Vineyard Soil Microbiome Dashboard

Bioinformatics Workflow

ONT amplicon sequencing pipeline for 16S rRNA and ITS analysis of vineyard soil microbiomes.

16S rRNA Pipeline

Bacterial Community Analysis

Full-length 16S amplicons (~1400 bp) processed through ONT basecalling, quality filtering, and taxonomic classification.

Basecalling	Dorado SUP
QC Filtering	Chopper (Q≥10, 1000-1600 bp)
Taxonomy	EMU (genus + species)
Functional	PICRUSt2 (KEGG KOs, MetaCyc)
Diversity	Shannon, Simpson, Bray-Curtis PCoA

ITS Pipeline

Fungal Community Analysis

ITS amplicons processed through ONT basecalling, ITSxRust extraction, and EMITS classification.

Basecalling	Dorado SUP
ITS Extraction	ITSxRust
Taxonomy	EMITS (genus + species)
Ecology	FUNGuild (curated)
Diversity	Shannon, Simpson, Bray-Curtis PCoA

Cross-Kingdom Integration

Both marker genes analyzed in parallel across 30 samples (20 Paine/Ramsey, 5 Elqui/Carignan, 5 Itata/Cinsault). Cross-kingdom analysis via Procrustes, Mantel tests, and Spearman correlations. Soil chemistry (10 parameters) integrated via CCA and Spearman-FDR analysis.

Study Overview

ONT amplicon sequencing (16S & ITS) of vineyard soils across three Chilean wine regions — Paine, Elqui & Itata — exploring bacterial, fungal, and functional community structure alongside soil chemistry.

Bacteria 16S

Top 10 Genera by Site

Fungi ITS

Top 10 Genera by Site

Key Finding

Three sites show highly distinct microbial communities. PCoA analysis reveals complete separation of Paine, Elqui and Itata in both bacterial and fungal datasets (Procrustes M²=0.23, p=0.001).

Quality Control

Sequencing statistics and Chopper filtering (1000–1600 bp, Q≥10).

Chopper Filtering

Raw vs Filtered Reads per Barcode

Statistics

QC Table

Bacterial Communities (16S rRNA)

Top genera identified via EMU from ONT long-read 16S amplicons.

Composition

Stacked Abundance per Sample — Top 15 Genera

Table

Genus Abundance (Variety Averages)

Bacterial Species (16S)

Top 20 species identified via EMU at species-level resolution.

Stacked Composition

Top 20 Species per Sample

Differential

Top 15 Differential Species

Species with greatest abundance difference between varieties.

Fungal Communities (ITS)

Top genera identified via EMITS from ONT long-read ITS amplicons.

Composition

Stacked Abundance per Sample — Top 15 Genera

Highlight

Mortierella dominates Itata/Cinsault at 73.6% average abundance — dramatically higher than Paine (10.9%) and Elqui (0.4%).

Table

Genus Abundance (Variety Averages)

Fungal Species (ITS)

Top 20 species identified via EMITS at species-level resolution.

Stacked Composition

Top 20 Species per Sample

Differential

Top 15 Differential Fungal Species

Alpha Diversity

Shannon, Simpson, Richness & Evenness across grape varieties.

Metric:

Bacteria 16S

Fungi ITS

Beta Diversity (PCoA — Bray-Curtis)

Principal Coordinates Analysis on genus-level abundance matrices.

Bacteria 16S

Fungi ITS

Observation

Both 16S and ITS PCoA plots show three perfectly separated clusters corresponding to the three vineyard sites, indicating highly distinct soil microbiome profiles.

Abundance Heatmaps

Top 30 genera × 30 samples intensity heatmaps for both bacterial and fungal communities.

Kingdom:

Bacteria 16S

Top 30 Genera × 30 Samples

Yellow/green = high abundance. Samples colored by site.

Functional Potential (PICRUSt2)

Predicted functional gene abundances from N, C, and S biogeochemical cycles across grape varieties.

Cycle:

Nitrogen Cycle

N-Cycle KOs by Process & Variety

Soil Chemistry

Physicochemical parameters across three vineyard sites (10 parameters).

Grouped Bar Chart

Parameters by Site (Mean ± SD)

Summary

Soil Chemistry Table

Soil Chemistry ↔ Microbiome Correlations

Spearman rank correlations with Benjamini-Hochberg FDR correction (q < 0.05). 27 of 150 tests significant.

27

FDR Significant

150

Total Tests

0.814

Max |ρ|

FDR Significant

Correlations (sorted by |ρ|)

Cross-Kingdom Analysis

Bacteria–Fungi co-occurrence patterns via Procrustes & Mantel tests.

0.230

Procrustes M²

0.001

Procrustes p

0.691

Mantel r

0.001

Mantel p

Interpretation

Low Procrustes M² (0.23) indicates high concordance between bacterial and fungal community ordinations. The strong Mantel correlation (r=0.69) confirms that sites with similar bacterial communities also harbor similar fungal communities.

Correlation Heatmap

Top 20 Bacteria × Top 20 Fungi (Spearman ρ)

Blue = positive co-occurrence, red = negative. Only significant correlations shown (p < 0.05).

Top Associations

Strongest Cross-Kingdom Genus Correlations

Fungal Ecological Guilds (FUNGuild)

Ecological guild assignment for ITS taxa based on known fungal ecology. Top 20 genera manually curated from literature (FUNGuild DB returned no matches for genus-level EMITS output).

20

Taxa Curated

529

Total ITS Taxa

73.6%

Abundance Covered

Ecological patterns

Saprotrophs dominate across all sites. Mortierella (soil saprotroph) alone accounts for 20% of all ITS reads, peaking at 73.6% in Itata/Cinsault. Pathotroph-saprotroph taxa like Alternaria, Fusarium, and Cladosporium are present but at lower abundances. Leohumicola (ericoid mycorrhizal) represents the only significant symbiotroph, found primarily in Cinsault soils.

Trophic Mode

Distribution by Variety

Proportion of each trophic mode per grape variety.

Global

Overall Trophic Distribution

Abundance-weighted across all 30 samples.

Top Taxa

Ecological Guild Assignments

Top 20 genera with manually curated guild assignments from established literature.

Co-occurrence Network

Cross-kingdom correlation network built from Spearman correlations between the top 30 bacterial and top 30 fungal genera. Edges shown for |ρ| > 0.6.

39

Genera (Nodes)

95

Associations

38

Positive (co-occur)

57

Negative (exclude)

How to read

Drag nodes to rearrange. Scroll to zoom. Green circles = bacteria, purple = fungi. Green lines = positive co-occurrence, red = mutual exclusion. Line width scales with |ρ|. Filter by association type.

Interactive Network

Cross-Kingdom Genera (|ρ| > 0.6)

Bacteria Fungi Positive (co-occur) Negative (exclude)

Bacteria ↔ Fungi Interactions

Spearman rank correlations between bacterial and fungal genera across all 30 samples. 328 total cross-kingdom pairs evaluated from the top 30 genera of each kingdom.

Key patterns

Strongest positive: Ktedonosporobacter–Saitozyma (ρ=0.92) — both enriched in Cinsault/Itata soils. Strongest negative: Desulfuromonas–Knufia (ρ=−0.80) — opposing site preferences. Mortierella shows strong positive correlations with Gemmata and Vicinamibacter, suggesting shared niche requirements in vineyard soils.

Bipartite View

Bacteria (left) ↔ Fungi (right)

Green = positive co-occurrence, red = mutual exclusion. Line width ∝ |ρ|. Top 30 pairs shown.

Strongest Positive Associations

Strongest Negative Associations

Biogeochemical Cycle Sankey Diagrams

Interactive Sankey diagrams showing predicted functional gene abundance flow (PICRUSt2) from grape varieties through biogeochemical processes (N, C, S cycles) to individual KEGG genes.

How to read

Left = grape varieties. Middle = biogeochemical processes. Right = individual genes (KOs). Ribbon width represents predicted abundance. Hover any node or ribbon for details. Use the tabs within the diagram to switch between Integrated, Nitrogen, Carbon, and Sulfur views.

Top MetaCyc Pathways

Top 30 predicted MetaCyc metabolic pathways (PICRUSt2) averaged by grape variety.

Top 30 MetaCyc

Average Predicted Abundance by Variety

Differential Taxa

Top 15 genera showing the greatest abundance differences between grape varieties (max – min variety average).

Bacteria 16S

Top 15 Differential Bacterial Genera

Fungi ITS

Top 15 Differential Fungal Genera

Figure Gallery

All generated figures from the analysis. Click any figure to view full-size.

Filter:

Summary of Key Findings

Major patterns identified across bacterial, fungal, functional, and soil chemistry analyses of the vineyard soil microbiome project.

Site Differentiation

Three distinct microbiome profiles. Both 16S and ITS PCoA show complete separation of Paine (Ramsey), Elqui (Carignan), and Itata (Cinsault) with high statistical support (Procrustes M²=0.23, p=0.001; Mantel r=0.69, p=0.001).

Bacteria 16S

Key Bacterial Patterns

Total genera	399
Dominant genus	Gemmatirosa
Shannon range	3.63 – 4.55
Most differential	Cand. Koribacter (Itata-enriched)
Elqui signature	Arthrobacter, Bacillus

Fungi ITS

Key Fungal Patterns

Total genera	529
Dominant genus	Mortierella (20% overall)
Shannon range	1.15 – 3.83
Itata dominance	Mortierella at 73.6%
Elqui signature	Cryptococcus, Naganishia

Soil Chemistry ↔ Microbiome

27 of 150 correlations significant (FDR q<0.05). Strongest: Potasio–K_transport (ρ=0.81). Soil pH strongly shapes fungal beneficial guilds and salinity tolerance genes. Copper (Cu) inversely correlates with phytohormone-related functions, potentially reflecting historical vineyard management.

Functional Highlights

PICRUSt2 Predicted Functions

N-cycle	Denitrification dominates (nosZ, norC highest in Ramsey). Nitrification (amoA/B/C) enriched in Carignan.
C-cycle	Calvin cycle genes (rbcL, PRK) present across all sites. Methanotrophy (pmo genes) only detected in Carignan.
S-cycle	Assimilatory sulfate reduction dominates. Sox pathway genes suggest active sulfur oxidation, particularly in Ramsey.
Top MetaCyc	Aerobic respiration, pyruvate fermentation, and branched-chain amino acid biosynthesis most abundant predicted pathways.

Cross-Kingdom

95 strong bacteria–fungi correlations (|ρ|>0.6). Ktedonosporobacter–Saitozyma (ρ=0.92) is the strongest positive pair, both Cinsault-enriched. Mortierella shows positive associations with Gemmata and Vicinamibacter, suggesting shared ecological niches.