ORIGINAL PAPER Gas chromatographic–mass spectrometric investigation of metabolites from the needles and roots of pine seedlings at early stages of pathogenic fungi Armillaria ostoyae attack Valery A. Isidorov Æ Pawel Lech Æ Anna _ Zo ´lciak Æ Magdalena Rusak Æ Lech Szczepaniak Received: 18 June 2007 / Revised: 30 November 2007 / Accepted: 15 January 2008 Ó Springer-Verlag 2008 Abstract An investigation was carried out of the com- position of metabolites in pine seedlings tissues at the initial stages of the infectious process caused by pathogenic fungi Armillaria ostoyae, which causes a root rot of trees and degradation of forest resources. With the help of suc- cessive extraction with organic solvents of different polarity, more than 190 metabolites were extracted from the needles and roots of the seedlings and then identified by GC–MS method. The composition of the extracts from control plants and those inoculated with Armillaria ostoyae were compared. It was established that part of secondary metabolites (glucosamines and free amino acids, carbohy- drates raffinose and trehalose) were present only in the tissues of inoculated plants. Possible roles of some of these compounds appearing in the roots of seedlings infected with the fungus are also discussed in the paper. Keywords Armillaria root disease  Scots pine seedlings  Metabolites composition  GC–MS analysis Introduction Fungi play an important role in the dynamics of forest ecosystems (Sinclair et al. 1987). Pathogenic root rot fungi are an integral part of forest ecosystems, but sometimes produce undesirable effects and cause degradation of forest resources. One of the most serious diseases of forest trees is the so called ‘‘armillaria root disease’’, caused by several species from Armillaria genus. The disease affects various forest tree and shrub species (Smith et al. 1994; Rizzo et al. 1995; Wargo 1996; Mallett and Mayrand 1998; Thomson et al. 1998; Ayres and Lombardero 2000; DeLong et al. 2005). The primary cause of armillaria root disease in European conifers is A. ostoyae (Romag.) Herink. The aboveground symptoms of the disease in coniferous trees are the following: thinning of shoots, growth delay due to the reduction of leading and lateral shoots, discol- oration and withering away of the needles, bleed of oleoresin from the butt-end (Rykowski 1975). However, not all infected trees show visible symptoms of the disease. According to Robinson et al. (2003), they appeared only in about 60% of the infected trees. Besides, these symptoms relate to the far advanced phase of the disease. Investigations carried out in recent years allowed the determination of numerous biochemical changes in the plants infected with different species of root rot fungi. In particular, it was established that plants react to fungal infection by producing a series of protective chemical compounds. Among these are phytoalexins (Bailey and Mansfield 1982), phenols (Wargo 1981; Shaw 1985; Entry et al. 1991; 1992; Myszewski et al. 2002), stilbenes (Woodwards and Pearce 1988; Harju et al. 2003) and pathogenesis related proteins (Bowles 1990; Robinson et al. 2000). However, there are almost no data in the lit- erature on biochemical changes in plant tissues at the initial stages of the infectious process. The aim of this pilot investigation was to work out methods for effective separation of a wide range of pine primary and secondary metabolites, which belong to Communicated by W. Oßwald. V. A. Isidorov (&)  M. Rusak  L. Szczepaniak Institute of Chemistry, Bialystok University, 15-399 ul. Hurtowa 1, Bialystok, Poland e-mail: isidorov@uwb.edu.pl P. Lech  A. _ Zo ´lciak Department of Forest Phytopathology, Forest Research Institute, 05-090 Raszyn, Poland 123 Trees DOI 10.1007/s00468-008-0213-z