1300 Regular Article Biol. Pharm. Bull. 33(8) 1300—1306 (2010) Vol. 33, No. 8

Diversity and Antimicrobial Activity of Endophytic Fungi Associated with the Alpine Plant Saussurea involucrata

a,b a a a a a Ya-li LV, Fu-sheng ZHANG, Juan CHEN, Jin-long CUI, Yong-mei XING, Xiang-dong LI, and ,a Shun-xing GUO* a Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College; Beijing 100193, P.R. China: and b Baotou Medical College of Inner Mongolia Science & Technology University; Baotou 014040, P.R. China. Received March 16, 2010; accepted May 26, 2010; published online May 28, 2010

Endophytic fungi are rich in species diversity and may play an important role in the fitness of their host plants. This study investigated the diversity and antimicrobial potential of endophytic fungi obtained from Saus- surea involucrata KAR. et KIR. A total of 49 endophytic fungi were isolated from S. involucrata and identified using morphological and molecular techniques. Extracts of fermentation broth from the 49 fungi were tested for antimicrobial activity against pathogenic microorganisms using the agar diffusion method. Forty-eight out of the 49 endophytic fungi were identified and grouped into 14 taxa. Cylindrocarpon sp. was the dominant species iso- lated from S. involucrata, followed by Phoma sp. and Fusarium sp. Among the 49 endophytic fungi, 9 root isolates having darkly pigmented, septate hyphae were identified as dark septate endophytic (DSE) fungus, and 12 fungi inhibited at least one test microorganism. Moreover, 5 strains showed a broader spectrum of antimicrobial activ- -ity and 4 strains displayed strong inhibition (؉؉؉) against pathogenic fungi. The results indicate that endo phytic fungi isolated from S. involucrata are diverse in species and a potential source of antimicrobial agents. Key words agar diffusion method; antimicrobial activity; endophytic fungi; dark septate endophytic fungi; Alpine plant; Saussurea involucrata

Since the discovery of endophytes in 1904, many re- evolved specific adaptations, some of which could also be of searchers have defined endophytic fungi. Bacon and White1) technical interest. For this reason, systematic investigation of give an inclusive and widely accepted definition of endo- endophytic fungi associated with S. involucrata is necessary, phytes: “microbes that colonize living, internal tissues of and will not only provide us with genetic information, but plants, without causing any immediate negative effect.” En- also may allow for new natural products with higher antimi- dophytic fungi have been associated with plants for more crobial activity to be found. than 400 million years2) and play an important role in natural The aims of this research study were: (1) to isolate endo- ecosystems by promoting plant growth,3—5) enhancing the phytic fungi from the medicinal plant S. involucrata, (2) to ability to resist disease,6) enhancing the ecophysiology of identify them by morphological and molecular techniques host plants, and enabling plants to counter abiotic stresses based on the 5.8S gene and internal transcribed spacer (ITS) such as drought7) and metal contamination.8) Endophytes sequence analysis and (3) to evaluate the antimicrobial activ- protect hosts to some extent against pathogens,9—11) reflect- ity of endophytic fungi colonizing S. involucrata. ing the production of secondary metabolites,12—17) fungal parasitism,18) or induction of systemic resistance.19,20) MATERIALS AND METHODS Since recognizing medicinal plants as a repository of fun- gal endophytes with novel metabolites of pharmaceutical im- Collection of Plant Material and Isolation of Endo- 21—24) portance, many studies of species of species composi- phytic Fungi Saussurea involucrata KAR. et KIR. (Fig. 1) tion, relative abundance and associations of endophytic fungi was collected from Tianshan Mountain, Xinjiang Uygur Au- have focused on medicinal plants.25—27) Alpine medicinal tonomous Region, People’s Republic of China. Roots, stems, plants is challenged by low temperature, a shorter vegetation leaves and flowers were respectively washed in de-ionized period, more snow, and harsher conditions by the rising number of weather-related extreme events.28) Despite great progress towards understanding mycorrhiza of these plants,29—31) the biodiversity and function of endophytic fungi associated with alpine medicinal plants remain largely unexplored. Saussurea involucrata KAR. et KIR., popularly known as snow lotus, grows wild in rocky habitats in the alpine zones of the Tianshan, A’er Tai and Kunlun mountain ranges of China at elevations of 2600 m or higher. S. involucrata is a perennial herbaceous plant of Asteraceae and is used to treat rheumatoid arthritis, cough due to lung infection, impotence, etc. in China.32,33) Endophytic fungi associated with S. involucrata which can grow on organic substrates in the harsher conditions, might include specialists that have Fig. 1. Saussurea involucrata KAR. et KIR.

∗ To whom correspondence should be addressed. e-mail: [email protected] © 2010 Pharmaceutical Society of Japan August 2010 1301 water, sterilized in 70% ethanol for 50 s followed by 0.1% Table 1. Endophytic Fungi Isolation from Different Tissues of S. involu- crata HgCl2 for 7 min, rinsed three times in de-ionized water, cut into segments approximately 1 mm in diameter and placed in Number of Number Colonization Isolation 90 mm Petri dishes containing potato dextrose agar (PDA). Organ segments of rate (%) rate The Petri dishes were sealed with a sealing film and incu- incubated isolates bated at 25 °C. Different mycelia growing out of the seg- ments were sub-cultured and individually maintained on Root 100 29 72.0 0.29 PDA. Colony morphology and growth of the isolates were Steam 100 11 43.0 0.11 Leaf 100 7 22.0 0.07 then studied on PDA. Growth and spore formation were also Flower 100 2 13.0 0.02 examined on PDA, oatmeal agar, and cornmeal agar. The iso- late strains were deposited at the Institute of Medical Science & Peking Union Medical College, where they were main- tained at low temperatures (4—5 °C). Endophytic Fungi Identification The sporulating strains were identified on the basis of morphology using perti- nent monographs,34—36) while the endophytic fungi that failed to sporulate were identified by molecular techniques. DNA was extracted using DNA extracting kit (Sangon). The inter- nal transcribed spacer (ITS) region and 5.8S gene were ampli- fied using primers ITS1 and ITS4.37) Polymerase chain reac- tion (PCR) was performed in 50 ml reaction volumes con- taining 2 ml of genomic DNA, 0.3 m M of each primer, 200 m M of each deoxyribonucleotide triphosphate (dNTP), 5 ml of ϫ 10 PCR buffer (containing MgCl2) and 2 units of Taq DNA polymerase (Sangon). PCR was performed in a MinicyclerTM (MJ Research, Reno, NV, U.S.A.) using the following pro- gram: 3 min initial denaturation at 95 °C, followed by 35 cy- cles of 1 min denaturation at 94 °C, 50 s primers annealing at Fig. 2. Light Micrographs of Endophytic Fungi Isolated from S. involu- 52 °C, 1 min extension at 72 °C, and a final 7 min extension at crata 72 °C. PCR products were analyzed in 1% agarose gels (a) EF-2: typical morphology of Cylindrocarpon sp. (b) EF-10-1: typical morphol- (mixed with Goldview DNA stain) by electrophoresis and vi- ogy of Ulocladium sp. (c) EF-4: typical morphology of Fusarium sp. (d) EF-37: typical sualized under UV light. The ITS1-5.8S-ITS4 sequences were morphology of DSE fungus. used to retrieve similar sequences from the GenBank se- quence database using the NCBI BLAST program.38) The se- the inhibition zones. Penicillin and amphotericin B were used quence alignments, maximum parsimony analysis and boot- as standard antibacterial and antifungal agents respectively, strap percentages used to assess the support for the branching while sterilized water was used as the negative control. Each topologies were calculated using MEGA version 4.0.39) The inhibition experiment was repeated three times. Kimura two-parameter model40) was used to estimate evolu- Data Analysis The colonization rate (CR, expressed as a tionary distance. The phylogenetic reconstruction was done percentage) was calculated as the total number of plant tissue using the neighbor-joining (NJ) algorithm, with bootstrap val- segments infected by fungi divided by the total number of ues performed with 1000 replications, using the software rou- segments incubated; this allowed for comparison of degrees tines included in the MEGA software. of infection of different tissues by endophytic fungi. The iso- Fermentation and Extraction The fresh mycelia of dif- lation rate (IR, expressed as a fraction) was determined by ferent endophytic fungi were grown on PDA plates at 25 °C dividing the number of isolates obtained from plant segments for 7 d. Three plugs (0.7 mm in diameter) of culture agar by the total number of segments incubated; this allowed for medium plus the adhering mycelium were subsequently measurement of fungal richness in a given sample of plant added to 500 ml flasks containing 200 ml of broth (wheat tissues in this study. The relative frequency (RF, expressed as bran 30 g, KH2PO4 3 g, MgSO4·7H2O 1.5 g, glucose 20 g, a percentage) was calculated as the total number of isolates de-ionized water 1000 ml), followed by shaking in an incuba- from a single taxa divided by the total number of taxa ob- tor at 110 rpm for 2 weeks at 25 °C. The fermentation broth tained from all tissues incubated. of each endophyte was filtered to separate the filtrate from the mycelia. Filtrate was extracted three times with ethyl ac- RESULTS etate, freeze-dried and then dissolved in sterilized water at a concentration of 1.0 mg·mlϪ1.41) A total of 49 endophytic fungi were isolated from 400 tis- Antimicrobial Assay The fungal extracts were screened sue segments (100 segments from each organ) of S. involu- using the agar diffusion method42) for antimicrobial activity crata. The total isolation rate was 12.25%. Among the 49 against potentially pathogenic bacteria [Escherichia coli isolates, 29 were from roots, 11 from stems, 7 from leaves (Ec), Staphylococcus aureus (Sa), and Bacillus subtilis (Bs)] and 2 from flowers. The colonization and isolation rates of and three fungi [Candida albicans (Ca), Cryptococcus endophytic fungi from roots were 72.0% and 0.29 respec- neoformans (Cn), and Aspergillus fumigatus (Af)]. Antimi- tively, which were higher than other tissue components tested crobial activity was assessed by the size (diameter in mm) of (Table 1). 1302 Vol. 33, No. 8

Table 2. Closest Relatives of S. involucrata Endophytic Fungus Isolates Based on BLAST Analyses and Data from Morphological Identification

Strain Organ Closest related species % similarity Microscopic identification

EF-1 Root Phoma sclerotioides 99 Non-sporulating EF-2 Root Cylindrocarpon sp. (Neonectria) EF-3 Root Cylindrocarpon sp. (Neonectria) 99 Non-sporulating EF-4 Root Fusarium sp. EF-5 Root Fusarium sp. EF-6 Root Cylindrocarpon sp. (Neonectria) 99 Non-sporulating EF-7 Root Fusarium sp. EF-8 Root Cistella grevillei 92 Non-sporulating EF-9 Root Cylindrocarpon sp. (Neonectria) 99 Non-sporulating EF-10-1 Flower Ulocladium sp. EF-10-2 Flower Ulocladium sp. EF-11 Leaf Phoma exigua 99 Non-sporulating EF-12 Root Tetracladium sp. 99 Non-sporulating EF-13 Root Tetracladium sp. 99 Non-sporulating EF-15 Leaf Phoma sclerotioides 99 Non-sporulating EF-16 Root Phoma chrysanthemicola 96 Non-sporulating EF-17 Root Phaeosphaeria avenaria 92 Non-sporulating EF-18 Root Leptosphaeria sp. 90 Non-sporulating EF-21 Leaf Fusarium sp. EF-22 Root Phoma glomerata 99 Non-sporulating EF-24 Root Cylindrocarpon sp. (Neonectria) 99 Non-sporulating EF-25 Root Phoma chrysanthemicola 99 Non-sporulating EF-26 Leaf Fusarium sp. EF-27 Leaf Phoma exigua 99 Non-sporulating EF-28 Leaf Fusarium sp. EF-29 Root Cladosporium sp. 100 Non-sporulating EF-32 Root Leptosphaeria sp. 91 Non-sporulating EF-33 Steam Rhizoctonia solani 91 Non-sporulating EF-36 Steam Non-identified Non-sporulating EF-37 Root Mycocentrospora acerina 95 Non-sporulating EF-38 Root Cylindrocarpon sp. (Neonectria) EF-39 Root Alternaria solani 99 Non-sporulating EF-40-1 Root Phoma chrysanthemicola 97 Non-sporulating EF-40-2 Root Phoma chrysanthemicola 97 Non-sporulating EF-41 Root Cladosporium sp. 99 Non-sporulating EF-43 Leaf Alternaria tenuissima 99 Non-sporulating EF-44 Root Phaeosphaeria avenaria 100 Non-sporulating EF-48 Root Cylindrocarpon sp. (Neonectria) 100 Non-sporulating EF-55 Steam Epicoccum nigrum 100 Non-sporulating EF-56 Steam Fusarium sp. EF-57 Steam Fusarium sp. EF-58 Steam Cylindrocarpon sp. (Neonectria) EF-59 Steam Rhizoctonia sp. 100 Non-sporulating EF-60 Root Leptodontidium orchidicola 98 Non-sporulating EF-62 Steam Rhizoctonia sp. 100 Non-sporulating EF-64 Steam Cylindrocarpon sp. (Neonectria) EF-65 Steam Rhizoctonia sp. 100 Non-sporulating EF-D Steam Cylindrocarpon sp. (Neonectria) EF-M Root Leptodontidium sp. 98 Non-sporulating

Based on their morphological characteristics, 15 isolates which were able to produce spores were identified as belong- ing to 3 genera: Fusarium sp., Ulocladium sp. and Cylindro- carpon sp. (Figs. 2a—c). Nine of the 49 isolates were from roots, and having darkly pigmented septate hyphae were de- fined as dark septate endophytic (DSE) fungus (Fig. 2d). Molecular analyses were required to characterize 34 of the endophytic fungi that failed to sporulate. ITS1-5.8S-ITS4 partial sequences from these 34 isolates were compared to ITS sequences of organisms represented in the GenBank database. Results from BLAST (Table 2) showed that 33 Fig. 3. Relative Frequency of Endophytic Fungi Isolated from S. involu- crata isolates might belong to 12 different genera: Cylindrocarpon (Neonectria), Phoma, Rhizoctonia, Phaeosphaeria, results, the 49 endophytic fungi were considered to belong Leptosphaeria, Alternaria, Cladosporium, Leptodontidium, to 14 different genera. The endophytic fungi with the high- Tetracladium, Epicoccum, Cistella, Mycocentrospora, while est RF were as follows: Cylindrocarpon sp. (20.4%)ϾPhoma one isolate could not be identified (Table 2). Combining all sp. (18.4%)ϾFusarium sp. (16.3%) (Fig. 3). August 2010 1303

Fig. 4. Ascomycota Phylogenetic Tree Generated from the ITS1-5.8S-ITS4 Sequences of 52 Taxa Showing the Closest Relationships of Endophytic Fungi Isolated from S. involucrata with Reference Taxa (Kimura Two-Parameter Model; Neighbor–Joining Algorithm and 1000 Replicate Bootstrap) The tree was rooted with T. melanosporum. Class: Do (Dothideomycetes), So (Sordariomycetes), Le (Leotiomycetes); Order: Pl (Pleosporales), Ca (Capnodiales), Hy (Hypo- creales) and He (Helotiales). 1304 Vol. 33, No. 8

Based on search results for similar ITS sequence from Representing the Order Hypocreales (Hy) within the Class GenBank using FASTA, the Ascomycota phylogenetic tree Sordariomycetes (So) isolates EF-3, EF-6, EF-9, EF-24 and (Fig. 4) showed that the 29 isolates were distinctly distributed EF-48 formed a monophyletic clade with three reference among three classes: Dothideomycetes, Sordariomycetes, species of Nectriaceae, [Cylindrocarpon sp. (Neonectria), Leotiomycetes and four orders: Pleosporales, Capnodiales, Neonectria radicicola, Neonectria macrodidyma], although Hypocreales, Helotiales. there was relatively low bootstrap support (Fig. 4). On the top of the tree (Fig. 4), 19 isolates were shown to Based on the phylogenetic tree (Fig. 4) the Order Helo- be related to the Order Pleosporales (Pl) and Capnodiales tiales (He) was represented by five endophytic fungi: EF-8, (Ca) within the Class Dothideomycetes (Do). Isolates EF-1, EF-12, EF-13, EF-60 and EF-M. Isolate EF-8 formed a EF-11, EF-15, EF-16, EF-22, EF-25 and EF-27 were related monophyletic clade with Cistella grevillei with a bootstrap to different species of the genus Phoma. Isolates EF-40-1 support of 71%. EF-12 and EF-13 were closely related (99% and EF-40-2 were related to Phoma sp. with 96—97% bootstrap) and formed a cluster supported by 100% bootstrap BLAST similarity (Table 2), but there was low bootstrap sup- with Tetracladium sp. EF-M formed a terminal cluster with port of 61%. Strains EF-18 and EF-32 formed a cluster sup- Leptodontidium orchidicola with a bootstrap support of 85% ported by 77% and 98% respectively with Leptosphaeria sp. and EF-60 formed the second subclade with a bootstrap sup- EF-44 formed a terminal cluster with Phaeosphaeria sp. port of 100% (Fig. 4). with a bootstrap support of 83% and EF-17 formed the sec- Based on search results for similar ITS sequences from ond subclade with a bootstrap support of 84%. Isolate EF-37 GenBank using FASTA, the Ceratobasidiaceae phylogenetic formed a monophyletic clade with Mycocentrospora acerina analyses (Fig. 5) showed that isolates EF-33, EF-59, EF-62 with a bootstrap support of 99%. Isolate EF-55 and Epicoc- and EF-65 were genotypically closely related and formed a cum nigrum clustered together with a bootstrap support of clad with Rhizoctonia sp. and then formed the second sub- 93% within the monophyletic clade. EF-39 grouped with Al- clade with a bootstrap support of 98% with Ceratobasidium ternaria solani and EF-43 grouped with Alternaria tenuis- sp. (Fig. 5). sima (99% bootstrap), which formed the second subclade All isolates were fermented and the crude extract of each with a bootstrap support of 100%. Isolates EF-29 and EF-41 fermentation broth was evaluated in vitro for antimicrobial were both clustered with Cladosporium sp. with strong sup- activity. Among the 49 isolates, 12 (24.5%) belonging to port (100% bootstrap) (Fig. 4). Fusarium sp., Phaeosphaeria sp., Cylindrocarpon sp., Lep- tosphaeria sp., Cladosporium sp. and Phoma sp. exhibited antimicrobial activity against at least one test microorganism whereas the remainder yielded no activity (Table 3). There were 6 strains having antimicrobial activity against Bacillus subtilis, 1 strain against Staphylococcus aureus, 9 strains against Candida albicans, 6 strains against Cryptococcus neoformans and 8 strains against Aspergillus fumigatus (Table 2). Moreover, 5 strains (EF-17, EF-18, EF-26, EF-28, EF-41) showed a broader spectrum of antimicrobial activity (zone of inhibition ϩ, ϩϩ or ϩϩϩ against test bacteria and Fig. 5. Ceratobasidiaceae Phylogenetic Tree Generated from the ITS1- fungi, respectively), and 4 strains (EF-21, EF-26, EF-28, EF- 5.8S-ITS4 Sequences of 9 Taxa Showing the Relationships of EF-33, EF-59, 58) displayed strong inhibition (ϩϩϩ) to pathogenic fungi. EF-62 and EF-65 Isolated from S. involucrata with Rhizoctonia sp. (Kimura None of the fungal broths inhibited the test bacteria E. coli Two-Parameter Model; Neighbor–Joining Algorithm and 1000 Replicate (Table 3). Bootstrap) The tree was rooted with P. byssina.

Table 3. Antimicrobial Activity of Metabolites from Endophytic Fungi

Escherichia Bacillus Staphylococcus Candida Cryptococcus Aspergillus Strain Taxa coli subtilis aureus albicans neoformans fumigatus

EF-17 Phaeosphaeria avenaria Ϫ ϩ Ϫ ϩϩ ϩϩ ϩ EF-18 Leptosphaeria sp. ϪϩϪϩϩϩϩ EF-21 Fusarium sp. ϪϪϪϩϩϩϪϩϩ EF-26 Fusarium sp. ϪϩϩϪϩϩϩϩϩϩϩϩ EF-28 Fusarium sp. Ϫ ϩ Ϫ ϩϩϩ ϩϩϩ ϩϩϩ EF-32 Leptosphaeria sp. ϪϩϩϪϩϩϪϪ EF-40-1 Phoma chrysanthemicola ϪϪϪϩϩϪϪ EF-41 Cladosporium sp. ϪϩϪϩϩϪ EF-44 Phaeosphaeria avenaria ϪϪϪϩϪϩ EF-48 Cylindrocarpon sp. ϪϪϩϪϪϩ EF-56 Fusarium sp. ϪϪϪϪϩϩϪ EF-58 Cylindrocarpon sp. ϪϪϪϪϪϩϩϩ Positive control Penicillin ϩϩϩϩϩϩϪϪϪ Amphotericin B ϪϪϪϩϩϩϩϩϩϩ Negative control Sterilized water ϪϪϪϪϪϪ

Ϫ: No antimicrobial activity. ϩ: The inhibition zone is less than 10 mm. ϩϩ: The inhibition zone is from 10 to 20 mm. ϩϩϩ: The inhibition zone is above 20 mm. August 2010 1305

DISCUSSION tation broth of most Fusarium sp. showed strong antimicro- bial activity. A total of 49 fungal isolates were obtained from health tis- Therefore, endophytic fungi are obviously a rich and reli- sues of S. involucrata. Those endophytic fungi were grouped able source for the biotechnological, medicinal, and agricul- in 14 species, being 44 Ascomycoa, four Basidiomycota and tural industries. Our results clearly confirm that endophytic one not yet identified. Cylindrocarpon sp. was the dominant fungi from S. involucrata are sources of novel and antimicro- species in the plants of S. involucrata, followed by Phoma sp. bial compounds. Further research work on endophytic fungi and Fusarium sp. It has been reported elsewhere that Col- from S. involucrata is in progress, such as the mechanism of letotrichum sp. and Phoma sp. were found at four of five sites mutual symbiosis between DSE and S. involucrata and the of 29 medicinal plant species and Colletotrichum sp. were identification of active compounds. the dominant endophytes.26) Also, Alternaria alternate was 43) found to be dominant in Pinus tabulaeformis CARR. This Acknowledgments We would like to thank Dr. Peter indicates that there is a high diversity of endophytic fungi as- Saranchuk for critically reviewing the manuscript. This re- sociated with different plants, and the composition and distri- search was financially supported by the International Science bution of endophytic fungi are conspicuously affected by the and Technology Cooperation Projects of China (No. environment, hosts and tissues. Although we were not able to 2009DFA32250), National S&T Major Special Project on identify one of the isolates in this study, the results still show Major New Drug Innovation (No. 2009ZX09301-003-3-3). the great diversity of these organisms in the internal plant en- vironment of S. involucrata. REFERENCES The colonization and isolation rates of endophytic fungi in this study were higher in roots than in other tissues of S. in- 1) Bacon C. W., White J. F. Jr., “Microbial Endophytes,” Marcel-Dekker volucrate, but it has been reported elsewhere that leaves, Inc., New York, Basel, 2000, p. 487. stems and fruits were colonized more by endophytic fungi 2) Krings M., Taylor T. N., Hass H., Kerp H., Dotzler N., Hermsen E. J., than roots.26) Possible reasons for this dissimilarity are that New Phytol., 174, 648—657 (2007). the structure and substrate are different between different tis- 3) Lyons P. C., Evans J. J., Bacon C. W., Plant Physiol., 92, 726—732 (1990). sues, which influence the colonization and distribution of en- 4) Lu H., Zou W. X., Meng J. C., Plant Sci., 151, 67—73 (2000). 44,45) dophytic fungi. Because the interrelation between endo- 5) Wu L. Q., Lv Y. L., Meng Z. X., Chen J., Guo S. X., Mycorrhiza, 20, phytic fungi and its host plants is complicated, the endo- 127—135 (2010). phytic fungi we isolated may not include all of the endo- 6) Clay K., Cheplick G. P., Wray S. M., Oncologia, 80, 374—380 (1989). phytic fungi in S. involucrata. However, we are the first to re- 7) Arechavaleta M., Bacon C. W., Hoveland C. S., Radcliffe D. E., Agron. J., 81, 83—90 (1989). port endophytic fungi being associated with S. involucrata, 8) Malinowski D. P., Belesky D. P., Crop Sci., 40, 923—940 (2000). which has established the basis for further investigation of 9) Danielsen S., Jensen D. F., Biocontrol. Sci. Technol., 9, 545—553 the interrelation between endophytic fungi and this alpine (1999). plant. 10) Narisawa K., Kawamata H., Currah R. S., Hashiba T., Eur. J. Plant Based on the morphological characteristics, 9 isolates Pathol., 108, 103—109 (2002). 11) Campanile G., Ruscelli A., Luisi N., Eur. J. Plant Pathol., 117, 237— were considered to be dark septate endophytic (DSE) fungi. 246 (2007). DSE fungi are distributed all over the world and in all kinds 12) Schulz B., Rommert A. K., Dammann U., Aust H. J., Strack D., Mycol. of plants, especially those living in harsh climatic conditions Res., 10, 1275—1283 (1999). such as alpine environments.46,47) DSE fungi have been found 13) Stinson M., Ezra D., Hess W. M., Sears J., Strobel G., Plant Sci., 165, to have a mutualistic, mycorrhiza-like relationship with their 913—922 (2003). 46) 14) Corrado M., Rodrigues K. F., J. Basic Microb., 44, 157—160 (2004). host plants. Among the 9 DSE fungi that we identified, EF- 15) Kim S., Shin D. S., Lee T., Oh K. B., J. Nat. Prod., 67, 448—450 37 and EF-M were found to be beneficial to the growth and (2004). development of their host plant S. involucrata in our prior re- 16) Atmosukarto I., Castillo U., Hess W. M., Sears J., Strobel G., Plant search.5,48) It suggested that DSE fungi might have important Sci., 169, 854—861 (2005). 17) Li Y., Song Y. C., Liu J. Y., Ma Y. M., Tan R. X., World J. Microb. role in the growth of S. involucrata, so more research about Biot., 21, 553—558 (2005). the interaction of DSE and its host plants should be carried 18) Samuels G. J., Pardo-Schultheiss R., Hebbar K. P., Lumsden R. D., out in our future study. Bastos C. N., Costa J. C., Bezerra J. L., Mycol. Res., 104, 760—764 Endophytic fungi living in the healthy tissues of plants are (2000). relatively unstudied and potential sources of novel natural 19) Vu T., Hauschild R., Sikora R. A., Nematology, 8, 847—852 (2006). 20) Song Y. C., Huang W. Y., Sun C., Wang F. W., Tan R. X., Biol. Pharm. products for exploitation in medicine, agriculture, and indus- Bull., 28, 506—509 (2005). 49) try. Upon exposure to virulent pathogens, endophyte- 21) Strobel G., Daisy B., Castillo U., Harper J., J. Nat. Prod., 67, 257— colonized plants activate host defenses more quickly 268 (2004). than non-symbiotic plants.50,51) In this study, 24.5% of strains 22) Wiyakrutta S., Sriubolmas N., Panphut W., Thongon N., Danwiserkan- had antimicrobial activity. Among those, Fusarium sp., jana K., Ruangrungsi N., Meevootisom V., World J. Microb. Biot., 20, 265—272 (2004). Phaeosphaeria sp., Leptosphaeria sp. and Cladosporium sp. 23) Kumar D. S. S., Hyde K. D., Fungal Divers., 17, 69—90 (2004). could be able to produce substances that have broader an- 24) Tejesvi M. V., Kini K. R., Prakash H. S., Subbiah V., Shetty H. S., timicrobial spectrum. Also, Fusarium sp. and Cylindrocar- Fungal Divers., 24, 37—54 (2007). pon sp. both displayed strong antifungal activity, which even 25) Raviraja N. S., Maria G. L., Sridhar K. R., Eng. Life Sci., 5, 515—520 (2006). exceeded that of the positive control amphotericin B against 26) Huang W. Y., Cai Y. Z., Hyde K. D., Corke H., Sun M., Fungal Candida albicans and Aspergillus fumigatus. This was simi- Divers., 33, 61—75 (2008). lar to the work of Wang et al.,52) which reported the fermen- 27) Sun J. Q., Guo L. D., Zang W., Ping W. X., Chi D. F., Sci. China C, Life 1306 Vol. 33, No. 8

Sci., 8, 751—759 (2008). 39) Tamura K., Dudley J., Nei M., Kumar S., Mol. Biol. Evol., 24, 1596— 28) Stocklin J., Kuss P., Pluess A. R., Bot. Helv., 119, 125—133 (2009). 1599 (2007). 29) Onipchenko V. G., Zobel M., Folia Geobot., 35, 1—11 (2000). 40) Kimura M., J. Mol. Evol., 16, 111—120 (1980). 30) Likar M., Bukovnik U., Kreft I., Chrungoo N. K., Regvar M., Mycor- 41) Sette D. L., Passarini M. R. Z., Delarmelina C., Salati F., Duarte M. C. rhiza, 18, 309—315 (2008). T., World J. Microb. Biot., 22, 1185—1195 (2006). 31) Lugo M. A., Ferrero M., Menoyo E., Estevez M. C., Sineriz F., Anton 42) Rios J. L., Recio M. C., Villar A., J. Ethnopharmacol., 23, 127—149 A., Microb. Ecol., 55, 705—713 (2008). (1988). 32) Li G. H., Zhao R. C., Acta Pharmaceut. Sin., 15, 368—369 (1989). 43) Guo L. D., Xu L., Zheng W. H., Hyde K. D., Fungal Divers., 16, 53— 33) Guo B., Gao M., Liu C. Z., Plant Cell Rep., 26, 261—265 (2007). 67 (2004). 34) Barnett H. L., Hunter B. B., “Illustrated Genera of Imperfect Fungi,” 44) Carroll G. C., Petrini O., Mycologia, 75, 53—63 (1983). 3rd ed., Burgess Publishing, Minnesota, 1972. 45) Rodrigues K. F., Mycologia, 86, 376—385 (1994). 35) Ellis M. B., “More Dematiaceous Hyphomycetes,” Commonwealth 46) Jumpponen A., Trappe J. M., New Phytol., 140, 295—310 (1998). Mycological Institute, Kew, England, 1976, p. 507. 47) Addy H. D., Piercey M. M., Currah R. S., Can. J. Bot., 83, 1—13 36) Nelson P. E., Toussoun T. A., Marasas W. F. O., “Fusarium Species: An (2005). Illustrated Manual for Identification,” Pennsylvania State University 48) Wu L. Q., Guo S. X., Mycorrhiza, 18, 79—85 (2008). Press, University Park, PA, 1983, pp. 1—193. 49) Strobel G., Daisy B., Microbiol. Mol. Biol. R., 67, 491—502 (2003). 37) White T. J., Bruns T. D., Lee S., Taylor J. W., “Amplification and Di- 50) Redman R. S., Freeman S., Clifton D. R., Morrel J., Brown G., Ro- rect Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics, driguez R. J., Plant Physiol., 119, 795—804 (1999). PCR Protocols: a Guide to Methods and Applications,” ed. by Innis M. 51) Rodriguez R. J., White J. F. Jr., Arnold A. E., Redman R. S., New Phy- A., Gelfand D. H., Sninsky J. S., White T. J., Academic Press, New tol., 182, 314—330 (2009). York, 1990, pp. 315—322. 52) Wang F. W., Jiao R. H., Cheng A. B., Tan S. H., Song Y. C., World J. 38) Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Microb. Biot., 23, 79—83 (2007). Miller W., Lipman D. J., Nucleic Acids Res., 25, 3389—3402 (1997).