Ann Microbiol (2015) 65:47–57 DOI 10.1007/s13213-014-0834-y ORIGINAL ARTICLE Muscodor tigerii sp. nov.-Volatile antibiotic producing endophytic fungus from the Northeastern Himalayas Sanjai Saxena & Vineet Meshram & Neha Kapoor Received: 22 August 2013 /Accepted: 3 February 2014 /Published online: 2 March 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Genus Muscodor came into existence with the dis- Introduction covery of Muscodor albus, a sterile endophytic fungus that produces a medley of volatile organic moieties possessing Endophytes comprise of an extremely diverse group of mi- strong antimicrobial activity. The current paper reports croorganisms that are ubiquitous in plants and maintain a Muscodor tigerii as a novel endophytic fungus from the stem symptomless and unobtrusive union with their hosts for at internal tissue of Cinnamomum camphora growing in the least a period of their life cycle (Stone et al. 2000; Kusari et al. Tiger Hill area of Darjeeling, West Bengal, India. M. tigerii 2012). Endophytes play an important role in plant symbiosis, exhibited distinct morphological, molecular and physiological rescuing their host from microbial infiltration and stressful features than previously reported Muscodor species. The fun- conditions (Shipunov et al. 2008). The genetic recombination gus possesses all the morphological features described till date of the endophytes with the host plant enables them to mimic in genus Muscodor making it remarkably unique. The strong the biological properties of their host and produce analogous fruity smell of the fungus is attributed to 22 volatile organic bioactive metabolites (Zhao et al. 2011; Kusari et al. 2012). compounds (VOCs), predominantly 4-Octadecylmorpholine, Thus, endophytic microorganisms are considered as a lucra- 1-Tetradecanamine,N,N-dimethyl and 1,2- tive source of bioactive metabolites with promising applica- Benzenedicarboxylic acid, mono(2-ethylhexyl) ester. The tions in the agrochemical and pharmaceutical industries in vitro VOC stress assay completely suppressed the growth (Strobel and Daisy 2003;Kudalkaretal.2012). of Alternaria alternata and Cercospora beticola while growth Muscodor is a genus of sterile endophytic fungi that has of other fungal species was inhibited in a range of 10 %-70 %. been isolated from tropical rainforest areas of Australia, Cen- The growth of Candida albicans in the presence of VOC was tral and South America and South-east Asia (Zhang et al. reduced by 50 %-65 % while in bacteria 50 %-80 % reduction 2010; Suwannarach et al. 2013). To date, twelve species have in growth was observed. Thus, M. tigerii stands as a potential been described in this genus based on ITS sequencing and candidate to be further developed into a biocontrol agent. volatile gas composition analysis and hyphal structures using a scanning electron microscope (SEM) (Suwannarach et al. 2013). The fungus has a remarkable ability to produce volatile Keywords Anamorphic fungus . Tiger hills . Cinnamomum antibiotics that are lethal against plant and human pathogens, camphora . ITS-rDNA nematodes and insects (Strobel 2006). Hence, Muscodor spp. stands out as a potential candidate as a biocontrol agent in post harvest technology with possible replacement of hazardous chemical fumigants like methyl bromide and; therefore, it is considered to be a promising source of eco-friendly antimi- S. Saxena (*) : V. Me sh ra m : N. Kapoor crobial compounds thus exhibiting mycofumigation potential Department of Biotechnology, Thapar University, Patiala, Punjab, (Strobel et al. 2001; Mercier et al. 2007). India 147001 The Northeastern Himalayas are located at the junction of e-mail: [email protected] Indo-Burma biodiversity hotspots. The hotspots cover the S. Saxena Indian states of Arunachal Pradesh, Sikkim and the Darjeeling e-mail: [email protected] district of West Bengal (Mcneely et al. 1990). The 48 Ann Microbiol (2015) 65:47–57 Northeastern Himalayas harbour a rich diversity of over 7,500 sterile plant segments were placed in the other quadrants plant species out of which 1,500 are endemic (Chatterjee et al. containing WA thereby exposing the plant segments to VOC’s 2006). Cinnamomum camphora is an evergreen plant native of M. albus arising in the plates. The fungi emerging out of the to India, China, Taiwan and Japan. The medicinial properties host tissue was aseptically subcultured onto a fresh PDA plate of the camphor plant have been explored since antiquity as a so as to obtain pure isolates which were further preserved on panacea for diarrhoea, muscular spasm, bronchitis, rheuma- PDA slants supplemented with 10 % glycerol (Ezra et al 2004; tism, etc. Camphor oil is used as an antiseptic, anti- Strobel et al. 2007;Mitchelletal.2008). inflammatory, diuretic, rubefacient, odontalgic, laxative and as well as an insecticide (Chelliah 2008). It also inhibits the Morphotaxonomy production of aflatoxin B1 produced by Aspergillus flavus (Singh et al. 2008). Microbial symbionts of Cinnamomum Morphological and microscopic characters of M. tigerii were camphora have been poorly studied. The plant harbours A- studied by growing the fungus on four different media com- spergillus fumigatus, Alternaria cineraria, Curvularia lunata, prising of PDA, CDA (Czapek Dox Agar), WA and SNA Nigrospora oryzae, Periconia sp. Pestalotiopsis sp. Phomopsis (Synthetischer Nahrstoffarmer agar). Morphotaxonomic stud- sp. and several sterile fungi as endophytes (Kharwar et al. ies of the endophytic fungal isolate was done by mounting the 2012). Another astonishing feature of the camphor plant is the culture in lactophenol cotton blue and then observing under a use of its endophytic fungal wealth like Colletotrichum, Nikon Stereozoom microscope (Nikon SMZ 745 T) coupled Fusarium and Aspergillus clavatus for synthesis of gold and with NIS element D 3.2 software and a Nikon Eclipse Com- silver nanoparticles (Verma et al. 2010). However, the current pound microscope (E100). Micrometry was done using ocular study reports Muscodor species #2 CCSTITD from the stem internal tissue of the camphor plant growing in the Tiger Hill area of Darjeeling, West Bengal, India. The endophytic fungus produces a unique set of volatile compounds, which possess antimicrobial activity and could be further used to identify the particular Muscodor species. Morphological, molecular and chemical characteristics of #2 CCSTITD distinguishes it from earlier reported Muscodor species. Based on its distinctive features, #2 CCSTITD is introduced as a new species of Muscodor genus for which the name Muscodor tigerii is proposed. Materials and methods Fungal isolation and preservation Healthy and mature plant parts (leaf and stems) of Cinnamomum camphora were collected from the Tiger hill area, Darjeeling, West Bengal during March 2011. Plant sam- ples were kept in sterile packets and stored at 4 °C till further use. The fungal isolation was done using Muscodor albus CZ620 as a screening tool as reported by Ezra et al. (2004). Briefly, Potato Dextrose Agar (PDA) was poured into one quadrant of the four sectioned commercially available Petri plates. An actively growing agar plug of M. albus CZ 620 was placed over the PDA medium while other quadrants of the Petriplates contained water agar (WA). The plates were then incubated at 24 °C for four days for Volatile Organic Com- pounds (VOCs) production by M. albus. The plant samples were washed under running tap water; air dried and cut into Plate 1 Morphological traits of Muscodor tigerii (#2 CCSTITD) over small segments of 5 mm, then the plant segments were surface PDA. (a) Morphological features, front view (b) Brown colored pigment production, reverse view. (c) Sterile hyphae (d) Ropy mycelium. (e) sterilized with 75 % ethanol for 30 s, 2 % sodium hypochlorite Swollen hyphae (f) Coil formation of fungal hyphae. Bar 1(a) and 1(b)= for 3 min and 95 % ethanol under a laminar flow hood. The 10mm, 1(c)-1(f)=10µm Ann Microbiol (2015) 65:47–57 49 Plate 2 Scanning electron micrograph of 10-day old Muscodor tigerii (#2 CCSTITD) including (a) Sterile mycelial web, bar- 100 µm (b) right angle branching, bar 10 µm (c) ropy mycelium with nondescript hyphal projection, Bar=10 µm (d) Close view of nondescript hyphal projections. Bar=2µm and stage scale and further confirmed by Image J software dNTP, 10 pmol/μl of each primer, 1.5 U of Taq DNA poly- with at least 30 observations per structure. The colony appear- merase (Bangalore Genei) in 10X Taq buffer of the 25 μl ance, color, colony growth rate, pigment and VOCs produc- reaction volume. Thermal cycling conditions used were pre- tion along with its microscopic structures like hyphal charac- viously described by Ezra et al. (2010). The PCR product of teristics and other cellular bodies were minutely observed and approximately 400-500 bp was purified by the Wizard® SV recorded (Guo et al. 1998;Mitchelletal.2008). Gel and PCR clean up system kit (Promega, USA). The purified amplicon was sequenced by Xcleris Genomics, DNA Extraction, Sequence Assembly and Phylogenetic Xcleris labs, Gujarat. Analysis The chromatograms of the isolate M. tigerii was edited and aligned in Sequencher, Version 5 (Gene Codes, Ann, Total fungal genomic DNA isolation was carried out with the Arbor, MI). The homology of the M. tigerii was ascertained Wizard® Genomic DNA purification kit (Promega, USA) as by subjecting the final sequence of the isolate to a sequence per instructions of the manufacturer. The ITS1-5.8S-ITS2 similarity search using BLAST against the database main- region was amplified using the M. albus specific primer pair tained by NCBI. Sequences showing the highest sequence as described by Ezra et al. (2010). The reaction mixture similarity with the sequence of M. tigerii were aligned composition for the amplification of the ITS region comprised using MUSCLE in MEGA5.2 (Tamura et al. 2011). Dis- of 1 μl of extracted genomic DNA, 25 mM MgCl2,2.5mM tance based analysis of the ITS region alignment was done 50 Ann Microbiol (2015) 65:47–57 Table 1 Synopsis of the advanced BLAST search homology analysis of consensus tree for the representation of evolutionary rela- ITS-5.8S ribosomal gene sequence of Muscodor tigerii tionship.