Wood-inhabiting microfungi isolated from Populus tremuloides from Alberta and northeastern British Columbia

Forty-nine taxa of sporulating microfungi were isolated in pure culture from the bark and wood of living and recently cut stems of trembling aspen (Populus tremuloides) from several sites in central Alberta and northeastern British Columbia. Stems were either healthy, blue stained, or decayed, or else possessed cankers or black galls. Several ecological groupings of fungi were recognized, including wood-staining fungi (e.g., Ophiostoma piliferum), black-gall-associated fungi (e.g., Hyphozyma lignicola, Knufia cryptophialidica,Phoma etheridgei), canker-causing pathogens (e.g., Nectria cinnabarina, Tympanis spermatiospora, Valsa sordida), and fungi that could be referred to as endophytes. This latter group consisted of an assortment of fungi normally found in niches other than wood (e.g., Ciborinia whetzelii, Sporormiella similis), microfungi associated with decayed wood (e.g., Cryptosphaeria lignyota, Phialocephala fusca, Phialophora alba, Phialophora bubakii, or canker-causing fungi isolated from healthy tissue (e.g., Hypoxylon mammatum, Leucostoma nivea.Key words: microfungi, endophytes, Populus, aspen, wood, Ascomycota, hyphomycetes, coelomycetes.

Inoculation methods for selecting Populus tremuloides resistant to Hypoxylon canker

Canker expansion and the amount of callus tissue formed were measured monthly on 60 ramets from each of five trembling aspen (Populus tremuloides Michx.) clones that had been inoculated in wounds with Entoleuca mammata (= Hypoxylon mammatum (Wahl.) Mill.) over a 12-month period. At the clone level, the prevalence of nonlethal cankers within clones prior to the study had no correlation with canker expansion with three of the five clones. Greenhouse inoculation of ramets derived from the same five clones resulted in the same resistance rankings as main-stem inoculations in the field. One isolate, Hm-27, produced longer cankers, and less callus developed on all clones compared with trees inoculated with the less aggressive strain, Hm-24. Cankers developed only on clones that were inoculated during the months of April through July with April inoculations resulting in the largest cankers. These results indicate that there is a limited time frame when wounds on aspen are susceptible to infection by artificial inoculation with the pathogen as many wound inoculations neither produced cankers nor wound callus. Comparing inoculation methods, either main-stem inoculations in the field or greenhouse inoculations coupled with the natural canker prevalence could be used to include or exclude clones for use in an aspen breeding program.

Wound response characteristics as related to phenylpropanoid enzyme activity and lignin deposition in resistant and susceptible Populus tremuloides inoculated with Entoleuca mammata (Hypoxylon mammatum)

Green internodal stem tissues of glasshouse grown Populus tremuloides were either wounded or wound-inoculated with Entoleuca mammata (Hypoxylon mammatum) and assayed for phenylalanine ammonia-lyase (PAL), caffeic acid - o-methyltransferase (CA-OMT), and cinnamyl - alcohol dehydrogenase (CAD) activity over a 96-h period. Lignin deposited in response to the treatments was analyzed by the Klason and the pyrolysis - gas chromatographic (GC) - mass spectroscopy (MS) methodologies. The wound-inoculated treatment resulted in a wound morphology congruent with a typical resistant and susceptible response to E. mammata. Wounding alone resulted in no morphological differences between the two genotypes. In wound-inoculated stem tissue PAL and CAD activities were substantially higher in the resistant relative to the susceptible genotype. Total Klason lignin was similar for both genotypes; however, pyrolysis-GC-MS analysis revealed a difference in the lignin monomeric composition between the two genotypes, with the susceptible genotype accumulating higher levels of hydroxyphenyl units relative to the resistant genotype. It is concluded that differences in PAL and CAD activity and the synthesis of distinct phenylpropanoid monomers distinguish the resistant from the susceptible aspen genotype. Alterations in boundary zone formation due to the differential synthesis of phenylpropanoid monomers and its effect on compartmentalization of the pathogen are discussed.Key words: aspen, Hypoxylon canker, phenylalanine ammonia-lyase, cinnamyl - alcohol dehydrogenase, caffeic acid - o-methyltransferase, disease resistance.

Infection of Populus tremuloldes by Hypoxylon mammatum ascospores through Saperda inornata gails

Branch galls resulting from oviposition wounds made by Saperdainornata Say on Populustremuloides Michx. were studied in relation to inoculation with Hypoxylonmammatum (Wahlenberg) J.H. Miller ascospores. The natural infection rate of galls in an aspen plantation in central Minnesota averaged 1.6% (range 0.8–2.6%) during a 4-year period. However, when S. inornata galls were inoculated with ascospores during a 3-year period, 9.1% (range 6.5–2.6%) of the branches became infected. When galls were inoculated with sterile water, 3.3% of the branches became infected, perhaps indicating that inoculum already was present in the galls. The average elapsed time from ascospore inoculation until the development of hypoxylon canker symptoms was 26 months. These results provide the first demonstration of canker development following inoculation of natural wounds on aspen with H. mammatum ascospores.

Antifungal compounds in aspen: effect of water stress

A thin layer chromatography bioassay was used to detect antifungal compounds in tissue culture plantlets and potted seedlings of aspen. Catechol and the phenolic glycosides salicin and salicortin were identified as inhibitory compounds; a fourth compound was observed but was not identified. Inhibitory compound levels were estimated in eight tissue culture clones grown on unamended media and media amended with 0.22 M mannitol to induce water stress. Aspen tissue culture plantlets grown under water stress conditions had significantly lower levels of catechol, salicortin, and salicin. Significant clonal variation in levels of catechol, salicin, and the unidentified compound was also observed. Catechol, salicortin, and salicin were inhibitory to Hypoxylon mammatum when tested at levels similar to those employed in the thin layer chromatography bioassay. These results suggest that a reduction in the levels of inhibitory compounds in water-stressed aspen may be a factor in the water stress induced susceptibility of aspen to H. mammatum. Key words: Populus tremuloides, Hypoxylon mammatum, water stress, tissue culture.

Genetic control of Populus tremuloides sensitivity to metabolites of Hypoxylon mammatum

Tissue culture plantlets derived from the vegetative buds of six aspen trees and from the seeds of controlled pollinations of these trees were bioassayed with culture filtrates from three isolates of Hypoxylon mammatum. Analysis of variance of parent and progeny bioassay responses revealed significant main effects and insignificant interaction, which suggests that metabolite sensitivity is a horizontally controlled trait. However, in certain crosses progeny response differed markedly from that of either parent. In these cases progeny were either far more or far less sensitive than the parents. This dramatic switching of sensitivity to the metabolites suggests that this trait is conditioned by a small number of genes. Key words: Populus tremuloides, Hypoxylon mammatum, phytotoxin.