CN114015623B - Burkholderia gladioli P8 and application thereof - Google Patents

Abstract

The invention discloses Burkholderia gladioli P8 and application thereof, belonging to the technical field of microorganisms, wherein the strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation date of 2021, 07, 13 days and the preservation registration number of CGMCC No.22825; the invention shows that the strain has the best effect of decomposing inorganic phosphorus when the temperature is 35 ℃, the pH is 7.5, the carbon source is glucose, the nitrogen source is ammonium sulfate, the C/N is 40 and the salt concentration is 1.5 percent, can secrete phosphatase and phytase, dissolve insoluble organic phosphorus and secrete phytohormone and iron carrier, 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 seedlings of the strelitzia.

Description

Burkholderia gladioli P8 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to Burkholderia gladioli P8 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 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 high, but the acid soil has a strong solidifying effect on phosphorus, so that most of 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 low, and only a small amount of effective phosphorus can be provided for plants. On average, most mineral nutrients in the soil solution are present in millimoles and phosphorus is present in micromoles or less, and when plants lack phosphorus, the plants suffer from slow root development, underdeveloped lateral roots, slow growth and weak resistance. 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 the 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 quassia tree (Parashorea chinensis Wang Hsie.) also called "Ongtian tree" is a kind of plants belonging to the genus Salix of the family Dipterocarpaceae, tropical rain forest marker species and endangered tree species, and is mainly used for protecting wild plants at the first national level, and Yunnan and Guangxi provinces are the main distribution areas. 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 growth of the tamarind tree is retarded, 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 of slow development and slow growth of the root system of the tamarind tree in the seedling stage and the young trees.

Disclosure of Invention

The invention aims to provide Burkholderia gladioli P8 and application thereof, so as to solve the problems in the prior art, the strain can effectively convert insoluble phosphorus into available phosphorus capable of being absorbed by plants, and can secrete plant hormones and siderophores to promote plant growth.

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

the invention provides Burkholderia gladioli P8, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC for short, the address is China academy of sciences institute of microbiology 3, north West Lu No.1 Hospital, chaoyang district, beijing), the preservation date is 2021 year, 07 month and 13 days, and the preservation registration number is CGMCC No.22825.

The invention also provides application of the Burkholderia gladioli P8 for converting indissolvable phosphorus in soil into available phosphorus capable of being absorbed by plants.

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

The invention also provides application of the Burkholderia gladioli P8 for the cultivation of the tamarind tree artificial forest.

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 Burkholderia gladioli P8.

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 inorganic phosphorus decomposition when the temperature is 35 ℃, the pH is 7.5, the carbon source is glucose, the nitrogen source is ammonium sulfate, the C/N is 40, and the salt concentration is 1.5%, can secrete phosphatase and phytase, dissolve insoluble organic phosphorus, secrete phytohormone and iron carrier, promote plant photosynthesis, respiration, substance and energy metabolism, and can be used for solving the problems of slow development of the root system of the seedling 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 influence of C/N values 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 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 the bacteria solution of inorganic phosphorus bacteria;

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

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

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

FIG. 21 shows the activity of degrading the acid phosphatase secreted by the organophosphorus bacteria;

FIG. 22 shows the activity of degrading the secretion of alkaline phosphatase by an organophosphorous bacterium;

FIG. 23 shows the degradation of phytase activity secreted by organophosphorus 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 rather as a more detailed description of certain aspects, features and embodiments of the invention.

Example 1 isolation, screening and identification of Phosphorus tabacum 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 organic phosphorus culture medium and a calcium phosphate inorganic phosphorus culture medium, 68 isolates are obtained, wherein 30 isolates can form obvious transparent rings on an organic phosphorus or inorganic phosphorus solid culture medium, the 30 strains are proved to have the capability of dissolving insoluble organic phosphorus or inorganic phosphorus, 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

As a result of determination of the ratio of the diameter (D) of the clearing zones to the colony diameter (D) of the organic and inorganic phosphorus solid medium into which 30 strains in total of P1 to P30 isolated as described above were inoculated, respectively, as shown in Table 1, in the organic phosphorus solid medium, 10 strains having D/D >2.0, P1, P2, P5, P7, P8, P14, P22, P27, P28, P30, which accounted for 33.3% of the total number of strains, indicating that the 10 strains had a strong ability to decompose organic phosphorus, 6 strains having D/D of 1.5< -2.0, 20% of the total number of strains, 14 strains having D/D < 1.0D/D <1.5, and 46.7% of the total number of strains, among the 30 organic phosphorus strains, the strain P14 had the largest D/D value on the organic phosphorus solid medium of 2.15, and the strain P23 had the smallest D/D value of 1.05.

In inorganic phosphorus medium, the D/D of different strains has obvious difference, 8 strains with D/D >2.0 are respectively P4, P8, P10, P12, P15, P23, P25 and P30, account for 26.7 percent of the total number of the strains, 8 strains with D/D <2.0 are restricted by 1.5, 26.7 percent of the total number of the strains, 14 strains with D/D <1.5 are restricted by 1.0, and 46.6 percent of the total number of the strains, in 30 strains, the D/D value of the strain P12 on the inorganic phosphorus medium is maximum 2.16, and the D/D value of the strain P13 is minimum 1.061.

TABLE 1 results of primary screening of phosphate-solubilizing bacteria

Note: D/D represents the diameter of the phosphate solubilizing circle of the phosphate solubilizing bacteria/the diameter of the phosphate solubilizing bacteria strain, the data in the table are mean values +/-standard errors, the same letter represents that the difference is not significant (P > 0.05) after the same column of numerical values, and different letters represent that the difference is significant (P < 0.05).

1.2.2 rescreening of phosphate solubilizing bacteria

In order to judge the phosphate-solubilizing ability of the strains more accurately, strains with stronger phosphate-solubilizing ability, namely 10 strains with D/D greater than 2.0 for decomposing organic phosphorus and 8 strains for decomposing inorganic phosphorus, which are obtained by primary screening are rescreened, 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 at 30 ℃ for 24h, inoculating a ring of activated thalli in an LB liquid culture medium, performing shaking culture at 30 ℃ for 24h 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 shaking culture of 7d and 7d, centrifuging the organic phosphorus culture solution at 12000r/min for 30min, performing inorganic phosphorus culture at 10000r/min for 15min, absorbing supernatant, measuring phosphorus content by using a molybdenum-antimony colorimetric method, and calculating phosphorus removal rate, wherein the result is 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 (6.38 mg/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 16.18mg/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 P7> P8> P5> P1> P28> P30> P2> P22> P27> P14 from large to small in sequence, the phosphorus-dissolving rate of 10 strains is between 1.96% and 7.04%, 4 strains with the phosphorus-dissolving rate larger 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 stock tree rhizosphere soil forested in 2019 years, the strains P5 and P7 are derived from the stock tree rhizosphere soil forested in 2018 years, and the strain P8 is derived from the stock tree rhizosphere soil forested 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 (21.44 mg/L), and the effective phosphorus content in the culture medium of different strains is significantly different. The effective phosphorus content in the culture solution of 8 inorganic phosphorus-decomposing bacteria is 305.33 mg/L-598.89 mg/L, and the phosphorus-decomposing rate is 5.68-11.55%. Compared with CK, the effective phosphorus increment is 283.89 mg/L-577.45 mg/L, and the amplification is 13.24-26.93 times of CK. The effective phosphorus content of each strain culture solution is P30, P8, P4, P12, P23, P15 and P10 from large to small in sequence, the phosphorus dissolving amount of the strains P4, P8, P12 and P30 is high, the effective phosphorus content of 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%, the phosphorus dissolving rate is more than 10%, the strains P4, P8, P12 and P30 are strong in inorganic phosphorus dissolving capacity, the strain P4 is from the stock tree root soil forested in 2019, the strains P8 and P12 are from stock tree root soil forested in 2017, the strain P30 is from stock tree root soil forested in 1987, and the 4 strains can be considered as key points for developing microbial fertilizers.

TABLE 2 organophosphorus bacteria rescreening

Year of afforestation	Strain number	Effective 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 bacterial rescreening of inorganic phosphorus

1.3 identification of phosphate-solubilizing bacteria

1.3.1 morphological and physiological-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 the cell morphology and colony characteristics of the 4 separated and screened strains P1, P5, P7, 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 the observation results of the 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 result shows that the strains P1 and P30 have the same characteristics, namely the strains can utilize citrate, can make gelatin hydrolysis and catalase reaction positive, can produce indole, can make starch hydrolysis and nitrate reduction, the strains P5, P7, P8, P4 and P12 have the same characteristics, can utilize citrate, can make gelatin hydrolysis and catalase reaction positive, can make nitrate reduction, and can preliminarily conclude that the strains P1 and P30 are possibly bacilli and the strains P5, P7, P8, P4 and P12 are possibly Burkholderia according to the morphological and physiological and biochemical characteristics of the strains.

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: + means positive, -means negative

In order to verify whether the guess of the strain according to morphological and physiological and biochemical characteristics is accurate or not, 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 high sequence similarity with Bacillus, strains P4, P5, P7, P8 and P12 have high sequence similarity with Burkholderia, and it can be seen from the phylogenetic tree of FIG. 1 that strains P5, P7 and P8 are located at the same branch as Burkholderia gladioli, strains P4 and P12 are located at the same branch as Burkholderia cepacia, and strains P1 and P30 are located at the same branch as Bacillus cereus and have close relationship, so that, by combining the morphological characteristics, physiological and biochemical characteristics and phylogenetic analysis results of the respective strains, strains P5, P7 and P8 are respectively identified as Burkholderia gladioli, strains P4 and P12 are respectively identified as Burkholderia cepacia (Burkholderia cepacia), and strains P1 and P30 are identified as Bacillus cereus (Bacillus cereus).

1.4 preservation of phosphate solubilizing bacteria

Burkholderia gladioli P7 is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of CGMCC, china academy of sciences, 3, west Lu No.1 Hospital, north Cheng, south China area, beijing) on 2021, 07.13 days, 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 phosphorus-solubilizing bacteria P1, P5, P7 and P8 and the inorganic phosphorus-solubilizing bacteria P4, P8, P12 and P30 screened out in the experiment of 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 phosphate solubilizing ability of phosphate solubilizing bacteria

Respectively preparing liquid culture media of organic phosphorus and inorganic phosphorus, 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 at the high temperature of 121 ℃ for 20min, wherein each bottle is 30mL. Respectively absorbing 1mL of organophosphorus bacteria seed solution to inoculate in organophosphorus liquid culture medium with different pH values, absorbing 1mL of inorganic phosphorus bacteria seed solution to inoculate in inorganic phosphorus liquid culture medium with different pH values, setting 3 times for each treatment, placing the culture solution in a shaking table at 180r/min and 30 ℃ for 7d and 7d, centrifuging the culture solution, absorbing supernatant, and measuring the effective phosphorus content in the culture solution, wherein the results are shown in figure 2 and figure 3.

As can be seen from FIG. 2, the phosphate-solubilizing abilities of the 4 organophosphorus bacteria are different under different pH conditions, the phosphate-solubilizing abilities of the 4 strains are lower 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 bacterial solutions of the strains tends to increase first and then decrease with the increase of the pH, the optimal pH range of the bacterial strain P5 is 6.0-7.0, the effective phosphorus content in the bacterial solution reaches up to 43.01mg/L when the pH is 6.5, which indicates that the phosphate-solubilizing ability of the bacterial strain P5 is stronger when the bacterial strain P5 is cultured in a slightly acidic environment. The optimum pH ranges of the strains P1, P7 and P8 are all 7.0-8.0, 3 strains have the best phosphate-solubilizing effect 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 show lower phosphate solubilizing abilities at pH 3.5, which indicates that the strains are not suitable for growth and performance of the phosphate solubilizing abilities under a strong acid environment, and the effective phosphorus content in the bacterial liquid of each strain tends to increase first and then decrease with the increase of 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 bacterial liquid of the strain P30 between pH4.5 and pH7.5 is not changed greatly, and the effective phosphorus content of the bacterial liquid is always very high, which shows that the strain P30 has stronger adaptability to the pH value of the environment and wider pH adaptation range. The optimum pH value of the strains P8 and P12 is 7.5, when the pH values of the two strains are 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 150mL triangular bottles, sterilizing at 121 ℃ for 20min, wherein each bottle is 30mL. 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 can be seen from FIG. 4, the effective phosphorus contents of the 4 organophosphorus bacteria in the bacterial solutions are different 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 removal 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 strains are 39.05mg/L, 42.40mg/L and 45.31mg/L respectively, the effective phosphorus contents of the strains are 30 ℃, the effective phosphorus contents of the strains are 33.39mg/L, 39.53mg/L and 41.58mg/L respectively, the phosphorus removal effect of the strain P1 is the best when the temperature of the strains is 30 ℃, the effective phosphorus contents of the strains are 33.08mg/L, and the effective phosphorus contents of the strains are 35 ℃ and 29.96mg/L respectively. 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 ℃, and is 548.53mg/L and 552.87mg/L respectively, the phosphorus-dissolving amounts of the strains P12 and P4 at 35 ℃ and 25 ℃ are 477.74mg/L and 500.98mg/L respectively, and the phosphorus-dissolving amounts at 25 ℃ are 437.71mg/L and 472.68mg/L respectively. The maximum content of available phosphorus in the bacterial liquid of the strain P30 at the temperature of 30 ℃ is 598.89mg/L, and the second content is 517.09mg/L at the temperature of 35 ℃. The maximum effective phosphorus content of the strain P8 in the bacterial liquid at 35 ℃ is 623.71mg/L, and the effective phosphorus content at 30 ℃ is 559.78mg/L. The effective phosphorus content of the bacterial liquid of the strains P12 and P4 is obviously higher than that of the respective strains 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 strains P30 and P8 is obviously higher than that of the respective strains at other temperatures at 30 ℃ and 35 ℃, 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 equal carbon content, subpackaging in a triangular flask with 150mL, sterilizing each flask at the high temperature of 121 ℃ for 20min, measuring the effective phosphorus content in the bacterial liquid after culturing for 7d, wherein the inoculation amount and shaking table culture conditions in the culture medium with different carbon sources are the same as 2.1, and the results are shown in fig. 6 and 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, wherein 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 the glucose, and the strain P1 selects the 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 phosphorus-dissolving amount of the strain P30 is 651.10mg/L at most when sucrose is used as a carbon source, the phosphorus-dissolving amounts of the strains P12 and P4 are 489.39mg/L and 500.98g/L respectively at most when lactose is used as the carbon source, the phosphorus-dissolving amount of the strain P8 is 559.78mg/L at most when glucose is used as the carbon source, and therefore the optimal carbon source of the strain P30 is sucrose, the optimal carbon sources of the strains P12 and P4 are lactose, and the optimal 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, sterilizing at 121 deg.C for 20min at 30mL each bottle. 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 and sodium nitrate, the maximum phosphate-solubilizing amounts of the strains P5, P8 and P7 are respectively 33.39mg/L, 39.53mg/L and 41.58mg/L when the ammonium sulfate is used as a nitrogen source and are obviously higher than those of other nitrogen sources, the maximum phosphate-solubilizing amount of the strain P1 is 32.98mg/L when the potassium nitrate is used as the nitrogen source, the minimum phosphate-solubilizing amount of the strain P5 is 10.56mg/L when the potassium nitrate is used as the nitrogen source, the minimum phosphate-solubilizing amounts of the strains P8 and P7 are respectively 16.61mg/L and 19.09mg/L when the sodium nitrate is used as the nitrogen source, the minimum phosphate-solubilizing amount of the strain P1 is 6.16mg/L when the urea is used as the nitrogen source, it can be clearly seen from the figure that ammonium nitrogen and nitrate nitrogen have different degrees of influence on the phosphate solubilizing capability of the strains, the phosphate solubilizing amounts of the strains P5, P8 and P7 are higher when ammonium sulfate and ammonium oxalate are used as nitrogen sources, the phosphate solubilizing effect is poorer when potassium nitrate and sodium nitrate are used as nitrogen sources, and the phosphate solubilizing effect of the strain P1 is better when potassium nitrate and ammonium sulfate are used as nitrogen sources, which indicates that the phosphate solubilizing effects of the strains P5, P8 and P7 are better than that of urea and nitrate nitrogen when ammonium nitrogen is used as a nitrogen source, and the effect of the strain P1 is more prominent in ammonium nitrogen and nitrate nitrogen. 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 was 598.89mg/L when ammonium sulfate was used as a nitrogen source, the highest phosphate solubilizing amount of the strain P12 and the highest phosphate solubilizing amount of the strain P4 were 461.45mg/L and 477.37mg/L when ammonium oxalate was used as a nitrogen source, and the highest phosphate solubilizing amount of the strain P8 was 559.78mg/L when ammonium sulfate was used as a nitrogen source, the potassium nitrate solubilizing amount of the strain P82 was 556.82mg/L and was slightly lower than the phosphate solubilizing amount of the strain P in ammonium sulfate. It can be seen from the figure that the strains P12, P8 and P4 all have better phosphate-solubilizing effect by using ammonium sulfate and ammonium oxalate as nitrogen sources, the urea is used as the nitrogen source, and the phosphate-solubilizing quantity is lowest when potassium nitrate and sodium nitrate are used as the nitrogen sources, which indicates that the strains P12, P8 and P4 have the best phosphate-solubilizing effect by using ammonium nitrogen as the nitrogen source, the strain P30 has better phosphate-solubilizing effect by using ammonium sulfate and potassium nitrate as the nitrogen sources, and indicates that the strain P30 can better utilize nitrate nitrogen and ammonium nitrogen, but the phosphate-solubilizing quantity in the ammonium sulfate is slightly higher than that in the potassium nitrate, and the effective phosphorus content in the urea is lowest. In conclusion, the optimal nitrogen source of the strains P30 and P8 is ammonium sulfate, the optimal 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

And (2) taking glucose and ammonium sulfate in an organic phosphorus and inorganic phosphorus liquid culture medium as a carbon source and a nitrogen source, adjusting the C/N ratio to be 40. 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 phosphate solubilizing ability of the strain P5 under the conditions of C/N of 20 and 40 is significantly higher than that of C/N of 8. The phosphorus dissolving amount of the strain P8 is remarkably increased along with the increase of C/N, when the C/N is 40. The phosphorus-dissolving amount of the strain P1 is 33.08mg/L at the C/N of 20, is obviously higher than that of the strain P1 under the conditions that the C/N is 8 and 40, and is 1.61 and 1.73 times of that of the strain P1 at the C/N of 8 and 40 respectively, and the difference of the phosphorus-dissolving amounts of the strain P1 at the C/N of 8. The phosphorus-dissolving amount of the strain P7 is 50.21mg/L at the C/N of 40, is obviously higher than that of the strain C/N of 8. From the above, the optimum C/N of each of the strains P5, P8 and P7 was 40.

As can be seen from fig. 11, the strains P30, P12 and P4 all have the highest phosphate solubilizing capacity at a C/N of 20, 581.89mg/L, 493.71mg/L and 501.44mg/L, respectively, and secondly 40. The phosphorus-dissolving amount of the strain P8 is 532.61mg/L at the C/N of 40, and is 20 in the second place, the phosphorus-dissolving amount is the lowest at the C/N of 8.

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%, 5%, subpackaging in 150mL triangular bottles, each bottle of 30mL, and sterilizing at 121 deg.C for 20min. 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 liquid was determined after 7 days of culture, and the results are shown in FIG. 12 and FIG. 13.

As can be seen from fig. 12, different salt concentrations have significant effects on the phosphate-solubilizing abilities of 4 organophosphorus bacteria, and the phosphate-solubilizing abilities of 4 strains are significantly reduced when the salt concentrations are 5% and 10%, which indicates that the phosphate-solubilizing abilities of the strains are significantly inhibited when the salt concentration is too high, in different salt concentrations, the strains P5, P8 and P1 show the same rule, the phosphate-solubilizing amounts are respectively 28.43mg/L,34.58mg/L and 27.27mg/L at the highest concentration of 1.5% and are significantly higher than those of the other respective salt concentrations, the differences between the phosphate-solubilizing amounts of the strains are not significant when the salt concentration is 0.5% and 1.5%, and the phosphate-solubilizing amounts of 3 strains are respectively 1.16mg/L,1.02mg/L and 1.13mg/L at the lowest concentration of 10%. The strain P7 is different from the other 3 strains, the phosphorus-solubilizing amount of the strain P7 at the salt concentration of 2.5% is 40.63mg/L which is significantly higher than that of the strain treated at other salt concentrations, and the minimum phosphorus-solubilizing amount is 1.25mg/L at the salt concentration of 10%, so that the optimum salt concentrations of the strains P5, P8 and P1 are 1.5%, and the optimum 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 the salt concentration of 2.5%, when the salt concentration continues to rise to 5%, the strain P30 still has a higher phosphorus solubilizing capability, the effective phosphorus content in the bacterial liquid is 117.79mg/L, and the effective phosphorus content in the bacterial liquids of the strains P12, P8 and P4 is significantly reduced, which are respectively 11.12mg/L, 16.76mg/L and 13.36mg/L, which indicates that the strain P30 shows a strong salt tolerance compared with the other 3 strains, and when the salt concentration reaches 10%, the effective phosphorus content of the 4 strains is all reduced to the lowest, so that the strain has a certain optimum salt concentration range, and the NaCl with an excessively high concentration can significantly inhibit the phosphorus solubilizing capability of the strain. As can be seen from the figure, the optimum salt concentration ranges of the strains P12, P8 and P4 are all 0-2.5%, wherein the strains P12 and P4 have the highest phosphate solubilizing capability when the salt concentration is 0.5%, the effective phosphate content in the bacterial liquid is 3235.35mg/L and 349.15mg/L respectively, which indicates that the optimum salt concentration of the strains P12 and P4 is 0.5%, the phosphate solubilizing amounts of the two strains when the salt concentration is 0.5% are 79.06 times of the phosphate solubilizing amount when the salt concentration is 10%, the strain P8 has the highest phosphate solubilizing capability when the salt concentration is 1.5%, the phosphate solubilizing amount is 400.70mg/L and is 66.67 times of the phosphate solubilizing amount when the salt concentration is 10%, which indicates that the optimum salt concentration of the strain P8 is 1.5%, the optimum salt concentration range of the strain P30 is 0-5%, wherein the phosphate solubilizing capability of the strain P30 when the salt concentration is 1.5%, the effective phosphate solubilizing capability of the bacterial liquid is 450.33%, the optimum salt concentration of the strain P30 is 40.47 times of the salt concentration. In summary, the optimum salt concentrations of the strains were different, and 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 medium at 30 ℃ for 24h, inoculating a ring of activated thalli into 30mL LB liquid medium, and culturing for 24h in a shaking table at the rotating speed of 180r/min and the temperature of 30 ℃. Absorbing 1mL of seed liquid of each strain cultured overnight and respectively inoculating the seed liquid into 30mL of organic phosphorus and inorganic phosphorus liquid culture media (the bacteria for removing organic phosphorus is inoculated into the organic phosphorus liquid culture media, and the bacteria for removing inorganic phosphorus is inoculated into the inorganic phosphorus liquid culture media), repeating each treatment for 3 times, carrying out shaking culture at 30 ℃ and 180r/min for 7d, regularly detecting the soluble phosphorus content and the pH value in the bacterial liquid every day, measuring the effective phosphorus content by adopting a molybdenum-antimony colorimetric method, measuring the pH value by using a pH meter, and recording the effective phosphorus content and the pH value of the bacterial liquid every day.

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

Respectively inoculating inorganic phosphorus-decomposing bacteria into inorganic phosphorus liquid culture medium, shake culturing at 7d, centrifuging the culture solution at 8000r/min for 15min, collecting supernatant and each organic acid standard, filtering with 0.22 μm filter membrane, removing impurities, ultrasonic degassing for 10min, and performing high performance liquid chromatography. 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 degrading bacteria into an organophosphorus liquid culture medium, performing shake culture for 7d, then sucking 1mL of supernatant, adding 4mL of buffer solution with pH 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 at 8000r/min for 15min, detecting the light absorption value of the supernatant at 420nm, and changing the buffer solution with pH 6.5 into the buffer solution with pH 11 in the same way as the determination of alkaline phosphatase. The enzyme activity was calculated by catalyzing disodium phenylphosphate to produce 0.1mg phenol as 1 phosphatase activity unit in 1mL of culture solution 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.

Sucking 1mL of organophosphorus bacteria liquid cultured for 7 days, centrifuging for 10min at 4000r/min, diluting the centrifuged supernatant, sucking 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 (trichloroacetic acid) into the solution after 30min to terminate the reaction, measuring the light absorption value of the solution at 710nm after constant volume, and calculating the soluble phosphorus content in the bacteria liquid at the same time, wherein the calculation formula of the phytase activity is as follows:

wherein, N: the dilution multiple 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 to 19, and it can be seen from the graphs that the phosphorus-solubilizing ability of the 4 organophosphorus-solubilizing bacteria is strongest when the strains P1 and P5 are cultured for 72h to 96h, the phosphorus-solubilizing ability of the strains P7 and P8 is strongest when the strains P7 and P8 are cultured for 48h to 72h, the effective phosphorus content of the 4 strains is highest when the strains are cultured for 120h, the phosphorus-solubilizing amount of the strain P7 is highest in the whole culture process, and the phosphorus-solubilizing amounts of the 4 strains and the pH of the strains 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 amounts of the 4 inorganic phosphorus-dissolving bacteria and the pH value of a bacterial liquid are in extremely obvious negative correlation.

(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 respectively represented by calcium phosphate > aluminum phosphate, and the dissolving amount of the strain P12 is represented by 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 the sequence, and the phosphorus dissolving amount of the 4 strains in an aluminum phosphate culture medium is P30> P12> P8> P4.

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

TABLE 6 kinds and contents of organic acids secreted from 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	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

Organic acid species	P30	P12	P8	P4
					Oxalic acid	2205.45±0.60a	70.83±5.72d	536.23±0.64b	85.64±5.83c
Tartaric acid	42.11±0.58c	-	106.17±2.89a	69.56±4.62b
					Malic acid	-	35.36±1.15c	95.91±5.77b	221.88±3.46a
Lactic acid	1472.44±3.46	-	-	-
					Acetic acid	-	503.30±1.73	131.42±1.73	-
Citric acid	100.58±5.78	-	-	-
					Succinic acid	430.57±5.89c	-	626.31±3.46b	818.55±4.62a
Propionic acid	637.44±2.89	1657.44±5.77	-	-
					Glutaric acid	-	-	-	-
Total amount of organic acid	4855.36±19.19a	2266.93±14.38b	1496.03±14.49c	1195.63±18.53d

TABLE 8 type and content of organic acids secreted by inorganic phosphorus-decomposing bacteria in iron 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 (AA)	-	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 acids	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 the calcium phosphate culture medium is in the order of P30> P8> P4> P12, the total content of organic acids secreted by the strains in the aluminum phosphate culture medium is in the order of P30> P12> P8> P4, the total content of organic acids secreted by the strains in the iron phosphate culture medium is in the order of P30> P12> P8> P4, and the total phosphate solubilizing amount of the strains in the other two culture media corresponds to the total content of organic acids secreted by the strains besides ferric phosphate, which indicates that the production of organic acids is an important factor for determining the phosphate solubilizing amount of the strains, and the total organic acid secreted by the strain P30 in the calcium phosphate, the aluminum phosphate and the aluminum phosphate is the highest, namely 9950.50mg/L,4855.36mg/L and 6556.17mg/L respectively. In a calcium phosphate culture medium, 9 organic acids are detected in a 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, 6 organic acids are generated by a strain P30, wherein the oxalic acid content is highest, the propionic acid content is highest in 4 organic acids generated by a strain P12, and the succinic acid content is highest in 5 organic acids secreted by strains P8 and P4 and in 4 organic acids secreted by the strains P8 and P4 respectively. 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 to 23, and it is known from the graphs that 4 strains of bacteria capable of dissolving organophosphorus 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 the bacterial liquid is 0.80U/mL, and as a whole, the amount of the strain dissolved organophosphorus is substantially in a positive correlation with the enzyme activity in the bacterial liquid.

Example 4 investigation of the ability of the Phosphorocarbus rhizosphere bacteria to secrete phytohormones and siderophores

4.1 test methods

Activating a test strain on a beef extract peptone agar slant culture medium at 30 ℃ for 24h, selecting a part of the strain with toothpicks, inoculating the selected strain into 30mL of LB liquid culture medium, and carrying out shake cultivation for 24h at the rotating speed of 180r/min and the temperature of 30 ℃ 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 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.

Inoculating 1mL of seed solution obtained by overnight culturing bacteria of organophosphorus and inorganic phosphorus in liquid culture medium of organophosphorus and inorganic phosphorus, shake-culturing at 30 deg.C for 7d, centrifuging to obtain 1mL of sample solution, measuring absorbance at 412nm, and determining gibberellin content.

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 of LB liquid culture medium, oscillating overnight at the rotation speed of 180r/min and the temperature of 30 ℃ to enable the bacterial liquid OD600=1, inoculating 1mL of bacterial liquid into the MKB liquid culture medium, performing shaking culture at the temperature of 30 ℃ and 180r/min for 48h, centrifuging the bacterial liquid at 5000r/min for 10min after 48h, sucking the centrifuged bacterial liquid and uniformly mixing the centrifuged bacterial liquid with 3mL of CAS detection liquid, measuring the absorbance at 630nm after reacting for 1h, taking the MKB liquid culture medium without the inoculated bacterial liquid as a reference, and adopting the calculation formula of an iron carrier 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 4 strains of the bacteria capable of releasing organophosphorus bacteria have the capability of secreting IAA, the IAA secretion amount is between 61.57mg/L and 100.75mg/L, and the IAA content of the 4 strains of the bacteria capable of releasing organophosphorus bacteria is P7> P8> P5> P1 in the order of magnitude. The highest secretion amount of the strain P7 IAA is 100.75mg/L, which is obviously higher than that of other strains, the next strain is the strain P8, the IAA contents secreted by the strains P5 and P1 are lower, the difference between the two strains is not obvious, and the secretion amounts of the strain P7 IAA are respectively 30.19%, 8.89% and 38.90% higher than those of the strains P5, P8 and P1. The 4 inorganic phosphorus-decomposing bacteria also have the capability of secreting IAA, and the IAA content of the 4 inorganic phosphorus-decomposing bacteria is P30> P8> P12> P4 in the sequence. Wherein the highest IAA secretion amount of the strain P30 is 103.27mg/L, the next strain is the strain P8, the IAA secretion amount of the strain P30 is 91.80mg/L, and the lowest IAA secretion amount of the strain P4 is 22.47mg/L. The secretion of the strain P30 IAA is 33.66 percent, 78.24 percent and 11.1 percent higher than that of the strains P12, P8 and P4 respectively, and the difference of the secretion of the IAA among the 4 strains is obvious.

As can be seen from FIG. 25, 4 organophosphorus degrading bacteria can secrete gibberellin, the gibberellin secretion amounts of the bacteria are 19.60 mg/L-57.20 mg/L, the gibberellin content of the 4 strains are 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 differences of the gibberellin secretion amounts are not significant, the gibberellin secretion amount of the strain P5 is 32.86mg/L, the gibberellin secretion amount of the strain P1 is lowest and is 19.60mg/L, the gibberellin secretion amount of the strain P7 is 37.9% and 63.0% higher than that of the strain P5 and P1, the gibberellin secretion amount of the strain P8 is 42.6% and 65.7% higher than that of the strain 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 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 of the strains P30 and P8 is higher and is respectively 57.65mg/L and 55.28mg/L and is remarkably higher than that of the other 2 strains, the gibberellin secretion of the strains P12 and P4 is respectively 23.17mg/L and 17.59mg/L, the gibberellin secretion of the strain P30 is higher than that of the strains P12 and P4 by 59.8 percent and 69.5 percent respectively, and the gibberellin secretion of the strain P8 is higher than that of the strains P12 and P4 by 58.1 percent and 68.2 percent 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 higher, respectively 59.32% and 61.47%, the siderophore activities of the two strains are significantly higher than those of the strains P5 and P1, the siderophore activities of the strains P5 and P1 are lower, respectively 24.14% and 24.04%, the siderophore activity difference is not significant, and in conclusion, the siderophore secretion abilities of the strains P7 and P8 in the 4 strains for decomposing the organophosphorus bacteria are stronger. The activity difference of the 4 inorganic phosphorus bacteria secreting the siderophore is obvious, the sequence of the siderophore secretion activity is P8> P30> P4> P12, the siderophore activity in the bacterial liquid of the strain P8 is 59.58 percent, the activity of the siderophore is P30, the activity of the siderophore is 48.28 percent, the activity of the siderophore of the strains P4 and P12 is 24.14 percent and 17.16 percent respectively, and the siderophore secretion ability of the strain P8 in the 4 inorganic phosphorus bacteria is the strongest.

According to 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 57.20mg/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 iron carrier in the bacterial liquid of the strain P8 is 59.58%. The strain screened by the invention has good capability of secreting IAA, gibberellin and siderophore, and can well solve the problems of low content of available phosphorus in soil, slow development of the root system of the seedlings of the strelitzia tree and slow growth of seedlings when the strain is prepared into a microbial fertilizer and applied to the soil.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> university of Guangxi; guangxi Zhuang autonomous region Nanning Liangfengjiang national forest park

<120> Burkholderia gladioli P8 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1440

<212> DNA

<213> Burkholderia gladioli glakholderia gladioli)

<400> 1

tcagacagat ctacgtggtg accgtcctcc ttgcggttag actagccact tctggtaaaa 60

cccactccca tggtgtgacg ggcggtgtgt acaagacccg ggaacgtatt caccgcggca 120

tgctgatccg cgattactag cgattccagc ttcatgcact cgagttgcag agtgcaatcc 180

ggactacgat cggttttctg ggattagctc cccctcgcgg gttggcgacc ctctgttccg 240

accattgtat gacgtgtgaa gccctaccca taagggccat gaggacttga cgtcatcccc 300

accttcctcc ggtttgtcac cggcagtctc cctagagtgc tcttgcgtag caactaagga 360

caagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacagc 420

catgcagcac ctgtgtatcg gttctctttc gagcaccctc ggatctctcc aaggttccga 480

ccatgtcaag ggtaggtaag gtttttcgcg ttgcatcgaa ttaatccaca tcatccaccg 540

cttgtgcggg tccccgtcaa ttcctttgag ttttaatctt gcgaccgtac tccccaggcg 600

gtcaacttca cgcgttagct acgttactaa ggaaatgaat ccccaacaac tagttgacat 660

cgtttagggc gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgtgcat 720

gagcgtcagt attggcccag ggggctgcct tcgccatcgg tattcctcca catctctacg 780

catttcactg ctacacgtgg aattctaccc ccctctgcca tactctagct tgccagtcac 840

caatgcagtt cccaggttga gcccggggat ttcacatcgg tcttaacaaa ccgcctgcgc 900

acgctttacg cccagtaatt ccgattaacg ctcgcaccct acgtattacc gcggctgctg 960

gcacgtagtt agccggtgct tattcttccg gtaccgtcat ccccgaagga tattagccct 1020

caggatttct ttccggacaa aagtgcttta caacccgaag gccttcttca cacacgcggc 1080

attgctggat caggctttcg cccattgtcc aaaattcccc actgctgcct cccgtaggag 1140

tctgggccgt gtctcagtcc cagtgtggct ggtcgtcctc tcagaccagc tactgatcgt 1200

cgccttggtg ggcctttacc ccaccaacta gctaatcagc catcggccaa ccctatagcg 1260

cgaggcccga aggtcccccg ctttcatccg tagatcgtat gcggtattaa tccggctttc 1320

gccgggctat cccccactac aggacatgtt ccgatgtatt actcacccgt tcgccactcg 1380

ccaccaggtg caagcacccg tgctgccgtt cgacttgcat ggtaaggctg tcgaccgccc 1440

Claims (6)

1. The Burkholderia gladioli P8 is characterized by being preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation date of 2021, 07, 13 days and the preservation registration number of CGMCC No.22825.

2. The use of Burkholderia gladioli P8 according to claim 1 for converting poorly soluble phosphorus in soil to available phosphorus for plant uptake.

3. Use according to claim 2, characterized in that the sparingly soluble phosphorus is a sparingly soluble organophosphorus.

4. The use of Burkholderia gladioli P8 according to claim 1 for the cultivation of an artificial forest of strelitzia.

5. The use according to claim 4 for enhancing the development of the roots of the trees at the seedling stage and young trees of the stretchy trees and for promoting growth.

6. A microbial agent comprising the burkholderia gladioli P8 according to claim 1.

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