Chitinases are hydrolytic enzymes that have been employed in biotechnology in attempts to increase plants' resistance against fungal pathogens. Genetically modified plants have given rise to concerns of the spreading of transgenes into the environment through vertical or horizontal gene transfer (HGT). In this study, chitinase-like sequences from silver birch (Betula pendula) EST-libraries were identified and their phylogenetic relationships to other chitinases were studied. Phylogenetic analyses were used to estimate the frequency of historical gene transfer events of chitinase genes between plants and other organisms, and the usefulness of phylogenetic analyses as a source of information for the risk assessment of transgenic silver birch carrying a sugar beet chitinase IV gene was evaluated. Thirteen partial chitinase-like sequences, with an approximate length of 600 bp, were obtained from the EST-libraries. The sequences belonged to five chitinase classes. Some bacterial chitinases from Streptomyces and Burkholderia, as well as a chitinase from an oomycete, Phytophthora infestans, grouped together with the class IV chitinases of plants, supporting the hypothesis that some class IV chitinases in bacteria have evolved from eukaryotic chitinases via horizontal gene transfer. According to our analyses, HGT of a chitinase IV gene from eukaryotes to bacteria has presumably occurred only once. Based on this, the likelihood for the HGT of chitinase IV gene from transgenic birch to other organisms is extremely low. However, as risk is a function of both the likelihood and consequences of an event, the effects of rare HGT event(s) will finally determine the level of the risk.

Roots of clonal birches (Betula pendula) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro. Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum, whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.

Field-grown trees of Alnus incana (L.) Moench, Alnus glutinosa (L.) Geartner and Betula pendula Roth displayed pronounced differences in responses of light-saturated net photosynthesis (Asat) to herbivory by the alder beetle (Agelastica alni L., Galerucinae), a specialized insect which primarily defoliates alders. We found that photosynthetic rates of grazed leaves increased following herbivory in Alnus but not in Betula. Area- and mass-based Asat of grazed leaves declined linearly with increasing amount of leaf perforation in B. pendula, by as much as 57%. By contrast Alnus glutinosa and Alnus incana increased area-based rates of Asat by 10–50% at all levels of leaf grazing. Given increased Asat in the remaining portion of grazed leaves, a net reduction in photosynthesis per leaf occurred only when the proportion of leaf area grazed exceeded 40% for Alnus incana and 23% for Alnus glutinosa. Since vein perforation by Agelastica alni was observed much more frequently in leaves of Betula than in Alnus, we hypothesized that declining Asat in herbivorized Betula was related to this disruption of water transport. A field experiment with artificial leaf perforation demonstrated a greater decline in Asat in vein-perforated Betula leaves than perforated leaves with midrib veins intact. However, regardless of leaf perforation regime, birch never showed post-perforation increases in Asat. In all species, rates of transpiration of grazed leaves linearly increased and water-use efficiency decreased with increased amount of leaf perforation. In grazed Alnus incana leaves, increasing leaf area consumption by Agelastica alni resulted in an increase of total phenols, a reduction in starch content and no changes in nitrogen concentration in the remaining portion. The increase in photosynthesis in Alnus incana might be related to declining leaf starch concentration or increasing stomatal conductance, but was unrelated to leaf nitrogen concentration. These gas exchange and leaf chemistry measurements suggest that in contrast to B. pendula, Alnus incana and Alnus glutinosa, which are the major host species for Agelastica alni, possess leaf-level physiological adaptations and defence mechanisms which can attenuate negative effects of herbivory by the alder leaf-beetle.