of air-borne, soil-borne and water-borne mycoflora isolated from different sampling sites of Samahni (Azad Kashmir).