Centre de recherche en biologie forestière, Université Laval, Québec, Qc, Canada, G1K 7P4

Background and objectives
Perennial Nectria canker, otherwise known as European canker, is caused by the ascomycete fungus Nectria galligena and occurs throughout the temperate zone where it causes important losses on pomaceous fruit trees and on a broad spectrum of decidous forest trees. Although the disease has been known for a long time, interactions between the pathogen and its hosts remain poorly understood. Thus, the objectives of the work described hereafter were: (i) to evaluate the pathogenic range of a population of N. galligena isolated from different hosts; (ii) to investigate pathogenic variability and to examine its genetic basis; and (iii) to develop an efficient DNA-mediated transformation system for a direct assessment of putative pathogenicity genes in N. galligena.

Materials and methods
Isolates of N. galligena were recovered from apple trees and from over 12 species of northern hardwoods. The identity of the isolates was confirmed by analysis of cultural characteristics and PCR amplification patterns. Pathogenicity tests were carried out on saplings of various hardwood species, on calli obtained from yellow birch (Betula alleghaniensis), and on Golden Delicious apples. The genetic basis of pathogenic variability was verified by inoculating sets of full-sib progeny from perithecia found at the margin of naturally occurring cankers or formed in vitro by crossing selected isolates with known levels of virulence. Hygromycin B was chosen as a selection marker for transformation since it was found to inhibit the growth of wild strains of N. galligena. Spheroplasts obtained by enzymatic treatment of germinated macroconidia were transformed by electroporation in the presence of undigested plasmid pPS57 containing the hygromycin phosphotransferase (hph) gene.

Results and conclusion
Inoculation of saplings with N. galligena isolates from various host species confirmed the polyphagous nature of the pathogen and showed the wide range of its pathogenic variability. This variability was quantitative rather than qualitative: there was no evidence for host specificity (with the possible exception of white ash [Fraxinus americana]), whereas virulence varied among isolates. Inoculation of yellow birch calli and Golden Delicious apples further confirmed the occurrence of pathogenic variability in N. galligena. Analysis of full-sib progeny showed that variations in virulence resulted from differences at several nuclear loci. One locus, Vir1, was identified following the recovery of two classes of progeny from a controlled cross between a highly and a moderately virulent isolates.

Digestion of germinated macroconidia with Novozym usually yielded ca 1X107 spheroplasts/g fresh fungal material. Following electroporation in the presence of pPS57, 1 hygromycin-resistant colony/2 ug plasmid/1X107 spheroplasts was recovered after eight days of incubation on selective medium, whereas the negative control treatment (no plasmid) yielded no colony. Putative transformants were assayed for the presence of sequences hybridizing to hph DNA. Work is under way for increasing spheroplast yield and optimizing the parameters for stable integration and expression of exogenous DNA.