Effects of an EPSPS-transgenic soybean line ZUTS31 on root-associated bacterial communities during field growth

Abstract

The increased worldwide commercial cultivation of transgenic crops during the past 20 years is accompanied with potential effects on the soil microbial communities, because many rhizosphere and endosphere bacteria play important roles in promoting plant health and growth. Previous studies reported that transgenic plants exert differential effects on soil microbial communities, especially rhizobacteria. Thus, this study compared the soybean root-associated bacterial communities between a 5-enolpyruvylshikimate-3-phosphate synthase -transgenic soybean line (ZUTS31 or simply Z31) and its recipient cultivar (Huachun3 or simply HC3) at the vegetative, flowering, and seed-filling stages. High-throughput sequencing of 16S rRNA gene (16S rDNA) V4 hypervariable region amplicons via Illumina MiSeq and real-time quantitative PCR (qPCR) were performed. Our results revealed no significant differences in the overall alpha diversity of root-associated bacterial communities at the three developmental stages and in the beta diversity of root-associated bacterial communities at the flowering stage between Z31 and HC3 under field growth. However, significant differences in the beta diversity of rhizosphere bacterial communities were found at the vegetative and seed-filling stages between the two groups. Furthermore, the results of next generation sequencing and qPCR showed that the relative abundances of root-associated main nitrogen-fixing bacterial genera, especially Bradyrhizobium in the roots, evidently changed from the flowering stage to the seed-filling stage. In conclusion, Z31 exerts transitory effects on the taxonomic diversity of rhizosphere bacterial communities at the vegetative and seed-filling stages compared to the control under field conditions. In addition, soybean developmental change evidently influences the main symbiotic nitrogen-fixing bacterial genera in the roots from the flowering stage to the seed-filling stage.

Fig 1. Principal coordinate analysis (PCoA) based on the Bray–Curtis distance metrics of bacterial communities between the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-transgenic soybean line ZUTS31 (Z31) and its recipient cultivar Huachun3 (HC3) at the vegetative stage (n = 32).

HC3BRh and Z31BRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the vegetative stage, respectively. HC3BSO and Z31BSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the vegetative stage, respectively. HC3ASO and Z31ASO represent the bulk soils before sowing seeds of the recipient cultivar HC3 and the transgenic line Z31, respectively.

Fig 2. PCoA based on the Bray–Curtis distance metrics of root-associated bacterial communities between Z31 and HC3 at the flowering stage (n = 32).

HC3CRt and Z31CRt represent the roots of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively. HC3CRh and Z31CRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively. HC3CSO and Z31CSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively.

Fig 3. PCoA based on the Bray–Curtis distance metrics of root-associated bacterial communities between Z31 and HC3 at the seed-filling stage (n = 36).

HC3DRt and Z31DRt represent the roots of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively. HC3DRh and Z31DRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively. HC3DSO and Z31DSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively.

Fig 4. Relative abundances of main nitrogen-fixing bacterial genera at the vegetative stage.

HC3BRh and Z31BRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the vegetative stage, respectively. HC3BSO and Z31BSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the vegetative stage, respectively. HC3ASO and Z31ASO represent the bulk soils before sowing seeds of the recipient cultivar HC3 and the transgenic line Z31, respectively. Error bars indicate the standard errors (SE) calculated by Metastats. Values are mean ± SE (n = 4 or 6);*, P < 0.05; **, P < 0.01.

Fig 5. Relative abundances of main nitrogen-fixing bacterial genera at the flowering stage.

HC3CRt and Z31CRt represent the roots of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively. HC3CRh and Z31CRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively. HC3CSO and Z31CSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the flowering stage, respectively. Error bars indicate SE calculated by Metastats. Values are mean ± SE (n = 4 or 6); *, P < 0.05; **, P < 0.01.

Fig 6. Relative abundances of main nitrogen-fixing bacterial genera at the seed-filling stage.

HC3DRt and Z31DRt represent the roots of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively. HC3DRh and Z31DRh represent the rhizospheric soils of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively. HC3DSO and Z31DSO represent the surrounding soils of the recipient cultivar HC3 and the transgenic line Z31 at the seed-filling stage, respectively. Error bars indicate SE calculated by Metastats. Values are mean ± SE (n = 6); *, P < 0.05; **, P < 0.01.

Fig 7. Relative abundance of the nifH gene in soybean root-associated bacterial communities of Z31 and HC3 at the flowering and seed-filling stages.

CRt and DRt represent the roots of soybean at the flowering and seed-filling stages, respectively. CRh and DRh represent the rhizospheric soils of soybean at the flowering and seed-filling stages, respectively. CSO and DSO represent the surrounding soils of soybean at the flowering and seed-filling stages, respectively. Levels of the nifH gene abundance were normalized to 16S rDNA abundance. A value of 1 was assigned to the detected value of the surrounding soil of HC3 at the flowering stage (HC3CSO). The error bars represent the standard deviation of four or six replicates of soil or root samples and each replicate with technical triplicate. *Indicates significant difference (P < 0.05) according to the one-way ANOVA.