Detection and Taxonomic Placement of Endophytic Fungi within Frond Tissues of Livistona chinensis Based on rDNA Sequences Liang Dong Guo, Kevin D. Hyde, 1 and Edward C. Y. Liew Centre for Research in Fungal Diversity, Department of Ecology and Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong, S. A. R. China Received February 8, 2000; revised October 2, 2000; published online June 6, 2001 The 5.8S gene and flanking internal transcribed spacers (ITS1 and ITS2) of the rDNA were amplified from total DNA extracted from frond tissues of Livis- tona chinensis with universal and fungal-specific primers. These amplified fragments were cloned and sequenced. Phylogenetic analysis based on the 5.8S gene sequences indicated that the six clone sequences obtained were of different origins. Five sequences, P1-9, P2-6, P4-4, P4-5, and P4-7, belonged to the fungi and one sequence, P3-2, belonged to the plants. P1-9 was inferred to belong to the Basidiomycota based on the phylogenetic analysis of the 5.8S gene sequences but could not be identified to lower taxonomic levels. Further identification of the other four fungal clones to lower taxonomic levels was attempted based on phylogenetic analysis and sequence comparison of both the conserved 5.8S gene and the variable ITS regions. The origin of P2-6 was identified to be Glomer- ella and its anamorph Colletotrichum, the origins of P4-5 and P4-7 were Mycosphaerella and its anamorph Cladosporium, and the origin of P4-4 was the Herpo- trichiellaceae. The direct approach to detection and taxonomic placement of endophytic fungi within host tissue without the need for conventional in vitro cul- turing is discussed. © 2001 Academic Press Key Words: molecular identification; molecular phy- logenetics; environmental DNA; mycelia sterilia; ribo- somal RNA gene. INTRODUCTION Techniques that have routinely been employed in previous endophyte studies generally involve three ba- sic steps: (1) surface sterilization of plant tissue to eliminate any fungi and other microorganisms on the host surface, (2) isolation of fungal endophytes growing from samples placed onto nutrient agar, and (3) iden- tification of the endophytes based on morphological characters in culture (Guo et al., 1998). The study of endophytes is generally regarded as a method-depen- dent process. To a great extent, fungal endophyte com- munities obtained from plants are subject to surface sterilization techniques, incubation conditions, and sporulation of isolates. Nonsporulating isolates are generally termed mycelia sterilia and can be identified to various taxonomic levels by molecular techniques (Guo et al., 2000). Due to the limitations of traditional isolation tech- niques, it is highly probable that some or even numer- ous endophytes are never isolated. This may be due to the fastidious nature of some endophytes, which do not grow on the artificial media that are routinely used. Based on results of previous studies, most of the endo- phytic fungi isolated are usually ascomycetes or their anamorphs. More evidence is needed to ascertain whether the isolation techniques preclude the isolation of other fungi or whether ascomycetes (and their ana- morphs) constitute the majority of the endophyte com- munities studied. To overcome this putative technical bias, a culture-independent approach to the analysis of endophyte communities of plants is needed. With this rationale, the use of molecular techniques to detect and identify endophytes directly within palm tissues is hence explored here. The use of molecular techniques for the direct detec- tion and identification of fungi within natural habitats has been reviewed by Liew et al. (1998). Molecular techniques have mainly been used in the detection and identification of mycorrhizal fungi (Gardes et al., 1990; Simon et al., 1993; Bonito et al., 1995; Clapp et al., 1995; Kraigher et al., 1995; Abbas et al., 1996; Harney et al., 1997; Chambers et al., 1998, 1999; Chelius and Triplett, 1999) and phytopathogenic fungi directly from within plant tissues (e.g., Schesser et al., 1991; Mills et al., 1992; Johanson and Jeger, 1993; Moukhamedov et al., 1994; Beck and Ligon, 1995; Sha- pira et al., 1996). In addition, molecular techniques have been employed to detect and identify fungi di- rectly from the grass clothing of the Iceman, a mum- mified human body of neolithic origin, (Rollo et al., 1 To whom correspondence should be addressed. Fax: (852) 2517 6082. E-mail: kdhyde@hkucc.hku.hk. Molecular Phylogenetics and Evolution Vol. 20, No. 1, July, pp. 1–13, 2001 doi:10.1006/mpev.2001.0942, available online at http://www.idealibrary.com on 1055-7903/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. 1