Yeast communities of a North American hybrid wine grape differ between organic and conventional vineyards.

AIMS: To compare the species diversity and composition of indigenous yeast communities of hybrid grapes from conventionally and organically cultivated vineyards of an emerging cool-climate wine producing region. METHODS AND RESULTS: Illumina MiSeq sequences from L'Acadie blanc grape musts were processed and filtered to characterize indigenous yeast communities in organic and conventional vineyards of the Annapolis Valley wine region in Nova Scotia, Canada. While cultivation practice was not associated with yeast diversity or species richness, there was a strong effect on yeast community composition, with conventional vineyards characterized by higher proportions of Sporidiobolales and Filobasidium magnum, and organic vineyards supporting Filobasidium species other than F. magnum and higher proportions of Symmetrospora. There was also variation in yeast community composition among individual vineyards, and from year to year. CONCLUSIONS: This is the first comprehensive assessment of yeasts associated with hybrid grapes grown using different cultivation practices in a North American cool climate wine region. Communities were dominated by basidiomycete yeasts and species composition of these yeasts differed significantly between vineyards employing organic and conventional cultivation practices. The role of basidiomycete yeasts in winemaking is not well understood, but some species may influence wine characteristics.

Yeast communities before and after spontaneous fermentation of wine grapes: a case study from Nova Scotia.

Wine fermentations are generally completed by the domestic yeast Saccharomyces cerevisiae, but many indigenous vineyard yeasts also influence wine flavour and aroma. Despite the flourishing wine industry in Nova Scotia, there has yet to be any systematic evaluation of these yeasts in Atlantic Canada. The yeast communities of pressed L'Acadie blanc grapes sampled from an organic vineyard in the Annapolis Valley in 2018 and 2019 were characterized before and after spontaneous fermentation by both Illumina and PacBio sequencing, to address and compare potential platform biases. Chemical and sensory evaluations were also conducted. Basidiomycete yeasts, including Vishniacozyma carnescens, Filobasidium globisporum, and Curvibasidium cygneicollum, dominated pre-fermentation diversity. Species of Saccharomyces made up ∼0.04% of sequences prior to fermentation, but 85%-100% after fermentation, with some replicates dominated by S. cerevisiae and some by S. uvarum. PacBio sequencing detected high proportions of Hanseniaspora uvarum, while Illumina sequencing did not. A better understanding of Nova Scotia vineyard yeast communities will allow local wine makers to make better use of non-traditional yeasts and spontaneous fermentations to produce high-quality wines unique to the region.

Fungal Succession During the Decomposition of Ectomycorrhizal Fine Roots.

Ectomycorrhizal (ECM) fine roots account for a substantial proportion of forest production and their decomposition releases large amounts of nutrients to the soil ecosystem. However, little is known about the fungi involved in ECM decomposition, including assemblages of fungal saprotrophs, endophytes, and the ECM fungi themselves. To follow fungal succession during the degradation of senescing fine roots, understory seedlings of Abies balsamea and Picea rubens at two sites in the Acadian forest of Nova Scotia were either severed at the root collar or left as controls. Root systems were collected sequentially over two growing seasons and assessed for fine root loss and ECM mantle integrity. ECM were identified by ITS-PCR and grouped into broad morphological categories. Fungal communities colonizing the senescing fine roots were also monitored by systematically constructing clone libraries over the course of the experiment. ECM with cottony, weakly pigmented mantles (e.g., Cortinarius) degraded within the first year. Those with cottony, but intensely pigmented mantles (Piloderma), and smooth mantles with weak pigmentation (Russulaceae) degraded more slowly. Smooth, melanized ECM (Cenococcum and Tomentella) generally maintained integrity over the course of the experiment. Rates of fine root loss and changes in ECM mantle integrity were positively correlated with soil temperature. ECM DNA was detected throughout the experiment, and was not replaced by that of saprotrophic species during the two seasons sampled. However, fungal root endophytes (e.g., Helotiaceae) initially increased in abundance and then decreased as mantles degraded, suggesting a possible role in ECM decomposition.

Fungal endophytes of wild and hybrid Vitis leaves and their potential for vineyard biocontrol.

Plants are colonized by diverse assemblages of fungal endophytes that have potential as biocontrol agents for a variety of crops, including grapevine. Although the diversity of symbionts can be very high in wild plants, the fungal endophytes of wild Vitis plants have not yet been investigated. We surveyed the fungal endophytes of 6 wild populations of Vitis riparia, as well as a cold-tolerant, hybrid grapevine in 5 vineyards (1 certified organic), using 454 pyrosequencing. We detected between 43 and 235 operational taxonomic units per sample, with the highest richness and diversity in the wild, the lowest in conventional vineyards, and intermediate levels in the organic vineyard. Wild plants supported a range of taxa not seen in the conventional vineyards, and vineyards were dominated by relatively few taxa. We also isolated fungi from the wild plants and tested them for their ability to inhibit pathogens of grapevine. Several wild isolates (e.g., Ramularia spp.) were strongly inhibitory to grapevine pathogens. We show that wild Vitis supports a distinct and highly diverse community of fungal endophytes and may represent a rich repository of potential vineyard biocontrol agents.

Acid protease production in fungal root endophytes.

Fungal endophytes are ubiquitous in healthy root tissue, but little is known about their ecosystem functions, including their ability to utilize organic nutrient sources such as proteins. Root-associated fungi may secrete proteases to access the carbon and mineral nutrients within proteins in the soil or in the cells of their plant host. We compared the protein utilization patterns of multiple isolates of the root endophytes Phialocephala fortinii s.l., Meliniomyces variabilis and Umbelopsis isabellina with those of two ectomycorrhizal (ECM) fungi, Hebeloma incarnatulum and Laccaria bicolor, and the wood-decay fungus Irpex lacteus at pH values of 2-9 on liquid BSA media. We also assessed protease activity using a fluorescently labeled casein assay and gelatin zymography and characterized proteases using specific protease inhibitors. I. lacteus and U. isabellina utilized protein efficiently, while the ECM fungi exhibited poor protein utilization. ECM fungi secreted metallo-proteases and had pH optima above 4, while other fungi produced aspartic proteases with lower pH optima. The ascomycetous root endophytes M. variabilis and P. fortinii exhibited intermediate levels of protein utilization and M. variabilis exhibited a very low pH optimum. Comparing proteolytic profiles between fungal root endophytes and fungi with well defined ecological roles provides insight into the ecology of these cryptic root associates.

The effects of fungal root endophytes on plant growth: a meta-analysis.

Fungal root endophytes are plant associates that colonize root tissue internally without causing any obvious harm to their host. Although ubiquitous, this relationship is not well understood. Our objectives were to determine the effects of fungal root endophyte inoculation on plant biomass and nitrogen concentration by conducting an extensive meta-analysis. We also explored the effects of experimental conditions on the host-endophyte relationship. We performed analyses weighted with non-parametric variance on plant response to root endophytes from the Ascomycetes (excluding the Clavacipitaceae), including categorical analyses of 21 experimental factors, ranging from the identity of the host and the endophyte, to the composition of the growing medium. The response of total biomass to endophyte inoculation was 18% lower than non-inoculated controls, while individually, root biomass, shoot biomass, and nitrogen concentration responses to endophyte inoculation were neutral. The identities of both the host and the endophyte had an influence, as did the original source of the endophyte (whether or not the isolate used originated from the same host species). Experimental conditions also influenced the plant-endophyte relationship, with the most important being the availability and sources of carbon and organic nitrogen, particularly peat moss. Although our analysis demonstrates that overall plant biomass and nitrogen concentration responses to ascomycetous root endophyte inoculation is neutral to negative, these results are somewhat confounded by among-study differences in experimental conditions, which undoubtedly contribute to the high levels of variability in plant response seen in the literature.

Host associations between fungal root endophytes and boreal trees.

Fungal root endophytes colonize root tissue concomitantly with mycorrhizal fungi, but their identities and host preferences are largely unknown. We cultured fungal endophytes from surface-sterilized Cenococcum geophilum ectomycorrhizae of Betula papyrifera, Abies balsamea, and Picea glauca from two boreal sites in eastern Canada. Isolates were initially grouped on the basis of cultural morphology and then identified by internal transcribed spacer ribosomal DNA sequencing or by PCR restriction fragment length polymorphism. Phylogenetic analysis of the sequence data revealed 31 distinct phylotypes among the isolates, comprising mainly members of the ascomycete families Helotiaceae, Dermateaceae, Myxotrichaceae, and Hyaloscyphaceae, although other fungi were also isolated. Multivariate analyses indicate a clear separation among the endophyte communities colonizing each host tree species. Some phylotypes were evenly distributed across the roots of all three host species, some were found preferentially on particular hosts, and others were isolated from single hosts only. The results indicate that fungal root endophytes of boreal trees are not randomly distributed, but instead form relatively distinct assemblages on different host tree species.

Species composition of saproxylic fungal communities on decaying logs in the boreal forest.

Coarse woody debris supports large numbers of saproxylic fungal species. However, most of the current knowledge comes from Scandinavia and studies relating the effect of stand or log characteristics on the diversity and composition of decomposer fungi have not been conducted in Northeastern Canada. Logs from five tree species were sampled along a decomposition gradient in nine stands representing three successional stages of the boreal mixed forest of Northwestern Quebec, Canada. Using a molecular fingerprinting technique, we assessed fungal community Shannon-Weaver diversity index, richness, and composition. We used linear mixed models and multivariate analyses to link changes in fungal communities to log and stand characteristics. We found a total of 33 operational taxonomic units (OTUs) including an indicator species for balsam fir (similar to Athelia sp.) and one found only in aspen stands (similar to Calocera cornea). Spruce logs supported the highest fungal Shannon-Weaver diversity index and OTU number. Our results support the hypothesis that log species influences fungal richness and diversity. However, log decay class does not. Stand composition, volume of coarse woody debris, and log chemical composition were all involved in structuring fungal communities. Maintaining the diversity of wood-decomposing communities therefore requires the presence of dead wood from diverse log species.

Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

Availability of ectomycorrhizal fungi to black spruce above the present treeline in Eastern Labrador.

Ectomycorrhizal fungi (ECMF) are an important biotic factor in the survival of conifer seedlings under stressful conditions and therefore have the potential to facilitate conifer establishment into alpine and tundra habitats. In order to assess patterns of ectomycorrhizal availability and community structure above treeline, we conducted soil bioassays in which Picea mariana (black spruce) seedlings were grown in field-collected soils under controlled conditions. Soils were collected from distinct alpine habitats, each dominated by a different ectomycorrhizal host shrub: Betula glandulosa, Arctostaphylos alpina or Salix herbacaea. Within each habitat, half of the soils collected contained roots of ectomycorrhizal shrubs (host (+)) and the other half were free of host plants (host(-)). Forest and glacial moraine soils were also included for comparison. Fungi forming ectomycorrhizae during the bioassays were identified by DNA sequencing. Our results indicate that ECMF capable of colonizing black spruce are widespread above the current tree line in Eastern Labrador and that the level of available inoculum has a significant influence on the growth of seedlings under controlled conditions. Many of the host(-) soils possessed appreciable levels of ectomycorrhizal inoculum, likely in the form of spore banks. Inoculum levels in these soils may be influenced by spore production from neighboring soils where ectomycorrhizal shrubs are present. Under predicted temperature increases, ectomycorrhizal inoculum in soils with host shrubs as well as in nearby soils without host shrubs have the potential to facilitate conifer establishment above the present tree line.