Two Endophytic Fungi in Different Tissues of Scots Pine Buds (Pinus sylvestris L.) A.M. Pirttila ¨, 1,2 H. Pospiech, 2 H. Laukkanen, 1 R. Myllyla ¨, 2 A. Hohtola 1 1 Department of Biology/Botany, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland 2 Department of Biochemistry, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland Received: 17 June 2002; Accepted: 4 October 2002; Online publication: 17 December 2002 A B S T R A C T Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant. Introduction Fungal endophytes are detected in the leaves of many grasses and trees [5, 35, 40]. Endophytic fungi are well studied in grasses, but the endophytes of trees are known in less detail. The fungal diversity is large in tree leaves, especially in conifers, where the needles are colonized locally by several endophytic fungal species [11, 12, 16, 18, 32, 45, 46]. In addition to leaves, endophytic fungi are found in other tree tissues, such as wood and bark [3, 8, 15, 22, 33]. However, plant buds have been scarcely studied for fungal endophytic presence. Although endophytic fungi are rarely detected from conifer needles soon after emergence [12, 14, 17], bud scales of some trees contain fungal endophytes [50]. In addition, fungi are frequently detected in plant tissue cultures that are initiated from the buds. The microbes appearing in plant tissue cultures create a particular problem in terms of regenerating the plant. Some mi- crobes may also remain latent in the plant tissue and are considered the most problematic ones, because of diffi- culties in their detection [27, 28]. Although many of these microbes may represent endophytes, they are usually re- garded as contaminants originating from outside the cul- Current address: A.M. Pirttila ¨, Department of Botany and Plant Pathol- ogy, Purdue University, 1155 Lilly Hall, West Lafayette, IN 47907-1155, USA Correspondence to: A.M. Pirttila ¨; E-mail: AM.Pirttila@oulu.fi Microb Ecol (2003) 45:53–62 DOI: 10.1007/s00248-002-1038-8 Ó 2002 Springer-Verlag New York Inc.