Jean A. Bérubé

A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete

An aquatic hyphomycete, Dwayaangam sp., was isolated from superficially sterilized black spruce (Picea mariana) needles submerged in aerated water in a small glass chamber (microcosm). The internal transcribed spacer (ITS) sequence of this fungus and of a commonly encountered foliar endophyte isolated from P. mariana showed a high degree of similarity. When sporulation was induced in the microcosm, both the aquatic hyphomycete and the endophyte isolate produced similar aquatic conidia after 30 days, which is longer than previously documented in similar studies. Without the use of molecular tools, the link between the aquatic and endophytic phases of the fungus would have gone unnoticed. This is the first time that a fungal endophyte of conifer needles has been shown to have an aquatic phase. Its presence both as a foliar endophyte and a sporulating aquatic fungus suggests an alternating life cycle between the two environments.

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

ABSTRACT The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.

Black spruce (Picea mariana) foliage hosts numerous and potentially endemic fungal endophytes

Fungal foliar endophytes (hereinafter endophytes) were isolated on malt agar from black spruce (Picea mariana (Mill.) BSP) needles from the Quebec boreal forest during 2 successive years to study their distribution along a latitudinal transect. Twenty sites were sampled in 2002, 8 of which were sampled again in 2003, in an area located between 47°N and 54°N in the province of Quebec. The endophytes were named whenever possible, using homologies of ITS rDNA sequences from GenBank. In 2002, 232 morphotypes were classified in 40 operational taxonomic units (OTUs), and in 2003, 93 morphotypes were classified in 16 OTUs, for a total of 44 OTUs. Isolation frequencies of the most common endophyte species were 28% for Lophodermium piceae (Fuchel) Hohn., 17% for Darkera parca Whitney, Reid & Piroz, and 9% for Dwayaangam colodena Sokolski & Berube. Of the 44 OTUs identified, 18 were unique to P. mariana and found on one site only, possibly constituting rare endemic species. There was some evidence that needles colo...

Morchella tomentosa: a unique belowground structure and a new clade of morels

Mechanisms involved in post-fire morel fructification remain unclear. A new undescribed belowground vegetative structure of Morchella tomentosa in a burned boreal forest was investigated north of Fairbanks, Alaska. The name “radiscisclerotium” is proposed to define this peculiar and elaborate below-ground vegetative structure of M. tomentosa. Bayesian and maximum parsimony analyses based on ITS rRNA regions and nLSU gene strongly supported a new clade composed of M. tomentosa within the genus Morchella.

Lophodermium macci sp. nov., a new species on senesced foliage of five-needle pines

The new species Lophodermium macci is described. It is similar in its morphology, habitat, geographic range and ecology to L. pini-excelsae, L. staleyi and L. nitens and often is misidentified as L. pinastri on Pinus strobus in herbaria. A modified technique was used to extract DNA from minute ascomata on herbarium specimens, and new primers were made to amplify the damaged DNA from these specimens. It provides added evidence to separate L. macci from L. pini-excelsae, its closest morphological taxon.

Alien fungal species on asymptomatic live woody plant material imported into Canada

Abstract Undescribed exotic and scientifically unknown fungal species with the potential to be pathogens are often difficult to detect on imported live plant material due to their inconspicuous nature and thus represent an important risk to Canadian forests. We have developed an early warning method based on a random sampling of asymptomatic woody live plant material imported into Canada to detect such potential alien fungal species. We received 150 asymptomatic sample lots collected by Canadian Food Inspection Agency (CFIA) inspectors. Samples were analysed by cloning and sequencing the PCR-amplified fungal nuclear ribosomal internal transcribed spacer (ITS) DNA from plant tissues to reveal fungal diversity. Out of 1845 fungal clones obtained, 267 fungal operational taxonomic units (OTUs) were identified. Using phylogenetic profiling methods, two fungal OTUs were categorized as having potential for a moderate impact on Canadian forests, 37 OTUs had a low impact and 18 OTUs could not be assessed given their low genetic similarity with other ITS sequences in GenBank. In all cases, the potential risk associated with these 57 fungal OTUs is based on (i) the fact that they can be considered unknown species to science; and (ii) they belong to orders, classes, genera and families in which pathogenic species are common. Fungal introductions with potential for a moderate impact on Canadian forests were observed at a very low frequency (0.2%) in the sampling units (clones). Only 1.3% of the CFIA samples had an OTU with a potential moderate impact on Canadian forests and 74% of the samples were free of fungal OTUs that could have any potential impact. This early warning method sheds light on the suite of exotic fungi that may enter Canada via plant material. Additionally, this method provides the tools to assess the potential risk that these fungi may post to Canadian trees and determines the magnitude of asymptomatic material that harbours fungal pathogens.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences

ABSTRACT Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence–based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

Incidence of Heterobasidion irregulare aerial basidiospores at different locations in southern Quebec

Abstract Heterobasidion irregulare (Underw.) Garbel. & Otrosina is an important fungal parasitic basidiomycete of coniferous trees throughout temperate regions of the world. In Canada, the disease was first reported on red pine in Ontario in 1955, then in Quebec in 1989. As infection probability is proportional to the density of its aerial basidiospores infecting freshly cut pine stumps, the purpose of this study was to quantify the H. irregulare aerial basidiospore deposition along a southern Quebec transect. Spore counts from automated rotary arm collectors were determined by using a ribosomal ITS TaqMan real-time PCR detection assay. Total count was performed on rods at each location, with the highest score of 706.3 spores in Harrington, then 459.6 spores in Cowansville, rapidly decreasing to 52.3 spores in Compton and reaching lows in St-Aubert and Rimouski, with an eastward direction on the transect. Clearly, plantations in southwestern Quebec are at a higher risk of infection than anywhere else in the province. With few exceptions, weekly spore depositions at all sites other than Harrington were greatly below 0.2 per m−2 h−1. Despite this very low spore deposition rate observed in one season, the large number of trees thinned annually warrants that stump treatment with Rotstop® C is still the best strategy, especially in valuable plantations, to prevent against infection by Heterobasidion.

Detection of Diplodia corticola spores in Ontario and Québec based on High Throughput Sequencing (HTS) methods

Abstract A total of 252 aerial spore samples from the provinces of Quebec and Ontario were obtained during an extensive research programme focusing on aerobiology of fungal spores. Samples were collected on 95 sticky rods from rotary arm spore collectors and 157 filters from passive rain collectors. DNA from samples was PCR-amplified for fungal ribosomal ITS and sequenced using High Throughput Sequencing (HTS) methods. After bioinformatics analysis of DNA sequences, the presence of Diplodia corticola, an emerging tree pathogen in North America, was observed. In total, 313 DNA sequence reads from aerial spores of D. corticola were found in the Illumina data set, and 199 DNA sequence reads were obtained from the Ion Torrent data set. DNA of D. corticola was found in 16 of the 32 sampled sites, always less than 10 reads per site, with the exception of three sites – Quebec City, Aylmer and Ottawa – where 287, 125 and 73 DNA reads, respectively, were detected. This is not a first report of the presence of D. corticola causing a tree disease in Canada as symptomatic trees have not been identified. Typically, like many other species of the Botryosphaeriaceae, this fungal pathogen is believed to be an opportunistic plant endophyte capable of living asymptomatically for several years before showing up as a pathogen when conducive conditions arise. Its presence as singletons in nearly half the sampled sites in Quebec and Ontario may be the result of long-distance spore dispersal originating from known infected sites in Massachusetts, Maine and other unknown sites in the north-eastern USA. However, its much higher read counts in three sites may indicate the possibility of a few trees being asymptomatically infected and spreading conidia locally.