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Assessment of species richness in Lake Dal, Kashmir, based on classical approach, physiological approach and rDNA ITS sequences from isolates
Microbial Pathogenesis 104 (2017) 303e309
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Assessment of species richness in Lake Dal, Kashmir, based on classical approach, physiological approach and rDNA ITS sequences from isolates Suhaib A. Bandh a, *, Azra N. Kamili a, Bashir A. Ganai a, Bashir A. Lone b a b
Microbiology Research Laboratory, Centre of Research for Development/Department of Environmental Science, University of Kashmir, Srinagar, India Parasitology Research Laboratory, Centre of Research for Development/Department, University of Kashmir, Srinagar, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2017 Received in revised form 30 January 2017 Accepted 31 January 2017 Available online 2 February 2017
As a first description to document the species richness in Dal Lake, a freshwater lake ecosystem in Kashmir valley, an extensive network of sixteen sampling stations with distinguishing features was sampled seasonally for two years. The identification process yielded fifty-one species probably first and new records for this area to date. The taxonomic groups observed were those with species from Ascomycetes (inclusive of yeasts), Basidiomycetes, Blastocladiomycetes, Zygomycetes, and Peronosporomycetes. Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In the filamentous fungal group, family Trichocomaceae was dominant followed by Saccharomycetaceae, Mucoraceae, Nectriaceae, Tremellaceae and Hypocreaceae. However, in the group of zoosporic & fungal like eukaryotes, family Saprolegniaceae was most dominant followed by Blastocladiaceae and Pythiaceae. A dramatic decrease in fungal load was observed in different seasons with highest colonial load in the summer season and lowest in the winter season. The observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) as well as zoosporic fungi & fungal like eukaryotes (p < 0.05). In order to assess biodiversity patterns of fungi more accurately, it is necessary to repeat such investigations in other areas and to improve the tools for taxonomic identification of these highly diverse but mostly microscopic organisms. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Dal lake Fungi ITS gene Kashmir
1. Introduction Fungal species reported from aquatic habitats range from those adapted to complete their life cycle in aquatic habitats and not found outside the aquatic environments (residents) to those occurring in water fortuitously by being washed or blown in (transients). Representatives of all fungal groups like Chytridiomycetes, Hyphochytridiomycetes, Peronosporomycetes (formerly Oomycetes), Ascomycetes and Mitosporic fungi have been found in or isolated from aquatic habitats. Freshwater fungi play an important role in freshwater ecosystems mainly due to their role in decomposing woody debris and leaves, with their mycelium and fruiting bodies being available to the animal food web [1e3]. In surface waters, fungal communities form an
* Corresponding author. E-mail address: [email protected] (S.A. Bandh). http://dx.doi.org/10.1016/j.micpath.2017.01.058 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
important component of biotic communities by decomposing and transforming organic material [4] and as a food source [5]. Ingold [6] isolated and described many filamentous fungi, whose spore morphologies were adapted to dispersal in running waters followed by attachment to plant materials like leaves which later came to be known as aquatic Hyphomycetes or ‘Ingoldian fungi’. Fungi, commonly known as chytrids, thrive and reproduce in fresh and marine environments, however, they can also be found commonly in habitats such as terrestrial soils [7,8]. Based on their evolutionary history, fungi in aquatic habitats are either primarily (Chytridiomycetes) or secondarily adapted to life in water (aquatic Hyphomycetes, Yeasts). In addition, primarily terrestrial forms (fungi imperfecti, endophytes) are found in lakes, suggesting that their occurrence is not necessarily restricted to a single habitat by stringent morphological or physiological adaptations. Like Peronosporomycetes (fungus-like organisms belonging to the Kingdom Chromista), Chytridiomycetes (Kingdom Fungi) possess chemotactic, flagellated zoospores, which can disperse in the water
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column [9]. Terrestrial fungi are often passively introduced into lakes in the form of high loads of fungal propagules via inflowing streams, rainwater, and wind [5]. Though fungi are often found in disturbed areas with high anthropogenic loads from various sources like domestic, industrial, municipal and sewage treatment plants [10,11]; many fungal species survive in oligotrophic and ultra-oligotrophic environments, such as in water distillation apparatus [12]. There are many reports of freshwater fungi from both lotic and lentic habitats in different areas [13e25] but nothing has been written on the subject in valley Kashmir. It is in this backdrop the present study was carried out to focus on the unexplored and undocumented fungal diversity of an important freshwater ecosystem of Kashmir valley. 2. Methods
prepared according to the recipes of Pitt [30]. Each filamentous fungal culture was inoculated in triplicate on each medium and incubated at three different temperatures (5 C, 25 C and 37 C) for a period of 7 days in the dark. In addition to these the morphological characteristics were studied by making slide cultures obtained by inoculating microfungi directly on a small square of agar medium [31]. Morphology was characterized by using a semiautomatic image analysis system consisting of an Olympus microscope (Olympus, New Hyde Park, NY, U.S.A.) operated as phase contrast, a charge coupled device (CCD) camera (Sony, Cambridge, U.K.) a PC with a frame-grabber, and the image analysis software (SIS, Olympus, Germany). Sample preparation and measurement was done as described by Papagianni et al. [32,33]. A magnification of 100 was applied for measurement of mycelial particles to estimate the individual mycelia and other micro-morphological features.
2.1. Study area and sites The study area Dal lake (N 34 070, E 74 520 , 1583e1600 m) a Himalayan lake in Srinagar, represents a unique ecosystem with respect to its species diversity, aesthetics and interaction with people living in the houseboats and the small hamlets within the lake. As it is a multi-basined lake with many inlets and outlets, an extensive network of sixteen sampling stations viz. Hazratbal Open (S1), Hazratbal littoral (S2), Nageen Open (S3), Nageen littoral (S4), Gagribal Open (S5), Gagribal littoral (S6), Nishat Open (S7), Near Centeur (S8), Dal Lock Gate-I (S9), Dal Lock Gate-II (S10), BoathallNallah-I (S11), Boathall Nallah-II (S12), Pokhribal Nallah-I (S13), Pokhribal Nallah-II (S14) Tailbal Nallah-I (S15) and Tailbal Nallah-II (S16), from both littoral zones as well as limnetic zones were selected. Among the selected sites, eight (8) sites were selected in four basins, four (4) sites were selected from two inlets and four (4) sites were selected from two outlets. 2.2. Sample collection Water samples from different sites of the lake, under consideration for exploring the species richness were collected on seasonal basis in Polyethylene (PET) bottles, which were previously carefully cleaned and rinsed three to four times with autoclaved distilled water. All the samples were collected just below the surface of lake water by plunging the open end of each bottle before turning it upright to fill. During collection of samples, extreme care was exercised to avoid contamination of the bottles and collected samples were processed for analysis using the standard methodology. 2.3. Isolation and identification of fungi For the isolation of fungi different approached like direct plating method [26] and baiting technique [7,27] were followed. Identification of the isolated fungal species was done on the basis of classical morphological and culture approach, biochemical testing approach and molecular approach. 2.4. Classical morphological and culture approach Identification of the species was performed mainly on the basis of the micro and macro-morphological features of colonies grown on differential media. Fungi were identified to genus level using Barnett and Hunter's work [28]. Cultures were identified to species level using various mycological texts like Penicillium LINK, Raper and Fennell [29] and PITT's monograph [30]. These species were grown on three different media {Czapek Yeast Agar (CYA), Malt Extract Agar (MEA), and 25% Glycerol nitrate Agar (G25A)} all
2.5. Biochemical testing approach Physiological profiling of a few yeast species was carried out by the biochemical tests included Physiological tests, Fermentation tests and Assimilation tests. The inoculated microplates were shaken (with a microplate shaker) just before automatic reading using a microplate reader. Absorbance values at 405 nm were transferred by cable (RS-232 through a serial port) to the computer and transformed by the BioloMICS software into negative, weak or positive results. The results of every test were transformed independently.
2.6. Molecular approach 2.6.1. Growth and harvesting cells from liquid cultures The fungal colonies were grown in Potato Dextrose Broth and Sabouraud Dextrose Broth containing 10 ml of Tween-80 as it keeps the fungal cells dispersed and helps in weighing the culture. After inoculation of fungal cultures, incubation at 30 C, preferably on a shaker was done.
2.6.2. DNA extraction The weighed fungal tissue was ground to a fine powder in a mortar and pestle using liquid nitrogen which was then allowed to evaporate and samples were not allowed to thaw. DNA was extracted using a HiPur A™ SP Fungal DNA Mini kit (Himedia) following the manufacturers protocol and the purity of DNA was measured by the absorbance ratio (A260/A280).
2.6.3. Polymerase chain reaction (PCR) ITS region of the rRNA operon was amplified using ITS1 and ITS4 primers [34]. Amplification was performed in 50 ml PCR reaction tubes containing 6 ml genomic DNA, 5 ml 10 thermophilic buffer, 2 ml MgCl2, 1.4 ml dNTP (10 mM), 1.2 ml (10 mM) ITS1, 1.2 ml (10 mM) ITS4, 6 ml (10u/ml) Taq (Promega) and 32 ml ddH2O in an Eppendorf Thermal Cycler with the following program for 40 cycles: initial denaturation temperature 94 C for 5 min, melt temperature 94 C for 50 s, annealing temperature 54.5 C for 45 s, extending temperature of 72 C for 45 s, final extension temperature 72 C for 10 min and hold temperature of 4 C. The ITS bands were identified by gel electrophoresis on a 2% agarose gel. PCR products obtained were sequenced and the sequences obtained from both primers were BLAST searched to conform the percent similarity to the ITSI, 5.8S rRNA gene and ITSII regions of the respective fungi in the Gene Bank (Fig. 4).
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Table 1 Systematic diversity of the isolated taxa. Kingdom
Phylum
Class
Fungi
Ascomycota
Chromista
Basidiomycota Blastocladiomycota Zygomycota Heterokontophyta
Number of taxa
Eurotiomycetes Sordariomycetes Ascomycetes Saccharomycetes Tremellomycetes Blastocladiomycetes Zygomycetes Peronosporomycetes
3. Results Species richness assessed in the water samples collected from Dal lake on seasonal basis from sixteen sites of the lake belonged to two groups viz. the filamentous fungi and zoosporic fungi and fungal like eukaryote group with twenty-eight (28) species belonging to the former and twenty-three (23) species belonging to the later. Species isolated and identified during the study belonged to five major phyla with twenty-five (25) species belonging to Ascomycota, one (1) species to Basidiomycota, four (4) species to Blastocladiomycota, two (2) species to Zygomycota and nineteen (19) species to Heterokontophyta. Phylum Ascomycota consisted of four major classes Eurotiomycetes, Sordariomycetes, Ascomycetes and Saccharomycetes; phylum Basidiomycota consisted of one class
Table 2 Total count (TC), % total count (%TC), number of cases of isolation (NCI) and occurrence remarks (OR) of filamentous fungi. Family and Species
TC
% TC
NCI
OR
Trichocomaceae Aspergillus Aspergillus flavus Aspergillus fumigatus Aspergillus japonicus Aspergillus niger Aspergillus oryzae Aspergillus terreus Aspergillus versicolor Aspergillus wentii Penicillium Penicillium caseicolum Penicillium chrysogenum Penicillium commune Penicillium dimorphosporum Penicillium funiculosum Penicillium lilacinum Penicillium olivicolor Penicillium spp. I Penicillium spp. II Penicillium spp. III Penicillium spp. IV Mucoraceae Mucor spp. Rhizopus stolonifer Nectriaceae Fusarium oxysporum Hypocreaceae Acremonium strictum Saccharomycetaceae Candida albicans Candida krusei Candida parapsilosis Candida glabrata Tremellaceae Cryptococcus neoformans Total
2517 1073 178 98 98 218 72 110 142 157 1444 123 334 63 128 236 78 15 100 84 136 147 186 94 92 80 80 36 36 282 84 83 9 106 56 56 3157
79.72 e 5.64 3.1 3.1 6.9 2.28 3.48 4.5 4.98 3.9 10.58 1.99 4.05 7.47 2.47 0.47 3.17 2.66 4.3 4.65 5.9 2.98 2.91 2.53 2.53 1.14 1.14 8.94 2.66 2.63 0.29 3.36 1.77 1.77 -
e e 15 14 15 16 11 10 15 14 e 14 16 7 14 16 7 4 14 12 14 11 15 9 11 e 11 11 14 3 10 6 e
H e H H H H H H H H e H H M H H M L H H H H e H H e H R e e H H L H e M e
Order
Genera
Species
1 1 1 1 1 1 1 2
2 3 1 1 1 1 2 6
17 3 1 4 1 4 2 19
Tremellomycetes, Blastocladiomycota of one class Blastocladiomycetes and Heterokontophyta of one class Peronosporomycetes, and Zygomycota of one class Zygomycetes (Table 1). Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In all genus Penicillium was represented by a maximum of eleven species with a relative abundance of 21.6% followed by genus Aspergillus eight species (15.7%), Achyla five species (9.8%), Candida, Allomyces, Brevilegnia and Pythium four species (8%) each, Aphanomyces three species (5.9%), Dictyuchus two species (3%), Mucor, Rhizopus, Fusarium, Acremonium, Cryptococcus and Saprolegnia one species (2%) each. The species isolated belonged to nine families with maximum contribution of nineteen species by family Trichocomaceae followed by fifteen species of family Saprolegniaceae, four species each by Saccharomycetaceae, Blastocladiaceae and Pythiaceae, two species by family Mucoraceae and one species each by family Nectriaceae, Hypocreaceae and Tremellaceae.
Table 3 Total count (TC), % total count (%TC), number of cases of isolation (NCI) and occurrence remarks (OR) of zoosporic fungi and fungal like eukaryotes. Family and Species
TC
% TC
NCI
OR
Blastocladiaceae Allomyces moniliformis Allomyces anomalus Allomyces arbuscula Allomyces spp. Saprolegniaceae Achyla Achyla klebsiana Achyla apiculata Achyla flagellate Achyla cornuta Achyla conspicua Brevilegnia Brevilegnia indica Brevilegnia linearis Brevilegnia diclina Brevilegnia subclavata Dictyuchus Dictyuchus monosporus Dictyuchus spp. Aphanomyces Aphanomyces laevis Aphanomyces cladogamous Aphanomyces spp. Saprolegnia Saprolegnia parasitica Pythiaceae Pythium proliferum Pythium elongatum Pythium debaryanum Pythium spp. Total
659 220 154 181 104 2474 791 131 85 105 261 209 679 136 143 221 179 401 133 268 323 76 107 140 280 280 650 204 124 206 116 3783
17.42 5.81 4.07 4.79 2.75 65.39 31.97 3.46 2.25 2.77 6.9 5.52 27.45 3.6 3.78 5.84 4.73 16.2 3.52 7.08 13.06 2.01 2.83 3.7 11.32 7.4 17.19 5.4 3.28 5.44 3.07 -
e 15 12 14 12 e 15 12 11 16 16 16 15 15 15 16 16 11 12 16 16 e 13 12 16 14 e
H H H H H e e H H H H H e H H H H e H H e H H H e H H H H H H e
H¼ High; M ¼ Medium; L ¼ Low.
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Fig. 1. Seasonal fluctuation of fungal load.
Tables 2 and 3 summarizing the family and species wise total count, % total count, number of cases of isolation and occurrence remarks, depicts that among the filamentous group, different species of family Trichocomaceae were isolated in a low to high frequency represent 79.72% of this group with a total colony count of 2517. Different species of genus Aspergillus constituting 33.99% of the whole group and 42.63% of the family Trichocomaceae showed a total count of 1073 colonies. Genus Penicillium showed a total count of 1444 colonies constituting 45.74% of the whole group and 57.37% of the family Trichocomaceae. Family Mucoraceae isolated in a high frequency with a total count of 186 colonies constituted 5.9% of this group. Families Nectriaceae, Hypocreaceae and Tremellaceae were represented respectively by F. oxysporum, A. strictum and C. neoformans. Family Saccharomycetaceae isolated with a low to high frequency was represented by four species. It constituted 8.94% of the group with a total colony count of 28. Among the different families of zoosporic fungi and fungal like eukaryotes, family Saprolegniaceae was most dominant with a maximum number of 2474 colonies of fifteen (15) species constituting 65.39% of the group followed by family Blastocladiaceae with 659 colonies of four (4) species constituting 17.42% and family Pythiaceae with 650 colonies of four (4) species 17.19%. Showing considerable seasonal variations (Fig. 1) at all the selected sites highest number of filamentous fungal colonies 1208 (38.26%) was registered in summer season followed by 972 colonies (30.79%) in spring, 660 colonies (20.91%) in autumn and the least number of 317 colonies (10.04%) in winter season. Similarly, maximum number of zoosporic fungal and fungal like eukaryote
colonies 1380 (36.48%) was observed in summer followed by 1251 colonies (33.07%) in spring, 709 colonies (18.74%) in autumn and least number of colonies 443 (11.71%) in winter. One-way analysis of variance (ANOVA) showed that the observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) and zoosporic and fungal like eukaryotic species (p < 0.05). Explanatory data analysis by Box and Whisker Plot model shown in Figs. 2 and 3 on the basis of all experimental data for both the groups, from all the selected microhabitats studied as an indicator of centrality, spread and similarity lined up side by side showing the main characteristics of their distribution shows that for filamentous fungi (Fig. 2) site 14 and 16 appear to have similar centers exceeding the rest of the sites while as sites 3, 4, 5, 6 and 12 appear to have similar centers lying towards the base of the graph. Site 14 appears to have the largest variability along with site 13 and 16 than the other thirteen sites. However, sites 4, 5, 6 and 12 are reasonably symmetric compared to rest of the sites. For zoosporic fungi and fungal like eukaryotes (Fig. 3) it can be seen that site 14 appears to have the center which exceed the rest of the sites. Here also site 13 and 14 appear to have largest variability along with site 8 than the other thirteen sites and the sites 4, 5, 6 and 12 are reasonably symmetric compared to rest of the sites.
4. Discussion Microorganisms play important roles in the environment. Studies of microbial community composition therefore, provide potentially descriptive information about ecosystem function [35]. Fungi and fungus-like organisms show a high capacity for adaptation to the environmental conditions, and are therefore widely spread in nature [36]. The fungal community present in the lake water was assessed using the traditional method of culture and baiting as well as the molecular techniques. The use of these techniques for the isolation of fungal communities from different water bodies and other environmental samples has been done in many other studies [37e44]. The majority of fungal isolates (50%) obtained from the lake belonged to Ascomycota and only a meager percentage (0.5%) Basidiomycota is due to the fact the water of Dal Lake is not that deficient in dissolved oxygen as proved by some limnological studies carried out recently [45] on this water body. The results here are well supported by the work of Shearer et al. [46] reporting that taxonomically, the aquatic fungi known as
Fig. 2. Box and Whisker Plot Model for filamentous fungi.
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Fig. 3. Box and Whisker Plot Model for zoosporic fungi and fungal like eukaryotes.
“aquatic hyphomycetes” found most commonly in clean and well oxygenated [47] are mainly associated with the Ascomycota, and only a small percentage is affiliated with the Basidiomycota. The predominance of Ascomycota in the aquatic ecosystems like Dal lake has been discussed by many authors [48e53]. The variations in the fungal community characteristics of the lake can be attributed to the heterogeneity in the water characteristics of the aquatic habitats as they are heterogeneous in time and space and greatly differ in their physico-chemical features. Favoring the current observation, a recent review of the ecology of fungi in lake ecosystems by Wurzbacher et al. [54] mentioned that the fungal communities differ significantly between and within the different water bodies as the aquatic habitats are characterized by a unique balance of allochthonous (external) and autochthonous (internal) organic matter supply, controlled largely by watershed characteristics, surface area and location. In aquatic systems, the fungal community structure greatly differs between substrates [55e59] and with the physico-chemical properties of the respective habitats, such as flow [4,60] dissolved oxygen concentration [61,62], nutrient concentrations [63,64], salinity [65,66], temperature [67] and depth [54]. The occurrence of zoosporic fungi in the lake waters is not surprising as they have been found to be universally present in all types of freshwater systems and occur as saprotrophs on a wide variety of substrata, playing a key role in those ecosystems as decomposers of organic materials [68]. Most studies dealing with zoosporic fungi have provided extensive inventories of taxa from specific sites or geographic regions, but often without characterizing the microhabitat, determining frequencies of occurrence or relative abundance of species [7,8,69,70]. The isolation of different fungal species belonging to both the zoosporic group and filamentous group of fungi in the present study corroborates well with the study of Rajanaika et al. [71]. Useful correlations could be made between temperature and the occurrence of Peronosporomycetes in this study as this was probably because the study extended over two years of essentially variable climatic conditions and because there was more variation in temperature within different seasons of a year. Seasonal variation observed in the fungal population was possibly due to the temperature variations, as the maximum percentage of fungi was found during high temperature (summer) and minimum during low temperature (winter) season. Similar results were obtained by Khulbe and Durgapal [72], in their study on the population dynamics of geofungi in a polluted freshwater body at Nainital. A preliminary
mycological study on the same lake by Bhat and Kamili [73] had also reported the presence of filamentous fungal genera especially Aspergillus and Penicillium with a similar fluctuation in their numbers during different seasons. The seasonal variation found in this study is also in consonance with the results of Sharma and Parveen [74]. The influence of temperature on the fungal load is further supported by the work of Stoll [75] mentioning that the occurrence and distribution of water molds is directly related to the water temperature. Well in support of the results of present study Ho [76] also found a positive correlation between the water temperature and the isolation frequency of water molds. The presence of the terrestrial molds like Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Acremonium spp., Penicillium chrysogenum, P. caseicolum, P. commune, P. lilacinum, Fusarium oxysporum and Rhizopus stolonifer in the Dal waters is due to the influence of catchment area like entry of litter, soil, dead plant parts, sewage and the influence of air as the occurrence of these terrestrial fungal species in aquatic systems are likely to originated from air [9] as well as from living or dead animal and plant parts, soil and litter being in contact with
Fig. 4. Amplified rDNA ITS regions of specified fungi. Lane no. 1 ¼ 100bp Ladder; 2 ¼ A. terreus; 3 ¼ A. flavus; 4 ¼ A. japonicas; 5 ¼ A. versicolor; 6 ¼ A. klebsiana; 7 ¼ A. flagelata; 8 ¼ A. apiculata; 9 ¼ A. moniliformis; 10 ¼ A. anomalous; 11 ¼ D. monosporus; 12 ¼ A. leivis.
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water [77]. Filamentous fungi belong to the genera Aspergillus, Penicillium and Fusarium in water bodies have also been reported in many studies p [78,79]. These frequently found fungi could be considered as indicators of the generic composition of the water body. The occurrence of these fungal species in water samples of Dal Lake is not new but it is surely a new report in the lake as no study has been carried out till now on the fungal community of the lake. Conflicts of interest We declare that the research doesn't have any conflict of interest. Acknowledgements S. Bandh was supported by PhD grant from University of Kashmir, Srinagar (Grant No. PhD-G/Env.Sc/KU/176-11). Research infrastructure was provided collectively by the Centre of Research for Development (CORD) and Department of Biochemistry of the university. Part of the identification work of the samples was carried out at Agharkar Reaserch Institute, Pune, India and the sequencing was performed at SciGenome Laboratory Kochin, India. References [1] L. Cai, K.F. Ji, K.D. Hyde, Variation between freshwater and terrestrial fungal communities on decaying bamboo culms, Antonie Leeuwenhoek 89 (2006) 293e301. [2] W.H. Ho, K.D. Hyde, I.J. Hodgkiss, Yanna, Fungal communities on submerged wood from streams in Brunei, Hong Kong, and Malaysia, Mycol. Res. 105 (2001) 1492e1501. [3] M. Bergbauer, M.A. Moran, R.E. Hodson, Decomposition of lignocellulose from a freshwater macrophyte by aero-aquatic fungi, Microb. Ecol. 32 (1992) 159e167. [4] V. Baldy, E. Chauvet, J.Y. Charcosset, M.O. Gessner, Microbial dynamics associated with leaves decomposing in the main-stem and flood-plain pond of a large river, Aquat. Microb. Ecol. 28 (1) (2002) 25e36. [5] N.N. Smirnov, On the quantity of allochthonous pollen and spores received by the Rybinsk reservoir, Hydrobiologia 24 (1964) 421e429. [6] C.T. Ingold, Endocoenobium eudorinae gen. et sp. nov., a chytridiaceous fungus parasitizing Eudorinaelegans Ehrenb, New Phytol. 39 (1940) 97e103. [7] F.K. Sparrow Jr., Aquatic Phycomycetes, second ed., University of Michigan Press, 1960. [8] J.S. Karling, Chytridiomycetarum iconographia, Lubrecht & Cramer Monticello N.Y., 1977. [9] F. Sparrow, Ecology of freshwater fungi, in: G. Ainsworth, A. Sussman (Eds.), The Fungi-an Advanced Treatise, The Fungal Population 3, 1968, pp. 41e93. [10] W. Cooke, Fungi in sewage, in: E. Jones (Ed.), Recent Advances in Aquatic Mycology, Elek Science, London, 1976, pp. 389e434. [11] S.D. Weber, A. Hofmann, M. Pilhofer, G. Wanner, The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses, FEMS Microbiol. Ecol. 68 (2009) 246e254. [12] M. Wainwright, Oligotrophic growth of fungi, in: 3rdedn, in: J. Dighton, J.F. White, P. Oudemans (Eds.), The Fungal Community: its Organization and Role in the Ecosystem, vol. 23, Taylor & Francis Group, Boca Raton, FL, 2005, pp. 643e658. [13] H.A. Raja, J.P. Schmit, C.A. Shearer, Latitudinal, habitat and substrate distribution patterns of freshwater ascomycetes in Florida Peninsula, Biodivers. Conserv. 18 (2009) 419e455. [14] L. Cai, C.K.M. Tsui, K. Zhang, K.D. Hyde, Aquatic fungi from Lake Fuxian, Yunnan, China, Fungal Divers. 9 (2002) 57e70. [15] L.G. Nikolcheva, T. Bourque, F. Barlocher, Fungal diversity during initial stages of leaf decomposition in a stream, Mycol. Res. 109 (2005) 246e253. [16] C. Pascoal, F. Cassio, Contribution of fungi and bacteria to leaf litter decomposition in a polluted river, Appl. Environ. Microbiol. 70 (2004) 5266e5273. [17] M. Rajashekhar, K.M. Kaveriappa, Diversity of aquatic hyphomycetes in the aquatic ecosystems of the Western Ghats of India, Hydrobiologia 501 (2003) 167e177. [18] C.K.M. Tsui, K.D. Hyde, I.J. Hodgkiss, Longitudinal and temporal distribution of freshwater ascomycetes and dematiaceous hyphomycetes on submerged wood in the Lam Tsuen River, Hong Kong, J. Natl. Am. Benthol. Soc. 20 (2001a) 533e549. [19] C.K.M. Tsui, K.D. Hyde, I.J. Hodgkiss, The effect of glyphosate on lignicolous freshwater fungi of Hong Kong, Sydowia 53 (2001b) 167e174. [20] C.K.M. Tsui, K.D. Hyde, Freshwater Mycology, Fungal Diversity Press, Hong Kong, 2003.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Assessment of species richness in Lake Dal, Kashmir, based on classical approach, physiological approach and rDNA ITS sequences from isolates Suhaib A. Bandh a, *, Azra N. Kamili a, Bashir A. Ganai a, Bashir A. Lone b a b
Microbiology Research Laboratory, Centre of Research for Development/Department of Environmental Science, University of Kashmir, Srinagar, India Parasitology Research Laboratory, Centre of Research for Development/Department, University of Kashmir, Srinagar, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2017 Received in revised form 30 January 2017 Accepted 31 January 2017 Available online 2 February 2017
As a first description to document the species richness in Dal Lake, a freshwater lake ecosystem in Kashmir valley, an extensive network of sixteen sampling stations with distinguishing features was sampled seasonally for two years. The identification process yielded fifty-one species probably first and new records for this area to date. The taxonomic groups observed were those with species from Ascomycetes (inclusive of yeasts), Basidiomycetes, Blastocladiomycetes, Zygomycetes, and Peronosporomycetes. Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In the filamentous fungal group, family Trichocomaceae was dominant followed by Saccharomycetaceae, Mucoraceae, Nectriaceae, Tremellaceae and Hypocreaceae. However, in the group of zoosporic & fungal like eukaryotes, family Saprolegniaceae was most dominant followed by Blastocladiaceae and Pythiaceae. A dramatic decrease in fungal load was observed in different seasons with highest colonial load in the summer season and lowest in the winter season. The observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) as well as zoosporic fungi & fungal like eukaryotes (p < 0.05). In order to assess biodiversity patterns of fungi more accurately, it is necessary to repeat such investigations in other areas and to improve the tools for taxonomic identification of these highly diverse but mostly microscopic organisms. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Dal lake Fungi ITS gene Kashmir
1. Introduction Fungal species reported from aquatic habitats range from those adapted to complete their life cycle in aquatic habitats and not found outside the aquatic environments (residents) to those occurring in water fortuitously by being washed or blown in (transients). Representatives of all fungal groups like Chytridiomycetes, Hyphochytridiomycetes, Peronosporomycetes (formerly Oomycetes), Ascomycetes and Mitosporic fungi have been found in or isolated from aquatic habitats. Freshwater fungi play an important role in freshwater ecosystems mainly due to their role in decomposing woody debris and leaves, with their mycelium and fruiting bodies being available to the animal food web [1e3]. In surface waters, fungal communities form an
* Corresponding author. E-mail address: [email protected] (S.A. Bandh). http://dx.doi.org/10.1016/j.micpath.2017.01.058 0882-4010/© 2017 Elsevier Ltd. All rights reserved.
important component of biotic communities by decomposing and transforming organic material [4] and as a food source [5]. Ingold [6] isolated and described many filamentous fungi, whose spore morphologies were adapted to dispersal in running waters followed by attachment to plant materials like leaves which later came to be known as aquatic Hyphomycetes or ‘Ingoldian fungi’. Fungi, commonly known as chytrids, thrive and reproduce in fresh and marine environments, however, they can also be found commonly in habitats such as terrestrial soils [7,8]. Based on their evolutionary history, fungi in aquatic habitats are either primarily (Chytridiomycetes) or secondarily adapted to life in water (aquatic Hyphomycetes, Yeasts). In addition, primarily terrestrial forms (fungi imperfecti, endophytes) are found in lakes, suggesting that their occurrence is not necessarily restricted to a single habitat by stringent morphological or physiological adaptations. Like Peronosporomycetes (fungus-like organisms belonging to the Kingdom Chromista), Chytridiomycetes (Kingdom Fungi) possess chemotactic, flagellated zoospores, which can disperse in the water
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column [9]. Terrestrial fungi are often passively introduced into lakes in the form of high loads of fungal propagules via inflowing streams, rainwater, and wind [5]. Though fungi are often found in disturbed areas with high anthropogenic loads from various sources like domestic, industrial, municipal and sewage treatment plants [10,11]; many fungal species survive in oligotrophic and ultra-oligotrophic environments, such as in water distillation apparatus [12]. There are many reports of freshwater fungi from both lotic and lentic habitats in different areas [13e25] but nothing has been written on the subject in valley Kashmir. It is in this backdrop the present study was carried out to focus on the unexplored and undocumented fungal diversity of an important freshwater ecosystem of Kashmir valley. 2. Methods
prepared according to the recipes of Pitt [30]. Each filamentous fungal culture was inoculated in triplicate on each medium and incubated at three different temperatures (5 C, 25 C and 37 C) for a period of 7 days in the dark. In addition to these the morphological characteristics were studied by making slide cultures obtained by inoculating microfungi directly on a small square of agar medium [31]. Morphology was characterized by using a semiautomatic image analysis system consisting of an Olympus microscope (Olympus, New Hyde Park, NY, U.S.A.) operated as phase contrast, a charge coupled device (CCD) camera (Sony, Cambridge, U.K.) a PC with a frame-grabber, and the image analysis software (SIS, Olympus, Germany). Sample preparation and measurement was done as described by Papagianni et al. [32,33]. A magnification of 100 was applied for measurement of mycelial particles to estimate the individual mycelia and other micro-morphological features.
2.1. Study area and sites The study area Dal lake (N 34 070, E 74 520 , 1583e1600 m) a Himalayan lake in Srinagar, represents a unique ecosystem with respect to its species diversity, aesthetics and interaction with people living in the houseboats and the small hamlets within the lake. As it is a multi-basined lake with many inlets and outlets, an extensive network of sixteen sampling stations viz. Hazratbal Open (S1), Hazratbal littoral (S2), Nageen Open (S3), Nageen littoral (S4), Gagribal Open (S5), Gagribal littoral (S6), Nishat Open (S7), Near Centeur (S8), Dal Lock Gate-I (S9), Dal Lock Gate-II (S10), BoathallNallah-I (S11), Boathall Nallah-II (S12), Pokhribal Nallah-I (S13), Pokhribal Nallah-II (S14) Tailbal Nallah-I (S15) and Tailbal Nallah-II (S16), from both littoral zones as well as limnetic zones were selected. Among the selected sites, eight (8) sites were selected in four basins, four (4) sites were selected from two inlets and four (4) sites were selected from two outlets. 2.2. Sample collection Water samples from different sites of the lake, under consideration for exploring the species richness were collected on seasonal basis in Polyethylene (PET) bottles, which were previously carefully cleaned and rinsed three to four times with autoclaved distilled water. All the samples were collected just below the surface of lake water by plunging the open end of each bottle before turning it upright to fill. During collection of samples, extreme care was exercised to avoid contamination of the bottles and collected samples were processed for analysis using the standard methodology. 2.3. Isolation and identification of fungi For the isolation of fungi different approached like direct plating method [26] and baiting technique [7,27] were followed. Identification of the isolated fungal species was done on the basis of classical morphological and culture approach, biochemical testing approach and molecular approach. 2.4. Classical morphological and culture approach Identification of the species was performed mainly on the basis of the micro and macro-morphological features of colonies grown on differential media. Fungi were identified to genus level using Barnett and Hunter's work [28]. Cultures were identified to species level using various mycological texts like Penicillium LINK, Raper and Fennell [29] and PITT's monograph [30]. These species were grown on three different media {Czapek Yeast Agar (CYA), Malt Extract Agar (MEA), and 25% Glycerol nitrate Agar (G25A)} all
2.5. Biochemical testing approach Physiological profiling of a few yeast species was carried out by the biochemical tests included Physiological tests, Fermentation tests and Assimilation tests. The inoculated microplates were shaken (with a microplate shaker) just before automatic reading using a microplate reader. Absorbance values at 405 nm were transferred by cable (RS-232 through a serial port) to the computer and transformed by the BioloMICS software into negative, weak or positive results. The results of every test were transformed independently.
2.6. Molecular approach 2.6.1. Growth and harvesting cells from liquid cultures The fungal colonies were grown in Potato Dextrose Broth and Sabouraud Dextrose Broth containing 10 ml of Tween-80 as it keeps the fungal cells dispersed and helps in weighing the culture. After inoculation of fungal cultures, incubation at 30 C, preferably on a shaker was done.
2.6.2. DNA extraction The weighed fungal tissue was ground to a fine powder in a mortar and pestle using liquid nitrogen which was then allowed to evaporate and samples were not allowed to thaw. DNA was extracted using a HiPur A™ SP Fungal DNA Mini kit (Himedia) following the manufacturers protocol and the purity of DNA was measured by the absorbance ratio (A260/A280).
2.6.3. Polymerase chain reaction (PCR) ITS region of the rRNA operon was amplified using ITS1 and ITS4 primers [34]. Amplification was performed in 50 ml PCR reaction tubes containing 6 ml genomic DNA, 5 ml 10 thermophilic buffer, 2 ml MgCl2, 1.4 ml dNTP (10 mM), 1.2 ml (10 mM) ITS1, 1.2 ml (10 mM) ITS4, 6 ml (10u/ml) Taq (Promega) and 32 ml ddH2O in an Eppendorf Thermal Cycler with the following program for 40 cycles: initial denaturation temperature 94 C for 5 min, melt temperature 94 C for 50 s, annealing temperature 54.5 C for 45 s, extending temperature of 72 C for 45 s, final extension temperature 72 C for 10 min and hold temperature of 4 C. The ITS bands were identified by gel electrophoresis on a 2% agarose gel. PCR products obtained were sequenced and the sequences obtained from both primers were BLAST searched to conform the percent similarity to the ITSI, 5.8S rRNA gene and ITSII regions of the respective fungi in the Gene Bank (Fig. 4).
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Table 1 Systematic diversity of the isolated taxa. Kingdom
Phylum
Class
Fungi
Ascomycota
Chromista
Basidiomycota Blastocladiomycota Zygomycota Heterokontophyta
Number of taxa
Eurotiomycetes Sordariomycetes Ascomycetes Saccharomycetes Tremellomycetes Blastocladiomycetes Zygomycetes Peronosporomycetes
3. Results Species richness assessed in the water samples collected from Dal lake on seasonal basis from sixteen sites of the lake belonged to two groups viz. the filamentous fungi and zoosporic fungi and fungal like eukaryote group with twenty-eight (28) species belonging to the former and twenty-three (23) species belonging to the later. Species isolated and identified during the study belonged to five major phyla with twenty-five (25) species belonging to Ascomycota, one (1) species to Basidiomycota, four (4) species to Blastocladiomycota, two (2) species to Zygomycota and nineteen (19) species to Heterokontophyta. Phylum Ascomycota consisted of four major classes Eurotiomycetes, Sordariomycetes, Ascomycetes and Saccharomycetes; phylum Basidiomycota consisted of one class
Table 2 Total count (TC), % total count (%TC), number of cases of isolation (NCI) and occurrence remarks (OR) of filamentous fungi. Family and Species
TC
% TC
NCI
OR
Trichocomaceae Aspergillus Aspergillus flavus Aspergillus fumigatus Aspergillus japonicus Aspergillus niger Aspergillus oryzae Aspergillus terreus Aspergillus versicolor Aspergillus wentii Penicillium Penicillium caseicolum Penicillium chrysogenum Penicillium commune Penicillium dimorphosporum Penicillium funiculosum Penicillium lilacinum Penicillium olivicolor Penicillium spp. I Penicillium spp. II Penicillium spp. III Penicillium spp. IV Mucoraceae Mucor spp. Rhizopus stolonifer Nectriaceae Fusarium oxysporum Hypocreaceae Acremonium strictum Saccharomycetaceae Candida albicans Candida krusei Candida parapsilosis Candida glabrata Tremellaceae Cryptococcus neoformans Total
2517 1073 178 98 98 218 72 110 142 157 1444 123 334 63 128 236 78 15 100 84 136 147 186 94 92 80 80 36 36 282 84 83 9 106 56 56 3157
79.72 e 5.64 3.1 3.1 6.9 2.28 3.48 4.5 4.98 3.9 10.58 1.99 4.05 7.47 2.47 0.47 3.17 2.66 4.3 4.65 5.9 2.98 2.91 2.53 2.53 1.14 1.14 8.94 2.66 2.63 0.29 3.36 1.77 1.77 -
e e 15 14 15 16 11 10 15 14 e 14 16 7 14 16 7 4 14 12 14 11 15 9 11 e 11 11 14 3 10 6 e
H e H H H H H H H H e H H M H H M L H H H H e H H e H R e e H H L H e M e
Order
Genera
Species
1 1 1 1 1 1 1 2
2 3 1 1 1 1 2 6
17 3 1 4 1 4 2 19
Tremellomycetes, Blastocladiomycota of one class Blastocladiomycetes and Heterokontophyta of one class Peronosporomycetes, and Zygomycota of one class Zygomycetes (Table 1). Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In all genus Penicillium was represented by a maximum of eleven species with a relative abundance of 21.6% followed by genus Aspergillus eight species (15.7%), Achyla five species (9.8%), Candida, Allomyces, Brevilegnia and Pythium four species (8%) each, Aphanomyces three species (5.9%), Dictyuchus two species (3%), Mucor, Rhizopus, Fusarium, Acremonium, Cryptococcus and Saprolegnia one species (2%) each. The species isolated belonged to nine families with maximum contribution of nineteen species by family Trichocomaceae followed by fifteen species of family Saprolegniaceae, four species each by Saccharomycetaceae, Blastocladiaceae and Pythiaceae, two species by family Mucoraceae and one species each by family Nectriaceae, Hypocreaceae and Tremellaceae.
Table 3 Total count (TC), % total count (%TC), number of cases of isolation (NCI) and occurrence remarks (OR) of zoosporic fungi and fungal like eukaryotes. Family and Species
TC
% TC
NCI
OR
Blastocladiaceae Allomyces moniliformis Allomyces anomalus Allomyces arbuscula Allomyces spp. Saprolegniaceae Achyla Achyla klebsiana Achyla apiculata Achyla flagellate Achyla cornuta Achyla conspicua Brevilegnia Brevilegnia indica Brevilegnia linearis Brevilegnia diclina Brevilegnia subclavata Dictyuchus Dictyuchus monosporus Dictyuchus spp. Aphanomyces Aphanomyces laevis Aphanomyces cladogamous Aphanomyces spp. Saprolegnia Saprolegnia parasitica Pythiaceae Pythium proliferum Pythium elongatum Pythium debaryanum Pythium spp. Total
659 220 154 181 104 2474 791 131 85 105 261 209 679 136 143 221 179 401 133 268 323 76 107 140 280 280 650 204 124 206 116 3783
17.42 5.81 4.07 4.79 2.75 65.39 31.97 3.46 2.25 2.77 6.9 5.52 27.45 3.6 3.78 5.84 4.73 16.2 3.52 7.08 13.06 2.01 2.83 3.7 11.32 7.4 17.19 5.4 3.28 5.44 3.07 -
e 15 12 14 12 e 15 12 11 16 16 16 15 15 15 16 16 11 12 16 16 e 13 12 16 14 e
H H H H H e e H H H H H e H H H H e H H e H H H e H H H H H H e
H¼ High; M ¼ Medium; L ¼ Low.
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Fig. 1. Seasonal fluctuation of fungal load.
Tables 2 and 3 summarizing the family and species wise total count, % total count, number of cases of isolation and occurrence remarks, depicts that among the filamentous group, different species of family Trichocomaceae were isolated in a low to high frequency represent 79.72% of this group with a total colony count of 2517. Different species of genus Aspergillus constituting 33.99% of the whole group and 42.63% of the family Trichocomaceae showed a total count of 1073 colonies. Genus Penicillium showed a total count of 1444 colonies constituting 45.74% of the whole group and 57.37% of the family Trichocomaceae. Family Mucoraceae isolated in a high frequency with a total count of 186 colonies constituted 5.9% of this group. Families Nectriaceae, Hypocreaceae and Tremellaceae were represented respectively by F. oxysporum, A. strictum and C. neoformans. Family Saccharomycetaceae isolated with a low to high frequency was represented by four species. It constituted 8.94% of the group with a total colony count of 28. Among the different families of zoosporic fungi and fungal like eukaryotes, family Saprolegniaceae was most dominant with a maximum number of 2474 colonies of fifteen (15) species constituting 65.39% of the group followed by family Blastocladiaceae with 659 colonies of four (4) species constituting 17.42% and family Pythiaceae with 650 colonies of four (4) species 17.19%. Showing considerable seasonal variations (Fig. 1) at all the selected sites highest number of filamentous fungal colonies 1208 (38.26%) was registered in summer season followed by 972 colonies (30.79%) in spring, 660 colonies (20.91%) in autumn and the least number of 317 colonies (10.04%) in winter season. Similarly, maximum number of zoosporic fungal and fungal like eukaryote
colonies 1380 (36.48%) was observed in summer followed by 1251 colonies (33.07%) in spring, 709 colonies (18.74%) in autumn and least number of colonies 443 (11.71%) in winter. One-way analysis of variance (ANOVA) showed that the observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) and zoosporic and fungal like eukaryotic species (p < 0.05). Explanatory data analysis by Box and Whisker Plot model shown in Figs. 2 and 3 on the basis of all experimental data for both the groups, from all the selected microhabitats studied as an indicator of centrality, spread and similarity lined up side by side showing the main characteristics of their distribution shows that for filamentous fungi (Fig. 2) site 14 and 16 appear to have similar centers exceeding the rest of the sites while as sites 3, 4, 5, 6 and 12 appear to have similar centers lying towards the base of the graph. Site 14 appears to have the largest variability along with site 13 and 16 than the other thirteen sites. However, sites 4, 5, 6 and 12 are reasonably symmetric compared to rest of the sites. For zoosporic fungi and fungal like eukaryotes (Fig. 3) it can be seen that site 14 appears to have the center which exceed the rest of the sites. Here also site 13 and 14 appear to have largest variability along with site 8 than the other thirteen sites and the sites 4, 5, 6 and 12 are reasonably symmetric compared to rest of the sites.
4. Discussion Microorganisms play important roles in the environment. Studies of microbial community composition therefore, provide potentially descriptive information about ecosystem function [35]. Fungi and fungus-like organisms show a high capacity for adaptation to the environmental conditions, and are therefore widely spread in nature [36]. The fungal community present in the lake water was assessed using the traditional method of culture and baiting as well as the molecular techniques. The use of these techniques for the isolation of fungal communities from different water bodies and other environmental samples has been done in many other studies [37e44]. The majority of fungal isolates (50%) obtained from the lake belonged to Ascomycota and only a meager percentage (0.5%) Basidiomycota is due to the fact the water of Dal Lake is not that deficient in dissolved oxygen as proved by some limnological studies carried out recently [45] on this water body. The results here are well supported by the work of Shearer et al. [46] reporting that taxonomically, the aquatic fungi known as
Fig. 2. Box and Whisker Plot Model for filamentous fungi.
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Fig. 3. Box and Whisker Plot Model for zoosporic fungi and fungal like eukaryotes.
“aquatic hyphomycetes” found most commonly in clean and well oxygenated [47] are mainly associated with the Ascomycota, and only a small percentage is affiliated with the Basidiomycota. The predominance of Ascomycota in the aquatic ecosystems like Dal lake has been discussed by many authors [48e53]. The variations in the fungal community characteristics of the lake can be attributed to the heterogeneity in the water characteristics of the aquatic habitats as they are heterogeneous in time and space and greatly differ in their physico-chemical features. Favoring the current observation, a recent review of the ecology of fungi in lake ecosystems by Wurzbacher et al. [54] mentioned that the fungal communities differ significantly between and within the different water bodies as the aquatic habitats are characterized by a unique balance of allochthonous (external) and autochthonous (internal) organic matter supply, controlled largely by watershed characteristics, surface area and location. In aquatic systems, the fungal community structure greatly differs between substrates [55e59] and with the physico-chemical properties of the respective habitats, such as flow [4,60] dissolved oxygen concentration [61,62], nutrient concentrations [63,64], salinity [65,66], temperature [67] and depth [54]. The occurrence of zoosporic fungi in the lake waters is not surprising as they have been found to be universally present in all types of freshwater systems and occur as saprotrophs on a wide variety of substrata, playing a key role in those ecosystems as decomposers of organic materials [68]. Most studies dealing with zoosporic fungi have provided extensive inventories of taxa from specific sites or geographic regions, but often without characterizing the microhabitat, determining frequencies of occurrence or relative abundance of species [7,8,69,70]. The isolation of different fungal species belonging to both the zoosporic group and filamentous group of fungi in the present study corroborates well with the study of Rajanaika et al. [71]. Useful correlations could be made between temperature and the occurrence of Peronosporomycetes in this study as this was probably because the study extended over two years of essentially variable climatic conditions and because there was more variation in temperature within different seasons of a year. Seasonal variation observed in the fungal population was possibly due to the temperature variations, as the maximum percentage of fungi was found during high temperature (summer) and minimum during low temperature (winter) season. Similar results were obtained by Khulbe and Durgapal [72], in their study on the population dynamics of geofungi in a polluted freshwater body at Nainital. A preliminary
mycological study on the same lake by Bhat and Kamili [73] had also reported the presence of filamentous fungal genera especially Aspergillus and Penicillium with a similar fluctuation in their numbers during different seasons. The seasonal variation found in this study is also in consonance with the results of Sharma and Parveen [74]. The influence of temperature on the fungal load is further supported by the work of Stoll [75] mentioning that the occurrence and distribution of water molds is directly related to the water temperature. Well in support of the results of present study Ho [76] also found a positive correlation between the water temperature and the isolation frequency of water molds. The presence of the terrestrial molds like Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Acremonium spp., Penicillium chrysogenum, P. caseicolum, P. commune, P. lilacinum, Fusarium oxysporum and Rhizopus stolonifer in the Dal waters is due to the influence of catchment area like entry of litter, soil, dead plant parts, sewage and the influence of air as the occurrence of these terrestrial fungal species in aquatic systems are likely to originated from air [9] as well as from living or dead animal and plant parts, soil and litter being in contact with
Fig. 4. Amplified rDNA ITS regions of specified fungi. Lane no. 1 ¼ 100bp Ladder; 2 ¼ A. terreus; 3 ¼ A. flavus; 4 ¼ A. japonicas; 5 ¼ A. versicolor; 6 ¼ A. klebsiana; 7 ¼ A. flagelata; 8 ¼ A. apiculata; 9 ¼ A. moniliformis; 10 ¼ A. anomalous; 11 ¼ D. monosporus; 12 ¼ A. leivis.
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water [77]. Filamentous fungi belong to the genera Aspergillus, Penicillium and Fusarium in water bodies have also been reported in many studies p [78,79]. These frequently found fungi could be considered as indicators of the generic composition of the water body. The occurrence of these fungal species in water samples of Dal Lake is not new but it is surely a new report in the lake as no study has been carried out till now on the fungal community of the lake. Conflicts of interest We declare that the research doesn't have any conflict of interest. Acknowledgements S. Bandh was supported by PhD grant from University of Kashmir, Srinagar (Grant No. PhD-G/Env.Sc/KU/176-11). Research infrastructure was provided collectively by the Centre of Research for Development (CORD) and Department of Biochemistry of the university. Part of the identification work of the samples was carried out at Agharkar Reaserch Institute, Pune, India and the sequencing was performed at SciGenome Laboratory Kochin, India. References [1] L. Cai, K.F. Ji, K.D. Hyde, Variation between freshwater and terrestrial fungal communities on decaying bamboo culms, Antonie Leeuwenhoek 89 (2006) 293e301. [2] W.H. Ho, K.D. Hyde, I.J. Hodgkiss, Yanna, Fungal communities on submerged wood from streams in Brunei, Hong Kong, and Malaysia, Mycol. Res. 105 (2001) 1492e1501. [3] M. Bergbauer, M.A. Moran, R.E. Hodson, Decomposition of lignocellulose from a freshwater macrophyte by aero-aquatic fungi, Microb. Ecol. 32 (1992) 159e167. [4] V. Baldy, E. Chauvet, J.Y. Charcosset, M.O. Gessner, Microbial dynamics associated with leaves decomposing in the main-stem and flood-plain pond of a large river, Aquat. Microb. Ecol. 28 (1) (2002) 25e36. [5] N.N. Smirnov, On the quantity of allochthonous pollen and spores received by the Rybinsk reservoir, Hydrobiologia 24 (1964) 421e429. [6] C.T. Ingold, Endocoenobium eudorinae gen. et sp. nov., a chytridiaceous fungus parasitizing Eudorinaelegans Ehrenb, New Phytol. 39 (1940) 97e103. [7] F.K. Sparrow Jr., Aquatic Phycomycetes, second ed., University of Michigan Press, 1960. [8] J.S. Karling, Chytridiomycetarum iconographia, Lubrecht & Cramer Monticello N.Y., 1977. [9] F. Sparrow, Ecology of freshwater fungi, in: G. Ainsworth, A. Sussman (Eds.), The Fungi-an Advanced Treatise, The Fungal Population 3, 1968, pp. 41e93. [10] W. Cooke, Fungi in sewage, in: E. Jones (Ed.), Recent Advances in Aquatic Mycology, Elek Science, London, 1976, pp. 389e434. [11] S.D. Weber, A. Hofmann, M. Pilhofer, G. Wanner, The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses, FEMS Microbiol. Ecol. 68 (2009) 246e254. [12] M. Wainwright, Oligotrophic growth of fungi, in: 3rdedn, in: J. Dighton, J.F. White, P. Oudemans (Eds.), The Fungal Community: its Organization and Role in the Ecosystem, vol. 23, Taylor & Francis Group, Boca Raton, FL, 2005, pp. 643e658. [13] H.A. Raja, J.P. Schmit, C.A. Shearer, Latitudinal, habitat and substrate distribution patterns of freshwater ascomycetes in Florida Peninsula, Biodivers. Conserv. 18 (2009) 419e455. [14] L. Cai, C.K.M. Tsui, K. Zhang, K.D. Hyde, Aquatic fungi from Lake Fuxian, Yunnan, China, Fungal Divers. 9 (2002) 57e70. [15] L.G. Nikolcheva, T. Bourque, F. Barlocher, Fungal diversity during initial stages of leaf decomposition in a stream, Mycol. Res. 109 (2005) 246e253. [16] C. Pascoal, F. Cassio, Contribution of fungi and bacteria to leaf litter decomposition in a polluted river, Appl. Environ. Microbiol. 70 (2004) 5266e5273. [17] M. Rajashekhar, K.M. Kaveriappa, Diversity of aquatic hyphomycetes in the aquatic ecosystems of the Western Ghats of India, Hydrobiologia 501 (2003) 167e177. [18] C.K.M. Tsui, K.D. Hyde, I.J. Hodgkiss, Longitudinal and temporal distribution of freshwater ascomycetes and dematiaceous hyphomycetes on submerged wood in the Lam Tsuen River, Hong Kong, J. Natl. Am. Benthol. Soc. 20 (2001a) 533e549. [19] C.K.M. Tsui, K.D. Hyde, I.J. Hodgkiss, The effect of glyphosate on lignicolous freshwater fungi of Hong Kong, Sydowia 53 (2001b) 167e174. [20] C.K.M. Tsui, K.D. Hyde, Freshwater Mycology, Fungal Diversity Press, Hong Kong, 2003.
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