CN113980875B - Bacillus cereus P1 and application thereof - Google Patents

Abstract

The invention discloses a bacillus cereus P1 and application thereof, belonging to the technical field of microorganisms, wherein the bacillus cereus is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2021 year, 07 month and 13 days, and the preservation registration number is CGMCC No. 22821; the invention shows that the strain has the best effect of dissolving organic phosphorus when the temperature is 30 ℃, the pH value is 7.5, the carbon source is sucrose, the nitrogen source is potassium nitrate, the C/N ratio is 20:1 and the salt concentration is 1.5 percent, can secrete phosphatase and phytase, dissolve insoluble organic phosphorus, secrete phytohormone and siderophores, promote photosynthesis, respiration, substance and energy metabolism of plants, and can be used for solving the problems of slow development of the root system of the seedling of the strelitzia.

Description

Bacillus cereus P1 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a bacillus cereus P1 and application thereof.

Background

Phosphorus plays an important role in plant growth, is a main nutrient element in metabolic processes such as plant energy transfer, signal conduction, macromolecular biosynthesis, photosynthesis, respiration and the like, is also an important nutrient element for promoting root growth and development, and is lack of the important nutrient element which seriously restricts the growth of plants. The phosphorus content of the acid soil is very high, but the acid soil has a strong solidifying effect on phosphorus, so that most of the phosphorus in the soil is combined by calcium, aluminum or iron minerals to form insoluble phosphate, and further, the effective phosphorus content in the soil is very low, and only a small amount of effective phosphorus can be provided for plants. On average, most mineral nutrients in soil solution are present in millimoles and phosphorus is present in micromoles or less, and when plants lack phosphorus, the development of root systems is retarded, lateral roots are not developed, and the plants grow slowly and are weak. The application of phosphate fertilizer to soil is an important way for supplementing the demand of plants on phosphorus, but the phosphate fertilizer is expensive and has low utilization rate, and the problems of environmental pollution, soil hardening and the like are caused by excessive application of phosphate fertilizer. Therefore, there is a need to find an environmentally friendly, economically feasible method to increase plant yield in low or phosphorus deficient soils. The phosphate solubilizing microorganisms are ubiquitous in most soils, particularly rhizosphere soils, are more in types and content, can convert insoluble phosphorus in the soils into available phosphorus which can be absorbed by plants, replace chemical fertilizers and solve the problem of low available phosphorus in the soils, and are promising biological fertilizers.

A large number of autotrophic and heterotrophic microorganisms exist in soil, and have the ability to dissolve poorly soluble phosphorus, and the types and amounts of phosphorus-solubilizing microorganisms vary depending on the soil, and the types of phosphorus-solubilizing microorganisms that have been reported include Pseudomonas (Pseudomonas), Enterobacter cloacae (Enterobacter), Bacillus (Bacillus), Rhizobium (Rhizobium), Agrobacterium (Agrobacterium), Erwinia (Erwinia), Streptomyces (Streptomyces), Nocardia (Nocardia), Aspergillus (Aspergillus), Penicillium (Penicillium), Trichoderma (Trichoderma), Anabaena (Anabaena), Candida (Nostoc), Nostox (Calothrix), and Pseudomyces (Scytonema). Although most of researches show that the screened phosphate solubilizing microorganisms have strong phosphate solubilizing capability, the phosphate solubilizing microorganisms do not have stable effect when applied to soil, and sometimes the yield of crops and the accumulation of phosphorus are not increased. The strain has better phosphate-solubilizing capability in a laboratory, but the phosphate-solubilizing capability is weakened or completely disappeared after the strain is applied into soil, and the main reasons are as follows: (1) the soil has a complex environment, and the phosphate-solubilizing microorganisms cannot survive and colonize in the soil after being applied to the soil; (2) competition between phosphate solubilizing microorganisms and soil microorganisms; (3) the utilization capacity of phosphate-solubilizing microorganisms on nutrient substances in soil; (4) degradation of phosphate-solubilizing capability of the phosphate-solubilizing microorganisms; (5) the texture, pH, water content, organic matter content, etc. of the soil can affect the growth and reproduction of phosphate-solubilizing microorganisms and the colonization effect thereof. The existence and successful colonization of the phosphate solubilizing microorganisms in the soil are related to environmental factors such as temperature, pH, salt concentration and the like in the soil, and meanwhile, nutrient substances such as a carbon source, a nitrogen source and the like in the soil can also influence the phosphate solubilizing capability of the phosphate solubilizing microorganisms by influencing the reproduction of phosphate solubilizing microorganism bodies and the content of organic acid secretion.

The strelitzia (Parashorea chinensis Wang Hsie) is also called "chongtian tree", which is a plant of the genus elaeagnus of the family dipterocarpaceae, a tropical rain forest marker species and an endangered tree species, and the first-level of the country mainly protects wild plants, and Yunnan and Guangxi are the main distribution areas thereof. The stretchtree has high scientific value and economic value, is high-quality wood manufactured by shipbuilding, buildings, bridges, musical instruments and the like, and has considerable application and development prospects. According to the previous investigation and experiments, the root system of the tamarind tree grows slowly when seedlings grow, lateral roots are not developed, the tamarind tree grows slowly when seedlings grow, the resistance is weak, the death rate is high, phosphorus is vital to the growth and development of the root system, and phosphate solubilizing microorganisms are a main way for improving the effective phosphorus of soil, so that the efficient phosphate solubilizing bacteria are separated and screened from the artificial forest of the tamarind tree, and the method is an effective method for solving the problems that the root system of the tamarind tree grows slowly and slowly.

Disclosure of Invention

The invention aims to provide a bacillus cereus P1 strain and application thereof, aiming at solving the problems in the prior art, the bacillus cereus strain can effectively convert insoluble phosphorus into available phosphorus which can be absorbed by plants, and can secrete phytohormone and siderophores to promote the growth of the plants.

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

the invention provides a Bacillus cereus (Bacillus cereus) P1, which is preserved in the China general microbiological culture Collection center (CGMCC for short, the address is China academy of sciences, 3, of Xilu No.1, Beijing, Chaoyang, North Chen, the collection date is 2021 year 07 month 13 days, and the preservation registration number is CGMCC No. 22821.

The invention also provides application of the bacillus cereus P1, which is used for converting phosphorus which is difficult to dissolve in soil into available phosphorus which can be absorbed by plants.

Further, the insoluble phosphorus is an organic phosphorus that is insoluble.

The invention also provides application of the bacillus cereus P1, which is used for the cultivation of the artificial forest of the strelitzia.

Further, the method is used for improving the growth of the seedling stage and the root system of the young tree of the tamarind tree and promoting the growth.

The invention also provides a microbial agent, which comprises the bacillus cereus P1.

The invention discloses the following technical effects:

the invention separates, screens and identifies the efficient phosphate solubilizing bacteria at the root of the tamarind tree from the tamarind tree artificial forests of different forestation years, and researches the phosphate solubilizing effect, the phosphate solubilizing mechanism, the optimal phosphate solubilizing condition and the capacity of the strain to secrete related plant hormones and iron carriers so as to evaluate the capacity of the phosphate solubilizing bacteria to convert the insoluble phosphorus in the soil into the available phosphorus which can be absorbed by plants. The invention shows that the strain has the best effect of dissolving the organic phosphorus when the temperature is 30 ℃, the pH value is 7.5, the carbon source is sucrose, the nitrogen source is potassium nitrate, the C/N ratio is 20:1 and the salt concentration is 1.5 percent, can secrete phosphatase and phytase, dissolve insoluble organic phosphorus, secrete phytohormone and siderophores, promote plant photosynthesis, respiration, substance and energy metabolism, and can be used for solving the problems of slow root system development and slow seedling growth of the strelitzia.

Drawings

FIG. 1 is the construction of phylogenetic trees of phosphate solubilizing bacteria;

FIG. 2 is the effect of pH on the phosphate solubilizing ability of organophosphorus bacteria;

FIG. 3 is the effect of pH value on the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 4 is a graph showing the effect of temperature on the phosphate solubilizing ability of organophosphorus-solubilizing bacteria;

FIG. 5 is the effect of temperature on the phosphate solubilizing ability of inorganic phosphate solubilizing bacteria;

FIG. 6 is the influence of carbon source on the phosphate solubilizing ability of organophosphorus bacteria;

FIG. 7 is the influence of carbon source on the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 8 is a graph showing the influence of nitrogen source values on the phosphate solubilizing ability of organophosphorus bacteria;

FIG. 9 is a graph showing the influence of nitrogen source values on the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 10 is a graph showing the effect of C/N on the phosphate solubilizing ability of organophosphorus bacteria;

FIG. 11 is a graph showing the effect of C/N values on the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 12 is a graph showing the effect of salt concentration on the phosphate solubilizing ability of organophosphorus-solubilizing bacteria;

FIG. 13 is a graph showing the effect of salt concentration on the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 14 is a dynamic change of the phosphate-solubilizing ability of organophosphorus bacteria;

FIG. 15 is a graph showing the dynamic change of the phosphate solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 16 is a graph showing the dynamic change of the pH of the organophosphorus bacteria liquid;

FIG. 17 shows the dynamic change of pH of inorganic phosphorus bacteria solution;

FIG. 18 is a graph showing the relationship between the phosphate solubilizing amount of organophosphorus bacteria and pH;

FIG. 19 is a graph showing the relationship between the phosphate-solubilizing amount of the inorganic phosphate-solubilizing bacteria and pH;

FIG. 20 shows the phosphorus-dissolving amount of inorganic phosphorus-dissolving bacteria in different phosphorus source media;

FIG. 21 shows the activity of degrading the secretion of acid phosphatase by organophosphorous bacteria;

FIG. 22 shows the activity of organophosphorous bacteria in secreting alkaline phosphatase;

FIG. 23 shows the release of phytase activity secreted by organophosphorous bacteria;

FIG. 24 shows the IAA content secreted by phosphate-solubilizing bacteria;

FIG. 25 shows the gibberellin content secreted by phosphate solubilizing bacteria;

FIG. 26 shows the siderophore secretion activity of phosphate-solubilizing bacteria.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

Example 1 isolation, screening and identification of Phosphorocarba rhizosphere bacteria

1.1 isolation of phosphate solubilizing bacteria

In 8 months in 2019, in Guangxi Nanning Trees gardens, the artificial quast forests for forestation in 1978, 2011, 2012, 2017, 2018 and 2019 are respectively selected, and the root systems of the quast trees and soil samples adhered to the rhizosphere are collected.

The phosphate solubilizing bacteria are separated from the soil of the root of the tamarind tree in different afforestation years by utilizing a lecithin organophosphorus culture medium and a calcium phosphate inorganic phosphate culture medium, 68 isolates are obtained, wherein 30 isolates can form obvious transparent rings on an organophosphorus or inorganic phosphate solid culture medium, the 30 strains are proved to have the capability of dissolving insoluble organophosphorus or inorganic phosphate, and the 30 strains capable of generating the phosphate solubilizing rings are numbered from P1 to P30.

1.2 screening of phosphate-solubilizing bacteria

1.2.1 Primary Screen for phosphate-solubilizing bacteria

The results of measuring the ratio of the diameter (D) of the transparent circle generated by inoculating 30 strains of the P1-P30 separated as described above to the organic and inorganic phosphorus solid culture media, respectively, and the diameter (D) of the colony, are shown in Table 1, wherein 10 strains of the organophosphorus solid culture media having D/D >2.0, P1, P2, P5, P7, P8, P14, P22, P27, P28 and P30, respectively, account for 33.3% of the total number of the strains, indicating that the 10 strains have strong organophosphorus resolving power, 6 strains of 1.5< D/D <2.0, account for 20% of the total number of the strains, 14 strains of 1.0< D/D <1.5, account for 46.7% of the total number of the strains, and among 30 strains, P14 has the largest D/D value on the organophosphorus solid culture media, 2.15, the smallest P23/D value, and smallest P23D/D value of 1.05.

In the inorganic phosphorus culture medium, the D/D of different strains has obvious difference, 8 strains with the D/D being more than 2.0 are P4, P8, P10, P12, P15, P23, P25 and P30 respectively, the total number of the strains is 26.7 percent, 8 strains with the D/D being less than 1.5< 2.0 are 1.7 percent, 14 strains with the D/D being less than 1.5 are 1.0< 1.5 are 46.6 percent, in 30 strains, the D/D value of the strain P12 on the inorganic phosphorus culture medium is 2.16 at most, and the P13D/D value of the strain is 1.061 at least.

TABLE 1 results of preliminary screening of phosphate-solubilizing bacteria

Note: D/D represents the diameter of the phosphate solubilizing ring of the phosphate solubilizing bacteria/the diameter of the strain of the phosphate solubilizing bacteria, the data in the table are mean values +/-standard error, the same letter represents no significant difference (P >0.05) after the same column of numerical values, and different letters represent significant difference (P < 0.05).

1.2.2 rescreening of phosphate solubilizing bacteria

The observation of the phosphate solubilizing rings generated by phosphate solubilizing strains on a solid culture medium is only one method for qualitatively determining the phosphate solubilizing capability of the strains, in order to more accurately judge the phosphate solubilizing capability of the strains, 10 strains with strong phosphate solubilizing capability, namely 10 strains with D/D larger than 2.0 and 8 strains with inorganic phosphate solubilizing capability, which are obtained by primary screening are subjected to secondary screening, and the effective phosphorus content in a strain culture solution is quantitatively determined by a liquid culture method.

Respectively preparing organic and inorganic phosphorus liquid culture media, inoculating a test strain to a beef extract peptone solid culture medium, activating for 24h at 30 ℃, inoculating a ring of activated thalli in an LB liquid culture medium, performing shaking culture for 24h at 30 ℃ to prepare a seed solution, accurately measuring 1mL of the seed solution by using a sterilized liquid transfer gun, inoculating the seed solution into the sterilized organic and inorganic phosphorus liquid culture media, taking out the bacteria after 3 times of inoculation by using a Contrast (CK) as an uninoculated bacterial solution, placing the bacteria in a shaking table after inoculation, wherein the temperature of the shaking table is 30 ℃, the rotating speed is 180r/min, performing shaking culture for 7d and 7d, centrifuging the organic phosphorus culture solution for 30min at 12000r/min, performing inorganic phosphorus culture for 10000r/min and 15min, absorbing a supernatant, measuring the phosphorus content by using a molybdenum-antimony colorimetric method, and calculating the phosphorus dissolution rate, wherein the results are shown in tables 2 and 3.

As can be seen from Table 2, in a culture medium taking lecithin as an organic phosphorus source, the effective phosphorus content in culture solutions of 10 strains is significantly higher than that of a control CK (6.38mg/L), which indicates that the 10 strains all have a certain phosphorus solubilizing capability in the organic phosphorus liquid culture medium, the amount of dissolved organic phosphorus of each strain is between 16.18 and 41.58mg/L, the phosphorus solubilizing amount difference between different strains is significant, the phosphorus solubilizing amount of the strain P7 is at most 41.58mg/L, and the phosphorus solubilizing amount of the strain P14 is at least 16.18 mg/L. Compared with CK, the increase of effective phosphorus in the culture solution is between 9.8mg/L and 35.20mg/L, and the increase is 1.54 to 5.52 times of CK. The effective phosphorus content of each strain culture solution is sequentially P7> P8> P5> P1> P28> P30> P2> P22> P27> P14 from large to small, the phosphorus-dissolving rate of 10 strains is 1.96-7.04%, 4 strains with the phosphorus-dissolving rate of more than 5% are respectively strains P1, P5, P7 and P8, and the 4 strains are proved to have strong organophosphorus-dissolving capacity, the strain P1 in the 4 strains is derived from the root soil of the Tamarindus indica forest planted in 2019 years, the strains P5 and P7 are derived from the root soil of the Tamarindus indica forest planted in 2018 years, and the strain P8 is derived from the root soil of the Tamarindus indica forest planted in 2017 years.

As can be seen from Table 3, in the liquid culture medium of inorganic phosphorus phosphate, the effective phosphorus content in the culture medium of 8 strains of inorganic phosphorus-decomposing bacteria is significantly higher than that of the control CK (21.44mg/L), and the difference of the effective phosphorus content in the culture medium of different strains is significant. The effective phosphorus content in the culture solution of 8 inorganic phosphorus-decomposing bacteria is between 305.33mg/L and 598.89mg/L, and the phosphorus-decomposing rate is between 5.68 percent and 11.55 percent. Compared with CK, the increase of available phosphorus is 283.89-577.45 mg/L, and the increase is 13.24-26.93 times of CK. The effective phosphorus content in each strain culture solution is sequentially P30> P8> P4> P12> P23> P15> P10> P25 from large to small, the phosphorus dissolving amount of the strains P4, P8, P12 and P30 is high, the effective phosphorus content in the culture solution is respectively up to 552.87mg/L, 559.78mg/L, 548.53mg/L and 598.89mg/L, the phosphorus dissolving rate is respectively 10.63%, 10.77%, 10.54% and 11.55%, and the phosphorus dissolving rate is more than 10%, which indicates that the strains P4, P8, P12 and P30 have strong inorganic phosphorus dissolving capacity, the strain P4 is from the oregano rhizosphere soil for forestation in 2019, the strains P8 and P12 are from the oregano rhizosphere soil for forestation in 2017, the strain P30 is from the oregano rhizosphere soil for forestation in 1987, and the strain P364 can be taken as a key fertilizer for the oregano strain.

TABLE 2 rescreening of organophosphorus bacteria

Year of afforestation	Strain number	Available phosphorus content (mg/L)	Phosphorus dissolution rate (%)
				2019	P1	33.08±1.02b	5.34
2019	P2	18.87±0.06de	2.50
				2018	P5	33.39±1.31b	5.40
2018	P7	41.58±0.64a	7.04
				2017	P8	39.53±0.79a	6.63
2017	P14	16.18±0.20f	1.96
				2011	P22	17.16±1.11ef	2.16
1978	P27	16.67±0.47ef	2.06
				1978	P28	21.52±0.42c	3.03
1978	P30	20.74±0.64cd	2.87
					CK	6.38±0.91g	0

TABLE 3 inorganic phosphorus bacteria rescreening

1.3 identification of phosphate-solubilizing bacteria

1.3.1 morphological and physiological and biochemical identification of bacteria

After streaking and purifying the screened phosphate-solubilizing bacteria, observing the characteristics of colony size, color and the like, and performing physiological and biochemical tests such as gram staining, oxidase test, catalase test, indole test, VP, citrate, gelatin liquefaction, hydrogen sulfide, methyl red and the like.

Observing cell morphology and colony characteristics of the 4 separated and screened strains P1, P5, P7 and P8 with strong organic phosphorus dissolving capacity and the 4 strains P4, P8, P12 and P30 with strong inorganic phosphorus dissolving capacity, wherein P8 has both organic phosphorus dissolving capacity and inorganic phosphorus dissolving capacity, and observing results of colony morphology characteristics of the strains are shown in Table 4.

TABLE 4 morphological Observation of phosphate-solubilizing bacteria

The physiological and biochemical identification items of the phosphate-solubilizing bacteria comprise: citrate test, gelatin hydrolysis test, catalase test, V-P reaction, indole test, starch hydrolysis test, nitrate reduction test, H production ₂ S test and the like, the results of each item of the strain test are shown in Table 5, each test item is repeated for 3 times, and the measured results are relatively stable. The experimental results show that the strains P1 and P30 have the same characteristics, namely the strains can utilize citrate, can hydrolyze gelatin, are positive in catalase reaction and can produce indole, can hydrolyze starch and reduce nitrate, the strains P5, P7, P8, P4 and P12 have the same characteristics, and are respectively capable of utilizing citrate, can hydrolyze gelatin, are positive in catalase reaction and can reduce nitrate, the strains P1 and P30 can be preliminarily judged to be bacillus according to the morphological and physiological and biochemical characteristics of the strains, and the strains P5, P7, P8, P4 and P12 can be Burkholderia.

TABLE 5 physiological and biochemical identification of phosphate-solubilizing bacteria

Test item

P1

P5

P7

P8

P4

P12

P30

Citric acid salt

+

+

+

+

+

+

+

Hydrolysis test of gelatin

+

+

+

+

+

+

+

Catalytic reaction

+

+

+

+

+

+

+

V-P reaction

+

-

-

-

-

-

+

Indole test

+

-

-

-

-

-

+

Starch hydrolysis test

+

-

-

-

-

-

+

Nitrate reduction test

+

+

+

+

+

+

+

H 2 S

-

-

-

-

-

-

-

Note: + positive, -negative

In order to verify whether the guessing of the strain according to the morphological and physiological and biochemical characteristics is accurate, the invention also identifies the strain in molecular biology. The 16SrDNA sequence (16 SrDNA base sequence of Burkholderia gladioli P7 of the invention, shown as SEQ ID No. 1) measured by the strain is compared with other 16SrDNA sequences in a GenBank nucleic acid database for homology, ClustalX is used for analysis, MEGA7.0 is used for constructing a phylogenetic tree by adopting a Neighbor Joining method, and 7 strains can find similar strain sequences with very high homology in the database through BLAST comparison. Strains P1 and P30 have a higher sequence similarity to Bacillus, strains P4, P5, P7, P8 and P12 have a higher sequence similarity to Burkholderia, it can be seen from the phylogenetic tree of FIG. 1 that strains P5, P7 and P8 are located in the same branch as Burkholderia gladioli, strains P4 and P12 are located in the same branch as Burkholderia cepacia, strains P1 and P30 are located in the same branch as Bacillus cereus and are closer in relative relation, and thus, strains P4 and P12 are identified as Burkholderia cepacia (Burkholderia gladioli), respectively, strains P5, P7 and P8 as Burkholderia cepacia (Burkholderia gladioli), strains P4 and P12 as Burkholderia cepacia (Burkholderia cepacia), strains P1 and P30 as Burkholderia gladiolus (Burkholderia gladiolus).

1.4 preservation of phosphate solubilizing bacteria

Burkholderia gladioli P7 is preserved in China general microbiological culture Collection center (CGMCC for short, the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaozhou, the area of the facing Yang, and the date of 2021, 13.07.13), and the preservation registration number is CGMCC No. 22824.

Example 2 screening of optimal phosphate solubilizing conditions for Tamarindus indica rhizosphere phosphate solubilizing bacteria

The organophosphorus-solubilizing bacteria P1, P5, P7 and P8 and the inorganic phosphorus-solubilizing bacteria P4, P8, P12 and P30 which are screened by the test in example 1 were used as test materials to further study the phosphorus solubilizing conditions of the phosphorus-solubilizing bacteria.

2.1 influence of pH value on the phosphate solubilizing ability of phosphate solubilizing bacteria

Respectively preparing organic phosphorus liquid culture medium and inorganic phosphorus liquid culture medium, setting the initial pH values to be 3.5, 4.5, 5.5, 6.5, 7.5 and 8.5, respectively, subpackaging in 150mL triangular bottles, sterilizing 30mL each bottle at 121 ℃ for 20 min. Respectively absorbing 1mL of organophosphorus bacteria seed solution to inoculate in organophosphorus liquid culture media with different pH values, absorbing 1mL of inorganic phosphorus bacteria seed solution to inoculate in inorganic phosphorus liquid culture media with different pH values, setting 3 times of repetition for each treatment, placing the bacteria solution in a shaking table at 180r/min and 30 ℃ for 7d, centrifuging the bacteria solution after 7d, absorbing supernatant, and determining the effective phosphorus content in the bacteria solution, wherein the results are shown in figure 2 and figure 3.

As can be seen from FIG. 2, the 4 organophosphorus bacteria have different phosphate solubilizing abilities under different pH conditions, and the 4 strains show lower phosphate solubilizing abilities when the pH is 3.5, which indicates that the phosphate solubilizing abilities of the strains are limited when the pH is 3.5, the effective phosphorus content in the bacterial solutions of the strains tends to increase and decrease with the increase of the pH, the optimal pH range of the strain P5 is 6.0-7.0, the effective phosphorus content in the bacterial solutions reaches the maximum of 43.01mg/L when the pH is 6.5, which indicates that the strain P5 has stronger phosphate solubilizing ability when cultured in a meta-acid environment. The optimal pH ranges of the strains P1, P7 and P8 are all 7.0-8.0, the phosphate-solubilizing effect of the 3 strains is the best when the pH is 7.5, and the effective phosphorus contents in the bacterial liquid are 41.86mg/L, 44.19mg/L and 45.43mg/L respectively, which indicates that the strains P1, P7 and P8 have stronger phosphate-solubilizing capability in a slightly alkaline environment.

As can be seen from FIG. 3, the phosphate solubilizing abilities of the 4 inorganic phosphate solubilizing bacteria are different under different pH conditions, and the 4 strains also show lower phosphate solubilizing abilities when the pH is 3.5, which indicates that the strains are not suitable for growth and exerting the phosphate solubilizing abilities under the strong acid environment, and the effective phosphate content in the bacterial liquid of each strain tends to increase and then decrease with the increase of the pH. The optimum pH values of the strains P30 and P4 are both 6.5, and when the pH value is 6.5, the effective phosphorus contents in the bacterial liquids of the strains P30 and P4 are the highest, and are 577.37mg/L and 551.83mg/L respectively. The effective phosphorus content of the strain P30 in the bacterial liquid between pH4.5-7.5 is not changed greatly, and the effective phosphorus content in the bacterial liquid is always high, which indicates that the strain P30 has strong adaptability to the pH value of the environment and a wide pH adaptation range. The optimum pH of the strains P8 and P12 is 7.5, and when the pH of the strains is 7.5, the effective phosphorus content in the bacterial liquid reaches the highest, namely 579.74mg/L and 551.90mg/L respectively, which indicates that the strains P8 and P12 have stronger phosphorus-dissolving capacity in a slightly alkaline environment.

2.2 Effect of temperature on the phosphate solubilizing ability of phosphate solubilizing bacteria

Respectively preparing liquid culture media of organic phosphorus and inorganic phosphorus, subpackaging in triangular bottles of 150mL, 30mL each, and sterilizing at 121 ℃ for 20 min. Respectively absorbing 1mL of organophosphorus bacteria seed solution to inoculate in an organophosphorus liquid culture medium, absorbing 1mL of inorganic phosphorus bacteria seed solution to inoculate in an inorganic phosphorus liquid culture medium, placing the inorganic phosphorus bacteria seed solution in a shake culture at 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ for 7d, repeating the processes for 3 times, centrifuging the bacterial solution after 7d, absorbing the supernatant, and determining the effective phosphorus content in the bacterial solution, wherein the results are shown in fig. 4 and 5.

As shown in FIG. 4, the bacteria liquid of 4 organophosphorus bacteria has different effective phosphorus contents under different temperature culture conditions, the effective phosphorus contents of the 4 strains are the lowest when the temperature of the 4 strains is 10 ℃, the phosphorus dissolving effects of the strains P5, P8 and P7 are the best when the temperature of the strains is 35 ℃, the effective phosphorus contents of the bacteria liquid are 39.05mg/L, 42.40mg/L and 45.31mg/L respectively, the effective phosphorus contents of the bacteria liquid are 30 ℃, the effective phosphorus contents of the bacteria liquid are 33.39mg/L, 39.53mg/L and 41.58mg/L respectively, the phosphorus dissolving effect of the strain P1 is the best when the temperature of the strains is 30 ℃, the effective phosphorus contents of the bacteria liquid are 33.08mg/L, the effective phosphorus contents of the strains are 35 ℃ and the effective phosphorus contents of the strains are 29.96 mg/L. The effective phosphorus content of the 4 organophosphorus bacteria in the bacterial liquid under the culture conditions of 30 ℃ and 35 ℃ is higher than that of the bacteria treated at other temperatures, which indicates that the temperature of 30-35 ℃ is the optimal phosphorus dissolving temperature range of the 4 organophosphorus bacteria, wherein the optimal phosphorus dissolving temperature of the strain P1 is 30 ℃, and the optimal phosphorus dissolving temperature of the strains P5, P7 and P8 is 35 ℃.

As can be seen from FIG. 5, the phosphorus-dissolving amounts of the strains P12 and P4 are both high between 25 ℃ and 35 ℃, the effective phosphorus content in the bacterial liquid is highest at 30 ℃, which is 548.53mg/L and 552.87mg/L respectively, then 35 ℃ and 25 ℃, the phosphorus-dissolving amounts of P12 and P4 are 477.74mg/L and 500.98mg/L respectively at 35 ℃, and the phosphorus-dissolving amounts are 437.71mg/L and 472.68mg/L respectively at 25 ℃. The maximum content of available phosphorus in the bacterial liquid of the strain P30 is 598.89mg/L at 30 ℃, and 517.09mg/L at 35 ℃. The maximum content of available phosphorus in the bacterial liquid of the strain P8 at 35 ℃ is 623.71mg/L, and the content of available phosphorus at 30 ℃ is 559.78 mg/L. The effective phosphorus content of the bacterial liquid of the strains P12 and P4 is obviously higher than that of the strains P12 and P4 treated at other temperatures at 25 ℃, 30 ℃ and 35 ℃, which indicates that the optimum phosphorus dissolving temperature range of the strains P12 and P4 is 25-35 ℃, the effective phosphorus content of the bacterial liquid of the strains P30 and P8 at 30 ℃ and 35 ℃ is obviously higher than that of the strains P30 and P8 treated at other temperatures, which indicates that the optimum phosphorus dissolving temperature range of the strains P30 and P8 is 30-35 ℃, the optimum phosphorus dissolving temperature of the strains P30, P12 and P4 in the 4 inorganic phosphorus dissolving bacteria is 30 ℃ and the optimum phosphorus dissolving temperature of the strain P8 is 35 ℃.

2.3 Effect of carbon sources on the phosphate solubilizing ability of phosphate solubilizing bacteria

Replacing glucose in the liquid culture medium of the organic phosphorus and the inorganic phosphorus with lactose, sucrose, soluble starch and mannitol with the same carbon content, subpackaging in 150mL triangular bottles, sterilizing at 121 ℃ for 20min by 30mL each bottle, measuring the effective phosphorus content in the bacterial liquid after culturing for 7d under the conditions that the inoculation amount in the culture medium with different carbon sources and the shake culture conditions are the same as 2.1, and measuring the result as shown in the graph 6 and the graph 7.

As can be seen from fig. 6, the different carbon sources have significant effects on the phosphorus-solubilizing abilities of 4 organophosphorus bacteria, and among 5 different carbon sources, the phosphorus-solubilizing abilities of 4 organophosphorus bacteria are: p5: glucose > sucrose > lactose > soluble starch > mannitol, P8: glucose > lactose > sucrose > soluble starch > mannitol, P1: sucrose > glucose > lactose > soluble starch > mannitol, P7: glucose, sucrose, lactose, soluble starch and mannitol, and on the whole, the phosphate-solubilizing effect of 4 strains is better when glucose, sucrose and lactose are used, the phosphate-solubilizing effect is poorer when the soluble starch and the mannitol are used as carbon sources, the phosphate-solubilizing amounts of the strains P5, P8 and P7 are the highest when the glucose is used as the carbon source and are respectively 33.39mg/L, 39.53mg/L and 41.58mg/L, and the phosphate-solubilizing amount of the strain P1 is 23.64mg/L when the sucrose is used as the carbon source, so that the strains P5, P8 and P7 select glucose, and the strain P1 selects sucrose as the optimal carbon source.

As can be seen from FIG. 7, the phosphate solubilizing abilities of 4 inorganic phosphate solubilizing bacteria under different carbon source conditions are different, and among 5 different carbon sources, the phosphate solubilizing abilities of 4 inorganic phosphate solubilizing bacteria are as follows: p30: sucrose > glucose > lactose > soluble starch > mannitol, P12: lactose > glucose > sucrose > soluble starch > mannitol, P8: glucose > lactose > sucrose > soluble starch > mannitol, P4: lactose, glucose, sucrose, soluble starch and mannitol, although the phosphate-solubilizing abilities of the 4 strains are different under different carbon sources, the graph shows that the phosphate-solubilizing effect of the 4 strains is better when the glucose, sucrose and lactose are used as carbon sources, the phosphate-solubilizing quantity is the lowest when the mannitol is used as a carbon source, and the law is the same as that of organophosphorus bacteria, so that the utilization of carbon sources by both organophosphorus bacteria and inorganic phosphorus bacteria is mainly monosaccharide and double ponds, and the utilization efficiency of polysaccharide is lower. The maximum phosphate-dissolving amount of the strain P30 is 651.10mg/L when sucrose is used as a carbon source, the maximum phosphate-dissolving amounts of the strains P12 and P4 are 489.39mg/L and 500.98g/L respectively when lactose is used as a carbon source, the maximum phosphate-dissolving amount of the strain P8 is 559.78mg/L when glucose is used as a carbon source, and therefore the optimum carbon source of the strain P30 is sucrose, the optimum carbon sources of the strains P12 and P4 are lactose, and the optimum carbon source of the strain P8 is glucose.

2.4 Effect of Nitrogen Source on phosphate solubilizing ability of phosphate solubilizing bacteria

Replacing ammonium sulfate in liquid culture medium of organic phosphorus and inorganic phosphorus with urea, potassium nitrate, sodium nitrate and ammonium oxalate with equal nitrogen content, subpackaging in 150mL triangular bottles, 30mL each, and sterilizing at 121 deg.C for 20 min. The inoculum size and shake culture conditions in different nitrogen source media were the same as 2.1, and the available phosphorus content in the bacterial liquid was determined after 7 days of culture, the results are shown in FIG. 8 and FIG. 9.

As can be seen from fig. 8, different nitrogen sources significantly affect the phosphate solubilizing ability of the strains, and of the 5 different nitrogen sources, the phosphate solubilizing ability of 4 strains of organic bacteria is as follows: p5: ammonium sulfate > ammonium oxalate > urea > sodium nitrate > potassium nitrate, P8: ammonium sulfate > ammonium oxalate > urea > sodium nitrate > potassium nitrate, P1: potassium nitrate > ammonium sulfate > ammonium oxalate > sodium nitrate > urea, P7: ammonium sulfate > ammonium oxalate > urea > potassium nitrate > sodium nitrate, the maximum phosphate-solubilizing amounts of all the strains P5, P8 and P7 are respectively 33.39mg/L, 39.53mg/L and 41.58mg/L when ammonium sulfate is used as a nitrogen source and are significantly higher than those of other nitrogen sources, the maximum phosphate-solubilizing amounts of all the strains P1 are 32.98mg/L when potassium nitrate is used as a nitrogen source, the minimum phosphate-solubilizing amounts of all the strains P5 are 10.56mg/L when potassium nitrate is used as a nitrogen source, the minimum phosphate-solubilizing amounts of all the strains P8 and P7 are respectively 16.61mg/L and 19.09mg/L when sodium nitrate is used as a nitrogen source, the minimum phosphate-solubilizing amounts of all the strains P1 are 6.16mg/L when urea is used as a nitrogen source, as can be obviously seen from the figure, ammonium nitrate and nitrate have different degrees of influence on the phosphate-solubilizing abilities of the strains, and the strains P5, P8 and P7 have poor effects when ammonium sulfate is used as a nitrogen source, the bacterial strain P1 has better phosphate-solubilizing effect when potassium nitrate and ammonium sulfate are used as nitrogen sources, which shows that the bacterial strains P5, P8 and P7 have better phosphate-solubilizing effect than urea and nitrate nitrogen when ammonium nitrogen is used as the nitrogen source, and the bacterial strain P1 can play better phosphate-solubilizing ability in ammonium nitrogen and nitrate nitrogen, particularly nitrate nitrogen, and has more outstanding effect. The most suitable nitrogen source for strains P5, P8 and P7 is therefore ammonium sulphate and the most suitable nitrogen source for strain P1 is potassium nitrate.

As can be seen from fig. 9, different nitrogen sources significantly affect the phosphate solubilizing ability of 4 inorganic phosphate solubilizing bacteria, and among 5 different nitrogen sources, the phosphate solubilizing ability of 4 inorganic phosphate solubilizing bacteria is as follows: p30: ammonium sulfate > potassium nitrate > sodium nitrate > ammonium oxalate > urea, P12: ammonium oxalate > ammonium sulfate > urea > potassium nitrate > sodium nitrate, P8: ammonium sulfate > ammonium oxalate > urea > potassium nitrate > sodium nitrate, P4: ammonium oxalate > ammonium sulfate > urea > sodium nitrate > potassium nitrate, the highest phosphate-solubilizing amount of the strain P30 is 598.89mg/L when ammonium sulfate is used as a nitrogen source, the phosphate-solubilizing amount of the strain P is 556.82mg/L and is slightly lower than that of the strain P in ammonium sulfate, the highest phosphate-solubilizing amounts of the strains P12 and P4 are 461.45mg/L and 477.37mg/L respectively when ammonium oxalate is used as a nitrogen source, and the highest phosphate-solubilizing amount of the strain P8 is 559.78mg/L when ammonium sulfate is used as a nitrogen source. It can be seen from the figure that all the strains P12, P8 and P4 have better phosphate-solubilizing effect by using ammonium sulfate and ammonium oxalate as nitrogen sources, the next to urea as nitrogen sources and the lowest phosphate-solubilizing quantity by using potassium nitrate and sodium nitrate as nitrogen sources, which indicates that the strains P12, P8 and P4 have the best phosphate-solubilizing effect by using ammonium nitrogen as nitrogen sources, the strain P30 has better phosphate-solubilizing effect by using ammonium sulfate and potassium nitrate as nitrogen sources, and the strain P30 can better utilize both nitrate nitrogen and ammonium nitrogen, but the phosphate-solubilizing quantity in ammonium sulfate is slightly higher than that in potassium nitrate, and the lowest available phosphorus content in urea. In conclusion, the optimum nitrogen source of the strains P30 and P8 is ammonium sulfate, the optimum nitrogen source of the strains P12 and P4 is ammonium oxalate, and the phosphate-solubilizing effect of the 4 strains is better when ammonium nitrogen is used as the nitrogen source.

2.5 influence of C/N ratio on phosphate solubilizing ability of phosphate solubilizing bacteria

Glucose and ammonium sulfate in an organic phosphorus and inorganic phosphorus liquid culture medium are used as a carbon source and a nitrogen source, the C/N ratio is adjusted to 40:1, 20:1 and 8:1, the mixture is subpackaged in 150mL triangular bottles, each bottle is 30mL, and the mixture is sterilized at the high temperature of 121 ℃ for 20 min. The inoculum size and shake culture conditions in different C/N media were the same as 2.1, and the effective phosphorus content in the broth was determined after 7 days of culture, the results are shown in FIG. 10 and FIG. 11.

As can be seen from FIG. 10, the phosphorus-solubilizing abilities of the strain P5 under the conditions of C/N of 20:1 and 40:1 are significantly higher than those of the strain C/N of 8:1, and the phosphorus-solubilizing amounts of 34.52mg/L under the conditions of C/N of 40:1 are slightly higher than those of 20:1, but the differences between the two are not significant, which indicates that the strain P5 has better phosphorus-solubilizing abilities under the conditions of C/N of 20:1 and 40:1, but the optimal C/N is 40: 1. The phosphorus-dissolving amount of the strain P8 is obviously increased along with the increase of C/N, the phosphorus-dissolving capacity of the strain P8 is the strongest when the C/N is 40:1, and the phosphorus-dissolving amount is 45.94mg/L, which is equivalent to 1.74 times when the C/N is 8: 1. The phosphorus-dissolving amount of the strain P1 is 33.08mg/L at most when the C/N is 20:1, is obviously higher than that under the conditions that the C/N is 8:1 and 40:1, is 1.61 and 1.73 times of that under the conditions that the C/N is 8:1 and 40:1 respectively, and the difference of the phosphorus-dissolving amounts of the strain P1 at 8:1 and 40:1 is not obvious. The phosphorus-dissolving amount of the strain P7 is 50.21mg/L at the maximum when the C/N is 40:1, which is obviously higher than the phosphorus-dissolving amounts when the C/N is 8:1 and 20:1, and the difference of the phosphorus-dissolving amounts of the strains at 8:1 and 20:1 is not obvious. As can be seen from the above, the optimum C/N ratios of the strains P5, P8 and P7 were all 40:1, and the optimum C/N ratio of the strain P1 was 20: 1.

As can be seen from FIG. 11, the phosphorus-solubilizing amounts of the strains P30, P12 and P4 were the highest at a C/N ratio of 20:1, 581.89mg/L, 493.71mg/L and 501.44mg/L, respectively, and then 40:1, the phosphorus-solubilizing amounts of the 3 strains were the lowest at a C/N ratio of 8:1, and the phosphorus-solubilizing amounts of the 3 strains were 2.07 times, 2.42 times and 2.28 times, respectively, at a C/N ratio of 20: 1. The phosphorus-dissolving amount of the strain P8 is 532.61mg/L at the highest when the C/N is 40:1, then 20:1, the phosphorus-dissolving amount is the lowest when the C/N is 8:1, and the phosphorus-dissolving amount of the strain P8 is 2.36 times of that of the strain P20: 1 when the C/N is 40:1 and 20:1, so that the 4 strains have better phosphorus-dissolving capacity when the C/N is 40:1 and 20:1, wherein the optimal C/N of the strains P30, P12 and P4 is 20:1, the optimal C/N of the strain P8 is 40:1, and the phosphorus-dissolving amount is the lowest when the C/N is 8:1, which indicates that the culture condition with the C/N of 8:1 is not favorable for the strains to exert the phosphorus-dissolving capacity.

2.6 Effect of salt concentration on the phosphate solubilizing ability of phosphate solubilizing bacteria

Preparing liquid culture medium of organic phosphorus and inorganic phosphorus with salt concentration of 0, 1%, 2%, 3%, 4% and 5%, subpackaging in 150mL triangular bottles, 30mL each, and sterilizing at 121 deg.C for 20 min. The inoculum size and shake culture conditions in the culture medium with different salt concentrations were the same as 2.1, and the effective phosphorus content in the bacterial solution was determined after 7 days of culture, the results are shown in FIG. 12 and FIG. 13.

As can be seen from fig. 12, different salt concentrations have a significant effect on the phosphorus-dissolving capacity of 4 organophosphorus-decomposing bacteria, and the phosphorus-dissolving capacity of 4 strains is significantly reduced when the salt concentrations are 5% and 10%, which indicates that the phosphorus-dissolving capacity of the strains is significantly inhibited when the salt concentration is too high, in different salt concentrations, the strains P5, P8 and P1 show the same rule, the phosphorus-dissolving amounts are 28.43mg/L, 34.58mg/L and 27.27mg/L at the highest level of 1.5% salt concentration, and are significantly higher than the phosphorus-dissolving amounts treated by other salt concentrations, the phosphorus-dissolving amounts of the strains are not significantly different when the salt concentrations are 0.5% and 1.5%, and the phosphorus-dissolving amounts of the 3 strains are 1.16mg/L, 1.02mg/L and 1.13mg/L at the lowest level of 10%. The strain P7 is different from other 3 strains, the phosphorus-solubilizing quantity of P7 is 40.63mg/L which is obviously higher when the salt concentration is 2.5% than that of the strain treated by other salt concentrations, and the phosphorus-solubilizing quantity is at least 1.25mg/L when the salt concentration is 10%, so that the optimal salt concentration of the strains P5, P8 and P1 is 1.5%, and the optimal salt concentration of the strain P7 is 2.5%.

As can be seen from fig. 13, when the salt concentration reaches 2.5%, the 4 strains of bacterial liquid still have a higher effective phosphorus content, which indicates that the 4 strains of bacterial liquid still have a better salt tolerance in a salt concentration of 2.5%, and when the salt concentration continues to rise to 5%, the strain P30 still has a higher phosphorus-solubilizing ability, the effective phosphorus content in the bacterial liquid is 117.79mg/L, and the effective phosphorus contents in the strains P12, P8, and P4 bacterial liquids are significantly reduced to 11.12mg/L, 16.76mg/L, and 13.36mg/L, respectively, which indicates that the strain P30 exhibits a strong salt tolerance compared with the other 3 strains, and when the salt concentration reaches 10%, the effective phosphorus contents of the 4 strains are all reduced to the minimum, so that the strains have a certain optimal salt concentration range, and NaCl with an excessively high concentration can significantly inhibit the phosphorus-solubilizing ability of the strains. As can be seen from the figure, the optimal salt concentration ranges of the strains P12, P8 and P4 are all 0-2.5%, wherein the phosphorus-dissolving capacity of the strains P12 and P4 is strongest when the salt concentration is 0.5 percent, the effective phosphorus content in the bacterial liquid is 3235.35mg/L and 349.15mg/L respectively, which shows that the optimum salt concentration of the strains P12 and P4 is 0.5 percent, the phosphorus-dissolving amount of the two strains when the salt concentration is 0.5 percent is 79.06 times of the phosphorus-dissolving amount when the salt concentration is 10 percent, the phosphorus-dissolving capacity of the strain P8 is strongest when the salt concentration is 1.5 percent, the phosphorus-dissolving amount is 400.70mg/L, which is 66.67 times of the phosphorus-dissolving amount when the salt concentration is 10 percent, which shows that the optimum salt concentration of the strain P8 is 1.5 percent, and the optimum salt concentration range of the strain P30 is 0-5 percent, the phosphorus-dissolving capacity of the P30 strain is strongest when the salt concentration is 1.5%, the effective phosphorus content in the bacterial liquid is up to 450.33mg/L, which is 47.40 times of that when the salt concentration is 10%, and the optimal salt concentration of the strain P30 is 1.5%. As a result, the optimum salt concentration of the strain P30 was 1.5%, the optimum salt concentrations of the strains P12 and P4 were 0.5%, and the optimum salt concentration of the strain P8 was 1.5%.

Example 3 the phosphate solubilizing mechanism of the phosphate solubilizing bacteria in the rhizosphere of the Tamarindus indica

3.1 test methods

3.1.1 measurement of phosphate solubilizing ability of phosphate solubilizing bacteria and pH of bacterial liquid

Activating a test strain on a beef extract peptone solid culture medium at 30 ℃ for 24h, inoculating a ring of activated thalli into 30mL LB liquid culture medium, and culturing for 24h in a shaking table at the rotating speed of 180r/min and the temperature of 30 ℃. 1mL of seed liquid of each strain cultured overnight is sucked and inoculated into 30mL of organic phosphorus and inorganic phosphorus liquid culture medium respectively (organic phosphorus bacteria are inoculated into the organic phosphorus liquid culture medium, inorganic phosphorus bacteria are inoculated into the inorganic phosphorus liquid culture medium), 3 times of treatment are repeated, the strain is subjected to shaking culture at 30 ℃ and 180r/min for 7d, the soluble phosphorus content and the pH value in the strain liquid are detected regularly every day, the effective phosphorus content is measured by adopting a molybdenum-antimony colorimetric method, the pH value is measured by using a pH meter, and the effective phosphorus content and the pH value of the strain liquid in the strain liquid every day are recorded.

3.1.2 determination of the type and content of organic acids secreted by phosphorus-solubilizing bacteria

Respectively inoculating inorganic phosphorus-decomposing bacteria into an inorganic phosphorus liquid culture medium, culturing for 7d by a shaking table, centrifuging the culture solution after 7d, wherein the centrifugation condition is 8000r/min, the centrifugation time is 15min, reserving the supernatant after centrifugation, filtering the collected supernatant and each organic acid standard substance by using a filter membrane of 0.22 mu m, removing impurities, performing ultrasonic degassing for 10min, and then performing high performance liquid chromatography analysis. Setting the same chromatographic condition as that of standard organic acid measurement, comparing the peak time of each organic acid in the bacterial liquid with the standard organic acid according to the rule to determine the type of the organic acid in the bacterial liquid, and comparing the standard curve between the standard organic acid concentration and the peak area according to the peak area of each organic acid in the bacterial liquid to determine the content of each organic acid in the bacterial liquid.

3.1.3 determination of Phosphomonoesterase Activity secreted by phosphate-solubilizing bacteria

Inoculating organophosphorus bacteria into an organophosphorus liquid culture medium, culturing for 7d in a shaking way, sucking 1mL of supernatant after 7d, adding 4mL of buffer solution with pH of 6.5, then adding 1mL of 0.025mol/L p-nitrophenyl disodium phosphate solution and 4mL of 0.5mol/L sodium hydroxide, uniformly mixing the above solutions, centrifuging for 15min at 8000r/min, detecting the light absorption value of the supernatant at 420nm, and determining alkaline phosphatase by changing the buffer solution with pH of 6.5 into the buffer solution with pH of 11. Calculation of enzyme Activity disodium phenylphosphate was catalyzed to produce 0.1mg phenol as 1 phosphatase activity unit in 1mL of culture medium for 12 h.

3.1.4 determination of Phytase Activity secreted by phosphate-solubilizing bacteria

The amount of enzyme required to release 1umol of soluble phosphorus from a 5mmol/L sodium phytate solution within 1min at 37 ℃ and pH5.5 is defined as one unit of enzyme activity.

Absorbing 1mL of organophosphorus bacteria liquid cultured for 7d, centrifuging for 10min at 4000r/min, diluting the centrifuged supernatant, absorbing 1mL of sodium phytate solution, adding the sodium phytate solution into a 25mL volumetric flask, adding the diluted bacteria liquid into the volumetric flask, adding 1mL of trichloroacetic acid into the diluted bacteria liquid in a contrast manner, reacting for 30min at 30 ℃, adding TCA after 30min to terminate the reaction, detecting the light absorption value of the bacteria liquid at 710nm after constant volume, and simultaneously calculating the content of soluble phosphorus in the bacteria liquid, wherein the calculation formula of the phytase activity is as follows:

wherein, N: the dilution times of the bacterial liquid; pi: diluting the soluble phosphorus content mg/L in the sample; p0: the soluble phosphorus content of the diluted sample control is mg/L.

3.2 results

(1) The results are shown in fig. 14-19, and it can be seen from the graphs that among the 4 organophosphorus degrading bacteria, the phosphorus-solubilizing abilities of the strains P1 and P5 are strongest when the strains are cultured for 72 h-96 h, the phosphorus-solubilizing abilities of the strains P7 and P8 are strongest when the strains are cultured for 48 h-72 h, the effective phosphorus content of the 4 strains is highest in the bacterial liquid cultured for 120h, the phosphorus-solubilizing amounts of the strains P7 are highest in the whole culture process, and the phosphorus-solubilizing amounts of the 4 strains and the pH of the bacterial liquid have no significant correlation. The phosphorus-dissolving capacity of the 4 inorganic phosphorus-dissolving bacteria is strongest when the bacteria are cultured for 48-72 h, the phosphorus-dissolving amount of the strain P30 is highest when the bacteria are cultured for 144h, the phosphorus-dissolving amounts of the strains P12, P8 and P4 are highest when the bacteria are cultured for 120h, the phosphorus-dissolving amount of the strain P30 is highest in the whole culture process, and the phosphorus-dissolving amount of the 4 inorganic phosphorus-dissolving bacteria is in extremely obvious negative correlation with the pH value of a bacterial liquid.

(2) The results are shown in fig. 20, and it can be seen that the phosphate solubilizing amounts of the strains P30, P8 and P4 in the three media are shown as calcium phosphate > iron phosphate > aluminum phosphate, and the dissolving amount of the strain P12 is shown as iron phosphate > aluminum phosphate > calcium phosphate. Compared with the phosphorus dissolving amount of 4 strains, the phosphorus dissolving amount of the 4 strains in a calcium phosphate culture medium and an iron phosphate culture medium is P30> P8> P4> P12 in sequence, and the phosphorus dissolving amount of the 4 strains in the aluminum phosphate culture medium is P30> P12> P8> P4.

(3) The results are shown in tables 6 to 8.

TABLE 6 type and content of organic acids secreted by inorganic phosphorus-decomposing bacteria in calcium phosphate medium

Organic acid species	P30	P12	P8	P4
					Oxalic acid	399.27±5.77a	119.07±0.28c	168.98±0.01b	96.01±0.01d
Tartaric acid	603.22±0.58a	135.32±0.58b	102.65±0.06c	79.97±0.01d
					Malic acid	80.85±0.01	-	60.07±0.01	-
Lactic acid	4287.96±5.20a	413.61±6.35c	1169.71±58.3b	392.13±6.35c
					Acetic Acid (AA)	1863.88±0.58	-	-	119.52±1.15
Citric acid	174.66±0.58	-	-	551.84±17.32
					Succinic acid	2145.63±0.52a	-	1056.03±9.24b	947.15±6.35c
Propionic acid	212.92±5.19a	135.23±8.66b	197.76±8.76a	-
					Glutaric acid	182.08±6.35b	1372.64±6.35a	179.75±0.58b	-
Total amount of organic acids	9950.50±5.12a	2175.88±22.52c	2934.95±59.55b	2186.61±31.19c

Note: the data in the table are mean values ± sd, different lower case letters indicate that the difference between the different strains is significant at the 0.05 level (P <0.05), "-" indicates that no corresponding organic acid was detected in the bacterial solution, the same applies below.

TABLE 7 species and content of organic acids secreted by inorganic phosphorus-decomposing bacteria in aluminum phosphate medium

TABLE 8 kinds and contents of organic acids secreted by inorganic phosphorus-decomposing bacteria in ferric phosphate medium

Organic acid species	P30	P12	P8	P4
					Oxalic acid	3602.58±2.31a	445.57±0.58c	863.67±17.32b	150.78±5.78d
Tartaric acid	-	336.09±4.62b	31.56±1.15c	469.07±6.93a
					Malic acid	345.49±1.73	-	-	-
Lactic acid	-	-	-	411.45±5.77
					Acetic acid	-	924.16±4.62	266.12±2.89	-
Citric acid	-	52.34±0.58	169.89±1.15	-
					Succinic acid	-	-	1109.29±0.58	350.85±2.89
Propionic acid	2448.65±1.15	-	-	-
					Glutaric acid	134.45±17.32	2560.29±3.46	-	-
Total amount of organic acid	6556.17±14.43a	4318.46±11.55b	2440.54±13.86c	1382.15±4.04d

As can be seen from the table, the total content of organic acids secreted by the strains in calcium phosphate culture medium is in the order of P30> P8> P4> P12, the total content of organic acids secreted by the strains in aluminum phosphate culture medium is in the order of P30> P12> P8> P4, the total content of organic acids secreted by the strains in iron phosphate culture medium is in the order of P30> P12> P8> P4, and besides ferric phosphate, the phosphate solubilizing quantity of the strains in other two culture media corresponds to the total content of organic acids secreted by the strains, which indicates that the production of organic acids is an important factor for determining the phosphate solubilizing quantity of the strains, and the total amount of organic acids secreted by the strains P30 in calcium phosphate, aluminum phosphate and ferric phosphate is the highest and is 9950.50mg/L, 4855.36mg/L and 6556.17mg/L respectively. In a calcium phosphate culture medium, 9 organic acids are detected in bacterial liquid of a strain P30, wherein the content of lactic acid is the highest, 5 organic acids are detected by a strain P12, the content of glutaric acid is the highest, 7 organic acids are detected by a strain P8, the content of lactic acid is the highest, 6 organic acids are detected by a strain P4, and the content of succinic acid is the highest. In an aluminum phosphate culture medium, a strain P30 produces 6 organic acids, wherein the oxalic acid content is highest, a strain P12 produces 4 organic acids, the propionic acid content is highest, and strains P8 and P4 secrete 5 organic acids and 4 organic acids respectively, and the succinic acid content is highest. In an iron phosphate culture medium, the oxalic acid content of 4 organic acids generated by a strain P30 is highest, the glutaric acid content of 5 organic acids generated by a strain P12 is highest, the succinic acid content of 5 organic acids generated by a strain P8 is highest, and the tartaric acid content of 4 organic acids generated by a strain P4 is highest.

(4) The results are shown in fig. 21-23, and as can be seen from the graphs, 4 organophosphorus-solubilizing bacteria can secrete phosphatase and phytase in the process of dissolving organophosphorus, wherein the activities of acid phosphatase and alkaline phosphatase secreted by strains P7 and P8 are significantly higher than those of P5 and P1, the activity of acid phosphatase secreted by strain P7 is 16.61U/mL, the activity of alkaline phosphatase is 11.26U/mL, the activity of acid phosphatase secreted by strain P8 is 15.08U/mL, the activity of alkaline phosphatase is 10.83U/mL, the activity of phytase secreted by strain P7 is significantly higher than that of other strains, the phytase activity in a bacterial liquid is 0.80U/mL, and the amount of organophosphorus dissolved by the strains is substantially in a positive correlation with the enzyme activity in the bacterial liquid as a whole.

Example 4 study of the ability of the Phosphorocarpus sp.rhizosphere of the Tamarindus to secrete phytohormones and siderophores

4.1 test methods

Activating the test strains on a beef extract peptone agar slant culture medium at 30 ℃ for 24h, selecting partial strains with toothpicks, inoculating the strains into 30mL LB liquid culture medium, and performing shake culture at the rotation speed of 180r/min and the temperature of 30 ℃ for 24h to prepare a seed solution.

Respectively taking 1mL of seed liquid obtained by culturing organophosphorus and inorganic phosphorus bacteria overnight, inoculating the seed liquid into organophosphorus and inorganic phosphorus liquid culture medium, and carrying out shake culture at 30 ℃ for 7d at 180 r/min. And then, centrifuging the bacterial liquid at 4000r/min for 20min, mixing the centrifuged supernatant with a Salkowsk colorimetric solution in equal volume, reacting for 30min in a dark place, and measuring the light absorption value at 540nm to obtain the IAA content.

Respectively taking 1mL of seed liquid obtained by decomposing bacteria of the organic phosphorus and the inorganic phosphorus to culture overnight, inoculating the seed liquid into liquid culture media of the organic phosphorus and the inorganic phosphorus, carrying out shaking culture for 7d at the temperature of 30 ℃ at 180r/min, centrifuging the seed liquid after 7d, taking 1mL of test solution, measuring the light absorption value at 412nm, and calculating the gibberellin content of the test solution.

Activating the test strain on beef extract peptone agar slant culture medium at 30 deg.C for 24 hr, and picking out partial bacteria with toothpick

Inoculating the strain into 30mL LB liquid culture medium, oscillating overnight at the rotation speed of 180r/min and the temperature of 30 ℃ to enable the bacterial liquid OD600 to be 1, inoculating 1mL bacterial liquid into the MKB liquid culture medium, culturing for 48h at the temperature of 30 ℃ and 180r/min in a shaking table, centrifuging the bacterial liquid for 10min at 5000r/min after 48h, sucking the centrifuged bacterial liquid, uniformly mixing the centrifuged bacterial liquid with 3mL CAS detection liquid respectively, reacting for 1h, measuring the absorbance at the position of 630nm, taking the MKB liquid culture medium without the inoculated bacterial liquid as a reference, and adopting an iron carrier calculation formula as follows:

U(％)＝(Ar-As)/Ar×100

wherein, U: activity of a siderophore; as: absorbance of the mixture at 630 nm; ar: absorbance of the MKB liquid medium from the non-inoculated solution.

4.2 results

The results are shown in FIGS. 24 to 26. As can be seen from FIG. 24, all the 4 organophosphorus bacteria have the capability of secreting IAA, the IAA secretion amount is between 61.57mg/L and 100.75mg/L, and the IAA secretion content of the 4 organophosphorus bacteria is P7, P8, P5 and P1 in the sequence. The highest secretion amount of the strain P7 IAA is 100.75mg/L, which is obviously higher than that of other strains, and then the strain P8, the IAA content secreted by the strains P5 and P1 is lower, the difference between the two strains is not obvious, and the secretion amount of the strain P7 IAA is respectively 30.19%, 8.89% and 38.90% higher than that of the strains P5, P8 and P1. The 4 strains of inorganic phosphorus-decomposing bacteria also have the capability of secreting IAA, and the IAA content of the 4 strains of inorganic phosphorus-decomposing bacteria is P30> P8> P12> P4 in the sequence. The highest IAA secretion of the strain P30 is 103.27mg/L, the next strain P8 is 91.80mg/L, and the lowest IAA secretion of the strain P4 is 22.47 mg/L. The IAA secretion amount of the strain P30 is 33.66 percent, 78.24 percent and 11.1 percent higher than that of the strains P12, P8 and P4 respectively, and the IAA secretion amount of the strains 4 is obviously different.

As can be seen from FIG. 25, all 4 organophosphorus degrading bacteria can secrete gibberellin, the gibberellin secretion amount of the bacteria is 19.60 mg/L-57.20 mg/L, the gibberellin content of the 4 strains is P8> P7> P5> P1 in the sequence, wherein the gibberellin secretion amounts of the strains P7 and P8 are higher and are respectively 52.99mg/L and 57.20mg/L, the difference between the gibberellin secretion amounts is not significant, the gibberellin secretion amount of the strains P5 is 32.86mg/L, the gibberellin secretion amount of the strains P1 is 19.60mg/L, the gibberellin secretion amount of the strains P7 is 37.9% and 63.0% higher than that of P5 and P1, the gibberellin secretion amount of the strains P8 is 42.6% and 65.7% higher than that of P5 and P1, and the gibberellin secretion amounts of the strains P8 and P7 in the 4 organophosphorus degrading bacteria are stronger. The gibberellin secretion amounts of the 4 inorganic phosphorus bacteria are remarkably different, the gibberellin content of the 4 strains is P30> P8> P12> P4 in the sequence, the gibberellin secretion amounts of the strains P30 and P8 are 57.65mg/L and 55.28mg/L respectively and are remarkably higher than those of the other 2 strains, the gibberellin secretion amounts of the strains P12 and P4 are 23.17mg/L and 17.59mg/L respectively, the gibberellin secretion amount of the strain P30 is 59.8% higher than that of the strains P12 and P4 and is 69.5% higher than that of the strains P8 and is 58.1% higher than that of the strains P12 and P4 and is 68.2% higher than that of the strains P12 and P4 respectively. In conclusion, the strains P30 and P8 in the 4 strains have stronger gibberellin secretion capability.

From fig. 26, the sizes of the siderophore secretion activities of the 4 strains are P7> P8> P5> P1, the siderophore activities of P7 and P8 are 59.32% and 61.47%, respectively, the siderophore activities of the 4 strains are significantly higher than those of the P5 and P1, the siderophore activities of the P5 and the P1 are lower than those of the P5 and P1, respectively are 24.14% and 24.04%, and the siderophore activities of the two strains are not significantly different, so that the siderophore secretion abilities of the P7 and the P8 in the 4 strains of the organophosphorus bacteria are stronger. The activity difference of the iron carrier secreted by the 4 inorganic phosphorus-decomposing bacteria is remarkable, the order of the activity of the iron carrier secreted by the 4 inorganic phosphorus-decomposing bacteria is P8, P30, P4 and P12, the strongest activity of the iron carrier in bacterial liquid of the strain P8 is 59.58 percent, the second activity of the iron carrier is P30, the activity of the iron carrier is 48.28 percent, the lower activities of the iron carriers of the strains P4 and P12 are 24.14 percent and 17.16 percent respectively, and in conclusion, the iron carrier secreted by the strain P8 in the 4 inorganic phosphorus-decomposing bacteria is strongest.

In the test, the screened phosphate solubilizing strains are analyzed for hormone secretion capacity, compared with 4 organophosphorus solubilizing bacteria, the IAA content and the siderophore activity generated by the strain P7 are highest and are respectively 100.75mg/L and 61.47%, and the gibberellin content secreted by the strain P8 is highest and is 57.20 mg/L. The IAA and gibberellin secreted by the strain P30 in the 4 inorganic phosphorus decomposing bacteria have the highest contents of 103.27mg/L and 57.65mg/L respectively, and the highest activity of the siderophore in the bacterial liquid of the strain P8 is 59.58%. The strain screened by the invention has better capability of secreting IAA, gibberellin and siderophores, and the strain prepared into the microbial fertilizer can well solve the problems of low effective phosphorus content of soil, slow development of the root system of the seedling of the strelitzia tree and slow growth of the seedling when applied to the soil.

The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Sequence listing

<110> Guangxi university of forest park in south Ning Liangfengjiang national of the Guangxi Zhuang nationality autonomous region

<120> bacillus cereus P1 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1449

<212> DNA

<213> Bacillus cereus (Bacillus cereus)

<400> 1

catcgtcact taggcggctg gctccaaaaa ggttacccca ccgacttcgg gtgttacaaa 60

ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120

gatccgcgat tactagcgat tccagcttca tgtaggcgag ttgcagccta caatccgaac 180

tgagaacggt tttatgagat tagctccacc tcgcggtctt gcagctcttt gtaccgtcca 240

ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct 300

tcctccggtt tgtcaccggc agtcacctta gagtgcccaa cttaatgatg gcaactaaga 360

tcaagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa 420

ccatgcacca cctgtcactc tgctcccgaa ggagaagccc tatctctagg gttttcagag 480

gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540

ttgtgcgggc ccccgtcaat tcctttgagt ttcagccttg cggccgtact ccccaggcgg 600

agtgcttaat gcgttaactt cagcactaaa gggcggaaac cctctaacac ttagcactca 660

tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcgcc 720

tcagtgtcag ttacagacca gaaagtcgcc ttcgccactg gtgttcctcc atatctctac 780

gcatttcacc gctacacatg gaattccact ttcctcttct gcactcaagt ctcccagttt 840

ccaatgaccc tccacggttg agccgtgggc tttcacatca gacttaagaa accacctgcg 900

cgcgctttac gcccaataat tccggataac gcttgccacc tacgtattac cgcggctgct 960

ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtgccagc ttattcaact 1020

agcacttgtt cttccctaac aacagagttt tacgacccga aagccttcat cactcacgcg 1080

gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140

agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200

gttgccttgg tgagccgtta cctcaccaac tagctaatgc gacgcgggtc catccataag 1260

tgacagccga agccgccttt caatttcgaa ccatgcagtt caaaatgtta tccggtatta 1320

gccccggttt cccggagtta tcccagtctt atgggcaggt tacccacgtg ttactcaccc 1380

gtccgccgct aacttcttga gagcaagctc tcaatccatc cgctcgactg catgtatagc 1440

cgccgcaca 1449

Claims (4)

1. A Bacillus cereus (Bacillus cereus) P1 is characterized in that it is preserved in China general microbiological culture Collection center, the preservation date is 2021 year, 07 months and 13 days, and the preservation registration number is CGMCC No. 22821.

2. The use of bacillus cereus P1 as claimed in claim 1, for converting organic phosphorus which is poorly soluble in soil into available phosphorus which can be absorbed by plants.

3. The use of bacillus cereus P1 as claimed in claim 1, for promoting the development of the root system of the seedlings and young trees of the stretchy trees.

4. A microbial inoculant comprising bacillus cereus P1 of claim 1.

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