Gaston Laflamme

Genetic differentiation within the European race of Gremmeniella abietina

Twelve random amplified polymorphic DNA markers were variable within the European race of Gremmeniella abietina var. abietina (GAA-EU) in Europe. Three distinct DNA amplification profiles (amplitypes) appeared to be correlated with ecotypic origin. The northern amplitype was present exclusively in northern Europe in plantations and natural stands of Pinus sylvestris and in plantations of P. contorta and apparently was adapted to the presence of deep, long-lasting snow cover in the winter. An alpine amplitype was found exclusively in the Alps at altitudes above 2000 m on P. cembra, P. mugo, P. sylvestris and Larix lyalli and also appears to represent an ecotype adapted to conditions of deep snow cover. The third amplitype, the European amplitype, was present throughout Europe and ranged from the Scandinavian countries and extended south to the Apennine mountains of northern Italy. Most of the GAA-EU samples tested from North America had RAPD profiles identical to those of the European amplitype indicating that the origin of this introduced pathogen could be central Europe. However, some of the samples from North America had RAPD profiles that did not match any found in Europe. The internal transcribed spacers of the ribosomal DNA repeat subunit were amplified and digested with restriction enzymes Hae III and Msp I. These restriction sites were polymorphic between the North American (GAA-NA) race and the EU race of G. abietina but were homogeneous among the three amplitypes described above. The rDNA restriction and RAPD profiles also indicated that GAA-NA was absent from the samples from Europe and that symptoms resembling those caused by GAA-NA were attributable to the northern and alpine amplitypes.

PCR detection of Gremmeniella abietina, the causal agent of Scleroderris canker of pine

Scleroderris canker of conifer is caused by Gremmeniella abietina var. abietina , which comprises several taxa, including races, varieties, and biotypes. The European race of G. abietina var. abietina was introduced into North America early in the century, most likely on asymptomatic infected pine seedlings, and is currently widespread in eastern North America. In order to detect latent infections and differentiate the North American and European races of the fungus, we developed oligonucleotide primers to amplify by PCR portions of the ITS of the ribosomal DNA of G. abietina var. abietina. The 417 bp amplified DNA fragment comprises two Msp 1 restriction sites in the NA race but only one in the EU race. DNA extractions directly from infected asymptomatic needles, or from single fruiting bodies, followed by PCR amplification and Msp 1 digestion allowed the detection and race identification of both races of G. abietina var. abietina from seedlings and branches of Pinus resinosa and P. banksiana. A nested PCR assay was sensitive enough to detect the equivalent of a single infected seedling in a bulk sample of 1000 healthy seedlings. Validation tests were conducted by comparing PCR and isolation assays with 104 fascicles. All samples for which the fungus was isolated yielded a positive PCR assay and there was no false negative, i.e. samples for which the fungus was isolated but not detected by PCR. Among the samples from which the fungus was not isolated, most yielded a negative PCR assay (71%), but a proportion (29%) yielded positive PCR assays. In several of those cases, aggressive contaminants had apparently overgrown the pathogen. The method described here can lead to the detection and race identification of the NA and EU races of G. abietina var. abietina directly from infected tissues without the need to culture the fungus and should find applications in nursery inspection and quarantine.

Molecular evidence of distinct introductions of the European race of Gremmeniella abietina into North America

ABSTRACT The presence of the European (EU) race of Gremmeniella abietina var. abietina, the causal agent of Scleroderris canker of conifers, was first reported in North America in 1975 in the northeastern United States and subsequently in southern Quebec and Newfoundland during the late 1970s, where it quickly became established. We analyzed DNA profiles in samples from a historic collection of G. abietina var. abietina that included some of the first isolates of the EU race reported in the United States to test hypotheses concerning the G. abietina var. abietina epidemic in North America. Genetic diversity was partitioned by an analysis of molecular variance with haplotype frequencies and distances. Genetic differentiation was high between populations in continental North America and Newfoundland (between region differentiation, Phi(ct) = 0.665, P < 0.001). This result was not consistent with the hypothesis of a single introduction of the pathogen into the northeastern United States followed by secondary spread into northeastern Canada. In contrast, small levels of genetic differentiation were observed among continental North American populations (Phi(ct) = 0.047, P = 0.079), suggesting gene flow among these populations. A single haplotype of G. abietina var. abietina dominated the continental populations (80% of the isolates) but was absent from Newfoundland and Europe. Five haplotypes were found in the New-foundland population, all of which were either absent or very rare on the continent. Populations from continental North America clustered together and were distinct from a second cluster composed of European and Newfoundland populations. A phylogenetic analysis of the haplotypes indicated that some of the rare haplotypes may have derived from somatic mutations, whereas others probably occurred as the result of new introductions. The results are consistent with a scenario of distinct primary introductions of this pathogen into Newfoundland and continental eastern North America followed by secondary asexual propagation.

Formation of Ligno-Suberized Tissues in Jack Pine Resistant to the European Race of Gremmeniella abietina.

ABSTRACT A histological study was conducted to provide insights into the defense mechanisms of Pinus banksiana resistant to the European (EU) race of Gremmeniella abietina in naturally infected sites. At the time of sampling, the only apparent symptom was a blight induced at the tip of the shoots. The identity of G. abietina during microscopic examinations was confirmed by an immunogold labeling method. Once the fungus had succeeded in penetrating the bracts through stomata, it invaded the stem cortex and the phloem cells and attained the vascular cambium. The progression of the pathogen to the pith was possible principally through intense colonization of needle traces but also by the invasion of the rays. Ligno-suberized tissues confining the pathogen within the necrotic area were revealed by histochemical tests. Well-defined boundaries were initiated at the base of healthy needles and at the vascular cambium level. They regularly formed one continuous suberized barrier completely crossing the shoot from one needle to the other. A nonlamellar form of suberin was observed in transmission electron microscopy. Restoration of cambial activities and tissue regeneration following necrophylactic periderm formation were suggested as essential factors in the defense system of P. banksiana against the EU race of G. abietina. To our knowledge, this is the first demonstration of an anatomical defense mechanism of a conifer against Scleroderris canker.

DNA polymorphism and molecular diagnosis in Inonotus spp

Specific polymerase chain reaction (PCR) primers were developed for the internal transcribed spacer (ITS) region of the rDNA gene of Inonotus tomentosus, the causal agent of tomentosus root rot of conifers. The primers were designed to specifically amplify DNA from I. tomentosus and allow its differentiation from Inonotus leporinus and from Phellinus pini s.l., which are morphologically very similar to I. tomentosus in culture. The PCR amplification was carried out successfully from DNA extracted from fruiting bodies and cultures and can potentially be used to detect the pathogen from environmental samples for survey and management purposes. The PCR assay was validated with 42 samples from seven coniferous hosts originating from eight provinces or states across the North American continent. No cross reaction was observed with DNA of several other species of the same genus, with Phellinus pini or with white spruce (Picea glauca), a conifer host of I. tomentosus. Phylogenetic analysis of the ITS region for six species of Inonotus suggests that these resulted from the adaptation of a generalist ancestor to different ecological niches. It also appears that divergent evolution of an ancestor occupying different ecological niches has driven the speciation process, which subsequently conferred specificity to either coniferous or deciduous trees.

Priority of Lophophacidium over Canavirgella: taxonomic status of Lophophacidium dooksii and Canavirgella banfieldii, causal agents of a white pine needle disease

In 2009 unusual white pine needle discoloration was observed in eastern Canada and northeastern USA. While the symptoms were similar in most pine stands, the disease was diagnosed as Canavirgella banfieldii in several locations and Dooks needle blight caused by Lophophacidium dooksii in others. Because of the similarities in symptom development and morphological characters of the causal agents, it was suspected that C. banfieldii and L. dooksii are either the same or closely related species. To test this hypothesis we examined several collections representing C. banfieldii and L. dooksii, including the two type specimens. Phylogenetic analyses of nuc internal transcribed spacer rDNA sequences confirm the synonymy of C. banfieldii with the earlier described L. dooksii and provide the first evidence of the close evolutionary relationship of L. dooksii to other pine pathogens.

Patterns of colonization and spread in the fungal spruce pathogen Onnia tomentosa.

The basidiomycetous fungus Onnia tomentosa is one of the most widespread root rot pathogens in North America. Although the disease is more severe on spruce and pine trees, this pathogen can infect several coniferous species. To study the population structure of O. tomentosa, we harvested 180 basidiocarps in a 45‐year‐old white spruce plantation in western Quebec in autumn 1997 and extracted DNA directly from individual basidiocarps. Using a combination of spatial coordinates and molecular data based on the analysis of two mitochondrial and three nuclear loci, we measured the average genet size and molecular diversity and assessed the relative contribution of basidiospores and vegetative growth to the stand colonization. Most of the sampled basidiocarps that clustered spatially belonged to the same genet. A total of 37 discrete multilocus genets of an average size of 3.42 m were obtained. The genet size distribution was skewed towards smaller genets (<3 m) that displayed higher diversity than the larger genets (>3 m). The nuclear loci were in Hardy–Weinberg equilibrium in the larger genets, but not in the smaller genets, which displayed a deficiency of heterozygotes. This suggests a Wahlund effect, whereby different colonization events resulted in expected heterozygosity higher than observed heterozygosity. Using an estimate of the growth rate of the fungus, only a few of the largest genets were approximately the age of the plantation. These observations are consistent with the colonization by basidiospores subsequent to site preparation and tree planting followed by secondary colonization events and vegetative spread.