curr-Antimicrobial activity and biodiversity

Antimicrobial Activity and Biodiversity of Endophytic Fungi in Dendrobium devonianum and Dendrobium thyrsiflorum from VietmanYong-Mei Xing •Juan Chen •Jin-Long Cui •Xiao-Mei Chen •Shun-Xing GuoReceived:23June 2010/Accepted:3December 2010/Published online:22December 2010ÓSpringer Science+Business Media,LLC 2010Abstract Endophytic fungi are rich in orchids and have great impacts on their host plants.53endophytes (30iso-lates from Dendrobium devonianum and 23endophytic fungi from D.thyrsiflorum )were isolated,respectively,from roots and stems of Dendrobium species.All the fungi were identified by way of morphological and/or molecular biological methods.30endophytic fungi in D .devonianum were categorized into 11taxa and 23fungal endophytes in D.thyrsiflorum were grouped into 11genera,respectively.Fusarium was the dominant species of the two Dendrobium species in common.Antimicrobial activity of ethanol extract of fermentation broth of these fungi was explored using agar diffusion test.10endophytic fungi in D.devo-nianum and 11in D.thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among 6pathogenic microbes (Escherichia coli ,Bacillus subtilis ,Staphylococcus aureus ,Candida albicans ,Cryp-tococcus neoformans ,and Aspergillus fumigatus ).Out of the fungal endophytes isolated from D.devonianum and D.thyrsiflorum ,Phoma displayed strong inhibitory activity (inhibition zones in diameter [20mm)against pathogens.Epicoccum nigrum from D.thyrsiflorum exhibited anti-bacterial activity even stronger than ampicillin sodium.Fusarium isolated from the two Dendrobium species was effective against the pathogenic bacterial as well as fungal pathogens.The study reinforced the assumption that endo-phytic fungi isolated from different Dendrobium species could be of potential antibacterial or antifungal resource.IntroductionDrug resistance in microorganisms and overuse of antibi-otics are becoming a serious concern around the world [1].Therefore,there is an urgent need to search for effective new antibacterial or antifungal agents in treatment of infectious diseases at present.Recently,actinomycetes,cyanobacteria,and uncultured bacteria have received con-siderable attention as new sources for new antibiotics dis-covery [11].Meanwhile,promising approaches of mining unexplored microbial niches for use as antibiotics have also been emerging [16].Endophytic fungi were defined as fungi residing the living plant tissue without causing any apparent diseases or injuries to the host plant [10].To date,many studies have elucidated the ubiquity of the endophytic fungi in plants and the mutualistic relationship between the fungal endo-phytes and the host plant [13].It was also documented that fungal endophytes could exert beneficial activities on host plants such as favorable,growth-promoting effects,increasing host fitness and contributing to effective host defense against pathogens,herbivores,or abiotic stress [2,12,19].Nowadays,fungal endophytes have been recog-nized as relatively unexplored and potential sources of bioactive secondary metabolites for exploitation in medi-cine,agriculture,and industry [9,17,22,34].Furthermore,antimicrobial substances that could be produced by endo-phytic fungi have drawn even greater attention [7,18,21,28].This gives rise to people’s interest in screening endophytic fungi for discovery of novel metabolites.Traditional medicines (mostly derived from plants)have profound health promoting benefits.However,the wild sources of most medicinal plants have been seriously depleted due to over-harvesting and inefficient protection.It was reported that endopytic fungi could produce similarY.-M.Xing ÁJ.Chen ÁJ.-L.Cui ÁX.-M.Chen ÁS.-X.Guo (&)Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences &Peking Union Medical College,No.151,Malianwa North Road,Haidian District,Beijing 100193,People’s Republic of Chinae-mail:sxguo2006@Curr Microbiol (2011)62:1218–1224DOI 10.1007/s00284-010-9848-2or the same bioactive metabolites as its host plant[20,30]. To the best of our knowledge,since fungal endophytes that reside in their host plants might produce unique metabo-lites with multifold functions,it is necessary to perform studies of plant-associated endophytic fungi.In China, Dendrobium genus,one of the large groups of Orchidaceae family plants,was widely used in traditional Chinese medicine bearing high medicinal value for curing cataracts, throat inflammation,and digestive system disorders [14,27].Thus,the present study was conducted to inves-tigate endophytic fungi associated with medicinal plants of D.devonianum and D.thyrsiflorum isolated from Longling of Vietman and to evaluate antimicrobial activity of the fungal endophytes against six human pathogens.Screening of endophytic fungi with inhibitory activities against pathogenic bacteria or fungi and systematic investigations of endophytic fungi related to Dendrobium species might provide us with new knowledge for interpreting interrela-tionship of fungal endophytes with Dendrobium under natural stress biotope and discover,if possible,novel nat-ural products with higher antimicrobial activity,as well. Materials and MethodsDendrobium devonianum and D.thyrsiflorum were col-lected from Longling,Vietnam.Stems and roots of each sample were rinsed with water and surface-disinfected by immersion in75%ethanol for1min,5min in5%sodium hypochlorite solution,and1min in sterile de-ionized water for three times.The samples were then surface-dried with sterilefilter paper[9].Roots and stems were cut into 0.5cm90.5cm pieces and placed in petri dishes(9cm in diameter)with potato dextrose agar(PDA)medium(g/l; dextrose-20,agar-15,potato infusion-200)and cultured at 25°C under dark[4].Identification of Endophytic Fungi with Antibacterial and Antifungal ActivitiesMycelia of fungi were soaked in10%KOH and observed with ZEISS Axio Imager A1microscope for morphological examination.Endophytic fungi failing to sporulate were identified by means of molecular biological analysis of the internal transcribed region(ITS)of ribosomal DNA.Uni-versal primers of ITS1(50-TCCGTAGGTGAACCTGC GG-30)and ITS4(50-TCCTCCGCTTATTGATATGC-30) were used[29]to amplify ribosomal internal transcribed spacer(ITS).DNA was extracted from21-day-old culture in PDA medium using E.Z.N.A.TM Fungal DNA Mini Kit (Omegabiotek,Norcross,USA).PCR was carried out as previously described[5].The PCR reaction mixture in a total volume of25l l contained1U(0.75l l)Taq DNA polymerase(Sangon Biological Engineering Technology& Services Co.Ltd,Shanghai,China)and2.5l l109buffer (with MgCl2),10mM(2l l)each dNTP,5l M(2l l)of each primer,13.75l l H2O and2l l undiluted DNA tem-plate.An initial reaction of3min at95°C was followed by 35cycles of94°C for1min,53°C for50s,and72°C for 1min,with afinal cycle at72°C for7min.PCR products were analyzed in1%agarose gels by electrophoresis and observed under UV light,after purified and then sequenced at Shanghai Sangon Biological Engineering Technology& Services Co.Ltd,the NCBI BLAST search program was used to search similar sequences from the GenBank sequence database for the5.8S-ITS sequence for fungi. Fungal Cultivation and Crude Extract Preparationof Fermentation Broth53endophytic fungi were cultured in500mlflasks,each containing200ml potato dextrose liquid medium(g/l; dextrose-20,potato infusion-200).Then three plugs(8mm in diameter)of each fungus were inoculated and cultured with shaking(120rpm)at25°C under dark conditions for1 week.After that,the cultures werefiltered to obtain the fermentation broth.Then the broth was extracted with four times the volume of ethanol for24h and further concen-trated in vacuum to remove organic solvent[26].The concentrate was volatilized later on60°C water bath to obtain dried residues prior to antimicrobial assays.EtOH extract was diluted with sterile distilled water at the con-centration of10mg/ml and was sterilized byfiltration through a0.22-l m Milliporefilter.Antibacterial and Antifungal activityAll the endophytic fungi of the two Dendrobium species were fermented and the crude EtOH extract was tested for the antimicrobial activity.Antimicrobial activity of the EtOH extract of the fungal fermentation broth at a con-centration of100l g/disk was conducted by way of agar diffusion method[31]against a panel of target pathogenic microorganisms,including Gram-positive,Gram-negative bacteria,and three fungi:S.aureus,E.coli,B.subtilis, C.albicans,C.neoformans,and A.fumigatus.Distilled water was used as negative control,while ampicillin sodium(100l g/disk)andfluconazole(25l g/disk)as positive control,were used as standard antibacterial and antifungal agents,respectively.Inhibition zones in diame-ter were measured to assess antimicrobial activity.Each inhibitory experiment was replicated three times.Calculation and Statistical AnalysisColonization rate(CR),expressed in percentage,which was calculated as the total number of plant tissue segments infected by fungi divided by the total number of segments incubated,was used to indicate comparison of degrees of different tissues infected by endophytic fungi.Isolation rate (IR)was used to demonstrate the fungal richness in a given sample of plant tissue which was counted as the number of isolates obtained from plant segments divided by the total number of segments incubated[8].The statistic analysis was analyzed with SPSS11.0(SPSS Inc.,Chicago,IL,USA). ResultsTotally,30endophytic fungi were isolated from100tissue segments(50segments from stem and root each)of D.devonianum,while23isolates were gained from D.thyrsiflorum.The total isolation rates were30and23%, respectively.The colonization and isolation rates of endophytic fungi from roots were higher than that of stem in D.devonianum,but in D.thyrsiflorum,it was the opposite.30endophytic fungi in D.devonianum belonged to11taxa and11genera of23isolates in D.thyrsiflorum were identified.For D.devonianum,Fusarium and Phoma was the dominant genera,and for D.thyrsiflorum,Fusar-ium,and Epicocuum was preponderant(Table1).Both in the two Dendrobium species,Phoma and Rhizopus were isolated from Dendrobium species for thefirst time.In addition,‘‘host-specificity’’was also observed,for Epi-cocuum and Arthrobotray might particularly resided in D.thyrsiflorum while Trichoderma preferred colonizing in D.devonianum.Based on morphological characteristics,Fusarium, Rhizopus,Acremonium were identified(Fig.1).None of the crude ethanol extract of the isolates showed inhibitory effects against C.neoformans.The proportion of endophytic fungi with antimicrobial activity to the total isolated fungi in D.devonianum was23%in percentage (10/23),and43.5%(11/23)in D.thyrsiflorum.Overall,4 isolates in D.devonianum and7endophytes from D.thyrsiflorum exhibited antagonistic effects against more than one pathogenic microorganism.Out of the53endo-phytic fungi,Epicoccum nigrum from root of D.thyrsi-florum displayed strongest antagonistic effect on S.aureus; Phoma from stem of D.devonianum showed greatest inhibitory activity against E.coli,while Phoma from root of D.thyrsiflorum exhibited strongest activity against B.subtilis and slightly inhibited S.aureus as well;Fusar-ium tricinctum from D.devonianum could inhibit both bacterial and fungal pathogens,while the same fungus from D.thyrsiflorum showed antagonistic actions against E.coli and B.subtilis.In addition,Epicoccum from root of D.thyrsiflorum exhibited slightly inhibitory activity against S.aureus,E.coli,and B.subtilis(Table2).DiscussionIn this study,endophytic fungi in D.devonianum and D.thyrsiflorum were diverse.Different endophytic fungi preferred in different Dendrobium species had performed ‘‘host-specificity’’.The remarkably diverse fungal species in association with the host plant may play an important role in ecological patterns and processes[24].Crude eth-anol extract of a large number of endophytes possessed antimicrobial activities againstfive human pathogenic microorganisms.Furthermore,active isolates from stem were more than fungal endophytes from root in D.devo-nianum and D.thyrsiflorum(Table2).However,some of the isolates from D.loddigesii which had been obtained from roots,showed antimicrobial activity against patho-genic microbes[6].Possible reasons for this incompati-bility are different orchid inhabitation,different environment which influence the distributions and coloni-zation of fungal endophytes.The difference of colonization and isolation rates of endophytic fungi in different tissues of Dendrobium spe-cies probably lied in different composition and distribution of endophytic fungi in survival of various environmental conditions and host plant.Fusarium as the dominant genera and the predominant genus in antimicrobial isolates in D.loddigesii had met with great attention[6].In our study,we also found that Fusarium was dominant in both D.devonianum and D.thyrsiflorum.Otherwise,Cladosporium were reported to be dominant in Azadirachta indica[25].This might be due to the fact that the fungal endophytes were isolated from different host plants.Fusarium could inhibit both bacteria and fungi in this study,which was compatible with the fact in our previous study that Fusarium isolated from Saussurea involucrata Kar.et Kir.,also performed broad antimicrobial spectrum [15].It has been reported that the compounds epicorazine A and B isolated from Epicoccum nigrum presented selective antibacterial activity against S.aureus[3].In our work, ethanol crude extract of Epicoccum nigrum showed stron-ger antagonistic activity against S.aureus even superior to Ampicillin.This would be of great importance as antibi-otics especially penicillin resistance has become an esca-lating problem worldwide.Bioactive substances of Epicocuum nigrum against S.aureus are now still under study.Zhang et al.[32]found that epicoccins A derived from Epicoccum nigrum displayed activity againstTable1Identification of endophytic fungi in D.devonianum and D.thyrsiflorumPlant species Strain no.Taxa GenBank accession numberD.devonianum CB-S a-1Fusarium sp1.–cCB-S-2Phoma HM486935CB-S-3Fusarium tricinctum HM486932CB-S-4Leptosphaerulina chartarum HM486931CB-S-5Arthrinium sp.HM486947CB-S-6Glomerella cingulata HM486938CB-S-7Phoma HM486941CB-S-8Pestlotiopsis microspora HM486934CB-S-9Xylaria sp1.HM486948CB-S-10Cladosporium cladosporioides HM486946CB-S-11Xylaria sp2.–CB-S-12Acremonium sp.–CB-S-13Fusarium sp2.–CB-R b-1Pestalotiopsis photiniae HM486945CB-R-2Fusarium sp3.–CB-R-3Acremonium sp.–CB-R-4Phoma HM486943CB-R-5Rhizopus sp.–CB-R-6Phoma sp.–CB-R-7Xylaria sp3.–CB-R-8Fusarium sp4.–CB-R-9Phoma sp.–CB-R-10Leptosphaerulina sp.–CB-R-11Fusarium sp5.–CB-R-12Acremonium sp.–CB-R-13Fusarium sp6.–CB-R-14Trichoderma sp.HM486933CB-R-15Fusarium sp7.–CB-R-16Trichoderma koningiopsis HM486944CB-R-17Fusarium sp8.–D.thyrsiflorum SC-S a-1Fusarium sp1.HM486951SC-S-2Leptosphaerulina chartarum HM486940SC-S-3Alternaria sp.–SC-S-4Colletotrichum sp.HM486939SC-S-5Fusarium sp2.–SC-S-6Colletotrichum gloeosporioides HM486936SC-S-7Pestlotiopsis sp.–SC-S-8Epicoccum nigrum HM486952SC-S-9Phoma sp.–SC-S-10Epicoccum sp1.–SC-S-11Alternaria sp.–D.thyrsiflorum SC-S-12Epicoccum sp2.–SC-S-13Glomerella acutata HM486937SC-R b-1Rhizopus sp.–SC-R-2Fusarium sp3.–SC-R-3Alternaria sp.–SC-R-4Fusarium sp4.–SC-R-5Xylaria sp.–B.subtilis .Furthermore,a new indole derivative from endophytic fungus of Colletotrichum sp.showed moderate antibacterial activity against Gram-positive bacteria of B.subtilis ,S.aureus and Sarcina lutea and Gram-negative bacterium Pseudomonas [23].In our work,extract of Epicoccum sp.from root of D.thyrsiflorum could slightly inhibit three pathogens of S.aureus ,E.coli ,and B.subtilis ,which illustrated the broad antimicrobial spectrum of the metabolites of endophytic fungi.It was speculated that the possible mechanism of inhibitory bacteria was not by way of suppressing cell wall synthesis but through inhibiting bacterial protein synthesis.Compounds of pyrenophorol derivatives from Phoma sp.presented antagonistic activity against E.coli ,Bacillus megaterium ,and Microbotryum violaceum [33].In the present study,it indicated that Phoma from different Dendrobium species exhibited antibacterial activity against different bacteria.This probably lied in that different spe-cies of Phoma were isolated from the two Dendrobium species.Gong and Guo [8]also found that different species of Fusarium showed different antimicrobial activity.Fusarium tricinctum isolated from D.devonianum and D.thyrsiflorum displayed inhibitory effect on different pathogens.Possible reasons for this difference are different origins from different plant species or different survival environment that affect the antimicrobial activity of the fungus.Indeed,endophytic fungi inside the host plants are a versatile reservoir of the various bioactive metabolites and can be of potential use to modern medicine,industryandFig.1Light micrographs of endophytic fungi isolated from D.devonianum andD.thyrsiflorum.a Fusarium isolated from stem ofD.devonianum .b Acremonium isolated from stem ofD.devonianum .c Rhizopus isolated from root ofD.thyrsiflorum.d Fusarium isolated from stem of D.thyrsiflorumTable 1continued Plant speciesStrain no.Taxa GenBank accession number SC-R-6Phoma sp .–SC-R-7Epicoccum sp3.HM486950SC-R-8Fusarium sp5.–SC-R-9Arthrobotray sp .HM486942SC-R-10Fusarium tricinctumHM486949a Endophytes isolated from stem of the Dendrobium speciesb Endophytic fungi isolated from root of the Dendrobium species cIdentified based on morphological characteristicsagriculture.Current work of characterizing the bioactive metabolites of the potent fungal strains is underway. 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