Unravelling the diversity of grapevine microbiome

Abstract

Vitis vinifera is one of the most widely cultivated fruit crops with a great economic impact on the global industry. As a plant, it is naturally colonised by a wide variety of both prokaryotic and eukaryotic microorganisms that interact with grapevine, having either beneficial or phytopathogenic effects, who play a major role in fruit yield, grape quality and, ultimately, in the evolution of grape fermentation and wine production. Therefore, the objective of this study was to extensively characterize the natural microbiome of grapevine. Considering that the majority of microorganisms are uncultivable, we have deeply studied the microflora of grapevine leaves using massive parallel rDNA sequencing, along its vegetative cycle. Among eukaryotic population the most abundant microorganisms belonged to the early diverging fungi lineages and Ascomycota phylum, whereas the Basidiomycota were the least abundant. Regarding prokaryotes, a high diversity of Proteobacteria, Firmicutes and Actinobacteria was unveiled. Indeed, the microbial communities present in the vineyard during its vegetative cycle were shown to be highly structured and dynamic. In all cases, the major abundant microorganisms were the yeast-like fungus Aureobasidium and the prokaryotic Enterobacteriaceae. Herein, we report the first complete microbiome landscape of the vineyard, through a metagenomic approach, and highlight the analysis of the microbial interactions within the vineyard and its importance for the equilibrium of the microecosystem of grapevines.

Figure 1. Rarefaction curves at a genetic distance of 3% for each sample (T1–T10).

D2 (a) and ITS2 (b) sequences both from the analysis of 26S rRNA and ITS regions of eukaryotic population present in the sample and V6 sequences (c) from the analysis of 16S rRNA of prokaryotic diversity.

Figure 2. Biodiversity dynamics associated with D2, ITS2 and V6 during the vegetative cycle of grapevine.

The means of Chao1 index ± SEM are represented in the graph. Significance was assessed with one-way analysis of variance (ANOVA) and p<0.05 was set as statistic significant level. No significant differences were obtained for D2, ITS2 and V6 regions among May, June and July.

Figure 3. Eukaryotic (a) and prokaryotic (b) microbial community distribution from T1 to T10.

Relative abundance of the 10 most abundant eukaryotic and prokaryotic microorganisms through the genus and family analysis, respectively.

Figure 4. PCA biplot diagram and loading plot of microbial community across sampling time (T1–T10), based on sequence abundance of eukaryotic genus and bacterial family.

(a) The PCA diagram is shown and the percentages of data variability explanation are shown in the upper of X and Y axis and more than 85% of the variability in the data is accounted. (b) Loading plot of principal component 1 showing variables that explain variability across eukaryotic genus and prokaryotic family during the vegetative cycle of grapevine (T1–T10). The significant differences were observed in Metastats for eukaryotic genus or prokaryotic families and are represented with asterisk (*) and microbial community that are identified with ## are considered false discovery rate.

Figure 5. Variation on the abundance of the significant eukaryotic genus and prokaryotic family (p<0.05) as affected by chemical treatment (a) and grapevine vegetative cycle (b).

The variation on population during the grapevine vegetative cycle could be interpreted through the variation of the square's color where the red square indicates a higher number of reads and a blue square denotes a reduce number of reads of a specific microorganism. Only the significant population is shown (p<0.05).