Vol. 7(36), pp. 2638-2644, 25 September, 2013 DOI: 10.5897/JMPR10.390 Journal of Medicinal Plant Research ISSN 1996-0875 © 2013 Academic Journals http://www.academicjournals.org/JMPR 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