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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 4  |  Page : 457-461
 

Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens


Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Chennai, Tamil Nadu, India

Date of Submission24-Jun-2016
Date of Acceptance14-Sep-2016
Date of Web Publication8-Dec-2016

Correspondence Address:
A J Kindo
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.195369

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 ~ Abstract 

Aim: Fungi are increasing in incidence as human pathogens and newer and rarer species are continuously being encountered. Identifying these species from growth on regular culture media may be challenging due to the absence of typical features. An indigenous and cheap medium, similar to the natural substrate of these fungi, was standardised in our laboratory as an aid to species identification in a conventional laboratory setting. Materials and Methods: Ripe banana peel pieces, sterilised in an autoclave at 121°C temperature and 15 lbs pressure for 15 min promoted good growth of hyphae and pycnidia or acervuli in coelomycetes, flabelliform and medusoid fruiting bodies of basidiomycetes and fruit bodies such as cleistothecium in ascomycetes. The growth from the primary isolation medium was taken and inoculated onto the pieces of double-autoclaved ripe banana peel pieces in a sterile glass Petri dish with some moisture (sprinkles of sterile distilled water). A few sterile coverslips were placed randomly inside the Petri dish for the growing fungus to stick on to it. The plates were kept at room temperature and left undisturbed for 15–20 days. At a time, one coverslip was taken out and placed on a slide with lactophenol cotton blue and focused under the microscope to look for fruit bodies. Results: Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum, Nattrassia mangiferae and Schizophyllum commune were identified by characteristic features from growth on banana peel culture. Conclusions: Banana peel culture is a cheap and effective medium resembling the natural substrate of fungi and is useful for promoting characteristic reproductive structures that aid identification.


Keywords: Ascomycetes, banana peel culture, basidiomycetes, black fungi, coelomycetes, fruit bodies


How to cite this article:
Kindo A J, Tupaki-Sreepurna A, Yuvaraj M. Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens. Indian J Med Microbiol 2016;34:457-61

How to cite this URL:
Kindo A J, Tupaki-Sreepurna A, Yuvaraj M. Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens. Indian J Med Microbiol [serial online] 2016 [cited 2017 Nov 22];34:457-61. Available from: http://www.ijmm.org/text.asp?2016/34/4/457/195369



 ~ Introduction Top


Owing to the increasing size of a susceptible population, fungi appear to be increasing in incidence in human disease. They are frequently acquired through inhalation or some type of traumatic implantation and are of particular concern in patients being maintained on long-term immunosuppressive therapy.[1] Newer and unfamiliar fungal pathogens are thus regularly being encountered in the diagnostic mycology laboratory. Although the recovery of these fungi in the laboratory is not particularly difficult, identification of some species remains laborious due to poorly defined, difficult to assess criteria and atypical characteristics displayed under artificial growth conditions. The right temperature, media and incubation time are extremely important in allowing the growth of the fungus with characteristic morphological features that make identification possible.

For example, in coelomycete fungi, the fungal hyphae germinate from conidia and form an aggregated mass until they form a fertile layer of densely packed conidiophores. Conidiophores are simple or branched hyphae that develop specialised cells (conidiogenous) from which sexual conidia are produced. It is this mass of hyphae that create the fruiting structure known as conidiomata. The conidiophores may either partially or completely cover the base of the fruiting structure. Since coelomycetes are mostly plant pathogens, they usually develop just beneath the plant tissue which can be colonised at various depths. They can develop immediately beneath the plant's cuticle (subcuticular), within the epidermal layer (intraepidermal) or just beneath the epidermal layer (subepidermal) depending on the type of coelomycetes.

Coelomycetes of the genus Colletotrichum and Pestalotiopsis are examples of fungi containing acervular fruiting structures. Pestalotiopsis spores which originate from an acervular conidiomata are occasionally seen in outdoor air samples. Since the fruiting structures of coelomycetes are not easily aerosolised, identification of a coelomycetous fungus in air samples may not be possible. Identifying the presence of coelomycetes on direct analyses and cultures, on the other hand, is easily accomplished since the growth on such substrates allows the presence of the fruiting structures. It is, however, difficult to identify coelomycetes to genus and species on substrates other than a plant host. This is because, their fruiting structures are much simpler in artificial media and lack many of the elaborate characteristics present in those growing on a natural plant host. As a result, identification to genus and species may be laborious if not impossible.

The ascomycete fungi or the sac fungi may possibly form the largest group among human fungal pathogens. These fungi can be heterothallic or homothallic, where an ascus develops from ascogenous hyphae. These fungi are also called spore shooters as sexual spores called ascospores develop within the sac-like asci and are forcibly discharged from time-to-time, up to a distance of 30 cm. Visualisation of characteristic fruit bodies such as cleistothecium helps identify the rarer ascomycete pathogens.

The basidiomycetes are club mushrooms containing a huge diversity of fungi but only rarely encountered as human pathogens. However, many of these fungi grow slowly in culture, requiring specific conditions and take a relatively long time to form fruiting structures.

Although molecular techniques are now available for easy detection of most fungal pathogens, there is still a long way to go in making it accessible to the regular mycology laboratory of a developing nation like India. The banana peel culture medium as used in our laboratory provides an effective alternate medium for the conventional laboratory at no cost.


 ~ Materials and Methods Top


Ripe banana peel was used as the substrate for the growth of human pathogens belonging to the coelomycetes, ascomycetes and basidiomycetes. The banana peel was taken and cut into half an inch pieces. It was autoclaved twice at 121°C and 15 lbs for 15 min on two separate days. The sterile pieces were spread in a sterile glass Petri dish, sprinkled with sterile distilled water to provide moisture. The unidentified fungi from the primary isolation medium such as Sabouraud dextrose agar or oat meal agar were inoculated in the banana peel pieces along with few sterile coverslips and covered and kept at room temperature [Figure 1]. An uninoculated separate plate of sterilised banana peel served as the negative control.
Figure 1: (a and b) Banana peel culture medium

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The plates were kept undisturbed for 10–15 days at room temperature and then one coverslip on which fungal growth would have adhered to was taken, and a drop of lactophenol cotton blue or normal saline was put on a sterile glass slide, and the cover slip was gently placed on it and observed under the microscope.

The presence of sac-like structures was looked for and if present, the coverslip was slightly pressed to enable the conidia to be released from the pycnidia.


 ~ Results Top


The identification of six of the rarer human pathogenic ascomycetes (Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum), coelomycetes (Nattrassia mangiferae) and basidiomycetes (Schizophyllum commune) fungi were done by this method in our laboratory.[2],[3],[4],[5]


 ~ Discussion Top


Ascomycetes and coelomycetes are predominantly found in tropical and subtropical regions where they mainly exist as plant pathogens affecting a variety of different plant species. However, they are also known to exist as saprobes or parasites of fungi, lichens and vertebrates. They require hot and humid conditions for optimal growth. Most commonly, these fungi grow on living and dead plant parts. Ascomycetes and coelomycetes fungi usually exhibit a moderate-to-rapid growth rate on a variety of media and are not particularly difficult to recover from specimens. The problem lies in promoting diagnostic reproductive structures necessary for characterisation of the isolates. Not only is a considerable amount of time required, particularly for the pycnidial species (it took 2 months in one of our isolates), it is also necessary to utilize a medium in which these pycnidia will develop.

Hence, we thought of a plant-based substrate which is easily available at no cost. The banana peel which is thrown away serves as a good culture medium to propagate this group of fungi with the formation of pycnidia or acervuli. [Figure 1] shows the culture media.

Following is a description of the species identified with banana peel culture in our laboratory till date. L. theobromae was one of the fungi for which this culture method was used to identify an isolate from maxillary sinusitis.[6] Known for almost a century, L. theobromae is a pleomorphic fungus, a member of the phylum Ascomycota and a common plant and wood pathogen in tropical and subtropical countries with a wide host range.[7] Formerly called Botryodiplodia theobromae, L. theobromae causes rotting and dieback in most species it infects. It is a common post-harvest fungus disease of citrus plants known as stem-end rot. In humans, L. theobromae is a rare cause of mycotic keratitis,[8],[9] maxillary sinusitis [6] and skin lesions [10] showing characteristic two-celled, pigmented, longitudinally striate pycnidioconidia. L. theobromae is a cosmopolitan, tropical saprophyte which forms pycnidia. Colonies produced hairy, dark brown round to oval structures which could be seen under the stereoscopic microscope resembling structure of pycnidia. Conidia released from the pycnidia were hyaline and non-septate when young but were septate and brown, with longitudinal striations, when mature [Figure 2]. Fungi, which have been considered simple organisms, are extremely adaptable to diverse environmental conditions. They can exhibit striking differences in appearance with temperature changes. Therefore, unless the essential conditions for culture are provided, it can be very difficult to accurately speciate the fungus.
Figure 2: Lasiodiplodia theobromae, (a) culture in Sabouraud dextrose agar showing brownish colour colonies with abundant aerial mycelium (b) conidia released from pycnidia were hyaline and non-septate when young but septate and brown with longitudinal striation when mature (×40)

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N. mangiferae [Figure 3] was isolated from a case of fungal keratitis.[2] The coelomycete (synanamorph Scytalidium dimidiatum) is a filamentous fungus, which was first described in 1916 as Dothiorella mangiferae. Until recently, it was classified as Hendersonula toruloidea. In the latest revision of the Hendersonula, it was reclassified in its own genus based on its pycnidial stage.[11] The previously reported human infections include deep infections with this organism in diabetic or steroid-treated patients, including subcutaneous infection of the arm in a steroid-dependent diabetic patient.[11] Disseminated infection with this fungus has also been reported in an immunosuppressed patient who underwent cadaveric kidney transplantation.[12] The identifying feature of N. mangiferae, although very slow to form, is a mature, versicoloured (darkened middle cell and pale end cells) arthroconidia, easily recognised by its distinctive features from Scytalidium lignicola and Scytalidium hyalinum synanamorphs.[2]
Figure 3: Nattrassia mangiferae, (a) crushed pycnidium, arrow showing immature hyaline conidia, (×400). (b) Slide culture showing brown hyaline septate barrel-shaped arthroconidia, arrow showing the median band (×400)

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C. murorum, another isolate from maxillary sinusitis, was also identified using banana peel culture in our laboratory. Belonging to the phylum Ascomycota, it is highly cellulolytic. The colonies rapidly grow in Sabouraud agar, cottony, initially white; on maturation, it becomes grey to olivaceous-brown. The banana peel culture showed ascomatal setae, long flexous, smooth ascospores with asci. Ascospores were ellipsoidal to broadly fusiform brown in colour with long bands and germinal pores on both ends [Figure 4]. As well as being a common fungal contaminant indoors, especially in damp ill-maintained buildings, Chaetomium species have been previously reported as causative agents in human onychomycosis (Chaetomium globosum)[13] and also invasive mycotic infections such as cerebral phaeohyphomycosis (Chaetomium perlucidum).[14]
Figure 4: Chaetomium murorum, (a) Mature culture in Sabouraud dextrose agar. (b and c) Large ellipsoidal ascospores with apical germ pores and flexuous/undulate ascomatal hairs seen (×400)

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M. phaseolina isolated from fungal keratitis belongs to the phylum Ascomycota of the Botryosphaeriaceae family. The previous reports of human infections include cutaneous infection in a child with acute myeloid leukaemia [15] and a disseminated fungal infection in a renal transplant recipient.[16]Botryosphaeria teleomorph is seldom seen in culture, whereas the anamorphs are common. Species differences are manifested in the anamorph while there is considerable overlapping in the continuous characters of the teleomorph such as spore sizes. For these reasons, anamorph characters often are considered important to identify species in this genus. [Figure 5] shows the typical identifying features. The cultures were subsequently sent for molecular identification.
Figure 5: Macrophomina phaseolina, (a) Sclerotium showing aggregates of cells brown in colour, appearing smooth and round (×1000). (b and c) Pycnidia and conidiation in sterilised banana peel culture, respectively (×400)

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Nigrospora species are a filamentous melanised (phaeoid or dematiaceous) group of fungi that belong to the phylum Ascomycota.[17]N. sphaerica has been previously isolated from keratitis and onychomycosis.[4],[18] Although N. sphaerica grew very rapidly in culture of the clinical specimen, sporulation took nearly 5 weeks [Figure 6] which would warrant prolonged incubation and usage of multiple techniques such as slide culture and banana peel culture for the purpose of accurate speciation of any isolate exhibiting delayed sporulation.[4]
Figure 6: Nigrospora sphaerica, (a) colonies with woolly texture and greyish black colour as seen on Sabouraud dextrose agar slope incubated at 25°C. (b) Lactophenol cotton blue mount of Nigrospora sphaerica showing septate hyphae and conidiophore with inflated apex and single black conidium (inset) (×400). (c) Older conidia showing horizontal flattening (×1000)

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S. commune was reported first as a human pathogen causing onychomycosis in 1950.[19] It has also been implicated as a causative agent of aseptic/atypical meningitis and brain abscess, lung infections including fungal ball and allergic bronchopulmonary syndromes and ulcerative lesions of the hard palate and maxillary sinusitis (including our isolate).[5],[20],[21],[22],[23],[24]S. commune produces characteristic fan-shaped basidiocarps with split gills [Figure 7]. A case of basidiomycetous endophthalmitis necessitating enucleation has been described where species identification was not possible because of absent fruiting bodies.[25] Non-clamped variants of S. commune have also posed diagnostic difficulties.[26]
Figure 7: Schizophyllum commune, (a) Fan-shaped fruiting bodies in banana peel culture (basidiocarps). (b) Lactophenol cotton blue mount shows hyaline septate hyphae, with clamp connections, seen in the inset (×400)

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 ~ Conclusion Top


Fungi usually exhibit a moderate-to-rapid growth rate on a variety of media and are not particularly difficult to recover from specimens. The problem lies in promoting diagnostic reproductive structures necessary for characterisation of the isolates. Not only is a considerable amount of time required, particularly for the pycnidial species (up to 2 months occasionally) but also it is necessary to utilise a medium in which these pycnidia will develop. Previously, media using plant substrates such as carnation leaf agar has been successfully used for the purpose, but the required raw materials may not be readily available in all regions of India. Using banana peel as the plant substrate was found to be easy, cheap and effective and is being practised successfully in our laboratory.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Sutton DA. Coelomycetous fungi in human disease. A review: Clinical entities, pathogenesis, identification and therapy. Rev Iberoam Micol 1999;16:171-9.  Back to cited text no. 1
    
2.
Kindo AJ, Anita S, Kalpana S. Nattrassia mangiferae causing fungal keratitis. Indian J Med Microbiol 2010;28:178-81.  Back to cited text no. 2
[PUBMED]  Medknow Journal  
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Premamalini T, Ambujavalli BT, Vijayakumar R, Rajyoganandh SV, Kalpana S, Kindo AJ. Fungal keratitis caused by Macrophomina phaseolina – A case report. Med Mycol Case Rep 2012;1:123-6.  Back to cited text no. 3
    
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Ananya TS, Kindo AJ, Subramanian A, Suresh K. Nigrospora sphaerica causing corneal ulcer in an immunocompetent woman: A case report. Int J Case Rep Images 2014;5:675-9.  Back to cited text no. 4
    
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Premamalini T, Ambujavalli BT, Anitha S, Somu L, Kindo AJ. Schizophyllum commune a causative agent of fungal sinusitis: a case report. Case Rep Infect Dis 2011;2011:821259.  Back to cited text no. 5
    
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Kindo AJ, Pramod C, Anita S, Mohanty S. Maxillary sinusitis caused by Lasiodiplodia theobromae. Indian J Med Microbiol 2010;28:167-9.  Back to cited text no. 6
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Saha S, Sengupta J, Banerjee D, Khetan A. Lasiodiplodia theobromae keratitis: a case report and review of literature. Mycopathologia 2012;174:335-9.  Back to cited text no. 9
    
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Willinger B, Kopetzky G, Harm F, Apfalter P, Makristathis A, Berer A, et al. Disseminated infection with Nattrassia mangiferae in an immunosuppressed patient. J Clin Microbiol 2004;42:478-80.  Back to cited text no. 12
    
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Kim DM, Lee MH, Suh MK, Ha GY, Kim H, Choi JS. Onychomycosis caused by Chaetomium globosum. Ann Dermatol 2013;25:232-6.  Back to cited text no. 13
    
14.
Barron MA, Sutton DA, Veve R, Guarro J, Rinaldi M, Thompson E, et al. Invasive mycotic infections caused by Chaetomium perlucidum, a new agent of cerebral phaeohyphomycosis. J Clin Microbiol 2003;41:5302-7.  Back to cited text no. 14
    
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Srinivasan A, Wickes BL, Romanelli AM, Debelenko L, Rubnitz JE, Sutton DA, et al. Cutaneous infection caused by Macrophomina phaseolina in a child with acute myeloid leukemia. J Clin Microbiol 2009;47:1969-72.  Back to cited text no. 15
    
16.
Tan DH, Sigler L, Gibas CF, Fong IW. Disseminated fungal infection in a renal transplant recipient involving Macrophomina phaseolina and Scytalidium dimidiatum: case report and review of taxonomic changes among medically important members of the Botryosphaeriaceae. Med Mycol 2008;46:285-92.  Back to cited text no. 16
    
17.
de Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of Clinical Fungi. 2nd ed. Utrecht, The Netherlands: Centraalbureau Voor Schimmelcultures; 2000. p. 777.  Back to cited text no. 17
    
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Fan YM, Huang WM, Li W, Zhang GX. Onychomycosis caused by Nigrospora sphaerica in an immunocompetent man. Arch Dermatol 2009;145:611-2.  Back to cited text no. 18
    
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Kligman AM. A basidiomycete probably causing onychomycosis. J Invest Dermatol 1950;14:67-70.  Back to cited text no. 19
    
20.
Rihs JD, Padhye AA, Good CB. Brain abscess caused by Schizophyllum commune: an emerging basidiomycete pathogen. J Clin Microbiol 1996;34:1628-32.  Back to cited text no. 20
    
21.
Cifferi R, Batista AC, Campos S. Isolation of Schizophyllum commune from a sputum. Atti Ist Bot Lab Crittogam Univ Pavia 1956;14:3-5.  Back to cited text no. 21
    
22.
Rosenthal J, Katz R, DuBois DB, Morrissey A, Machicao A. Chronic maxillary sinusitis associated with the mushroom Schizophyllum commune in a patient with AIDS. Clin Infect Dis 1992;14:46-8.  Back to cited text no. 22
    
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Kern ME, Uecker FA. Maxillary sinus infection caused by the homobasidiomycetous fungus Schizophyllum commune. J Clin Microbiol 1986;23:1001-5.  Back to cited text no. 23
    
24.
Chowdhary A, Randhawa HS, Gaur SN, Agarwal K, Kathuria S, Roy P, et al. Schizophyllum commune as an emerging fungal pathogen: A review and report of two cases. Mycoses 2013;56:1-10.  Back to cited text no. 24
    
25.
Bartz-Schmidt KU, Tintelnot K, Steffen M, Ozel M, Kirchhof B, Heimann K. Chronic basidiomycetous endophthalmitis after extracapsular cataract extraction and intraocular lens implantation. Graefes Arch Clin Exp Ophthalmol 1996;234:591-3.  Back to cited text no. 25
    
26.
Sigler L, de la Maza LM, Tan G, Egger KN, Sherburne RK. Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung. J Clin Microbiol 1995;33:1979-83.  Back to cited text no. 26
    


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