Fungal Diversity Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia species Huang, W.Y.1, Cai, Y.Z.1, Surveswaran, S.1, Hyde, K.D.2, Corke, H.1 and Sun M.1* 1School of Biological Sciences, the University of Hong Kong, Pokfulam Road, Hong Kong, PR China 2School of Science, Mae Fah Luang University, Chiang Rai, Thailand Huang, W.Y., Cai, Y.Z., Surveswaran, S., Hyde, K.D., Corke, H. and Sun, M. (2009). Molecular phylogenetic identification of endophytic fungi isolated from three Artemisia species. Fungal Diversity 36: 69-88. Diverse fungal species live inside plant tissues, some of which presumably occur in a mutualistic association. Some fungal endophytes are widespread and can be found in many different plant species, whereas others are highly specific to single hosts. In this study, we investigated the taxonomic identities and phylogenetic relationships of fungal endophytes isolated from three plant species, Artemisia capillaris, A. indica, and A. lactiflora, using a combination of morphological and molecular approaches. Morphological differences among the fungal isolates indicate that diverse distinct morphotypes might be present within the hosts. Thirty-four fungal isolates were selected for further molecular phylogenetic analysis using nuclear ribosomal DNA sequences, including both the internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene region. The 34 endophytes were identified to various taxonomic levels, and some to the species level based on fungal sequences with known identities in GenBank. Our results suggest that Alternaria, Colletotrichum, Phomopsis, and Xylaria species are the dominant fungal endophytes in the Artemisia hosts, and some of these endophytes exhibit host and tissue specificity. This aspect can be further explored to understand the relationships between plant hosts and their fungal endophytes. Key words: Artemisia, endophytic fungi, molecular identification, phylogenetic analysis. Article Information Received 22 February 2008 Accepted 20 October 2008 Published online 31 May 2009 *Corresponding author: Mei Sun; e-mail: [email protected] Introduction Hyde and Soytong, 2008; Mitchell et al., 2008). Much attention is now being paid to Endophytes commonly refer to a group endophytic biodiversity, the chemistry and of fungi that reside asymptomatically inside the bioactivity of endophytic metabolites, and the living plant tissues (Sánchez Márquez et al., relationship between endophytes and their host 2007, 2008; Hyde and Soytong, 2008). Recent plants (Tan and Zou, 2001; Schulz et al., 2002; surveys of various host plants have demon- Kumar et al., 2005; Rungjindamai et al., 2008; strated that fungal endophytes are ubiquitous in Tao et al., 2008). plant species (Kumar et al., 2004; Zhang et al., The genus Artemisia (Asteraceae) is 2006; Sánchez Márquez et al., 2007; Huang et widely distributed in the world, with about 185 al., 2008; Oses et al., 2008) and even lichens species in China and is highly valued for its (Li et al., 2007). Globally, there are at least one medicinal properties (Zou et al., 2000). million species of endophytic fungi (Ganley et Artemisia capillaris is a traditional oriental al., 2004), which represent an important medicinal herb, and has been used for genetic resource for biotechnology. Endo- treatment of various liver diseases such as phytes have been recognized as potential hepatitis, jaundice and fatty liver (Hong et al., sources of novel natural products for 2004). Artemisia indica allegedly possesses pharmaceutical, agricultural and industrial uses, anti-malarial activity, and has been reported in especially those secondary metabolites the guideline of Thai medicinal plants used in produced by fungal endophytes colonizing primary health care (Chanphen et al., 1998). medicinal plants (Strobel and Daisy, 2003; Artemisia lactiflora, an edible plant in 69 Southeast Asia, has inhibitory effects on a Sánchez Márquez et al., 2007; Morakotkarn et variety of tumor promoter-induced biological al., 2007). Most of the endophytic fungi were responses such as oxidative stress as well as detected and identified by comparative tumor promotion in ICR mouse skin analyses of the ribosomal DNA sequences, (Nakamura et al., 1999). As part of our especially the ITS region. For example, Harney ongoing research towards finding novel anti- et al. (1997) identified arbuscular mycorrhizal oxidants from natural resources, we investi- fungi from Artemisia californica using the ITS gated the secondary metabolites of endophytes region. Also based on ribosomal DNA isolated from these three Artemisia medicinal sequences, Guo et al. (2000) and Lacap et al. plants and found that some of the endophytic (2003) evaluated the endophytic fungal fungal isolates exhibited strong anti-oxidant ‘morphotype’ concept concerning mycelia capacity (Huang et al., 2007). It is important to sterilia. A high diversity of endophytic fungal further identify these bioactive fungi for communities was revealed from either potential utilization using morphological and/or Heterosmilax japonica or Livistona chinensis molecular techniques. using a cultivation-independent approach by Fungal taxonomy is traditionally based analyzing fungal DNA sequences extracted on comparative morphological features (e.g., from plant tissues (Guo et al., 2001; Gao et al., Lodge et al., 1996; Sette et al., 2006; Crous et 2005). Ganley et al. (2004) studied morpho- al., 2007; Zhang et al., 2008). However, logically similar endophytes and parasites special caution should be taken when closely based on the ITS region, and found that the related or morphologically similar endophytes endophytic fungi in west white pine were are identified, because the morphological actually most closely related to, but distinct characteristics of some fungi are medium- from, the parasites. Peintner et al. (2003) first dependent and cultural conditions can recorded ectomycorrhizal Cortinarius species substantially affect vegetative and sexual from tropical India and established their compatibility (Zhang et al., 2006; Hyde and phylogenetic position using ITS sequences. Soytong, 2007). Furthermore, the conventional Queloz et al. (2005) monitored the spatial and methods cannot be applied for identifying temporal dynamics of the tree-root endophyte fungal isolates that fail to sporulate in culture, Phialocephala fortinii using the restriction which are categorized as mycelia sterilia fragment length polymorphism (RFLP) (Lacap et al., 2003). Various optimization of analysis. growth conditions have been used to promote In the present study, we isolated a total of sporulation of these fungi, such as different 108 endophytic fungal isolates from different culture media, potato dextrose agar (PDA), tissues of three medicinal Artemisia species (A. malt extract agar (MEA), corn meal agar capillaris, A. indica, and A. lactiflora). These (CMA), potato carrot agar (PCA), and water fungal endophytes were identified using a agar (WA), as well as the inclusion of host combination of morphological and molecular tissues in plate cultures (Guo et al., 2000). methods. Thirty-four representative isolates of Nevertheless, a large number of fungi still do various morphological endophytes were not sporulate in culture, and these mycelia selected for DNA sequence analysis using the sterilia are considerably frequent in the whole ITS region, including ITS1, 5.8S, and endophyte studies (Lacap et al., 2003). ITS2. Combining both morphological charac- In contrast, molecular techniques exhibit ters and DNA sequence data, we identified high sensitivity and specificity for identifying some of these endophytes to the species level microorganisms and can be used for classifying and the others at least to the family level, and microbial strains at diverse hierarchical evaluated phylogenetic relationships among the taxonomic levels (Sette et al., 2006). Several endophytes. This study represents the first step recent studies have shown that genetic methods for linking the biodiversity of endophytes, their can be successfully used in the studies of taxonomic identities and phylogenetic positions endophytic fungi (Gao et al., 2005; Wang et al., with their bioactivities in the medicinal plant 2005; Arnold et al., 2007; Ligrone et al., 2007; species. 70 Fungal Diversity Materials and methods 1990). The amplified fragment includes ITS1, 5.8S and the ITS2 of rDNA. Amplification was Plant material performed in a 50 μl reaction mixture Healthy plants of three medicinal containing 10 mM Tris–HCl (pH 9.0), 50 mM Artemisia species growing in Hong Kong were KCl, 0.1% Triton X 100, 2 mM MgCl2, 0.2 sampled from June to October, 2005. Artemisia mM dNTPs, 0.4 μM primers, 0.5 unit of Taq capillaris was collected from Ham Tin Wan, A. polymerase, and 2μl of fungal genomic DNA. indica from Kadoorie Farm and Botanic The thermal cycling program was as follows: 2 Garden, and A. lactiflora from the Hong Kong min initial denaturation at 94˚C, followed by Island. Fresh samples were taken to the 35 cycles of 30 s denaturation at 94˚C, 30 s laboratory and treated within 8 hours. Digital primer annealing at 55˚C, and 45˚s extension at photographs were taken of each species, and all 72˚C, and 5 min at 72˚C for a final extension. the species samples were deposited in School The PCR product was purified with High Pure of Biological Sciences, the University of Hong PCR Product Purification Kit (Roche Kong. Diagnostics GmbH, Germany). Direct DNA sequencing was performed using primers ITS3 Isolation of endophytic