Isolation of Endophytic Fungi from Huperzia Serrata Grown in Guangxi Province, China

Vol. 7(36), pp. 2638-2644, 25 September, 2013

Full Length Research Paper

Isolation of endophytic fungi from Huperzia serrata grown in Guangxi Province, China

X. Y. Chen1, C. Sui2, J. H. Wei12*, B. C. Gan1, D. L. Wang1 and J. D. Feng1

1Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College (Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine), Wanning, 571533, China. 2Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 1001193, China.

Accepted 12 May, 2011

In this study, 53 strains of endophytic fungi with different colonial morphologies were isolated from the healthy tissues of Huperzia serrata (Thunb. ex Murray) Trev, collected from Guangxi province of China. Of these 53 strains, 25 were identified based on the morphological characteristics and ITS (internal transcribed spacer) sequences, the others with non-sporulation based on the ITS sequence analysis. Fifty-one strains were identified at the genus level, 1 at the order level, and 1 at the family level. Fifty-one strains belonged to Ascomycota, and the other two respectively belonged to Basidiomycota and Eukaryota. Sixteen identified strains belonged to the genus of Glomerella and its anamorph Colletotrichum, which are most frequently isolated from H. serrata. Taken together, different types and quantities of endophytic fungi are distributed in the stems, leaves and roots of H. serrata.

Key words: Huperzia serrata (Thunb. ex Murray) Trev., endophytic fungi, morphological characteristics, ITS rDNA sequences, identification.

INTRODUCTION

Huperzia serrata (Thunb. ex Murray) Trev. (formerly dementia (VaD) (Ma et al., 2007; Wang et al., 2000). known as Lycopodium serratum, Huperziaceae) is a Because of its medicinal value, the wild resources of H. fern-ally plant distributed in the eastern, southern and serrata have been over-exploited (Ai and Zhang, 2005; southeastern areas of Asia, as well as in the Oceania and Ma et al., 2006). To ensure its sustainable utilization, the the Central America (Huang and He, 2010). In China, it H. serrata cultivation is necessary. distributed from the subtropical region to the tropical Endophytic fungi spend the whole or part of their region. This tiny club moss usually grows at the bryophyte lifecycle by inter- and/or intra- cellular colonization inside layer of shady and humid habitats in the forest, and its the healthy tissues of their host plants, typically causing growth into a young plant takes three to four years. The no apparent symptom of disease (Li et al., 2008). Some whole plant has long been used in traditional Chinese endophytic fungi stimulate plant growth and produce medicine for the treatment of swelling, fever and blood bioactive metabolites with active functions (Rakotoniriana disorders (Zangara, 2003). Huperzine A, one of the et al., 2008; Shipunov et al., 2008). Some endophytic alkaloid constituents of H. serrata, has been shown to be fungi have been found as potential sources of drugs (Lin implicated in alleviating the learning and memory et al., 2007; Márquez et al., 2007). For example, a strain impairment in Alzheimer’s disease (AD) and vascular of Acremonium sp. from H. Serrata was found producing

*Corresponding author. E-mail: [email protected]., Tel/Fax: +86-10-62818841.

Chen et al. 2639

Huperzine A at a higher yield than its host plant (Li et al., Mycelia of each fungus were obtained by vacuum ﬁltration. Genomic 2007). Wang et al. (2010) isolated and characterized the DNA was extracted by the plant genomic DNA kit (Tiangen, Beijing). endophytic fungi which produced Huperzine A from H. The PCR amplification of the rDNA ITS region was performed using primers ITS1 (5′-TCCGTATGGTGAACCTGCGG-3′) and ITS4 serrata grown in Jiangxi Province of China. In our (5′-TCCTCCGCTTATTGATATGC-3′). The conditions for the previous work, endophytic fungi were isolated from H. amplification were according to the reference (Chen et al., 2011). serrata grown in Hainan Province of China (Chen et al., The PCR products were purified using the DNA fragment purification 2011). To obtain more strains of endophytic fungi hosted kit (Tiangen, Beijing) and sequenced using the primer pair ITS1 and in H. serrata which occurs in different environment and to ITS4 on an ABI 3730 XL sequencer. Strain sequences and their similar sequences obtained by blasting in GenBank, subsequently, get some enlightenment for further identification of useful were aligned with CLUSTAL X. Phylogenetic analysis of the aligned endophytic fungi in H. serrata cultivation and utilization, sequences was performed by the maximum-parsimony method the endophytic fungi were isolated from H. serrata grown using the heuristic search algorithm of the Phylogeny Analysis Using in Guangxi Province of China in this study. Most of the Parsimony (PAUP*) program version 4.0. Bootstrap analysis was strains isolated were identified by the morphological conducted with 1000 replicates of the heuristic search with the characteristics and rDNA internal transcribed spacer (ITS) Tree-Bisection-Reconnection (TBR) branch swapping, the ACCelerated TRANsformation (ACCTRAN) optimization, and sequences. In addition, the stains of endophytic fungi random taxon addition. The MaxTree was set at 1000. The isolated in this study were compared with those which phylogenetic analysis was performed on 69 taxa, including were isolated in previous reports. reference taxa (Table 2).

MATERIALS AND METHODS RESULTS

Plant materials Endophytic fungi were identified based on the

The wild samples of H. serrata plant were collected in August, 2008 morphological characteristics from the Napo County of Guangxi Province, China. The taxonomical identification of the plant material was conducted by Dr. Yaodong Qi. Fifty-three strains with different colonial morphologies The voucher (Zhu2008002) was deposited in the Herbarium of the were isolated from 60 stems, leaves and roots fragments Institute of Medicinal Plant Development, Chinese Academy of of H. serrata grown in Guangxi Province (Table 1). Among Medical Sciences. these strains, 25 were identified into ten genera by their morphological characteristics and ITS sequences, Isolation and culture of endophytic fungi including Glomerella (Phyllachoraceae), Plectosphaerella (Phyllachoraceae), Gibberella (Nectriaceae), Trichoderma The plant surface was sterilized according to Dobranic et al. (1995) (Hypocreaceae), Coniochaeta (Coniochaetaceae), with some modifications. The plant was thoroughly washed under Pestalotiopsis (Amphisphaeriacea), Periconia running tap water, immerged in 70% ethanol for 3 to 5 min, and then in 10% sodium hypochlorite for 5 min. Finally, the plant was rinsed in (Halosphaeriaceae), Cryptosporiopsis (Dermateaceae), sterile distilled water three times. Each sample of the stems, roots Alternaria (Pleosporaceae) and Pythium (Pythiaceae). and leaves was cut into 10 small fragments and repeated twice. The The others could not be identified based on their stems and roots were cut into 5 mm segments, and the leaves were morphological characteristics due to non-sporulation. cut into 5 mm ⅹ 5 mm segments by a flame-sterilized cork borer. Thus they were grouped as mycelia sterilia. These segments were placed in a 90 mm diameter petri dish containing the potato dextrose agar (PDA) medium with streptomycin sulfate (200 mg/L) to suppress bacterium contamination. After an incubation of five days, new fungal colonies Mycelia sterilia was identified based on the ITS were monitored and picked out every day for 30 days. Individual sequences and phylogenetic analysis fungal colonies were picked out and transferred into PDA. After the subculture, these fungi were stored in the National Medicinal Plant The maximum parsimony analysis of 28 taxa and their Gene Bank of IMPLAD. similarity sequences containing 600 sites was performed with 500 bootstrap replications. A major rule tree with Identification of endophytic fungi 1793 steps was obtained with a consistency index (CI) of 0.5176 and a retention index (RI) of 0.8746 (Figure 1). The hyphae or spores of endophytic fungi were spread on slides and Based on the analysis results of the similar sequences identified based on the cultural, conidial characters; their taxonomic and phylogenetic tree, GXG8, GXJ44, GXJ35 and GXJ30 position was determined by previous report (Shao et al., 1984) and confirmed by ITS sequences. The remaining cultures of (one code represents one strain) had relatively high non-sporulation were identified based on the ITS sequences sequence similarities with Glomerella and its anamorph included DNA extraction, PCR amplification and phylogenetic Colletotrichum and formed a monophyletic clade with ten analysis. They specified as follows: After the subculture of fungal representative species (Colletotrichum gloeosporioides, strains that had been grown in PDA for 5 days, fresh mycelia were Colletotrichum boninense, Glomerella cingulata, inoculated into a 100 ml Erlenmeyer flask containing 50 ml of the liquid potato dextrose (PD) media and cultured in a spinning Colletotrichum truncatum, Glomerella lindemuthiana, incubator at 120 rpm/min and 28°C in darkness for 5 to 10 days. Colletotrichum destructivum, Colletotrichum higginsianum,

2640 J. Med. Plants Res.

Table 1. Fungal taxa and the number of endophytic fungi isolated from the H. serrata grown in Guangxi Province.

Tissues Total Phylum Class Order Family Genus Stems Leaves Roots strains Glomerella 10 2 0 12 Phyllachorales Phyllachoraceae Plectosphaerella 2 0 0 2 Nectriaceae Gibberella 1 1 0 2 Sordariomycetes Hypocreales Hypocreaceae Trichoderma 1 1 1 3 Ascomycota Coniochaetales Coniochaetaceae Coniochaeta 1 0 0 1

Xylariales Amphisphaeriaceae Pestalotiopsis 1 0 0 1 Microascales Halosphaeriaceae Periconia 0 0 1 1 Leotiomycetes Helotiales Dermateaceae Cryptosporiopsis 0 0 1 1 Dothideomycetes Pleosporales Pleosporaceae Alternaria 0 1 0 1 Pythium 1 0 0 1 Eukaryota Oomycetes Pythiales Pythiaceae Mycelia sterilia 17 8 3 30 Total strains 34 13 5 53

Colletotrichum sp. E5T9, G. cingulata and C. three representative species (Cyphellophora Endophytic fungi distributed in stems, leaves gloeosporioides f. sp.) of the Phyllachoraceae with laciniata, Phialophora europaea and Phialophora and roots of H. serrata the bootstrap support of 100%. GXJ17, GXJ36, reptans) formed a monophyletic clade with GXJ3, GXY3, Gibberella and its anamorph bootstrap support of 100%. Within this clade, The quantity of endophytic fungi in H. serrata Fusarium clustered together with the bootstrap GXJ43, GXY21 and Cyphellophora laciniata demonstrated significant variations in different support of 100%. GXJ8 and Nigrospora oryzae clustered with the bootstrap support of 94% (94% tissues. Thirty-four strains were isolated from the formed a subclade with the bootstrap support of similar blast). GXJ45 and three representative stems (64.15%), 13 from the leaves (24.52%), and 99%. GXJ42, GXY10, GXG9, GXJ37, GXJ2 with species (Phaeosphaeria sp. HKC12, 5 from the roots (9.43%) (Table 1). Strains types Diaporthe and its anamorph Phomopsis of Phaeosphaeriopsis musae and Stagonospora sp.) were also found different in the stems, leaves and Valsaceae formed a group with a bootstrap of Pleosporales formed a monophyletic clade with roots. Glomerella sp. (Colletotrichum sp.), support of 80% (83% similarity blast). GXJ25, the 100% bootstrap support. GXJ12 and three Diaporthe sp. (Phomopsis sp.) and Trichoderma Guignardia camelliae, Guignardia mangiferae and representative species (Leptosphaeria sp. 3813, sp. were simultaneously found in the stems, Phyllosticta sp. MSR_1 of Botryosphaeriaceae Leptosphaeriaceae sp. HKC16, leaves and roots. Cyphellophora sp., Podospora clustered together with the bootstrap support of Leptosphaeriaceae sp. GF014) clustered together sp., Rhizoctonia sp. and Gibberella sp. were 100%. GXG2, Mycorrhizal fungal sp. and with the bootstrap support of 100%. GXJ46, simultaneously found in the stems and leaves. Lecythophora mutabilis clustered together with the GXJ15, GXY15, GXY22, GXY7 and the genus of Plectosphaerella sp., Pestalotiopsis sp., 100% bootstrap value. GXY8 and two Podospora clustered with the bootstrap support of Coniochaeta sp., Nigrospora sp., Leptosphaeria representative species Aspergillus niger and 100%. GXJ28, GXY14 and Rhizoctonia sp. AG_A, sp., Pythium sp., Leptosphaeria sp., Aspergillus foetidus clustered together with the Rhizoctonia solani clustered together at the Botryosphaeriaceae sp. and Pleosporales sp. bootstrap support of 100%. GXJ43, GXY21 and bootstrap support of 100%. were distributed only in stems, Alternaria sp. and

Chen et al. 2641

Table 2. Closest relatives of endophytic fungi from the GenBank for the phylogenetic analysis.

Closest related species Accession number Closest related species Accession number C. boninense AB042313 Phyllosticta sp. MSR-1 AF532314 G. cingulata EU520087 G. camelliae FJ462743 C. higginsianum AB042303 G. mangiferae EU273524 Colletotrichum sp. E5T9 FJ480405 Mycorrhizal fungal sp. pkc01e AY394920 G. cingulata FJ459940 L. mutabilis DQ093680 C. gloeosporioides f. sp. AJ301986 A. niger EU386133 C.gloeosporioides EU847425 A. foetidus AY373850 C.destructivum EU070911 C. laciniata EU035416 G. lindemuthiana EF608059 P. reptans EU514699 C. truncatum AF451902 P. europaea EF540756 Fusarium sp. CCN1 DQ993637 Stagonospora sp. SS-1575 AM262366 G. moniliformis EU364856 Phaeosphaeria sp. HKC12 DQ092527 F. proliferatum EU091039 P. musae GQ169764 G. fujikuroi EU326195 Leptosphaeria sp. 3813 FN394721 N. oryzae EU436680 Leptosphaeriaceae sp. HKC16 DQ092530 F. cerealis EU214569 Leptosphaeriaceae sp. GF014 DQ342361 F. aethiopicum FJ240310 Podospora sp. XSD-39 EU273519 F. proliferatum FJ648201 Podospora sp. Ppf7 EF495232 Diaporthe sp. SAB_2009 FJ799941 Rhizoctonia sp. AG-A AY927362 Phomopsis sp. Yonghe 1 EU512922 R. solani FJ492158

Aspergillus sp. only in leaves, and Cryptosporiopsis sp., In this study, the endophytic fungi from Guangxi province Periconia sp. and Lecythophora sp. only in roots (Table 3). were isolated for the first time. Fifty-three strains were isolated and classified into 19 genera, showing a great diversity of taxa in H. serrata from Guangxi Province and DISCUSSION Plectosphaerella sp., Pestalotiopsis sp., Cryptosporiopsis sp., Nigrospora sp., Diaporthe sp. (Phomopsis sp.), The results showed that the quantity of endophytic fungi Cyphellophora sp., Leptosphaeria sp. of endophytic fungi from H. serrata varied in different tissues: stem first reported in H. serrata from Guangxi Province (Figure (64.15%) > leave (24.52%) > root (9.43%), similar to the 4). Glomerella sp. (Colletotrichum sp.), Diaporthe sp. previous results. Gong et al. (2007) isolated 180 (Phomopsis sp.) and Trichoderma sp. were endophytic fungi from the H. serrata grown in Anhui simultaneously found in the stems, leaves and roots of H. Province, China and found 55.6% fungi in stems, 40.6% in serrara and the genus of Glomerella sp. (Colletotrichum leaves and 3.8% in roots. Zhao (2006) isolated endophytic sp.), which are most frequently isolated in our study. Our fungi from the H. serrata samples collected from January previous studies showed that Glomerella sp. to December in Jiuhua Mountain and found them mainly (Colletotrichum sp.) and Hypoxylon sp. were frequently exist in stems. Their results showed that stems are the isolated in H. serrata from Hainan Province (Chen et al., most preferred location for endophytic fungi of H. serrata. 2011). Gong et al (2007) study showed that the Phoma sp. However, Ma et al. (2007) isolated 80 endophytic fungi and the Phacodium sp. were dominate genus in H. serrata from the stems, leaves and roots of Eucommia ulmoides, from Anhui provinces. Wang et al (2010) isolated 127 but with the largest quantity of endophytic fungi found in strains and grouped into 19 genera and the Aspergillus the roots. Taechowisan et al. (2003) also found that sp., Podospora sp., Penicillium sp., Colletotrichum sp., endophytic fungi existed mainly in roots. The results and Acremonium sp. were the frequently genera from H. showed that different quantities of endophytic fungi are serrata in Jiangxi Province. Glomerella sp. (Colletotrichum distributed in different parts of a plant. sp.) were the dominate genus from H. Serrata in Guangxi Many endophytic fungi were isolated from H. serrata in Province, Hainan Province and Jiangxi Province. China, such as Hainan province (Chen et al., 2011), Comparing the endophytic fungi isolated from different Jiangxi province (Wang et al., 2010), Anhui province (Shi regions of China indicated that endophytic fungi in H. et al., 2005; Gong et al., 2007), Hunan province (Huang et serrata are diverse and abundant. The quantity and the al., 2009; Zhang et al., 2010), Fujian province (Xie, 2009). population from H.serrata in different regions, maybe the

2642 J. Med. Plants Res.

Table 3. Endophytic fungi distributed in the stems, leaves and roots of H. serrata.

Taxa Strain Position Glomerella sp. (Colletotrichum Sp.) 16 Stems, Leaves and Roots Diaporthe sp. (Phomopsis sp.) 5 Stems, Leaves and Roots Trichoderma sp. 3 Stems, Leaves and Roots Cyphellophora sp. 1 Stems and Leaves Podospora sp. 5 Stems and Leaves Rhizoctonia sp. 2 Stems and Leaves Gibberella sp. (Fusarium sp.) 7 Stems and Leaves Plectosphaerella sp. 2 Stems Coniochaeta sp. 1 Stems Pestalotiopsis sp. 1 Stems Nigrospora sp. 1 Stems Botryosphaeriaceae sp. 1 Stems Pleosporales sp. 1 Stems Leptosphaeria sp. 1 Stems Pythium sp. 1 Stems Alternaria sp. 1 Leaves Aspergillus sp. 1 Leaves Cryptosporiopsis sp. 1 Roots Periconia sp. 1 Roots Lecythophora sp. 1 Roots

sample were collected different or the different serrata grown in south Hunan Province, China, and composition of the endophytic fungi from different identified only one of them as Cladosporium sp. Mycelia regions.Glomerella sp. (Colletotrichum sp.)，Trichoderma sterilia has been found not only in H. serrata but also in sp., Lecythophora sp., Coniochaeta sp. simultaneously other species. Cao et al. (2002) reported that 10% found in Guangxi Province and Hainan province. endophytic fungi isolated from Musa acuminata had no Glomerella sp. (Colletotrichum sp.), Trichoderma sp., spores. Huang et al. (2007) isolated 42 endophytic fungi Fusarium sp., Aspergillus sp. simultaneously found in from Nerium oleander L., and found half of them lacked Guangxi Province and Anhui Province. Colletotrichum sp., the sporulating structures. DNA sequence analysis, Fusarium sp., Alternaria sp. and Rhizoctonia sp. especially the ITS, has been widely used for identifying simultaneously found in Guangxi Province and Fujian non-sporulation fungi. In this study, 28 strains were Province. Glomerella sp. (Colletotrichum sp.) are identified by the ITS sequences analysis from H. serrata. simultaneously found in this four regions. The results For the strains identifiable only at the genus level, not at showed that Glomerella sp. (Colletotrichum sp.) were the species level, the adoption of the ITS sequences was frequently found in H.serrata from different regions. limited. Thus it is necessary to develop a more effective Glomerella (Colletotrichum) species always cause method, such as high-throughput sequencing method, to anthracnose which leads to considerable damage to the identify the non-sporulation fungi at species level. production of crops. Recent research has indicated that Colletotrichum species produce phytotoxins and thus have bioherbicidal potential (García-Pajón and Collado, ACKNOWLEDGEMENTS 2003) (Table 4). The authors are grateful to D. L. Zhu from the Dalian Institute of Drug Control for providing the experimental Conclusion materials and thank Dr. Y. D. Qi from the Medicinal Plant Development, Chinese Academy of Medical Sciences, for Morphological characteristics are a conventional factor critical reading of the manuscript. This research was used for the identification of endophytic fungi in H. serrata supported by the National Key Project of Scientific and (Shi et al., 2005; Gong et al., 2007; Zhang et al., 2010; Yu Technological Supporting Programs Funded by the et al., 2009; Xie, 2009). Some endophytic fungi in H. Ministry of Science ＆ Technology of China serrata are unidentifiable because they are mycelia (2007BAI27B01), and the Special Fund in Basic Scientific sterilia. Li et al. (2007) isolated 17 strains but could not Research for Non-Profit Research Institutes Financed by identify them. Xu et al. (2008) isolated 9 strains from H. the Ministry of Finance of China (2007HNB003).

Chen et al. 2643

Table 4. Comparison of endophytic fungi isolated from different regions of China.

Location and Identification Frequently Isolates Fungi isolated and identified Fungi firstly reported references methods genera Glomerella sp. (Colletotrichum sp.) Trichoderma sp., Gibberella sp. (Fusarium sp.) Lecythophora sp.,Coniochaeta sp., Glomerella sp. Plectosphaerella sp., Pestalotiopsis Morphological Rhizoctonia sp., Alternaria sp., (Colletotrichum sp., Cryptosporiopsis sp., Nigrospora Guangxi province characteristics and 53 Aspergillus sp., Plectosphaerella sp., sp.) and sp., Diaporthe (Phomopsis) sp., ITS sequence (This study) Pestalotiopsis sp., Periconia sp., Cyphellophora sp. and Leptosphaeria analysis Gibberella.sp Cryptosporiopsis sp., Pythium sp., (Fusarium.sp) sp. Nigrospora sp., Guignardia sp., Diaporthe sp. (Phomopsis sp.) Cyphellophora sp., Leptosphaeria sp., Podospora sp. Glomerella sp. (Colletotrichum sp), Hypocrea. sp., Trichoderma sp., Pleurostoma sp., Chaetomium sp., Glomerella Coniochaeta sp., Lecythophora sp., Hainan province ITS sequences and Lecythophora sp., Coniochaeta sp., sp.(Colletotrichu 52 Daldinia sp., Xylaria sp., Hypoxylon phylogenetic analysis Pleurostoma sp., Chaetomium sp., m sp.) and (Chen et al., 2011) sp., Nodulisporium sp., Cazia sp., and Daldinia sp., Xylaria sp., Hypoxylon sp., Hypoxylon sp. Nodulisporium sp., Cazia sp. and Phellinus sp. Phellinus sp. Colletotrichum Morphological sp. Aspergillus Podospora sp., Coniothyrium sp., Jiangxi province characteristics and sp., Podospora Paraphaeosphaeria sp., 127 All strains were identified to 19 genera (Wang et al., 2010) ITS sequence sp., Penicillium Leptosphaeria sp., Mortierella sp., analysis sp. and Capronia sp. and Chaunopycnis sp. Acremonium sp. Colletotrichum sp., Trichoderma sp., Fusarium sp., Aspergillus sp., Alternaria Anhui province Morphological sp., Cephalosporium sp. Guignardia sp. Phoma sp., 180 - (Gong et al.,2007) characteristics Penicillium sp. Diaporthe sp., Phoma sp. Phacodium sp. Verticilliun sp. Phacodium sp. Paecilomyces sp. and mycelia sterilia Anhui province Morphological Cephalosporium sp., Plasmoparad sp., 4 - - (Shi et al., 2005) characteristics Saccharomyces sp. and Penicillium sp. Morphological Hunan province characteristics and Cladosporium sp., Penicillium sp., , 4 - - (Huang et al.,2009) ITS sequence Fusarium sp., and Coniothyrium sp. analysis

2644 J. Med. Plants Res.

Table 4. Contd.

Peziza sp., Coniothecium sp., Hunan province Morphological 7 Paecilomyces sp., Penicillium sp., - - (Zhang et al.2010) characteristics Saccardia sp., Aspergillus sp., Zhejiang province Morphological Cephalosporium sp., Saccharomyces 6 - - (Yu et al.,2009) characteristics sp., Meria Vaill and Penicillium sp. Colletotrichum sp., Fusarium sp., Fujian province Morphological 12 Alternaria sp. Rhizoctonia sp., Glomus - - (Xie et al.,2009 ) characteristics sp.

REFERENCES condition for production of huperzine A from endophytic 19:381-385. fungus 2F09P03B of Huperzia serrata. Chin. Med. Wang LM, Han YF, Tang XC (2000). Huperzine A improves Ai JG, Zhang YQ (2005). Study on community property of Biotechnol. 2:254-259. cognitive deficits caused by chronic cerebral hypoperfusion Huperzia serrata habitat in Tianmushan nature reserves. J. Lin X, Lu CH, Huang YJ, Zheng ZH, Su WJ, Shen YM (2007). in rats. Eur. J. Pharmacol. 398:65-72. Zhejiang Sci. Technol. 25:14-17. Endophytic fungi from a pharmaceutical plant, Camptotheca Wang Y, Zeng QG, Zhang ZB, Yan RM, Wang LY, Zhu D Cao LX, You JL, Zhou SN (2002). Endophytic fungi from Musa acuminata: isolation, identiﬁcation and bioactivity. World J. (2010). Isolation and characterization of endophytic acuminata leaves and roots in South China. World J. Microb. Microbiol. Biotechnol. 23:1037–1040. huperzine A-producing fungi from Huperzia serrata. DOI Biot. 18:169-171. Ma XQ, Tan CH, Zhu DY, Gang DR (2006). A survey of 10.1007/s10295-010-0905-4. J. Ind. Microbiol. Biotechnol. Chen XY, Qi YD, Wei JH, Zhang Z, Wang DL, Feng JD, Gan potential huperzine A natural resources in China: The 38:1267-1278 BC (2011). Molecular identification of endophytic fungi from Huperziaceae. J. Ethnopharmacol. 104:54-67. Xie DF (2009). The isolation and identification of endophytic medicinal plant Huperzia serrata based on rDNA ITS Ma XQ, Tan CH, Zhu DY, Gang DR, Xiao PG (2007). Huperzine fungi and the detection of Huperzine A in Fujian Huperzia analysis. World J. Microbiol. Biotechnol. 27(3):495-503. A from Huperzia species-An ethnopharmacolgical review. J. serrata (Thunb.) Trev. dissertation, Fujian Agri. Forest. Univ, Dobranic JK, Johnson JA, Alikhan QR (1995). Isolation of Ethnopharmacol. 113:15-34. Fujian, China. endophytic fungi from eastern larch (Larix laricina) leaves Ma YM, Zhu HL, Su YQ, Shi QH, Li N (2007). Isolation and Xu QY, He XB, Tang KH, Tang YJ, Li j, Tian XJ (2008). Isolation from New Brunswick, Canada. Can. J. Microbiol. identification of endophytic fungi from Eucommia ulmoides. of endophytic fungi from Huperzia Serrata in Western Hunan l41:194-198. International Symposium on Eucommia ulmoides 1: 82-85. province. Life Sci. Res. 12:340-342. García-Pajón CM, Collado IG (2003). Secondary metabolites Márquez S, Bills GF, Zabalgogeazcoa I (2007). The endophytic Yu C, Luo HH, Qi N, Hu Y, Wang ZH (2009). Isolation and isolated from Colletotrichum species. Nat. Prod. Rep. mycobiota of the grass Dactylis glomerata. Fungal Divers. 27: identification of endophytic fungi of Huperzia Serrata. 20:426-431. 171-195. Jiangsu Agric. Sci. 3:381-384. Gong YX, Jiang JH, Chen FM, Zhang XP (2007). Isolation and Rakotoniriana EF, Munaut F, Decock C, Randriamampionona D, Zangara A (2003). The psychopharmacology of huperzine A: an screening of endophytic fungi from Huperzia Serrata and Andriambololoniaina M, Rakotomalala T, Rakotonirina EJ, alkaloid with cognitiveenhancing and neuroprotective their antifungal activity. J. Anhui. Norm. Univ. 30:689-692. Rabemanantsoa C, Cheuk K, Ratsimamanga SU, Mahillon J, properties of interest in the treatment of Alzheimer’s disease. Huang J, He CZ (2010). Population structure and genetic El-Jaziri M, Quetin-Leclercq J, Corbisier AM (2008). Pharmacol. Biochem. Be. 75:675-686. diversity of Huperzia serrata (Huperziaceae) based on Endophytic fungi from leaves of Centella asiatica: Zhao XD (2006). Study on the relationship between the habitats amplified fragment length polymorphism (AFLP) markers. occurrence and potential interactions within leaves. Anton. of Huperzia serrata and seasonal fluctuating accumulation of Biochem. Syst. Ecol. 38(6):1137-1147. Van. Leeuw. 93:27–36. Huperzine A. dissertation, Hefei Univ.Tech., Hefei, China. Huang LH, Feng JQ, Zhou SL, Hong YK (2009). Isolation and Shao LP, Sheng RX, Zhang SX (1984). Fungal taxonomy. Zhang L, Duan ZY, Geng X, Li KG, Wei Z, Hu SP (2010). identification of endophytic fungi of Huperzia Serrata. Prog. Chinese Forestry Press. Beijing. Isolation and identification of endophytic fungi of Huperzia Mod. Biomed. 9:2641-2644. Shipunov A, Newcombe G, Raghavendra AKH, Anderson CL Serrata. J. Jishou. Univ. 31:79-84. Huang WY, Cai YZ, Hyde KD, Corke H, Sun M (2007) (2008). Hidden diversity of endophytic fungi in an invasive Endophytic fungi from Nerium oleander L. (Apocynaceae): plant. Am. J. Bot. 95:1096-1108. main constituents and antioxidant activity. World J. Microbiol. Shi W, Luo JP, Ting ZH, Wu J (2005). Isolation and Biotechnol. 23:1253-1263. identification of endophytic fungi of Huperzia serrata. Chin. Li J, Zhao JL, Xu LJ, Zhou LG, Li X (2008). Endophytic fungi Tradit. Herbal Drugs 36:281-283. from rhizomes of Paris polyphylla var. yunnanensis. World J. Taechowisan T, Peberdy JF, Lumyong S (2003). Isolation of Microbiol. Biotechnol. 24:733-737. endophytic actinomycetes from selected plants and their Li WK, Zhou JY, Lin ZW, Hu ZB (2007). Study on fermentation antifungal activity. World J. Microbiol. Biotechnol.