Bt-Modified Transgenic Rice May Shift the Composition and Diversity of Rhizosphere Microbiota

Abstract

Plants significantly shape root-associated microbiota, making rhizosphere microbes useful environmental indicator organisms for safety assessment. Here, we report the pyrosequencing of the bacterial 16S ribosomal RNA in rhizosphere soil samples collected from transgenic cry1Ab/cry1Ac Bt rice Huahui No. 1 (GM crop) and its parental counterpart, Minghui63. We identified a total of 2579 quantifiable bacterial operational taxonomic units (OTUs). Many treatment-enriched microbial OTUs were identified, including 14 NonGM-enriched OTUs and 10 GM-enriched OTUs. OTUs belonging to the phyla Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, Nitrospirae, Chlorobi and GN04 were identified as statistically different in abundance between GM and the other two treatments. Compared with the different impacts of different rice varieties on microbiota, the impact of rice planting on microbiota is more obvious. Furthermore, Huahui No. 1 transgenic Bt rice had a greater impact on the rhizosphere bacterial communities than Minghui63. Early developmental stages of the transgenic Bt rice had a significant impact on many Bacillaceae communities. Soil chemical properties were not significantly altered by the presence of transgenic Bt rice. The peak concentration level of Bt protein products was detected during the seedling stage of transgenic Bt rice, which may be an intriguing factor for bacterial diversity variations. Based on these findings, we conclude that transgenic Bt rice has a significant impact on root-associated bacteria. This information may be leveraged in future environmental safety assessments of transgenic Bt rice varieties.

Keywords: 16S ribosomal RNA; genetically modified rice; microbial communities; operational taxonomic units; transgenic Bt rice variety.

Figure 2

Root microbiota comparisons of GM, NonGM, and CK. The ternary plots depicted the relative occurrence of individual OTUs (circles) in samples of GM and NonGM compared with CK samples (A), in samples of GM and CK compared with NonGM samples (B), and in samples of NonGM and CK compared with GM samples (C), respectively. Colored circles depict significantly different (FDR < 0.1) OTUs between two treatments at the bottom of the triangles. The mean abundance of individual OTUs was calculated in each treatment and plotted ranked by average OTU abundance across all treatments (D). OTU scores of principle coordinate analysis of ACM OTUs (E) and scores of principle coordinate analysis for all samples based on Bray–Curtis distance (F), constrained by treatments and based on Bray–Curtis compositional dissimilarities among all samples. The arrows pointed to the centroid of the constrained factor. Circle size corresponded to relative abundance of OTUs/samples, and colors were assigned to different phyla. The percentage of variation explained by each axis referred to the fraction of the total variance in the data explained by treatments.

Figure 1

Beta diversity of the ACM. Between-sample diversity was calculated for ACMs using the weighted UniFrac distance metric (phylogeny-based and sensitive to the sequence abundances) on 800 sequences per sample.

Figure 3

Stage-specific accumulations of thirteen Bacillaceae ACM OTUs in GM rice. Note: Nine Bacillaceae OTUs were changed significantly (FDR < 0.05) in GM ACM between stage I and stage II. OTU103\512\93\304\312\10 were very significant between Stage I and Stage II (FDR < 0.01). OTU4\1\257 were significant between Stage I and Stage II (FDR < 0.05). OTU4 was also significant between Stage III and IV, and between Stage IV and V (FDR < 0.05). RA, relative abundance; *, significant difference (FDR < 0.05); **, very significant difference (FDR < 0.01).

Figure 4

Identification and classification of significantly differed GM bacterial communities. Abundance of thirty OTUs was significantly different in GM, comparing with both CK and NonGM (A). Taxonomical profiles at the phylum rank and number of OTUs were shown (B). Proteobacteria OTUs were found to be significantly more abundant in stage II than in stage I (C), and Nitrospirae OTUs were found to be significantly more abundant in stage III than in stage II (D). ‘GM ∅ CK’ and ‘GM ∅ NonGM’ stand for significant OTUs (FDR < 0.1) between GM and corresponding treatments. *, significant difference (FDR < 0.05).

Figure 5

Abundance of ten GM-enriched OTUs. GM-enriched OTUs were defined as OTUs that are very significant in abundance (FDR < 0.01) between GM and CK, and between GM and NonGM. Minimum mean abundance log2(RA+1)) of GM OTUs ≥ 1. Comparing to CK and NonGM, the minimum increased changing fold of GM OTUs ≥ 1.5-fold. RA, relative abundance; f_, family; o_, order; g_, genus. **, very significant difference (FDR < 0.01).

Figure 6

Concentration of Cry1Ac protein in Huahui No.1 transgenic Bt rice and its parental counterpart Minghui63 rice. The level of Cry1Ac protein was gradually decreased as rice was developing to higher stages. Bt, positive control; CK, negative control; S1, stage I; S2, stage II; S3, stage III, S4, stage IV; F, Minghui63 rice; T, Huahui No.1 transgenic Bt rice; **, very significant difference (FDR < 0.01).

Figure 7

DDGE validation of 16S rDNA. Each column represents a pooled sample from the same treatment in same sampling time. Numbers adhered to gel bands corresponded to cloned sequences annotated in Supplementary Table S10. Note: K1~K5, CK samples from stage I to stage V; F1~F5, NonGM samples from stage I to stage V; T1~T5, GM samples from stage I to stage V.

Figure 8

Hypothesized impact mechanism of planting GM Cry1Ab/Cry1Ac rice on rhizosphere bacteria.