Microbial dysbiosis in roots and rhizosphere of grapevines experiencing decline is associated with active metabolic functions

When grapevine decline, characterized by a premature decrease in vigor and yield and sometimes plant death, cannot be explained by pathological or physiological diseases, one may inquire whether the microbiological status of the soil is responsible. Previous studies have shown that the composition and structure of bacterial and fungal microbial communities in inter-row soil are affected in areas displaying vine decline, compared to areas with non-declining vines within the same plot. A more comprehensive analysis was conducted in one such plot. Although soil chemical parameters could not directly explain these differences, the declining vines presented lower vigor, yield, berry quality, and petiole mineral content than those in non-declining vines. The bacterial and fungal microbiome of the root endosphere, rhizosphere, and different horizons of the bulk soil were explored through enzymatic, metabolic diversity, and metabarcoding analysis in both areas. Despite the lower microbial diversity and richness in symptomatic roots and soil, higher microbial activity and enrichment of potentially both beneficial bacteria and pathogenic fungi were found in the declining area. Path modeling analysis linked the root microbial activity to berry quality, suggesting a determinant role of root microbiome in the berry mineral content. Furthermore, certain fungal and bacterial taxa were correlated with predicted metabolic pathways and metabolic processes assessed with Eco-Plates. These results unexpectedly revealed active microbial profiles in the belowground compartments associated with stressed vines, highlighting the interest of exploring the functional microbiota of plants, and more specifically roots and rhizosphere, under stressed conditions.


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Supplementary Tables Supplementary Table S1.Primers for 16S and 18S rRNA amplification for q-PCR, as well as for 16S rRNA gene and ITS sequencing.Specific overhang Illumina adapters are in italic and underlined.

Primer
Figure S6.Composition of the root endosphere and rhizosphere microbiome in symptomatic (orange, S) and asymptomatic (green, AS) conditions (n = 5).(A) Shared OTUs represented by Venn diagram for 16S and ITS sequencing, with significant overlaps detected using hyper-geometric tests.(B) α-diversity of both root and rhizosphere associated to their phyla.P-values, determined with t or Wilcoxon tests, depending on the normality hypothesis, are indicated.Supplementary Figure S10.Comparisons between symptomatic (S, orange) and asymptomatic (AS, green) conditions for each compartment of the 13 detected guilds from FUNGuild database.In bold are indicated significant differences based on either student t or Mann-Whitney tests.

Table S3 .
Physicochemical characteristics of the different depth soils from the studied plot with (S) and without (AS) decline symptoms.Data shown are the values obtained after pooling 3

Table S4 .
Physicochemical characteristics of the inter-row soils from the studied plot with (S) and without (AS) decline symptoms.Numbers represents means ± SE (n = 3).In bold are indicated significant differences based on either student t or Mann-Whitney tests.

Table S5 .
Cultivable population levels of bacteria and fungi, and Eco-Plates measurements (AUC, Simpson's index, family compounds consumed, and functional richness at 96 hours post-incubation) within the symptomatic (S) and asymptomatic (AS) rhizosphere and bulk soils.Means ± SE are presented for bacterial and fungal counts (n = 5), as well as for Eco-Plates measurements (n = 3).Letter a represents variables in log (CFUs / g of dry soil), while b represents variables calculated based on AWCD values from the Eco-Plates.

Table S6 .
Factors effects related to compartment (bulk, rhizosphere, root endosphere) and soil composition (S, AS) on richness, diversity, and β-diversity related to bacterial, fungal, and Glomeromycota communities.Significances were assessed through a Type II ANOVA for richness and diversity, while PERMANOVA (n=999) was used for distance dissimilarities.Significant P-values (<0.05) were represented in bold.

Table S7 .
Reports of α-diversities metrics represented by Chao1 and Simpson for each of the conditions among the bacterial and fungal communities.Different letters indicate significant differences among the communities (pairwise test, p < 0.05).

Table S8 .
Abundances (± SE) in percentage of fungal OTUs potentially associated with grapevine diseases across compartment × soil status (S: Symptomatic; AS: Asymptomatic) conditions.Percentages indicate proportions of sequences affiliated with pathogenic fungi relative to total sequences.Significant differences were detected using student t-tests or Wilcoxon-tests, depending on the normality and variance (n=5).P values below 0.05 are highlighted in bold.