CN113234630B - A cadmium-resistant microbacterial strain and its application - Google Patents

Abstract

The present invention relates to a Microbacterium sp. strain PLR25 and an application thereof. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 11, 2021, and the preservation number is GDMCC.NO: 61652. The strain has the characteristics of acid resistance, cadmium resistance, phosphorus solubility, siderophore and auxin secretion. Soybean inoculation with this Exiguobacterium PLR25 can increase soybean biomass, chlorophyll SPAD value and phosphorus content; and can overcome the inhibitory effect of cadmium on soybean growth, especially the inhibitory effect of cadmium on soybean root growth, which has high application value.

Description

A cadmium-resistant microbacterium strain and its application

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to Exiguobacterium strain PLR25 and its application.

Background technique

Cadmium is not an element necessary for plant growth and development. When its body content exceeds a certain concentration, plants will be seriously harmed, such as plant wilting and yellowing, weak growth and other symptoms. Plant roots can easily absorb cadmium, and then transport it to the shoot, which will poison plants, affect the absorption of nutrient elements, and then inhibit plant growth. Cadmium can also damage root structure, thereby affecting plant nutrient uptake.

Phosphorus is one of the most important essential nutrients for plants and a macronutrient essential for key metabolic processes such as plant cell division, energy generation, macromolecular biosynthesis, membrane integrity, signal transduction, and photosynthesis. It also plays a role in plant respiration and nitrogen fixation in legumes. Although soil contains a lot of phosphorus compounds, very little soluble phosphorus is actually available to plants. Because most of the phosphorus in the soil exists in insoluble form, and only the phosphorus source in the form of phosphate can be absorbed by plants.

Microorganisms and plants are closely related, and microorganisms either positively or negatively affect the growth and development of plants. Affected by plant root exudates, the microorganisms in the soil establish a stable symbiotic relationship with the plant rhizosphere and root system. Some bacteria can infect the plant root system and colonize the interior of the plant. Chinese invention patent, CN112625967A, discloses a kind of Exiguobacterium acetylide to adsorb cadmium ion in aqueous solution. However, the strain does not coexist with plants and cannot solve the problems of low phosphorus stress and cadmium toxicity in plants.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a new Microbacterium sp. strain PLR25, which is symbiotic with plants and can solve the technical problems of low phosphorus stress and cadmium poisoning stress in plants. The strain has the characteristics of auxin secretion, acid resistance, cadmium resistance, phosphorus solubility and siderophore production. Soybean inoculation with the Exiguobacterium can significantly increase soybean biomass (dry weight), chlorophyll SPAD value and phosphorus content; and can overcome the inhibitory effect of cadmium on soybean growth, especially the inhibitory effect of cadmium on soybean root growth.

The object of the present invention is to provide a Microbacterium sp. strain PLR25.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in cultivating cadmium-resistant plants.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in reducing the inhibition of plant growth by cadmium.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in promoting the synthesis of chlorophyll in plant leaves.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in promoting the absorption of phosphorus by plants.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in the preparation of cadmium-resistant microbial pesticides, chlorophyll biosynthesis promoters or phosphorus-absorbing inoculants suitable for plants.

Another object of the present invention is to provide the application of Microbacterium sp. strain PLR25 in dissolving insoluble phosphorus.

Another object of the present invention is to provide a soybean planting method.

To achieve the above object, the present invention provides the following technical solutions:

The applicant team obtained a plant with the characteristics of secreting auxin, acid resistance, cadmium resistance, phosphorus solubility and siderophore production from the peanut rhizosphere in Huangyewu Village, Qujiang District, Shaoguan City through artificial separation and purification, which can significantly improve soybean biological activity. amount, phosphorus content, chlorophyll SPAD value, and strains that make soybean resistant to cadmium. The sequencing results of this strain were completed by Guangzhou Ruibo Biotechnology Co., Ltd. The sequencing results of its 16S rDNA were subjected to Blast multiple sequence alignment analysis in the NCBI database, thereby confirming that the isolated strain was a new strain of Microbacterium sp. , named PLR25. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 11, 2021. The deposit address is: Guangdong, China, and the deposit number is GDMCC.NO: 61652.

The morphological characteristics of the strain PLR25 are as follows: the optimum growth temperature is 37 degrees Celsius, on the LB plate medium, it forms approximately round, smooth, protruding, orange colonies, short rods under the light microscope, and Gram stain is purple, Gram spp. positive bacteria.

The study showed that the Exiguobacterium strain PLR25 significantly promoted the absorption of phosphorus in soybean and improved the ability of soybean to tolerate cadmium. The present invention therefore claims:

The application of Exiguobacterium strain PLR25 in cultivating cadmium-resistant plants, especially for cultivating cadmium-resistant soybean plants, to overcome the inhibitory effect of cadmium on the root growth of soybean plants.

The Exiguobacterium strain PLR25 is used in reducing the inhibition of plant growth by cadmium, especially in promoting the biomass increase and root growth of soybean plants under the stress of cadmium toxicity, or in the preparation of products capable of attenuating the inhibition of plant growth by cadmium.

The application of Exiguobacterium strain PLR25 in promoting the synthesis of chlorophyll in plant leaves, or the application in preparing a product capable of promoting the synthesis of chlorophyll in plant leaves, especially the chlorophyll in soybean leaves.

The application of Exiguobacterium strain PLR25 in promoting the absorption of phosphorus by plants, especially the application of promoting the absorption of phosphorus by soybean plants, or the application of preparing a product capable of promoting the absorption of phosphorus by plants.

The application of Exiguobacterium strain PLR25 in dissolving insoluble phosphorus, wherein, preferably, the insoluble phosphorus is Ca ₃ (PO ₄ ) ₂ .

The application of Exiguobacterium strain PLR25 in the preparation of growth promoters, anti-cadmium microbial pesticides, chlorophyll biosynthesis promoters or phosphorus absorption-promoting bacteria agents suitable for plants.

Wherein, preferably, the plant is a soybean plant.

A method for planting soybean, utilizing the bacterial suspension of Exiguobacterium strain PLR25 described in claim 1 or watering to process soybean seedlings;

Specifically preferably, the treatment method is watering;

More preferably, it specifically includes the following steps: after the soybean seed is raised, the bacterial suspension of the Exiguobacterium strain PLR25 described in claim 1 is irrigated in the cultivation medium, and then irrigated once 1, 3, and 5 days after the seedling transplant; wherein Preferably, the amount of each watering is 80-120 mL per seedling; preferably, the OD ₆₀₀ of the bacterial suspension is 0.6-1.0.

Wherein, as an optional embodiment, the dispersion medium of the bacterial suspension is soybean nutrient solution, and its formula is as follows: 2.5mM KNO ₃ , 2.5mM Ca(NO ₃ ) ₂ ·4H ₂ O, 0.08mM Fe -Na _- EDTA, 0.25 mM _K2SO4 , ₁ mM _MgSO4.7H2O , _4.5 x ^10-3 mM _MnCl2.4H2O , 0.3 x ^10-3 mM _ZnSO4.7H2O , 0.16 x ^10-3 mM ^CuSO4.5H2O , ^0.16x10-3 mM _{( NH4 ) 6Mo7O24.4H2O} , _{20x10-3 mM} ^H3BO3 , _{50x10-3 mM KH2PO4 .}

The present invention has the following beneficial effects:

The present invention provides a cadmium-resistant microbacterium strain PLR25, which is symbiotic with plants and can:

(1) Promote plant growth, promote plant absorption of phosphorus content, increase its biomass and chlorophyll SPAD, especially suitable for soybean plants.

(2) Improve the cadmium poisoning ability of plants, and overcome the growth inhibition effect of cadmium on plants, especially the growth inhibition effect of cadmium on soybean.

(3) It has the characteristics of secreting auxin, acid resistance, barrier resistance, dissolving phosphorus and producing siderophore.

Description of drawings

Figure 1 shows the phosphatisol zone of Exiguobacterium tumefaciens PLR25 strain.

Figure 2 shows that Exiguobacterium tumefaciens PLR25 produces IAA at different tryptophan concentrations.

Figure 3 shows a quantitative graph of IAA production by Exiguobacterium tumefaciens PLR25.

Figure 4 shows the cadmium tolerance curve (OD ₆₀₀ - cadmium concentration) of Exiguobacterium tumefaciens PLR25.

Figure 5 shows the acid resistance curve (OD ₆₀₀ -pH) of Exiguobacterium tumefaciens PLR25.

Figure 6 shows the developmental phylogenetic tree of Exiguobacterium tumefaciens PLR25.

Figure 7 shows the changes of plant dry weight (A) and SPAD value (B) of soybean inoculated with Exiguobacterium PLR25 under cadmium toxicity.

Figure 8 shows the changes of root length (A), root surface area (B), root diameter (C) and root volume (D) of soybean inoculated with Exiguobacterium PLR25 under cadmium toxicity.

Figure 9 shows the changes of the shoot cadmium concentration (A) and the root cadmium concentration (B) of soybean inoculated with Exiguobacterium PLR25 under cadmium toxicity stress.

Figure 10 shows the changes of phosphorus content in roots (A) and phosphorus content (B) in shoots of soybeans inoculated with Exiguobacterium PLR25 under cadmium toxicity.

In the figure *: 0.01<P<0.05, **: 0.001<P<0.01, ***: P<0.001.

Detailed ways

The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

Example 1: Isolation and purification of strains

1. Isolation and purification of strains

A peanut rhizosphere isolate of peanut (Yueyou 45) grown on cadmium-contaminated soil in Huangyewu Village, Qujiang District, Shaoguan City. Cut 5g of peanut roots respectively, use a small brush to remove the surface soil, rinse with sterile water for several times until there is no attached soil, and place them in a 250mL numbered triangular bottle containing 100mL of sterilized water, 180 r/min, 37 ℃ shaker, shake for 30min. Add 10-20 glass beads to the conical flask to help break up the soil, so that the bacteria can be released from the soil. After the shaking, let the soil suspension stand for 10 minutes to obtain a soil suspension, take the supernatant, and carry out 10 times Serial concentration gradient dilution, dilute to 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , and then draw 0.1 mL of the dilution solution and apply it to the LB plate. Each concentration gradient is repeated three times. Invert the culture at 37°C, observe the growth of the colonies, pick single clones of different phenotypes, number the back of each colony, and record the colony morphology and the time of colony growth. Shake the bacteria in a 1.5mL centrifuge tube, culture at 180r/min and 37°C, and then streak and purify 3-4 times in LB solid medium (until the colony shape under the microscope is consistent, indicating that the purification has been completed), the concentration added is: 25% sterilized glycerol was stored at -80°C until use.

Results 40 strains were isolated in the peanut rhizosphere, numbered PLR1-40, which were isolated and purified strains to be tested.

Example 2: strain screening

2.1 Primary screening of phosphorus-dissolving bacteria

The screened strains were inoculated on the PKO solid medium, each plate was connected three times, and cultivated in an incubator at 28°C. On the 7th day, the size of the outer diameter (D) and colony diameter (d) of the phosphate-solubilizing circle of the strain was measured and photographed. The phosphorus-dissolving ring can dissolve phosphorus. Then compare the ratio of (D/d) to judge the ability of the strain to dissolve phosphorus.

Draw 1mL of the bacterial liquid of the strain with phosphorus-dissolving circle, inoculate it in 20mL LB liquid medium (50mL centrifuge tube), after 24h of culture, determine the OD ₆₀₀ to be about 1.0, take 1mL of bacterial re-suspension and inoculate it in 20mL PKO liquid culture In the base (50 mL centrifuge tube), each strain was repeated three times, and the CK control group was inoculated with an equal amount of sterile water, placed at 28°C, and cultured in a shaker at 180 r/min. On the 7th day, the pH value of the PKO liquid medium was measured, and 1 mL of the supernatant (1200 r/min, centrifuged for 5 min) was transferred to a 25 mL volumetric flask to be tested. , after the reaction for 30min, use a microplate reader to measure the absorbance value at 880nm (in order to remove the influence of the color of the medium, use 880nm measurement, through the conversion formula, get the standard curve (y=0.4833x+0.0002 (R ² =0.9997) .Convert to get the corresponding phosphorus concentration (mg/L).

Results: It was found that PLR25 performed poorly in the phosphorus dissolution plate test, with a phosphorus dissolution circle ratio of only 1.25 (Figure 1 and Table 1). However, in the quantitative test of phosphorus dissolution, PLR25 showed a strong ability to dissolve phosphorus, and it could dissolve phosphorus to 135.47±7.19mg/mL.

Table 1

strain number D(cm) d(cm) D/d Dissolved Phosphorus Concentration (mg/mL) PLR25 0.71 0.57 1.25 135.47±7.19

2.2 Screening of secreted IAA

Preparation containing L-tryptophan concentration was 0, 100, 200, 500mg/L LB liquid medium respectively, and the strains screened out were inoculated in the liquid medium, and each strain was repeated three times (with the same medium without inoculation). As a blank control), cultured at 28°C for 2 days at 180 r/min, 200 μL of supernatant was taken from each replicate, 200 μL of Salkowski chromogenic solution was added to a 96-well microtiter plate, and the same liquid medium without bacteria was added. Compared with 200 μL of Salkowski chromogenic solution, the wavelength was measured at 530 nm using a microplate reader after being placed in the dark for 20 min at room temperature. Find the corresponding IAA yield on the standard curve. IAA production units are mg/mL.

The qualitative results showed (Fig. 2) that the strain PLR25 showed a very clear pink color and had the ability to produce IAA. PLR25 showed that in the range of 0-500mg/L tryptophan concentration, the release ability of strain IAA was enhanced with the increase of tryptophan concentration. At the same time, we can observe that the PLR25 strain is still pink even under the condition of 0 tryptophan concentration, indicating that PLR25 can use the non-tryptophan pathway to produce IAA. The quantitative results showed (Fig. 3) that PLR25 could produce IAA without tryptophan, and could produce 57.10±4.76 mg/L at a tryptophan concentration of 500 mg/L.

2.3 Screening of siderophore capacity

Pipette 1 mL of the bacterial suspension of the strain to be tested and inoculate it in MKB liquid medium. Incubate at 28°C for 48h at 180r/min. The culture medium was centrifuged for 10 min (1200 r/min), and 200 μL of the supernatant was taken (the reference value (Ar) was the addition of 200 μL of uninoculated MKB liquid medium during the measurement), which was mixed with the CAS test solution at a ratio of 1:1. After 1 h of reaction at room temperature, the OD value (A) at the wavelength of 630 nm was measured by a microplate reader. In the experiment, if the siderophore was not produced, the CAS liquid medium was blue like the control, and if the strain produced siderophore, the CAS liquid medium changed to orange. The ratio of A/Ar is used to represent the relative content of siderophore in the sample. The smaller the value, the stronger the ability of the strain to produce siderophore. The ratio of (Ar-A)/Ar represents the activity unit of siderophore in the sample. higher, the stronger the iron carrier capacity.

The results showed that the PLR25 strain had the highest activity of siderophore among the PLR1-40 strains, reaching 58.35±5.77%.

2.4 Screening of cadmium resistance

Six LB liquid culture mediums with cadmium concentrations of 0, 4, 8, 12, 16, and 20 mg/L were prepared respectively, and sterilized at 121° C. for 20 min. Prepare two 96-well sterilized cell culture plates, and pipette 200 μL of the prepared LB liquid medium with different cadmium concentrations into the culture plate. After the strains to be tested were activated and cultured for 24 hours, 5 μL of each strain was taken into the culture wells with different cadmium concentration values, and each strain was repeated 4 times, cultured at 37°C and 180 r/min for 48 hours, and the absorbance value of the bacterial liquid at 600 nm was determined.

Results: Under the condition of 0-20mg/L Cd concentration, all strains could grow, but the growth rate was still different. PLR25 grows faster under low concentration (below 4 mg/L) cadmium, but grows slowly under high concentration Cd (Fig. 4), but it outperforms other PLR growth-promoting strains obtained by isolation and purification.

5. Screening for acid resistance

Six LB mediums with pH of 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 were prepared, respectively, and sterilized at 121 °C for 20 min. Prepare two 96-well sterilized cell culture plates, and pipette 200 μL of the prepared LB liquid medium with different pH into the culture plate. After the strains to be tested were activated and cultured for 24h, each strain was pipetted into 5 μL of culture wells with different pH values, and each strain was repeated 4 times, cultured at 37°C, 180r/min for 48h, and the absorbance of the bacterial solution at 600nm was determined.

The strain PLR25 had the fastest growth rate between pH 5.5 and 7.0 (Fig. 5), indicating that this strain can also grow well under mildly acidic conditions.

Example 3 Identification of strains

After comprehensive consideration of various aspects, the screened strain PLR25 was identified.

3.1 Morphological identification of strains

The strain PLR25 was inoculated on LB solid medium for cultivation and observed and recorded. After culturing for 5 to 7 days in the optimum growth conditions (pH 7.0, temperature 37°C), the isolated and purified strain PLR25 was subjected to single colony state observation, mainly including colony size, color, colony surface state and colony edge state, etc. . On the other hand, for the strain PLR25 in the logarithmic growth phase, the morphology of the cells was observed with an optical microscope after smear staining.

On the LB plate medium, approximately round, smooth, protruding, orange colonies formed, short rods under the light microscope, and Gram-stained purple, Gram-positive bacteria.

3.2 Molecular identification

After multiple screening and comprehensive consideration, the strain PLR25 was screened for identification and continued research. The strains were inoculated in LB medium at 37°C and cultured at 180 r/min for 24 hours. The total DNA of the strains was extracted using a bacterial total DNA extraction kit and sent to a biological company for sequencing. The obtained strain sequences were compared in the NCBI database for Blast sequence comparison. For analysis, a phylogenetic tree was constructed using MEGA 7.0 (Figure 6).

According to the sequencing results, PLR25 is a Microbacterium (Microbacterium.sp), and its 16S rDNA sequence is shown in SEQ ID NO: 1; and the Microbacterium sp. strain PLR25 was deposited in Guangdong Province on May 11, 2021 Microorganism Culture Collection Center, the deposit number is GDMCC.NO: 61652.

Example 4 Test of back-linking soybeans of Exiguobacterium tumefaciens PLR25

4.1 Tie-back test

In this study, a matrix soil pot experiment was used. Three cadmium concentration groups were set up in the experiment, 0, 10, and 20 mg/kg CdCl ₂ ·5/2H ₂ O, respectively.

Mixed substrate treatment method of substrate and vermiculite: Weigh the fertilizer-free substrate and vermiculite and mix them in a ratio of 3:1 (0.5 kg mixed soil = 0.125 kg vermiculite + 0.375 kg substrate), sterilize at 121 °C for 40 minutes, and repeat after 24 hours. Sterilize and store for one week. Take a flowerpot with a capacity of 2L, soak it in 10% sodium hypochlorite overnight, rinse it with clean water several times, and air-dry it for later use. Weigh a certain amount of cadmium chloride and dissolve it in water, prepare a solution of 0, 10, 20 mg/kg, measure a certain volume and pour it into the mixed matrix, and at the same time mix 125 mg Ca ₃ (PO4) ₂ (equivalent to pure Phosphorus 50mg/kg), weighed and mixed in each pot, and let stand for a week for later use.

The seeds were sterilized with hydrochloric acid-sodium hypochlorite to generate chlorine gas for 4 hours, and the seedlings were raised with the mixed sand of coarse sand: medium sand=1:2. The bacterial suspension was irrigated 1, 3, and 5 days after the seedlings were transplanted, 100 mL at a time, 3 times in total.

Preparation of bacterial suspension nutrient solution: the bacterial strains were inoculated into 250 mL of LB medium, in a shaker at 37 °C, and incubated at 180 r/min for about 18 hours. Resuspend in low-phosphorus nutrient solution, and use a sterilized pipette tip to remove the surface matrix of the seedling roots. The bacteria resuspended nutrient solution was poured into the roots, and the bacteria were watered into the substrate with the nutrient solution.

The formula of the low phosphorus nutrient solution is: 2.5mM KNO ₃ , 2.5mM Ca(NO ₃ ) ₂ ·4H ₂ O, 0.08mM Fe-Na-EDTA, 0.25mM K ₂ SO 4 , 1mM MgSO ₄ ·7H ₂ O , 4.5×10 ^-3 mM MnCl ₂ ·4H ₂ O, 0.3×10 ^-3 mMZnSO ₄ ·7H ₂ O, 0.16×10 ^-3 mM CuSO ₄ ·5H ₂ O, 0.16×10 ^-3 mM(NH4) ₆ Mo 7O ₂₄ ·4H ₂ O, 20× ₁₀ ⁻³ mM H ₃ BO ₃ , 50×10 ⁻³ mM KH ₂ PO ₄ .

4.2 Indicator detection

Soybeans were harvested after 30 days of cultivation. Under the condition of sufficient light, the SPAD value of different parts of the lower three leaves of the plant was measured with a SPAD instrument and the average value was taken. After rinsing with tap water, put it in the cold storage first, and scan it in time within a week. Use a desktop scanner (Epson1460XL) to scan the root system, spread the root system as flat as possible so that it does not overlap. If the root system is too large, it can be cut and scanned without affecting the scanning result. Cover the blue shading plate. After the scanning is completed, the root system is analyzed. The software WinRHIZO (RegentInstruments Inc., Canada) was used to analyze the root traits of each sample root.

The shoots and roots of the crops were separated, and the fresh weights of the shoots and roots were weighed. The shoots were placed in a 105°C oven for 30 minutes, dried in the oven, taken out and placed at room temperature for 10 minutes to cool and weighed for dry weight. The roots were first scanned with a root scanner and weighed, and the rest were also dried and weighed dry.

Then soak the soybean roots in 10mmol/L Na ₂ -EDTA solution for 5min to remove the Cd ²⁺ adsorbed on the surface, then rinse with secondary water, and separate the roots and stems and leaves at 75°C to dry and weigh the dry weight. The phosphorus content and cadmium concentration in the shoots were measured; the roots were placed in cold storage for root sweeping, dried and weighed after sweeping, and the phosphorus content and cadmium concentration in the roots were measured. Phosphorus was detected by ultraviolet spectrophotometry, and cadmium was detected by atomic absorption spectrophotometer flame absorption method.

The result is as follows:

(1) With the increase of cadmium concentration, the dry weight of plants decreased significantly. Compared with 0Cd, the plant biomass of no inoculation treatment (CK group) under the conditions of 10mg/kg Cd ²⁺ and 20mg/kg Cd ²⁺ (10Cd and 20Cd group), decreased by 31% and 55%, respectively, and soybeans inoculated with strain PLR25 decreased by 31% and 41%. At 20Cd level (high concentration of cadmium), the dry weight of soybean plants inoculated with strain PLR25 was 49% higher than that of soybeans not inoculated (Fig. 7A).

(2) Inoculation treatment significantly affected the SPAD value of soybean leaves (0.01＜P＜0.05), and there was a significant interaction between cadmium treatment and inoculation treatment (0.01＜P＜0.05). The SPAD value of soybean inoculated with PLR25 was significantly higher than that in the non-inoculated CK treatment by 20% (Fig. 7B).

(3) Cd treatment significantly affected the total root length of soybean (P<0.001). Inoculation treatment significantly affected the total root length of soybean (0.01＜P＜0.05). There was a very significant interaction between cadmium treatment and inoculation (P＜0.001). Under the conditions of 10 mg/kg Cd ²⁺ and 20 mg/kg Cd ²⁺ , the root length of soybeans treated with no inoculation (CK group) decreased by 46% and 48%, respectively, while the total root length of soybean treated with Exiguobacterium PLR25 was respectively decreased by 36% and 32% (Fig. 8A). In addition, Exiguobacterium tumefaciens PLR25 attenuated the inhibitory effect of cadmium on root growth at high concentrations of cadmium (Fig. 8B).

(4) Different cadmium concentration treatments significantly affected the cadmium concentration of soybean shoots and roots (P<0.001), inoculation treatment significantly affected the cadmium concentrations of soybean shoots and roots (0.01<P<0.05), cadmium treatment and inoculation treatment There was a significant interaction (0.01<P<0.05). The data showed that with the increase of cadmium concentration, the cadmium concentration in soybean shoots and roots increased significantly. At 0Cd and 10Cd levels, the cadmium concentrations in the shoots and roots of soybeans inoculated with strain PLR25 did not change significantly compared to those without CK inoculation, but at 20Cd levels, the cadmium concentrations in shoots of soybeans inoculated with strain PLR25 decreased by 16% (Fig. 9A); root cadmium concentration was reduced by 72% (FIG. 9B).

(5) Different cadmium concentrations significantly affected the phosphorus content of soybean shoots (P<0.001), and there was a significant interaction between the inoculation treatment and the cadmium treatment (0.01<P<0.05). The data showed that with the increase of cadmium concentration, the phosphorus content in the shoots of the plants increased significantly. At the level of 0Cd, the phosphorus content in the shoots of soybeans inoculated with strain PLR25 increased by 181% compared to that without CK inoculation. Under the 20Cd level, the phosphorus content of the shoots inoculated with PLR25 increased by 74%. The results showed that the inoculation of PLR25 strain could increase the phosphorus content of soybean shoots under the condition of low phosphorus and no cadmium addition, and at 20Cd level, the inoculation of PLR25 strain could significantly increase the phosphorus content of soybean shoots (Figure 10).

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

SEQUENCE LISTING

<110> South China Agricultural University

<120> A cadmium-resistant microbacterium strain and its application

<130> YGZS214947

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1370

<212> DNA

<213> Microbacterium sp. strain PLR25

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gcgcagatat caggaggaac accgatggcg aaggcagatc tctgggccgt aactgacgct 660

gaggagcgaa agggtgggga gcaaacaggc ttagataccc tggtagtcca ccccgtaaac 720

gttgggaact agttgtgggg tccattccac ggattccgtg acgcagctaa cgcattaagt 780

tccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg gggacccgca 840

caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc aaggcttgac 900

atatagagga aacgtctgga aacagtcgcc ccgcaaggtc tctatacagg tggtgcatgg 960

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aagtcggtaa cacctgaagc cggtggccca acccttgtgg aggagccgtc 1370

Claims (9)

1. A strain of micro-bacillusMicrobacteriumsp.) strain PLR25, which is deposited at 11.5.2021 in Guangdong province of microbial cultures Collection with the following deposition number GDMCC No.: 61652.

2. the 16S rDNA of the Microbacterium strain PLR25 of claim 1, wherein the sequence is as shown in SEQ ID No. 1.

3. Use of the microbacterium strain PLR25 of claim 1 for breeding cadmium poison resistant soybean plants.

4. The use of the microbacterium strain PLR25 of claim 1 for reducing cadmium to inhibit the growth of soybean plants, promoting the synthesis of chlorophyll by leaves of soybean plants, promoting the absorption of phosphorus by soybean plants and the dissolution of insoluble phosphorus; or preparing products which can weaken cadmium to inhibit the growth of soybean plants, promote the soybean plants to absorb phosphorus and dissolve insoluble phosphorus.

5. Use of the microbacterium strain PLR25 of claim 1 in the preparation of cadmium poison resistant microbial pesticides, chlorophyll biosynthesis promoters or phosphate uptake promoting microbial agents suitable for soybean plants.

6. A planting method for promoting soybean plant growth in a heavy metal stress-resistant environment, which is characterized in that soybean seedlings are irrigated by using a bacterial suspension containing the microbacterium strain PLR25 in claim 1.

7. The planting method of claim 6, wherein the planting method comprises the following steps: after soybean seeds were cultivated, a bacterial suspension containing the microbacterium strain PLR25 of claim 1 was poured into the cultivation medium, and then poured once each 1, 3, and 5 days after the seedlings were transplanted.

8. The planting method of claim 7, wherein the amount of each irrigation is 80-120 mL per seedling.

9. The growing method of claim 7, wherein said bacterial suspension OD ₆₀₀ 0.6 to 1.0.

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