CN112143687B - Bacillus megaterium and application of fermentation liquor thereof in promoting plant germination - Google Patents
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Abstract
The invention is applicable to the technical field of microorganisms, and provides a bacillus megaterium and application of fermentation liquor thereof in promoting plant germination. Specifically, after the bacillus megaterium and the fermentation liquor of the bacillus megaterium provided by the invention are properly diluted, the activity of anti-reverse enzymes in seed germination can be improved, and the content of malonaldehyde can be reduced.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus megaterium and application of fermentation liquor thereof in promoting plant germination.
Background
Phosphorus is one of three essential nutrients for plant growth, and phosphorus deficiency has influence on plant photosynthesis, respiration and biosynthesis. At present, the total phosphorus in different types of soil in China contains 0.31 to 1.72 g.Kg -1 The content of the quick-acting phosphorus is between 0.1 and 228.8 mg.Kg -1 The available phosphorus accounts for less than 5 percent of the phosphorus content of the soil, so that the requirement of the growth and development of crops cannot be met, the phosphorus can be supplemented by applying a phosphate fertilizer, the utilization rate of the phosphate fertilizer in China is only 10 to 25 percent at present, most phosphate ions can be fixed when the phosphate fertilizer is applied for a long time, the soil is hardened and the fertility is reduced, and a series of environmental problems such as water pollution, soil pollution and the like can be caused, so that the increase of the phosphate fertilizer application is a high-input and low-output way.
Phosphate solubilizing bacteria in soil, also called as phosphate solubilizing bacteria, such as bacillus megaterium, can convert phosphorus-containing compounds from an insoluble state into a soluble state through self metabolism, and thus, the phosphorus-containing compounds can be absorbed and utilized by plants. The microbial fertilizer prepared by reasonably applying the phosphate-solubilizing bacteria can improve the utilization efficiency of the phosphorus of plants, reduce the application of chemical fertilizers, improve the soil fertility and restore the ecological environment, and is one of effective ways for improving the crop yield and solving the problem of quick-acting phosphorus deficiency of soil.
However, only the phosphate solubilizing capability of strains is concerned in the currently studied phosphate solubilizing bacterial agents such as bacillus megaterium, and the reports of the growth promoting and antagonistic properties of the strains are rare.
Disclosure of Invention
The embodiment of the invention aims to provide a bacillus megaterium and application of fermentation liquor thereof in promoting plant germination, and aims to solve the problems in the background art.
The embodiment of the invention is realized by the application of the bacillus megaterium and the fermentation liquor thereof in promoting plant germination.
As a preferable embodiment of the present invention, the plant is mung bean.
As another preferred embodiment of the present invention, the Bacillus megaterium and its fermentation broth are used for the production of auxin and/or siderophore.
As another preferable scheme of the embodiment of the invention, the bacillus megaterium and the fermentation liquor thereof are used for improving the activity of anti-retrozyme in seed germination and/or reducing the content of malondialdehyde.
As another preferred scheme of the embodiment of the invention, the test isolate Dq-1 is Bacillus megaterium JL-1, and the preservation number is CCTCC NO: m2019928.
As another preferable scheme of the embodiment of the invention, the preparation method of the fermentation liquor of the bacillus megaterium comprises the following steps:
and (3) taking the bacillus megaterium strain in the slant culture medium, inoculating the bacillus megaterium strain into an LB culture medium, and performing shake culture in a shaking table to obtain the fermentation liquor of the bacillus megaterium.
In another preferred embodiment of the present invention, the temperature of the shaking culture in the step is 29 to 31 ℃.
As another preferred embodiment of the present invention, the LB medium comprises the following components per liter: 4-6 g of yeast powder, 8-12 g of sodium chloride and 8-12 g of peptone.
The bacillus megaterium and the fermentation liquor thereof provided by the embodiment of the invention have strong phosphate-solubilizing capability and also have the capability of producing auxin and siderophin, so that the potential growth-promoting and antagonistic functions of the strain are shown, and the bacillus megaterium and the fermentation liquor thereof can be used for promoting the germination of plants such as mung beans.
Drawings
FIG. 1 is a graph showing the change in phytase activity at different times in example 1.
FIG. 2 is a graph showing the change of inorganic phosphorus content in the medium at different times in example 1.
FIG. 3 is a graph showing the results of the assay on a siderophore plate.
FIG. 4 is a graph comparing the productivity of strain IAA with time.
FIG. 5 is a graph showing the effect of different treatments on the SOD activity of the hypocotyl of mung bean.
FIG. 6 is a graph showing the effect of different treatments on the SOD activity of mung bean radicle.
FIG. 7 is a graph showing the effect of different dilution times on SOD activity in mung bean germination.
FIG. 8 is a graph showing the effect of different treatments on the activity of mung bean hypocotyl POD.
FIG. 9 is a graph showing the effect of different treatments on the POD activity of mung bean radicles.
FIG. 10 is a graph showing the effect of different concentrations on POD activity in mung bean germination.
FIG. 11 is a graph showing the effect of different treatments on the MDA content of the hypocotyl of mung beans.
FIG. 12 is a graph showing the effect of different treatments on the MDA content of mung bean radicles.
FIG. 13 is a graph showing the effect of different concentrations on MDA content in mung bean germination.
Detailed Description
In order to clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention, it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
The embodiment provides an experimental method for measuring phytase production capacity of Bacillus megatherium JL-1, wherein an isolated strain Dq-1 adopted in an experiment, namely Bacillus megatherium JL-1 (Bacillus megatherium JL-1) is preserved in a China center for type culture collection in 2019, 11 months and 13 days, and the address is China, wuhan university, the preservation number is CCTCC NO: m2019928.
In addition, the phytase-producing ability was determined as follows:
taking the Bacillus megaterium JL-1 fermentation liquor 6000r/min, centrifuging for 5min, diluting properly, taking 0.4mL, adding 1mL of 5mmol/L sodium phytate solution prepared by using pH 4.4 acetic acid buffer solution, preserving heat for 30min at 37 ℃, finally adding 1.0mL of color termination mixed solution, 700nm, and determining the content of inorganic phosphorus. As a control, 1.0mL of the color termination mixture was added, 0.4mL of the centrifugally diluted fermentation broth was added, and 1mL of a 5mmol/L sodium phytate solution prepared with pH 4.4 acetate buffer (Lambrchts et al 1992) was added. Definition of enzyme activity: the amount of enzyme required to hydrolyze 5mmol/L sodium phytate per minute to 1nmol soluble phosphorus was hydrolyzed at 37 ℃ and pH 4.4. The preparation method of the fermentation liquor of the bacillus megatherium JL-1 comprises the following steps: inoculating the bacillus megaterium JL-1 strain in the slant culture medium into 50mL of LB culture medium, and performing shake culture in a shaking table at 29 ℃ for 24 hours to obtain a bacterial suspension of the bacillus megaterium JL-1; the bacterial suspension is used for being inoculated into a modified PKO culture medium which takes calcium phytate as the only phosphorus source in an inoculation amount of 1 percent, and is subjected to shaking culture in a shaker at 29 ℃, so that fermentation liquor for sampling and determining the phytase activity at different times is obtained. Each liter of LB culture medium comprises 4g of yeast powder, 8g of sodium chloride and 8g of peptone, the pH is adjusted to 7.0, the LB culture medium is sterilized at the temperature of 121 ℃ for 20min, and the pH value of the LB culture medium is adjusted to 0.11MPa; the modified PKO calcium phytate culture medium comprises: 25.0g of calcium phytate, 10.0g of glucose, mgCl 2 5.0g，MgSO 4 ·7H 2 O 0.1g，KCl 0.2g，(NH 4 ) 2 SO 4 1g, naCl 0.2g, pH adjusted to 7.0, adding distilled water, stirring to constant volume to 1L,0.1MPa,Sterilizing at 121 deg.C for 20min.
The measurement results are shown in FIG. 1. As can be seen from the figure, the phytase-producing ability of the phosphobacteria JL-1 strain is measured in the modified PKO culture medium with calcium phytate as the only phosphorus source, and in the 8d measurement, the phytase-producing ability of the strain gradually increases along with the increase of the measurement time from 1d to 5 d. The phytase production peaks at 5d and declines slightly after 5d, but the difference in the yield from 5d is not significant.
In addition, the ability of this strain to dissolve calcium phosphate is shown in FIG. 2, and it can be seen from the graph that the ability of Bacillus megaterium JL-1 to release inorganic phosphorus was measured in modified PKO medium with calcium phosphate as the sole source of phosphorus, and in 8d of the measurement, the trend was similar to phytase production, i.e., 1d to 5d, the ability of the strain to release soluble phosphorus in the supernatant was gradually increased as the measurement time was extended. The peak was highest at 5d and slightly declined after 5d, but the yield was not significantly different from 5 d. The formula of the PKO culture medium is as follows: ca 3 (PO) 4 25.0g, 10.0g glucose, mgCl 2 5.0g，MgSO 4 ·7H 2 O 0.1g，KCl 0.2g，(NH 4 ) 2 SO 4 1g, naCl 0.2g, pH value adjusted to 7.0, and distilled water added to stir evenly and fix the volume to 1L.
Example 2
This example provides an experimental method for determining the ability of bacillus megaterium to produce auxin (indoleacetic acid, IAA) and siderophore, comprising the following steps:
(1) IAA production capacity assay: inoculating 1mL of fermentation liquid into a King liquid culture medium for 24h, then starting to measure, taking 2mL of bacterial liquid for 12000r/min, centrifuging for 3min, taking 1mL of supernatant into a test tube, adding 2mL of Sakowski reagent, reacting for 30min in the dark at room temperature, measuring the absorbance at 530nm after the reaction, and taking the King liquid culture medium without inoculation as a blank control.
IAA production = n × (A) i -A 0 -0.0213)/0.0308。
(2) And (3) determining the siderophore production ability of the strain: after the test strain is subjected to shake culture in an MKB culture medium for 48 hours, the fermentation liquor is centrifuged for 5min at the rotating speed of 10000r/min. And (3) adding an equal volume of CAS detection solution into 3mL of supernatant, fully and uniformly mixing, standing for 1h, measuring the light absorption value at the wavelength of 630nm by using a spectrophotometer, and taking double distilled water for zero adjustment. Another 3mL CAS detection solution was mixed well with 3mL uninoculated MKB medium supernatant, and absorbance was determined as a control Ar by the same method. Relative content of siderophore = [1- (a/Ar) ] × 100%. Relative content of siderophiles according to the criteria of Manjanatha: 2 to 0.4 is a low-yield strain, 0.4 to 0.6 is a medium-yield strain, 0.6 to 0.8 is a high-yield strain, and 0.8 to 1.0 is a very high-yield strain.
Wherein, the CAS detection solution for producing siderophin:
a, 60.5mg of chromium azure S is dissolved in 50mL of deionized water; b:10mL of ferric iron solution (1 mmoL/L FeCl) 3 .6H 2 O,10mmoL/L hydrochloric acid is taken as a solvent); c72.9 mg CTAB was dissolved in 40mL deionized water. Mixing the above 3 solutions, diluting to 100mL, adjusting pH to neutral, and sterilizing at 121 deg.C for 20min.
MKB Medium: 5g of complex protein amino acid, 15mL of glycerol, 1.5g of dipotassium phosphate, 1.5g of magnesium sulfate, 1000mL of double distilled water and pH7.2.
Salkowski reagent: contains 2% of 0.5mol FeCl 3 The solution was mixed with 500mL of 35% perchloric acid solution before use, and the mixture was stored in the dark.
Auxin production LB medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 200mg of L-tryptophan and 1000mL of deionized water; mixing well, loading into test tube (5 mL/tube), and sterilizing at 121 deg.C for 20min.
Auxin production King medium: peptone 20g, glycerol 15mL, dipotassium hydrogen phosphate 1.5g, magnesium sulfate 1.5g, distilled water 1L, ph7.2, 121 ℃ sterilization for 20min.
The results of the detection of the production of the siderophore and the determination of the content of the siderophore are shown in FIG. 3, and the orange chelate ring is generated around the strain, which shows that the strain has the capability of producing the siderophore. The relative content of the siderophin in the strain fermentation liquor is 38.10 +/-2.12%, and the strain is a low-yield siderophin strain with the relative content of the siderophin of 0.2-0.4 according to the Manjanatha standard.
Further, the results of the measurement of the indole acetic acid-producing ability of the strain are shown in Table 1 and FIG. 4.
TABLE 1
The assay results of the indoleacetic acid content in the King medium in table 1 show that: the strain can produce indoleacetic acid, the productivity of the strain is increased along with the time extension, and the productivity of the strain is not reduced when the measuring time is 120 hours, which shows that the productivity of the strain in the King culture medium is very strong and durable.
In addition, as can be seen from fig. 4, the strain has the capability of secreting IAA in an LB-tryptophan culture medium, the IAA yield of the strain is continuously increased in the measuring process from 48h to 120h and reaches a maximum value in 120h, and although the IAA yield is reduced in 144h, the difference is not obvious, so that the strain has strong capability of producing IAA and is maintained for a long time.
Example 3
The embodiment provides a preparation method of fermentation liquor of bacillus megaterium JL-1, which comprises the following steps: inoculating the bacillus megaterium JL-1 strain in the slant culture medium into 50mL LB culture medium, and carrying out shake culture in a shaking table at 31 ℃ for 24h to obtain a fermentation liquid of the bacillus megaterium JL-1; each liter of the LB culture medium comprises 6g of yeast powder, 12g of sodium chloride and 12g of peptone, the pH value is adjusted to 7.0, the LB culture medium is 0.1MPa, and the LB culture medium is sterilized for 20min at 121 ℃.
Example 4
The embodiment provides an experiment for influence of bacillus megaterium and fermentation liquor thereof on mung bean seed germination, which specifically comprises the following steps:
s1, inoculating bacillus megaterium JL-1 strain in a slant culture medium into 50mL of LB culture medium, carrying out shake culture in a shaking table at 30 ℃ for 24 hours, centrifuging at 12000r/min for 3 minutes, separating fermentation supernatant (LB) and thalli (Dq-1), washing the thalli once with distilled water, diluting the thalli and the fermentation broth supernatant by 50 times, carrying out a mung bean seed germination test, placing a piece of filter paper in a culture dish, placing 40 mung bean seeds activated by hot water in each culture dish, soaking the mung bean seeds in 3mL of diluent of the thalli and the supernatant respectively, using water as a contrast, pouring the liquid when the mung bean seeds just sprout, watering once a day, and after the culture of each flat dish of 1mL and 7d is finished, separately freezing and storing the roots and the hypocotyls for later use.
S2, preparing an LB culture medium, and diluting the fermentation liquor (Dq-1) and the culture medium (LB) into sterile water with concentration gradients of 20 times, 40 times, 60 times, 80 times and 100 times respectively by taking the sterile water as a reference. Disinfecting the surfaces of mung bean seeds by using hot water at 70 ℃, and soaking the treated mung beans in diluent with corresponding concentration gradient for 4 hours. The seeds were transferred in a sterile room to pre-sterilized filter paper-lined petri dishes. 40 seeds were inoculated to the green beans in each plate. And 2mL of dilution solution corresponding to the gradient was added to each group. Culturing with 2mL of diluent with corresponding gradient every day for 7d, and measuring parameters of root and hypocotyl with plant root system scanner. Three replicates for each treatment were required. The seeds soaked in sterile water are used as negative control, and LB culture solution without inoculation is used as control. In the process of cultivation, the growth condition of the mung beans is recorded every day, after the cultivation is finished, 20 mung bean seedlings with good growth vigor are selected from the mung beans, the hypocotyl length and the root length of the mung beans are measured, and the number of roots is counted. After the measurement, 1g of each of the samples was repeatedly taken for each treatment and stored for further use.
Wherein each liter of LB culture medium comprises 6g of yeast powder, 12g of sodium chloride and 12g of peptone, the pH value is adjusted to 7.0,0.1MPa, and the LB culture medium is sterilized at 121 ℃ for 20min.
S3, the influence of the fermentation liquor on the contents of the adverse enzymes and the MDA in the mung bean seed germination process is as follows:
superoxide Dismutase (SOD): taking 50 mu L of enzyme solution, putting into a test tube, adding 3mL of SOD reaction solution, setting three times for each sample, taking a phosphate buffer solution with pH 7.8 as a reference, illuminating for 30min under strong light at the same time, taking the phosphate buffer solution as a reaction system, adjusting zero, and carrying out color comparison at the wavelength of 560 nm.
Peroxidase (Peroxidase, POD): 50 μ L of enzyme solution was placed in a tube, 3mL of PO D reaction solution was added, triplicate for each sample, and read every minute at 470nm for five times, using phosphate buffer pH 7.8 as a control.
Malondialdehyde (MDA) assay: adding 1mL of enzyme solution into a test tube, adding 2mL of MDA reaction solution, setting three times for each sample, taking a phosphate buffer solution with pH 7.8 as a reference, covering a test tube plug, carrying out water bath in boiling water for 15min, rapidly cooling, centrifuging at 6000r/min for 15min, taking supernate, and carrying out color comparison at three wavelengths of 600nm, 532nm and 450 nm.
Wherein, in order to better explain the experimental result, the germinated mung beans with good and bad germination effects are selected in different treatments, and the beard number, hypocotyl length and root length are measured. It can be seen from table 2 that the influence of the strain fermentation broth on the mung bean germination is mainly reflected on the hypocotyl and the number of roots, and when the dilution factor of the fermentation broth is 60 times and 80 times, the hypocotyl of the germinated mung bean seeds tends to increase and the increase difference is obvious compared with the water treatment and the culture medium treatment with the same dilution factor; meanwhile, the number of the germinated mung bean seeds is relatively obviously increased, but the difference is not obvious when the dilution multiple is increased to 100 times. It is shown that proper dilution of the fermentation broth can promote the extension of hypocotyl and increase the number of the hypocotyl in the germination process. Specifically, the results of the effects of different treatment methods on the germination capacity of mung bean seeds are shown in table 2.
TABLE 2
The results of detection of superoxide dismutase (SOD) are shown in FIGS. 5 to 7. Compared with the water control, the SOD enzyme activity in the mung bean hypocotyls treated by the strains (Dq-1) and the fermentation supernatant (LB) is different, wherein the differences of the fermentation supernatant treatment (LB) and the strain treatment (Dq-1) and the water treatment are obvious; in the germinated radicle, the activity of the thallus (Dq-1) and the water treatment enzyme is not different; the enzyme activity of the fermentation supernatant (LB) is the highest among the processes, and the difference is obvious. Namely, the fermentation supernatant and the thalli both have certain effect of improving the enzyme activity; considering that the different concentrations of the fermentation liquid have different effects, and thus the treatment of the fermentation liquid (Dq-1) and the culture medium (LB) with different concentrations is increased, the test results show that: in different concentrations, the enzyme activity of the fermentation liquor treated by diluting 80 times is improved most obviously compared with that of the fermentation liquor treated by the same dilution times, the enzyme activity of the fermentation liquor treated by diluting 100 times is lowest, and the treatment is also obviously different. The results show that the fermentation liquor shows certain improvement effect on the SOD enzyme activity of the mung bean in a certain range.
In addition, the measurement results of Peroxidase (POD) are shown in the attached figures 8-10, and it can be seen in hypocotyl measurement that the enzyme activities of fermentation supernatant (LB) treatment are all higher, while the enzyme activity of thallus treatment (Dq-1) is the lowest, and the enzyme activity difference is not obvious in the fermentation liquid treatment of different dilution concentrations.
In the radiculoenzyme activity determination, the enzyme activity of POD (peroxidase) of mung bean hypocotyl and radicle can be improved by treating the strain (Dq-1) and treating the fermentation supernatant (LB) compared with the enzyme activity of POD (peroxidase) treated by water, wherein the enzyme activity of the fermentation supernatant (LB) is the highest, which shows that the POD activity of the mung bean hypocotyl and radicle can be improved by treating the fermentation supernatant (LB). However, the enzyme activity of the fermentation liquor diluted by 60 times is the highest at different concentrations.
In addition, as shown in FIGS. 11 to 13, the results of measurement of Malondialdehyde (MDA) showed that there was no significant difference between the fermentation supernatant (LB) treatment and water in the hypocotyl measurement, and the MDA content in the cell body (Dq-1) treatment was significantly reduced as compared with water.
In germinated radicles, the MDA content of each group treated is obviously different, and the MDA content of the fermentation supernatant (L B) is obviously increased compared with that of water. The MDA content of the cells (Dq-1) treated with the cells was the lowest. The MDA content of the mung bean hypocotyl and radicle can be obviously reduced by treating the thalli, and the mung bean hypocotyl and radicle show a certain protection effect on seed germination. In the treatment of fermentation liquor (Dq-1) with different dilution times and a culture medium (LB), the thallus fermentation liquor with the same dilution time has the effect of reducing the MDA content of the mung bean roots compared with the culture medium, and the result shows that the thallus fermentation liquor has a certain protection effect on plants when diluted by 80 times.
In conclusion, the phosphorus dissolving and dissolving effects of the isolate Dq-1, namely the bacillus megaterium JL-1 strain are verified by measuring the phytase producing and inorganic phosphorus releasing capacities; meanwhile, the growth promoting substance experiment of the strain is tested to prove that the strain has IAA and siderophore secretion capability.
In addition, after the thalli and fermentation supernatant and fermentation liquids with different concentrations are applied in seed germination, the measurement and calculation of the length of hypocotyls, radicles and the number of roots in mung bean seed germination are carried out, the promotion effect of the fermentation liquids on the hypocotyls and the roots is found, auxin (IAA) is a plant hormone and has an important effect on the growth processes of plant embryonic development, organogenesis, gametocyte formation and the like, and the change of the hypocotyls and the number of the roots in a test can be related to the growth process.
Superoxide dismutase (SOD) is the only one discovered so far, and can specifically eliminate superoxide anion free radicals, prevent oxygen free radicals from destroying the composition, structure and function of cells, protect cells from oxidative damage, and prevent and treat related diseases caused by the superoxide dismutase (SOD). Peroxidase (POD) is involved in the metabolism of auxin, respiration, synthesis of cell walls and nociceptive reactions. Catalase (CAT) is one of the key enzymes of the biological defense system, which promotes the decomposition of hydrogen peroxide into molecular oxygen and water, protecting cells from the toxic effects of hydrogen peroxide. Malondialdehyde (MDA) content is the embodiment of the peroxidation degree of plant cytoplasmic membrane, and the damage to the cytoplasmic membrane is serious due to the high MDA content. The invention determines the contents of the adversity-resistance enzyme and the MDA in the seed germination, and reflects that the activity of the adversity-resistance enzyme in the seed germination can be improved and the MDA content can be reduced after the thalli and the strain fermentation liquor are properly diluted to a certain extent.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (5)
1. The application of the bacillus megaterium and the fermentation liquor thereof in promoting plant germination is characterized in that the plant is mung bean; the bacillus megaterium and the fermentation liquor thereof are used for improving the activity of anti-retroenzymes in seed germination and/or reducing the content of malondialdehyde; the culture name of the bacillus megaterium is bacillus megaterium JL-1, and the preservation number is CCTCC NO: m2019928.
2. The use of bacillus megaterium and its fermentation broth for promoting germination of plants as claimed in claim 1, wherein the bacillus megaterium and its fermentation broth are used for producing auxin and/or siderophin.
3. The use of bacillus megaterium and the fermentation broth thereof for promoting plant germination according to any one of claims 1 to 2, wherein the preparation method of the bacillus megaterium fermentation broth comprises the following steps:
and (3) taking the bacillus megaterium strain in the slant culture medium, inoculating the bacillus megaterium strain into an LB culture medium, and performing shaking culture in a shaking table to obtain the fermentation liquor of the bacillus megaterium.
4. The use of Bacillus megaterium and its fermented liquid for promoting plant germination as claimed in claim 3, wherein the temperature of shaking culture is 29-31 ℃.
5. The use of Bacillus megaterium and its fermentation broth for promoting plant germination as claimed in claim 3, wherein each liter of the LB medium comprises the following components: 4-6 g of yeast powder, 8-12 g of sodium chloride and 8-12 g of peptone.
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