ITS-RFLP and ITS sequence analysis of a foliar endophytic Phyllosticta from different tropical trees Ajay K. PANDEY 1 , M. Sudhakara REDDY 1 * and Trichur S. SURYANARAYANAN 2 1 Department of Biotechnology, Thapar Institute of Engineering and Technology, Patiala 147 001, India. 2 Post graduate and Research Department of Botany, Ramakrishna Mission, Vivekananda College, Chennai 600 004, India. E-mail : vasu70@yahoo.com Received 20 August 2002; accepted 6 February 2003. Different isolates of a foliar endophytic species of Phyllosticta were isolated from different tropical tree species in India to examine genetic variation among the isolates. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis did not detect any variation among the isolates, suggesting that they all belong to the same species. Sequence data of the ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) supported the identity of the present fungal isolates as P. capitalensis. These results show that P. capitalensis (teleomorph Guignardia endophyllicola ?) is an ubiquitous foliar endophyte that can infect tree hosts from different families and habitats. INTRODUCTION A guild of mitosporic and teleosporic ascomycetes, en- dophytic fungi, cause symptomless infections in leaves of vascular plants. Several plants in the temperate reg- ion have been studied for their endophyte assemblages (Petrini 1991), and only recently have tropical plants been investigated for their endophyte associations (Lodge, Fisher & Sutton 1996, Rodrigues & Petrini 1997, Suryanarayanan, Kumaresan & Johnson 1998, Arnold et al. 2000, Fro¨hlich, Hyde & Petrini 2000, Surya- narayanan, Senthilarasu & Muruganandam 2000). Most of studies on endophytes of tropical plants centered around one or a few individual hosts; few address the occurrence and distribution of endophytes in a tropical plant community (Arnold, Maynard & Gilbert 2001, Kumaresan & Suryanarayanan 2001, Cannon & Simmons 2002, Suryanarayanan, Murali & Venkatesan 2002). Among the genera isolated as endophytes from the leaves of various tropical plants, the coelomycete genus Phyllosticta is often present (Stone, Sherwood & Carroll 1996, Suryanarayanan, Kumaresan & Johnson 2001). Conversely, it has been demonstrated that in temperate coniferous hosts (Carroll & Carroll 1978), evergreen shrubs (Petrini, Stone & Carroll 1982) and mangrove trees (Kumaresan & Suryanarayanan 2001) certain fungal endophytes are host-specific. Such host specificity for endophytes has been recorded for many temperate plants, including Salix fragilis, Quercus rubur, and Alnus rubra (Petrini & Fisher 1990, Sieber, Sieber-Canavesi & Dorworth 1991, Arnold et al. 2001). In the course of an ecological study of foliar endo- phytes of trees in different tropical forests of south India, isolates of Phyllosticta were obtained from vari- ous hosts. These could not be differentiated on the basis of cultural characters or morphology, and so were suspected to belong to the same species. A mol- ecular approach using internal transcribed spacer- restriction fragment length polymorphism (ITS-RFLP) patterns was used to compare the isolates and test this hypothesis. The nuclear rDNA repeat unit is a useful area of the genome to examine for polymorphisms, because of the juxtaposition of conserved and variable regions and its high copy number. Analysis of rDNA regions, through the use of PCR and RFLPs, has been applied to many systematic questions (Bruns, White & Taylor 1991). For analysis of fungal taxa at or below the species level, the more variable ITS region is commonly used (Pryor & Gilbertson 2000, Anderson, Chambers & Cairney 2001). This region has been used to examine phylo- genetic relationships among the Phyllosticta isolates from different hosts in our study. The ITS sequence of rDNA of a Phyllosticta isolate was also determined with the objective of providing information on the genetic variability among the isolates and to compare the sequence to other isolates described in the litera- ture. This study aims to provide a better understanding * Corresponding author. Mycol. Res. 107 (4): 439–444 (April 2003). f The British Mycological Society 439 DOI: 10.1017/S0953756203007494 Printed in the United Kingdom.