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Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India)
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MYCMED-815; No. of Pages 5 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
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Original article/Article original
Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India) S.K. Deshmukh a,*, S.A. Verekar b, Y.G. Chavan c a
TERI-Deakin Nano Biotechnology Centre, The Energy and Resources Institute, Darbari Seth Block, IHC Complex, Lodhi Road, 110003 New Delhi, India MicroChem Silliker Pvt. Ltd., Microchem House, A513, TTC Ind. Area, MIDC, Mahape, 400701 Navi Mumbai, India c geneOmbio Technologies Pvt. Ltd., Vedant, No 39/3, Yogi Park, Baner, 411045 Pune, Maharashtra, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 January 2018 Received in revised form 2 May 2018 Accepted 6 June 2018 Available online xxx
Forty soil samples were collected from seven sites in the vicinity of Sambhar lake Rajasthan, India and screened for the presence of keratinophilic fungi using hair baiting techniques for isolation. Seventeen isolates were recovered and identified. The cultures were identified by recognition of their macro- and micro- morphological features. Their identification was confirmed by BLAST using ITS1-5.8S-ITS2 rDNA region and sequences have been deposited in NCBI data base. A total of 34 species belonging to 29 genera were isolated. Among the dermatophytes and related keratinophilic fungi Chrysosporium indicum was predominant followed by Ctenomyces serratus, C. tropicum, Keratinophyton durus, Auxarthron conjugatum, Gymnascella dankaliensis, Gymnoascoideus petalosporus and Uncinocarpus reesii. Twenty-six species belonging to 22 genera represented other species. Our study indicates that keratinophilic fungi and species are found in the soils at the vicinity of the Sambhar Lake, and human activities can be the potential source of pathogenic fungi. C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Sambhar salt lake Salt pan Soil fungi Keratinophilic fungi India Alkaline saline environment
1. Introduction Sambhar Lake is situated at 60 km west of Jaipur in Rajasthan, India and is the largest salt lake (27*00’N, 75*00’E) covering 24,000 ha area. It is a shallow wetland whose depth ranges between 0.5 and 2 meters. Four main streams feed the lake, which is roughly elliptical in shape, from a drainage area of about 268,800 ha. On the eastern end, the lake is divided by a 5-km long dam made of stone. Further east of the dam is salt evaporation ponds where salt has been farmed for thousands of years. This part also has a railroad, built by the British (before India’s independence) that provides access from Sambhar lake city to the salt works. Sambhar lake brine is somewhat unique with a very low potassium concentration. The vegetation present in the catchment area is mostly xerophytic. The waters of Sambhar Lake have been used for centuries to make salt. There is, however, another distinctive feature of this extensive saline wetland. The waters here are glacially still edged with a glittering frost of salt. There is a sharp briny tang in the air that takes one straight back to coastal
* Corresponding author. E-mail addresses: [email protected] (S.K. Deshmukh), [email protected] (S.A. Verekar), [email protected] (Y.G. Chavan).
fish markets. The Sambhar lake city is the largest of human settlements around the lake. Sambhar Lake is famous for harboring large number of flamingos. Together with adjacent saline wetlands, Phulera and Deedwana, the lake is probably the most important wintering area for flamingoes (both Phoniconaias minor and Phoenicopterus roseus) in India, second only to the Rann of Kutch, where they breed. These beautiful tall birds flock the lake and enjoy the feast of large numbers of algae that swarm the lake. Pelican is another species of birds that one can see at the lake. Some other birds at the lake are storks, redshanks, sandpipers, coots, black-winged stilts, and shovelers. However, Sambhar’s ferocious brine is too saline for many species as birds can be found in the freshwater ponds in the surrounding areas. The Naliasar Pond, just 4 km south of Salt Lake City, is crammed with waterfowl-shovelers, common teals, pintails, common pochards, tufted pochards, gadwalls, graylag and bar-headed geese and even busty shelduck that fly swiftly overhead. The terrestrial fauna confined to the catchment area includes rare/threatened species such as the Uromastix, saw-scaled viper, desert cat and desert fox. This ecological diversity makes it a potential area of interest to study the distribution of keratinophilic fungi from soils of Sambhar Lake. The present investigation reports the isolation of these fungi from soils of Sambhar Lake.
https://doi.org/10.1016/j.mycmed.2018.06.002 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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2. Materials and methods Forty soil samples were collected from seven sites of Sambhar lake, Rajasthan state India April 2013. The samples were taken from the banks of the lake: approximately 10 feet from water line (dried crust of soil with dried salt layers), bottom sediments: from 1 feet depth of shallow water, adjacent marshy meadows: semimoist region with wild xerophytic plants and weed around some peripheral region of the lake (radish salty soil), salt pan, salt storage areas and dropped off feathers. Soil samples were collected by scraping a layer of soil not exceeding 5 cm in depth. The bottom sediments samples were also obtained from different layer of soil from bank. The samples were brought to the laboratory and processed promptly. The brines had pH of 9.5 0.2 and a total salt content ranging from 7% (w/v) to more than 30% (w/v). Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sulphate were the principal salts present in these brines that lacked divalent cations (calcium and magnesium). Sambhar has a tropical climate. The summers can be scorching with mercury crossing 45 8C, whereas winters are moderately chilled and the temperature can fall below 10 8C. The hair bait technique of Vanbreuseghem [1] was used to isolate keratinophilic fungi. For this purpose, sterile petri dishes were half filled with the soil samples and moistened with water and baited by burying sterile human hairs in the soil. These dishes were incubated at room temperature and examined daily after five days for fungal growth over a period of four weeks. After observing the growth under a stereoscopic binocular microscope it was cultured on Sabouraud’s dextrose agar (HiMedia) amended with chloramphenicol (HiMedia) (50 mg/L) in one set and Sabouraud’s dextrose agar amended with chloramphenicol (50 mg/L) and cycloheximide (HiMedia) (500 mg/L) in other set. These fungi were identified based on the monographs of Domsch et al. [2], Oorchschot [3], Sigler and Carmichael [4], Currah [5], Cano and Guarro [6], Arx von [7], Domsch et al. [8], Seifert [9]. The molecular identification of isolated colony from each culture was performed using DNA sequencing of the ITS1-5.8SITS2 region. Genomic DNA was extracted by the miniprep protocol of Lee and Taylor [10]. The ITS1-5.8S-ITS2 rDNA was amplified using primers ITS1 and ITS4 as the forward and reverse primers as described by White et al. [11]. Amplification was performed in 100 mL reaction volumes containing 10 buffer 10 ml, MgCl2 (25 mM) 2 ml, dNTP (10 mM) 2 ml, ITS1 primer (20 pm) 2 ml, ITS4 primer (20 pm) 2 ml, Taq Polymerase (2.5U) 1 ml, DNA Sample (5 mg/ml) 3 ml, and Milli Q Water 78 ml. The PCR reaction was carried out using a Thermal Cycler (M.J. Research, PTC 200) with conditions as follows: denaturation for five minutes at 94 8C, 34 cycles of (30 s at 94 8C, 30 s at 55 8C, 1 min at 72 8C) extension for four minutes at 72 8C and storage at 4 8C. Negative controls were used in each set of reactions. The final products were analyzed by electrophoresis on 2.0% agarose gel (Sigma). The PCR products were purified using Gel extraction kit (Qiagen, CAT No. 28704) and then sequenced using ITS1 and ITS4 primers at geneOmbio Technologies Pvt Ltd, Pune, India, using Applied Biosystems 3730 DNA analyzer. Phylogenetic analysis: Similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank [12]. For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using MEGA6 [13].
3. Results and discussion A total of 34 fungi belonging to 29 genera were isolated from the soil samples collected from various sites of Sambhar Lake and %
frequency occurrence of keratinophilic fungi isolated is given in Table 1. Of these 8 fungi were dermatophyte and related fungi, which accounted for 26.32% of the total fungal flora, whereas the other 26 fungi contributed 73.64%. Amongst the keratinophilic fungi Chrysosporium indicum was most frequent (100%) followed by Ctenomyces serratus, C. tropicum, Keratinophyton durus, Auxarthron conjugatum, Gymnascella dankaliensis, Gymnoascoideus petalosporus and Uncinocarpus reesii. Among other fungi Cladosporium cladosporioides (100%) occurred most frequently and contributed 7.89% to the fungal flora. Whereas Acremonium sp., Aspergillus aculeatus, Aspergillus versicolor, Fusarium nematophilum, Fusarium solani, Penicillium verruculosum, Sarocladium strictum, Syncephalastrum racemosum were recovered from only one site with a least of 1.32% contribution each. The DNA fragments amplified using PCR by primers ITS1 and ITS4 vary between 509 and 548 bp in length. The fragment contained the 30 end of 18S rDNA, ITS1, 5.8S rDNA, and ITS2 and the 50 end of 28S rDNA. The presence and similarity of the sequence in thirty four different isolates was ascertained using the CLUSTAL W multiple sequence alignment available at www.ebi.ac.uk/Tools/ msa/clustalw2. The similarity based on the ITS region (ITS1, 5.8S rDNA, and ITS2 regions) between these isolates varied from 59 to 100% as per the alignment. Fig. 1 displays the Maximum Composite Likelihood tree constructed by comparing the sequence identities of the ITS regions in different isolates. The eight different isolates sequences were identified by BLAST analysis. Most significant BLAST hit obtained was considered as reference sequence for each sequence to be used in construction of phylogenetic tree. In the present study, C. indicum was the most frequently isolated species with 7.89% distribution. Its high percentage of distribution indicated that it is well adapted to Indian climatic conditions, as it can tolerate higher temperature, pH and salt. In our previous report, we have found that keratinophilc fungi isolated from Lonar crater can tolerate 5% salt concentration [14]. C. serratus was found next to C. indicum. It has been reported from various part of India [15–19]. Chrysosporium tropicum comprised 7.5% in distribution. It is a cosmopolitan species and has been reported from different parts of India [20–23]. In the present study, A. conjugatum was 2.63% in distribution. It is reported from India’s plains [24–26]. Other fungi isolated were K. durus (2.63%), Gymnoascella dankaliensis (1.32%), U. reesii (1.32%) G. petalosporus (1.32%). K. durus was previously recorded from meteorite crater of Lonar [14], Gir Forest National Park and Wildlife Sanctuary, Gujarat, (India) [26], Sanjay Gandhi National Park (SGNP) [27], Kaziranga National Park [19]. Gymnoascella dankaliensis was reported from usar soils of Uttar Pradesh [30]. Ajello and Padhye [29] isolated U. reesii as Gymnoascus reesii from the nesting sites of blue footed boobies and greater frigate birds from the xeric low lying coastal areas of the Galapagos Islands. It has also been isolated from Chilka Lake, which is the largest saline lake in India [28] and from salt pan soils of Mumbai [18]. G. petalosporus was previously recorded from Chilka Lake soil [30], soils from Vedanthangal Water Bird Sanctuary [31], and feathers of birds of Orissa [32], and Soils of Vidharbha [33]. We are reporting for the first time the presence of keratinophilic fungi from Sambhar Lake Rajasthan (India). The fungi other than dermatophytes and related species, 26 fungal species belonging to 22 genera were recovered from the soil samples collected and were 73.64% in distribution. Among the other fungi C. cladosporioides (7.89%), was predominant followed by Exophiala spinifera (5.26%), Trichoderma virens (5.26%), Aureobasidium pullulans (3.95%), Chaetomium globosum (3.95%), Fusarium oxysporum (3.95%), Nigrospora oryzae (3.95%), Alternaria daucifolii (2.63%), Bipolaris rostrate (2.63%), Bipolaris stenospila (2.63%), Cochliobolus verruculosus (2.63%), Curvularia spicifera (2.63%), Humicola fuscoatra (2.63%), Myrothecium verru-
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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Table 1 Distribution of keratinophilic fungi and % frequency in soil samples of Sambhar Lake. Genus and species Dermatophytes and closed related species Auxarthron conjugatum Chrysosporium indicum Chrysosporium tropicum Ctenomyces serratus Gymnoascoideus petalosporus Gymnoascella dankaliensis Keratinophyton durus Uncinocarpus reesii Other species Acremonium sp. Alternaria daucifolii Aspergillus aculeatus Aspergillus versicolor Aureobasidium pullulans Bipolaris rostrate Bipolaris stenospila Chaetomium globosum Cladosporium cladosporioides Cochliobolus verruculosus Curvularia spicifera Exophiala spinifera Fusarium nematophilum Fusarium oxysporum Fusarium solani Humicola fuscoatra Myrothecium verrucaria Nigrospora oryzae Penicillium verruculosum Sarocladium implicatum Sarocladium strictum Syncephalastrum racemosum Trichoderma virens Thielavia subthermophila Torulaher barum Yunnania penicillata Total sp./site Frequency occurrence at site
GenBank accession number (s)
Bank of the lake
MF996764 MF996760 MF996758 MF996761 MF996762 MF996760 MF996759 MF996763
+ + + + – + – –
MG551554 MG551556 MG551557 MG551559 MG551558 MG551564 MG551561 MG685669 MG685670 MG685671 MG551562 MG685672 MG551567 MG551565 MG551566 MG551568 MG685673 MG685674 MG551571 MG551553 MG551555 MG551574 MG551576 MG551575 MG685675 MG551572
– – – – + – + – + – + + – + – + – – + – – – – + + + 16 47.06
Banks of salt pans
Bottom sediments
Adjacent marshy meadows
Burrows
Dropped off feathers
Total
% distribution
% frequency
– – – + –
+ + + + – – – +
– + – + – – + –
– + – – – – – –
– + + + + – – –
2 6 3 4 1 1 2 1
2.63 7.89 3.95 5.26 1.32 1.32 2.63 1.32
33.33 100 50 66.67 16.67 16.67 33.33 16.67
– + + – + + – – + + + – – – – – – + – + – – – – – – 11 32.35
– + – – – – – + + – – + – – – – + – – – + – + – – – 12 35.29
– – – – + + – – + – – + + + – – – + – – – – + + – – 12 35.29
– – – + – – + + + – – + – + + – – – – – – + + – + – 11 32.35
+ – – – – – – + + + – – – – – + + + – + – – + – – + 14 41.18
1 2 1 1 3 2 2 3 6 2 2 4 1 3 1 2 2 3 1 2 1 1 4 2 2 2 76
1.32 2.63 1.32 1.32 3.95 2.63 2.63 3.95 7.89 2.63 2.63 5.26 1.32 3.95 1.32 2.63 2.63 3.95 1.32 2.63 1.32 1.32 5.26 2.63 2.63 2.63 100
16.67 33.33 16.67 16.67 50 33.33 33.33 50 100 33.33 33.33 66.67 16.67 50 16.67 33.33 33.33 50 16.67 33.33 16.67 16.67 66.67 33.33 33.33 33.33
+
caria (2.63%), Sarocladium implicatum (2.63%), Thielavia subthermophila (2.63%), Torula herbarum (2.63%), Yunnania penicillata (2.63%), Acremonium sp. (1.32%), A. aculeatus (1.32%), A. versicolor (1.32%), F. nematophilum (1.32%), F. solani (1.32%), P. verruculosum (1.32%), S. strictum (1.32%) and S. racemosum (1.32%). There are reports on various types of infections caused by species of Chrysosporium in human and animals particularly in immuno-compromised patients [34]. For example Chrysosporium zonatum was reported to cause disseminated infection in a patient with chronic granulomatous disease [35]. In Japan, C. zonatum strains were isolated from bronchial lavage from a female in Chiba and from a male in Kyushu. Both patients presented pulmonary cavity sites [36]. Chrysosporium tropicum was reported from comb lesion in two different breeds of chicken in India [37]. G. dankaliensis was reported from superficial infections in human beings [38]. Iwen et al. [39] reported Gymnascella hyalinospora from the invasive pulmonary infection in a patient with acute myelogenous leukemia. Other than dermatophytes and related species, 26 fungal species belonging to 22 genera were recovered from the soil samples collected. Potential human fungi isolated in the present study were E. spinifera, C. cladosporioides, Curvularia spicifera, etc. E. spinifera has been reported to cause phaeohyphomycosis and chromoblastomycosis in humans [40]. Cladosporium is rarely reported from invasive infections [41]. Bentz and Sautter [42] reported a mixed disseminated infection by Aspergillus fumigatus and C. cladosporioides in an immunocompromised patient. C. cladosporioides has been reported from two clinical cases
involving the central nervous system [43]. Gugnani et al. [44] reported a case of subcutaneous phaeohyphoymycosis caused by C. cladosporioides in a 25-year-old male. The other fungi include Curvularia, Fusarium, etc. The fungal genus Curvularia includes some emerging opportunistic pathogens of humans. Curvularia spicifera is reported as important agents of opportunistic infections in vertebrates [45]. In humans, Fusarium species cause a broad spectrum of infections, including superficial (such as keratitis and onychomycosis), locally invasive, or disseminated infections, with the last occurring almost exclusively in severely immunocompromised patients [46]. Fusarium species may also cause allergic diseases (sinusitis) in immunocompetent individuals [47] and also in Fusarium Infections in Immunocompromised Patients [48]. As such, there is no published report of fungal infection from Sambhar lake. However, there are few reports on dermatophytic and non-dermatophytic infections from Rajasthan where the Sambhar lake is situated [49–51]. Apart from dermatophytic infections, there are reports of non-dermatophytic infections caused by Curvularia spicifera, Cladosporium cladosporioides, Exophiala spinifera, Fusarium sp. Aspergillus sp. from various parts of India [52–56]. Nath et al. [52] reported two cases of subcutaneous mycosis from Assam, north-east India, caused by C. cladosporioides and Bipolaris cynodontis in immunocompetent individuals clinically presenting as chromoblastomycosis. A case of cutaneous ulcers due to Bipolaris spicifera in a patient with diabetes and alcohol abuse from a 47-year-old male who is resident of rural area of Amritsar, Punjab, India was reported by Sharma et al.
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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Fig. 1. Phylogenetic relationship of the nine isolates of fungi based on ITS1, 5.8S rDNA, and ITS2 regions of DNA sequences. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of transitional substitutions per site. The analysis involved 67 nucleotide sequences. Evolutionary analyses were conducted in MEGA6. Sequence obtained from each sample show maximum similarity with its closed phylogenetic member as depicted in this tree. Distinct subclades for various organisms distinguishes each fungal isolates based on evolutionary distance. Isolate Gymnoscoideus petalosporus (MF996762) has smallest length (298 bases) of ITS sequence whereas isolate Trichoderma virens (MG551576) has longest length (561 bases) of ITS sequence amongst all isolates. Bar scale represents evolutionary distance.
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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[55]. There are reports on Phaeohyphomycosis caused by E. spinifera was reported from various parts of India [54]. There are some reports on the Skin and Nail Infections caused by Fusarium solani: similarly by species of Aspergillus including A. niger, A. flavus, and A. fumigatus are the causative agent of non-dermatophytic mycoses in human in India [57–59]. It appears from this study that the keratinophilic flora of Sambhar Lake is different from other parts of India. This may be attributed to the climatic and other environmental condition prevailing in this area. Disclosure of interest The authors declare that they have no competing interest. References [1] Vanbreuseghem R. Technique biologique pour 1’isolement des dermatophytes du sol. Ann Soc Belge Med Trop 1952;32:173–8. [2] Domsch KH, Games W, Traute-Heidi A. Compendium of soil fungi. London Sanfrasco: Academic Press; 1980. [3] van Oorschot CAN. A revision of Chrysosporium and allied genera. Stud Mycol 1980;20:1–89. [4] Sigler L, Carmichael JW. Taxonomy of Malabranchea and some other hyphomycetes with arthroconidia. Mycotaxon 1976;4:349–488. [5] Currah RS. Taxonomy of Onygenales: Arthrodermaceae, Gymnoascaceae, Myxotrichaceae and Onygenaceae. Mycotaxon 1985;24:1–216. [6] Cano J, Gurrao J. The genus Aphanoascus. Mycol Res 1990;94:355–77. [7] Von Arx Von JA. The ascomycetes genus Gymnoascus. Persoonia 1986;13:173–83. [8] Domsch KH, Gams W, Anderson TH. Compendium of soil fungi. APS Press; 2007. p. 672. [9] Seifert K, Morgan-Jones G, Gams W, Kendrick B. The Genera of Hyphomycetes. CBS Biodiversity Series no. 9; 1–997. Utrecht, Netherlands: CBS-KNAW Fungal Biodiversity Centre; 2011. [10] Lee SB, Taylor JW. Isolation of DNA from fungal mycelium single cells. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press; 1990. p. 282–7. [11] White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis A, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press; 1990. p. 315–22. [12] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10. [13] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725–9. [14] Deshmukh SK, Verekar SA. Keratinophilic fungi from the vicinity of meteorite crater soils of Lonar (India). Mycopathologia 2006;162:303–6. [15] Randhawa HS, Sandhu RS. A survey of soil inhabiting dermatophytes and related keratinophilic fungi of India. Sabouraudia 1965;4:71–9. [16] Deshmukh SK, Agrawal SC. Prevalence of dermatophytes and other keratinophilic fungi in soils of Madhya Pradesh (India). Mykosen 1983;26:574–7. [17] Deshmukh SK, Agrawal SC, Jain PC. Isolation of Dermatophytes and other keratinophilic fungi from soils of Mysore (India). Mycoses 2000;43:55–7. [18] Deshmukh SK. Isolation of Dermatophytes and other Keratinophilic fungi from the vicinity of salt pan soils of Mumbai (India). Mycopathologia 2004;157:265–7. [19] Deshmukh SK, Verekar SA, Chavan YG. Incidence of Keratinophilic fungi from the selected soils of Kaziranga National Park Assam, (India). Mycopathologia 2017;182:371–7. [20] Carmichael JW. Chrysosporium and some other aleuriosporic hyphomycetes. Can J Bot 1962;40:1137–73. [21] Kushwaha RKS, Agarwal SC. Some keratinophilic fungi and related dermatophytes from soils. Proc Indian Natn Sci Acad 1976;42:102–10. [22] Deshmukh SK, Agrawal SC. Isolation of keratinophilic fungi from coastal habitats of Goa (India). Kavaka 1983;11:53–4. [23] Deshmukh SK. Incidence of Dermatophytes and other keratinophilic fungi in the soils of Kerala (India). Mycopathologia 2002;156:177–81. [24] Jain PC, Agrawal SC. Some addition to Indian Malbranchea. Kavaka 1979;7:69–72. [25] Deshmukh SK, Verekar SA, Shrivastav A. Prevalence of Keratinophilic Fungi in selected soils of Ladakh (India). Nat Sci 2010;2:1147–52. [26] Deshmukh SK, Verekar SA. Isolation of keratinophilic fungi from selected soils of The Gir Forest National Park and Wildlife Sanctuary, Gujarat, (India). Kavaka 2014;43:7–11. [27] Deshmukh SK, Verekar SA. Isolation of keratinophilic fungi from selected soils of Sanjay Gandhi National Park Mumbai (India). J Mycol Med 2014;24:319–27. [28] Deshmukh SK, Verekar SA. Prevalence of Keratinophilic fungi in ‘Usar’ Soils of Uttar Pradesh, India. Microbiol Res 2011;3:e15. http://dx.doi.org/10.4081/ mr.2011.e15.
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[45] de Hoog GS, Guarro J, Gene´ J, Figueras MJ. Atlas of clinical fungi, 2nd ed., Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures; 2000. [46] Nucci M, Anaissie E. Cutaneous infection by Fusarium species in healthy and immunocompromised hosts: implications for diagnosis and management. Clin Infect Dis 2002;35:909–20. [47] Wickern GM. Fusarium allergic fungal sinusitis. J Allergy Clin Immunol 1993;92:624–5. [48] Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clin Microbiol Rev 2007;20:695–704. [49] Sharma M, Sharma R. Profile of dermatophytic and other fungal infections inJaipur. Indian J Microbiol 2012;52:270–4. [50] Lakshmanan A, Ganeshkumar P, Mohan SR, Hemamalini M, Madhavan R. Epidemiological and clinical pattern of dermatomycoses in rural India. Indian J Med Microbiol 2015;33(Suppl S1):134–6. [51] Kumar Y, Singh K, Kanodia S, Singh S, Yadav N, Chidambaram N. Clinicoepidemiological profile ofsuperficial fungal infections in Rajasthan. MedPulse Int Med J 2015;2(3):139–43. [52] Nath R, Barua S, Barman J, Swargiary P, Borgohain M, Saikia L. SubcutaneousMycosis Due to Cladosporium cladosporioides and Bipolaris cynodontis from Assam, North-East India and Review of Published Literature. Mycopathologia 2015;180:379–87. [53] Anandi V, Vishwanathan P, Sasikala S, Rangarajan M, Subramaniyan CS, Chidambaram N. Fusarium solani breast abscess. Indian J Med Microbiol 2005;23:198–9. [54] Radhakrishnan D, Jayalakshmi G, Madhumathy A, Banu ST, Geethalakshmi S, Sumathi G. Subcutaneous phaeohyphomycosis due to Exophiala spinifera in an immunocompromised host. Indian J Med Microbiol 2010;28:396–9. [55] Sharma P, Gill PK, Aggarwal A. Bipolaris spicifera, an unusual cause of nonhealing cutaneous ulcer in an immunocompromised patient. J Microbiol Infect Dis 2014;4:33–5. [56] Raghavendra K, Yadav D, Kumar A, Sharma M, Bhuria J, Chand A. The nondermatophyte molds: emerging as leading cause of onychomycosis in SouthEast Rajasthan. 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Original article/Article original
Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India) S.K. Deshmukh a,*, S.A. Verekar b, Y.G. Chavan c a
TERI-Deakin Nano Biotechnology Centre, The Energy and Resources Institute, Darbari Seth Block, IHC Complex, Lodhi Road, 110003 New Delhi, India MicroChem Silliker Pvt. Ltd., Microchem House, A513, TTC Ind. Area, MIDC, Mahape, 400701 Navi Mumbai, India c geneOmbio Technologies Pvt. Ltd., Vedant, No 39/3, Yogi Park, Baner, 411045 Pune, Maharashtra, India b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 January 2018 Received in revised form 2 May 2018 Accepted 6 June 2018 Available online xxx
Forty soil samples were collected from seven sites in the vicinity of Sambhar lake Rajasthan, India and screened for the presence of keratinophilic fungi using hair baiting techniques for isolation. Seventeen isolates were recovered and identified. The cultures were identified by recognition of their macro- and micro- morphological features. Their identification was confirmed by BLAST using ITS1-5.8S-ITS2 rDNA region and sequences have been deposited in NCBI data base. A total of 34 species belonging to 29 genera were isolated. Among the dermatophytes and related keratinophilic fungi Chrysosporium indicum was predominant followed by Ctenomyces serratus, C. tropicum, Keratinophyton durus, Auxarthron conjugatum, Gymnascella dankaliensis, Gymnoascoideus petalosporus and Uncinocarpus reesii. Twenty-six species belonging to 22 genera represented other species. Our study indicates that keratinophilic fungi and species are found in the soils at the vicinity of the Sambhar Lake, and human activities can be the potential source of pathogenic fungi. C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Sambhar salt lake Salt pan Soil fungi Keratinophilic fungi India Alkaline saline environment
1. Introduction Sambhar Lake is situated at 60 km west of Jaipur in Rajasthan, India and is the largest salt lake (27*00’N, 75*00’E) covering 24,000 ha area. It is a shallow wetland whose depth ranges between 0.5 and 2 meters. Four main streams feed the lake, which is roughly elliptical in shape, from a drainage area of about 268,800 ha. On the eastern end, the lake is divided by a 5-km long dam made of stone. Further east of the dam is salt evaporation ponds where salt has been farmed for thousands of years. This part also has a railroad, built by the British (before India’s independence) that provides access from Sambhar lake city to the salt works. Sambhar lake brine is somewhat unique with a very low potassium concentration. The vegetation present in the catchment area is mostly xerophytic. The waters of Sambhar Lake have been used for centuries to make salt. There is, however, another distinctive feature of this extensive saline wetland. The waters here are glacially still edged with a glittering frost of salt. There is a sharp briny tang in the air that takes one straight back to coastal
* Corresponding author. E-mail addresses: [email protected] (S.K. Deshmukh), [email protected] (S.A. Verekar), [email protected] (Y.G. Chavan).
fish markets. The Sambhar lake city is the largest of human settlements around the lake. Sambhar Lake is famous for harboring large number of flamingos. Together with adjacent saline wetlands, Phulera and Deedwana, the lake is probably the most important wintering area for flamingoes (both Phoniconaias minor and Phoenicopterus roseus) in India, second only to the Rann of Kutch, where they breed. These beautiful tall birds flock the lake and enjoy the feast of large numbers of algae that swarm the lake. Pelican is another species of birds that one can see at the lake. Some other birds at the lake are storks, redshanks, sandpipers, coots, black-winged stilts, and shovelers. However, Sambhar’s ferocious brine is too saline for many species as birds can be found in the freshwater ponds in the surrounding areas. The Naliasar Pond, just 4 km south of Salt Lake City, is crammed with waterfowl-shovelers, common teals, pintails, common pochards, tufted pochards, gadwalls, graylag and bar-headed geese and even busty shelduck that fly swiftly overhead. The terrestrial fauna confined to the catchment area includes rare/threatened species such as the Uromastix, saw-scaled viper, desert cat and desert fox. This ecological diversity makes it a potential area of interest to study the distribution of keratinophilic fungi from soils of Sambhar Lake. The present investigation reports the isolation of these fungi from soils of Sambhar Lake.
https://doi.org/10.1016/j.mycmed.2018.06.002 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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2. Materials and methods Forty soil samples were collected from seven sites of Sambhar lake, Rajasthan state India April 2013. The samples were taken from the banks of the lake: approximately 10 feet from water line (dried crust of soil with dried salt layers), bottom sediments: from 1 feet depth of shallow water, adjacent marshy meadows: semimoist region with wild xerophytic plants and weed around some peripheral region of the lake (radish salty soil), salt pan, salt storage areas and dropped off feathers. Soil samples were collected by scraping a layer of soil not exceeding 5 cm in depth. The bottom sediments samples were also obtained from different layer of soil from bank. The samples were brought to the laboratory and processed promptly. The brines had pH of 9.5 0.2 and a total salt content ranging from 7% (w/v) to more than 30% (w/v). Sodium chloride, sodium carbonate, sodium bicarbonate and sodium sulphate were the principal salts present in these brines that lacked divalent cations (calcium and magnesium). Sambhar has a tropical climate. The summers can be scorching with mercury crossing 45 8C, whereas winters are moderately chilled and the temperature can fall below 10 8C. The hair bait technique of Vanbreuseghem [1] was used to isolate keratinophilic fungi. For this purpose, sterile petri dishes were half filled with the soil samples and moistened with water and baited by burying sterile human hairs in the soil. These dishes were incubated at room temperature and examined daily after five days for fungal growth over a period of four weeks. After observing the growth under a stereoscopic binocular microscope it was cultured on Sabouraud’s dextrose agar (HiMedia) amended with chloramphenicol (HiMedia) (50 mg/L) in one set and Sabouraud’s dextrose agar amended with chloramphenicol (50 mg/L) and cycloheximide (HiMedia) (500 mg/L) in other set. These fungi were identified based on the monographs of Domsch et al. [2], Oorchschot [3], Sigler and Carmichael [4], Currah [5], Cano and Guarro [6], Arx von [7], Domsch et al. [8], Seifert [9]. The molecular identification of isolated colony from each culture was performed using DNA sequencing of the ITS1-5.8SITS2 region. Genomic DNA was extracted by the miniprep protocol of Lee and Taylor [10]. The ITS1-5.8S-ITS2 rDNA was amplified using primers ITS1 and ITS4 as the forward and reverse primers as described by White et al. [11]. Amplification was performed in 100 mL reaction volumes containing 10 buffer 10 ml, MgCl2 (25 mM) 2 ml, dNTP (10 mM) 2 ml, ITS1 primer (20 pm) 2 ml, ITS4 primer (20 pm) 2 ml, Taq Polymerase (2.5U) 1 ml, DNA Sample (5 mg/ml) 3 ml, and Milli Q Water 78 ml. The PCR reaction was carried out using a Thermal Cycler (M.J. Research, PTC 200) with conditions as follows: denaturation for five minutes at 94 8C, 34 cycles of (30 s at 94 8C, 30 s at 55 8C, 1 min at 72 8C) extension for four minutes at 72 8C and storage at 4 8C. Negative controls were used in each set of reactions. The final products were analyzed by electrophoresis on 2.0% agarose gel (Sigma). The PCR products were purified using Gel extraction kit (Qiagen, CAT No. 28704) and then sequenced using ITS1 and ITS4 primers at geneOmbio Technologies Pvt Ltd, Pune, India, using Applied Biosystems 3730 DNA analyzer. Phylogenetic analysis: Similarity analysis of the nucleotides was performed by BLAST searches against sequences available in GenBank [12]. For phylogenetic tree construction, multiple sequences were obtained from GenBank and the alignments were performed using MEGA6 [13].
3. Results and discussion A total of 34 fungi belonging to 29 genera were isolated from the soil samples collected from various sites of Sambhar Lake and %
frequency occurrence of keratinophilic fungi isolated is given in Table 1. Of these 8 fungi were dermatophyte and related fungi, which accounted for 26.32% of the total fungal flora, whereas the other 26 fungi contributed 73.64%. Amongst the keratinophilic fungi Chrysosporium indicum was most frequent (100%) followed by Ctenomyces serratus, C. tropicum, Keratinophyton durus, Auxarthron conjugatum, Gymnascella dankaliensis, Gymnoascoideus petalosporus and Uncinocarpus reesii. Among other fungi Cladosporium cladosporioides (100%) occurred most frequently and contributed 7.89% to the fungal flora. Whereas Acremonium sp., Aspergillus aculeatus, Aspergillus versicolor, Fusarium nematophilum, Fusarium solani, Penicillium verruculosum, Sarocladium strictum, Syncephalastrum racemosum were recovered from only one site with a least of 1.32% contribution each. The DNA fragments amplified using PCR by primers ITS1 and ITS4 vary between 509 and 548 bp in length. The fragment contained the 30 end of 18S rDNA, ITS1, 5.8S rDNA, and ITS2 and the 50 end of 28S rDNA. The presence and similarity of the sequence in thirty four different isolates was ascertained using the CLUSTAL W multiple sequence alignment available at www.ebi.ac.uk/Tools/ msa/clustalw2. The similarity based on the ITS region (ITS1, 5.8S rDNA, and ITS2 regions) between these isolates varied from 59 to 100% as per the alignment. Fig. 1 displays the Maximum Composite Likelihood tree constructed by comparing the sequence identities of the ITS regions in different isolates. The eight different isolates sequences were identified by BLAST analysis. Most significant BLAST hit obtained was considered as reference sequence for each sequence to be used in construction of phylogenetic tree. In the present study, C. indicum was the most frequently isolated species with 7.89% distribution. Its high percentage of distribution indicated that it is well adapted to Indian climatic conditions, as it can tolerate higher temperature, pH and salt. In our previous report, we have found that keratinophilc fungi isolated from Lonar crater can tolerate 5% salt concentration [14]. C. serratus was found next to C. indicum. It has been reported from various part of India [15–19]. Chrysosporium tropicum comprised 7.5% in distribution. It is a cosmopolitan species and has been reported from different parts of India [20–23]. In the present study, A. conjugatum was 2.63% in distribution. It is reported from India’s plains [24–26]. Other fungi isolated were K. durus (2.63%), Gymnoascella dankaliensis (1.32%), U. reesii (1.32%) G. petalosporus (1.32%). K. durus was previously recorded from meteorite crater of Lonar [14], Gir Forest National Park and Wildlife Sanctuary, Gujarat, (India) [26], Sanjay Gandhi National Park (SGNP) [27], Kaziranga National Park [19]. Gymnoascella dankaliensis was reported from usar soils of Uttar Pradesh [30]. Ajello and Padhye [29] isolated U. reesii as Gymnoascus reesii from the nesting sites of blue footed boobies and greater frigate birds from the xeric low lying coastal areas of the Galapagos Islands. It has also been isolated from Chilka Lake, which is the largest saline lake in India [28] and from salt pan soils of Mumbai [18]. G. petalosporus was previously recorded from Chilka Lake soil [30], soils from Vedanthangal Water Bird Sanctuary [31], and feathers of birds of Orissa [32], and Soils of Vidharbha [33]. We are reporting for the first time the presence of keratinophilic fungi from Sambhar Lake Rajasthan (India). The fungi other than dermatophytes and related species, 26 fungal species belonging to 22 genera were recovered from the soil samples collected and were 73.64% in distribution. Among the other fungi C. cladosporioides (7.89%), was predominant followed by Exophiala spinifera (5.26%), Trichoderma virens (5.26%), Aureobasidium pullulans (3.95%), Chaetomium globosum (3.95%), Fusarium oxysporum (3.95%), Nigrospora oryzae (3.95%), Alternaria daucifolii (2.63%), Bipolaris rostrate (2.63%), Bipolaris stenospila (2.63%), Cochliobolus verruculosus (2.63%), Curvularia spicifera (2.63%), Humicola fuscoatra (2.63%), Myrothecium verru-
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Table 1 Distribution of keratinophilic fungi and % frequency in soil samples of Sambhar Lake. Genus and species Dermatophytes and closed related species Auxarthron conjugatum Chrysosporium indicum Chrysosporium tropicum Ctenomyces serratus Gymnoascoideus petalosporus Gymnoascella dankaliensis Keratinophyton durus Uncinocarpus reesii Other species Acremonium sp. Alternaria daucifolii Aspergillus aculeatus Aspergillus versicolor Aureobasidium pullulans Bipolaris rostrate Bipolaris stenospila Chaetomium globosum Cladosporium cladosporioides Cochliobolus verruculosus Curvularia spicifera Exophiala spinifera Fusarium nematophilum Fusarium oxysporum Fusarium solani Humicola fuscoatra Myrothecium verrucaria Nigrospora oryzae Penicillium verruculosum Sarocladium implicatum Sarocladium strictum Syncephalastrum racemosum Trichoderma virens Thielavia subthermophila Torulaher barum Yunnania penicillata Total sp./site Frequency occurrence at site
GenBank accession number (s)
Bank of the lake
MF996764 MF996760 MF996758 MF996761 MF996762 MF996760 MF996759 MF996763
+ + + + – + – –
MG551554 MG551556 MG551557 MG551559 MG551558 MG551564 MG551561 MG685669 MG685670 MG685671 MG551562 MG685672 MG551567 MG551565 MG551566 MG551568 MG685673 MG685674 MG551571 MG551553 MG551555 MG551574 MG551576 MG551575 MG685675 MG551572
– – – – + – + – + – + + – + – + – – + – – – – + + + 16 47.06
Banks of salt pans
Bottom sediments
Adjacent marshy meadows
Burrows
Dropped off feathers
Total
% distribution
% frequency
– – – + –
+ + + + – – – +
– + – + – – + –
– + – – – – – –
– + + + + – – –
2 6 3 4 1 1 2 1
2.63 7.89 3.95 5.26 1.32 1.32 2.63 1.32
33.33 100 50 66.67 16.67 16.67 33.33 16.67
– + + – + + – – + + + – – – – – – + – + – – – – – – 11 32.35
– + – – – – – + + – – + – – – – + – – – + – + – – – 12 35.29
– – – – + + – – + – – + + + – – – + – – – – + + – – 12 35.29
– – – + – – + + + – – + – + + – – – – – – + + – + – 11 32.35
+ – – – – – – + + + – – – – – + + + – + – – + – – + 14 41.18
1 2 1 1 3 2 2 3 6 2 2 4 1 3 1 2 2 3 1 2 1 1 4 2 2 2 76
1.32 2.63 1.32 1.32 3.95 2.63 2.63 3.95 7.89 2.63 2.63 5.26 1.32 3.95 1.32 2.63 2.63 3.95 1.32 2.63 1.32 1.32 5.26 2.63 2.63 2.63 100
16.67 33.33 16.67 16.67 50 33.33 33.33 50 100 33.33 33.33 66.67 16.67 50 16.67 33.33 33.33 50 16.67 33.33 16.67 16.67 66.67 33.33 33.33 33.33
+
caria (2.63%), Sarocladium implicatum (2.63%), Thielavia subthermophila (2.63%), Torula herbarum (2.63%), Yunnania penicillata (2.63%), Acremonium sp. (1.32%), A. aculeatus (1.32%), A. versicolor (1.32%), F. nematophilum (1.32%), F. solani (1.32%), P. verruculosum (1.32%), S. strictum (1.32%) and S. racemosum (1.32%). There are reports on various types of infections caused by species of Chrysosporium in human and animals particularly in immuno-compromised patients [34]. For example Chrysosporium zonatum was reported to cause disseminated infection in a patient with chronic granulomatous disease [35]. In Japan, C. zonatum strains were isolated from bronchial lavage from a female in Chiba and from a male in Kyushu. Both patients presented pulmonary cavity sites [36]. Chrysosporium tropicum was reported from comb lesion in two different breeds of chicken in India [37]. G. dankaliensis was reported from superficial infections in human beings [38]. Iwen et al. [39] reported Gymnascella hyalinospora from the invasive pulmonary infection in a patient with acute myelogenous leukemia. Other than dermatophytes and related species, 26 fungal species belonging to 22 genera were recovered from the soil samples collected. Potential human fungi isolated in the present study were E. spinifera, C. cladosporioides, Curvularia spicifera, etc. E. spinifera has been reported to cause phaeohyphomycosis and chromoblastomycosis in humans [40]. Cladosporium is rarely reported from invasive infections [41]. Bentz and Sautter [42] reported a mixed disseminated infection by Aspergillus fumigatus and C. cladosporioides in an immunocompromised patient. C. cladosporioides has been reported from two clinical cases
involving the central nervous system [43]. Gugnani et al. [44] reported a case of subcutaneous phaeohyphoymycosis caused by C. cladosporioides in a 25-year-old male. The other fungi include Curvularia, Fusarium, etc. The fungal genus Curvularia includes some emerging opportunistic pathogens of humans. Curvularia spicifera is reported as important agents of opportunistic infections in vertebrates [45]. In humans, Fusarium species cause a broad spectrum of infections, including superficial (such as keratitis and onychomycosis), locally invasive, or disseminated infections, with the last occurring almost exclusively in severely immunocompromised patients [46]. Fusarium species may also cause allergic diseases (sinusitis) in immunocompetent individuals [47] and also in Fusarium Infections in Immunocompromised Patients [48]. As such, there is no published report of fungal infection from Sambhar lake. However, there are few reports on dermatophytic and non-dermatophytic infections from Rajasthan where the Sambhar lake is situated [49–51]. Apart from dermatophytic infections, there are reports of non-dermatophytic infections caused by Curvularia spicifera, Cladosporium cladosporioides, Exophiala spinifera, Fusarium sp. Aspergillus sp. from various parts of India [52–56]. Nath et al. [52] reported two cases of subcutaneous mycosis from Assam, north-east India, caused by C. cladosporioides and Bipolaris cynodontis in immunocompetent individuals clinically presenting as chromoblastomycosis. A case of cutaneous ulcers due to Bipolaris spicifera in a patient with diabetes and alcohol abuse from a 47-year-old male who is resident of rural area of Amritsar, Punjab, India was reported by Sharma et al.
Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002
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Fig. 1. Phylogenetic relationship of the nine isolates of fungi based on ITS1, 5.8S rDNA, and ITS2 regions of DNA sequences. The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of transitional substitutions per site. The analysis involved 67 nucleotide sequences. Evolutionary analyses were conducted in MEGA6. Sequence obtained from each sample show maximum similarity with its closed phylogenetic member as depicted in this tree. Distinct subclades for various organisms distinguishes each fungal isolates based on evolutionary distance. Isolate Gymnoscoideus petalosporus (MF996762) has smallest length (298 bases) of ITS sequence whereas isolate Trichoderma virens (MG551576) has longest length (561 bases) of ITS sequence amongst all isolates. Bar scale represents evolutionary distance.
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Please cite this article in press as: Deshmukh SK, et al. Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India). Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.002