Insights into the effects of transgenic glyphosate-resistant semiwild soybean on soil microbial diversity
Plant materials and molecular identification The four soybean varieties used in this study were glyphosate-resistant semiwild soybean (DT-1), glyphosate-resistant soybean variety Hujiao698 (HJ698), cultivated soybean variety Dongnong50 (D50) and wild soybean (WS-1). Fresh soybean leaves were placed in EP tubes, liquid nitrogen was added, and the samples were returned to the laboratory. DNA was extracted via the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0. The extracted genomic DNA was used as a template for PCR amplification. The primer pairs used to amplify the CP4-epsps gene (mCP4ES-F 5′-ACG GTG AYC GTC TTC CMG TTA C-3′ and mCP4ES-R 5′-GAA CAA GCA RGG CMG CAA CCA-3′) were added . The results of electrophoresis on a 1% agarose gel are shown in S1 (see Supplementary Materials), with “M” representing the marker, and showed that the glyphosate-resistant semi-wild soybean contained the EPSPS gene, validating its status as a genetically modified material. Experimental design and sample collection The field experiment was conducted at the transgenic test base of Northeast Agricultural University, Heilongjiang Province, China (45°74′ N, 126°73′ E). Isolation belts and fences were set up in the test area, and a specific individual was assigned to supervise. Three replicate plots for each of the four cultivars were established in a randomized design. A total of 48 samples (4 soybean varieties × 3 replications × 4 stages) were collected in this study. The rhizosphere soils of the four soybean varieties were sampled at the seedling (10 June), flowering (15 July), pod (25 August), and mature (30 September) stages. The samples were collected following the methods described by Babujia et al. and Lu et al. . The soil adhering to the roots was removed, and the rhizosphere soil was collected in a plastic bag. The soil samples were placed in a cool place to air dry, ground, sieved, and then divided into two portions. One portion was used to quantify the soil microbial community, and the other was stored at −80 °C for DNA extraction. Quantitative analysis of rhizosphere soil microorganisms In this study, the plate culture method was used to quantify the bacteria and fungi in the rhizosphere soil. The calculation formula for determining the quantity of microorganisms was as follows: Number of microorganisms per gram of soil = average number of colonies × dilution ratio/weight of the soil sample . Test Method: First, 10 g of rhizosphere soil was weighed, and 100 mL of sterile water was added, followed by 15 min of oscillation. A total of 1 mL of the soil suspension was added to 9 mL of sterile water. Dilution ratios were 10 −5 , 10 −6 , and 10 −7 for bacteria and 10 −3 , 10 −4 , and 10 −5 for fungi. Each diluted soil suspension was inoculated according to the mixed bacterial method, repeated five times. The bacteria were cultured at 28 °C for 48 h, and the fungi were cultured for 3 d. The colonies of bacteria and fungi at each dilution ratio were counted to quantify the bacteria and fungi. Bacteria were cultured on a beef extract peptone medium. Fungi were cultured on Martin’s medium, to which 0.3 mL of a 1% streptomycin solution was added for every 100 mL of medium in order to inhibit bacterial growth . DNA extraction and high-throughput sequencing analysis A soil DNA extraction kit from Suzhou Keming Biotechnology Co., Ltd. was used in this study according to the kit instructions. A 250 mg soil sample was added to the tube, 750 µL of SL1 buffer and 150 µL of SX buffer were added, and the mixture was ground for 5 min before incubation for 13 min at 65 °C. Then, the mixture was centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was added to a centrifuge tube containing SL3, mixed well, and incubated at −20 °C for 5 min. The mixture was subsequently centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was added to the Inhibitor Removal Column and centrifuged at 12,000 rpm for 2 min at room temperature. Next, 250 µL of SB buffer was added, and the mixture was mixed, transferred to a binding column, and centrifuged at 12,000 rpm for 2 min at room temperature. After 700 µL of SW1 and SW2 were added for washing, 100 µL of eluent SE was added, and the mixture was incubated at room temperature for 5 min and centrifuged at 12,000 rpm for 5 min to elute the DNA. Next-generation sequencing library preparation and Illumina MiSeq sequencing were performed at BGI (Shenzhen, China). PCR amplification of 16 S rRNA gene fragments was carried out with the primers 341 F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT −3′), as described by Ding et al. . PCR amplification of the ITS region gene fragments was carried out with the primers its1 (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and its2 (5′-GCT GCG TTC TTC ATC GAT GC-3′), as described by Chen et al. . Bioinformatics analysis The composition of the microbial communities in different soybean rhizosphere soil samples was analysed via the MISEQ PE300 sequencing platform. This included 16 treatment groups: seedling stage for D50 (AA), transgenic glyphosate-resistant semiwild soybean (BA), HJ698 (CA), and wild soybean (DA); flowering stage for D50 (AB), transgenic glyphosate-resistant semiwild soybean (BB), HJ698 (CB), and wild soybean (DB); pod stage for D50 (AC), transgenic glyphosate-resistant semiwild soybean (BC), HJ698 (CC), and wild soybean (DC); and mature stage for D50 (AD), transgenic glyphosate-resistant semiwild soybean (BD), HJ698 (CD), and wild soybean (DD). Each group had three independent biological replicates, resulting in a total of 48 libraries. The filtered data were assembled into sequences using FLASH (v1.2.11) by merging paired-end reads based on their overlap, resulting in high-quality tags. OTUs were clustered at 97% similarity using UPARSE, and representative sequences for each OTU were obtained. Chimeric sequences generated by PCR amplification were removed from the representative OTU sequences using UCHIME (v4.2.40). After the OTU representative sequences were obtained, PyNAST software was used to align these sequences against the Greengenes database for taxonomic annotation. MUSCLE software was used to generate phylogenetic trees and standardize the OTU abundance information. The α and β diversity indices were conducted via QIIME software (V1.7.0) and R software (V3.4.1). The LEfSe online analysis platform was employed to estimate the impact of species abundance on differential effects and to identify species with significant differences between groups. Statistical analysis IBM SPSS (version 22.0, USA) was used to analyse the quantitative data for statistical significance at a confidence level of P < 0.05 or P < 0.01. The figures were generated via Origin (version 2021, USA) software.   The four soybean varieties used in this study were glyphosate-resistant semiwild soybean (DT-1), glyphosate-resistant soybean variety Hujiao698 (HJ698), cultivated soybean variety Dongnong50 (D50) and wild soybean (WS-1). Fresh soybean leaves were placed in EP tubes, liquid nitrogen was added, and the samples were returned to the laboratory. DNA was extracted via the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0. The extracted genomic DNA was used as a template for PCR amplification. The primer pairs used to amplify the CP4-epsps gene (mCP4ES-F 5′-ACG GTG AYC GTC TTC CMG TTA C-3′ and mCP4ES-R 5′-GAA CAA GCA RGG CMG CAA CCA-3′) were added . The results of electrophoresis on a 1% agarose gel are shown in S1 (see Supplementary Materials), with “M” representing the marker, and showed that the glyphosate-resistant semi-wild soybean contained the EPSPS gene, validating its status as a genetically modified material.   The field experiment was conducted at the transgenic test base of Northeast Agricultural University, Heilongjiang Province, China (45°74′ N, 126°73′ E). Isolation belts and fences were set up in the test area, and a specific individual was assigned to supervise. Three replicate plots for each of the four cultivars were established in a randomized design. A total of 48 samples (4 soybean varieties × 3 replications × 4 stages) were collected in this study. The rhizosphere soils of the four soybean varieties were sampled at the seedling (10 June), flowering (15 July), pod (25 August), and mature (30 September) stages. The samples were collected following the methods described by Babujia et al. and Lu et al. . The soil adhering to the roots was removed, and the rhizosphere soil was collected in a plastic bag. The soil samples were placed in a cool place to air dry, ground, sieved, and then divided into two portions. One portion was used to quantify the soil microbial community, and the other was stored at −80 °C for DNA extraction.   In this study, the plate culture method was used to quantify the bacteria and fungi in the rhizosphere soil. The calculation formula for determining the quantity of microorganisms was as follows: Number of microorganisms per gram of soil = average number of colonies × dilution ratio/weight of the soil sample . Test Method: First, 10 g of rhizosphere soil was weighed, and 100 mL of sterile water was added, followed by 15 min of oscillation. A total of 1 mL of the soil suspension was added to 9 mL of sterile water. Dilution ratios were 10 −5 , 10 −6 , and 10 −7 for bacteria and 10 −3 , 10 −4 , and 10 −5 for fungi. Each diluted soil suspension was inoculated according to the mixed bacterial method, repeated five times. The bacteria were cultured at 28 °C for 48 h, and the fungi were cultured for 3 d. The colonies of bacteria and fungi at each dilution ratio were counted to quantify the bacteria and fungi. Bacteria were cultured on a beef extract peptone medium. Fungi were cultured on Martin’s medium, to which 0.3 mL of a 1% streptomycin solution was added for every 100 mL of medium in order to inhibit bacterial growth .   A soil DNA extraction kit from Suzhou Keming Biotechnology Co., Ltd. was used in this study according to the kit instructions. A 250 mg soil sample was added to the tube, 750 µL of SL1 buffer and 150 µL of SX buffer were added, and the mixture was ground for 5 min before incubation for 13 min at 65 °C. Then, the mixture was centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was added to a centrifuge tube containing SL3, mixed well, and incubated at −20 °C for 5 min. The mixture was subsequently centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was added to the Inhibitor Removal Column and centrifuged at 12,000 rpm for 2 min at room temperature. Next, 250 µL of SB buffer was added, and the mixture was mixed, transferred to a binding column, and centrifuged at 12,000 rpm for 2 min at room temperature. After 700 µL of SW1 and SW2 were added for washing, 100 µL of eluent SE was added, and the mixture was incubated at room temperature for 5 min and centrifuged at 12,000 rpm for 5 min to elute the DNA. Next-generation sequencing library preparation and Illumina MiSeq sequencing were performed at BGI (Shenzhen, China). PCR amplification of 16 S rRNA gene fragments was carried out with the primers 341 F (5′-ACT CCT ACG GGA GGC AGC AG-3′) and 806R (5′-GGA CTA CHV GGG TWT CTA AT −3′), as described by Ding et al. . PCR amplification of the ITS region gene fragments was carried out with the primers its1 (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and its2 (5′-GCT GCG TTC TTC ATC GAT GC-3′), as described by Chen et al. .   The composition of the microbial communities in different soybean rhizosphere soil samples was analysed via the MISEQ PE300 sequencing platform. This included 16 treatment groups: seedling stage for D50 (AA), transgenic glyphosate-resistant semiwild soybean (BA), HJ698 (CA), and wild soybean (DA); flowering stage for D50 (AB), transgenic glyphosate-resistant semiwild soybean (BB), HJ698 (CB), and wild soybean (DB); pod stage for D50 (AC), transgenic glyphosate-resistant semiwild soybean (BC), HJ698 (CC), and wild soybean (DC); and mature stage for D50 (AD), transgenic glyphosate-resistant semiwild soybean (BD), HJ698 (CD), and wild soybean (DD). Each group had three independent biological replicates, resulting in a total of 48 libraries. The filtered data were assembled into sequences using FLASH (v1.2.11) by merging paired-end reads based on their overlap, resulting in high-quality tags. OTUs were clustered at 97% similarity using UPARSE, and representative sequences for each OTU were obtained. Chimeric sequences generated by PCR amplification were removed from the representative OTU sequences using UCHIME (v4.2.40). After the OTU representative sequences were obtained, PyNAST software was used to align these sequences against the Greengenes database for taxonomic annotation. MUSCLE software was used to generate phylogenetic trees and standardize the OTU abundance information. The α and β diversity indices were conducted via QIIME software (V1.7.0) and R software (V3.4.1). The LEfSe online analysis platform was employed to estimate the impact of species abundance on differential effects and to identify species with significant differences between groups.   IBM SPSS (version 22.0, USA) was used to analyse the quantitative data for statistical significance at a confidence level of P < 0.05 or P < 0.01. The figures were generated via Origin (version 2021, USA) software.   Quantitative analysis of rhizosphere soil microbial communities The data presented in figure S2 demonstrates that the rhizosphere soil culturable bacteria and fungi counts of DT-1 during the seedling, pod, and mature stages of soybean growth did not significantly differ from those of D50, HJ698, and WS-1. However, at the flowering stage, the rhizosphere soil culturable bacteria count of DT-1 was significantly different from that of D50. Nevertheless, there was no significant difference in the rhizosphere soil culturable bacteria counts of DT-1 and WS-1. During the flowering stage, the rhizosphere soil culturable fungi count of DT-1 differed significantly from that of WS-1 and HJ698. However, there was no significant difference in the rhizosphere soil culturable fungi counts of DT-1 and D50 (see Supplementary Materials). Effects of transgenic glyphosate-resistant semi-wild soybean on the compositions of the soil bacterial and Fungi communities Bacterial and fungal community compositions at the flowering stage The relative abundance at the phylum level of the bacteria and fungi in the rhizosphere soil of the different soybeans at the flowering stage are shown in Fig. a and c. Compared with those of D50, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 22.11% and 8.68–23.31% and 8.98%, respectively, whereas those of Acidobacteria decreased from 40.39 to 37.34%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 66.17% and 6.08–76.58% and 10.93%, respectively, whereas that of Mortierellomycota decreased from 7.88 to 7.44%. Compared with those of HJ698, the relative abundances of Acidobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 32.36% and 6.65–37.34% and 8.98%, respectively, whereas that of Proteobacteria decreased from 28.68 to 23.31%. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 75.83%, 7.51%, and 6.02–76.58%, 10.93%, and 7.44%, respectively. Compared with that of WS-1, the relative abundance of Acidobacteria in the rhizosphere soil of DT-1 increased from 29.08 to 37.34%, whereas those of Proteobacteria and Bacteroidetes decreased from 27.43% and 12.60–23.31% and 8.98%, respectively. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 51.64%, 6.00%, and 6.18–76.58%, 10.93%, and 7.44%, respectively. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Steroidobacter , Lysobacter , Pseudomonas , Chitinophaga , Niabella , Stenotrophomonas , and Aquicella and the fungi Acremonium , Guehomyces , Herpotrichia , Nectria , Podospora , Pseudogymnoascus , Tetracladium , Ustilago , Kochiomyces , Chloridium , and Phialemonium were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Kaistobacter , Sphingomonas , Ramlibacter , Methylibium , Segetibacter , Bradyrhizobium , Hymenobacter , Phormidium , Novosphingobium , Agrobacterium , Janthinobacterium , Leptolyngbya , and Rubrivivax and the fungi Alternaria , Ascobolus , Bipolaris , Cladosporium , Hannaella , Phaeosphaeria , Plectosphaerella , Schizothecium , Setosphaeria , Thanatephorus , Trebouxia , Paraphoma , Coniosporium , and Nigrospora were lower in the rhizosphere soil of DT-1. Bacterial and fungal community composition at the mature stage The relative abundances at the phylum level of the rhizosphere bacteria and fungi in the different soybeans at the mature stage are shown in Fig. a and c. Compared with those of D50, the relative abundance of Proteobacteria in the rhizosphere soil of DT-1 increased from 34.70 to 36.54%, whereas those of Acidobacteria and Bacteroidetes decreased from 25.72% and 13.21–24.82% and 11.61%, respectively. The relative abundance of Ascomycota in the rhizosphere soil of DT-1 increased from 68.73 to 79.00%, whereas those of Basidiomycota and Mortierellomycota decreased from 14.59% and 7.03–7.24% and 6.40%, respectively. Compared with those of HJ698, the relative abundances of Proteobacteria and Acidobacteria in the rhizosphere soil of DT-1 increased from 35.29% and 18.63–36.54% and 24.82%, respectively, whereas that of Bacteroidetes decreased from 22.23 to 11.61%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 72.05% and 6.90–79.00% and 7.24%, respectively, whereas that of Mortierellomycota decreased from 8.08 to 6.40%. Compared with those of WS-1, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 30.62% and 9.60–36.54% and 11.61%, respectively, whereas that of Acidobacteria decreased from 30.85 to 24.82%. The relative abundances of Ascomycota and Mortierellomycota in the rhizosphere soil of DT-1 increased from 70.99% and 6.01–79.00% and 6.40%, respectively, whereas that of Basidiomycota decreased from 11.42 to 7.24%. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Bradyrhizobium , Nitrospira , Rhodoplanes , DA101, Segetibacter , Hymenobacter , Dyadobacter , Bdellovibrio , and Aeromicrobium and the fungi Cladosporium , Clonostachys , Edenia , Gibberella , Guehomyces , Nectria , Nigrospora , Phaeosphaeria , Plectosphaerella , and Solicoccozyma were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Ramlibacter , Rubrobacter , and Duganella and the fungi Leptosphaeria , Preussia , Pseudogymnoascus , Toninia , Trichocladium , and Mrakiella were lower in the rhizosphere soil of DT-1. In this study, the population of delayed Rhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean during the flowering period decreased compared to that of the other three soybeans. However, the population of Bradyrhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean at the mature stage increased compared to that of the other three soybeans. Diversity of the bacterial and fungi community Diversity of the bacterial community The results of the bacterial diversity analysis are shown in Figs. and a-d. The Shannon and Simpson diversity indices were used to evaluate microbial diversity . Both indices indicated that the rhizosphere bacteria of DT-1 were similar to those of the other three soybeans at all stages. At the pod stage, the Shannon index of Hujiao 698 was significantly lower than that of wild soybean ( p < 0.05), indicating a reduction in the species diversity of soil bacteria in the rhizosphere due to the root exudates of Hujiao 698. However, there was no significant difference in the Shannon index between the transgenic EPSPS-resistant semiwild soybean and wild soybean, suggesting that the decline in the species diversity of the soil bacteria in the rhizosphere was not caused by the EPSPS protein. Diversity of the fungi community The results of the fungal diversity analysis are shown in Figs. and a-d. There was no significant difference between the Shannon index of the transgenic semiwild soybean and those of Dongnong 50 and Hujiao 698 at the flowering stage ( p > 0.05), but the Shannon index of the transgenic semiwild soybean significantly decreased compared with that of wild soybean ( p < 0.05). There was no significant difference ( p > 0.05) in the Simpson index of fungi in the rhizosphere soil of the transgenic herbicide-resistant semiwild soybean at the flowering stage compared with that of Hujiao 698, but it was significantly lower ( p < 0.05) than those of Dongnong 50 and wild soybean. LEfSe analyses of the bacterial and fungi communities LEfSe analysis of the bacterial community The results of the linear discriminant analysis of the bacterial community are shown in Fig. a-c. Figure a shows that, Rhizobiaceae, Pseudomonadaceae, Pseudonocardiaceae, Dyadobacter and Novosphingobium were more prominent in DT-1 than in D50. As depicted in Fig. b, Proteobacteria , Alphaproteobacteria, Pseudonocardiaceae, Sinobacteraceae, Solitalea and DA101 were prominent in DT-1 than in HJ698. As depicted in Fig. c, Chitinophaga and Dyadobacter were more predominant in DT-1 than in WS-1. LEfSe analysis of the fungal community The results of the linear discriminant analysis of the fungal community are shown in Fig. a-c. Figure a shows that, Glomerellales, Capnodiales, Chaetomiaceae, Phaeosphaeriaceae and Cantharellales fam incertae sedis were more prominent in DT-1 than in D50. As depicted in Fig. b, Basidiomycota, Glomerellales, Phaeosphaeriaceae, Nectria and Schizothecium were more prominent in DT-1 than in HJ698. As depicted in Fig. c, Trichosphaeriales, Hypocreales, Cantharellales fam incertae sedis, Phaeosphaeriaceae and Curvularia were more predominant in DT-1 than in WS-1.   The data presented in figure S2 demonstrates that the rhizosphere soil culturable bacteria and fungi counts of DT-1 during the seedling, pod, and mature stages of soybean growth did not significantly differ from those of D50, HJ698, and WS-1. However, at the flowering stage, the rhizosphere soil culturable bacteria count of DT-1 was significantly different from that of D50. Nevertheless, there was no significant difference in the rhizosphere soil culturable bacteria counts of DT-1 and WS-1. During the flowering stage, the rhizosphere soil culturable fungi count of DT-1 differed significantly from that of WS-1 and HJ698. However, there was no significant difference in the rhizosphere soil culturable fungi counts of DT-1 and D50 (see Supplementary Materials).   Bacterial and fungal community compositions at the flowering stage The relative abundance at the phylum level of the bacteria and fungi in the rhizosphere soil of the different soybeans at the flowering stage are shown in Fig. a and c. Compared with those of D50, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 22.11% and 8.68–23.31% and 8.98%, respectively, whereas those of Acidobacteria decreased from 40.39 to 37.34%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 66.17% and 6.08–76.58% and 10.93%, respectively, whereas that of Mortierellomycota decreased from 7.88 to 7.44%. Compared with those of HJ698, the relative abundances of Acidobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 32.36% and 6.65–37.34% and 8.98%, respectively, whereas that of Proteobacteria decreased from 28.68 to 23.31%. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 75.83%, 7.51%, and 6.02–76.58%, 10.93%, and 7.44%, respectively. Compared with that of WS-1, the relative abundance of Acidobacteria in the rhizosphere soil of DT-1 increased from 29.08 to 37.34%, whereas those of Proteobacteria and Bacteroidetes decreased from 27.43% and 12.60–23.31% and 8.98%, respectively. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 51.64%, 6.00%, and 6.18–76.58%, 10.93%, and 7.44%, respectively. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Steroidobacter , Lysobacter , Pseudomonas , Chitinophaga , Niabella , Stenotrophomonas , and Aquicella and the fungi Acremonium , Guehomyces , Herpotrichia , Nectria , Podospora , Pseudogymnoascus , Tetracladium , Ustilago , Kochiomyces , Chloridium , and Phialemonium were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Kaistobacter , Sphingomonas , Ramlibacter , Methylibium , Segetibacter , Bradyrhizobium , Hymenobacter , Phormidium , Novosphingobium , Agrobacterium , Janthinobacterium , Leptolyngbya , and Rubrivivax and the fungi Alternaria , Ascobolus , Bipolaris , Cladosporium , Hannaella , Phaeosphaeria , Plectosphaerella , Schizothecium , Setosphaeria , Thanatephorus , Trebouxia , Paraphoma , Coniosporium , and Nigrospora were lower in the rhizosphere soil of DT-1. Bacterial and fungal community composition at the mature stage The relative abundances at the phylum level of the rhizosphere bacteria and fungi in the different soybeans at the mature stage are shown in Fig. a and c. Compared with those of D50, the relative abundance of Proteobacteria in the rhizosphere soil of DT-1 increased from 34.70 to 36.54%, whereas those of Acidobacteria and Bacteroidetes decreased from 25.72% and 13.21–24.82% and 11.61%, respectively. The relative abundance of Ascomycota in the rhizosphere soil of DT-1 increased from 68.73 to 79.00%, whereas those of Basidiomycota and Mortierellomycota decreased from 14.59% and 7.03–7.24% and 6.40%, respectively. Compared with those of HJ698, the relative abundances of Proteobacteria and Acidobacteria in the rhizosphere soil of DT-1 increased from 35.29% and 18.63–36.54% and 24.82%, respectively, whereas that of Bacteroidetes decreased from 22.23 to 11.61%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 72.05% and 6.90–79.00% and 7.24%, respectively, whereas that of Mortierellomycota decreased from 8.08 to 6.40%. Compared with those of WS-1, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 30.62% and 9.60–36.54% and 11.61%, respectively, whereas that of Acidobacteria decreased from 30.85 to 24.82%. The relative abundances of Ascomycota and Mortierellomycota in the rhizosphere soil of DT-1 increased from 70.99% and 6.01–79.00% and 6.40%, respectively, whereas that of Basidiomycota decreased from 11.42 to 7.24%. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Bradyrhizobium , Nitrospira , Rhodoplanes , DA101, Segetibacter , Hymenobacter , Dyadobacter , Bdellovibrio , and Aeromicrobium and the fungi Cladosporium , Clonostachys , Edenia , Gibberella , Guehomyces , Nectria , Nigrospora , Phaeosphaeria , Plectosphaerella , and Solicoccozyma were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Ramlibacter , Rubrobacter , and Duganella and the fungi Leptosphaeria , Preussia , Pseudogymnoascus , Toninia , Trichocladium , and Mrakiella were lower in the rhizosphere soil of DT-1. In this study, the population of delayed Rhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean during the flowering period decreased compared to that of the other three soybeans. However, the population of Bradyrhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean at the mature stage increased compared to that of the other three soybeans.   The relative abundance at the phylum level of the bacteria and fungi in the rhizosphere soil of the different soybeans at the flowering stage are shown in Fig. a and c. Compared with those of D50, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 22.11% and 8.68–23.31% and 8.98%, respectively, whereas those of Acidobacteria decreased from 40.39 to 37.34%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 66.17% and 6.08–76.58% and 10.93%, respectively, whereas that of Mortierellomycota decreased from 7.88 to 7.44%. Compared with those of HJ698, the relative abundances of Acidobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 32.36% and 6.65–37.34% and 8.98%, respectively, whereas that of Proteobacteria decreased from 28.68 to 23.31%. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 75.83%, 7.51%, and 6.02–76.58%, 10.93%, and 7.44%, respectively. Compared with that of WS-1, the relative abundance of Acidobacteria in the rhizosphere soil of DT-1 increased from 29.08 to 37.34%, whereas those of Proteobacteria and Bacteroidetes decreased from 27.43% and 12.60–23.31% and 8.98%, respectively. The relative abundances of Ascomycota, Basidiomycota, and Mortierellomycota in the rhizosphere soil of DT-1 increased from 51.64%, 6.00%, and 6.18–76.58%, 10.93%, and 7.44%, respectively. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Steroidobacter , Lysobacter , Pseudomonas , Chitinophaga , Niabella , Stenotrophomonas , and Aquicella and the fungi Acremonium , Guehomyces , Herpotrichia , Nectria , Podospora , Pseudogymnoascus , Tetracladium , Ustilago , Kochiomyces , Chloridium , and Phialemonium were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Kaistobacter , Sphingomonas , Ramlibacter , Methylibium , Segetibacter , Bradyrhizobium , Hymenobacter , Phormidium , Novosphingobium , Agrobacterium , Janthinobacterium , Leptolyngbya , and Rubrivivax and the fungi Alternaria , Ascobolus , Bipolaris , Cladosporium , Hannaella , Phaeosphaeria , Plectosphaerella , Schizothecium , Setosphaeria , Thanatephorus , Trebouxia , Paraphoma , Coniosporium , and Nigrospora were lower in the rhizosphere soil of DT-1.   The relative abundances at the phylum level of the rhizosphere bacteria and fungi in the different soybeans at the mature stage are shown in Fig. a and c. Compared with those of D50, the relative abundance of Proteobacteria in the rhizosphere soil of DT-1 increased from 34.70 to 36.54%, whereas those of Acidobacteria and Bacteroidetes decreased from 25.72% and 13.21–24.82% and 11.61%, respectively. The relative abundance of Ascomycota in the rhizosphere soil of DT-1 increased from 68.73 to 79.00%, whereas those of Basidiomycota and Mortierellomycota decreased from 14.59% and 7.03–7.24% and 6.40%, respectively. Compared with those of HJ698, the relative abundances of Proteobacteria and Acidobacteria in the rhizosphere soil of DT-1 increased from 35.29% and 18.63–36.54% and 24.82%, respectively, whereas that of Bacteroidetes decreased from 22.23 to 11.61%. The relative abundances of Ascomycota and Basidiomycota in the rhizosphere soil of DT-1 increased from 72.05% and 6.90–79.00% and 7.24%, respectively, whereas that of Mortierellomycota decreased from 8.08 to 6.40%. Compared with those of WS-1, the relative abundances of Proteobacteria and Bacteroidetes in the rhizosphere soil of DT-1 increased from 30.62% and 9.60–36.54% and 11.61%, respectively, whereas that of Acidobacteria decreased from 30.85 to 24.82%. The relative abundances of Ascomycota and Mortierellomycota in the rhizosphere soil of DT-1 increased from 70.99% and 6.01–79.00% and 6.40%, respectively, whereas that of Basidiomycota decreased from 11.42 to 7.24%. The distribution of the bacterial and fungal community compositions at the genus level is illustrated in Fig. b and d. Compared with those of the other three soybeans, the abundances of the bacteria Bradyrhizobium , Nitrospira , Rhodoplanes , DA101, Segetibacter , Hymenobacter , Dyadobacter , Bdellovibrio , and Aeromicrobium and the fungi Cladosporium , Clonostachys , Edenia , Gibberella , Guehomyces , Nectria , Nigrospora , Phaeosphaeria , Plectosphaerella , and Solicoccozyma were greater in the rhizosphere soil of DT-1. In contrast, compared with those of the other three soybeans, the abundances of the bacteria Ramlibacter , Rubrobacter , and Duganella and the fungi Leptosphaeria , Preussia , Pseudogymnoascus , Toninia , Trichocladium , and Mrakiella were lower in the rhizosphere soil of DT-1. In this study, the population of delayed Rhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean during the flowering period decreased compared to that of the other three soybeans. However, the population of Bradyrhizobium associated with symbiotic nitrogen fixation in the rhizosphere soil of DT-1 soybean at the mature stage increased compared to that of the other three soybeans.   Diversity of the bacterial community The results of the bacterial diversity analysis are shown in Figs. and a-d. The Shannon and Simpson diversity indices were used to evaluate microbial diversity . Both indices indicated that the rhizosphere bacteria of DT-1 were similar to those of the other three soybeans at all stages. At the pod stage, the Shannon index of Hujiao 698 was significantly lower than that of wild soybean ( p < 0.05), indicating a reduction in the species diversity of soil bacteria in the rhizosphere due to the root exudates of Hujiao 698. However, there was no significant difference in the Shannon index between the transgenic EPSPS-resistant semiwild soybean and wild soybean, suggesting that the decline in the species diversity of the soil bacteria in the rhizosphere was not caused by the EPSPS protein. Diversity of the fungi community The results of the fungal diversity analysis are shown in Figs. and a-d. There was no significant difference between the Shannon index of the transgenic semiwild soybean and those of Dongnong 50 and Hujiao 698 at the flowering stage ( p > 0.05), but the Shannon index of the transgenic semiwild soybean significantly decreased compared with that of wild soybean ( p < 0.05). There was no significant difference ( p > 0.05) in the Simpson index of fungi in the rhizosphere soil of the transgenic herbicide-resistant semiwild soybean at the flowering stage compared with that of Hujiao 698, but it was significantly lower ( p < 0.05) than those of Dongnong 50 and wild soybean.   The results of the bacterial diversity analysis are shown in Figs. and a-d. The Shannon and Simpson diversity indices were used to evaluate microbial diversity . Both indices indicated that the rhizosphere bacteria of DT-1 were similar to those of the other three soybeans at all stages. At the pod stage, the Shannon index of Hujiao 698 was significantly lower than that of wild soybean ( p < 0.05), indicating a reduction in the species diversity of soil bacteria in the rhizosphere due to the root exudates of Hujiao 698. However, there was no significant difference in the Shannon index between the transgenic EPSPS-resistant semiwild soybean and wild soybean, suggesting that the decline in the species diversity of the soil bacteria in the rhizosphere was not caused by the EPSPS protein.   The results of the fungal diversity analysis are shown in Figs. and a-d. There was no significant difference between the Shannon index of the transgenic semiwild soybean and those of Dongnong 50 and Hujiao 698 at the flowering stage ( p > 0.05), but the Shannon index of the transgenic semiwild soybean significantly decreased compared with that of wild soybean ( p < 0.05). There was no significant difference ( p > 0.05) in the Simpson index of fungi in the rhizosphere soil of the transgenic herbicide-resistant semiwild soybean at the flowering stage compared with that of Hujiao 698, but it was significantly lower ( p < 0.05) than those of Dongnong 50 and wild soybean.   LEfSe analysis of the bacterial community The results of the linear discriminant analysis of the bacterial community are shown in Fig. a-c. Figure a shows that, Rhizobiaceae, Pseudomonadaceae, Pseudonocardiaceae, Dyadobacter and Novosphingobium were more prominent in DT-1 than in D50. As depicted in Fig. b, Proteobacteria , Alphaproteobacteria, Pseudonocardiaceae, Sinobacteraceae, Solitalea and DA101 were prominent in DT-1 than in HJ698. As depicted in Fig. c, Chitinophaga and Dyadobacter were more predominant in DT-1 than in WS-1. LEfSe analysis of the fungal community The results of the linear discriminant analysis of the fungal community are shown in Fig. a-c. Figure a shows that, Glomerellales, Capnodiales, Chaetomiaceae, Phaeosphaeriaceae and Cantharellales fam incertae sedis were more prominent in DT-1 than in D50. As depicted in Fig. b, Basidiomycota, Glomerellales, Phaeosphaeriaceae, Nectria and Schizothecium were more prominent in DT-1 than in HJ698. As depicted in Fig. c, Trichosphaeriales, Hypocreales, Cantharellales fam incertae sedis, Phaeosphaeriaceae and Curvularia were more predominant in DT-1 than in WS-1.   The results of the linear discriminant analysis of the bacterial community are shown in Fig. a-c. Figure a shows that, Rhizobiaceae, Pseudomonadaceae, Pseudonocardiaceae, Dyadobacter and Novosphingobium were more prominent in DT-1 than in D50. As depicted in Fig. b, Proteobacteria , Alphaproteobacteria, Pseudonocardiaceae, Sinobacteraceae, Solitalea and DA101 were prominent in DT-1 than in HJ698. As depicted in Fig. c, Chitinophaga and Dyadobacter were more predominant in DT-1 than in WS-1.   The results of the linear discriminant analysis of the fungal community are shown in Fig. a-c. Figure a shows that, Glomerellales, Capnodiales, Chaetomiaceae, Phaeosphaeriaceae and Cantharellales fam incertae sedis were more prominent in DT-1 than in D50. As depicted in Fig. b, Basidiomycota, Glomerellales, Phaeosphaeriaceae, Nectria and Schizothecium were more prominent in DT-1 than in HJ698. As depicted in Fig. c, Trichosphaeriales, Hypocreales, Cantharellales fam incertae sedis, Phaeosphaeriaceae and Curvularia were more predominant in DT-1 than in WS-1.   The insertion of foreign genes in transgenic crops has the potential to alter both the composition and quantity of root exudates , and the exudates released from the roots of these plants may consequently impact the abundance of soil microbial communities . The hybrid offspring formed after the genetic drift of the glyphosate-tolerant transgenic soybean gene contained the glyphosate tolerance gene, and its secretions caused microbial changes, which can reflect the impact of transgenic crops on the soil ecological environment. These results indicated that DT-1 produced by genetic drift had no effect on rhizosphere soil bacteria or fungi. However, some results have shown a significant increase in the total bacterial biomass in soil with transgenic soybean, in contrast to that in soil cultivated with nontransgenic soybean . This study revealed that the species diversity of fungi in the rhizosphere soil of transgenic herbicide-resistant semiwild soybean plants was significantly lower than that in the rhizosphere soil of wild soybean plants at the flowering stage of soybean growth. However, there was no significant difference in the species diversity of fungi in the rhizosphere soil of the transgenic herbicide-resistant semiwild soybean compared with those of the other three soybeans at the other growth stages, which indicates that the species diversity of fungi in the rhizosphere soil of the transgenic herbicide-resistant semiwild soybean has temporal specificity and does not affect the soil environment. Most studies have consistently demonstrated that there are no significant differences in the diversities of bacterial communities between transgenic glyphosate-resistant soybeans and nontransgenic soybeans at various stages , . One study reported that GR significantly increased the abundance and diversity of the soil rhizospheric bacterial community at the pod stage . The glyphosate-tolerant soybean affecting the phylogenetic diversity of rhizosphere bacterial communities together with a significant difference in the relative abundances of some rhizosphere bacteria at different classification levels compared with those of the control cultivar . The maturation of soybeans influences the composition of key microorganism communities in soil . The results of this study indicate that Proteobacteria was found to be advantageous over different types of soybeans during maturity. This is consistent with the findings of Nie et al. and Wang et al. , These results indicated that Proteobacteria, Acidobacteria and Actinobacteria are the predominant phyla. Compared with that in nontransgenic soybean, the relative abundance of Proteobacteria in transgenic glyphosate-resistant soybean was lower by 5.28% at the mature stage . However, our experiment revealed that the transgenic glyphosate-resistant semiwild soybean presented a greater abundance of Proteobacteria compared to other soybeans. The abundance of Bradyrhizobium associated with symbiotic nitrogen fixation in the transgenic glyphosate-resistant semiwild soybean decreased during the flowering stage but increased during the maturity stage compared to those in the other soybeans. Rhizobacteria have the ability to establish symbiotic nodules specifically with leguminous plants, and the slow-growing root nodule bacteria in soybeans serve as nitrogen-fixing symbionts. Therefore, compared with nontransgenic soybeans, transgenic herbicide-resistant semiwild-type soybean with root nodules exhibit increased nitrogen fixation . Legume‒rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture . At present, there are few reports on the diversity of fungi in the rhizosphere soil of transgenic glyphosate-resistant soybean. This study provides basic data on the diversity of rhizosphere fungi in transgenic soybeans. There was no significant difference in the microbial abundance of transgenic cotton compared with that of nontransgenic cotton in studies by Qi and others . The flowering stage represents the most vigorous period of soybean plant growth and has the greatest effect on soil microorganisms . This study revealed that at the flowering stage of soybean growth, the genetic diversity of fungi in the rhizosphere soil of transgenic glyphosate-resistant semiwild soybean was significantly lower than that in the rhizosphere soil of wild soybean. The sequencing results revealed that during different periods of soybean growth, the rhizosphere fungi of the rhizosphere soil of the transgenic glyphosate-resistant semiwild soybean were dominated by Basidiomycota over those of the cultivated Dongnong 50 and wild soybeans. Therefore, the predominant phylum associated with the rhizosphere fungi of genetically modified glyphosate-resistant semiwild soybean is Basidiomycota.   This study investigated the impact of transgenic glyphosate-resistant semiwild soybean on the root zone soil microbial community through detailed field experiments and high-throughput sequencing technology. The results revealed that the bacterial and fungal populations in the rhizosphere soil of the transgenic glyphosate-resistant semiwild soybean at the seedling, pod, and mature stages were not significantly different from those in the rhizosphere soil of the other soybeans. However, the fungal species diversity in the rhizosphere soil of the transgenic glyphosate-resistant semiwild soybean was significantly lower than that in the rhizosphere soil of the wild soybean at the flowering stage. Additionally, the abundance of Proteobacteria was greater in the mature stage of transgenic glyphosate-resistant semiwild soybean than in other soybeans, whereas the number of nitrogen-fixing root nodules associated with soybeans decreased in the flowering stage and increased in the mature stage. These findings provide a new perspective for understanding the impact of transgenic soybean genetic drift on the soil microenvironment and provide important scientific evidence for future assessments of the environmental risk of transgenic crops.   Below is the link to the electronic supplementary material. Supplementary Material 1   