1Wood Research Institute, Kyoto University, Uji, Kyoto, 611-0011 Japan; 2Kansai Research Center, FFPRI, Momoyama, Fushimi, Kyoto, 612-0855 Japan

Background and objectives
Japanese pine trees (Pinus thunbergii and P. densiflora) been suffered from pine wilt diseases that are caused by wood nematodes, Bursaphelenchus xylophilus. Pinosylvin and its monomethyl ether are common stilbenoids in the pine heartwood, and show anti-fungal and nematicidal activities. They are inducible under biotic and abiotic stress, although stilbenoids are, in some cases, constitutive components. In theory, pine trees with stilbenoid formation have the potential to kill the nematodes; in practice, however, they easily suffer from the diseases. The aim of this study was to investigate how pine trees form stilbenoids and why nematodes are able to get around the defence systems of the host trees.

Pinosylvin synthase in pine trees is a key enzyme in stilbenoid formation. It is also inducible by stimuli or stress, and controlled by a gene called the stilbene synthase gene (sts). Here we report sts expression in pine trees in relation to pine wilt diseases.

Materials and methods
Plant materials: 15-day-old seedlings, a few year-old nursery stocks and 24-year-old trees of Japanese red pine, Pinus densiflora, were used. Sodium salicylate (10 mm) or its analogues was sprayed over the 15-day-old seedlings or the 7-year-old nursery stocks as a stress treatment.
RNA extraction and RT-PCR: total RNA was extracted and immediately reverse-transcribed [1]. After denaturation, PCR was performed (94C for 1 min followed by 58-65C for 2 min) by repeating the two-step reactions depending on the primer pairs used. Several sts primer pairs for PCR were designed with reference to a reported sts (Acc. No. X60753 in GenBank) by using computer-assisted software.
Southern hybridization: the RT-PCR products were vacuum-transferred onto positively charged nylon membrane after electrophoresis and detected with DIG-labelled probes (Boehringer).
Determination of base sequences: the Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin Elmer) and an ABI PRISM 377 DNA Sequencer were mainly used for sequence analyses.

Results and conclusions
We have already established how to amplify an sts fragment exclusively, even if chalcone synthase genes co-exist in the sample template [1, 2]. We have also demonstrated that Japanese red pine has at least two quite similar sts genes that are not localized on an allelic pair, and are both expressed simultaneously [2]. We analysed total sts expression by RT-PCR.

In the seedlings, we found successive colour variations from red to white in the hypocotyls. Under the stress applied, the red seedlings showed rather high sts expression compared with the white ones. However, sts was expressed only in roots, not in hypocotyls or in cotyledons. This suggests no direct correlation between the colour and sts expression.

In the 24-year-old trunks, expression depended on seasons and on the radial positions in the core harvested. High apparent expression was detected in autumn and is restricted in the transition zone and heartwood but not distributed in the sapwood. The transcribed RNAs of sts was detected as hnRNA and mRNA, suggesting the hnRNA might be formed at high levels and that processing might not catch up with transcription during heartwood formation.

As described above, we showed that not all organs or cells can express sts, even under stressed conditions. This suggests that wood nematodes may attack only cells that do not produce toxic stilbenoids. Actually, in the early stage of disease the nematodes move only in resin canals that are surrounded by epithelial cells. Those cells produce high amounts of terpenoids, while they may not produce stilbenoids.

1. Kubota K, Kuroda H, Sakai F, 1996. Wood Research 83, 17-20.
2. Yamauchi Y, Kuroda H, Sakai F, 1997. Wood Research 84, 15-18.