Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.

Fungi play important roles in forest ecosystems and understanding fungal diversity is crucial to address essential questions about species conservation and ecosystems management. Changes in fungal diversity can have severe impacts on ecosystem functionality. Unfortunately, little is known about fungal diversity in northern temperate and boreal forests, and we have yet to understand how abiotic variables shape fungal richness and composition. Our objectives were to make an overview of the fungal richness and the community composition in the region and identify their major abiotic drivers. We sampled 262 stands across the northern temperate and boreal Quebec forest located in the region of Abitibi-Témiscamingue, Mauricie, and Haute-Mauricie. At each site, we characterized fungal composition using Illumina sequencing, as well as several potential abiotic drivers (e.g., humus thickness, soil pH, vegetation cover, etc.). We tested effects of abiotic drivers on species richness using generalized linear models, while difference in fungal composition between stands was analyzed with permutational multivariate analysis of variance and beta-diversity partitioning analyses. Fungi from the order Agaricales, Helotiales, and Russulales were the most frequent and sites from the north of Abitibi-Témiscamingue showed the highest OTUs (Operational Taxonomic Unit) richness. Stand age and moss cover were the best predictors of fungal richness. On the other hand, the strongest drivers of fungal community structure were soil pH, average cumulative precipitation, and stand age, although much of community variance was left unexplained in our models. Overall, our regional metacommunity was characterized by high turnover rate, even when rare OTUs were removed. This may indicate strong environmental filtering by several unmeasured abiotic filters, or stronger than expected dispersal limitations in soil fungal communities. Our results show how difficult it can be to predict fungal community assembly even with high replication and efforts to include several biologically relevant explanatory variables.

Lobster mushroom is a wild edible mushroom with potential commercial value. It is the product resulting of the infection, most commonly of Russula brevipes, by Hypomyces lactifluorum. This study undertook quantitative polymerase chain reaction analysis of tissues sampled at different infection stages to investigate R. brevipes – H. lactifluorum interaction. We followed the colonization of R. brevipes sporocarps by H. lactifluorum that leads to the edible lobster mushrooms. In parallel, metabolomics analysis was performed to detect differences in metabolite profile among non-infected R. brevipes sporocarp and lobster mushroom. The results show that H. lactifluorum DNA is not restricted to the margin but is distributed relatively evenly across the sporocarp of the lobster mushroom. Russula brevipes DNA was also present throughout the sporocarp but was less abundant at the margins and increased inwards. Russula brevipes DNA also declined as the infection progressed. Metabolomics analysis revealed that the flesh of lobster mushroom, which remains identical in appearance to the flesh of the host, undergoes transformation that alters its metabolite profile, most notably of lipids and terpene compounds. These results define a parasitic relationship between the two species that entails a decline of R. brevipes DNA and a modification of its metabolite profile.

Boreal forests provide important ecosystem services, most notably being the mitigation of increasing atmospheric CO2 emissions. Microbial biodiversity, particularly the local diversity of fungi, has been shown to promote multiple functions of the boreal forests of Northeastern China. However, this microbial biodiversity-multifunctionality relationship has yet to be explored in Northeastern Canada, where historical environment have shaped a different regional pool of microbial diversity. This study focuses on the relationship between the soil microbiome and ecosystem multifunctionality, as well as the influence of pH and redox potential (Eh) on the regulation of such relationship. Structural equation modelling (SEM) was used to explore the different causal relationships existing in the studied ecosystems. In a managed part of the Canadian boreal forest, 156 forest polygons were sampled to (1) estimate the α- and β-diversity of fungal and bacterial communities and (2) measure 12 ecosystem functions mainly related to soil nutrient storage and cycling. Both bacteria and fungi influenced ecosystem multifunctionality, but on their own respective functions. Bacterial β-diversity was the most important factor increasing primary productivity and soil microbial biomass, while reducing soil emitted atmospheric CO2. Environmental characteristics, particularly low levels of organic matter in soil, were shown to have the strongest positive impact on boreal ecosystem multifunctionality. Overall, our results were consistent with those obtained in Northeastern China; however, some differences need to be further explored especially considering the history of forest management in Northeastern Canada.