Table 1.
The treatments used for bacterial community analysis.
Fig 1.
Germination rates of P. brassicae resting spores incubated in non-sterile and sterile soil suspensions from an oilseed rape field after 5 and 7 days of incubation.
Rhizosphere soil was collected from the roots of field-grown oilseed rape cv. Bender at BBCH 18 (eight-leaf stage). Bulk soil was collected from a neighbouring field without plants. Error bars represent standard deviations. Different letters represent significant differences among the treatments (Tukey test. P < 0.05, n = 3).
Table 2.
Germination rates of P. brassicae resting spores incubated in autoclaved and non-autoclaved soil with different levels of moisture.
Fig 2.
Germination rates of non-sterile resting spores of P. brassicae incubated with different bacterial suspensions or filtrate with Hoagland solution or sdH2O after 7 days.
Error bars represent standard deviations. Different letters represent significant differences among the treatments (Tukey test. P < 0.05, n = 3).
Fig 3.
Germination rates of non-sterile resting spores of P. brassicae incubated with Iso4 bacterial suspension in various solutions after 7 days.
The positive control of 1/10 strength Hoagland solution (H) is indicated in yellow. The negative control of sdH2O (sd) is indicated in green. Error bars represent standard deviation s. Different letters represent significant differences among the treatments (Wilcoxon rank sum test. P < 0.05, n = 3).
Fig 4.
15N enrichment (Δδ15N) of P. brassicae resting spores incubated with potassium nitrate with (+ E. coli) or without (- E. coli) the presence of E. coli suspension and corresponding germination rates (G%) after different incubation times.
Error bars indicate standard deviations. Different letters represent significant differences among the treatments (Tukey test. P < 0.05, n = 3).
Fig 5.
The shift of bacterial community composition after 7 days of incubation.
Comparison of bacterial communities differently treated and inducing ‘high’ or ‘low’ germination rates of P. brassicae, in relation to the ‘initial’ untreated non-sterile resting spores. (A) Germination rate of resting spores of P. brassicae in each sample used for 16S rRNA gene amplicon sequencing. (B) Shannon index of the microbiota from ‘high’, ‘low’ and ‘initial’ groups. (C) Principal coordinate analysis (PCoA) based on the Unifrac distance showing that the bacteria of ‘high’ germination rate group separate from those of ‘low’ germination and the initial groups (P < 0.001, PERMANOVA by Adonis). (D) Chord diagram visualizing community structure in different groups at class-level. HB: B. subtilis cell suspension with 1/10 strength Hoagland solution; KB: B. subtilis cell suspension with 6 mM potassium nitrate; A4f: filtrate of bacterial strain A4 with 1/10 strength Hoagland solution; sd: sterilized deionized water; H: 1/10 strength Hoagland solution; NS: non-sterile resting spore suspension (initial bacterial community).
Fig 6.
Taxonomic and functional characteristics of differential bacteria in ‘high’ and ‘low’ germination rate groups.
(A) Relative abundance of the corresponding ASVs enriched in ‘high’ and ‘low’ germination rate groups (Wilcoxon rank sum test, FDR adjusted P < 0.05). (B) Cladograms generated by LEfSe indicating differences in the bacterial taxa between ‘high’ and ‘low’ germination groups. Red bars indicate taxa enriched in ‘high’ germination rate group, green bars indicate taxa enriched in ‘low’ germination group. (C) LDA scores for the bacterial taxa differentially abundant in ‘high’ and ‘low’ groups. Red bars indicate taxa enriched in high germination rate group, and green bars indicate taxa in low germination group. (D) Metabolic and ecological functions of ASVs enriched in ‘high’ and ‘low’ groups based on FAPROTAX (Welch’s t-test, P < 0.05).
Fig 7.
Bacterial genera related to the stimulation of P. brassicae resting spore germination.
(A) Correlation between germination rates and relative abundance of bacteria (Spearman’s ρ, P < 0.0001 ****; P < 0.001 ***; P < 0.01 **; P < 0.05 *; ns, not significant). (B) Co-occurrence network of ‘high’ and ‘low’ germination rate groups based on the Spearman correlation algorithms. Each node represents a genus and is colored by modularity. The node size indicates the relative abundance of each genus per group, and the thickness of the line represents the Spearman coefficient (Rs > 0.8 or < -0.8, P < 0.01). Red links stand for positive, and blue links for negative correlations between nodes.
Fig 8.
Germination rates of P. brassicae resting spores incubated with various sugars (A) and L-amino acids (B) in the presence of nitrate. Non-sterile spores were incubated with 50 mM potassium nitrate for 14 days. The germination rate in the water control was 0%. Error bars indicate standard deviations. Different letters indicate significant differences among samples (Tukey test, P < 0.05, n = 3).
Fig 9.
A multifactorial pathobiome model of factors affecting the germination of Plasmodiophora brassicae resting spores.
Root exudates collected under sterile conditions cannot stimulate the germination of resting spores, while they may serve as a carbon source to modulate the microbial community under non-sterile conditions. The initial microbial community is reshaped by environmental factors (e.g. carbon sources, nutrients and soil moisture) to a community stimulating the germination of P. brassicae resting spores.