CN113234630A

CN113234630A - Cadmium-resistant growth-promoting microbacterium strain and application thereof

Info

Publication number: CN113234630A
Application number: CN202110531112.XA
Authority: CN
Inventors: 王秀荣; 王幼娟; 罗莎莉; 陆星
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-05-16
Filing date: 2021-05-16
Publication date: 2021-08-10
Anticipated expiration: 2041-05-16
Also published as: CN113234630B

Abstract

The invention relates to a micro bacillus (Microbacterium sp.) strain PLR25 and application thereof, wherein the strain is preserved in Guangdong province microorganism strain preservation center at 2021, 5 and 11 days, and the preservation number is GDMCC No: 61652. the strain has the characteristics of acid resistance, cadmium resistance, phosphorus dissolution, siderophore production and auxin secretion. The inoculation of the micro-bacillus PLR25 on soybean can improve the biomass, chlorophyll SPAD value and phosphorus content of the soybean; but also can overcome the effect of cadmium on the growth inhibition of the soybean, especially the effect of cadmium on the growth of soybean root systems, and has high application value.

Description

Cadmium-resistant growth-promoting microbacterium strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a microbacterium strain PLR25 and application thereof.

Background

Cadmium is not an essential element for plant growth and development. When the content in the plant exceeds a certain concentration, the plant is seriously damaged, such as wilting and yellowing, weak growth and the like. The plant root system can easily absorb cadmium and then transport the cadmium to the overground part, so that the plant can be poisoned, the absorption of nutrient elements is influenced, and the growth of the plant is inhibited. Cadmium also destroys root structure, thereby affecting the absorption of plant nutrients.

Phosphorus is one of the most important essential nutrient elements of plants, and is a macronutrient element 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 the soil contains a large amount of phosphorus compounds, plants can actually utilize very little soluble phosphorus. Because most of the phosphorus in the soil exists in a poorly soluble form, only the phosphorus source in the form of phosphate can be absorbed by the plants.

Microorganisms are closely related to plants, and microorganisms either positively or negatively affect the growth and development of plants. Under the influence of plant root secretion, microorganisms in soil establish stable symbiotic relationship with plant rhizosphere and root systems, and some bacteria can infect the plant root systems and colonize inside plants. The Chinese patent of invention, CN112625967A, discloses a method for adsorbing cadmium ions in an aqueous solution by using microbacterium acetylicum. However, the strain does not symbiotic with plants, and can not solve the problems of low phosphorus stress and cadmium toxicity stress of plants.

Disclosure of Invention

The invention aims to provide a novel Microbacterium sp strain PLR25 which is symbiotic with plants and can solve the technical problems of low phosphorus stress and cadmium toxicity stress of plants. The strain has the characteristics of auxin secretion, acid resistance, cadmium resistance, phosphorus dissolution and siderophore production. The micro-bacillus is inoculated on the soybeans, so that the biomass (dry weight), chlorophyll SPAD value and phosphorus content of the soybeans can be obviously improved; but also can overcome the effect of cadmium on the growth inhibition of the soybean, in particular the effect of cadmium on the growth of soybean roots.

The invention aims to provide a micro bacillus sp strain PLR 25.

Another object of the present invention is to provide the use of the Microbacterium sp strain PLR25 for cultivating cadmium virus resistant plants.

It is another object of the present invention to provide the use of the strain of Microbacterium sp PLR25 for reducing cadmium in inhibiting plant growth.

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

It is another object of the present invention to provide the use of the strain of micro-bacterium sp PLR25 for promoting the uptake of phosphorus by plants.

Another objective of the invention is to provide application of a Microbacterium sp strain PLR25 in preparation of cadmium toxin resisting microbial pesticides, chlorophyll biosynthesis promoters or phosphorus absorption promoting microbial agents suitable for plants.

Another object of the present invention is to provide the use of the Microbacterium sp strain PLR25 for solubilizing poorly soluble phosphorus.

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

In order to achieve the purpose, the invention provides the following technical scheme:

an applicant team obtains a strain which has the characteristics of secreting auxin, acid resistance, cadmium resistance, phosphorus dissolution and siderophores, can obviously improve the biomass of soybeans, the phosphorus content and the chlorophyll SPAD value of the soybeans and enables the soybeans to be cadmium-resistant through artificial separation and purification from the rhizosphere of peanuts in yellow leaf house village in the Yangtze area of Yanoguan city. The sequencing result of the strain is completed by Guangzhou Rui Bo biotechnology, and the sequencing result of the 16S rDNA is subjected to Blast multiple sequence alignment analysis in NCBI database, so as to determine that the separated strain is a new strain of Microbacterium sp, which is named as PLR 25. 2021, 5, 11 days, and is preserved in Guangdong province microorganism culture collection center, the preservation address is: china Guangdong, the number of the deposit is GDMCC NO: 61652.

the strain PLR25 was morphologically characterized as follows: the optimal growth temperature is 37 ℃, and on an LB plate culture medium, a round, smooth, protruding and orange colony is formed, and the colony is in a short rod shape under an optical microscope, and gram-positive bacteria are purple in gram stain.

Researches show that the micro bacillus strain PLR25 can remarkably promote the soybeans to absorb phosphorus and improve the cadmium resistance of the soybeans. The invention therefore claims:

the application of the microbacterium strain PLR25 in cultivating cadmium toxicity-resistant plants, in particular to cultivating cadmium toxicity-resistant soybean plants, and overcoming the inhibition effect of cadmium on the root growth of the soybean plants.

The application of the microbacterium strain PLR25 in weakening cadmium inhibition plant growth, in particular to the application in promoting the biomass increase and root growth of soybean plants under cadmium toxicity stress or preparing products capable of weakening cadmium inhibition plant growth.

Application of a microbacterium strain PLR25 in promoting synthesis of chlorophyll by plant leaves or preparing products capable of promoting synthesis of chlorophyll by plant leaves, especially chlorophyll of leaves of plants in the family of soybean.

Application of the microbacterium strain PLR25 in promoting phosphorus absorption of plants, especially in promoting phosphorus absorption of plants in the family of Soaceae, or in preparing products capable of promoting phosphorus absorption of plants.

Application of microbacterium strain PLR25 in dissolving insoluble phosphorus, wherein preferably the insoluble phosphorus is Ca₃(PO₄)₂。

The application of the micro bacillus strain PLR25 in the preparation of growth promoter, cadmium toxin resisting microbial pesticide, chlorophyll biosynthesis promoter or phosphorus absorption promoting microbial inoculum for plants.

Wherein, preferably, the plant is a plant of the family glycines.

A method for planting soybean, wherein soybean seedlings are treated by using a bacterial suspension or pouring of the micro-bacillus strain PLR25 as defined in claim 1;

particularly preferably, the mode of treatment is irrigation;

more preferably, the method specifically comprises the following steps: watering the cultivation medium after soybean seed seedling1, then irrigating once in 1, 3 and 5 days after seedling transplanting; wherein the amount of each irrigation is preferably 80-120 mL/seedling; preferably, the bacterial suspension OD₆₀₀0.6 to 1.0.

As an alternative embodiment, the dispersion medium of the bacterial suspension is soybean nutrient solution, and the formula of the soybean nutrient solution is as follows: 2.5mM KNO₃，2.5mM Ca(NO₃)₂·4H₂O，0.08mM Fe-Na-EDTA， 0.25mM K₂SO4，1mM MgSO₄·7H₂O，4.5×10^-3mM MnCl₂·4H₂O，0.3×10^-3mM ZnSO₄·7H₂O，0.16×10^-3mM CuSO₄·5H₂O，0.16×10^-3mM(NH4)₆Mo₇O₂₄·4H₂O， 20×10^-3mM H₃BO₃，50×10^-3mM KH₂PO₄。

The invention has the following beneficial effects:

the invention provides a cadmium-tolerant growth-promoting microbacterium strain PLR25, which is symbiotic with plants and can:

(1) promoting plant growth, promoting phosphorus absorption of plant, increasing biomass and chlorophyll SPAD, and is especially suitable for soybean plant.

(2) Improving the cadmium toxicity resistance of plants, overcoming the growth inhibition of cadmium on plants, in particular the growth inhibition of cadmium on soybeans.

(3) Has the characteristics of auxin secretion, acid resistance, phosphorus dissolution and siderophore production.

Drawings

FIG. 1 shows the phosphate solubilizing circle of the Microbacterium strain PLR 25.

FIG. 2 shows that Microbacterium PLR25 produced IAA at different tryptophan concentrations.

FIG. 3 shows a quantitative graph of IAA production by Microbacterium PLR 25.

FIG. 4 shows the cadmium resistance curve (OD) of Microbacterium PLR25₆₀₀-cadmium concentration).

FIG. 5 shows acid-tolerant koji of Microbacterium PLR25Line (OD)₆₀₀-pH)。

FIG. 6 shows a developmental clade of Microbacterium PLR 25.

FIG. 7 shows the variation of plant dry weight (A) and SPAD value (B) of P.minutus PLR25 inoculated soybeans under cadmium toxicity stress.

FIG. 8 shows the change in root length (A), root surface area (B), root diameter (C) and root volume (D) of P.minutus-inoculated soybeans PLR25 under cadmium toxicity stress.

FIG. 9 shows the change of the overground part cadmium concentration (A) and the root cadmium concentration (B) of a soybean inoculated with Microbacterium strain PLR25 under cadmium toxicity stress.

FIG. 10 shows the change in root phosphorus content (A) and overground phosphorus content (B) of P25 inoculated soybeans under cadmium toxicity stress.

0.01< P <0.05, 0.001< P <0.01, P < 0.001.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1: separation and purification of bacterial strains

1. Isolation and purification of the strains

In Huangye Wucun in Qujiang district, Shaoguan city, the strain was isolated from the peanut rhizosphere of peanuts (Yue oil 45) planted in cadmium contaminated soil. Respectively cutting 5g peanut roots, brushing surface soil with a small brush, rinsing with sterile water for multiple times until no attached soil exists, placing in a 250mL triangular bottle with serial number containing 100mL sterile water, shaking at 37 ℃ for 30min at 180 r/min. Adding 10-20 glass beads in the conical flask to help break soil and release bacteria from soil, standing the soil suspension for 10min after shaking to obtain soil suspension, taking supernatant, diluting with 10 times of gradient concentration, and diluting to 10 times of gradient concentration^-1、10^-2、10^-3、10^-4、 10^-5、10^-6Then 0.1mL of diluent is sucked and coated on an LB plate, each concentration gradient is repeated for three times, inverted culture is carried out at 37 ℃, the growth condition of colonies is observed, monoclonals with different phenotypes are picked, each colony is numbered on the back of a culture medium, the colony morphology and the time for growing the colony are recorded, each type of monoclonals is picked into a 1.5mL centrifuge tube to be shaken, the monoclonals are cultured at 37 ℃ at 180r/min, and then streaked and purified for 3-4 times by an LB solid culture medium (until the colony morphology under a microscope is consistent, the purification is finished), and sterilized glycerol with the concentration of 25 percent is added for preservation at-80 ℃.

And (4) separating 40 strains at the peanut rhizosphere, wherein the strains are numbered PLR 1-40, and the strains are separated and purified to be tested.

Example 2: strain screening

2.1 preliminary screening of phosphorus-solubilizing bacteria

And (3) inoculating the screened strains on a PKO solid culture medium, inoculating each plate for three times, culturing in an incubator at 28 ℃, measuring the sizes of the outer diameter (D) and the colony diameter (D) of a phosphorus-dissolving ring of the strains at the 7 th D, and taking pictures, wherein the phosphorus-dissolving ring can dissolve phosphorus. Then comparing the ratio of (D/D) to judge the phosphorus dissolving capacity of the strain.

1mL of the bacterial solution of the strain having the phosphorus-solubilizing loop was aspirated, inoculated into 20mL of LB liquid medium (50mL centrifuge tube), cultured for 24 hours, and then OD was measured₆₀₀About 1.0, 1mL of the bacterial suspension was inoculated into 20mL of PKO liquid medium (50mL centrifuge tube), each strain was replicated three times, and the CK control group was inoculated with an equal amount of sterile water and cultured in a shaker at 28 ℃ and 180 r/min. The pH value of the PKO liquid culture medium is measured at the 7 th day, 1mL of supernatant (1200R/min, 5min of centrifugation) is sucked into a 25mL volumetric flask to be measured, 5mL of molybdenum-antimony anti-chromogenic solution is sucked, secondary water is used for constant volume to 25mL, after reaction for 30min, an absorbance value at 880nm is measured by using a microplate reader (in order to remove the influence of the color of the culture medium, 880nm measurement is applied, and a standard curve (y is 0.4833x +0.0002(R is 0.4833x + 0.0002) is obtained through a conversion formula²0.9997). Converted to obtain the corresponding phosphorus concentration (mg/L).

As a result: PLR25 was found to perform poorly in the phosphorus melt plate test with a phosphorus melt circle ratio of only 1.25 (fig. 1 and table 1). However, in the phosphorus dissolution quantitative test, PLR25 shows stronger phosphorus dissolution capacity, and can dissolve phosphorus of 135.47 +/-7.19 mg/mL.

TABLE 1

Strain numbering	D(cm)	d(cm)	D/d	Concentration of dissolved phosphorus (mg/mL)
					PLR25	0.71	0.57	1.25	135.47±7.19

2.2 screening for secreted IAA

LB liquid culture medium with L-tryptophan concentration of 0, 100, 200 and 500mg/L respectively is prepared, the screened strains are inoculated into the liquid culture medium, each strain is repeatedly cultured for three times (the same culture medium without inoculation is used as blank control) at 28 ℃ and 180r/min for 2d, 200 mu L of supernatant is taken out for each repetition, 200 mu L of Salkowski developing solution is added into a 96-hole enzyme label plate, the same liquid culture medium with the added non-inoculated strain and 200 mu L of Salkowski developing solution are used as control, the temperature is kept away from the sun for 20min at room temperature, and then the wavelength is measured at 530nm by using an enzyme label instrument. The corresponding IAA production was checked on the standard curve. IAA production is in mg/mL.

The qualitative results show (FIG. 2) that strain PLR25 exhibits a very pronounced pink color with IAA-producing ability. PLR25 showed that in the tryptophan concentration range of 0-500mg/L, the releasing ability of strain IAA was increased with the increase of tryptophan concentration. At the same time, we can observe that the strain PLR25 still presents pink color even under the condition of 0 tryptophan concentration, which indicates that PLR25 can utilize the non-tryptophan pathway to produce IAA. Quantitative results show (FIG. 3), that PLR25 was able to produce IAA under the condition of no tryptophan, 57.10 + -4.76 mg/L under the condition of tryptophan concentration of 500mg/L,

2.3 screening for siderophore production Capacity

1mL of bacterial suspension of the strain to be tested is sucked and inoculated into MKB liquid culture medium. Culturing at 28 deg.C and 180r/min for 48 h. The culture was centrifuged for 10min (1200r/min), 200. mu.L of the supernatant (200. mu.L of uninoculated MKB liquid medium was added for the reference value (Ar)) was taken and mixed with the CAS detection solution at a ratio of 1: 1. After reacting for 1h at normal temperature, the OD value (A) at the wavelength of 630nm is measured by an enzyme-labeling instrument. In the experiment, if no siderophore production occurred, the CAS broth appeared blue as in the control, and if the strain produced siderophore production, the CAS broth changed to orange. The relative content of the siderophore in the sample is expressed by the ratio of A/Ar, the smaller the value is, the stronger the siderophore production capacity of the strain is expressed, the higher the activity unit of the siderophore in the sample is expressed by the ratio of (Ar-A)/Ar, the stronger the siderophore production capacity is.

As a result, the activity unit of the PLR25 strain for producing the siderophore in the PLR 1-40 strains is the highest and reaches 58.35 +/-5.77%.

2.4 screening for cadmium tolerance

Preparing 6 LB liquid culture media with cadmium concentration of 0, 4, 8, 12, 16, 20mg/L, sterilizing at 121 deg.C for 20 min. 2 96-well sterilized cell culture plates were prepared, and 200. mu.L each of the prepared LB liquid media with different cadmium concentrations was pipetted onto the culture plates. After the strains to be detected are activated and cultured for 24h, each strain absorbs 5 mu L of the strains to culture holes with different cadmium concentration values, each strain is repeatedly cultured for 4 times at 37 ℃ for 48h at 180r/min, and the light absorption value of the bacterial liquid at 600nm is measured.

As a result: under the condition of Cd concentration of 0-20mg/L, each strain can grow, but the growth rates of the strains are still different. PLR25 grows faster at low concentration (below 4 mg/L) of cadmium, but slowly at high concentration of Cd (FIG. 4), but shows better performance than other PLR growth-promoting strains obtained by separation and purification.

5. Screening for acid resistance

6 LB culture media with pH values of 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 are prepared respectively, and sterilized at 121 ℃ for 20 min. 2 96-well sterilized cell culture plates were prepared, and 200. mu.L each of prepared LB liquid media of different pH was pipetted onto the culture plates. After the strains to be detected are activated and cultured for 24h, each strain absorbs 5 mu L of the strains to culture holes with different pH values, each strain is cultured for 48h at 37 ℃ and 180r/min repeatedly for 4 times, and the light absorption value of the bacterial liquid under 600nm is measured.

The strain PLR25 grew most rapidly at pH 5.5-7.0 (FIG. 5), indicating that the strain grew well under mildly acidic conditions.

Example 3 Strain identification

The screened strain PLR25 is identified through comprehensive consideration in many aspects.

3.1 morphological characterization of the strains

The strain PLR25 was inoculated on LB solid medium for culture and the record was observed. After culturing for 5-7 days under the optimal growth conditions (pH 7.0 and temperature 37 ℃), observing the state of a single colony of the separated and purified strain PLR25, wherein the state mainly comprises the size, the color, the surface state of the colony, the edge state of the colony and the like. On the other hand, the strain PLR25 in the logarithmic growth phase was stained by smear, and the morphology of the cells was observed by an optical microscope.

On an LB plate culture medium, an approximately round, smooth, protruding and orange colony is formed, and the colony is in a short rod shape under an optical microscope, and gram stain is purple and gram positive bacteria.

3.2 molecular characterization

After various screening and comprehensive consideration, the strain PLR25 is screened for identification and continued research. Inoculating the strain into LB culture medium, culturing at 37 deg.C and 180r/min for 24 hr, extracting total DNA of the strain with total DNA extraction kit, sequencing with biological company, performing Blast sequence comparison analysis on the obtained strain sequence in NCBI database, and constructing phylogenetic tree with MEGA7.0 (FIG. 6).

The sequencing result shows that the PLR25 is a microbacterium (Microbacterium. sp), and the 16S rDNA sequence of the Microbacterium is shown as SEQ ID NO. 1; and the micro bacillus (Microbacterium sp.) strain PLR25 was deposited at 11/5/2021 in Guangdong province collection of microorganisms with the deposition number GDMCC No: 61652.

Example 4 Bacterium Microbacterium PLR25 Bacteria Linked Soybean test

4.1 Tie-back test

This study used a matrix soil potting test. Three cadmium concentration groups are set in the experiment, and are respectively 0, 10 and 20mg/kg CdCl₂·5/2H₂O, inoculated with Microbacterium PLR25 and low phosphorus treatment was performed per pot, 4 replicates per treatment, as follows.

The mixed matrix treatment method of the matrix and the vermiculite comprises the following steps: weighing the fertilizer-free matrix and vermiculite according to the ratio of 3: 1 (0.5kg mixed soil is 0.125kg vermiculite +0.375kg substrate), sterilizing at 121 ℃ for 40min, repeating the sterilization once after 24h intervals, and standing for one week for later use. A flowerpot with the capacity of 2L is taken, soaked overnight in 10% sodium hypochlorite, then fully washed with clear water for a plurality of times, and air-dried for later use. Weighing quantitative cadmium chloride, dissolving in water, preparing 0, 10, 20mg/kg solution, weighing certain volume, pouring into mixed matrix, and mixing 125mg Ca per basin (0.5kg)₃(PO4)₂(equivalent to 50mg/kg of pure phosphorus), weighing and uniformly mixing each pot, and standing for one week for later use.

Seeds were sterilized with hydrochloric acid-sodium hypochlorite generating chlorine for 4h, with grit: medium sand is 1: 2, breeding the seedlings by using the mixed sand, selecting soybean plants (Brazil 10) with basically consistent growth after one week of breeding, and transplanting one soybean plant in each pot. After 1, 3 and 5 days of seedling transplantation, the bacterial suspension is irrigated for 3 times, wherein the bacterial suspension is 100mL for one time.

Preparing a bacterium suspension nutrient solution: inoculating the strain into LB culture solution containing 250mL, shaking and culturing at 37 ℃ for about 18h at 180r/min, centrifugally collecting the strain, resuspending the strain with low-phosphorus nutrient solution when the OD of the turbid strain is between 0.6 and 1.0, and poking the matrix on the root surface of the seedling with a sterilizing gun head. And pouring the bacteria heavy suspension nutrient solution into the roots, and pouring the bacteria into the matrix along with the nutrient solution.

Wherein the formula of the low-phosphorus nutrient solution is 2.5mM KNO₃，2.5mM Ca(NO₃)₂·4H₂O， 0.08mM Fe-Na-EDTA，0.25mM K₂SO4，1mM MgSO₄·7H₂O，4.5×10^-3mM MnCl₂·4H₂O，0.3×10^-3mM ZnSO₄·7H₂O，0.16×10^-3mM CuSO₄·5H₂O，0.16×10^-3 mM(NH4)₆Mo₇O₂₄·4H₂O，20×10^-3mM H₃BO₃，50×10^-3mM KH₂PO₄。

4.2 index detection

The soybeans were cultured for 30 days and then harvested. Measuring SPAD values of different parts of the inverted three leaves of the plant by using a SPAD instrument under the condition of sufficient illumination, and then averaging. The tap water is flushed and then is put into a refrigeration house, and scanning is carried out in time within one week. Scanning the root system by a desk type scanner (Epson1460XL), spreading the root system as flat as possible to prevent the root system from overlapping, cutting and scanning the overlarge root system without influencing the scanning result, covering a blue light shielding plate, after the scanning is finished, analyzing the root system characters of each sample root by root system analysis software WinRHIO (Regent Instruments Inc., Canada),

separating the overground part and the root of the crop, weighing the fresh weight of the overground part and the root, putting the overground part into a 105 ℃ oven for deactivation of enzymes for 30min, drying in the oven, taking out, standing at room temperature for 10min, cooling, and weighing the dry weight. The roots are scanned by a root scanner and weighed, and the rest parts are dried after being de-enzymed and weighed as dry weights.

Soaking the soybean root in 10mmol/L Na₂-EDTA solution for 5min to remove surface adsorbed Cd²⁺Then, washing the root system and the stem leaf part by secondary water, drying the root system and the stem leaf part at 75 ℃, weighing dry weight, and then measuring the phosphorus content and cadmium concentration of the overground part; and the root is put in a cold storage for root sweeping, dried and weighed after the root sweeping is finished, and the phosphorus content and the cadmium concentration of the root are measured. The phosphorus detection adopts an ultraviolet spectrophotometry method, and the cadmium detection adopts a flame absorption method of an atomic absorption spectrophotometer.

The results are as follows:

(1) with the increase of cadmium concentration, the dry weight of the plants is obviously reduced, and compared with 0Cd, the biomass of the plants without inoculation treatment (CK group) is 10mg/kg Cd²⁺And 20mg/kg Cd²⁺Under the conditions (10Cd and 20Cd groups), the reduction was 31% and 55%, respectively, and the reduction of soybeans inoculated with the strain PLR25 was 31% and 41%. At the 20Cd level (high concentration of cadmium), soybean plants inoculated with strain PLR25 had a dry weight that was 49% higher than that of uninoculated soybeans (FIG. 7A).

(2) The inoculation treatment obviously influences the SPAD value of the soybean leaves (P is more than 0.01 and less than 0.05), and the cadmium treatment and the inoculation treatment have obvious interaction (P is more than 0.01 and less than 0.05). SPAD value of PLR25 inoculated soybean is significantly higher than CK non-inoculated soybean by 20% (FIG. 7B)

(3) Cadmium treatment significantly affected the total root length of soybeans (P < 0.001). The inoculation treatment has a significant influence on the total root length of the soybean (P is more than 0.01 and less than 0.05). There was a very significant interaction (P < 0.001) between cadmium treatment and inoculation. Non-inoculated (CK group) soybeans at 10mg/kg Cd²⁺And 20mg/kg Cd²⁺Under these conditions, the root length decreased by 46% and 48%, respectively, while the total root length of soybeans treated with Microbacterium strain PLR25 decreased by 36% and 32%, respectively (FIG. 8A). In addition, the growth inhibition of cadmium on roots was reduced by the presence of high concentrations of cadmium in the Bacillus pumilus PLR25 (FIG. 8B).

(4) The cadmium concentration of the overground part and the root of the soybean is greatly influenced by different cadmium concentration treatments (P is less than 0.001), the cadmium concentration of the overground part and the root of the soybean is obviously influenced by the inoculation treatment (P is more than 0.01 and less than 0.05), and the cadmium treatment and the inoculation treatment have obvious interaction (P is more than 0.01 and less than 0.05). The data show that as the cadmium concentration increases, the cadmium concentration in the upper and root parts of the soybean increases significantly. At the levels of 0Cd and 10Cd, the sum of the cadmium concentrations of the overground part and the root part of the soybean inoculated with the PLR25 strain was not significantly changed compared with that of the CK inoculated with no strain, but at the level of 20Cd, the cadmium concentration of the overground part of the soybean inoculated with the PLR25 strain was reduced by 16% (FIG. 9A); the root cadmium concentration was reduced by 72% (fig. 9B).

(5) The phosphorus content of the ground upper part of the soybeans is greatly influenced by treating the soybeans with different cadmium concentrations (P is less than 0.001), and the significant interaction exists between the inoculation treatment and the cadmium treatment (P is more than 0.01 and less than 0.05). The data show that the overground part phosphorus content of the plants increases significantly with the increase of the cadmium concentration. At the 0Cd level, the phosphorus content in the above-ground portion was increased 181% for soybeans inoculated with strain PLR25 compared to CK non-inoculated. At 20Cd levels, the phosphorus content of the overground part treated with inoculated PLR25 increased by 74%. The results show that the inoculated PLR25 strain can increase the phosphorus content of the overground part of the soybean under the condition of low phosphorus and no cadmium, and the inoculated PLR25 strain can significantly increase the phosphorus content of the overground part of the soybean under the 20Cd level (figure 10).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> southern China university of agriculture

<120> cadmium-resistant growth-promoting microbacterium strain and application thereof

<130> YGZS214947

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1370

<212> DNA

<213> Microbacterium sp strain PLR25

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gttgggaact agttgtgggg tccattccac ggattccgtg acgcagctaa cgcattaagt 780

tccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg gggacccgca 840

caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc aaggcttgac 900

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cagttcggat tgaggtctgc aactcgacct catgaagtcg gagtcgctag taatcgcaga 1260

tcagcaacgc tgcggtgaat acgttcccgg gtcttgtaca caccgcccgt caagtcatga 1320

aagtcggtaa cacctgaagc cggtggccca acccttgtgg aggagccgtc 1370

Claims

1. A micro bacillus (Microbacterium sp.) strain PLR25, which is preserved in Guangdong province microorganism culture collection center at 11/5/2021, and the preservation number is 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 micro-bacterium strain PLR25 according to claim 1 for breeding cadmium virus resistant plants.

4. Use of the microbacterium strain PLR25 as claimed in claim 1 for attenuating cadmium inhibition of plant growth or for preparing a product capable of attenuating cadmium inhibition of plant growth.

5. Use of the micro-bacterium strain PLR25 according to claim 1 for promoting synthesis of chlorophyll in plant leaves, or for preparing a product capable of promoting synthesis of chlorophyll in plant leaves.

6. Use of the micro-bacterium strain PLR25 of claim 1 for promoting phosphorus uptake by plants or for the preparation of a product capable of promoting phosphorus uptake by plants.

7. Use of the micro-bacterium 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 plants.

8. The use of claim 7, wherein the plant is a plant of the family Sojae.

9. Use of the micro-bacterium strain PLR25 according to claim 1 for solubilizing poorly soluble phosphorus or for preparing a product capable of solubilizing poorly soluble phosphorus.

10. A method for planting soybean, characterized in that soybean seedlings are treated by using a bacterial suspension or irrigation of the Microbacterium strain PLR25 described in claim 1;

particularly preferably, the mode of treatment is irrigation;

more preferably, the method specifically comprises the following steps: after soybean seeds are cultured, irrigating a bacterial suspension of the microbacterium strain PLR25 of claim 1 in a culture medium, and then irrigating once each 1, 3 and 5 days after seedling transplantation; wherein the amount of each irrigation is preferably 80-120 mL/seedling; preferably, the bacterial suspension OD₆₀₀0.6 to 1.0.