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Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea

Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp.... Ann Microbiol (2013) 63:793–800 DOI 10.1007/s13213-012-0534-4 ORIGINAL ARTICLE Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea Srichandan Padhi & Kumanand Tayung Received: 23 March 2012 /Accepted: 22 August 2012 /Published online: 21 September 2012 # Springer-Verlag and the University of Milan 2012 Abstract An endophytic fungus displaying considerable end were found to be unique to our isolate. These structural antimicrobial activity was isolated from stem tissue of an features can also be used as potential barcodes for this invasive plant species, Ipomoea carnea. The fungus was fungus. The findings indicate that invasive plant species identified as Quambalaria sp. and confirmed by ITS rDNA can be a reliable source of novel endophytes with rich sequence analysis. A BLAST search result of the sequence antimicrobial metabolites. The study also validates the indicated 97 % homology with Quambalaria cyanescens. assumption that endophytes can become parasites and share Crude metabolites of the fungus showed considerable anti- a close affinity. microbial activity against a panel of clinically significant . . microorganisms. The metabolites showed highest in vitro Keywords Ipomoea carnea Quambalaria sp. . . activity against Shigella dysenteriae followed by Escheri- Antimicrobial activity Phylogenetic analysis ITS2 RNA chia coli and Candida albicans. Optimum metabolites pro- secondary structures duction required neutral pH and a 15-day incubation period. Bark extracts amended with fungal media demonstrated higher antimicrobial activity. Optimum metabolites activity Introduction wasrecordedin Czapek Doxbroth amendedwithleaf extracts (CDB + LE) of the host plant. The metabolites Endophytic fungi that colonized inner plant tissues have been showed UV λ-max in ethyl acetate at 284.6 nm with an found to be associated with every plant species so far inves- absorbance value of 1.093. Phylogenetic tree generated by tigated. They are asymptomatic and often considered as the Maximum Parsimony method showed clustering of our mutualistic, benefiting the host by producing plant growth isolate with Q. cyanescens with supported bootstrap of regulatory, antimicrobial, antiviral and insecticidal substances 65 %. Species of Quambalaria are pathogens to Eucalyptus (Carroll 1988). It has been suggested that studies of endophyt- and occurrence of this fungus as endophytes support it to be ic fungi should be directed to plants growing in unique envi- a latent pathogen. Sequence base analysis and RNA second- ronmental settings, especially those with an unusual biology ary structure study also confirmed such a relationship. Sec- and possessing novel strategies for survival or plants with ondary structural features like two hinges and a 5’ dangling medicinal uses, as they are expected to harbor novel endo- phytes that may produce unique metabolites having diverse Electronic supplementary material The online version of this article applications (Strobel and Daisy 2003). Many workers gener- (doi:10.1007/s13213-012-0534-4) contains supplementary material, ally give priority to studying endophytic fungi associated with which is available to authorized users. medicinal plants because these microbes make the same bio- S. Padhi active natural products or derivatives that are more bioactive Department of Bioinformatics, North Orissa University, than those of their respective host (Strobel 2001). Neverthe- Takatpur, less, invasive plant species, even though having unique sur- Baripada 757003, India vival features, have been less studied for their endophytic K. Tayung (*) fungi. It is believed that associative endophytes of invasive Department of Botany, North Orissa University, plants could contribute to their greater competitiveness and Takatpur, may also produce novel allelochemicals that might have Baripada 757003, India inhibitory effects (Shipunov et al. 2008). Further endophytic e-mail: kumanandnou@yahoo.com 794 Ann Microbiol (2013) 63:793–800 fungi colonize such plants, which means they have evolved Fungal cultivation and metabolites extraction special mechanisms for survival and may provide new sources of secondary metabolites for biotechnological applications. The endophyte was cultivated on Potato Dextrose Broth We studied endophytic fungi associated with Ipomoea (PDB) by placing agar blocks of pure culture (3 mm in carnea tissues, a very common invasive species of India diameter) of actively growing culture in 250-ml Erlenmeyer and screened them for their antimicrobial potentials. A flask containing 100 ml of the medium. The flask was very rare endophytic fungus, Quambalaria sp., with con- incubated in a BOD shaking incubator for 3 weeks at 27± siderable antimicrobial activity was isolated from the inner 1 °C with periodic shaking at 150 rpm. The culture was bark tissue of I. carnea. A literature survey of the fungus filtered through sterile cheesecloth to remove the mycelial revealed it to be a pathogen causing diseases in Eucalyp- mats. The liquid broth was collected and extracted with an tus (Simpson 2000;Beeretal. 2006). Endophytes are equal volume of ethyl acetate in a separating funnel by generally regarded as symbiotic or mutualistic, but there vigorous shaking for 10 min. The cell mass was separated are also many examples showing endophytes to be latent and the solvent so obtained was collected. The ethyl acetate pathogens (Hyde and Soytong 2008). Several fungal taxa was evaporated off and the resultant compound was dried reported as endophytes closely resemble plant pathogenic with MgSO and concentrated to yield the crude extracts. fungi. This indicates close relationships exist between The crude extract was then dissolved in dimethyl sulphoxide endophytes and pathogens, but the molecular basis of (DMSO) for antimicrobial bioassay. switching of lifestyle from endophyte to parasite and the relationships they share are still to be elucidated. The aim Antimicrobial activity assay of the present work was to investigate the antimicrobial potential of endophytic Quambalaria sp. against some Antimicrobial activity of the crude metabolites was deter- clinically significant pathogens, and to elucidate the rela- mined by the agar cup diffusion method against six bacteria tionship among the pathogenic and endophytic strains (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, based on internal transcribed spacer (ITS) sequence and Salmonella typhi, Pseudomonas fluorescens, and Shigella RNA secondary structure analysis. dysenteriae) and three fungi (Trichosporon rubrum, Aspergil- lus fumigatus,and Candida albicans) as test pathogens. All the test microorganisms were obtained from the Institute of Materials and methods Microbial Technology (IMTECH), Chandigarh, India. Nutri- ent agar plates were inoculated with an overnight culture of Isolation of source organism each bacterial suspension. Similarly for the fungal pathogens, PDA plates were inoculated with each fungal suspension. The The endophytic fungus was isolated from the inner bark of plates with the inoculated organisms were evenly spread out an invasive plant, Ipomoea carnea, collected from the Uni- with sterile cotton swabs. Agar cups were prepared by scoop- versity campus of North Orissa, India. The procedure used ing out the media with a sterile cork borer (7 mm in diameter). to isolate this organism include collection of healthy stem The cups were then filled with 100 μl of the crude metabolites tissues, cutting it into several small segments (3–4 cm) and dissolved in DMSO to get a concentration of 1 mg/ml. The surface-sterilization by immersing sequentially in 70 % eth- plates were then incubated at 36±1 °C for 24 and 48 h, anol for 2 min and 0.5 % sodium hypochloride (NaOCl) for respectively, for bacteria and fungal pathogens. After the 5 min, then rinsing thoroughly with sterile distilled water. specified incubation period, the zone of inhibition was The surface-sterilized stems were then air-dried under lam- recorded and compared with the control (i.e. a cup filled with inar air flow chamber. Using a sterile blade, the outer tissues just DMSO solution). Three replicates were maintained in were gently removed and the inner tissues (0.5×0.5 cm ) each case. were dissected out and plated on potato dextrose agar (PDA) and water agar (WA) media. The plates were sealed with Determination of minimum inhibitory concentration parafilm and incubated in a BOD incubator at 30±1 °C for 2 weeks. The plated segments were observed once a day for The minimum inhibitory concentration (MIC) was deter- the growth of endophytic fungi. Hypal tips growing out the mined by micro-broth dilution assay in sterile 96-well plate. plated segments were immediately transferred into PDA A two-fold dilution of the crude metabolites with the con- slants, purified, and maintained at 4 °C. The source centration ranging from 1,000 to 62.5 μg/ml was made. The organism was among several endophytic isolates that wells were filled with 90 μl of each test bacterial and fungal displayed antimicrobial activity against some clinically suspension (approx. 10 CFU/ml). Crude metabolites (10 μl) significant microorganisms and, therefore, was selected of different concentrations were added into each well to give for further study. a final volume of 100 μl. Medium containing 10 % DMSO Ann Microbiol (2013) 63:793–800 795 was used as negative control. After incubation at 37 °C for to check on 1 % agarose gel. DNA sequencing was performed 24 h for bacteria and 48 h for fungal pathogens, a solution using an ABI 3730 sequencer. (10 μl) of triphenyl tetrazolium chloride (TTC) was added to each well as microbial growth indicator, and the micro- Taxon sampling and phylogenetic analysis plates were incubated for an additional 30 min. MIC was determined as the lowest concentration of the crude metab- The sequence of the fungus so obtained was annotated using olites at which no pink color appeared. All the determina- sequin software and submitted to the NCBI GenBank data- tions were conducted in duplicate. base. A total of 155 sequences of Quambalaria were also retrieved from Genbank (on 15 June 2011). The sequences Process optimization for metabolites production were filter-searched and altogether 53 sequences having 18S partial, ITS1, 5.8S, ITS2, 28S partial rRNA genes (ITS The endophyte was grown in different cultural conditions rDNA) were selected for phylogenetic analysis. Similarly, and the effect of various media—media amended with leaf the sequences were also trimmed for ITS2 using annotation extract, different pHs and incubation periods—were studied tools based on Hidden Markov Model (Keller et al. 2008) for optimum metabolite production. Different media were and only 9 sequences with complete ITS2 region were used, viz. potato dextrose broth (PDB), czapek dox dextrose obtained and selected for secondary structure analyses. For broth (CDB), malt extract (ME), and yeast extract (YE), into phylogenetic analysis, multiple sequence alignments were which leaf extracts (LE) of the host plant were added. For performed using CLUSTALW software utilizing default preparation of leaf extracts, 100 gm of leaves were boiled in settings, and trees were generated by the character state 250 ml of distilled water for 10 min and a decoction of Maximum Parsimony method using MEGA 4.0 (Tamura et 100 ml was added to 250 ml of each medium. pH range of al. 2007). 3–10 and incubation time up to 28 days in PDB were undertaken for optimum metabolite production. RNA secondary structure analysis Estimation of crude metabolites Altogether, 9 Quambalaria spp. (Table 1) with ITS2 sequences that consisted of two endophytic and seven path- The λ-max in ethyl acetate of the crude metabolites pro- ogenic strains were selected to generate the RNA secondary duced by the endophyte was determined by using a UV structure using mfold web server (Zuker 2003) with a preset spectrophotometer (SPECORD 210). A standard curve of folding temperature of 37 °C and the following default the bioactive metabolites was calibrated and the concentra- options: ionic conditions, 1 M NaCl, no divalent ions; tions of the bioactive metabolites produced were determined maximum number of nucleotides in a bulge or loop, 30; by comparing the optical density at 230 nm. maximum asymmetry of an interior/bulge loop, 30; percent- age suboptimality number, 5; upper bound on number of Isolation of genomic DNA, PCR amplification computed foldings, 50; and the structure chosen from dif- and sequencing ferent output files was the one with the highest negative free energy if various similar structures were obtained. The fungus was cultured on potato dextrose agar medium and a small amount of the mycelia was suspended in 40 μl MQ water. Genomic DNA was isolated by CTAB method. A Results and discussion portion of the genomic DNA was diluted to 50 ng/μl for use in PCR. The nuclear ribosomal DNA and ITS region of the Endophytic fungi are microbes that colonize inner healthy isolate were amplified using the universal primers ITS5 (5′- tissues of plants without causing disease. Strobel and Daisy GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′- (2003) are of the opinion that, considering the myriad of TCCTCCGCTTATTGATATGC-3′). The PCR was set up plants in the world, there is a great opportunity to find new using the following components: 2.5 μl buffer (10×), and interesting endophytic fungi. Some of the endophytes 1.5 μlMgCl (25 mM), 2.5 μldNTPs (2 mM), 0.2 μl are known to be host- and tissue-specific while others are Promega Taq (5 U/μl), 1.0 μl each of forward and reverse found colonizing various tissues and organs in different primers (5 pm/μl), and 6.0 μl DNA from diluted extract. hosts. There are several published reports indicating their The PCR condition was run with initial denaturation at 94 °C ecological role and elucidating their biological activities. for 3 min. Denaturation, annealing and extension were done at Endophytic fungi have been extensively investigated from 96 °C for 10 s, 55 °C for 10 s and 72 °C for 30 s, respectively, medicinal plants, but invasive plants are generally over- in 45 cycles. Final extension was done at 72 °C for 10 min and looked for endophytic study despite their uses in some traditional medicines and the uniqueness of their survival held at 4 °C. After the PCR cycle, 2 μl of the product was used 796 Ann Microbiol (2013) 63:793–800 Table 1 ITS2 sequences of Species Source Lifestyle Country GenBank Accession No. Quambalaria spp. used in the present study with their isolation Quambalaria sp. Ipomea carnea Endophyte India HQ316147.1 sources, lifestyles and GenBank accession numbers Q. pitereka Leptospermum juniperinum Endophyte Australia GQ258351.1 Q. eucalypti Myrceugenia glaucescens Pathogen Uruguay EU439923.1 Q. eucalypti M. glaucescens Pathogen Uruguay EU439922.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535498.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535495.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535494.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535493.1 Accession no. of the fungus Quambalaria sp. Eucalyptus Pathogen Brazil AJ535492.1 under study features. Research carried out on some invasive plant spe- 27±1 °C with periodic shaking at 150 rpm. After incuba- cies in United States for endophytic fungi has indicated their tion, crude metabolites of the fungus were extracted by a rich diversity in different tissues (Shipunov et al. 2008). In solvent extraction procedure using ethyl acetate as organic our present investigation, we isolated a rare and interesting solvent. The crude extract was evaluated for antimicrobial endophytic fungus from stem tissues of a very common activity against some clinically significant microorganisms invasive plant species of India, Ipomoea carnea. The mor- following agar cup diffusion assay. The metabolites dis- phological traits of the isolate were whitish, slow-growing played moderate to strong antimicrobial activity against all the test pathogens. The metabolites showed highest in and compact, producing diffusible reddish coloration pigment on PDA medium (ESM Fig. 1a, b). The organism was very vitro activity against Shigella dysenteriae followed by similar to Streptomyces sp. in cultural morphology. The overall Escherichia coli and Candida albicans (Table 2,ESM morphology of the isolate fitted best with an Quambalaria sp. Fig. 2). The ability to inhibit both bacterial and fungal describedbyAminetal. (2010), who first reported this species pathogens indicated the metabolites to be broad spec- as an endophyte in tissues of Leptospermum juniperinum.To trum in nature and suggested that this fungus as an our knowledge, this is the second report of this fungus as an endophyte may protect the host against invading patho- endophyte from Ipomoea carnea tissue. Furthermore, molecu- gens. Similarly, an endophytic Quambalaria sp. isolated lar identification was carried out based on ITS rDNA sequence from tissues of Leptospermum juniperinum has also analysis. A BLAST search result of the sequence indicated demonstrated significant biological activity against a 97 % homology with Quambalaria cyanescens (strain IMI wide range of bacterial and fungal pathogens (Amin et 298177) having GenBank accession no. AJ535500. The se- al. 2010). The antimicrobial property of this fungus may quence of this fungus has also been deposited in GenBank with be due to the development of an extracellular dark-red accession no. HQ316147.1. violet diffusible pigment which consisted of a mixture The fungus was grown aerobically on potato dextrose of volatile organic compounds, and one such compound broth (PDB) in a BOD shaking incubator for 3 weeks at sesquiterpenoid (+) globulol with antimycotic property has been reported from Q. cyanescens (Stodulkova et al. 2008). Thus, it can be assumed that the ability of I. Table 2 Antimicrobial activity of the crude metabolites of endophytic carnea to grow better and to have greater competitive- fungus, Quambalaria sp. ness in its invaded range may be due to the association Test organism Zone of inhibition Minimum inhibitory of such endophyte that produces novel allelochemicals, (mm ± SD) concentration (μg/ml) inhibitory to invading pathogens, and which may also help in competing with other fungi which occur in the Escherichia coli 27.3±0.57 62.5 same habitat. Bacillus subtilis 19.0±1.40 250 The effects of different media, pH, and incubation period Staphylococcus aureus 17.6±0.57 250 were studied for optimum metabolites production. The activ- Salmonella typhi 16.6±1.15 250 ity of the metabolites was tested against B. subtilis and E. coli. Pseudomonas fluorescens 23.6±1.52 125 Overall, increases in metabolite activity was observed in me- Shigella dysenteriae 28.0±2.00 >62.5 dia amended with leaf extract of the host plant (Fig. 1). The Trichosporon rubrum 18.3±1.52 250 strain showed maximum activity in CDB and PDB media Aspergillus fumigatus 06.3±1.52 <500 amended with leaf extract against E. coli while maximum Candida albicans 25.0±0.00 125 activity against B. subtilis was recorded in YE and CDB media amended with leaf extract. Lowest activity was Values are mean of three replicates Ann Microbiol (2013) 63:793–800 797 B. subtilis E. coli 369 12 15 18 21 24 28 Days Fig. 3 Effect of incubation period on the activity of metabolites Fig. 1 Effect of different media and combinations on the activity of produced by Quambalaria sp. metabolites produced by Quambalaria sp. PDB potato dextrose broth; ME malt extract; YE yeast extract; LE leaf extract isolated from Orthosiphon stamineus in which increased anti- microbial activity was observed after addition of water extract observed in YE medium against E. coli. Optimum metabolite of the host plant (Tong et al. 2011). Such increased metabolite activity involved 7.0 pH (Fig. 2) and a 15-day incubation activity may be due to some active principles in the host tissues period (Fig. 3). Many workers have also demonstrated that which might have induced the production of the fungal metab- maximum metabolite production in fungi needs neutral pH. olites. Incubation time is one of the crucial parameters in Rubini et al. (2005) reported growth and production of anti- metabolite production. Biomass production is often related to microbial metabolites in fungi at neutral pH. Similarly, opti- metabolite activity. In most fungi, maximum biomass produc- mum enzyme production of an endophytic fungus, tion takes place after 9–10 days of incubation (Gogoi et al. Pestalotiopsis sp., has been reported at neutral and slightly 2008). We found that optimum metabolite production was alkaline pH (Maria et al. 2005). However, some workers observed after 15 days of incubation. Strobel et al. (2008) have have reported acidic pH to be suitable for mycelial growth also reported similar results in an endophytic fungus, Gliocla- and metabolite production in fungi belonging to ascomycetes dium roseum, in which volatile antimicrobial metabolites were and basidiomycetes, including Cordyceps sp. (Park et al. 2001; produced after 18 days of incubation. The optimum metabolite Hsieh et al. 2005). In many instances, it has been observed that production at longer incubation periods may be due to the fact decreases in pH minimized metabolite production are probably that increased incubation might decrease fungal inhibitory because of toxic metabolite accumulation at low pH during the activities. The crude metabolites were partially characterized fermentation process. Nevertheless, optimum metabolite pro- by spectrophotometric analysis. The metabolites showed UV duction may be at variable pH depending upon the fungal λ-max (peak wavelength) in ethyl acetate at 284.6 nm with an species. Different media were used to observe the metabolite absorbance value of 1.093 (Fig. 4). production. It was found that metabolite production increased Phylogenetic analysis of the fungus was carried out in when the media were amended with leaf extract of the host relation to ITS rDNA sequences of different Quambalaria plant. Similar results were obtained in endophytic fungi spp. available in the GenBank database. The tree generated by the MP method out of 191 most parsimonious trees E. coli B. subtilis 25 Dry weight 2.5 20 2 15 1.5 10 1 5 0.5 0 0 34567 8 9 10 pH Fig. 2 Effect of pH on the activity of metabolites and biomass pro- Fig. 4 Standardization of UV λ max in ethyl acetate of the crude duction by Quambalaria sp. metabolites produced by endophytic Quambalaria sp. Inhibition zone (mm) Dry weight (mg/ml) Inhibition zone (mm) 798 Ann Microbiol (2013) 63:793–800 Fig. 5 Phylogenetic tree showing evolutionary relationship of 37 omitted for the final dataset. The evolutionary history was inferred Quambalaria spp. representing five species available with ITS rDNA using the Maximum Parsimony method. *Represents our own isolate seqeuences in GenBank database. Out of the total 53 sequences, 16 are and numbers indicate GenBank accession numbers Ann Microbiol (2013) 63:793–800 799 showed our sequence clustering with Q. cyanescens with a such as environmental stress, senescence of the plant, and supported bootstrap of 65 % (Fig. 5). The placement of our the plant defense response to infection. Such observations isolate within the clade of Q. cyanescens indicates its close indicate that endophytes could be pathogenic under certain affinity. Sequence analysis of the ITS region of nuclear circumstances. Clustering of our endophytic isolate within ribosomal DNA has been widely used for taxonomic place- the clade of Q. cyanescens which are mostly pathogens ment (Guo et al. 2001; Wang et al. 2005) and phylogenetic indicated such a relationship. ITS2 of the ITS region has study of fungi (Tejesvi et al. 2009). In the present study, the been foundto be highlyconserved and tovaryin both fungus isolated as the endophyte from I. carnea stem tissue primary sequences and secondary structures (Selig et al. showed closest homologue to Q. cyanescens, which is a 2008). It has been used as a possible marker in molecular pathogenic fungus belonging to the division basidiomyco- systematic and phylogenetic reconstruction by several tina causing diseases to Eucalyptus (Beer et al. 2006). This researchers (Coleman 2007; Miao et al. 2008; Schultz and indicated a close affinity between endophytes and pathogens Wolf 2009). Since the structure is more conserved than the as suggested by several workers (Sieber 2007; Brown et al. sequence, we generated a RNA secondary structure of en- 1998). According to Schulz and Boyle (2005), the mutual- dophytic and pathogenic strains of Quambalaria to validate istic interaction of endophytes may only be temporary and is their relationship. The result showed that there is close subject to change over time, depending on several factors resemblances in RNA secondary structural features of en- dophytic and pathogenic strains (Fig. 6). Such findings suggest that host–microbe interactions can range from mu- tualism through commensalism to parasitism in a continu- ous manner, and agree with the assumption that endophytes can become parasites under certain condition and vice versa. Further, among the generated secondary structures, some unique structure features were obtained which distinguished our isolate from other Quambalaria isolates used in the present study (Fig. 7). These structural features can also be used as potential barcodes for our isolate. One of the unique features was the long 5' dangling end with 9 bases. It has (a) AJ545393.1 (b) AJ545398.1 (c) GQ258351.1 (d) HQ316147.1* Fig. 6 ITS2 RNA secondary structures of pathogenic (a, b)and endophytic (c, d) strains of Quambalaria spp. The pathogenic and Fig. 7 ITS2 RNA secondary structural features of Quambalaria sp. endophytic strains considered are from different hosts as indicated in (HQ316147.1) showing junctions/hinges (a, b) and a 5’ dangling end Table 1 (c) which is different from secondary structures of the other 8 isolates 800 Ann Microbiol (2013) 63:793–800 Miao M, Warrenb A, Songa W, Wangc S, Shanga H, Chena Z (2008) been reported that a long RNA dangling end has large Analysis of the Internal Transcribed Spacer 2 (ITS2) region of energetic contribution to duplex stability (Ohmichi et al. Scuticociliates and Related Taxa (Ciliophora, Oligohymeno- 2002). Although endophytes are found colonizing every phorea) to infer their evolution and phylogeny. Protist 159:519– plant species, the vast majority of plants are still to be 533 Ohmichi T, Nakano S, Miyoshi D, Sugimoto N (2002) Long RNA explored for endophytic microbes. Our study suggests that dangling end has large energetic contribution to duplex stability. J invasive plants could be a rich and reliable source of genetic Am Chem Soc 124:35 diversity and novel endophytes. Production of biologically Park JP, Kim SW, Hwang HJ, Yun JW (2001) Optimization of sub- active metabolites capable of clinically significant inhibition merged culture conditions for the mycelial growth and exo- biopolymer production by Cordyceps militaris. Lett Appl Micro- also indicates that such endophytes could be a rich source of biol 33:76–81 antimicrobial metabolites and chemically novel compounds Rubini MR, Rute TSR, Pomella AW, Cristina SM, Arajo LW, Santos with great potential for development of new antimicrobial DRD, Azevedo JL (2005) Diversity of endophytic fungal com- agents. munity of cocao (Thebroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches’ Broom disease. Int J Biol Sci 1:24–33 Acknowledgements The authors are grateful to the coordinator, Schultz J, Wolf M (2009) ITS2 sequence-structure analysis in phylo- Bioinformatics Infrastructure facility (BIF), and the Head, Department genetics: A how to manual for molecular systematics. Mol Phy- of Botany, North Orissa University, India, for providing necessary facil- logenet Evol 52:520–523 ities to carry out the work. Financial support in the form of studentship by Schulz B, Boyle C (2005) The endophytic continuum. 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Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea

Padhi, Srichandan; Tayung, Kumanand
Annals of Microbiology , Volume 63 (2) – Sep 21, 2012
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Springer Journals
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Copyright © 2012 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-012-0534-4
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Abstract

Ann Microbiol (2013) 63:793–800 DOI 10.1007/s13213-012-0534-4 ORIGINAL ARTICLE Antimicrobial activity and molecular characterization of an endophytic fungus, Quambalaria sp. isolated from Ipomoea carnea Srichandan Padhi & Kumanand Tayung Received: 23 March 2012 /Accepted: 22 August 2012 /Published online: 21 September 2012 # Springer-Verlag and the University of Milan 2012 Abstract An endophytic fungus displaying considerable end were found to be unique to our isolate. These structural antimicrobial activity was isolated from stem tissue of an features can also be used as potential barcodes for this invasive plant species, Ipomoea carnea. The fungus was fungus. The findings indicate that invasive plant species identified as Quambalaria sp. and confirmed by ITS rDNA can be a reliable source of novel endophytes with rich sequence analysis. A BLAST search result of the sequence antimicrobial metabolites. The study also validates the indicated 97 % homology with Quambalaria cyanescens. assumption that endophytes can become parasites and share Crude metabolites of the fungus showed considerable anti- a close affinity. microbial activity against a panel of clinically significant . . microorganisms. The metabolites showed highest in vitro Keywords Ipomoea carnea Quambalaria sp. . . activity against Shigella dysenteriae followed by Escheri- Antimicrobial activity Phylogenetic analysis ITS2 RNA chia coli and Candida albicans. Optimum metabolites pro- secondary structures duction required neutral pH and a 15-day incubation period. Bark extracts amended with fungal media demonstrated higher antimicrobial activity. Optimum metabolites activity Introduction wasrecordedin Czapek Doxbroth amendedwithleaf extracts (CDB + LE) of the host plant. The metabolites Endophytic fungi that colonized inner plant tissues have been showed UV λ-max in ethyl acetate at 284.6 nm with an found to be associated with every plant species so far inves- absorbance value of 1.093. Phylogenetic tree generated by tigated. They are asymptomatic and often considered as the Maximum Parsimony method showed clustering of our mutualistic, benefiting the host by producing plant growth isolate with Q. cyanescens with supported bootstrap of regulatory, antimicrobial, antiviral and insecticidal substances 65 %. Species of Quambalaria are pathogens to Eucalyptus (Carroll 1988). It has been suggested that studies of endophyt- and occurrence of this fungus as endophytes support it to be ic fungi should be directed to plants growing in unique envi- a latent pathogen. Sequence base analysis and RNA second- ronmental settings, especially those with an unusual biology ary structure study also confirmed such a relationship. Sec- and possessing novel strategies for survival or plants with ondary structural features like two hinges and a 5’ dangling medicinal uses, as they are expected to harbor novel endo- phytes that may produce unique metabolites having diverse Electronic supplementary material The online version of this article applications (Strobel and Daisy 2003). Many workers gener- (doi:10.1007/s13213-012-0534-4) contains supplementary material, ally give priority to studying endophytic fungi associated with which is available to authorized users. medicinal plants because these microbes make the same bio- S. Padhi active natural products or derivatives that are more bioactive Department of Bioinformatics, North Orissa University, than those of their respective host (Strobel 2001). Neverthe- Takatpur, less, invasive plant species, even though having unique sur- Baripada 757003, India vival features, have been less studied for their endophytic K. Tayung (*) fungi. It is believed that associative endophytes of invasive Department of Botany, North Orissa University, plants could contribute to their greater competitiveness and Takatpur, may also produce novel allelochemicals that might have Baripada 757003, India inhibitory effects (Shipunov et al. 2008). Further endophytic e-mail: kumanandnou@yahoo.com 794 Ann Microbiol (2013) 63:793–800 fungi colonize such plants, which means they have evolved Fungal cultivation and metabolites extraction special mechanisms for survival and may provide new sources of secondary metabolites for biotechnological applications. The endophyte was cultivated on Potato Dextrose Broth We studied endophytic fungi associated with Ipomoea (PDB) by placing agar blocks of pure culture (3 mm in carnea tissues, a very common invasive species of India diameter) of actively growing culture in 250-ml Erlenmeyer and screened them for their antimicrobial potentials. A flask containing 100 ml of the medium. The flask was very rare endophytic fungus, Quambalaria sp., with con- incubated in a BOD shaking incubator for 3 weeks at 27± siderable antimicrobial activity was isolated from the inner 1 °C with periodic shaking at 150 rpm. The culture was bark tissue of I. carnea. A literature survey of the fungus filtered through sterile cheesecloth to remove the mycelial revealed it to be a pathogen causing diseases in Eucalyp- mats. The liquid broth was collected and extracted with an tus (Simpson 2000;Beeretal. 2006). Endophytes are equal volume of ethyl acetate in a separating funnel by generally regarded as symbiotic or mutualistic, but there vigorous shaking for 10 min. The cell mass was separated are also many examples showing endophytes to be latent and the solvent so obtained was collected. The ethyl acetate pathogens (Hyde and Soytong 2008). Several fungal taxa was evaporated off and the resultant compound was dried reported as endophytes closely resemble plant pathogenic with MgSO and concentrated to yield the crude extracts. fungi. This indicates close relationships exist between The crude extract was then dissolved in dimethyl sulphoxide endophytes and pathogens, but the molecular basis of (DMSO) for antimicrobial bioassay. switching of lifestyle from endophyte to parasite and the relationships they share are still to be elucidated. The aim Antimicrobial activity assay of the present work was to investigate the antimicrobial potential of endophytic Quambalaria sp. against some Antimicrobial activity of the crude metabolites was deter- clinically significant pathogens, and to elucidate the rela- mined by the agar cup diffusion method against six bacteria tionship among the pathogenic and endophytic strains (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, based on internal transcribed spacer (ITS) sequence and Salmonella typhi, Pseudomonas fluorescens, and Shigella RNA secondary structure analysis. dysenteriae) and three fungi (Trichosporon rubrum, Aspergil- lus fumigatus,and Candida albicans) as test pathogens. All the test microorganisms were obtained from the Institute of Materials and methods Microbial Technology (IMTECH), Chandigarh, India. Nutri- ent agar plates were inoculated with an overnight culture of Isolation of source organism each bacterial suspension. Similarly for the fungal pathogens, PDA plates were inoculated with each fungal suspension. The The endophytic fungus was isolated from the inner bark of plates with the inoculated organisms were evenly spread out an invasive plant, Ipomoea carnea, collected from the Uni- with sterile cotton swabs. Agar cups were prepared by scoop- versity campus of North Orissa, India. The procedure used ing out the media with a sterile cork borer (7 mm in diameter). to isolate this organism include collection of healthy stem The cups were then filled with 100 μl of the crude metabolites tissues, cutting it into several small segments (3–4 cm) and dissolved in DMSO to get a concentration of 1 mg/ml. The surface-sterilization by immersing sequentially in 70 % eth- plates were then incubated at 36±1 °C for 24 and 48 h, anol for 2 min and 0.5 % sodium hypochloride (NaOCl) for respectively, for bacteria and fungal pathogens. After the 5 min, then rinsing thoroughly with sterile distilled water. specified incubation period, the zone of inhibition was The surface-sterilized stems were then air-dried under lam- recorded and compared with the control (i.e. a cup filled with inar air flow chamber. Using a sterile blade, the outer tissues just DMSO solution). Three replicates were maintained in were gently removed and the inner tissues (0.5×0.5 cm ) each case. were dissected out and plated on potato dextrose agar (PDA) and water agar (WA) media. The plates were sealed with Determination of minimum inhibitory concentration parafilm and incubated in a BOD incubator at 30±1 °C for 2 weeks. The plated segments were observed once a day for The minimum inhibitory concentration (MIC) was deter- the growth of endophytic fungi. Hypal tips growing out the mined by micro-broth dilution assay in sterile 96-well plate. plated segments were immediately transferred into PDA A two-fold dilution of the crude metabolites with the con- slants, purified, and maintained at 4 °C. The source centration ranging from 1,000 to 62.5 μg/ml was made. The organism was among several endophytic isolates that wells were filled with 90 μl of each test bacterial and fungal displayed antimicrobial activity against some clinically suspension (approx. 10 CFU/ml). Crude metabolites (10 μl) significant microorganisms and, therefore, was selected of different concentrations were added into each well to give for further study. a final volume of 100 μl. Medium containing 10 % DMSO Ann Microbiol (2013) 63:793–800 795 was used as negative control. After incubation at 37 °C for to check on 1 % agarose gel. DNA sequencing was performed 24 h for bacteria and 48 h for fungal pathogens, a solution using an ABI 3730 sequencer. (10 μl) of triphenyl tetrazolium chloride (TTC) was added to each well as microbial growth indicator, and the micro- Taxon sampling and phylogenetic analysis plates were incubated for an additional 30 min. MIC was determined as the lowest concentration of the crude metab- The sequence of the fungus so obtained was annotated using olites at which no pink color appeared. All the determina- sequin software and submitted to the NCBI GenBank data- tions were conducted in duplicate. base. A total of 155 sequences of Quambalaria were also retrieved from Genbank (on 15 June 2011). The sequences Process optimization for metabolites production were filter-searched and altogether 53 sequences having 18S partial, ITS1, 5.8S, ITS2, 28S partial rRNA genes (ITS The endophyte was grown in different cultural conditions rDNA) were selected for phylogenetic analysis. Similarly, and the effect of various media—media amended with leaf the sequences were also trimmed for ITS2 using annotation extract, different pHs and incubation periods—were studied tools based on Hidden Markov Model (Keller et al. 2008) for optimum metabolite production. Different media were and only 9 sequences with complete ITS2 region were used, viz. potato dextrose broth (PDB), czapek dox dextrose obtained and selected for secondary structure analyses. For broth (CDB), malt extract (ME), and yeast extract (YE), into phylogenetic analysis, multiple sequence alignments were which leaf extracts (LE) of the host plant were added. For performed using CLUSTALW software utilizing default preparation of leaf extracts, 100 gm of leaves were boiled in settings, and trees were generated by the character state 250 ml of distilled water for 10 min and a decoction of Maximum Parsimony method using MEGA 4.0 (Tamura et 100 ml was added to 250 ml of each medium. pH range of al. 2007). 3–10 and incubation time up to 28 days in PDB were undertaken for optimum metabolite production. RNA secondary structure analysis Estimation of crude metabolites Altogether, 9 Quambalaria spp. (Table 1) with ITS2 sequences that consisted of two endophytic and seven path- The λ-max in ethyl acetate of the crude metabolites pro- ogenic strains were selected to generate the RNA secondary duced by the endophyte was determined by using a UV structure using mfold web server (Zuker 2003) with a preset spectrophotometer (SPECORD 210). A standard curve of folding temperature of 37 °C and the following default the bioactive metabolites was calibrated and the concentra- options: ionic conditions, 1 M NaCl, no divalent ions; tions of the bioactive metabolites produced were determined maximum number of nucleotides in a bulge or loop, 30; by comparing the optical density at 230 nm. maximum asymmetry of an interior/bulge loop, 30; percent- age suboptimality number, 5; upper bound on number of Isolation of genomic DNA, PCR amplification computed foldings, 50; and the structure chosen from dif- and sequencing ferent output files was the one with the highest negative free energy if various similar structures were obtained. The fungus was cultured on potato dextrose agar medium and a small amount of the mycelia was suspended in 40 μl MQ water. Genomic DNA was isolated by CTAB method. A Results and discussion portion of the genomic DNA was diluted to 50 ng/μl for use in PCR. The nuclear ribosomal DNA and ITS region of the Endophytic fungi are microbes that colonize inner healthy isolate were amplified using the universal primers ITS5 (5′- tissues of plants without causing disease. Strobel and Daisy GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′- (2003) are of the opinion that, considering the myriad of TCCTCCGCTTATTGATATGC-3′). The PCR was set up plants in the world, there is a great opportunity to find new using the following components: 2.5 μl buffer (10×), and interesting endophytic fungi. Some of the endophytes 1.5 μlMgCl (25 mM), 2.5 μldNTPs (2 mM), 0.2 μl are known to be host- and tissue-specific while others are Promega Taq (5 U/μl), 1.0 μl each of forward and reverse found colonizing various tissues and organs in different primers (5 pm/μl), and 6.0 μl DNA from diluted extract. hosts. There are several published reports indicating their The PCR condition was run with initial denaturation at 94 °C ecological role and elucidating their biological activities. for 3 min. Denaturation, annealing and extension were done at Endophytic fungi have been extensively investigated from 96 °C for 10 s, 55 °C for 10 s and 72 °C for 30 s, respectively, medicinal plants, but invasive plants are generally over- in 45 cycles. Final extension was done at 72 °C for 10 min and looked for endophytic study despite their uses in some traditional medicines and the uniqueness of their survival held at 4 °C. After the PCR cycle, 2 μl of the product was used 796 Ann Microbiol (2013) 63:793–800 Table 1 ITS2 sequences of Species Source Lifestyle Country GenBank Accession No. Quambalaria spp. used in the present study with their isolation Quambalaria sp. Ipomea carnea Endophyte India HQ316147.1 sources, lifestyles and GenBank accession numbers Q. pitereka Leptospermum juniperinum Endophyte Australia GQ258351.1 Q. eucalypti Myrceugenia glaucescens Pathogen Uruguay EU439923.1 Q. eucalypti M. glaucescens Pathogen Uruguay EU439922.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535498.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535495.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535494.1 Quambalaria sp. Eucalyptus Pathogen Brazil AJ535493.1 Accession no. of the fungus Quambalaria sp. Eucalyptus Pathogen Brazil AJ535492.1 under study features. Research carried out on some invasive plant spe- 27±1 °C with periodic shaking at 150 rpm. After incuba- cies in United States for endophytic fungi has indicated their tion, crude metabolites of the fungus were extracted by a rich diversity in different tissues (Shipunov et al. 2008). In solvent extraction procedure using ethyl acetate as organic our present investigation, we isolated a rare and interesting solvent. The crude extract was evaluated for antimicrobial endophytic fungus from stem tissues of a very common activity against some clinically significant microorganisms invasive plant species of India, Ipomoea carnea. The mor- following agar cup diffusion assay. The metabolites dis- phological traits of the isolate were whitish, slow-growing played moderate to strong antimicrobial activity against all the test pathogens. The metabolites showed highest in and compact, producing diffusible reddish coloration pigment on PDA medium (ESM Fig. 1a, b). The organism was very vitro activity against Shigella dysenteriae followed by similar to Streptomyces sp. in cultural morphology. The overall Escherichia coli and Candida albicans (Table 2,ESM morphology of the isolate fitted best with an Quambalaria sp. Fig. 2). The ability to inhibit both bacterial and fungal describedbyAminetal. (2010), who first reported this species pathogens indicated the metabolites to be broad spec- as an endophyte in tissues of Leptospermum juniperinum.To trum in nature and suggested that this fungus as an our knowledge, this is the second report of this fungus as an endophyte may protect the host against invading patho- endophyte from Ipomoea carnea tissue. Furthermore, molecu- gens. Similarly, an endophytic Quambalaria sp. isolated lar identification was carried out based on ITS rDNA sequence from tissues of Leptospermum juniperinum has also analysis. A BLAST search result of the sequence indicated demonstrated significant biological activity against a 97 % homology with Quambalaria cyanescens (strain IMI wide range of bacterial and fungal pathogens (Amin et 298177) having GenBank accession no. AJ535500. The se- al. 2010). The antimicrobial property of this fungus may quence of this fungus has also been deposited in GenBank with be due to the development of an extracellular dark-red accession no. HQ316147.1. violet diffusible pigment which consisted of a mixture The fungus was grown aerobically on potato dextrose of volatile organic compounds, and one such compound broth (PDB) in a BOD shaking incubator for 3 weeks at sesquiterpenoid (+) globulol with antimycotic property has been reported from Q. cyanescens (Stodulkova et al. 2008). Thus, it can be assumed that the ability of I. Table 2 Antimicrobial activity of the crude metabolites of endophytic carnea to grow better and to have greater competitive- fungus, Quambalaria sp. ness in its invaded range may be due to the association Test organism Zone of inhibition Minimum inhibitory of such endophyte that produces novel allelochemicals, (mm ± SD) concentration (μg/ml) inhibitory to invading pathogens, and which may also help in competing with other fungi which occur in the Escherichia coli 27.3±0.57 62.5 same habitat. Bacillus subtilis 19.0±1.40 250 The effects of different media, pH, and incubation period Staphylococcus aureus 17.6±0.57 250 were studied for optimum metabolites production. The activ- Salmonella typhi 16.6±1.15 250 ity of the metabolites was tested against B. subtilis and E. coli. Pseudomonas fluorescens 23.6±1.52 125 Overall, increases in metabolite activity was observed in me- Shigella dysenteriae 28.0±2.00 >62.5 dia amended with leaf extract of the host plant (Fig. 1). The Trichosporon rubrum 18.3±1.52 250 strain showed maximum activity in CDB and PDB media Aspergillus fumigatus 06.3±1.52 <500 amended with leaf extract against E. coli while maximum Candida albicans 25.0±0.00 125 activity against B. subtilis was recorded in YE and CDB media amended with leaf extract. Lowest activity was Values are mean of three replicates Ann Microbiol (2013) 63:793–800 797 B. subtilis E. coli 369 12 15 18 21 24 28 Days Fig. 3 Effect of incubation period on the activity of metabolites Fig. 1 Effect of different media and combinations on the activity of produced by Quambalaria sp. metabolites produced by Quambalaria sp. PDB potato dextrose broth; ME malt extract; YE yeast extract; LE leaf extract isolated from Orthosiphon stamineus in which increased anti- microbial activity was observed after addition of water extract observed in YE medium against E. coli. Optimum metabolite of the host plant (Tong et al. 2011). Such increased metabolite activity involved 7.0 pH (Fig. 2) and a 15-day incubation activity may be due to some active principles in the host tissues period (Fig. 3). Many workers have also demonstrated that which might have induced the production of the fungal metab- maximum metabolite production in fungi needs neutral pH. olites. Incubation time is one of the crucial parameters in Rubini et al. (2005) reported growth and production of anti- metabolite production. Biomass production is often related to microbial metabolites in fungi at neutral pH. Similarly, opti- metabolite activity. In most fungi, maximum biomass produc- mum enzyme production of an endophytic fungus, tion takes place after 9–10 days of incubation (Gogoi et al. Pestalotiopsis sp., has been reported at neutral and slightly 2008). We found that optimum metabolite production was alkaline pH (Maria et al. 2005). However, some workers observed after 15 days of incubation. Strobel et al. (2008) have have reported acidic pH to be suitable for mycelial growth also reported similar results in an endophytic fungus, Gliocla- and metabolite production in fungi belonging to ascomycetes dium roseum, in which volatile antimicrobial metabolites were and basidiomycetes, including Cordyceps sp. (Park et al. 2001; produced after 18 days of incubation. The optimum metabolite Hsieh et al. 2005). In many instances, it has been observed that production at longer incubation periods may be due to the fact decreases in pH minimized metabolite production are probably that increased incubation might decrease fungal inhibitory because of toxic metabolite accumulation at low pH during the activities. The crude metabolites were partially characterized fermentation process. Nevertheless, optimum metabolite pro- by spectrophotometric analysis. The metabolites showed UV duction may be at variable pH depending upon the fungal λ-max (peak wavelength) in ethyl acetate at 284.6 nm with an species. Different media were used to observe the metabolite absorbance value of 1.093 (Fig. 4). production. It was found that metabolite production increased Phylogenetic analysis of the fungus was carried out in when the media were amended with leaf extract of the host relation to ITS rDNA sequences of different Quambalaria plant. Similar results were obtained in endophytic fungi spp. available in the GenBank database. The tree generated by the MP method out of 191 most parsimonious trees E. coli B. subtilis 25 Dry weight 2.5 20 2 15 1.5 10 1 5 0.5 0 0 34567 8 9 10 pH Fig. 2 Effect of pH on the activity of metabolites and biomass pro- Fig. 4 Standardization of UV λ max in ethyl acetate of the crude duction by Quambalaria sp. metabolites produced by endophytic Quambalaria sp. Inhibition zone (mm) Dry weight (mg/ml) Inhibition zone (mm) 798 Ann Microbiol (2013) 63:793–800 Fig. 5 Phylogenetic tree showing evolutionary relationship of 37 omitted for the final dataset. The evolutionary history was inferred Quambalaria spp. representing five species available with ITS rDNA using the Maximum Parsimony method. *Represents our own isolate seqeuences in GenBank database. Out of the total 53 sequences, 16 are and numbers indicate GenBank accession numbers Ann Microbiol (2013) 63:793–800 799 showed our sequence clustering with Q. cyanescens with a such as environmental stress, senescence of the plant, and supported bootstrap of 65 % (Fig. 5). The placement of our the plant defense response to infection. Such observations isolate within the clade of Q. cyanescens indicates its close indicate that endophytes could be pathogenic under certain affinity. Sequence analysis of the ITS region of nuclear circumstances. Clustering of our endophytic isolate within ribosomal DNA has been widely used for taxonomic place- the clade of Q. cyanescens which are mostly pathogens ment (Guo et al. 2001; Wang et al. 2005) and phylogenetic indicated such a relationship. ITS2 of the ITS region has study of fungi (Tejesvi et al. 2009). In the present study, the been foundto be highlyconserved and tovaryin both fungus isolated as the endophyte from I. carnea stem tissue primary sequences and secondary structures (Selig et al. showed closest homologue to Q. cyanescens, which is a 2008). It has been used as a possible marker in molecular pathogenic fungus belonging to the division basidiomyco- systematic and phylogenetic reconstruction by several tina causing diseases to Eucalyptus (Beer et al. 2006). This researchers (Coleman 2007; Miao et al. 2008; Schultz and indicated a close affinity between endophytes and pathogens Wolf 2009). Since the structure is more conserved than the as suggested by several workers (Sieber 2007; Brown et al. sequence, we generated a RNA secondary structure of en- 1998). According to Schulz and Boyle (2005), the mutual- dophytic and pathogenic strains of Quambalaria to validate istic interaction of endophytes may only be temporary and is their relationship. The result showed that there is close subject to change over time, depending on several factors resemblances in RNA secondary structural features of en- dophytic and pathogenic strains (Fig. 6). Such findings suggest that host–microbe interactions can range from mu- tualism through commensalism to parasitism in a continu- ous manner, and agree with the assumption that endophytes can become parasites under certain condition and vice versa. Further, among the generated secondary structures, some unique structure features were obtained which distinguished our isolate from other Quambalaria isolates used in the present study (Fig. 7). These structural features can also be used as potential barcodes for our isolate. One of the unique features was the long 5' dangling end with 9 bases. It has (a) AJ545393.1 (b) AJ545398.1 (c) GQ258351.1 (d) HQ316147.1* Fig. 6 ITS2 RNA secondary structures of pathogenic (a, b)and endophytic (c, d) strains of Quambalaria spp. The pathogenic and Fig. 7 ITS2 RNA secondary structural features of Quambalaria sp. endophytic strains considered are from different hosts as indicated in (HQ316147.1) showing junctions/hinges (a, b) and a 5’ dangling end Table 1 (c) which is different from secondary structures of the other 8 isolates 800 Ann Microbiol (2013) 63:793–800 Miao M, Warrenb A, Songa W, Wangc S, Shanga H, Chena Z (2008) been reported that a long RNA dangling end has large Analysis of the Internal Transcribed Spacer 2 (ITS2) region of energetic contribution to duplex stability (Ohmichi et al. Scuticociliates and Related Taxa (Ciliophora, Oligohymeno- 2002). Although endophytes are found colonizing every phorea) to infer their evolution and phylogeny. Protist 159:519– plant species, the vast majority of plants are still to be 533 Ohmichi T, Nakano S, Miyoshi D, Sugimoto N (2002) Long RNA explored for endophytic microbes. Our study suggests that dangling end has large energetic contribution to duplex stability. J invasive plants could be a rich and reliable source of genetic Am Chem Soc 124:35 diversity and novel endophytes. Production of biologically Park JP, Kim SW, Hwang HJ, Yun JW (2001) Optimization of sub- active metabolites capable of clinically significant inhibition merged culture conditions for the mycelial growth and exo- biopolymer production by Cordyceps militaris. Lett Appl Micro- also indicates that such endophytes could be a rich source of biol 33:76–81 antimicrobial metabolites and chemically novel compounds Rubini MR, Rute TSR, Pomella AW, Cristina SM, Arajo LW, Santos with great potential for development of new antimicrobial DRD, Azevedo JL (2005) Diversity of endophytic fungal com- agents. munity of cocao (Thebroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches’ Broom disease. Int J Biol Sci 1:24–33 Acknowledgements The authors are grateful to the coordinator, Schultz J, Wolf M (2009) ITS2 sequence-structure analysis in phylo- Bioinformatics Infrastructure facility (BIF), and the Head, Department genetics: A how to manual for molecular systematics. Mol Phy- of Botany, North Orissa University, India, for providing necessary facil- logenet Evol 52:520–523 ities to carry out the work. Financial support in the form of studentship by Schulz B, Boyle C (2005) The endophytic continuum. 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Journal

Annals of Microbiology – Springer Journals

Published: Sep 21, 2012

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