Acinetobacter calcoaceticus and application thereof

Abstract

Acinetobacter calcoaceticus and application thereof, relates to the field of microorganisms, in particular to an acinetobacter calcoaceticus and application thereof. It aims to solve the problems of soil environment pollution and soil condition destruction caused by using carbendazim to inhibit soybean root rot and applying phosphate fertilizer. The strain is acinetobacter calcoaceticus HDP-02, which is preserved in China Center for Type Culture Collection on July 5, 2022, and the preservation number is CCTCC NO: M 20221027. The strain has the ability to dissolve phosphorus, can effectively alleviate the damage caused by soybean root rot to the roots of soybean plants, and can promote the increase of the biomass of soybean plants. In addition, acinetobacter calcoaceticus HDP-02 can degrade carbendazim. The strain of the application is used for inhibiting soybean root rot and degrading carbendazim.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to a strain of Acinetobacter calcitrinum and its applications. Background Technology

[0002] Soybean [Glycine max (L.) Merr.] is an important food and economic crop worldwide, with a vast planting area in my country. However, due to limited land resources and profit-driven motives, continuous cropping and pesticide overuse are prevalent in soybean-growing areas of my country. Continuous cropping of soybeans leads to problems such as deterioration of soil physical and chemical properties, imbalance of soil fertility, changes in microbial community composition, and increased severity of soil-borne diseases, resulting in reduced soybean yield and deteriorated quality. In agricultural production, farmers mainly rely on the application of chemical fertilizers to increase soybean yield under continuous cropping conditions. However, long-term application of chemical fertilizers damages soil structure, leads to soil nutrient imbalance, accumulation of autotoxic substances, and exacerbates soybean root rot.

[0003] Soybean root rot is mainly caused by soil-borne pathogens infecting the roots of soybean seedlings, leading to stunted growth, wilting leaves, and brittle plants. This results in fewer pods, weaker pod development, and ultimately reduced yield. Control methods for soybean root rot often involve using pesticides such as carbendazim to kill the pathogens at the root and reduce the incidence of the disease. However, carbendazim is a benzimidazole fungicide, which degrades slowly in the natural environment, mainly remaining in the rhizosphere soil and on the crop plants. Ingestion by humans and livestock can cause poisoning symptoms such as dizziness, nausea, and vomiting. Carbendazim residues in the soil may also affect the growth, reproduction, and metabolism of native microorganisms, thereby impacting soil biochemical processes.

[0004] Phosphorus is one of the three essential nutrients for plant growth, and most crops currently use phosphate fertilizers to alleviate soil phosphorus deficiency. However, about 90% of the phosphate fertilizer applied to the soil is chemically fixed, resulting in low utilization and causing a series of serious problems such as damage to soil physical structure, decreased fertility, and water and soil pollution. Summary of the Invention

[0005] The present invention aims to address the problems of existing methods for using carbendazim to suppress soybean root rot, which pollutes the soil environment, and the application of phosphate fertilizers which damages soil conditions. The invention provides a strain of Acinetobacter calcium acetate and its application.

[0006] This invention provides a strain of Acinetobacter calcoaceticus, HDP-02, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, on July 5, 2022, with accession number CCTCC NO: M 20221027.

[0007] The Acinetobacter calciacetate HDP-02 of this invention is a Gram-negative bacterium. The strain is a short rod-shaped, single-arranged bacterium, 1.6-2.4 μm long and 1.0-1.3 μm wide, with rounded ends, no spores, and no flagella. The colonies are regular in shape, with smooth edges, light yellow in color, flat, dull, opaque, and easy to pick up.

[0008] The *Acinetobacter calcifera* HDP-02 strain of this invention showed positive results for glucose fermentation, sucrose fermentation, lactose fermentation, fructose fermentation, and maltose fermentation; negative results for mannitol, citrate utilization, methyl red, VP, gelatin liquefaction, starch hydrolysis, indole, phenylalanine, hydrogen sulfide, catalase, nitrate reduction, growth temperature of 50°C, and salt tolerance to 3% NaCl.

[0009] The 16S rDNA sequencing results of Acinetobacter calcoaceticus HDP-02 of this invention were submitted to the NCBI database. BLAST analysis and comparison were performed, and the highest similarity (99%) was found with Acinetobacter calcoaceticus IIPRDSCP-11 (MT436393.1). Based on the comprehensive morphological observation results, strain HDP-02 was identified as Acinetobacter calcoaceticus and named Acinetobacter calcoaceticus HDP-02.

[0010] The present invention also provides the application of Acinetobacter calcitriol HDP-02 in the degradation of insoluble phosphorus, wherein the insoluble phosphorus is calcium phytate, aluminum phosphate, iron phosphate, lecithin and calcium phosphate.

[0011] This invention also provides the application of Acinetobacter calcitrinum HDP-02 in inhibiting soybean root rot.

[0012] The present invention also provides the application of Acinetobacter calcitrinum HDP-02 in the degradation of plant fungicides.

[0013] Furthermore, the plant fungicide is carbendazim.

[0014] This invention provides the application of Acinetobacter calcitriol HDP-02 in promoting the increase of soybean plant biomass.

[0015] Furthermore, the biomass includes aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, plant height, stem diameter, root length, weight per 100 seeds, yield per plant, and number of pods per plant.

[0016] The beneficial effects of this invention are:

[0017] This invention screened a strain of Acinetobacter calciacetate HDP-02 from the rhizosphere soil of soybean plants. This strain has phosphorus-solubilizing ability and can alleviate the problem of soil condition damage caused by the application of phosphate fertilizer.

[0018] It can effectively resist the invasion of soil pathogens on soybean plant roots, effectively alleviate the damage to soybean root rot, and synergistically promote the increase of various biomass of soybean plants with other beneficial microorganisms. In addition, Acinetobacter calciacetate HDP-02 can significantly reduce the residue of carbendazim, indicating that this strain can degrade the broad-spectrum plant fungicide carbendazim, thus avoiding pollution of the soil environment. Attached Figure Description

[0019] Figure 1 This is a morphological diagram of Acinetobacter calcium acetate HDP-02 strain.

[0020] Figure 2 The graph shows the changes in phosphorus solubility of Acinetobacter calcitriol HDP-02 in NBRIP liquid medium with different poorly soluble phosphorus sources.

[0021] Figure 3 The graph shows the changes in phosphorus solubility of Acinetobacter calcitrinum HDP-02 in NBRIP liquid medium at different pH values. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0023] Example 1:

[0024] In this embodiment, the Acinetobacter calcoaceticus is HDP-02, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, on July 5, 2022, with accession number CCTCC NO: M 20221027.

[0025] The method for obtaining Acinetobacter calciacetate HDP-02 in this embodiment is as follows:

[0026] The strain HDP-02 was obtained by selecting rhizosphere soil from soybean plants in the experimental field of Hanan 19th Road, core area of ​​Hanan Industrial New City, Harbin, Heilongjiang Province, and using traditional microbial isolation and purification methods (dilution plating method and streak plate separation method).

[0027] Example 2: Identification of strain HDP-02

[0028] According to the "Handbook for Systematic Identification of Common Bacteria", strain HDP-02 was subjected to Gram staining and physiological and biochemical identification. Strain HDP-02 was identified as a Gram-negative bacterium. The strain individuals are short rods, arranged singly, 1.6–2.4 μm long and 1.0–1.3 μm wide, with rounded ends, no spores, and no flagella. Colonies are regular in shape, with smooth edges, pale yellow in color, flat, dull, opaque, and easily picked up.

[0029] Strain HDP-02 showed positive results for glucose fermentation, sucrose fermentation, lactose fermentation, fructose fermentation, and maltose fermentation; negative results for mannitol, citrate utilization, methyl red, VP, gelatin liquefaction, starch hydrolysis, indole, phenylalanine, hydrogen sulfide, catalase, nitrate reduction, growth temperature of 50℃, and salt tolerance to 3% NaCl.

[0030] 16S rDNA identification of strain HDP-02:

[0031] DNA from the purified strain HDP-02 was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal bacterial primers 27F / 1492R. The PCR amplification system consisted of 20 μL of: 10 ng of HDP-02 template DNA, 2 μL of 2.5 mmol / L dNTPs, and 5×FastPfu buffer (containing Mg2+). 2+ 4 μL of 5 μmol / L primers, 0.8 μL each of primers, 0.4 μL of FastPfu Polymerase, and deionized water were added to a final volume of 20 μL. The PCR amplification program was set as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for 35 cycles; 72℃ final extension for 5 min, and storage at 4℃. Sequencing of the PCR amplification products showed that the 16S rDNA of strain HDP-02 had the highest similarity (99%) to Acinetobacter calcoaceticus IIPRDSCP-11 (MT436393.1).

[0032] Strain HDP-02 shares the same physiological and biochemical characteristics as the type species Acinetobacter calcoaceticus. Based on the results of various physiological and biochemical tests and 16S rDNA identification, strain HDP-02 was identified as Acinetobacter calcoaceticus and named Acinetobacter calcoaceticus HDP-02.

[0033] Example 3: Phosphate solubilization experiment of Acinetobacter calcitonin HDP-02

[0034] Acinetobacter calcifera HDP-02 was inoculated into LB liquid medium and then fermented at 170 rpm and 28°C for 24 h to obtain the Acinetobacter calcifera HDP-02 bacterial suspension. The viable count of the Acinetobacter calcifera HDP-02 bacterial suspension reached 1×10⁻⁶. 7 ~1×10 8 cfu / mL.

[0035] Add 125 mL of NBRIP liquid culture medium containing different insoluble phosphorus sources (calcium phytate, aluminum phosphate, ferric phosphate, lecithin, or calcium phosphate) to 250 mL Erlenmeyer flasks. Collect the Acinetobacter acetate HDP-02 bacterial suspension by centrifugation at 5000 rpm to prepare a bacterial suspension with a concentration of 1.0 × 10⁻⁶. 8 A bacterial suspension of [number] cells / mL was prepared and then inoculated at a 1.0% inoculum into NBRIP liquid medium containing different poorly soluble phosphorus sources. The control group was not inoculated with bacterial suspension but instead received the same volume of sterile water. Each treatment group was replicated in triplicate. The cultures were then incubated at 28°C with shaking at 170 rpm for 144 h, with samples taken every 24 h for analysis. After sampling, the culture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The water-soluble phosphorus content in the supernatant was calculated using the molybdenum-antimony colorimetric method.

[0036] NBRIP liquid medium is prepared by adding 10.0g glucose, 5.0g insoluble phosphorus source, 0.25g magnesium sulfate, 0.2g potassium chloride, 0.1g ammonium sulfate and 1L water, with a pH of 7.0-7.2, and sterilized at 121℃ for 20min.

[0037] The changes in phosphorus solubility of Acinetobacter calcitonin HDP-02 in NBRIP liquid medium containing different poorly soluble phosphorus sources are as follows: Figure 2 As shown, Figure 2The symbols ■ represent calcium phosphate, ● represent calcium phytate, ▲ represent ferric phosphate, ▼ represent aluminum phosphate, and ◆ represent lecithin, showing significant differences in phosphorus solubility in the culture medium within a certain time range. *Acinetobacter acetate* HDP-02 exhibited the strongest phosphorus solubility for calcium phosphate, followed by calcium phytate, while ferric phosphate, aluminum phosphate, and lecithin showed weaker solubility. After 24 hours of culture, *Acinetobacter acetate* HDP-02 showed phosphorus solubility of 196.6 mg / L and 88.8 mg / L in NBRIP liquid medium with calcium phosphate and calcium phytate as phosphorus sources, respectively, and 20.2 mg / L, 19.0 mg / L, and 15.2 mg / L for ferric phosphate, aluminum phosphate, and lecithin, respectively. After 48 hours of culture, *Acinetobacter acetate* HDP-02 showed a phosphorus solubility of 264.6 mg / L for calcium phosphate and the highest phosphorus solubility of 96.4 mg / L for calcium phytate. After 72 hours of culture, *Acinetobacter calcoaceticus* HDP-02 showed a phosphorus solubility of 273.2 mg / L for calcium phosphate, a slightly lower phosphorus solubility of 95.4 mg / L for calcium phytate, and the highest phosphorus solubility for ferric phosphate and aluminum phosphate, at 31.4 mg / L and 26.5 mg / L, respectively. After 96 hours of culture, the highest phosphorus solubility of calcium phosphate (279.9 mg / L) was observed, while the solubility for calcium phytate, ferric phosphate, and aluminum phosphate decreased to 81.4 mg / L, 20.0 mg / L, and 20.7 mg / L, respectively. After 144 hours of culture, the phosphorus solubility of calcium phosphate and calcium phytate decreased to 196.1 mg / L and 82.4 mg / L, respectively, while the phosphorus solubility for aluminum phosphate and lecithin was weak.

[0038] Acinetobacter calciacetate HDP-02 can convert phosphorus in the soil that is difficult for plants to absorb and utilize directly into a form that is easily absorbed and utilized, thereby increasing the absorption and utilization of phosphorus by crops. It can also promote the absorption and utilization of other nutrients by plants, thereby improving soil structure, promoting the secretion of growth hormones by crops, inhibiting the growth of pathogens, and increasing crop yield.

[0039] Example 4: Phosphate solubilization experiment of Acinetobacter calcitonin HDP-02 under different pH conditions:

[0040] Using 1 mol / L NaOH and HCl as acid-base adjusters, the pH of NBRIP liquid medium (with calcium phosphate as the phosphorus source) was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and sterilized at 121℃ for 20 min. A suspension of Acinetobacter calciacetate HDP-02 was then inoculated into NBRIP liquid medium at the above different pH values ​​at a 1.0% inoculation rate. The control group was not inoculated with Acinetobacter calciacetate HDP-02 suspension but was replaced with the same volume of sterile water. Each treatment group was replicated in triplicate. The medium was then incubated at 28℃ and 170 rpm with shaking for 144 h, with samples taken every 24 h for analysis. After sampling, the culture medium was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The water-soluble phosphorus content in the supernatant was calculated using the molybdenum-antimony colorimetric method.

[0041] The phosphate-solubilizing ability of *Acinetobacter calcifera* HDP-02 to calcium phosphate is significantly affected by pH changes (e.g., ...). Figure 3 As shown, Figure 3 The symbols ■ indicate pH = 4.0, ● indicate pH = 5.0, ▲ indicate pH = 6.0, ▼ indicate pH = 7.0, and ◆ indicate pH = 8.0. (Indicates pH=9.0). At initial pH values ​​of 6.0, 8.0, and 9.0, *Acinetobacter calcifera* HDP-02 exhibited significantly higher phosphorus-solubilizing capacity for calcium phosphate than at pH values ​​of 4.0, 5.0, and 7.0. After 48 h of culture, the phosphorus-solubilizing capacity in media with pH values ​​of 6.0, 8.0, and 9.0 reached 269.2 mg / L, 273.9 mg / L, and 282.5 mg / L, respectively, and tended to stabilize with increasing culture time. After 96 h of culture, the phosphorus-solubilizing capacity in media with pH values ​​of 4.0 and 5.0 decreased to 107.4 mg / L and 153.8 mg / L, respectively. After 120 h of culture, the phosphorus-solubilizing capacity reached its peak in media with pH values ​​of 6.0–9.0, at 271.9 mg / L, 256.7 mg / L, 274.2 mg / L, and 284.4 mg / L, respectively. However, after 144 hours of cultivation, the amount of dissolved phosphorus in the culture media with different pH values ​​all showed a significant decreasing trend.

[0042] Example 5: Experiment on the effects of Acinetobacter calcifera HDP-02 on disease resistance and biomass of soybean plants:

[0043] The experiment employed a randomized block design with four treatments: (1) Acinetobacter calciacetate HDP-02 inoculated under natural soil conditions (PN); (2) Acinetobacter calciacetate HDP-02 not inoculated under natural soil conditions (control, CkN); (3) Acinetobacter calciacetate HDP-02 inoculated under sterilized soil conditions (PM); and (4) Acinetobacter calciacetate HDP-02 not inoculated under sterilized soil conditions (control, CkM). Each pot contained 12 kg of soil, and each treatment had five biological replicates. The experiment was conducted at a temperature of 23±1℃, a day-day cycle of 8 h, and a humidity of 50±5%.

[0044] The soil sterilization conditions were as follows: sterilization at 121℃ for 2 hours, followed by sterilization again under the same conditions after an interval of 24 hours.

[0045] Soybean seeds are surface disinfected in 75% ethanol for 5 minutes, then rinsed with sterile distilled water at least 10 times. The disinfected soybean seeds are then sown in pots (6 seedlings per pot, 3 seedlings after germination) and watered regularly.

[0046] Acinetobacter calciacetate HDP-02 cells were collected by centrifugation, washed twice with sterile water, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶ with sterile water. 8 The concentration of *Acinetobacter calcium acetate* HDP-02 inoculum is calculated as 5 mL / mL. The inoculum is applied to the soil via root drenching, with each soybean seed inoculated with 5 mL of HDP-02. A control inoculation consists of 5 mL of sterile water.

[0047] 1. Effects of Acinetobacter calcoaceticus HDP-02 on the soybean root rot disease severity index

[0048] Intact root systems of soybean plants under different experimental treatments were randomly selected, rinsed with tap water, and allowed to air dry naturally. The soybean root rot disease severity index was assessed according to Table 1, with three replicates for each treatment.

[0049] Table 1. Soybean root rot disease severity index

[0050]

[0051] The effects of different experimental treatments on the root rot disease index of potted soybean plants are shown in Table 2. Table 2 shows that inoculation with *Acinetobacter calcareae* HDP-02 significantly reduced the soybean root rot disease index. The root rot disease index in the uninoculated group was 1.55 times that of the inoculated group, indicating that *Acinetobacter calcareae* HDP-02 can effectively alleviate the damage to soybean roots caused by root rot.

[0052] Table 2

[0053]

[0054] 2. Effects of Acinetobacter calciacetate HDP-02 on soybean plant biomass

[0055] After soybeans matured, three soybean plants under each of the different treatments were randomly selected, and their above-ground fresh weight, above-ground dry weight, underground fresh weight, underground dry weight, plant height, stem diameter, root length, 100-seed weight, yield per plant, and number of pods per plant were measured.

[0056] ① Fresh weight of aboveground parts: The aboveground parts of soybean plants were taken and their weight was measured using the connection point between the stem and the root as a marker. The weight was measured in grams, and each treatment was repeated three times.

[0057] ② Dry weight of aboveground parts: Using the connection point between the stem and root as a marker, the aboveground parts of soybean plants were taken, blanched at 105℃ for 10 min, dried at 80℃ to constant weight, and the dry weight was measured in g. Each treatment was repeated 3 times.

[0058] ③ Fresh weight of underground parts: The root system of soybean plants was taken, marked by the connection point between the stem and the root. The soil on the surface of the root system was washed away with running water and the roots were naturally dried. The weight was measured in grams. Each treatment was repeated 3 times.

[0059] ④Dry weight of underground parts: Using the connection point between the stem and the root as a marker, soybean plant roots were taken, the soil on the surface of the roots was washed off with running water, blanched at 105℃ for 10 min, and dried at 80℃ to constant weight before measuring the dry weight in grams. Each treatment was repeated 3 times.

[0060] ⑤ Plant height: The distance from the junction of the soybean stem and root to the highest point of the soybean plant, measured in cm. Each treatment was repeated 3 times.

[0061] ⑥ Stem diameter: The diameter at the junction of the soybean plant root and stem, expressed in mm. Each treatment was repeated 3 times.

[0062] ⑦ Root length: The distance from the end of the soybean plant's root system to the junction of the stem and root, measured in cm.

[0063] ⑧ 100-seed weight: 100 soybean seeds were randomly selected from each treatment and their weight was measured in grams, accurate to 0.01 g. Each treatment was repeated 3 times.

[0064] ⑨ Yield per plant: One soybean plant was randomly selected from each treatment, and the total weight of all soybean seeds was measured in grams, accurate to 0.01 g. Each treatment was repeated 3 times.

[0065] ⑩ Number of pods per plant: Randomly select one soybean plant from each treatment and measure the total number of pods, in units of pods. Each treatment is repeated 3 times.

[0066] The effects of different experimental treatments on the biomass of potted soybean plants are shown in Table 3. Under the inoculation condition of Acinetobacter calcifera HDP-02, all biomass indicators of soybean plants in natural soil were higher than those in sterilized soil. The biomass indicators of soybean plants in the experimental group inoculated with Acinetobacter calcifera HDP-02 were all higher than those in the control group, indicating that Acinetobacter calcifera HDP-02 can not only effectively resist the invasion of soil pathogens on soybean plant roots, but also synergistically promote the increase of various biomass indicators in soybean plants with other beneficial microorganisms.

[0067] Table 3

[0068]

[0069]

[0070] Example 4: Experiment on the effect of Acinetobacter calciacetate HDP-02 on carbendazim residues in soybean seeds and rhizosphere soil of soybean plants

[0071] The experiment employed a randomized block design with eight treatments: (1) soybeans sown in natural soil (NCK); (2) soybeans inoculated with Acinetobacter calcifera HDP-02 in natural soil (NA); (3) soybeans sown in natural soil and sprayed with carbendazim (NDK); (4) soybeans inoculated with Acinetobacter calcifera HDP-02 in natural soil and sprayed with carbendazim (NDA); (5) soybeans sown in sterilized soil (MCK); (6) soybeans inoculated with Acinetobacter calcifera HDP-02 in sterilized soil (MA); (7) soybeans sown in sterilized soil and sprayed with carbendazim (MDK); and (8) soybeans inoculated with Acinetobacter calcifera HDP-02 in sterilized soil and sprayed with carbendazim (MDA). Each pot contained 12 kg of soil, and each treatment had five biological replicates. The experiment was conducted at a temperature of 23±1℃, a day-day cycle of 8 h, and a humidity of 50±5%.

[0072] Soil sterilization conditions: Sterilize at 121℃ for 2 hours, and then sterilize again under the same conditions after an interval of 24 hours.

[0073] Soybean seeds are surface disinfected in 75% ethanol for 5 minutes, then rinsed with sterile distilled water at least 10 times. The disinfected soybean seeds are then sown in pots (6 seedlings per pot, 3 seedlings after germination) and watered regularly.

[0074] Acinetobacter calciacetate HDP-02 cells were collected by centrifugation, washed twice with sterile water, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶ with sterile water. 8The concentration of *Acinetobacter calcium acetate* HDP-02 inoculum is calculated as 5 mL / mL. The inoculum is applied to the soil via root drenching, with each soybean seed inoculated with 5 mL of HDP-02. A control inoculation consists of 5 mL of sterile water.

[0075] Application method of carbendazim in the experiment: Take 1.5g of carbendazim powder and add 900mL of sterile water to dissolve it completely. The concentration of carbendazim is 0.002g / mL. Spray it around the soil around the roots of soybean plants at 30d and 60d after the emergence of soybean plants. Spray 22.5mL per pot.

[0076] The method for detecting carbendazim residues in this embodiment:

[0077] (1) Preparation of standard solutions

[0078] Preparation of standard solution: Weigh 50 mg of carbendazim standard (accurate to 0.0001 g) accurately using an analytical balance, place it in a 100 mL volumetric flask, add 5 mL of methanol and 1 mL of glacial acetic acid, then dilute to the mark with methanol, shake well and place in an ultrasonic cleaner for 5 min, then set aside.

[0079] (2) Sample preparation

[0080] Sample preparation: Soybean seeds were crushed until no obvious particles were visible and placed in a glass container. Soil samples were sieved through a 40-mesh sieve to ensure no excessively large particles were present. Then, 10g of both soybean seed and soil samples were accurately weighed using an analytical balance (accurate to 0.0001g). The samples were placed in a stoppered Erlenmeyer flask, and 50mL of a mixed solvent of methanol and glacial acetic acid (methanol:glacial acetic acid = 9.5:0.5) was added. The mixture was sonicated for 30min, then washed with methanol and filtered into a centrifuge tube. The mixture was centrifuged at 2500r / min for 20min until the solution was clear. 1mL of the supernatant was accurately transferred to a 10mL volumetric flask, and the volume was adjusted to the mark with methanol. The flask was then shaken well and placed in an ultrasonic cleaner for 5min to ensure complete dissolution of the active ingredients. Before liquid chromatography, the sample was filtered through a 0.45μm organic filter membrane to prevent column clogging.

[0081] (3) Chromatographic conditions

[0082] Mobile phase: V(methanol):V(water) = 55:45; Flow rate: 1.0 mL / min; Detection wavelength: 280 nm; Injection volume: 10 μL; Column temperature: 27 °C; Retention time: 7.5 min.

[0083] (4) Calculation of carbendazim residue

[0084] After setting the chromatographic conditions on the high phase liquid chromatograph, place the standard sample and the sample to be tested into the autosampler respectively. After the baseline stabilizes, select the autosampler mode and inject the sample three times consecutively. The residual amount of carbendazim in the sample to be tested is determined by the retention time and peak area of ​​the standard.

[0085] The carbendazim content in the sample to be tested is expressed as a mass fraction X:

[0086]

[0087] In the formula: a1--------------- Area of ​​the carbendazim peak in the standard sample

[0088] a2--------------- Area of ​​the carbendazim peak in the sample to be tested

[0089] m1----------------Mass of carbendazim in the sample to be tested (g)

[0090] m2----------------Mass of carbendazim in the sample to be tested (g)

[0091] p-----------------The mass fraction (%) of carbendazim in the standard.

[0092] The effects of different experimental treatments on carbendazim residues in mature soybean seeds and rhizosphere soil are shown in Table 4. Table 4 shows that no carbendazim residues were detected in soybean seeds or rhizosphere soil in the control groups that were not sprayed with carbendazim. After inoculation with Acinetobacter calcitriol HDP-02, the carbendazim residues in soybean seeds and rhizosphere soil were significantly lower than those in the uninoculated and inoculated groups, indicating that inoculation with Acinetobacter calcitriol HDP-02 can significantly reduce carbendazim residues. Furthermore, the carbendazim residues in soybean seeds and rhizosphere soil in the sterilized soil treatment groups (MDK and MDA) were higher than the corresponding values ​​in the natural soil treatment groups (NDK and NDA), indicating that indigenous microorganisms in natural soil can also slowly degrade carbendazim to some extent, and can synergistically degrade carbendazim with Acinetobacter calcitriol HDP-02.

[0093] Table 4

[0094]

[0095]

Claims

1. A strain of Acinetobacter calcium acetate, characterized in that... The Acinetobacter calcium acetate bacillus is Acinetobacter calcium acetate ( Acinetobacter calcoaceticus HDP-02, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, on July 5, 2022, with accession number CCTCC NO: M 20221027.

2. The application of Acinetobacter calciacetate HDP-02 as described in claim 1 in the degradation of insoluble phosphorus, wherein the insoluble phosphorus is calcium phytate, aluminum phosphate, iron phosphate, lecithin, and calcium phosphate.

3. The application of Acinetobacter calcifera HDP-02 as described in claim 1 in inhibiting soybean root rot.

4. The application of Acinetobacter calcifera HDP-02 as described in claim 1 in the degradation of carbendazim.

5. The application of Acinetobacter calcifera HDP-02 as described in claim 1 in promoting the increase of soybean plant biomass.

6. The application according to claim 5, characterized in that... The biomass is defined as aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, plant height, stem diameter, root length, weight per 100 seeds, yield per plant, and number of pods per plant.

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