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Article

Responses of Soybean Biomass and Bacterial Community Diversity of AMF Spore-Associated and Soybean Rhizosphere Soil to Microbial Inoculation and Chlorothalonil

by
Weiguang Jie
1,2,* and
Min Zhang
1
1
Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education & Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region & Key Laboratory of Microbiology, College of Heilongjiang Province & School of Life Sciences, Heilongjiang University, Harbin 150080, China
2
School of Food Engineering, Heilongjiang East University, Harbin 150066, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(3), 738; https://doi.org/10.3390/agronomy15030738
Submission received: 12 February 2025 / Revised: 12 March 2025 / Accepted: 18 March 2025 / Published: 19 March 2025
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

:
Arbuscular mycorrhizal fungi (AMF) and phosphorus-solubilizing bacteria (PSB) play crucial roles in enhancing crop growth, increasing yields, and improving the soil microbial environment. The aim of this study was to investigate the effects of microbial inoculation and chlorothalonil on the AMF colonization rate in soybean roots, AMF spore density, nodule number, soybean biomass, and the composition of bacterial communities associated with soybean rhizosphere soil and AMF spores. The results indicated that the AMF colonization rate in soybean roots, AMF spore density, nodule number, and soybean biomass in the treatment inoculated with both Rhizophagus intraradices and Acinetobacter calcoaceticus were significantly greater than those in the other treatments. Inoculation with R. intraradices and A. calcoaceticus and spraying with chlorothalonil could influence the bacterial diversity in the rhizosphere soil of soybean. Compared with that in the control treatment, the relative abundance of Firmicutes in the rhizosphere soil of soybean plants inoculated with R. intraradices increased by 1.40%. In addition, both spraying with chlorothalonil and inoculation with A. calcoaceticus influenced the composition of AMF spore-associated bacterial communities. The relative abundance of Proteobacteria in AMF spore from soybean rhizosphere soil inoculated with R. intraradices and A. calcoaceticus increased by 12.42% compared to that in samples inoculated solely with A. calcoaceticus. This study provides a theoretical basis for microbial inoculation in improving the microenvironment of soybean rhizosphere soil and increasing soybean biomass.

1. Introduction

Soybean (Glycine max L.), one of the most important food crops worldwide, is rich in protein, oil, carbohydrates, vitamins, and minerals. It plays a crucial role in China’s agricultural output and national economy [1]. The soybean planting areas in China are distributed mainly in Northeast China and the Huang-Huai-Hai region. Despite the continued expansion of the soybean planting area, the prevalence of soybean root rot disease often leads to reduced soybean production or even abrogates production completely. Chemical pesticides, including chlorothalonil, can effectively prevent and control soybean root rot and have been widely applied. Chlorothalonil is an effective, efficient, and broad-spectrum organochlorine pesticide. It inhibits the activity of glycerol 3-phosphate dehydrogenase in fungi, providing excellent control over diseases [2]. After application, chlorothalonil can accumulate on the soil surface or enter the soil through plant residues. This can affect soil microbial diversity and function, potentially leading to soil ecosystem dysfunction [3,4]. It has been reported that some microorganisms can degrade soil pesticide residues, improve the soil ecological environment and increase crop biomass [5,6].
Arbuscular mycorrhizal fungi (AMF) exist widely in nature. They are oligotrophic microorganisms that can form symbiotic relationships with more than 80% of land plants [7,8]. AMF utilize the carbon fixed by plants to produce specific secretions that attract the mycelial microbiome [9]. They mediate interactions between crops and mycorrhizal microorganisms, influence the diversity of bacterial communities, promote crop growth, improve crop stress resistance, and reduce pesticide residues in soil and plants [8,9]. Jie et al. [10] showed that inoculation of Rhizophagus intraradices could promote soybean growth and increase soybean biomass. In addition, AMF can increase the absorption of nitrogen (N) and phosphorus (P) in soybean [11,12]. They create a suitable ecological niche for the growth and colonization of rhizobia, thereby increasing the number of soybean root nodules and increasing soybean biomass [13]. Furthermore, phosphorus can promote the formation and growth of plant roots, increase plant disease resistance, and improve crop quality [14]. Owing to the lack of phytase-encoding genes in AMF, organophosphorus cannot be directly utilized. However, by altering the composition of the soil microbial communities and increasing the relative abundance of bacteria that possess phosphatase-encoding genes, AMF can facilitate phosphorus mineralization and increase the organophosphorus utilization rate in soil [15].
Phosphorus-solubilizing bacteria (PSB) are microorganisms that convert insoluble phosphorus to soluble forms via the activity of secreted phosphatases [16]. PSB can increase the uptake of phosphorus by plants, inhibit the growth of pathogenic microorganisms, and increase crop biomass [17]. Acinetobacter calcoaceticus can convert insoluble phosphorus in soil into soluble inorganic phosphorus. This conversion helps enhance soil fertility, increase crop yield, improve crop quality, improve plant photosynthetic capacity and root morphology [18]. Foughalia et al. [19] reported that inoculation with A. calcoaceticus significantly promoted root and shoot elongation in tomato seedlings, increased plant fresh weight, and significantly improved the resistance of tomato seedlings to Botrytis cinerea infection. As biofertilizers, PSB can increase the effectiveness of phosphorus uptake, reduce the need for chemical fertilizers, and contribute positively to environmental protection and sustainable agricultural development. Our previous study showed that inoculation with R. intraradices and A. calcoaceticus can significantly increase soybean growth and reduce carbendazim residue [10]. However, few studies have investigated the effects of AMF and PSB on the bacterial community diversity of AMF spore-associated and soybean rhizosphere soil.
The aim of this study was to explore the effects of R. intraradices, A. calcoaceticus, and chlorothalonil on the AMF colonization rate, AMF spore density, nodule number, soybean biomass, and the bacterial community diversity of AMF spore-associated and soybean rhizosphere soil. The following hypotheses were tested in this study. (1) The AMF colonization rate, AMF spore density, nodule number, and soybean biomass of inoculation R. intraradices and A. calcoaceticus would significantly higher than those of control treatment. (2) Inoculation with R. intraradices, A. calcoaceticus and/or chlorothalonil could affact the alpha and beta diversity, the relative abundance of the bacterial communities in soybean rhizosphere soil. (3) A. calcoaceticus and/or chlorothalonil could affact the alpha and beta diversity, the relative abundance of the bacterial communities in AMF spore. The purpose of this study was to provide a theoretical basis for promoting microbial growth in soybean, increasing soybean yield, reducing the negative effects of chlorothalonil on soil microorganisms and soybean, and contributing to the sustainable development of agriculture. This study provides a solid theoretical basis for developing eco-friendly microbial inocula, reducing the dependence on chemical pesticides and enhancing food security, and can contribute to the sustainable development of agricultural ecology.

2. Materials and Methods

2.1. Soybean Cultivar

In this study, the nontransgenic soybean variety Heinong 48 (a disease-sensitive and high-protein variety with an average protein content of 45.23% and an average fat content of 19.50%, abbreviated as HN48) was used as the experimental material. The soybean cultivar was purchased from Heilongjiang Academy of Agricultural Sciences in Harbin, China.

2.2. AMF Inoculum and Phosphorus-Solubilizing Inoculum

Rhizophagus intraradices is the dominant AMF collected by our research group from soybean field in Heilongjiang Province. Alfalfa was selected as the host plant, and a mixture of sterile soil, sand, and vermiculite (5:2:3, v/v/v) was utilized as the substrate. The plants were harvested after 4 months of pot propagation. The AM colonization level (93.7%) and spore density (500 per 10 g of air-dried soil) were determined after harvest. The collected roots and soil served as the R. intraradices inoculum.
Acinetobacter calcoaceticus was isolated from soybean fields in Heilongjiang Province by members of the research team. A. calcoaceticus was inoculated in LB liquid medium (shaking at 170 rpm at 28 °C for 24 h), and the culture was then centrifuged at 5000 rpm for 5 min. The bacteria were collected, washed with sterile water twice, and then diluted with sterile water to create a bacterial suspension with a concentration of 1 × 107 CFU/mL. This suspension was used as the A. calcoaceticus inoculum.

2.3. Pesticides

Chlorothalonil, with the chemical name tetrachloroisophthalonitrile (C8N2Cl4), is a highly efficient, low-toxicity, broad-spectrum fungicide. It interacts with glyceraldehyde-3-phosphate dehydrogenase in fungal cells by binding to cysteine residues in the enzyme. This interaction disrupts the enzyme’s activity, leading to the disruption of fungal cell metabolism and eventually to cell death [2,20]. Because of its effectiveness in the control of fungal diseases, it is extensively utilized in agricultural production.

2.4. Experimental Design

Potted plants were used in the experiment. Each pot was filled with 12 kg of soil sourced from a soybean field in Pingfang District, Harbin, Heilongjiang Province (45°66′ N, 126°61′ E). The soil type was typical black soil (Mollisols, classified by the USDA Soil Taxonomy). The initial soil properties were as follows: pH 7.19, organic matter content 27.23 g⋅kg−1, total nitrogen 1.32 g⋅kg−1, total phosphorus 0.86 g⋅kg−1, total potassium 24.19 g⋅kg−1, loam particle content 33.95%, clay particle content 23.18%, and sand particle content 42.87%. This test set included six treatments: blank control (CK), Chlorothalonil (C); R. intraradices (R); R. intraradices and A. calcoaceticus (RA); Chlorothalonil and R. intraradices (CR); and Chlorothalonil, R. intraradices, and A. calcoaceticus (CRA). Each treatment was set up with nine replicates, and the experiment was designed with a completely randomized block design. All the soybean plants were grown under controlled conditions: temperature of 23 ± 1 °C, photoperiod of 12 h and humidity > 60%. The production management mode was the same as that used in the soybean field to ensure the practical relevance of the test results.
In the AMF inoculum treatment, the R. intraradices inoculum (6 g per soybean seed) was evenly spread on the topsoil. The soil was subsequently spread evenly to a thickness of 1–2 cm. Six soybean seeds were sown in each pot, followed by a uniform layer of soil on top of the soybean seeds with a thickness of 1–2 cm. The other treatments were not inoculated with R. intraradices inoculum, but all other steps remained the same as those described above. One soybean plant was maintained after germination. Seven days after soybean emergence, A. calcoaceticus was injected into the treated soil via the root irrigation method. The inoculation amount was 5 mL per plant; the blank control was injected with 5 mL of sterile water. A 5 mg/mL solution of chlorothalonil was evenly sprayed on the surface of soybean rhizosphere soil 60 days and 90 days after soybean emergence, and the spraying volume was 10 mL per plant. The management mode was the same as the conventional management method for growing field crops.

2.5. Sample Collection

The soybean plants were removed from the pots 120 days after emergence. The soil in each pot was thoroughly mixed to immediately determine the spore density of AMF. The roots were kept intact, and the roots were shaken to collect rhizosphere soil. The rhizosphere soil was stored at −80 °C for the analysis of bacterial community composition. The plant roots were rinsed with running water to remove soil particles and other impurities. After the water was absorbed by filter paper and the samples were naturally air-dried, the corresponding parameters were immediately measured. Soybean roots were used for analysis of AMF colonization rate, nodule number, and plant biomass; soybean stems and seeds were also used for biomass analysis. Three biological replicates were used for each treatment.

2.6. Determination of the AMF Colonization Rate

The alkali separation-acid fuchsin method was used to determine the AMF colonization rate. Roots were washed and cut into 1 cm fragments which were partially digested in 10% KOH for 30–60 min at 90 °C. Digested samples were rinsed, soaked for 3–5 min in 5% lactic acid solution, and stained with lactic acid-fuchsin dye mixture for 30–60 min at 90 °C. The AMF colonization rate was estimated according to the formula: AMF colonization rate = number of colonized root segments/numbers of detected root segments [21].

2.7. Determination of the Spore Density of AMF and Spore Selection

The spore density of AMF was measured via wet sieve decanting and 50% sucrose centrifugation. Sievings from each sieve (230 µm and 106 µm) collected separately into small beakers was transferred again into the fine sieve (45 µm). The filtrate was rinsed through a gridded filter funnel using water to eliminate excess moisture. A pre-marked filter paper was folded to position the grid pattern as the collection surface. Post-filtration, the filter was carefully transferred to a large Petridish and examined under a stereomicroscope. The total number of viable AMF spores were calculated, and the result was expressed as number 50 g−1 of the air-dried soil [22].
AMF spores were screened, with 50 ± 10 spores examined per sample. The selected AMF spores were washed several times with sterile water until no soil attached and stored at −80 °C for analysis of the AMF spore-associated bacterial communities. Three biological replicates were used for each treatment.

2.8. Determination of Soybean Plant Biomass

The methods used for determining the plant height, stem diameter, root length, 100-grain weight, pod number, aboveground dry weight, underground dry weight, aboveground fresh weight, and underground fresh weight of soybean plants 120 days after emergence were previously described by Yang et al. [23].

2.9. Determination of Nodule Number in Soybean Plants

The soil that adhered to the surface of the intact roots of the soybean plants was removed. The roots were then gently washed with distilled water several times until no soil remained attached. The samples were subsequently allowed to dry naturally. The nodule number was determined via direct counting.

2.10. DNA Extraction, Amplification and 16S rRNA Gene Amplicon Sequencing

The DNA from the soybean rhizosphere soil and AMF spores was extracted via the PowerSoil DNA Isolation Kit (MOBIO Laboratories Inc., Carlsbad, CA, USA). The V3-V4 variable region of the bacterial 16S rRNA gene was amplified from genomic DNA via the universal bacterial primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) [24]. Each PCR reaction was conducted in a total volume of 20 μL containing template DNA (10 ng/μL), 0.8 μL of each primer (5 µM), 2.0 μL of dNTP (2.5 mM), 4.0 μL of FastPfu Buffer, 0.4 μL of FastPfu Polymerase, and ddH2O in a total volume of 20 μL. The PCR cycling conditions were as follows: 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 45 s, with an extension at 72 °C for 10 min and a final hold at 10 °C until termination. The PCR products were purified via the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), quantified with QuantiFluor™-ST (Promega, Madison, WI, USA), and then pooled at equal concentrations. Amplicon sequencing was conducted on the PacBio platform (Majorbio Biopharm Technology Co., Ltd., Shanghai, China). The data were analyzed on the online platform of the Majorbio Cloud Platform. All raw reads have been deposited into the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA1163012 and PRJNA1162954.

2.11. Sequence Analysis

The original sequence was processed using QIIME v1.9.1 [25]. FASTP v0.19.6 [26] and FLASH v1.2.11 [27] were used for quality control and paired-end sequence merging, respectively. Low-quality sequences < 200 bp with an average base quality score < 20 or containing ambiguous bases were removed before further analysis. Chimera removal was performed using USEARCH v11 [28]. High-quality sequences with a similarity of ≥97% were clustered into one operational taxonomic unit (OTU) [29,30]. OTUs were classified and identified via the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 October 2024) [31] algorithm through a search in GenBank. The Ace [32], Chao1 [33], Shannon [34], Simpson [35], and Good’s Coverage [36] were used to evaluate the microbial community richness and diversity and were measured using MOTHUR v1.30 [37]. Venn diagrams showing the distribution of common and unique OTUs based on 97% sequence similarity were constructed. Beta diversity was analyzed via the Bray–Curtis distance calculation method and visualized via principal coordinates analysis (PCoA). Histograms were created to visualize the composition of the bacterial communities at the phylum and genus levels. Two heatmaps illustrating the relative variances in genus-level abundance within samples were created via the R language v3.3.1 pheatmap 1.0.8 package [38].

2.12. Statistical Analysis

Analysis of variance (ANOVA) and Duncan’s test (honestly significant difference, HSD) were applied to evaluate significant differences between treatments (p < 0.05) via SPSS 27.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Effects of Different Treatments on the AMF Colonization Rate

As illustrated in Table 1, the AMF colonization rate of soybean roots in the chlorothalonil spraying treatment was significantly lower than that in the control treatment (p < 0.05). These results suggest that chlorothalonil may disrupt the soil microbial environment, leading to harmful effects on soybean plant roots and reducing the AMF colonization rate of soybean roots. The AMF colonization rate inoculated with R. intraradices alone was significantly higher than that of the control treatment. However, the AMF colonization rate in the treatment sprayed with chlorothalonil and inoculated with R. intraradices and A. calcoaceticus was significantly higher than that in the chlorothalonil spraying treatment. The effect of R. intraradices and A. calcoaceticus could alleviate the harmful effects of chlorothalonil. Furthermore, the AMF colonization rate inoculated with R. intraradices and A. calcoaceticus was higher than that of the control treatment, indicating that A. calcoaceticus enhanced the symbiotic relationship between AMF and soybean roots.

3.2. Effects of the Different Treatments on AMF Spore Density

AMF spore density reflects the reproductive capacity of AMF in the soil. As shown in Table 1, inoculation with R. intraradices significantly increased AMF spore density. However, the effect was not as pronounced as that of inoculation with both R. intraradices and A. calcoaceticus (p < 0.05). R. intraradices and A. calcoaceticus could act synergistically with indigenous AMF to promote spore germination and reproduction of AMF in rhizosphere soil. Among the chlorothalonil treatments, the treatment sprayed with chlorothalonil alone presented the lowest density of AMF spores in the soil.

3.3. Effects of Different Treatments on the Number of Soybean Root Nodules

Root colonization by rhizobia can increase the nitrogen fixation ability of soybean plants, improve nitrogen nutrition, and increase soybean yield. As illustrated in Table 1, compared with the control treatment, the treatment inoculated with R. intraradices presented an increase in the number of soybean root nodules, although the effect was less pronounced than that observed in the treatment inoculated with both R. intraradices and A. calcoaceticus. The results indicated that R. intraradices and A. calcoaceticus promoted the colonization of soybean roots by rhizobia and had a positive synergistic effect with indigenous rhizobia. The number of soybean root nodules in the chlorothalonil spraying treatments were significantly lower than that in the non-spraying chlorothalonil treatments (p < 0.05).

3.4. Effects of Different Treatments on Soybean Plant Biomass

The plant height, stem diameter, root length, aboveground dry weight, underground dry weight, pod number, aboveground fresh weight, underground fresh weight, and 100-grain weight of soybean in the control treatment were 66.40 ± 0.61 cm, 6.38 ± 0.19 mm, 22.78 ± 0.20 cm, 25.04 ± 0.61 g, 2.70 ± 0.09 g, 33.00 ± 3.00, 66.86 ± 0.70, 10.83 ± 0.71 g, and 20.22 ± 0.13 g, respectively. Compared to the control treatment, inoculation with AMF inoculum (R and RA treatments) increased all the soybean plant biomass. In addition, the biomass of the mixed microbial inoculum treatment (RA) increased significantly, with the plant height, stem diameter, and 100-grain weight increasing by 26.37%, 22.78%, and 35.44%, respectively, compared with those of the blank control (CK) (Figure 1A,B,I). The results revealed that the synergistic effect of R. intraradices and A. calcoaceticus was beneficial for promoting soybean plant growth and increasing soybean yield. Under the same inoculation conditions, the plant height, stem diameter, root length, aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and 100-grain weight were significantly lower in the chlorothalonil spraying treatments than in the non-chlorothalonil treatments.

3.5. Diversity of the Bacterial Communities Associated with Rhizosphere Soil and AMF Spores

High-throughput sequencing was performed by the V3–V4 region of the 16S rRNA gene of bacteria associated with soybean rhizosphere soil and AMF spores under various treatments. After splice and quality control of the original data, 1, 484, 107 and 673, 413 original sequences were obtained from soybean rhizosphere soil and AMF spores, respectively. During the quality control process, 735, 336 and 490, 224 valid sequences were retained for subsequent analysis. USEARCH v11 software was used to perform cluster analysis of effective sequences at a 97% similarity level and obtain OTUs. After annotation against the SILVA v138 taxonomic database and OTU analysis, highly diverse bacterial communities with up to 3, 693 to 4, 123 OTUs were obtained from the rhizosphere soil samples (Table 2). Additionally, bacterial communities with up to 193 to 299 OTUs were obtained from AMF spores in rhizosphere soil (Table 3). In the Good’s Coverage of the rhizosphere soil and AMF spore-related bacteria was greater than 0.9760, indicating that the sequencing depth of all the samples was sufficient to represent the number of OTUs in the rhizosphere soil (Table 2) and AMF spores (Table 3). As depicted in Table 2, the number of OTUs and the Chao 1 and Ace indices of the bacterial communities in the rhizosphere soil of the soybean plants sprayed with chlorothalonil and inoculated with R. intraradices were greater than those in the other treatments. The results indicated that the composition of the soil bacterial communities in this treatment was relatively rich, indicating the presence of a greater number of bacterial species. The Simpson index analyses of the bacterial community composition in the soybean rhizosphere soil revealed that the Simpson index of the treatment inoculated with R. intraradices and A. calcoaceticus were lower than that of the control treatment. Furthermore, the Shannon index of the treatment inoculated with R. intraradices and A. calcoaceticus were higher than that of the control treatment. Interestingly, the Shannon index exhibited a remarkably low standard deviation across treatments, indicating that the observed bacterial diversity was highly consistent and stable under the same treatment. Alpha-diversity index difference may be attributed to the effect of R. intraradices and A. calcoaceticus on the soil bacterial community composition, leading to a significant increase in the diversity of the bacterial communities. As shown in Table 3, chlorothalonil and A. calcoaceticus had no effect on the richness and diversity of AMF spore-related bacteria.
Figure 2 shows the results of the PCoA of the beta-diversity of the soybean rhizosphere soil and AMF spore-associated bacteria. As illustrated in Figure 2A, the scatter points representing the rhizosphere soil bacterial community compositions of the different treatments all exhibited a certain projection distance on principal coordinate 1 (PC1). Among them, the treatment that was sprayed with chlorothalonil and inoculated with R. intraradices was more distant from the other treatments. These results suggest that the composition of the rhizosphere soil bacterial communities was significantly influenced by the combination of chlorothalonil and R. intraradices (p = 0.001). As shown in Figure 2B, there was no significant difference in the distribution of scatter points representing related bacterial communities on the surface of AMF spores among the different treatments along PC1. The results indicate that the composition of AMF spore-associated bacteria in the different treatments was similar. The projection distance on PC2 of the R. intraradices treatment was smaller than that of the other treatments. These findings indicate that A. calcoaceticus and chlorothalonil influence the composition of the AMF spore-associated bacterial communities.
A Venn diagram was used to illustrate differences in the distributions of common or unique OTUs differences between treatments (Figure 3). As illustrated in Figure 3A, there were 2, 729 common OTUs in the soybean rhizosphere soil samples, and the OTUs varied among the different samples. Consistent with the alpha diversity, there were more shared OTUs in the soybean rhizosphere soil samples. Among the samples of AMF spores in the soybean rhizosphere soil, there were 126 common OTUs (Figure 3B), with fewer shared OTUs among all treatments and more unique OTUs in each treatment. These results suggested that chlorothalonil and A. calcoaceticus might influence the distribution of AM spore-associated bacterial communities.
Histograms illustrating the relative abundance at the phylum level depict the bacterial phylum composition across various treatments in soybean rhizosphere soil (Figure 4A). Compared with that in the control treatment, the relative abundance of Myxococcota increased by 0.62% in the treatment inoculated with R. intraradices, indicating that R. intraradices promoted the growth of Myxococcota species. The relative abundance of Patescibacteria and Verrucomicrobiota increased significantly in the treatment inoculated with R. intraradices and A. calcoaceticus compared to the treatment inoculated solely with R. intraradices. Conversely, the relative abundance of Firmicutes and Myxococcota decreased significantly. The relative abundance of Patescibacteria and Verrucomicrobiota in the treatment inoculated with R. intraradices and A. calcoaceticus was higher than that in the other treatments. In addition, compared with those in the control treatment, the relative abundance of Firmicutes, Actinobacteriota, and Verrucomicrobiota in the chlorothalonil spraying treatment were reduced by 0.28%, 0.62%, and 0.38%, respectively. These findings suggest that chlorothalonil may inhibit the growth of these three bacterial phyla. At the genus level (Figure 4B), the relative abundances of Sphingomonas increased by 0.26% in the treatment sprayed with chlorothalonil and inoculated with R. intraradices compared with the treatment inoculated with only R. intraradices. The relative abundance of Arthrobacter and Aeromicrobium in the treatment inoculated with R. intraradices and A. calcoaceticus increased by 0.74% and 1.79%, respectively, compared to that in the treatment inoculated solely with R. intraradices. These results indicate that A. calcoaceticus positively influences the growth of both Arthrobacter and Aeromicrobium. The relative abundance of Rubrobacter increased in the treatment inoculated with R. intraradices; However, the effect was not as pronounced as that observed in the treatment inoculated with both R. intraradices and A. calcoaceticus. These findings suggest that the interaction between the two inoculated species is beneficial for the growth and reproduction of Rubrobacter. Compared with that in the control treatment, the relative abundance of Bradyrhizobium in the spray treatment decreased significantly, indicating that chlorothalonil may inhibit the growth of Bradyrhizobium.
According to the heatmap diagram of the bacterial communities at the genus level, the six soybean rhizosphere soil samples were divided into two clusters: The treatment inoculated with R. intraradices and the treatment sprayed with chlorothalonil clustered together; The other treatments clustered together, indicating that the bacterial communities among the four rhizosphere soil samples were similar (Figure 5). The results also demonstrated that the chlorothalonil, R. intraradices and A. calcoaceticus could affect the dominant genera and their relative abundances in the six soybean rhizosphere soil samples.
The spore walls and cytoplasm of AMF harbor a diverse array of bacteria. These bacteria can enhance ecological adaptability and establish symbiotic relationships with AMF by influencing spore germination, root colonization and the biological control of soil-borne diseases. Figure 6 illustrates the effects of various treatments on AMF spore-associated bacteria. At the phylum level (Figure 6A), Proteobacteria was the most dominant phylum within the communities of bacteria associated with AMF in the soybean rhizosphere soil. Since A. calcoaceticus belongs to the phylum Proteobacteria, the relative abundance of Proteobacteria in the treatment inoculated with both R. intraradices and A. calcoaceticus was significantly greater than that in the treatment inoculated solely with R. intraradices. The treatment that was sprayed with chlorothalonil and inoculated with R. intraradices was compared to the treatment inoculated with R. intraradices alone. The relative abundance of Firmicutes and Cyanobacteria decreased by 5.18% and 0.26%, respectively, indicating that chlorothalonil may inhibit the growth and reproduction of these microbial treatments. The relative abundance of Actinobacteriota in the treatment sprayed with chlorothalonil and inoculated with R. intraradices was 2.15% greater than that in the treatment sprayed with chlorothalonil and inoculated with both R. intraradices and A. calcoaceticus. These results indicate that A. calcoaceticus can promote the growth and reproduction of Actinobacteriota and is beneficial for mitigating the inhibitory effects of chlorothalonil on these microorganisms. At the genus level (Figure 6B), Acinetobacter was the most abundant genus, followed by Perlucidibaca and Pelomonas. The relative abundance of Perlucidibaca and Pelomonas in the treatment inoculated with R. intraradices and A. calcoaceticus increased by 2.17% and 0.37%, respectively, compared with their levels in the treatment inoculated with R. intraradices alone. The results indicated that A. calcoaceticus could influence the relative abundance of Acinetobacter species as well as other genera. The abundance of Zoogloea in the treatments inoculated with R. intraradices alone and those inoculated with both R. intraradices and A. calcoaceticus was 3.95% and 4.86%, respectively. However, the relative abundance of Zoogloea was nearly zero following the application of chlorothalonil. These results indicate that chlorothalonil can effectively inhibit or eliminate Zoogloea species. Zoogloea is a genus of microorganisms that can form bacterial micelles by secreting extracellular polymeric substances. This process enhances soil structural stability and improves the physical properties of the soil. Moreover, Zoogloea species can fix nitrogen, converting atmospheric nitrogen to a form that is accessible to plants. This process plays a crucial role in the nitrogen cycle within the soil.
The top 50 genera divided the four AMF spore samples into the following two treatments: The treatment inoculated with R. intraradices did not cluster with other AMF spore samples; The other three treatments of AMF spore samples clustered together, indicating their similar core function in shaping the composition of bacteria communities (Figure 7). Moreover, it also showed that both the microbial inoculation and chlorothalonil had effects on the dominant bacterial genera and their relative abundances in the four AMF spore samples.

4. Discussion

This study was conducted to investigate the effects of microbial inoculation and chlorothalonil on soybean biomass and bacterial community diversity of AMF spore-associated and soybean rhizosphere soil. This study showed that R. intraradices and A. calcoaceticus may increase the colonization rate of AMF, increase the spore density of AMF, and increase the number of soybean root nodules. As shown in Table 1, the AMF colonization rate, AMF spore density, and number of soybean root nodules in the AMF-inoculated treatments (R and RA) were significantly greater than those in the control treatment. Compared to the control treatment, the AMF colonization rate, AMF spore density, and the number of soybean root nodules of AMF in the treatment inoculated with R. intraradices and A. calcoaceticus increased by 140%, 696% and 74%, respectively. Importantly, there is a synergistic metabolic mechanism between AMF and hyphosphere bacteria. Fructose in hyphal secretions acts as a key signaling molecule to activate the expression of bacterial phosphatase genes [39]. Although AMF cannot directly secrete phosphatase, they can specifically enrich the microorganisms containing phosphatase coding genes and drive the enzymatic mineralization of organophosphates [15]. This transboundary regulation enables AMF to break through metabolic restrictions and establish a highly efficient phosphorous activation pathway. The interactions between fungi and bacteria not only shape the hyphosphere microbiome, but also significantly regulate the functions of AMF. The realization of mycorrhizal symbiosis depends on the synergy of specific microbial communities [40]. A. calcoaceticus can stimulate the release of flavonoids from crops and increase the formation of mycorrhizal structures [41]. R. intraradices and A. calcoaceticus synergistically enhanced the growth and propagation of AMF, increased the spore density of AMF, and increased the colonization rate of AMF in soybean roots. Compared with those in the treatment sprayed with chlorothalonil alone, the AMF colonization rate, AMF spore density, and node number increased by 157%, 164%, and 91%, respectively, in the treatment sprayed with chlorothalonil and inoculated with R. intraradices and A. calcoaceticus. The results indicated that R. intraradices and A. calcoaceticus could mitigate the adverse effects of chlorothalonil while enhancing the AMF colonization rate and promoting the reproduction of AMF and rhizobia. Jie et al. [10] reported that the synergistic effect of AMF and PSB can significantly reduce carbendazim pesticide residues in soybean seeds and rhizosphere soil. Furthermore, this combination enhances the colonization rate of AMF and increases the density of AMF spores.
As shown in Figure 1, the soybean plant biomass in the treatment sprayed with chlorothalonil and inoculated with R. intraradices and A. calcoaceticus was significantly greater than that in the control treatment. Furthermore, the synergistic interactions between AMF and PSB enhance phosphorus acquisition efficiency and promote nutrient absorption in plants. At present, the extensive use of chemical fertilizers and pesticides in soybean cultivation in China is leading to soil acidification, nutrient imbalance, organic pollution, and a decrease in microbial activity. AMF and PSB can improve the soil microbial environment and promote the nutrient absorption in crops [42]. PSB increase the availability of phosphorus, which is subsequently absorbed by AMF and transported to plants. This process promotes plant growth and increases biomass [43]. Pan et al. [44] reported that the combination of AMF and PSB significantly increased plant biomass accumulation, increased plant height and stem diameter, promoted root growth, and improved root structure. Compared with that in the control treatment, the biomass in the AMF treatments (R and RA) significantly increased (Figure 1). The most notable increases were observed in the treatment inoculated with R. intraradices and A. calcoaceticus, wherein plant height, stem diameter, and 100-grain weight increased by 26%, 23%, and 35%, respectively. The soybean plant biomass in the treatments that received chlorothalonil and AMF inoculum (CR and CRA) was significantly lower than that in the treatments (R and RA) that received the AM inoculum alone. The results indicated that chlorothalonil may impede the interaction between R. intraradices, A. calcoaceticus, and rhizosphere microorganisms and plants, leading to insufficient nutrient acquisition and reduced plant biomass.
Soil bacteria play crucial roles in ecological processes, including the carbon and nitrogen cycles, as well as the decomposition of organic matter. However, the composition of soil bacterial communities is influenced by various factors. Among these, microbial fertilizers significantly alter soil microbial composition, enhance diversity, and boost metabolic activity, which promotes the accumulation of microbial metabolites. Additionally, enriched microbial diversity induces the biosynthesis of defense-related compounds, such as antioxidant enzymes, chitinases, phytoalexins, and phenolics, thereby improving crop resistance and soil health [45]. Our findings revealed that R. intraradices, whether alone or in combination with A. calcoaceticus and/or chlorothalonil, influenced the composition of the bacterial communities. As shown in Figure 4A, the relative abundance of Proteobacteria, Firmicutes, and Myxococcota in the treatment inoculated with R. intraradices was significantly greater than that in the control treatment. Myxococcota species feed on pathogens and other harmful microorganisms in the soil, thereby reducing the incidence of soil-borne diseases and helping to maintain the ecological balance within the ecosystem [46,47]. The AMF colonization rate, AMF spore density, nodule number, and soybean biomass in the treatment inoculated with R. intraradices were significantly greater than those in the control treatment. AMF have access to nutrient patches outside the rhizosphere by producing an extensive network of fine hyphae, beyond the influence of the root and in soil pores inaccessible to roots [43]. The extraradical hyphae of AMF can transport PSB to organic phosphorus (P) patches and promote organic P mineralization [48]. The relative abundance of Acidobacteriota and Chloroflexi in the treatment inoculated with R. intraradices and A. calcoaceticus was significantly greater than that in the treatment inoculated solely with R. intraradices. This variation may be attributed to A. calcoaceticus potentially occupying or altering the ecological niches of certain microorganisms, thereby influencing the ecological dynamics of other soil microorganisms and subsequently affecting the composition of the soil bacterial communities. The soybean biomass in the treatment inoculated with R. intraradices and A. calcoaceticus was significantly greater than that in the treatment inoculated solely with R. intraradices, indicating that the combination of R. intraradices and A. calcoaceticus had the greatest beneficial effect. This improvement may be attributed to the influence of R. intraradices and A. calcoaceticus on enhancing the composition of the soil bacterial communities. Compared to the treatment inoculated with R. intraradices and A. calcoaceticus, the relative abundance of Proteobacteria in the treatment sprayed with chlorothalonil and inoculated with R. intraradices and A. calcoaceticus increased significantly. The application of chlorothalonil exerts selective pressure on soil microorganisms, leading to the enrichment of specific microbial groups that can degrade or tolerate pesticides, while the populations of sensitive bacterial groups decline. Baćmaga et al. [49] reported that chlorothalonil caused modifications in the abundance and biological diversity of soil microorganisms. It stimulated the growth of heterotrophic bacteria and actinobacteria and inhibited the growth of fungi. Rubrobacter species can thrive in extreme environments, play a significant role in soil carbon cycling, and can degrade organic compounds [50,51]. Compared with that in the control treatment, the relative abundance of Rubrobacter significantly increased following the application of chlorothalonil. Arthrobacter and Aeromicrobium may be sensitive to chlorothalonil, and their relative abundances decreased significantly following the application of chlorothalonil. Inoculation with R. intraradices and A. calcoaceticus could regulate the composition of soil bacterial communities, facilitating the involvement of diverse microorganisms in the exchange of nutrient elements between plants and soil. This process promotes plant growth and enhances crop biomass.
AMF spore-associated bacteria are located within AMF spores, adhering to the spore walls and the cytoplasm [52]. These bacterial communities usually have a variety of plant growth-promoting functions (plant hormones, antibiotics, iron carrier production, etc.) or mycorrhizal auxiliary functions (mycorrhizal colonization, AMF spore germination, AMF mycelial growth, etc.) [53]. In this study, the primary AMF spore-associated bacteria were identified as Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota, among others. Pseudomonas and Bacillus can promote AMF hyphal elongation and branching and improve AMF colonization efficiency within roots [54]. These interactions consequently contribute to improved plant growth and increased resistance to stress. In AMF spores, Proteobacteria was the most dominant treatment, with a relative abundance of more than 73%, followed by Firmicutes, which had a relative abundance exceeding 6%. A. calcoaceticus is a bacterium that belongs to the phylum Proteobacteria. Inoculating soil with A. calcoaceticus can increase the activities of soil phosphatase, catalase, and urease and influence the composition of the bacterial communities associated with AMF spores [55]. In addition, the results of the pot experiment revealed that the AMF colonization rate in soybean roots, AMF spore density (Table 1), and soybean biomass (Figure 1) in the treatment inoculated with R. intraradices and A. calcoaceticus were significantly greater than those in the other treatments. This increase may be attributed to the notable increase in the relative abundance of Proteobacteria among AMF spore-associated bacteria following inoculation with A. calcoaceticus (Figure 6A). At the genus level, the relative abundance of Acinetobacter and Pelomonas in AMF spore samples increased significantly following inoculation with R. intraradices and A. calcoaceticus (Figure 6B). Acinetobacter plays a crucial role in degrading organic pollutants, promoting plant growth, enhancing crop disease resistance, facilitating phosphorus cycling, and remediating heavy metal contamination [19]. Pelomonas can participate in the decomposition of organic matter, the nutrient cycle and the inhibition of pathogenic fungal activity [56]. Inoculation with A. calcoaceticus could enhance the soil microbial environment and significantly increase the AMF colonization rate, as well as the density of AMF spores (Table 1) and their biomass (Figure 1). In addition, the AMF spore-associated bacteria could also be influenced by soil environmental factors, including pH, temperature, and humidity. Pesticides can inhibit the growth of pathogenic fungi; However, they may also have detrimental effects on the soil environment. AMF can recruit stress-resistant bacteria to help mitigate the adverse impacts of pesticides. This study revealed that the abundance of Sphingomonas species on AMF spores was significantly greater in the treatment sprayed with chlorothalonil and inoculated with R. intraradices than in the treatment only inoculated with R. intraradices. Sphingomonas species can utilize a variety of organic pollutants, including polycyclic aromatic hydrocarbons, pesticides, and dyes, as sources of carbon and energy. This process helps mitigate the harmful effects of these substances on plants and the environment [57]. AMF spores recruit soil bacteria by releasing secretions rich in organic carbon compounds, including sugars and amino acids. These secretions not only provide a nutrient source for soil bacteria but also stimulate their growth and activity [48]. In summary, both the application of chlorothalonil and inoculation with A. calcoaceticus influence the composition of AM spore-associated bacteria. However, the mechanisms underlying these effects require further investigation.
Microbial inocula exhibit significant potential in sustainable agriculture by reducing reliance on chemical pesticides while enhancing crop productivity and soil health. Nevertheless, their efficacy is constrained by environmental factors such as soil pH, temperature, humidity, and microbial stability. Effective regulation of microbial-plant–soil interactions holds promise for advancing green agriculture, ensuring food security, and promoting ecological sustainability.

5. Conclusions

Inoculation with R. intraradices and A. calcoaceticus significantly increased the colonization rate of AMF, the spore density of AMF, the nodule number, and the biomass of soybean plants. Moreover, inoculation with R. intraradices and A. calcoaceticus could increase the microbial diversity of soybean rhizosphere soil and influence the composition of AM spore-associated bacteria. Proteobacteria was the most dominant phylum of AM spore-associated bacteria, whereas Actinobacteriota was the most dominant bacterial phylum found in the rhizosphere soil of soybean. Chlorothalonil may decrease the AMF colonization rate and reduce AMF spore density. Inoculation with R. intraradices and A. calcoaceticus could enhance soybean biomass and improve the bacterial community diversity of AMF spore-associated and soybean rhizosphere soil. This approach may offer both technical support and theoretical guidance for achieving sustainable agricultural development.

Author Contributions

W.J.: Conceptualization, writing—original draft preparation, methodology, and writing—review and editing. M.Z.: formal analysis and writing—original draft preparation. All authors have read and agreed to the published version of the manuscript.

Funding

This research and the APC was funded by Natural Science Foundation of Heilongjiang Province grant number LH2023C087.

Data Availability Statement

All raw reads have been deposited into the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA1163012 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1163012) accessed on 19 September 2024 and PRJNA1162954 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1162954) accessed on 1 October 2024.

Acknowledgments

Thanks to Haobo Yang for providing assistance in this research work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of different treatments on biomass of soybean plants. Plant height (A); stem diameter (B); root length (C); aboveground dry weight (D); underground dry weight (E); pod number (F); aboveground fresh weight (G); underground fresh weight (H); 100-grain weight (I). The values presented in (A–I) were all processed as follows: (treatment—blank control)/blank control %. The error bars represent the 95% confidence interval (CI). When the 95% CI did not overlap with zero, the response of each variable to different treatments was considered statistically significant. C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 1. Effects of different treatments on biomass of soybean plants. Plant height (A); stem diameter (B); root length (C); aboveground dry weight (D); underground dry weight (E); pod number (F); aboveground fresh weight (G); underground fresh weight (H); 100-grain weight (I). The values presented in (A–I) were all processed as follows: (treatment—blank control)/blank control %. The error bars represent the 95% confidence interval (CI). When the 95% CI did not overlap with zero, the response of each variable to different treatments was considered statistically significant. C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Figure 2. Principal coordinates analysis (PCoA) of the composition of soybean rhizosphere soil bacterial communities (A) and arbuscular mycorrhizal fungi spore-associated bacterial communities (B) was obtained by Bary–Curtis distance algorithm. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus; SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus; R-value: Significant differences within treatments; p-value: Significant differences between treatments; Ellipse: 95% confidence intervals.
Figure 2. Principal coordinates analysis (PCoA) of the composition of soybean rhizosphere soil bacterial communities (A) and arbuscular mycorrhizal fungi spore-associated bacterial communities (B) was obtained by Bary–Curtis distance algorithm. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus; SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus; R-value: Significant differences within treatments; p-value: Significant differences between treatments; Ellipse: 95% confidence intervals.
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Figure 3. The VENN diagrams of the composition of soybean rhizosphere soil bacterial communities (A) and arbuscular mycorrhizal fungi spore-associated bacterial communities (B). CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus; SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 3. The VENN diagrams of the composition of soybean rhizosphere soil bacterial communities (A) and arbuscular mycorrhizal fungi spore-associated bacterial communities (B). CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus; SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Figure 4. The composition of soybean rhizosphere soil bacterial communities at the phylum (A) and genus (B) levels. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 4. The composition of soybean rhizosphere soil bacterial communities at the phylum (A) and genus (B) levels. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Figure 5. Heatmap of the 50 most abundant soybean rhizosphere soil bacterial genera. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 5. Heatmap of the 50 most abundant soybean rhizosphere soil bacterial genera. CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Figure 6. The composition of arbuscular mycorrhizal fungi spore-associated bacterial communities at the phylum (A) and genus (B) levels. SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 6. The composition of arbuscular mycorrhizal fungi spore-associated bacterial communities at the phylum (A) and genus (B) levels. SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Figure 7. Heatmap of the 50 most abundant arbuscular mycorrhizal fungi spore-associated bacterial genera. SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
Figure 7. Heatmap of the 50 most abundant arbuscular mycorrhizal fungi spore-associated bacterial genera. SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus.
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Table 1. Effects of different treatments on the colonization rate and spore density of arbuscular mycorrhizal fungi and the number of soybean root nodules.
Table 1. Effects of different treatments on the colonization rate and spore density of arbuscular mycorrhizal fungi and the number of soybean root nodules.
TreatmentsCKCRRACRCRA
Arbuscular mycorrhizal fungi colonization rate0.40 ± 0.06 de0.35 ± 0.02 e0.93 ± 0.06 ab0.96 ± 0.01 a0.87 ± 0.06 bc0.90 ± 0.03 abc
Arbuscular mycorrhizal fungi spore density per gram of soil4.05 ± 6.03 c3.61 ± 5.02 c23.70 ± 16.18 b32.22 ± 29.38 a7.37 ± 6.28 c9.54 ± 7.91 c
Number of soybean root nodules per plant64.33 ± 2.08 e53.00 ± 1.00 f82.67 ± 3.21 c111.67 ± 3.06 a76.67 ± 1.53 d101.00 ± 3.06 b
Note: CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: Chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus. Values are means ± standard deviation with three replicates. Different letters indicate significant differences from different treatments (p < 0.05).
Table 2. Alpha-diversity of the bacterial communities in soybean rhizosphere soil.
Table 2. Alpha-diversity of the bacterial communities in soybean rhizosphere soil.
TreatmentsCKCRRACRCRA
OTU3757 ± 61.44 de3693 ± 35.85 e3910 ± 27.62 b3849 ± 21.79 bc4123 ± 48.69 a3819 ± 28.83 cd
Chao 14498.59 ± 107.81 c4513.74 ± 54.92 bc4558.84 ± 31.18 bc4632.91 ± 82.06 ab4741.82 ± 56.07 a4562.80 ± 38.78 bc
Shannon6.5671 ± 0.0137 e6.7059 ± 0.0288 d6.7838 ± 0.0160 c6.7611 ± 0.0134 c6.9920 ± 0.0135 a6.8575 ± 0.0084 b
Ace4654.08 ± 108.86 bc4599.21 ± 71.57 c4749.24 ± 35.34 b4769.68 ± 79.85 b4908.67 ± 69.75 a4682.67 ± 15.42 bc
Coverage0.9761 ± 0.0004 a0.9784 ± 0.0019 a0.9776 ± 0.0009 a0.9764 ± 0.0013 a0.9762 ± 0.0013 a0.9769 ± 0.0014 a
Simpson0.0078 ± 0.0001 a0.0042 ± 0.0001 c0.0042 ± 0.0002 c 0.0046 ± 0.0001 b0.0033 ± 0.0001 e0.0039 ± 0.0001 d
Note: CK: Blank control; C: Chlorothalonil; R: R. intraradices; RA: R. intraradices and A. calcoaceticus; CR: Chlorothalonil and R. intraradices; CRA: Chlorothalonil, R. intraradices and A. calcoaceticus. Values are means ± standard deviation with three replicates. Different letters indicate significant differences from different treatments (p < 0.05).
Table 3. Alpha-diversity of the arbuscular mycorrhizal fungi spore-related bacterial communities.
Table 3. Alpha-diversity of the arbuscular mycorrhizal fungi spore-related bacterial communities.
TreatmentsSRSRASCRSCRA
OTU283 ± 105.19 a193 ± 52.94 a229 ± 96.96 a299 ± 154.98 a
Chao 1290.72 ± 109.61 a201.70 ± 58.40 a238.92 ± 101.34 a316.46 ± 163.83 a
Shannon3.3961 ± 0.2992 a2.8866 ± 0.3387 a3.1025 ± 0.4394 a3.2732 ± 0.6613 a
Ace291.13 ± 108.69 a200.36 ± 54.74 a237.54 ± 99.81 a310.88 ± 155.06 a
Coverage0.9998 ± 0.0001 a0.9998 ± 0.0001 a0.9998 ± 0.0001 a0.9997 ± 0.0001 a
Simpson0.1010 ± 0.0295 a0.1530 ± 0.0625 a0.1259 ± 0.0355 a0.1354 ± 0.0420 a
Note: SR: R. intraradices; SRA: R. intraradices and A. calcoaceticus; SCR: Chlorothalonil and R. intraradices; SCRA: Chlorothalonil, R. intraradices and A. calcoaceticus. Values are means ± standard deviation with three replicates. Different letters indicate significant differences from different treatments (p < 0.05).
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Jie, W.; Zhang, M. Responses of Soybean Biomass and Bacterial Community Diversity of AMF Spore-Associated and Soybean Rhizosphere Soil to Microbial Inoculation and Chlorothalonil. Agronomy 2025, 15, 738. https://doi.org/10.3390/agronomy15030738

AMA Style

Jie W, Zhang M. Responses of Soybean Biomass and Bacterial Community Diversity of AMF Spore-Associated and Soybean Rhizosphere Soil to Microbial Inoculation and Chlorothalonil. Agronomy. 2025; 15(3):738. https://doi.org/10.3390/agronomy15030738

Chicago/Turabian Style

Jie, Weiguang, and Min Zhang. 2025. "Responses of Soybean Biomass and Bacterial Community Diversity of AMF Spore-Associated and Soybean Rhizosphere Soil to Microbial Inoculation and Chlorothalonil" Agronomy 15, no. 3: 738. https://doi.org/10.3390/agronomy15030738

APA Style

Jie, W., & Zhang, M. (2025). Responses of Soybean Biomass and Bacterial Community Diversity of AMF Spore-Associated and Soybean Rhizosphere Soil to Microbial Inoculation and Chlorothalonil. Agronomy, 15(3), 738. https://doi.org/10.3390/agronomy15030738

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