Effects of Rhizophagus intraradices and Acinetobacter calcoaceticus on Soybean Growth and Carbendazim Residue

Abstract

In agricultural production, carbendazim and other pesticides are used to prevent soybean root rot. However, carbendazim degrades slowly and affects a series of biochemical processes such as soil biological nitrogen fixation and soil nutrient activation in the natural environment. This study mainly investigated the effects of Rhizophagus intraradices, Acinetobacter calcoaceticus, and carbendazim on soybean biomass, the incidence of root rot, the total number of bacterial colonies and phosphorus-solubilizing bacteria in rhizosphere soil, and carbendazim residue in soybean grains and rhizosphere soil. The results showed that the arbuscular mycorrhizal fungi (AMF) spore density, AMF infection rate, soybean biomass, nodule number, total bacterial colonies, and phosphorus-solubilizing bacteria colonies in the soybean rhizosphere soil were the highest in the R. intraradices and A. calcoaceticus treatment groups under natural soil conditions. Moreover, the incidence of root rot and carbendazim residue in soybean grains and rhizosphere soil were the lowest in the R. intraradices and A. calcoaceticus treatment group under natural soil conditions. This result indicated that R. intraradices and A. calcoaceticus can effectively reduce carbendazim residue in soybean grains and rhizosphere soil. This study provided theoretical support for the development of microbial fertilizer and microbial degradation of pesticide residues and improved the practical basis for ensuring food safety.

1. Introduction

Soybean is one of the seven major grains and four major oil crops in China. Soybean is rich in protein, carbohydrates, fats, crude fiber, various minerals, and vitamins [1,2]. Heilongjiang Province is one of the main producing areas of soybean and occupies an important position in soybean production in China. In recent years, the soybean planting area has been expanding daily, but soybean root rot has become one of the most important factors leading to a decrease in soybean yield [3]. Soybean root rot is a widely distributed soil-borne disease that causes serious harm and is difficult to control, which leads to soybean yield reduction and quality deterioration, and it has become a major obstacle restricting the development of the soybean industry [4]. The pathogenic fungi that cause soybean root rot include Fusarium oxysporum, F. avenaceum, F. solanacearum, F. merismoides, Phytophthora sojae, and Pythium ultimum [5]. Chemical pesticides, including carbendazim, can effectively prevent and control soybean root rot and have been widely applied. Carbendazim, which is a benzimidazole fungicide, has endosorption and protective effects and has a better control effect on root rot caused by fungal infection [6]. However, carbendazim degrades slowly in the natural environment, and its residue in soil may affect the growth, reproduction, and metabolism of indigenous microorganisms and thus affect soil biochemical processes, such as biological nitrogen fixation, nitrification and denitrification, decomposition of organic matter, sulfur oxidation, and activation of soil nutrients. In addition, pesticide residues in soil pollute the soil environment and are not easily degraded, and soil nutrients decrease with increasing pollution degree [6,7]. Microbial fertilizers are known to promote crop growth, improve crop resistance, reduce pesticide use, reduce environmental pollution, and increase crop yields [8,9].

Arbuscular mycorrhizal fungi (AMF) are important and ubiquitous soil fungi that can form symbionts with more than 80% of terrestrial plants [10]. AMF can enhance crop disease resistance, promote crop growth, improve physical and chemical properties of soil, and degrade pesticide residue [11,12,13]. AMF can significantly increase alfalfa biomass and N and P absorption [14]. It has been shown that AMF colonization reduces the application rate of flonicamid and the residue level of flonicamid in soil [15]. In addition, because AM fungi lack the ability to secrete phosphatases, they cannot directly utilize organophosphorus nutrients, and their organic nutrients mainly come from mineralization driven by other soil microorganisms [16].

Phosphorus-solubilizing bacteria refer to a class of microorganisms that can activate insoluble inorganic phosphorus and organophosphorus in soil by secreting protons, organic acid cations, phosphatase, and other metabolites and convert phosphorus in soil, which is difficult for plants and AMF to absorb and utilize, into phosphorus that can be absorbed and utilized by plants and AMF [17]. Bargaz et al. studied the structure, function, and transcription of phosphorus-solubilizing bacteria. They found that phosphorus-solubilizing bacteria dissolved and mineralized insoluble phosphorus by releasing hydrolase and other substances into phosphorus-solubilizing medium and affected phytohormone signaling in crop root development to provide better growing conditions for crops [18]. Long et al. isolated a novel carbendazim-degrading strain, Rhodococcus sp. CX-1, from soil contaminated with carbendazim that can utilize carbendazim as its only carbon source and energy source [19].

However, there are few studies on the effects of AMF and phosphorus-solubilizing bacteria on soybean growth, root rot control, and pesticide residue degradation. This study mainly investigated the effects of AMF, phosphorus-solubilizing bacteria, and carbendazim on soybean biomass, root rot incidence, the total number of bacteria and phosphorus-solubilizing bacteria in plant rhizosphere soil, and carbendazim residue in soybean grains and plant rhizosphere soil. The purpose of this study is to provide a necessary theoretical basis for the study of microorganisms promoting crop growth, improving crop disease resistance, and degrading pesticide residue. At the same time, it contributes to the development of the soybean industry in our country, upgrading the production status of organic green soybean and increasing soybean yield in Heilongjiang Province.

2. Materials and Methods

2.1. Soybean Variety

The soybean variety Heinong 48 (disease-sensitive high-protein variety, protein content of approximately 45.23%, fat content of approximately 19.05%), purchased from Heilongjiang Academy of Agricultural Sciences, is the main soybean variety grown in Heilongjiang Province, China.

2.2. AMF Inocula and Phosphorous-Solubilizing Inocula

The AMF fungus (Rhizophagus intraradices) was obtained from a continuous soybean cropping field in Heilongjiang Province by members of our research group. The AM fungus was propagated in a pot culture with alfalfa plants grown in sterilized vermiculite, river sand, and soil (3: 2: 5, v/v/v) for approximately 5 months. The AM colonization (92.5%) and spore density (510 per 10 g of air-dried soil) were determined after harvest according to Jie et al. [20].

Phosphorus-solubilizing bacteria (Acinetobacter calcoaceticus) were obtained from a continuous soybean cropping field in Heilongjiang Province through screening by members of our research group and named PSB strains. The purified PSB was inoculated into LB liquid medium, incubated at 28 °C at 170 r/min for 24 h with shaking, and centrifuged at 5000 r/min for 5 min. The bacteria were collected and washed twice with sterile water, and the concentration of the bacterial solution was adjusted to 1.0 × 10⁸ CFU/mL with sterile water, which was considered to be the bacterial agent of PSB.

2.3. Pesticides

Carbendazim, chemically known as benzimidazole-2-methyl carbamate (C₉H₉N₃O₂), is a highly effective internal bactericide that can interfere with the formation of the mitotic spindle of pathogenic bacteria, affect cell division, and play a bactericidal role [21]. Carbendazim has a good control effect on root rot caused by fungi such as mycelia and polyascomycetes.

2.4. Experimental Design

Potted plants were used in the experiment, and the soil used was collected from the experimental field of Heilongjiang Oriental University (126°65′ E, 45°57′ N). The soil used in the experiment was divided into sterilized soil (121 °C intermittent sterilization for 1 h) and natural soil, and a total of 16 experimental treatment groups was established.

Sterilized soil treatment group: Blank control (MCK); R. intraradices (MR); A. calcoaceticus (MA); R. intraradices and A. calcoaceticus (MRA); carbendazim (MDCK); carbendazim and R. intraradices (MDR); carbendazim and A. calcoaceticus (MDA); carbendazim, R. intraradices and A. calcoaceticus (MDRA).

Natural soil treatment group: Blank control (NCK); R. intraradices (NR); A. calcoaceticus (NA); R. intraradices and A. calcoaceticus (NRA); carbendazim (NDCK); carbendazim and R. intraradices (NDR); carbendazim and A. calcoaceticus (NDA); carbendazim, R. intraradices, and A. calcoaceticus (NDRA).

Soybean seeds were superficially disinfected according to Jie et al. [20]. The disinfected soybean seeds were placed in sterile wet cotton and kept in darkness at 28 °C until the soybean radicle reached about 2 cm.

Nine replicates were prepared in each treatment group, each pot was loaded with 12 kg soil, 6 plants were seeded in each pot, and 3 plants were kept after germination. Pots of the same size were placed around the bowls as protective rows to reduce the marginal effect. All plants were grown at the temperature of 23 ± 1 °C, a photoperiod of 12 h, and humidity > 60%. The production management mode was the same as that of the adjacent field to ensure the practical significance of the test results.

Inoculant method: R. intraradices inocula (90 g per pot) was evenly spread on the topsoil of the corresponding treatment group. A soil thickness of 1–2 cm was evenly spread on R. intraradices inocula, and then the soybean seeds were evenly spread on the soil layer. Finally, a soil thickness of 1–2 cm was evenly spread on the soybean seeds. The phosphorus-solubilizing bacteria agent was injected into the corresponding treated soil by root irrigation; the inoculum amount was 5 mL of phosphorus-solubilizing bacteria agent in the rhizosphere soil of each soybean seed, and 5 mL of sterile water was injected into the control group at 7 d after the emergence of soybean plants.

Carbendazim spray method: Carbendazim at a concentration of 2 mg/mL was evenly sprayed on the rhizosphere soil of soybean 30 and 60 days after emergence, and the amount of spraying was 22.5 mL per pot.

2.5. Sample Collection

Three plants and their rhizosphere soil, root, stem, and soybean seeds were randomly selected under different processing conditions 120 d after the emergence of soybean plants.

2.6. Determination of the AMF Infection Rate and AMF Spore Density in the Rhizosphere Soil of Soybean

At 120 days after soybean emergence, the rate of AMF infection was determined by the alkali separation–acid fuchsin method, and the density of AMF spores in soybean rhizosphere soil was determined by wet sieve decanting and sucrose centrifugation according to Zhou et al. [22].

2.7. Determination of the Incidence of Soybean Root Rot

The disease index of soybean root rot was determined according to Zhou et al. [22].

2.8. Determination of Nodule Number in Soybean Plants

The soybean root was completely removed by the traditional digging method, and the soil attached to the surface was gently removed. Then, the soybean root was rinsed slowly with clean water, and the water was controlled dry after rinsing. All nodules were removed from the root of each plant, and the nodule number was counted according to Zhou et al. [22].

2.9. Analysis of Soybean Growth

The plant height, stem diameter, fresh weight, dry weight, root to shoot ratio, root length, pod number, and 100-grain weight of soybean plants were determined according to Jie et al. [20].

2.10. Determination of Total Bacterial Colonies and Phosphorus-Solubilizing Bacterial Colonies in Soybean Rhizosphere Soil

Samples (25 g) from the natural soil treatment group were weighed and placed in a triangular flask containing 225 mL sterile water and shaken at 28 °C and 180 r/min for 30 min. The samples were mixed into 10⁻³, 10⁻⁴, and 10⁻⁵ dilution soil sample homogenates with sterile water, and 0.2 mL of each dilution soil sample homogenate was absorbed. It was uniformly coated on the surface of beef extract peptone medium (3 g beef extract, 10 g peptone, 5 g NaCl, 20 g agar, 1 L water, pH 7.4–7.6) and cultured at 28 °C for 48 h. The total number of culturable bacterial colonies was counted. A total of 0.2 mL of the above soil samples diluted to 10⁻³, 10⁻⁴, and 10⁻⁵ was absorbed and evenly applied to Montana organophosphorus medium (glucose 10 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, lecithin 0.2 g, sodium chloride 0.3 g, potassium chloride 0.3 g, ammonium sulfate 0.5 g, calcium carbonate 3.0 g, agar 20 g, water 1 L, pH 7.2–7.4) and Montana inorganic phosphorus culture medium (glucose 10 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, magnesium sulfate 0.3 g, sodium chloride 0.3 g, potassium chloride 0.3 g, ammonium sulfate 0.5 g, calcium phosphate 3.0 g, yeast powder 0.4 g, agar 20 g, water 1 L, pH 7.2–7.4) and cultured at 28 °C for 5–7 days.

2.11. Determination of Carbendazim Residue in Soybean Grains and Rhizosphere Soil

Fifty milligrams of carbendazim standard (accurate to 0.0001 g) was accurately weighed and placed in a 100 mL volumetric bottle, and 5 mL methanol and 1 mL methanol ice acetic acid were added to 100 mL, mixed, shaken with ultrasonic waves for 5 min, and then set aside. After thoroughly crushing the soybean grains and passing the soil samples through a 40-mesh screen, 10 g of soybean grain samples and soil samples (accurate to 0.0001 g) was accurately weighed and added to 50 mL methanol and glacial acetic acid mixed solvent (methanol:glacial acetic acid = 9.5:0.5), ultrasonicated for 30 min, washed and filtered with methanol at 2500 r/min, and centrifuged for 20 min, and 1 mL of supernatant was transferred to a 10 mL volumetric bottle. The methanol volume was fixed at 10 mL. After mixing, the mixture was placed in an ultrasonic cleaning instrument and shaken for 5–10 min to fully dissolve the active ingredients. The samples were filtered using a 0.45 μm organic filter membrane. An Agilent HPLC1260 liquid chromatograph was used. Chromatographic operating conditions: the mobile phase was methanol and distilled water (55:45, v/v); flow rate: 1.0 mL/min; detection wavelength: 280 nm; sample size: 10 μL; column temperature: 27 °C; retention time: 7.5 min.

Carbendazim residue calculation:

Carbendazim in the sample to be tested is calculated by mass fraction X:

$$X=\frac{a_2{m}_{1}p}{a_1{m}_2}\times 100$$

In the formula: a₁—Peak area of carbendazim in the standard,

a₂—Peak area of carbendazim in the sample to be tested;

m₁—Mass of carbendazim in the sample to be tested, g;

m₂—Mass of carbendazim in the sample to be tested, g;

p—Mass fraction of carbendazim in the standard, %.

2.12. Statistical Analysis

Data processing and analysis of variance were completed by SPSS 22.0 (SPSS Inc., Chicago, IL, USA), and the obtained test data are expressed as the means ± standard deviations of three replicates; the significance level was p < 0.05.

3. Results

3.1. Effects of Different Treatments on the AMF Infection Rate and AMF Spore Density

The AMF infection rate is the direct manifestation of the symbiotic relationship between soybean roots and AMF.

Table 1. Effects of different treatments on the AMF infection rate, AMF spore density, incidence of soybean root rot, and the number of rhizosphere nodules in soybean rhizosphere soil.

Treatments	AMF Infection Rate (%)	AMF Spore Density Per Gram of Soil	Incidence of Soybean Root Rot (%)	Number of Rhizosphere Nodules per Plant
NCK	0.49 ± 0.04 e	1.20 ± 0.09 e	0.70 ± 0.02 a	64.00 ± 6.24 d
NR	0.93 ± 0.02 b	3.40 ± 0.07 c	0.27 ± 0.05 d	86.00 ± 6.24 c
NA	0.65 ± 0.02 d	2.14 ± 0.14 d	0.33 ± 0.05 cd	72.00 ± 8.73 cd
NRA	0.95 ± 0.01 a	4.28 ± 0.16 a	0.07 ± 0.01 e	175.00 ± 6.34 a
NDCK	0.43 ± 0.04 f	0.62 ± 0.14 f	0.47 ± 0.05 b	42.00 ± 6.16 e
NDR	0.81 ± 0.01 c	1.32 ± 0.06 e	0.27 ± 0.05 d	64.00 ± 7.13 d
NDA	0.52 ± 0.03 e	1.02 ± 0.04 e	0.33 ± 0.06 cd	61.00 ± 5.73 d
NDRA	0.85 ± 0.02 bc	2.44 ± 0.13 cd	0.13 ± 0.01 e	124.00 ± 4.49 b
MR	0.87 ± 0.04 bc	2.28 ± 0.10 d	-	-
MRA	0.89 ± 0.03 ab	3.70 ± 0.19 b	-	-
MDR	0.81 ± 0.03 c	0.60 ± 0.08 f	-	-
MDRA	0.84 ± 0.03 bc	1.28 ± 0.17 e	-	-

Note: N: natural soil; M: sterilized soil; CK: blank control; A: inoculated with A. calcoaceticus; R: inoculated with R. intraradices; RA: inoculated with R. intraradices and A. calcoaceticus; D: spraying carbendazim. Different letters indicate significant differences from different treatments (p < 0.05).

shows that under the same soil type, R. intraradices or A. calcoaceticus alone can improve the infection rate of AMF, but the effect was not as good as that of the R. intraradices- and A. calcoaceticus-treated group, and the influence difference was significant (p = 0.002). These results indicated that R. intraradices can form a symbiotic relationship with plants to increase the AMF infection rate and that A. calcoaceticus can promote AMF colonization. Under different treatment conditions, the effect of carbendazim spraying on the AMF infection rate was significantly different (p = 0.001). The infection rate of AMF sprayed with carbendazim in natural soil was significantly lower than that in the control group, which suggests that carbendazim disrupts the soil microenvironment. The toxicity of carbendazim can affect the roots of soybean plants to some extent, thus inhibiting the infection of AMF in soybean roots and reducing the infection rate of AMF.

The AMF spore density reflects the ability of AMF to reproduce in soil. Table 1 shows that R. intraradices or A. calcoaceticus alone can increase the spore density of AMF, but the effect was not as good as that of the R. intraradices and A. calcoaceticus group, and the influence difference was significant (p = 0.032). The results indicated that R. intraradices and A. calcoaceticus can enhance the development of symbionts and increase the mycorrhizal infection area, thus increasing the spore density of AMF. Under different treatment conditions, carbendazim had significant effects on the spore density of AMF (p = 0.016), and the spore density of AMF sprayed with carbendazim in natural soil was lower than that in the control group, which indicates that carbendazim may inhibit the growth of beneficial microorganisms, slow root growth, and reduce the fecundity of AMF while killing pathogenic microorganisms in the soil. However, after inoculation of R. intraradices and A. calcoaceticus in natural soil, they can mutually interact with other indigenous microorganisms, which can promote the development and reproduction of AMF and thus increase the spore density of AMF.

3.2. Effects of Different Treatments on the Incidence of Soybean Root Rot

R. intraradices or A. calcoaceticus alone can reduce the incidence of root rot, but the effect was not as good as that of the R. intraradices- and A. calcoaceticus-treated group, and the difference was significant (p = 0.011) (Table 1). The results indicated that simultaneous inoculation of R. intraradices and A. calcoaceticus can inhibit the growth of some pathogens in soil more effectively and reduce the incidence of soybean root rot. In addition, the incidence of root rot in the R. intraradices- and A. calcoaceticus-treated group was lower than that in the carbendazim-treated groups, and the difference was significant (p = 0.028). The reason for this result may be that carbendazim kills soil pathogenic bacteria, inhibits the growth of other beneficial microorganisms, damages the soil microenvironment, and worsens the growth environment of the rhizosphere of the plant.

3.3. Effects of Different Treatments on Nodule Numbers

R. intraradices or A. calcoaceticus alone can increase the nodule number, but the effect was not as good as that of the R. intraradices- and A. calcoaceticus-treated groups, and the difference was significant (p = 0.001) (Table 1). The results indicated that R. intraradices and A. calcoaceticus can form a good synergistic effect with indigenous rhizobia and effectively promote the growth of soybean root nodules and increase the number of soybean root nodules. Spraying the carbendazim treatment group can improve the effect of nodule number but not to the degree of that of inoculation with R. intraradices and A. calcoaceticus (Table 1), showing that carbendazim in the soil inhibits or kills some of the rhizosphere microorganisms, including rhizobia, causing a decline in the number of root nodules. However, R. intraradices and A. calcoaceticus in the soil can promote the growth of beneficial microorganisms and then promote the growth and development of the soybean rhizosphere, improve the number of soil rhizobia, enhance the ability of rhizobia to fix nitrogen, and increase the number of soybean root nodules.

3.4. Effects of Different Treatments on Soybean Plant Growth

The R. intraradices or A. calcoaceticus treatment groups increased the plant height, stem diameter, root length, fresh weight, dry weight, pod number, 100-grain weight, and root to shoot ratio of soybean, but the effect was lower than that of the R. intraradices and A. calcoaceticus treatment groups, and the influence difference was significant (p = 0.003) (

Table 2. Effects of different treatments on plant height, stem diameter, root length, fresh weight, dry weight, pod number, 100-grain weight, and root to shoot ratio of soybean plants.

Treatments	Plant Height (cm)	Stem Diameter (mm)	Root Length (cm)	Fresh Weight (g)	Dry Weight (g)	Pod Number Per Plant	100-Grain Weight (g)	Root/Shoot Ratio (%)
NCK	56.94 ± 4.08 de	7.25 ± 0.30 bcde	15.24 ± 0.37 h	62.55 ± 1.17 g	24.04 ± 0.59 fgh	23 ± 1.63 cd	20.09 ± 0.24 fgh	6.33 ± 0.30 defg
NR	61.16 ± 2.09 bcd	7.75 ± 0.26 abc	19.07 ± 0.59 cde	76.66 ± 0.83 b	31.45 ± 1.18 b	28 ± 2.82 ab	24.18 ± 0.38 b	6.07 ± 0.13 fg
NA	58.66 ± 3.08 cde	7.73 ± 0.22 abc	18.71 ± 0.20 cde	75.87 ± 0.63 b	29.91 ± 1.50 bc	26 ± 1.63 bc	22.71 ± 0.57 cd	6.21 ± 0.05 efg
NRA	68.02 ± 1.32 a	8.04 ± 0.14 a	25.66 ± 0.60 a	85.39 ± 0.97 a	34.74 ± 0.34 a	32 ± 1.24 a	26.23 ± 0.83 a	5.88 ± 0.22 gh
NDCK	56.79 ± 1.10 de	7.14 ± 0.28 cde	13.15 ± 0.43 i	56.95 ± 1.02 h	22.19 ± 1.55 hi	22 ± 1.41 cd	18.34 ± 0.26 ij	6.89 ± 0.07 bcd
NDR	59.35 ± 2.39 cde	7.58 ± 0.22 abcd	18.12 ± 0.59 defg	69.97 ± 0.65 d	28.39 ± 0.77 cd	24 ± 1.41 bcd	21.14 ± 0.27 ef	6.26 ± 0.19 bcde
NDA	59.82 ± 1.95 cde	7.46 ± 0.25 abcde	17.77 ± 0.67 dfg	67.25 ± 1.48 ef	26.83 ± 0.95 de	25 ± 2.16 bc	20.08 ± 0.29 fgh	6.57 ± 0.30 cdef
NDRA	63.72 ± 0.98 abc	7.87 ± 0.18 ab	22.87 ± 0.62 b	77.75 ± 0.69 b	31.21 ± 1.04 b	28 ± 2.16 ab	22.99 ± 0.15 bcd	6.26 ± 0.19 defg
MCK	57.26 ± 3.23 de	7.01 ± 0.35 de	12.94 ± 0.33 i	56.60 ± 1.07 h	20.88 ± 1.15 ij	22 ± 1.41 cd	17.02 ± 0.85 j	7.21 ± 0.32 bc
MR	60.36 ± 3.35 cd	7.65 ± 0.20 abcd	18.33 ± 0.57 def	67.46 ± 0.91 de	25.21 ± 0.82 efg	25 ± 2.16 bc	20.63 ± 0.79 efg	6.63 ± 0.34 cdef
MA	58.46 ± 3.13 cde	7.53 ± 0.23 abcde	17.64 ± 0.69 fg	64.71 ± 1.48 fg	23.94 ± 1.43 fgh	23 ± 1.63 cd	19.27 ± 0.75 ghi	7.33 ± 0.27 b
MRA	66.79 ± 1.77 ab	7.65 ± 0.20 abcd	19.87 ± 0.83 c	72.61 ± 1.73 c	29.73 ± 0.98 bc	26 ± 1.63 bc	23.54 ± 0.94 bc	5.41 ± 0.09 h
MDCK	53.90 ± 1.45 e	6.90 ± 0.28 e	12.69 ± 0.45 i	49.13 ± 0.44 i	18.67 ± 1.11 j	20 ± 1.41 d	15.52 ± 0.26 k	7.98 ± 0.58 a
MDR	56.19 ± 3.23 de	7.22 ± 0.28 bcde	17.11 ± 0.45 fg	58.28 ± 0.86 h	23.09 ± 0.53 ghi	23 ± 0.81 cd	19.01 ± 0.60 hi	6.64 ± 0.12 cdef
MDA	57.92 ± 3.53 cde	7.08 ± 0.22 de	16.27 ± 0.44 g	56.04 ± 0.71 h	21.70 ± 1.45 hi	24 ± 2.16 bcd	17.85 ± 0.53 ij	6.55 ± 0.27 def
MDRA	64.08 ± 1.62 abc	7.36 ± 0.38 bcde	19.21 ± 0.49 cd	63.94 ± 0.91 g	26.31 ± 0.84 def	25 ± 1.41 bc	21.77 ± 0.43 de	6.55 ± 0.17 def

Note: N: natural soil; M: sterilized soil; CK: blank control; A: inoculated with A. calcoaceticus; R: inoculated with R. intraradices; RA: inoculated with R. intraradices and A. calcoaceticus; D: spraying carbendazim. Different letters indicate significant differences from different treatments (p < 0.05).

). The results indicated that R. intraradices could expand the range of nutrients absorbed by plants when inoculating with R. intraradices alone. As a channel of nutrient exchange, R. intraradices could provide some N nutrients to plants and improve crop yield; A. calcoaceticus could improve the root activity of plants; promote the absorption of N, P, and K in plants; improve the nutrient level of the rhizosphere soil of plants; and thus promote the growth and development of plants and increase the yield. Simultaneous inoculation with R. intraradices and A. calcoaceticus could enhance the uptake and utilization of nutrient elements in the underground part of the plant and effectively promote the growth and development of the plant. Under the same soil type, carbendazim spraying could increase plant height, stem diameter, root length, fresh weight, dry weight, pod number, 100-grain weight, and root to shoot ratio of soybean, but the effect was not as good as that of the R. intraradices and A. calcoaceticus treatment groups alone or in combined inoculation, and the effects of carbendazim on soybean plant biomass were significantly different (p = 0.006). This difference may be related to the fact that carbendazim reduces soil beneficial microorganisms to some extent but also inhibits the colonization of soil pathogenic flora. However, R. intraradices forms symbionts with the plant rhizosphere and expands the range of nutrients absorbed by the plant rhizosphere, thus making the rhizosphere more fully developed. A. calcoaceticus converts insoluble phosphorus from soil to soluble phosphorus, enhances the absorption of available phosphorus in the rhizosphere of plants, and then promoting root growth. Therefore, the soil nutrient content is increased and balanced, so plant growth and development are better.

3.5. Effects of Different Treatments on the Total Number of Culturable Bacterial Colonies and Phosphorus-Solubilizing Bacterial Colonies in Soybean Rhizosphere Soil

3.5.1. Effects of Different Treatments on the Total Number of Culturable Bacterial Colonies in Rhizosphere Soil of Soybean

Under natural soil conditions, inoculation with R. intraradices and A. calcoaceticus alone or in combination with or without carbendazim had significant effects on the total number of soil bacterial colonies (p = 0.025). Under the same treatment, inoculation with R. intraradices and A. calcoaceticus was significantly better than that of the other treatments, increasing by 67% and 104% compared with NCK and NDCK, respectively. Inoculation with R. intraradices was increased by 22% and 20% compared with NCK and NDCK, respectively. Inoculation with A. calcoaceticus was increased by 40% and 38% compared with NCK and NDCK, respectively (

Table 3. Changes in the total number of soil bacterial colonies and the total number of phosphorus-solubilizing bacteria colonies.

Treatments	Total Number of Bacterial Colonies (CFU/g)	Total Number of Soluble Organic Phosphorus Bacteria Colonies (CFU/g)	Percentage of Soluble Organic Phosphorus Bacteria (%)	Total Number of Soluble Inorganic Phosphorus Bacteria Colonies (CFU/g)	Percentage of Soluble Inorganic Phosphorus Bacteria (%)
NCK	(1.14 ± 0.16) × 106 d	(5.46 ± 0.16) × 104 ef	5%	(3.33 ± 0.16) × 104 f	3%
NR	(1.56 ± 0.22) × 106 b	(7.50 ± 0.13) × 104 de	5%	(4.40 ± 0.13) × 104 e	3%
NA	(1.36 ± 0.16) × 106 c	(2.34 ± 0.18) × 105 b	17%	(7.26 ± 0.21) × 104 b	5%
NRA	(1.90 ± 0.16) × 106 a	(5.34 ± 0.18) × 105 a	28%	(8.83 ± 0.13) × 104 a	5%
NDCK	(3.51 ± 0.12) × 105 h	(3.42 ± 0.14) × 104 h	10%	(1.85 ± 0.11) × 104 h	5%
NDR	(5.97 ± 0.16) × 105 f	(5.19 ± 0.20) × 104 gh	9%	(2.87 ± 0.15) × 104 g	5%
NDA	(5.20 ± 0.14) × 105 g	(8.77 ± 0.15) × 104 e	17%	(5.32 ± 0.18) × 104 d	10%
NDRA	(7.15 ± 0.16) × 105 e	(2.03 ± 0.18) × 105 c	28%	(6.34 ± 0.17) × 104 c	9%

Note: N: natural soil; CK: blank control; A: inoculated with A. calcoaceticus; R: inoculated with R. intraradices; RA: inoculated with R. intraradices and A. calcoaceticus; D: spraying carbendazim. Different letters indicate significant differences from different treatments (p < 0.05).

). The results showed that inoculation of R. intraradices and A. calcoaceticus can effectively increase the number of culturable bacteria in the rhizosphere soil of plants, improve the microbial community structure, and increase the soybean yield. Spraying carbendazim had significant effects on the total number of culturable bacterial colonies in the soybean rhizosphere soil (p = 0.001). The total number of bacterial colonies in the NDCK treatment group was 31% lower than that in the NCK treatment group, and the total number of bacterial colonies in the NR group was 161% higher than that in the NDR group. The total number of bacterial colonies in the NA treatment group was 162% higher than that in the NDA treatment group, and the total number of bacterial colonies in the NRA treatment group was 166% higher than that in the NDRA treatment group (Table 3). The results showed that spraying carbendazim had a large effect on the total number of bacterial colonies in the rhizosphere soil. Although carbendazim can effectively inhibit the growth of soil pathogens, it can also reduce the total number of soil bacterial colonies, destroy the biological cycle of soil bacteria and change the genetic diversity of bacteria.

3.5.2. Effects of Different Treatments on the Total Number of Culturable Phosphorus-Solubilizing Bacteria Colonies in the Rhizosphere Soil of Soybean

Under natural soil planting conditions, the total number of organic phosphorus-soluble and inorganic phosphorus-soluble bacteria in soybean rhizosphere soil in the R. intraradices or A. calcoaceticus treatment groups was higher than that in the NCK treatment groups, and the difference was significant (p = 0.026). The total number of soluble organic phosphorus and soluble inorganic phosphorus bacteria in the rhizosphere soil of plants inoculated with both R. intraradices and A. calcoaceticus was the highest, and the results showed that the synergistic effect of R. intraradices and A. calcoaceticus was more conducive to promoting the growth of phosphorus-solubilizing bacteria in soybean rhizosphere soil (Table 3). The total number of soluble organic phosphorus and soluble inorganic phosphorus bacteria in the carbendazim treatment group was significantly lower than that in the non-carbendazim treatment group, and the difference was significant (p = 0.004). The results showed that carbendazim not only inhibited soil pathogenic microorganisms but also inhibited the total number of soluble organic phosphorus and soluble inorganic phosphorus bacteria in the soybean rhizosphere, which led to a decrease in the total number of soluble organic phosphorus and soluble inorganic phosphorus bacteria in the soybean rhizosphere (Table 3). The total number of NDCK soluble organic phosphorus and soluble inorganic phosphorus bacteria in the carbendazim treatment group was lower than that in the carbendazim treatment group inoculated with R. intraradices and A. calcoaceticus alone or mixed, and the difference was significant (p = 0.007) Inoculation with R. intraradices and A. calcoaceticus in the treatment group showed more significant growth of soluble organic phosphorus and soluble inorganic phosphorus bacteria, indicating that the synergistic effect of R. intraradices and A. calcoaceticus was beneficial to the growth of soluble organic phosphorus and soluble inorganic phosphorus bacteria.

3.6. Effects of Different Treatments on Carbendazim Residue in Soybean Grains and Rhizosphere Soil

Under the same inoculation conditions, the carbendazim residue in soybean grains and rhizosphere soil in the sterilized soil treatment group was higher than that in the natural soil treatment group, and the influence of soil type on the carbendazim residue in soybean grains and rhizosphere soil was significant (p = 0.019) (

Table 4. Effects of different treatments on carbendazim residue in soybean grains and rhizosphere soil.

Treatments	Carbendazim Residue in Soybean Grains (μg/mL)	Carbendazim Residue in Rhizosphere Soil (μg/mL)
NCK	0.00 ± 0.00 i	0.00 ± 0.00 i
NR	0.00 ± 0.00 i	0.00 ± 0.00 i
NA	0.00 ± 0.00 i	0.00 ± 0.00 i
NRA	0.00 ± 0.00 i	0.00 ± 0.00 i
NDCK	11.39 ± 0.32 b	6.77 ± 0.01 b
NDR	3.27 ± 0.08 g	1.38 ± 0.01 g
NDA	6.40 ± 0.02 e	2.85 ± 0.01 e
NDRA	1.63 ± 0.01 h	0.88 ± 0.01 h
MCK	0.00 ± 0.00 i	0.00 ± 0.00 i
MR	0.00 ± 0.00 i	0.00 ± 0.00 i
MA	0.00 ± 0.00 i	0.00 ± 0.00 i
MRA	0.00 ± 0.00 i	0.00 ± 0.00 i
MDCK	14.05 ± 0.01 a	9.36 ± 0.03 a
MDR	8.91 ± 0.03 d	3.99 ± 0.01 d
MDA	9.48 ± 0.01 c	5.61 ± 0.01 c
MDRA	5.48 ± 0.01 f	2.14 ± 0.01 f

Note: N: natural soil; M: sterilized soil; CK: blank control; A: inoculated with A. calcoaceticus; R: inoculated with R. intraradices; RA: inoculated with R. intraradices and A. calcoaceticus; D: spraying carbendazim. Different letters indicate significant differences from different treatments (p < 0.05).

), mainly because the microbial richness in natural soil is high. When carbendazim enters the soil, some microorganisms can release enzymes into the environment to degrade carbendazim so that the residual amount of carbendazim in the soil is reduced, and the uptake of carbendazim in the rhizosphere of plants is reduced, so the residual amount of carbendazim in the soil and grains is low. Sterilization in the soil treatment group led to the absence or singular presence of microorganisms, and carbendazim was difficult to degrade, causing the residues of soybean grains and rhizosphere soil to be higher.

Table 4 shows that under the same soil type, the carbendazim residue in the soybean grains and rhizosphere soil of the R. intraradices or A. calcoaceticus inoculation groups were significantly lower than those of the blank control group (p = 0.031). R. intraradices and A. calcoaceticus inoculation had the lowest carbendazim residue in soybean grains and rhizosphere soil. Inoculation of R. intraradices and A. calcoaceticus alone or in combination had degradation effects on carbendazim in soybean grains and rhizosphere soil. Simultaneous inoculation of R. intraradices and A. calcoaceticus had synergistic effects, which were conducive to the degradation of carbendazim in soil to minimize the residual carbendazim in rhizosphere soil and soybean grains.

4. Discussion

As shown in Table 1, inoculation with R. intraradices and A. calcoaceticus and spraying with carbendazim had significant effects on the spore density and infection rate of AMF. Under the same inoculation conditions, the AMF spore density and AMF infection rate of soybeans planted in natural soil were higher than those in sterilized soil, indicating that under the natural environment, the soil microbial flora was rich, the microenvironment was relatively stable, the plant resistance was strong, and the AMF spore density and AMF infection rate were relatively high. This conclusion is consistent with the results of Nacoon et al. [23]. Inoculation with R. intraradices and A. calcoaceticus significantly increased the AMF spore density and AMF infection rate in soybean roots. It showed that AMF can change the physical and chemical properties of soil by changing the exudates of crop roots, which leads to the improvement in AMF spore density and AMF infection rate [24]. To date, there have been few studies on the effects of R. intraradices and A. calcoaceticus on the number of soybean nodules and the incidence of root rot. The R. intraradices and A. calcoaceticus interaction can make rhizosphere development more complete, and the rhizosphere has a stronger ability to form root nodules, which is conducive to increasing the number of root nodules. AMF enhance soybean nodule growth by recruiting plant growth-promoting rhizobacteria (PGPR) to promote rhizosphere bacteria and changing root-associated microbial communities in a host-dependent manner [25]. Under the same inoculation conditions, the incidence of soybean root rot in the natural treatment group was significantly lower than that in the sterilized treatment group, and spraying carbendazim can also reduce the incidence of root rot. Under natural soil conditions, the rate of root rot in the NRA plants in the group inoculated with R. intraradices and A. calcoaceticus was the lowest. Although carbendazim has a certain effect on the control of plant root rot disease, carbendazim is an environmental endogenic fungicide that causes a certain amount of pesticide pollution in the process of its use [26]. After inoculation of R. intraradices and A. calcoaceticus, the synergistic effect between them enhanced the infection of AMF on soybean, regulated the related physiological and biochemical reactions of the plant, and enhanced the resistance of soybean to root rot pathogens to resist the invasion of pathogenic microorganisms in the rhizosphere of the plant, significantly reducing the incidence of root rot.

Our previous study showed that inoculation with R. intraradices can significantly increase soybean biomass and improve the microbial flora structure of soybean roots and rhizosphere soil [20,27]. It showed that AMF can significantly improve the absorption of nutrient elements and increase soybean biomass [28]. Compared with the group without inoculant, the R. intraradices- and A. calcoaceticus-treated groups significantly increased the soybean biomass. The synergy in the R. intraradices- and A. calcoaceticus-treated group can change the transfer and utilization of soil available phosphorus by plants, enhance the absorption and utilization of nutrient elements, promote the efficient growth and development of plants, and improve soybean biomass. Bacillus cereus YL6 can dissolve soil insoluble phosphorus by secreting organic acid and phosphatase and improve soil fertility and soybean plant biomass, providing a solid basis for the mechanism of PSB affecting plant growth [29]. It also showed that PSB can increase the content of soil organic phosphorus, improve the absorption of phosphorus by plants, and promote an increase in crop biomass. PSB can increase the biomass of AMF by promoting mycelial growth and proliferation and spore germination outside the root [30]. PSB can promote extra-root mycelial growth and proliferation and spore germination of AMF, thereby improving plant biomass. Moreover, PSB can indirectly provide a source of soluble phosphorus for mycorrhizae and AMF mycelia [31]. Under the same inoculation method, soybean biomass planted in natural soil was significantly higher than that in the sterilized soil treatment group, mainly because there were more beneficial microbial species in the natural soil, which was conducive to soybean growth. Under natural soil conditions, spraying the carbendazim treatment group increased soybean biomass compared with the blank control group, indicating that when carbendazim enters the soil, it can inhibit the growth of soil pathogens, reduce plant morbidity, provide a good growth environment for plants, and promote crop growth. However, carbendazim reduced soil microorganism abundance to some extent, and the effect of carbendazim spraying was lower than that of the groups inoculated with R. intraradices and A. calcoaceticus alone or together. When R. intraradices and A. calcoaceticus were inoculated together, they could improve the structure of root and rhizosphere soil microbial flora, stabilize the rhizosphere microecological environment and promote soybean growth and development, which was consistent with the results of Shubhangi’s study [32].

AMF help promote soil enzyme activity, and their propagules have the ability to synthesize and release soil enzymes [33]. In addition, that PSB colonization on the surface of AMF hyphae could enhance the activity of PSB-releasing phosphatase and promote organic phosphate mineralization and turnover in soil [34]. Under natural soil conditions, inoculation with R. intraradices and A. calcoaceticus significantly increased the total number of bacterial colonies in the rhizosphere soil of soybean plants. This finding indicates that R. intraradices and A. calcoaceticus can cooperate to increase soil available P content, enhance the absorption and utilization of soil P by plants, and then change plant root exudates to increase the total number of bacterial colonies in rhizosphere soil. At the same time, AMF and PSB can secrete enzymes to inhibit the growth of pathogenic microorganisms and increase the number of beneficial bacteria in the soil to maintain soil fertility and promote plant growth and development [31,33]. In this study, spraying carbendazim inhibited the growth and development of bacteria in the rhizosphere soil of soybean plants and reduced the total number of colonies. The total number of bacteria in the rhizosphere soil of soybean plants was also lower in the group inoculated with R. intraradices and A. calcoaceticus and sprayed with carbendazim. In addition, carbendazim treatment groups yielded lower phosphorus-solubilizing bacterial numbers than the other groups, showing that after carbendazim enters the soil, it affects the mitosis of soil microorganisms and inhibits the growth of microorganisms; carbendazim has an antiseptic effect that may reduce the diversity of soil microbes, change the composition of soil microbial flora, and reduce the number of phosphorus-solubilizing bacteria [35,36].

The residual problem of carbendazim in farmland and its degradation methods have attracted increasing attention. Many research studies showed that AMF can increase the activity of plant rhizosphere soil microorganisms to secrete pesticide-degrading enzymes and reduce environmental pesticide residue [15,37,38]. Huang et al. added AMF to soil contaminated by atrazine pesticide; atrazine residue in plants decreased, and the colonization of AMF enhanced the metabolic capacity of plants [39]. Other research showed that PSB improved soil enzyme activity and the soil phosphorus utilization rate by secreting enzymes, and the host plant decomposed pesticides through interactions with PSB, reducing pesticide residue in the soil environment [19,40,41]. In this study, carbendazim was sprayed to treat soybean seeds and rhizosphere soil residue to different degrees. The R. intraradices- and A. calcoaceticus-treated group had the lowest carbendazim residue in the soybean grains and rhizosphere soil, indicating that R. intraradices and A. calcoaceticus can degrade carbendazim.

In this study, by applying R. intraradices and A. calcoaceticus to potted soybeans and spraying with carbendazim, the effects of different soils, different inoculation methods and carbendazim spraying on AMF spore density, AMF infection rate, soybean biomass, root rot incidence, total number of culturable bacteria and phosphorus-solubilizing bacteria colonies in soybean rhizosphere soil, and carbendazim residue in soybean grains and plant rhizosphere soil were studied. This study provides a scientific theory and practical basis for the development and application of high-efficiency microbial agents to promote crop growth, improve crop disease resistance, and reduce pesticide residue. In addition, it provides a scientific basis for the sustainable development of the soybean industry in China.

5. Conclusions

This work first demonstrated that R. intraradices and A. calcoaceticus can directly alter the growth of soybean plants and carbendazim residue. R. intraradices or A. calcoaceticus can significantly increase the spore density, AMF infection rate, soybean biomass, nodule number, total number of bacterial colonies, and phosphorus-soluble bacterial colonies in the rhizosphere soil of soybean plants. The incidence of soybean root rot and residual carbendazim in soybean grains and plant rhizosphere soil were reduced, and the effect of inoculation with R. intraradices and A. calcoaceticus was the most significant. Although carbendazim spraying reduced the incidence of soybean root rot and increase soybean biomass, the effect was lower than that of the R. intraradices and A. calcoaceticus treatment group. Natural soil was more conducive to the accumulation of the main biomass of soybean plants, the increase in nodule number, and the degradation of pesticide residue. The purpose of this project is to provide the necessary theoretical basis for the biological control of soybean root rot disease and at the same time to promote the development of the Chinese soybean industry, improve the status of organic green soybean production in Heilongjiang Province, increase soybean yield, and reduce pesticide residue in crops and rhizosphere soil.