Cadmium-tolerant phosphate-solubilizing bacterium and application thereof

Abstract

The invention relates to a cadmium-tolerant phosphate-solubilizing bacterium and application thereof, the cadmium-tolerant phosphate-solubilizing bacterium comprises a bacillus which is tolerant to Cd and has a growth promoting effect, the bacillus is a bacillus siamensis R27 strain, and the preservation number of the strain is CCTCC NO: M 2023603. The cadmium-tolerant phosphate-solubilizing bacterium can be applied to a Cd solution to adsorb Cd, dissolve inorganic phosphorus, promote vegetable growth and reduce vegetable Cd enrichment. Experimental results show that the cadmium-tolerant phosphate-solubilizing bacteria can dissolve inorganic phosphorus under the condition of Cd stress, promote growth and development of vegetable seedlings and relieve adverse effects of Cd stress on vegetables.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a cadmium-resistant phosphate-solubilizing bacterium and an application thereof. Background Art

[0002] Vegetables can provide a variety of essential minerals, vitamins, crude fiber and other nutrients for the human body, and are an indispensable food for people's daily diet. With the rapid development of industry, cadmium pollution in vegetable planting bases has become increasingly serious. Vegetables have been contaminated with Cd to varying degrees, even exceeding the food safety limit, seriously affecting the quality of vegetables. Cd enriched in the edible part of vegetables will eventually enter the human body through the food chain and endanger human health. Therefore, it is particularly important to take effective measures to prevent vegetables from absorbing and enriching Cd to ensure the safety of the "vegetable basket".

[0003] Seeking low-carbon, energy-saving, environmentally friendly, and economical measures to effectively control cadmium pollution has become one of the hot topics for students at home and abroad. In recent years, the research and application of plant growth-promoting rhizobacteria (PGPR) in bioremediation has also attracted more and more attention. PGPR is a type of rhizosphere beneficial bacteria that colonizes in the rhizosphere of plants and can promote plant growth through direct or indirect action. Studies have found that in addition to the growth-promoting properties, some PGPR also have the function of reducing plant enrichment of heavy metals. Zhou et al. found that strains T77 and T36 can reduce the Cd content in the aboveground tissues of tomatoes by 33% and 23%, respectively, and the Cd content in the root tissues by 24% and 17%, respectively. Zhang et al. found that Enterobacter Zm-123 can reduce the Cd content in the edible part of rapeseed by 11% to 36%, and can increase the biomass of rapeseed by 9% to 20%. Summary of the invention

[0004] The purpose of the present invention is to provide a cadmium-resistant phosphate-dissolving bacterium, which can dissolve inorganic phosphorus under Cd stress conditions, promote the growth and development of vegetable seedlings, and alleviate the adverse effects of Cd stress on vegetables.

[0005] The first technical solution adopted by the present invention is: a cadmium-resistant phosphate-solubilizing bacterium, which is a Cd-tolerant and growth-promoting bacillus, and the bacillus is a Bacillus siamensis R27 strain, and the strain collection number is CCTCC NO: M 2023603.

[0006] The second technical solution adopted by the present invention is: the use of the cadmium-resistant phosphate-solubilizing bacteria described in the first technical solution to adsorb Cd in a Cd solution.

[0007] The third technical solution adopted by the present invention is: the use of the cadmium-resistant phosphate-dissolving bacteria described in the first technical solution in dissolving inorganic phosphorus.

[0008] The fourth technical solution adopted by the present invention is: the use of the cadmium-resistant phosphate-dissolving bacteria described in the first technical solution to dissolve inorganic phosphorus under Cd conditions.

[0009] Furthermore, the inorganic phosphorus is Ca3(PO4)2.

[0010] The fifth technical solution adopted by the present invention is: use of the cadmium-resistant phosphate-solubilizing bacteria described in the first technical solution in promoting the growth of vegetables.

[0011] Furthermore, the vegetables include lettuce and spinach.

[0012] The sixth technical solution adopted by the present invention is: the use of the cadmium-resistant phosphate-solubilizing bacteria described in the first technical solution in reducing Cd enrichment in vegetables.

[0013] Furthermore, the vegetables include lettuce, spinach and bitter lettuce.

[0014] The beneficial effect of the present invention is that the cadmium-resistant phosphate-solubilizing bacteria can dissolve insoluble Ca3(PO4)2 under Cd stress conditions. Experimental results show that the phosphate-solubilizing amount of the cadmium-resistant phosphate-solubilizing bacteria is up to 213 mg / L under Cd concentration of 250 mg / L, which can promote the growth and development of vegetable seedlings, reduce the use of chemical fertilizers, alleviate the adverse effects of Cd stress on vegetables, and play a role in protecting the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a rendering of the ability of the cadmium-resistant phosphate-solubilizing bacteria described in the embodiment of the present invention to remove Cd from a liquid culture medium;

[0017] Figure 2 This is a diagram showing the effect of the phosphate-solubilizing circle formed by the cadmium-resistant phosphate-solubilizing bacteria described in an embodiment of the present invention in the NBRIP solid culture medium. DETAILED DESCRIPTION

[0018] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments disclosed below.

[0019] An embodiment of the present invention provides a cadmium-resistant phosphate-solubilizing bacterium, wherein the cadmium-resistant phosphate-solubilizing bacterium is a Bacillus that tolerates Cd and has a growth-promoting effect, and the Bacillus is a Bacillus siamensis R27 strain, and the strain collection number is CCTCC NO: M 2023603. It was deposited in the China Center for Type Culture Collection on April 24, 2023, and the address of the depository unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0020] The cadmium-resistant phosphate-solubilizing bacteria, hereinafter referred to as strain R27, were isolated from the soil around the Jiangxi 721 uranium mine. The specific isolation and identification methods are as follows:

[0021] Place 5 g of soil sample in a triangular flask containing 45 mL of sterile water and glass beads, add CdCl2 to a CdCl2 concentration of 25 mg / L, and shake incubate for 24 hours. Increase the Cd concentration to 100 mg / L and continue shaking incubation for 24 hours.

[0022] The method adopts a 10-fold serial dilution method and is coated on a beef extract peptone medium containing a Cd concentration of 100 mg / L. The beef extract peptone medium includes 3 g of beef extract, 10 g of peptone, 10 g of NaCl and 20 g of agar. The pH value of the beef extract peptone medium is 7.0-7.2. The culture is carried out at 30°C for 3-5 days. Single colonies of different morphologies are selected and streaked on the beef extract peptone solid medium for purification to obtain a Cd-resistant strain.

[0023] The bacterial genomic DNA was extracted according to the conventional method, and the bacterial 16S rRNA was used to perform molecular identification of the strain. After sequencing and comparison in the NCBI database, the strain was identified as Bacillus siamensis R27.

[0024] Example 1 Effect of R27 strain on Cd removal

[0025] In a liquid beef extract peptone medium containing 200 mg / L Cd, the R27 strain seed liquid was inoculated at a 1% inoculation rate and cultured at a constant temperature of 30°C and 200 rpm. The adsorption of Cd by each component was measured every 5 hours until 60 hours. After the reaction solution was centrifuged, the supernatant was taken and the residual Cd concentration in the supernatant was determined by atomic absorption spectrometry. The removal rate R (%) was calculated by the following formula:

[0026]

[0027] Where R represents the Cd removal rate, C0 is the initial concentration of Cd, and Ce is the remaining Cd concentration at time t. The concentration units are all mg / L.

[0028] Set up 3 replicate groups, and based on the average of the experimental results of the 3 groups, we can get the following Figure 1 The R27 strain is shown in Figure 2 to remove Cd from liquid culture medium. Figure 1 It can be seen that the removal rate of Cd by R27 strain gradually increased from 10 to 45 hours, and the removal rate was the highest at 45 hours, reaching 80.1%, after which the removal rate of Cd tended to balance.

[0029] Example 2 Determination of phosphate solubilization ability of R27 strain

[0030] Example 2 NBRIP solid culture medium was used to determine the phosphorus solubility characteristics of the strain. In Example 2, the solubility of R27 strain for inorganic phosphorus, specifically the solubility of Ca3(PO4)2, was determined. The experimental method is: after the strain was cultured overnight on beef extract peptone medium, the bacterial solution was inoculated on NBRIP solid culture medium, wherein the NBRIP solid culture medium includes 10g of glucose, 0.5g of (NH4)2SO4, 0.3g of NaCl, 0.3g of KCl, 0.3g of MgSO4·7H2O, 10g of Ca3(PO4)2, 0.03g of FeSO4·7H2O, 0.03g of MnSO4·4H2O, 20g of agar and 1000mL of distilled water, and the culture was carried out at a constant temperature of 30°C for 3 to 5 days, and the size of the phosphorus solubility circle was observed.

[0031] Using the molybdenum antimony colorimetric method, the bacterial suspension of R27 strain was inoculated into 100 mL of liquid NBRIP medium, and cultured at 30°C and 180 rpm for 5 days. The soluble phosphorus content was measured every 12 hours. The results showed that Figure 2 As shown, the R27 strain produced an obvious phosphate solubilization zone, and its maximum phosphate solubilization amount could reach 385 mg / L.

[0032] Example 3 Phosphate solubilization of R27 strain under different Cd concentrations

[0033] The purpose of Example 3 is to detect the phosphate solubilization ability of R27 strain in NBRIP liquid culture medium containing different Cd concentrations. The specific experimental method is:

[0034] The molybdenum antimony colorimetric method was used to inoculate the bacterial liquid of R27 strain into 100 mL of liquid NBRIP medium containing different Cd concentrations. In Example 3, five Cd concentrations were set, namely 50, 100, 150, 200 and 250 mg / L. The culture was carried out at 30 ° C and 180 rpm for 5 days. The soluble phosphorus content was measured every 12 hours. The experimental results are shown in Table 1. Cd has a certain inhibitory effect on the phosphorus solubilizing ability of R27 strain. As the Cd concentration increases, the maximum phosphorus solubilization amount gradually decreases.

[0035] Table 1 Maximum phosphorus solubilization capacity of strain R27 at different Cd concentrations

[0036] Cd concentration (mg / L) 50 100 150 200 250 Maximum phosphorus solubilization amount (mg / L) 330.5±6.2 305.1±8.3 282.7±11.2 253±12.4 213±11.7

[0037] Example 4 Effect of strain R27 on promoting vegetable growth and reducing Cd content in leaves

[0038] Example 4 used potted plants for the experiment, and the experimental vegetables were lettuce, spinach and bitter lettuce.

[0039] (1) Experiment on the growth-promoting effect of strain R27 on lettuce

[0040] The test soil was sieved through a 2 mm sieve, sprayed with CdCl2 solution and mixed to prepare a soil with a Cd concentration of 8.0 mg / kg. The soil was potted after being balanced and stable for one month, with 400 g per pot. The test plant was lettuce (Lactuca sativa L.), and one lettuce seedling of uniform size and a growth period of 25 days was transplanted into each pot. The potted soil was divided into two parts, one with 20 mL and a concentration of 1×10 7 The roots of lettuce were watered with the bacterial suspension of R27 strain with a CFU / mL, which was set as the inoculation group; the other part was watered with the same amount of sterilized suspension, which was set as the control group, and 15 replicates were set in each group. During the plant growth period, an appropriate amount of water was irrigated to ensure the normal growth of the plants, and the plants were harvested after 30 days. After taking out the plants, they were rinsed with clean water and dried, and the fresh weight of the aboveground parts and roots was weighed. The chlorophyll content was determined by the method recorded by Ashraf et al. in "Drought stressinduced changes in some organic substances in nodules and other plant partsof two potential legumes differing in salt tolerance"; the plants were sterilized at 105℃, dried at 75℃, and the dry weight of the aboveground parts and roots was weighed again. The measurement results are shown in Table 1.

[0041] As shown in Table 2, compared with the control group (CK), the fresh weight of the aboveground and roots of the inoculated group (R27) treated with the R27 strain increased significantly by 29.4% and 40.2%, respectively, and the dry weight of the aboveground and roots increased by 47.8% and 38.5%, respectively; among them, the increase in the aboveground dry weight of the R27 strain reached an extremely significant level (p<0.01). In addition, the R27 strain increased the chlorophyll content of lettuce by 21.7%.

[0042] Table 2 The growth-promoting effect of strain R27 on lettuce

[0043]

[0044] Among them, “*” is used to indicate the results of variance analysis; “*” indicates that the experimental results between the inoculated group (R27) and the control group (CK) are significantly different at the 0.05 level; “**” indicates that the experimental results between the inoculated group (R27) and the control group (CK) are extremely significantly different at the 0.01 level.

[0045] (2) Experiment on the growth-promoting effect of R27 strain on spinach

[0046] The specific experimental method is the same as that of lettuce, except that the test plant is replaced by spinach. The specific measurement results are shown in Table 3.

[0047] As shown in Table 3, compared with the control group (CK), the fresh weight of the aboveground part and root of the spinach group treated with the inoculated group (R27) increased significantly by 36.1% and 33.3%, the dry weight of the aboveground part and root increased by 29.6% and 37.5%, and the chlorophyll content increased by 24.2%.

[0048] Table 3 Growth-promoting effect of strain R27 on spinach

[0049]

[0050] (3) Experiment on the growth-promoting effect of R27 strain on bitter chrysanthemum

[0051] The specific experimental method is the same as that of lettuce, except that the test plant is replaced by bitter lettuce. The specific measurement results are shown in Table 4.

[0052] As shown in Table 4, compared with the control group (CK), there was no difference in the fresh weight, dry weight and chlorophyll content of the aboveground part and roots of the bitter lettuce group treated with the inoculated group (R27), indicating that the inoculated group (R27) had no obvious growth-promoting effect on the growth of bitter lettuce.

[0053] Table 4 The growth-promoting effect of strain R27 on bitter chrysanthemum

[0054]

[0055] (4) Experiment on reducing Cd in vegetables using R27 strain

[0056] The control group (CK) and the inoculated group (R27) in the lettuce group, spinach group and bitter chrysanthemum group were digested with HNO3-HClO4, and the Cd content was determined by flame atomic absorption spectrometry. The measurement results are shown in Table 5.

[0057] As shown in Table 5, compared with the control group (CK), the cadmium content in the aboveground part of lettuce in the inoculated group (R27) was reduced by 50%, the cadmium content in the aboveground part of spinach was reduced by 38.9%, and the cadmium content in the aboveground part of bitter lettuce was reduced by 32%.

[0058] Table 5 Effect of strain R27 on reducing Cd in vegetables

[0059]

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cadmium-resistant phosphate-solubilizing bacterium, It is characterized in that The cadmium-resistant phosphate-dissolving bacteria is a bacillus that tolerates Cd and has a growth-promoting effect. The bacillus is a Bacillus siamensis R27 strain, and the strain collection number is CCTCC NO: M2023603.

2. Use of the cadmium-resistant phosphate-solubilizing bacteria according to claim 1 in adsorbing Cd in a Cd solution.

3. Use of the cadmium-resistant phosphate-solubilizing bacteria described in claim 1 in dissolving inorganic phosphorus.

4. Use of the cadmium-resistant phosphate-dissolving bacteria described in claim 1 to dissolve inorganic phosphorus under Cd conditions.

5. The use according to claim 3 or 4, It is characterized in that The inorganic phosphorus is Ca 3 (PO 4 ) 2 .

6. Use of the cadmium-resistant phosphate-solubilizing bacteria described in claim 1 in promoting the growth of vegetables.

7. The use according to claim 6, It is characterized in that The vegetables include lettuce and spinach.

8. Use of the cadmium-resistant phosphate-solubilizing bacteria according to claim 1 in reducing Cd accumulation in vegetables.

9. The use according to claim 8, It is characterized in that The vegetables include lettuce, spinach and bitter lettuce.

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