Orchard Conditions and Fruiting Body Characteristics Drive the Microbiome of the Black Truffle Tuber aestivum

Abstract

Truffle fungi are well known for their enticing aromas partially emitted by microbes colonizing truffle fruiting bodies. The identity and diversity of these microbes remain poorly investigated, because few studies have determined truffle-associated bacterial communities while considering only a small number of fruiting bodies. Hence, the factors driving the assembly of truffle microbiomes are yet to be elucidated. Here we investigated the bacterial community structure of more than 50 fruiting bodies of the black truffle Tuber aestivum in one French and one Swiss orchard using 16S rRNA gene amplicon high-throughput sequencing. Bacterial communities from truffles collected in both orchards shared their main dominant taxa: while 60% of fruiting bodies were dominated by α-Proteobacteria, in some cases the β-Proteobacteria or the Sphingobacteriia classes were the most abundant, suggesting that specific factors (i.e., truffle maturation and soil properties) shape differently truffle-associated microbiomes. We further attempted to assess the influence in truffle microbiome variation of factors related to collection season, truffle mating type, degree of maturation, and location within the truffle orchards. These factors had differential effects between the two truffle orchards, with season being the strongest predictor of community variation in the French orchard, and spatial location in the Swiss one. Surprisingly, genotype and fruiting body maturation did not have a significant effect on microbial community composition. In summary, our results show, regardless of the geographical location considered, the existence of heterogeneous bacterial communities within T. aestivum fruiting bodies that are dominated by three bacterial classes. They also indicate that factors shaping microbial communities within truffle fruiting bodies differ across local conditions.

Keywords: Tuber aestivum; amplicon sequencing; bacterial communities; mating type; microbiome; multilocus genotype.

FIGURE 1

Location of truffle samples within the French and Swiss orchards. Location of truffles, their maturity, mating type, collection year, and identical multilocus genotypes (MLGs, connected by lines) are shown in the French and Swiss orchards, along the position of truffle-mycorrhized trees (small gray dots) and fruit trees surrounding the orchard (crosses). Black arrows in the lower left corner of each orchard represent a distance of 5 m.

FIGURE 2

Bacterial community structure within T. aestivum fruiting bodies collected from two orchards. Each heat tree represents bacterial community structure as a taxonomic hierarchy up to genus level. The gray tree serves as a key for the smaller unlabeled trees, node labels highlight the most abundant taxa detected at both orchards. The smaller colored trees show community structure for each orchard, assessed with different amplicon sequencing technologies [MiSeq for France (FR), 454 for Switzerland (SW)]. Node and edge sizes are proportional to the number of OTUs within each taxon, whereas color represents taxon abundances (square root of read numbers).

FIGURE 3

The microbiome of T. aestivum. Distribution of major bacterial classes (top panel) and genera (intermediate and bottom panels) in the French and Swiss truffle fruiting bodies analyzed here. Each column represents a single sample. For each sample, three pile-up plots are given: the relative distribution of reads among the major bacterial classes (top panel) and of the different genera forms the Bacteroidetes (intermediate panel) and Proteobacteria phyla (bottom panel). Samples were ordered according to the year and month of collection, and maturity degree (from low to high).

FIGURE 4

Microbial community similarities among truffles within orchards. (A, FR; B, SW) Non-metric multidimensional scaling (NMDS) ordinations based on Bray–Curtis dissimilarities of the OTU composition of microbiomes in truffle samples. The closer the samples, the more similar their microbiomes are. Different symbols denote mating type, symbol sizes represent maturity of fruiting bodies, and symbol colors represent collection year. Points linked with lines are fruiting bodies belonging to the same MLG. (C, FR; D, SW) Correlograms showing correlation of microbiome similarity among samples (y-axis, Mantel’s R) with spatial distance (x-axis). Solid and empty points denote significant (P < 0.05) and non-significant correlations for each distance class based on Bonferroni adjustment for multiple testing, indicating that space affects the truffles microbiomes in the Swiss but not the French site.