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Characterization of compounds was made by using various methods: two-dimensional thin layer chromatography
(TLC) on cellulose plates to observe fluorescence and to test specific chemical reagents, observation of spectral characteristics
in U.V., cochromatography in HPLC with spots obtained from TLC and with various standards. The presence of glycosidic
or ester links was tested with acid or alkaline hydrolysis, respectively.
RESULTS AND DISCUSSION
Comparison Between Unwounded and Inoculated Phloem
Fourteen peaks were observed, of which eight were characterized at least at the chemical phenol family level (Fig. 1).
Considerable differences existed between unwounded and inoculated phloem (Fig. 2). The most striking concerned the
appearance of stilbenes, pinosylvin (Ps) and its monomethylether (Psme), and of one flavonoid, pinocembrin (Pc), in the
reaction zone of all trees, while these compounds were always totally absent in the unwounded phloem. In all trees, the
concentration of all compounds of the hydroxycinnamic acid group or its derivatives decreased during tree reaction. The
concentration of flavonoids other than Pc increased or decreased depending on clone.
Hydroxycinnamie
Flavonoid Stilbene acid group or its
group group derivatives
(a) (a) (a)
Figure 2.mVariations in the concentration of the phenolic compounds, between unwounded phloem and reactive phloem of
different Scots pine clones, after inoculation with Ophiostoma brunneo-ciliatum. (a) = compound appearing in the
reaction zone. One arrow means that the direction of variation was the same for all clones; several arrows mean that
the direction of variation depended on the clone. Txf = taxifolin; Txfgl = taxifolin glycosid; Pc = pinocembrin;
Ps = pinosylvin; Psme = pinosylvin monomethylether; Pcae = p-coumaric acid ester; Acphe = acetophenone glycoside.
These results confirm the preliminary study by Lieutier et al. (1991a) in Scots pine. Phloem phenolic response to
inoculations differed greatly according to compound, which caused dramatic modifications in the relative composition of this
tissue. These results differ completely from the observations on terpenes in any conifer species (Russel and Berryman 1976;
Raffa and Berryman 1982a,b; Delorme and Lieutier 1990; Lieutier et al. 1991b; Lfingstr6m et al. 1992), and makes certain
phenols good candidates to play a role in arresting invaders and to be used as biochemical markers of tree resistance.
Stilbenes and Pc, which were characteristic of phloem induced reactions, could play a particular role in reaction
efficiency, and consequently in a tree's induced resistance to bark beetle and fungi attacks. However, similar results have
been reported in needles after pollution by ozone (Rosemann et al. 1991), as well as in the sapwood of various pine species in
response to several kinds of aggression (Ref. in Kuc and Shain 1977, Hart and Shrimpton 1979, Shain 1979, Hart 1981,
Kemp and Burden 1986). In addition, similar results have also been obtained with a phloem wound without fungus (Lieutier
and Yart unpubl.). Lieutier et al. (1991a) also mentioned the non-specificity of the phloem phenolic response after various
aggressions. Tree response to bark beetles and their associated fungi are thus more a response to wounding than a response to
particular aggressors themselves (Lieutier 1993). Mullick (1977) has suggested previously that a tree's response to aggres.
sion is more concerned with tissue restoration than with defense.
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