CN112094771B - Bacillus cereus B-28 and application thereof - Google Patents
Bacillus cereus B-28 and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus cereus B-28 and application thereof. The bacillus cereus B-28 is obtained by separation and screening, and is preserved in Guangdong province microorganism strain preservation center in 7 and 13 months in 2020, and the preservation number is GDMCC NO:61089. the strain has strong heavy metal Cd passivation capacity and capacity of reducing the absorption of heavy metal Cd by plants, can repair soil polluted by heavy metal Cd, and can remarkably promote the increase of chlorophyll content of corn leaves and the enhancement of CAT and POD enzyme activities of roots, thereby promoting the increase of biomass of each tissue of corn; therefore, the strain has very wide application prospects in remediation of heavy metal Cd polluted soil, promotion of plant growth and prevention and control of plant absorption of heavy metal Cd.
Description
Technical Field
The invention belongs to the technical field of soil pollution biological treatment. More particularly, relates to bacillus cereus B-28 and application thereof.
Background
Cadmium is one of the main sources of heavy metal pollution of farmland soil in China, and has the characteristics of high biotoxicity, high mobility and the like, so that the cadmium poses serious threats to the safety of agricultural products. How to reduce the risk of cadmium pollution of soil and reduce the absorption of cadmium by crops becomes an urgent task. As a promising approach, microbial inactivation is a technique that allows microorganisms to convert into Cr (III) and SeO via valence state conversion (e.g., cr (VI) 4 2- Conversion to Se), biological adsorption (such as microbial extracellular polymer adsorption), chemical precipitation and volatilization (such as methyl mercury is converted to mercury vapor) and the like, so that the chemical form of the heavy metal is converted or the toxic effect of the heavy metal on organisms is reduced, and further the heavy metal pollution is repaired. However, the technology has harsh application conditions and high popularization cost, and is difficult to realize effective utilization. The combined application of microorganisms and plants to soil heavy metal pollution has become a hot direction. Heavy metals in soil are reduced and stabilized by modes of absorption and adsorption of plant root systems, complexation of root secretion and the like. The inoculation of the microorganism can not only assist in passivating the heavy metals in the soil, but also enhance the tolerance of plants to the heavy metals. And the root exudates released by the plants can provide good nutrient substances for the growth of microorganisms. The plant-microorganism combined passivation technology is proved to be capable of effectively reducing the migration rate of lead, arsenic, cadmium, chromium, copper and zinc in soil and lightening the ecological risk of polluted fields, particularly for areas with high heavy metal concentration and lack of natural vegetation.
It is generally considered that the reduction of the effectiveness of the heavy metal is mainly the conversion of exchangeable heavy metal in the soil to carbonate-bound state under the action of the growth-promoting rhizobacteria. It has been found that the carboxyl groups in the cell wall and extracellular polymeric substances of Bacillus subtilisThe hydroxyl, carbonyl, amido and phosphate groups can be complexed with Pb on the surface of the strain, and Pb is formed at the same time 5 (PO 4 ) 3 OH、Pb 5 (PO 4 ) 3 Cl and Pb 10 (PO 4 ) 6 (OH) 2 Precipitate, stabilizing lead in the soil (Bai J, yang X, du R, et al, biosorption mechanisms included in immobilized of soil Pb by Bacillus subtilis DBM in a multi-metal-stabilized soil [ J]Journal of Environmental Sciences 2014, 26 (10): 2056-2064). In addition, microorganisms enhance disease resistance and stress resistance of plants by producing antibiotics and other pathogen-inhibiting substances (such as siderophores and chelators), plant hormones (such as indoleacetic acid and cytokinin), and specific enzyme (ACC deaminase-producing) activities, etc., thereby promoting plant growth.
At present, chinese patent invention with 2016, 9, 7 and publication No. CN105925507A discloses Bacillus cereus B19 with heavy metal passivation and plant growth promotion functions; however, when studying that bacillus cereus promotes plant growth and prevents and controls plants from absorbing Cd, the prior art is directly carried out through soil culture, and has a lot of influencing factors in soil, complex bacterial colonies, and other strains possibly act besides bacillus cereus, and experimental conditions cannot be strictly controlled. Therefore, the method is not influenced by other factors in soil, has accurate and reliable results, and can effectively prevent and control the absorption of heavy metal Cd by plants and promote the growth of the plants.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provides a bacillus cereus B-28 strain and application thereof. According to the invention, a sand culture experiment is utilized, the cleaned and sterilized quartz sand is used for culturing plants, and the microbial inoculum only containing the bacillus cereus B-28 is applied, so that the influence factors are single, the experiment variables are perfectly controlled, and the result is more accurate and reliable.
The invention aims to provide a strain of Bacillus cereus B-28.
The invention also aims to provide application of the bacillus cereus B-28 in remediation of soil polluted by heavy metal Cd.
The invention also aims to provide application of the bacillus cereus B-28 in promoting plant growth or preparing a plant growth promoter.
The invention also aims to provide application of the bacillus cereus B-28 in inhibiting and controlling heavy metal Cd absorption of plants.
The invention also aims to provide a method for preventing and controlling the absorption of heavy metal Cd by plants.
Still another object of the present invention is to provide a plant growth promoter.
The above purpose of the invention is realized by the following technical scheme:
the invention separates Bacillus cereus B-28 from corn rhizosphere soil, the strain is preserved in Guangdong province microorganism strain preservation center in 7-13 th of 2020, and the preservation number is GDMCC NO:61089, preservation address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
The research of the invention finds that the bacillus cereus B-28 has strong heavy metal Cd passivating capability and capability of reducing the absorption of plants to the heavy metal Cd, can repair the soil polluted by the heavy metal Cd, and can promote the growth of the plants; therefore, the following applications should be within the scope of the present invention:
the application of the bacillus cereus B-28 in repairing heavy metal Cd polluted soil is provided.
The application of the bacillus cereus B-28 in promoting plant growth or preparing a plant growth promoter.
The application of the bacillus cereus B-28 in inhibiting and controlling the absorption of heavy metal Cd by plants.
Preferably, the application is that the bacillus cereus B-28 can passivate heavy metal Cd and reduce the absorption of the heavy metal Cd by plants.
The invention also provides a method for preventing and controlling the absorption of heavy metal Cd by plants, which directly inoculates the bacillus cereus B-28 at the rhizosphere of the plants.
Preferably, the plant is a plant 4 to 6 days after seedling transplantation.
More preferably, the plant is a plant 5 days after seedling.
Preferably, the inoculation frequency is once every 6 to 8 days.
More preferably, the inoculation frequency is every 7 days.
Preferably, the plant is maize.
In addition, the invention also provides a plant growth promoter, which comprises the bacillus cereus B-28 or the bacterial suspension thereof.
The invention has the following beneficial effects:
the invention provides a bacillus cereus B-28 and application thereof. According to the method, a strain of bacillus cereus B-28 capable of efficiently promoting corn growth and reducing Cd absorption is screened from corn rhizosphere soil, and through a sand culture experiment, a soil culture experiment and a shaking experiment, the strain is found to be capable of complexing or adsorbing Cd through a functional group so as to reduce the concentration of water-soluble Cd, so that the bioavailability of Cd is remarkably reduced, the absorption of Cd by corn is reduced, and the content of Cd in each tissue of corn is obviously reduced; meanwhile, the strain can remarkably promote the chlorophyll content of the corn leaves to be increased and the CAT and POD enzyme activities of roots to be enhanced, so that the biomass of each tissue of the corn to be increased is promoted; therefore, the strain has very wide application prospects in remediation of heavy metal Cd polluted soil, promotion of plant growth and prevention and control of plant absorption of heavy metal Cd.
Drawings
FIG. 1 is a graph showing the adsorption effect of Bacillus cereus B-28 on water-soluble Cd; wherein (a) is the result of the pH value of the culture medium and the growth condition of the bacillus cereus B-28 strain; (b) The figure shows the results of culture medium supernatant, cell wall adsorption of Bacillus cereus B-28 strain and intracellular Cd accumulation concentration.
FIG. 2 is a result graph of Cd contents in corn tissues under different conditions of a sand culture experiment; wherein, the graph (a) is the content of Cd in the corn root; (b) the diagram shows the content of Cd in the corn stalks; the graph is the Cd content in the maize leaves; (d) the diagram shows the Cd content in the corn grain; (e) the diagram shows the Cd content in the ear of corn; (f) the figure is the water soluble Cd content of the rhizosphere sand;
note: the difference in lower case letters at the same sand culture Cd concentration treatment indicated a significant difference (p < 0.05) between the inoculated B-28 strain of bacillus cereus compared to the uninoculated control.
FIG. 3 is a graph showing the results of inoculation with Bacillus cereus B-28 with direct effect on maize growth; wherein, the picture (a) is the fresh weight of the corn root; (b) the figure is the fresh weight of the corn stalks; (c) the graph shows the fresh weight of corn leaves; (d) the figure shows the fresh weight of the corn kernels; (e) the picture is the fresh weight of the corn ear; (f) the figure is the maize plant height;
note: under the same Cd concentration treatment, the difference of lower case letters indicates that the difference between the Bacillus cereus B-28 inoculation treatment and the non-inoculation control is significant (p < 0.05); in the treatment of inoculating the bacillus cereus B-28 strain or in the treatment without inoculating the bacillus cereus, the difference of capital letters indicates that the difference of different sand culture Cd concentration treatment is obvious (p is less than 0.05).
Detailed Description
The present invention is further illustrated by the following specific examples, 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.
The cadmium-tolerant bacteria B-28 separated from the corn rhizosphere soil is identified as Bacillus cereus by 16s rDNA sequence analysis and API reagent strip reaction, is named as Bacillus cereus B-28 and is preserved in Guangdong province microorganism strain preservation center in 7-13 days of 2020, and the preservation number is GDMCC NO:61089, preservation address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
Example 1 adsorption Effect of Bacillus cereus B-28 on Water-soluble Cd
1. Experimental methods
Inoculating Bacillus cereus B-28 into LB culture medium, culturing overnight, centrifuging at 6000rpm for 10min, discarding supernatant, washing thallus with sterile water for 2 times, re-suspending in sterile water to obtain a stock solution, inoculating 0.5mL of the stock solution into 100mL of LB culture medium containing 1mg/L Cd (added in the form of Cd (NO 3) 2), and inoculating into Cd-free LB culture mediumControl, each treatment was in triplicate. The mixture was placed in a shaker at 180rpm and 30 ℃. Sampling once on days 1, 3, 5 and 7 of strain culture, and determining OD of bacterial liquid 600 And (4) centrifuging the bacterial liquid at 6000rpm for 10min to obtain bacterial precipitates. And (3) washing the harvested thalli twice by using sterile water, then adding 20mL of 10mM EDTA solution, stirring for 10 minutes to wash and remove Cd adsorbed on the cell surface, and collecting a washing solution for determining the content of Cd adsorbed outside the cell. Adding 8mL of nitric acid into the thallus precipitate for digestion, and determining the intracellular Cd content of the strain.
2. Results of the experiment
The adsorption effect graph of the bacillus cereus B-28 on water-soluble Cd is shown in FIG. 1, and it can be seen that when the initial concentration of Cd in LB culture medium is 1mg/L, the 1 st day of inoculation of the bacillus cereus B-28 is sharply reduced to 6.8. Mu.g/L, the 3 rd day is reduced to the lowest value of 5.0. Mu.g/L, the adsorption efficiency is 99.5%, and then the 5 th day and the 7 th day are respectively increased to 29.8. Mu.g/L and 34.1. Mu.g/L; as can be seen, the adsorption efficiency of the bacillus cereus B-28 is highest on the 3 rd day, and the Cd concentration of the supernatant is gradually increased, but the Cd adsorption efficiency is still more than 95%.
The extracellular Cd concentration of Bacillus cereus B-28 was reduced from 42.1. Mu.g/L at day 1 of culture to 38.4. Mu.g/L at day 3, and then gradually increased to 67.7. Mu.g/L at day 7; and the concentration of intracellular Cd in Bacillus cereus B-28 increased from 0.80mg/L on day 1 to 0.92mg/L on day 5, and then decreased to 0.85mg/L on day 7; it can be seen that the intracellular Cd concentration of the Bacillus cereus B-28 is significantly higher than that of the extracellular strain, which indicates that the adsorption of the strain to Cd is mainly intracellular (95%), and the cell wall adsorption only accounts for about 5% of the Cd adsorption.
Example 2 Effect of Bacillus cereus B-28 inoculation on Cd uptake by maize
1. Experimental methods
According to the research, 5 different Cd concentrations are set through a sand culture experiment, and the influence of bacillus cereus B-28 on the growth of the corn and the absorption of cadmium is researched. The experiment was run for 10 treatments, each: 0.03J (adding quartz sand with Cd concentration of 0.03mg/kg in pot culture, then, inoculating bacteria), 0.06J (Cd concentration of 0.06mg/kg, inoculating bacteria), 0.09J (Cd concentration of 0.09mmg/kg, inoculating bacteria), 0.12J (Cd concentration of 0.12mg/kg, inoculating bacteria), 0.15J (Cd concentration of 0.15mg/kg, inoculating bacteria) and 5 non-inoculating control of each Cd concentration: 0.03CK, 0.06CK, 0.09CK, 0.12CK and 0.15CK. A total of 40 pots containing 4kg of silica sand per pot were set in parallel with 4 treatments each.
The corn seeds are subjected to 10 percent before use 2 O 2 Sterilized for 0.5h, and then washed with sterile water 2 times and soaked for 24h to promote germination. The soaked corn seeds are sowed in a seedling raising plate fully paved with wet quartz sand, and water absorption paper is covered to keep a dark environment so as to ensure the germination of the seeds. After the corn seeds grow two green leaves, selecting corn seedlings with the same growth vigor and transplanting the corn seedlings into pots, and transplanting the corn seedlings for 5 days to ensure that 3 seedlings with uniform growth vigor are remained in each pot. Seedling and sand culture pot experiments are carried out in a plant artificial climate incubator, the humidity is set to be 65%, the time of the day is 14h, the temperature is 30 ℃, and the illumination intensity is 20000lux; the time is 10 hours at night, the temperature is 25 ℃, and no light is emitted. 50mL of 1/3 Hoagland nutrient solution is poured every day to meet the requirements of corn growth on different elements. Add 0.15mg Cd (as Cd (NO) to the basin 3 ) 2 Added as a solution with a sand culture Cd concentration of 0.15mg/kg dry quartz sand).
The potting experiment was carried out in a greenhouse with an average temperature of 32 ℃ during the day and a humidity of 45%, and an average temperature of 22 ℃ at night and a humidity of 70%. Inoculation treatment was started 7 days after the transplantation, and the corn rhizosphere was inoculated with Bacillus cereus B-28 strain solution 8 times in total during the experiment.
2. Results of the experiment
The result graphs of the Cd contents of the corn tissues under different conditions of the sand culture experiment are shown in FIG. 2, and it can be seen that when the Cd concentrations of the sand culture experiment are 0.09mg/kg, 0.12mg/kg and 0.15mg/kg, the Cd contents of different corn tissues can be obviously reduced by inoculating bacillus cereus B-28, wherein the Cd concentrations of roots are reduced by 30.5-39.9%, the Cd concentrations of stems are reduced by 17.0-27.5%, the leaf concentrations are reduced by 21.1-26.5%, the Cd concentrations of corn kernels (edible parts) are reduced by 20.9-33.2%, and the Cd concentrations of corn cobs are reduced by 25.7-42.5%; however, when the Cd concentrations in the sand culture experiments are 0.03mg/kg and 0.06mg/kg, the Cd contents of various tissues of the corn under the treatment of non-inoculation and the treatment of inoculation of Bacillus cereus B-28 have no significant change.
Example 3 direct Effect of Bacillus cereus B-28 inoculation on corn growth
1. Experimental method
Harvesting is carried out after the corn grows for 100 days. At harvest, the height of the corn plants was measured, and the corn roots were shaken to separate the rhizosphere sand, washed with deionized water and washed with 0.5mM CaCl 2 Soaking for 0.5h to remove Cd attached to the root surface of the corn, and wiping the surface with absorbent paper to remove moisture attached to the root surface. The fresh weight of the corn tissue divided into roots, stems, leaves, corn kernels and corn cobs is respectively weighed, each reserved part of fresh sample is immediately quenched and stored in a refrigerator (Haier DW-86L 626) at-80 ℃ by using liquid nitrogen for measuring the activity of the plant enzyme and chlorophyll, the rest tissue sample is put into an envelope and is placed in a constant temperature oven for deactivation of enzymes at 105 ℃ for 0.5h, and then the tissue sample is dried to constant weight at 80 ℃.
2. Results of the experiment
The result chart of the direct influence of the inoculated bacillus cereus B-28 on the corn growth is shown in fig. 3, and it can be seen that the bacillus cereus B-28 can obviously promote the increase of the corn biomass and the plant height under the treatment of different Cd concentrations, wherein the fresh weight of roots is increased by 25.6-64.3%, the fresh weight of stems is increased by 21.2-32.9%, the fresh weight of leaves is increased by 14.5-41.0%, corn kernels (edible parts) are increased by 22.8-52.6%, and the cob is increased by 11.1-50.5%; when the concentration of Cd in sand culture is 0.09mg/kg, the maximum fresh weight growth rate of each tissue is 64.3 percent of root, 32.9 percent of stem, 41.0 percent of leaf, 52.6 percent of corn grain and 50.5 percent of corn cob; compared with the method without inoculating the bacillus cereus B-28, the height of the corn plant under the inoculation treatment is also obviously increased by 7.0-11.8%.
The above results show that: under different Cd concentrations, the growth of the corn can be remarkably promoted by inoculating the bacillus cereus B-28 (p is less than 0.05).
Example 4 Indirect Effect of Bacillus cereus B-28 inoculation on corn growth
1. Experimental method
(1) Measuring the chlorophyll content: selecting clean and complete fresh corn leaf tissues of a corn sample quenched by liquid nitrogen, shearing the fresh corn leaf tissues by using scissors, accurately weighing 0.1g of the sample, putting the sample into a mortar, simultaneously adding 5mL of 80% acetone, fully grinding the sample until no leaf tissues exist, transferring the sample into a centrifuge tube, adding 1mL of 80% acetone into the mortar to rinse the inner wall of the mortar for 3 times, finally fixing the volume of grinding fluid to 8mL, wrapping tin foil paper, and transferring the wrapped tin foil paper to a dark place for leaching for 12 hours at normal temperature. When the slurry was observed to whiten the precipitate, it was centrifuged at 8000rpm for 5min, and the chlorophyll content in the supernatant was measured. The whole process of the chlorophyll extraction and determination experiment is carried out under dark and dark conditions.
(2) POD enzyme and CAT enzyme activity assay: the POD enzyme activity was measured by the guaiacol method based on the following principle: guaiacol is oxidized by peroxide in plant tissue to produce dark brown substance with specific absorbance at 470nm, and POD enzyme activity in plant tissue can be obtained by measuring the change of the product amount in unit time. Catalase (CAT) Activity by H 2 O 2 Determination of the decomposition rate, H at a wavelength of 240nm 2 O 2 The absorption of visible light at this wavelength is strong, and therefore the CAT enzyme activity can be calculated from the rate of decrease in absorbance.
2. Results of the experiment
The results of the chlorophyll content and the antioxidant enzyme activity of roots of corn leaves treated in a sand culture experiment are shown in table 1, and it can be seen that under treatment of different Cd concentrations, the chlorophyll A (Chl a), the chlorophyll B (Chl B) and the Total chlorophyll content (Total Chl) of corn are obviously higher than those of corn treated without inoculation, and the chlorophyll A/B and the Total chlorophyll content are respectively increased by 17.5-45.5%, 14.0-49.9% and 20.5-44.4%; except that the concentration of Cd is 0.03mg/kg, the CAT enzyme activity of the corn root is obviously higher than that of non-inoculated treatment under the inoculation treatment, the growth rate is 9.4-75.0%, wherein when the concentration of Cd in sand culture is 0.15mg/kg, the maximum growth amplitude is 75.0%. The POD enzyme activity of the corn roots is higher than that of the corn roots subjected to non-inoculation treatment after the inoculation treatment, and the increase range is 13.4-32.0%; wherein, when the concentration of Cd is 0.09mg/kg and 0.15mg/kg, the activity of POD enzyme at the root part is obviously increased by 27.9 percent and 32.0 percent (p is less than 0.05), which is higher than that of other Cd.
TABLE 1 results of sand culture experiments on chlorophyll content and antioxidant enzyme activity of roots of treated maize leaves
Note: j represents the treatment of inoculating Bacillus cereus B-28, CK represents the control treatment without inoculating bacteria; data represent mean ± SD, n =4. By Duncan multiple comparison, different lower case letters indicate that the inoculated bacteria and the non-inoculated bacteria have obvious difference (p < 0.05) under the same Cd concentration treatment, and different upper case letters indicate that the different Cd concentration treatments have obvious difference (p < 0.05) under the inoculated bacteria or non-inoculated bacteria treatment.
The above results show that: inoculating bacillus cereus B-28 in sand culture experiments with different Cd concentrations, wherein the chlorophyll a/B and the total chlorophyll content of the corn leaves are obviously increased compared with those of an inoculation-free control; the bacillus cereus B-28 can relieve chlorophyll damage of corn caused by Cd stress, and has positive effects on photosynthesis of corn leaves and plant growth, so that the biomass of the corn is improved.
Compared with inoculation-free bacillus cereus, the activity of antioxidant enzymes (CAT and POD) at the roots of the corn can be obviously improved by inoculating bacillus cereus B-28 under different Cd concentrations; the active oxygen scavenging system has the advantages that after bacillus cereus B-28 is inoculated to the corn roots, the activity of the active oxygen scavenging system is enhanced, the oxidation resistance of plants is obviously improved, the stress of soil Cd on the corn roots can be effectively relieved, so that the normal metabolism of plant root cells is maintained, the biomass of corn is indirectly improved, and the growth of the corn is promoted.
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.
Claims (6)
1. A strain of Bacillus cereus B-28 is preserved in Guangdong province microorganism strain preservation center in 7 and 13 months in 2020, and the preservation number is GDMCC NO:61089.
2. the use of bacillus cereus B-28 according to claim 1 for promoting the growth of corn or preparing a corn growth promoter.
3. Use of bacillus cereus B-28 of claim 1 for inhibiting heavy metal Cd uptake in corn.
4. The application of claim 3, wherein the application is that the bacillus cereus B-28 can passivate heavy metal Cd and reduce the absorption of the heavy metal Cd by corn.
5. A method for preventing and controlling the absorption of heavy metal Cd by plants is characterized in that the bacillus cereus B-28 of claim 1 is directly inoculated at the rhizosphere of the plants; the plant is corn after 4-6 days of seedling transplantation, and the inoculation frequency is once every 6-8 days.
6. A plant growth promoter comprising bacillus cereus B-28 or a bacterial suspension thereof according to claim 1.
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