p-Coumaric acid influenced cucumber rhizosphere soil microbial communities and the growth of Fusarium oxysporum f.sp. cucumerinum Owen

Abstract

Background: Autotoxicity of cucumber root exudates or decaying residues may be the cause of the soil sickness of cucumber. However, how autotoxins affect soil microbial communities is not yet fully understood.

Methodology/principal findings: The aims of this study were to study the effects of an artificially applied autotoxin of cucumber, p-coumaric acid, on cucumber seedling growth, rhizosphere soil microbial communities, and Fusarium oxysporum f.sp. cucumerinum Owen (a soil-borne pathogen of cucumber) growth. Abundance, structure and composition of rhizosphere bacterial and fungal communities were analyzed with real-time PCR, PCR-denaturing gradient gel electrophoresis (DGGE) and clone library methods. Soil dehydrogenase activity and microbial biomass C (MBC) were determined to indicate the activity and size of the soil microflora. Results showed that p-coumaric acid (0.1-1.0 µmol/g soil) decreased cucumber leaf area, and increased soil dehydrogenase activity, MBC and rhizosphere bacterial and fungal community abundances. p-Coumaric acid also changed the structure and composition of rhizosphere bacterial and fungal communities, with increases in the relative abundances of bacterial taxa Firmicutes, Betaproteobacteria, Gammaproteobacteria and fungal taxa Sordariomycete, Zygomycota, and decreases in the relative abundances of bacterial taxa Bacteroidetes, Deltaproteobacteria, Planctomycetes, Verrucomicrobia and fungal taxon Pezizomycete. In addition, p-coumaric acid increased Fusarium oxysporum population densities in soil.

Conclusions/significance: These results indicate that p-coumaric acid may play a role in the autotoxicity of cucumber via influencing soil microbial communities.

Figure 1. Effects of p-coumaric acid on cucumber radicle elongation.

Data are represented as the means of three independent replicates with standard error bars. Different letters indicate significant difference between treatments (P<0.05, Tukey’s HSD test).

Figure 2. Effects of p-coumaric acid on bacterial community structure.

(A) DGGE profiles of partial bacterial 16S rRNA gene sequences. (B) Principal component analysis (PCA) of bacterial community based on DGGE profiles. B and W represent soil sample before cucumber planting and control, respectively. T1, T2, T3 and T4 represent soil treated with 0.1, 0.25, 0.5, 1.0 µmol p-coumaric acid/g soil, respectively.

Figure 3. Effects of p-coumaric acid on fungal community structure.

(A) DGGE profiles of partial fungal ITS regions. (B) Principal component analysis (PCA) of fungal community based on DGGE profiles. B and W represent soil sample before cucumber planting and control, respectively. T1, T2, T3 and T4 represent soil treated with 0.1, 0.25, 0.5, 1.0 µmol p-coumaric acid/g soil, respectively.

Figure 4. Rarefaction analysis of bacterial 16S rRNA and fungal ITS gene clone libraries.

The total number of sequences per library is plotted against the number of unique OTUs encountered within the same library. OTUs were defined at the 97% similarity level.

Figure 5. Effects of p-coumaric acid on bacterial and fungal community composition.

(A) relative abundances of bacterial 16S rRNA gene phylotypes. (B) relative abundances of fungal ITS gene phylotypes. Bacterial phylum Proteobacteria and fungal phylum Ascomycota are subdivided into class. Shown fractions indicate the relative percentage to the total number of clones. For bacteria: Acido, Acidobacteria; Spiro, Spirochaetes; Tene, Tenericutes; Actino, Actinobacteria; Fibro, Fibrobacteres; Arma, Armatimonadetes; Bacter, Bacteroidetes; UN, Unclassified; Chlo, Chloroflexi; Cyano, Cyanobacteria; Elusi, Elusimicrobia; Firmi, Firmicutes; Gemma, Gemmatimonadetes; Nitro, Nitrospirae; Plancto, Planctomycetes; Alpha, Alphaproteobacteria; Delta, Deltaproteobacteria; Gamma, Gammaproteobacteria; Beta, Betaproteobacteria; CTM7, Candidate division; TM7 Verru, Verrucomicrobia.

Figure 6. Effects of p-coumaric acid on F. oxysporum growth.

(A) F. oxysporum colony diameter. (B) F. oxysporum population in the soil. Data are represented as the means of three independent replicates with standard error bars. Different letters indicate significant difference between treatments (P<0.05, Tukey’s HSD test). CFU colony forming unit.