Vanillic acid changed cucumber (Cucumis sativus L.) seedling rhizosphere total bacterial, Pseudomonas and Bacillus spp. communities

Abstract

Soil microorganisms are key drivers of plant productivity in terrestrial ecosystems, yet controls on their diversities and abundances are not fully elucidated. Phenolic acids, released through plant root exudation and residues decomposition, are usually referred as autotoxins of several crops, including cucumber. In this study, effects of vanillic acid (VA) on cucumber rhizosphere microbial communities were investigated by treating cucumber seedlings with VA every two days for five times. Amplicon sequencing, PCR-denaturing gradient gel electrophoresis and quantitative PCR were used to analyzed the 16S rRNA genes of total bacterial, Pseudomonas and Bacillus spp. communities. Results showed that VA at 0.05 μmol g^-1 soil changed total bacterial community diversity and composition. In particular, VA inhibited the relative abundances of genera with plant-beneficial potentials, such as Bacillus and Lysobacter spp. Moreover, VA changed Pseudomonas and Bacillus spp. community compositions by altering the number and/or relative abundances of their OTUs; and decreased Bacillus spp. community abundance at 0.02 to 0.2 μmol g^-1 soil and Pseudomonas spp. community abundance at 0.2 μmol g^-1 soil. Overall, VA changed cucumber seedling rhizosphere total bacterial, Pseudomonas and Bacillus spp. communities, which maybe be associated with the adverse effects of VA on cucumber growth under soil conditions.

Figure 1

Relative abundances of main bacterial phyla (a) and classes (b) in cucumber rhizosphere soils treated with water (W) or vanillic acid at 0.05 μmol g⁻¹ soil (VA). Bacterial phyla and classes with average relative abundances >0.5% in at least one treatment were shown. Data are represented as the means of three independent replicates.

Figure 2

Cladograms, generated from LEfSe analysis, represent the polygenetic distribution of cucumber rhizosphere soil bacterial taxa. LEfSe analysis was based on the data of three independent replicates of each treatment. Bacterial taxa that are significantly enriched in each treatment with LDA scores larger than 2.0 are shown. Significantly discriminant taxon nodes are colored: red for cucumber rhizosphere soils treated with water (W), green for vanillic acid at 0.05 μmol g⁻¹ soil (VA). Yellow circles represent non-significant differences in abundance between treatments for that particular taxon. Each circle’s diameter is proportional to the taxon’s abundance. Labels are shown of the phylum, class and order levels. The LDA scores of each significantly discriminant taxon from the phylum to genus levels are shown in Figure S2.

Figure 3

Heat map showing the relative abundances of dominant classified bacterial genera in cucumber rhizosphere soils treated with water (W) or vanillic acid at 0.05 μmol g⁻¹ soil (VA). Classified bacterial genera with average relative abundances >0.3% were identified in each sample by colors deduced from the raw Z-scores. Hierarchical clustering of samples was performed using the average clustering method with the Euclidean distances.

Figure 4

Alpha diversity and beta diversity indices of bacterial communities in cucumber rhizosphere soils treated with water (W) or vanillic acid at 0.05 μmol g⁻¹ soil (VA). For alpha diversity, number of OTUs observed (No. of OTUs), ACE, Chao, Shannon and Inverse Simpson indices were calculated using random subsamples of 24,245 16S rRNA gene sequences per sample. OTUs were delineated at 97% sequence similarity. The box plot shows median (black line), first quartile–third quartile percentiles (box range) and 1.5× the interquartile range (whiskers). There were three independent replicates of each treatment. Different letters indicate significant difference based on Welch’s t test (P < 0.05). For beta diversity, differences in Bray-Curtis (b) and UniFrac distances (c) of bacterial communities at the OTU level were visualized by principal coordinates analyses. Ellipses indicate 95% confidence interval for replicates.

Figure 5

Principal component analysis of Bacillus (a) and Pseudomonas (b) spp. communities in cucumber rhizosphere soils based on PCR-DGGE analysis. W represents cucumber rhizosphere soils treated with water. 0.02, 0.05, 0.1 and 0.2 represent cucumber rhizosphere soils treated with vanillic acid at 0.02, 0.05, 0.1, 0.2 μmol g⁻¹ soil concentrations, respectively.

Figure 6

Abundances of Bacillus (a) and Pseudomonas (b) spp. communities in cucumber rhizosphere soils as determined by quantitative PCR. W represents cucumber rhizosphere soils treated with water. 0.02, 0.05, 0.1 and 0.2 represent cucumber rhizosphere soils treated with vanillic acid at 0.02, 0.05, 0.1, 0.2 μmol g⁻¹ soil concentrations, respectively. Data are represented as the means of three independent replicates with standard error bars. Different letters indicate significant difference based on Tukey’s HSD test test (P < 0.05).