CN114015625B - Bacillus cereus P30 and application thereof - Google Patents

Abstract

The invention discloses bacillus cereus P30 and application thereof, belonging to the technical field of microorganisms, wherein the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation date is 2021, 07 and 13 days, and the preservation registration number is CGMCC No.22827; the invention shows that the strain has the best effect of inorganic phosphorus decomposition at the temperature of 30 ℃ and the pH of 6.5, the carbon source is sucrose, the nitrogen source is ammonium sulfate, the C/N is 20:1, the salt concentration is 1.5 percent, the strain can secrete organic acid to dissolve insoluble inorganic phosphorus, and lactic acid, oxalic acid and oxalic acid are key organic acids in a calcium sulfate culture medium, an aluminum phosphate culture medium and an iron phosphate culture medium respectively, dissolve the insoluble inorganic phosphorus, secrete plant hormone and siderophore, promote plant photosynthesis, respiration, substances and energy metabolism, and can be used for solving the problems of slow root system development and slow seedling growth of the sapling in hopeful days.

Description

Bacillus cereus P30 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus cereus P30 and application thereof.

Background

Phosphorus plays an important role in plant growth, is a main nutrient element in metabolic processes such as plant energy transmission, signal transmission, macromolecular biosynthesis, photosynthesis and respiration, and the like, and meanwhile, phosphorus is an important nutrient element for promoting root growth and development, and the lack of the phosphorus element severely restricts the growth of plants. The acid soil itself has a very high phosphorus content, but because the acid soil has a strong solidification effect on phosphorus, most of the phosphorus in the soil is combined by calcium, aluminum or iron minerals to form insoluble phosphate, so that 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 solutions are present in millimoles, while phosphorus is present only in micromoles or less, and plants lacking phosphorus can develop retarded root development, underdeveloped lateral roots, slow plant growth, and weak resistance. The application of phosphate fertilizer to soil is an important way to supplement the demand of plants for phosphorus, but the phosphate fertilizer is expensive and has low utilization rate, and the excessive application of phosphate fertilizer can cause the problems of environmental pollution, soil hardening and the like. Thus, there is a need to find an environmentally friendly, economically viable method to increase plant yield in low-phosphorus or phosphorus-deficient soil. Phosphate-dissolving microorganisms are commonly present in most of the soil, especially in rhizosphere soil, and the variety and content of the phosphate-dissolving microorganisms are more, so that the phosphate-dissolving microorganisms can convert insoluble phosphorus in the soil into effective phosphorus which can be absorbed by plants, and the phosphate-dissolving microorganisms replace chemical fertilizers to solve the problem of low effective phosphorus in the soil, thereby being a very promising biological fertilizer.

There are a large number of autotrophic and heterotrophic microorganisms in soil, which have the ability to solubilize poorly soluble phosphorus, and the variety and number of phosphate-solubilizing microorganisms in different soils are different, and the variety of phosphate-solubilizing microorganisms that have been reported so far includes 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), nostoc (Nostoc), stratana (calix), pseudocladium (scents), and the like. Although most researches show that the screened phosphate-solubilizing microorganisms have strong phosphate-solubilizing ability, many phosphate-solubilizing microorganisms have no stable effect when applied into soil, and sometimes do not increase crop yield and accumulation of phosphorus. The strain has better phosphate-dissolving capacity in a laboratory, but the phosphate-dissolving capacity is weakened or completely disappeared after the strain is applied to soil, and the main reasons are as follows: (1) Soil has a complex environment, and 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-dissolving microorganisms on nutrient substances in soil; (4) degradation of the phosphate solubilizing ability of the phosphate solubilizing microorganism; (5) The texture of the soil, pH, water content, organic matter content and the like can influence the growth and propagation of phosphate solubilizing microorganisms and the colonization effect thereof. The phosphate-dissolving microorganism can survive and successfully colonize in the soil, and is related to environmental factors such as temperature, pH, salt concentration and the like in the soil, and simultaneously, nutrient substances such as carbon sources, nitrogen sources and the like in the soil can influence the phosphate-dissolving capability of the phosphate-dissolving microorganism by influencing the propagation of the phosphate-dissolving microorganism thallus and the content of secreted organic acid.

The hopus japonicus (Parashorea chinensis Wang Hsie.) is also known as the Optimus qingtian tree, which is a plant of the genus Liuan of the family Dryopteraceae, a tropical rainforest marker species and an endangered tree species, the first-order important protection of wild plants in China, and Yunnan and Guangxi are the main distribution areas. The hoping tree has high scientific and economic values, is high-quality wood for manufacturing ships, buildings, bridges, musical instruments and the like, and has considerable application and development prospects. According to the previous investigation and experiment, the growth of root system is slow, lateral root is not developed when the tree is grown, so that the tree is slow in growth in seedling period, weak in resistance and high in death rate, phosphorus is important to the growth and development of root system, and phosphorus-dissolving microorganism is a main way for improving the effective phosphorus of soil, so that the efficient phosphorus-dissolving bacteria are separated and screened from the artificial forest of the tree, and the method is an effective method for solving the problems of slow growth and slow growth of the tree seedling period and the young root system.

Disclosure of Invention

The invention aims to provide bacillus cereus P30 and application thereof, which are used for solving the problems of the prior art, and the strain can effectively convert insoluble phosphorus into effective phosphorus which can be absorbed by plants, can secrete plant hormone and siderophores and promote plant growth.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides Bacillus cereus P30 which is preserved in China general microbiological culture Collection center (CGMCC) with the address of China center for type 1 and 3 academy of China center for general microbiological study (CGMCC) in the North Chenxi Lu No. 1 of the Korean area of Beijing, and the preservation date is 2021, 07, 13 and the preservation registration number is CGMCC No.22827.

The invention also provides application of the bacillus cereus P30, which is used for converting insoluble phosphorus in soil into effective phosphorus which can be absorbed by plants.

Further, the poorly soluble phosphorus is a poorly soluble inorganic phosphorus.

The invention also provides application of the bacillus cereus P30 in cultivation of artificial forests of the hopus japonicus.

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

The invention also provides a microbial agent, which comprises the bacillus cereus P30.

The invention discloses the following technical effects:

according to the invention, the efficient phosphorus-decomposing bacteria of the rhizosphere of the zenia are separated, screened and identified from the zenia manmade forest of different forestation years, and the phosphorus-decomposing effect, the phosphorus-decomposing mechanism, the optimal phosphorus-decomposing condition and the capacity of the strain to secrete related plant hormone and siderophore are researched so as to evaluate the capacity of the phosphorus-decomposing bacteria to convert insoluble phosphorus in soil into effective phosphorus which can be absorbed by plants. The invention shows that the strain has the best effect of inorganic phosphorus decomposition at the temperature of 30 ℃ and the pH of 6.5, the carbon source is sucrose, the nitrogen source is ammonium sulfate, the C/N is 20:1, the salt concentration is 1.5 percent, the strain can secrete organic acid to dissolve insoluble inorganic phosphorus, and lactic acid, oxalic acid and oxalic acid are key organic acids in a calcium sulfate culture medium, an aluminum phosphate culture medium and an iron phosphate culture medium respectively, dissolve the insoluble inorganic phosphorus, secrete plant hormone and siderophore, promote plant photosynthesis, respiration, substances and energy metabolism, and can be used for solving the problems of slow root system development and slow seedling growth of the sapling in hopeful days.

Drawings

FIG. 1 is the construction of a phylogenetic tree of phosphate-solubilizing bacteria;

FIG. 2 is a graph showing the effect of pH on the phosphate solubilizing ability of an organophosphorus-solubilizing bacterium;

FIG. 3 is a graph showing the effect of pH 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 an organophosphorus-solubilizing bacterium;

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

FIG. 6 is a graph showing the effect of carbon source values on the phosphate-solubilizing ability of an organophosphorus-solubilizing bacterium;

FIG. 7 is a graph showing the effect of carbon source values on the phosphate-solubilizing ability of inorganic phosphate-solubilizing bacteria;

FIG. 8 is a graph showing the effect of nitrogen source values on the phosphate solubilizing ability of an organophosphorus-solubilizing bacterium;

FIG. 9 is a graph showing the effect 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 values on the phosphate solubilizing ability of an organophosphorus-solubilizing bacterium;

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 an organophosphorus-solubilizing bacterium;

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

FIG. 14 is a dynamic change in the phosphate solubilizing ability of an organophosphorus-solubilizing bacterium;

FIG. 15 is a dynamic change in the phosphate solubilizing ability of inorganic phosphate solubilizing bacteria;

FIG. 16 shows the dynamic pH change of the organophosphorus decomposing bacterial liquid;

FIG. 17 shows the dynamic pH change of the inorganic phosphate-solubilizing bacterial liquid;

FIG. 18 is a graph showing the relationship between the amount of phosphate to be decomposed by an organic phosphate-decomposing 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 phosphate solubilizing bacteria in different phosphate source media;

FIG. 21 is a graph showing the activity of acid phosphatase secreted by organophosphorus-degrading bacteria;

FIG. 22 is a graph showing alkaline phosphatase activity secreted by organophosphorus-degrading bacteria;

FIG. 23 shows the activity of a phytase secreted by organophosphorus-degrading bacteria;

FIG. 24 is a graph showing IAA secretion by phosphate solubilizing bacteria;

FIG. 25 is a graph showing gibberellin content of phosphate-solubilizing bacteria;

FIG. 26 is a graph showing the secretory siderophore activity of phosphate solubilizing bacteria.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

Example 1 isolation, screening and identification of the rhizosphere phosphate-solubilizing bacteria of the Hospital Tree

1.1 isolation of phosphate-solubilizing bacteria

In month 8 of 2019, in Guangxi nan Ning tree garden, the artificial forests of the forestation in 1978, 2011, 2012, 2017, 2018 and 2019 are respectively selected, and the root system of the forestation and the soil sample adhered to the rhizosphere are collected.

The phosphobacteria are separated from the rhizosphere soil of the zene tree in different forestation 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 have the capability of dissolving insoluble organic phosphorus or inorganic phosphorus, and the strains capable of producing the phosphorus-dissolving rings are numbered as P1-P30.

1.2 screening of phosphate-solubilizing bacteria

1.2.1 preliminary screening of phosphate-solubilizing bacteria

The total of 30 strains of the separated P1-P30 are respectively inoculated into organic and inorganic phosphorus solid culture media, the ratio between the diameter (D) of a transparent ring and the colony diameter (D) is measured, and the result is shown in table 1, 10 strains of which the D/D is more than 2.0 are respectively P1, P2, P5, P7, P8, P14, P22, P27, P28 and P30, which account for 33.3 percent of the total number of the strains, and the 10 strains have stronger capacity of dissolving organic phosphorus, 6 strains of which the D/D is less than 2.0 and account for 20 percent of the total number of the strains, 14 strains of which the D/D is less than 1.5 and account for 46.7 percent of the total number of the strains, and the D/D value of the strains P14 on the organic phosphorus solid culture media is the largest, 2.15 and the P23D/D value of the strains is the smallest and is 1.05.

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

TABLE 1 results of preliminary screening of phosphate solubilizing bacteria

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

1.2.2 Compound screening of phosphate-solubilizing bacteria

The observation of phosphate solubilizing ring produced by phosphate solubilizing strain on solid culture medium is only one method of qualitative determination of phosphate solubilizing ability of strain, in order to judge phosphate solubilizing ability of strain accurately, strain with strong phosphate solubilizing ability, i.e. 10 strains of phosphate solubilizing strain with D/D >2.0 and 8 strains of phosphate solubilizing strain with inorganic phosphate are re-screened, and the effective phosphate content in the strain culture solution is quantitatively determined by liquid culture method.

Preparing organic and inorganic phosphorus liquid culture media respectively, inoculating a strain to be tested to a beef extract peptone solid culture medium, activating for 24 hours at 30 ℃, inoculating a loop of activated thalli to an LB liquid culture medium, carrying out shaking culture for 24 hours at 30 ℃ to prepare a seed liquid, accurately weighing 1mL of the seed liquid by a sterilized pipette, inoculating the seed liquid to the sterilized organic and inorganic phosphorus liquid culture medium, inoculating 3 times of the seed liquid to a reference (CK) liquid without inoculating bacteria, placing the inoculated bacteria in a shaking table, placing the inoculated bacteria in the shaking table at the temperature of 30 ℃ and the rotation speed of 180r/min, carrying out shaking culture for 7d and 7d, taking out, centrifuging the organic phosphorus culture liquid for 12000r/min for 30min, centrifuging the inorganic phosphorus culture liquid for 10000r/min for 15min, absorbing supernatant, measuring phosphorus content by a molybdenum-antimony colorimetric method, and calculating the phosphorus resolution ratio, wherein the result is shown in a table 2 and a table 3.

As shown in Table 2, in the culture medium using lecithin as an organophosphorus source, the effective phosphorus content in the culture solution of 10 strains is significantly higher than that of a control CK (6.38 mg/L), which indicates that the 10 strains have certain phosphorus dissolving capacity in the organophosphorus liquid culture medium, the amount of organophosphorus dissolved by each strain is between 16.18 and 41.58mg/L, the phosphorus dissolving difference among different strains is significant, the phosphorus dissolving amount of the strain P7 is 41.58mg/L at the highest, and the phosphorus dissolving amount of the strain P14 is 16.18mg/L at the lowest. Compared with CK, the increment of effective phosphorus in the culture solution is between 9.8mg/L and 35.20mg/L, and the increment is 1.54-5.52 times of CK. The effective phosphorus content in the culture solution of each strain is P7> P8> P5> P1> P28> P30> P2> P22> P14 in turn from large to small, the phosphorus dissolution rate of 10 strains is between 1.96% and 7.04%, 4 strains with the phosphorus dissolution rate of more than 5% are respectively strains P1, P5, P7 and P8, which indicate that the 4 strains have stronger organophosphorus dissolution capability, the strain P1 in the 4 strains is derived from the root soil of the forestation in 2019, the strains P5 and P7 are both derived from the root soil of the forestation in 2018, and the strain P8 is derived from the root soil of the forestation in 2017.

As shown in Table 3, in the inorganic phosphorus liquid medium of calcium phosphate, the effective phosphorus content in the 8 inorganic phosphorus decomposing bacteria culture solutions is significantly higher than that of the control CK (21.44 mg/L), and the effective phosphorus content in the culture solutions of different strains is significantly different. The effective phosphorus content in the 8 inorganic phosphorus decomposing bacteria culture solution is 305.33 mg/L-598.89 mg/L, and the phosphorus decomposing rate is 5.68-11.55%. Compared with CK, the increment of available phosphorus is 283.89 mg/L-577.45 mg/L, and the increment is 13.24-26.93 times of CK. The effective phosphorus content in the culture solution of each strain is P30> P8> P4> P12> P23> P15> P10> P25 in turn from large to small, the phosphorus dissolving amount of the strains P4, P8, P12 and P30 is higher, the effective phosphorus content in the culture solution is up to 552.87mg/L, 559.78mg/L, 548.53mg/L and 598.89mg/L respectively, the phosphorus dissolving rate is 10.63%, 10.77%, 10.54% and 11.55%, the phosphorus dissolving rate is more than 10%, which means that the capacity of the strains P4, P8, P12 and P30 for dissolving inorganic phosphorus is stronger, the strain P4 is derived from the root soil of the forestation in 2019, the strains P8 and P12 are derived from the root soil of the forestation in 2017, the strain P30 is derived from the root soil of the forestation in 1987, and the 4 strains can be considered as key strains for developing microbial fertilizers.

TABLE 2 Compound screening for organophosphorus bacteria

Year of forestation	Strain number	Content of available phosphorus (mg/L)	Phosphate 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-decomposing bacterial double screen

1.3 identification of phosphate-solubilizing bacteria

1.3.1 identification of the morphology of the bacteria and of the physiological and biochemical identification

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, contact enzyme test, indole test, VP, citrate, gelatin liquefaction, hydrogen sulfide, methyl red and the like.

The cell morphology and colony characteristics of 4 strains P1, P5, P7, P8 and 4 strains P4, P8, P12 and P30 which are screened by the separation and screening and have strong capability of decomposing organic phosphorus are observed, wherein P8 has the capability of decomposing organic phosphorus and inorganic phosphorus, and the colony morphology characteristic observation results of each strain are shown in Table 4.

TABLE 4 morphological observations of phosphate solubilizing bacteria

Various physiological and biochemical identification items of phosphate solubilizing bacteria include: citrate test, gelatin hydrolysis test, contact enzyme test, V-P reaction, indole test, starch hydrolysis test, nitrate reduction test, H-production ₂ S test, etc., the results of each test item of the strain test are shown in Table 5, 3 repetitions were made for each test item, and the measurement results were relatively stable. The experimental results show that the strains P1 and P30 are the same in characteristics, can utilize citrate, can make gelatin hydrolyze and contact enzyme react positively, can produce indole, can hydrolyze starch and reduce nitrate, the strains P5, P7, P8, P4 and P12 are the same in characteristics, can utilize citrate, can make gelatin hydrolyze and contact enzyme react positively, can reduce nitrate, and can preliminarily judge that the strains P1 and P30 are possibly of bacillus and the strains P5, P7, P8, P4 and P12 are possibly of Burkholderia according to the morphological and physiological biochemical characteristics of the strains.

TABLE 5 physiological and biochemical identification of phosphate solubilizing bacteria

Test item

P1

P5

P7

P8

P4

P12

P30

Citrate salt

+

+

+

+

+

+

+

Gelatin hydrolysis test

+

+

+

+

+

+

+

Contact enzyme reaction

+

+

+

+

+

+

+

V-P reaction

+

-

-

-

-

-

+

Indole test

+

-

-

-

-

-

+

Starch hydrolysis test

+

-

-

-

-

-

+

Nitrate reduction test

+

+

+

+

+

+

+

H 2 S

-

-

-

-

-

-

-

Note that: + indicates positive, -indicates negative

In order to verify whether the guess made for the strain is accurate according to morphological and physiological biochemical characteristics, the invention also makes molecular biology identification for the strain. The 16SrDNA sequence (16 SrDNA base sequence of Burkholderia gladioli P7 of the invention is shown as SEQ ID No. 1) detected by the strain is subjected to homology comparison with other 16SrDNA sequences in a GenBank nucleic acid database, clustalX is used for analysis, MEGA7.0 is used for constructing a phylogenetic tree by adopting a Neighbor operation method, and the similar strain sequences with very high homology can be found in the database by BLAST comparison of 7 strains. Strains P1 and P30 have higher sequence similarity with Bacillus, strains P4, P5, P7, P8 and P12 have higher sequence similarity with Burkholderia, as can be seen from the phylogenetic tree of FIG. 1, strains P5, P7 and P8 are in the same branch as Burkholderia gladioli, strains P4 and P12 are in the same branch as Burkholderia cepacia, strains P1 and P30 are in the same branch as Bacillus cereus, and the relatedness is closer, therefore, strains P5, P7 and P8 are identified as Burkholderia glabra (Burkholderia gladioli), strains P4 and P12 are identified as Burkholderia cepacia (Burkholderia cepacia), and strains P1 and P30 are identified as Bacillus cereus (Bacillus cereus) respectively, by combining morphological characteristics, physiological and biochemical characteristics and phylogenetic analysis results of each strain.

1.4 preservation of phosphate-solubilizing bacteria

Burkholderia tangutica P7 is preserved in China general microbiological culture Collection center (CGMCC) for the 13 th month of 2021, and has a preservation registration number of CGMCC No.22824.

Example 2 screening of optimal phosphorus-solubilizing conditions for the rhizosphere phosphorus-solubilizing bacteria of the hopus japonicus

The organophosphorus-degrading bacteria P1, P5, P7 and P8 screened by the test of example 1, and the inorganic-phosphate-degrading bacteria P4, P8, P12 and P30 were used as test materials to further investigate the phosphate-degrading conditions of the organophosphorus-degrading bacteria.

2.1 Effect of pH on the phosphate solubilizing ability of phosphate-solubilizing bacteria

Respectively preparing organic phosphorus and inorganic phosphorus liquid culture media, respectively setting initial pH to 3.5, 4.5, 5.5, 6.5, 7.5 and 8.5, respectively, subpackaging into 150mL triangular bottles, 30mL each bottle, and sterilizing at 121deg.C for 20min. Respectively sucking 1mL of organophosphorus bacteria seed solution, inoculating the organophosphorus bacteria seed solution into organophosphorus liquid culture mediums with different pH values, sucking 1mL of organophosphorus bacteria seed solution, inoculating the organophosphorus bacteria seed solution into the organophosphorus liquid culture mediums with different pH values, setting 3 repetitions of each treatment, placing 180r/min and shaking table culture at 30 ℃ for 7d and 7d, centrifuging the bacterial solution, sucking the supernatant, and measuring the effective phosphorus content in the bacterial solution, wherein the results are shown in figures 2 and 3.

As shown in FIG. 2, 4 organophosphorus bacteria have different phosphorus-decomposing capacities under different pH conditions, and 4 strains have lower phosphorus-decomposing capacities at the pH of 3.5, which means that the phosphorus-decomposing capacity of the strains is limited at the pH of 3.5, the effective phosphorus content in bacterial solutions of all the strains is in a trend of rising first and then reducing with the rising of the pH, the optimal pH range of the strain P5 is between 6.0 and 7.0, the maximum effective phosphorus content in the bacterial solution reaches 43.01mg/L at the pH of 6.5, which means that the phosphorus-decomposing capacity of the strain P5 is stronger when the bacterial solution is cultured in a meta-acidic environment. The optimal pH ranges of the strains P1, P7 and P8 are 7.0-8.0,3, the phosphorus dissolving effect is best when the pH is 7.5, and the effective phosphorus content in the bacterial liquid is 41.86mg/L, 44.19mg/L and 45.43mg/L respectively, which indicates that the strains P1, P7 and P8 have stronger phosphorus dissolving capability in a slightly alkaline environment.

As can be seen from fig. 3, the 4 inorganic phosphorus decomposing bacteria have different phosphorus decomposing capacities under different pH conditions, and the 4 bacterial strains also have lower phosphorus decomposing capacities at the pH of 3.5, which indicates that the bacteria are unsuitable for growth of the bacterial strains and exert the phosphorus decomposing capacities under the strong acid environment, and the effective phosphorus content in bacterial solutions of each bacterial strain has a tendency of increasing firstly and then decreasing as the pH increases. The optimal pH of the strain P30 and the strain P4 is 6.5, and the effective phosphorus content in the bacterial solutions of the strain P30 and the strain P4 is highest at the pH of 6.5 and is 577.37mg/L and 551.83mg/L respectively. The effective phosphorus content of the strain P30 in the bacterial liquid is not greatly changed and is always high in the bacterial liquid between pH4.5 and 7.5, which indicates that the strain P30 has stronger adaptability to the pH value of the environment and wider pH adaptation range. The optimal pH value of the strains P8 and P12 is 7.5, and when the pH values of the 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 strong phosphorus dissolving capability in a alkalescent environment.

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

Respectively preparing organic phosphorus and inorganic phosphorus liquid culture medium, subpackaging in 150mL triangular bottles, and sterilizing at 121deg.C for 20min. Respectively absorbing 1mL of the organophosphorus-degrading bacterial seed solution, inoculating the organophosphorus-degrading bacterial seed solution into an organophosphorus liquid culture medium, absorbing 1mL of the organophosphorus-degrading bacterial seed solution, inoculating the organophosphorus-degrading bacterial seed solution into an inorganic phosphorus liquid culture medium, placing the organophosphorus-degrading bacterial seed solution into 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ for shake cultivation for 7d, repeating each treatment for 3 times, centrifuging the bacterial solution after 7d, absorbing the supernatant, and measuring the effective phosphorus content in the bacterial solution, wherein the result is shown in figures 4 and 5.

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

As shown in FIG. 5, the phosphorus-decomposing amount of the strains P12 and P4 is higher at 25-35 ℃, the effective phosphorus content in the bacterial liquid is highest at 30 ℃, the effective phosphorus-decomposing amount is 548.53mg/L and 552.87mg/L respectively, the phosphorus-decomposing amount of the strains P12 and P4 at 35 ℃ is 477.74mg/L and 500.98mg/L respectively, and the phosphorus-decomposing amount at 25 ℃ is 437.71mg/L and 472.68mg/L respectively. The highest effective phosphorus content of the bacterial liquid of the strain P30 at 30 ℃ is 598.89mg/L, and the second is 517.09mg/L at 35 ℃. The highest effective phosphorus content of the bacterial liquid of the strain P8 at 35 ℃ is 623.71mg/L, and the next effective phosphorus content of the bacterial liquid at 30 ℃ is 559.78mg/L. The effective phosphorus content of the bacterial solutions of the bacterial strains P12 and P4 at the temperature of 25 ℃ and the temperature of 30 ℃ and 35 ℃ is obviously higher than that of other temperature treatments respectively, which shows that the optimal phosphorus dissolving temperature range of the bacterial strains P12 and P4 is 25-35 ℃, the effective phosphorus content of the bacterial solutions of the bacterial strains P30 and P8 at the temperature of 30 ℃ and the temperature of 35 ℃ is obviously higher than that of other temperature treatments respectively, which shows that the optimal phosphorus dissolving temperature range of the bacterial strains P30 and P8 is 30-35 ℃, the optimal phosphorus dissolving temperature of the bacterial strains P30, P12 and P4 in the inorganic phosphorus dissolving bacteria of the bacterial strain 4 is 30 ℃ and the optimal phosphorus dissolving temperature of the bacterial strain P8 is 35 ℃.

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

Glucose in the organic phosphorus and inorganic phosphorus liquid culture medium is replaced by lactose, sucrose, soluble starch and mannitol with equal carbon content, and the glucose is subpackaged in 150mL triangular flasks, 30mL of each flask is sterilized at the high temperature of 121 ℃ for 20min, the inoculation amount and shake culture conditions in different carbon source culture mediums are the same as 2.1, and the effective phosphorus content in the bacterial liquid is measured after 7d of culture, and the results are shown in figures 6 and 7.

As can be seen from fig. 6, the different carbon sources have significant influence on the phosphorus-decomposing ability of 4 strains of organophosphorus-decomposing bacteria, and among the 5 different carbon sources, the phosphorus-decomposing ability of 4 strains of organophosphorus-decomposing bacteria is: 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 > mannitol, and overall, 4 strains have better phosphate-dissolving effect when glucose, sucrose and lactose, have poorer phosphate-dissolving effect when soluble starch and mannitol are used as carbon sources, the phosphate-dissolving amounts of the strains P5, P8 and P7 are respectively 33.39mg/L, 39.53mg/L and 41.58mg/L when glucose is used as the carbon source, the phosphate-dissolving amount of the strain P1 is 23.64mg/L when sucrose is used as the carbon source, so 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 4 inorganic phosphorus-decomposing bacteria have different phosphorus-decomposing abilities under different carbon sources, and among the 5 different carbon sources, the 4 inorganic phosphorus-decomposing bacteria have the following phosphorus-decomposing abilities in order: p30: sucrose > glucose > lactose > soluble starch > mannitol, P12: lactose > glucose > sucrose > soluble starch > mannitol, P8: glucose > lactose > sucrose > soluble starch > mannitol, P4: although 4 strains have different phosphorus dissolving capability under different carbon sources, the figure shows that 4 strains have better phosphorus dissolving effect when glucose, sucrose and lactose are taken as carbon sources, and the phosphorus dissolving amount is the lowest when mannitol is taken as the carbon source, which is the same as the rule of the bacteria for dissolving organic phosphorus, so that the utilization of the carbon sources by the bacteria for dissolving organic phosphorus and the bacteria for dissolving inorganic phosphorus is mainly based on monosaccharides and double ponds, and the utilization efficiency of the polysaccharide is lower. The highest phosphorus dissolving amount of the strain P30 is 651.10mg/L when sucrose is taken as a carbon source, the highest phosphorus dissolving amount of the strain P12 and the highest phosphorus dissolving amount of the strain P4 are 489.39mg/L and 500.98g/L respectively when lactose is taken as a carbon source, the highest phosphorus dissolving amount of the strain P8 is 559.78mg/L when glucose is taken as a carbon source, and the best carbon source of the strain P30 is sucrose, the best carbon source of the strain P12 and the best carbon source of the strain P4 are lactose, and the best carbon source of the strain P8 is glucose.

2.4 Effect of Nitrogen Source on the phosphate-solubilizing Capacity of phosphate-solubilizing bacteria

And replacing ammonium sulfate in the organic phosphorus and inorganic phosphorus liquid culture medium with urea, potassium nitrate, sodium nitrate and ammonium oxalate with equal nitrogen content, subpackaging in 150mL triangular bottles, and sterilizing at 121 ℃ for 20min at 30mL each bottle. The inoculum size and shaking culture conditions in the different nitrogen source culture media were the same as those of 2.1, and the effective phosphorus content in the bacterial liquid was measured after 7d of culture, and the results are shown in FIG. 8 and FIG. 9.

As can be seen from fig. 8, the phosphorus-decomposing ability of the strain is significantly affected by different nitrogen sources, and among 5 different nitrogen sources, the phosphorus-decomposing ability of 4 strains of organic bacteria is: 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 highest phosphate dissolving amount of each of the strains P5, P8 and P7 is 33.39mg/L, 39.53mg/L and 41.58mg/L respectively and is obviously higher than that of other nitrogen sources, the highest phosphate dissolving amount of the strain P1 is 32.98mg/L when the potassium nitrate is taken as the nitrogen source, the lowest phosphate dissolving amount of the strain P5 is 10.56mg/L when the potassium nitrate is taken as the nitrogen source, the lowest phosphate dissolving amount of each of the strains P8 and P7 is 16.61mg/L and 19.09mg/L when the sodium nitrate is taken as the nitrogen source, the lowest phosphate dissolving amount of the strain P1 when the urea is taken as the nitrogen source is 6.16mg/L respectively, as can be clearly seen from the figure, the ammonium nitrogen and the nitrate nitrogen have different degrees of influence on the phosphate dissolving capacity of the strain, the phosphate dissolving capacity of the strain P5, the strain P8 and the strain P7 is higher when the ammonium sulfate and the ammonium oxalate are taken as nitrogen sources, the phosphate dissolving effect is poor when the potassium nitrate and the sodium nitrate are taken as nitrogen sources, the phosphate dissolving effect of the strain P1 when the potassium nitrate and the ammonium sulfate are taken as nitrogen sources is better, the phosphate dissolving effect of the strain P5, the strain P8 and the strain P7 when the ammonium nitrogen is taken as nitrogen sources is better than that of urea and the nitrate nitrogen, and the strain P1 can exert better phosphate dissolving capacity in both the ammonium nitrogen and the nitrate nitrogen, especially the nitrate nitrogen, and the effect is more outstanding. Therefore, the most suitable nitrogen source for strains P5, P8 and P7 is ammonium sulfate and the most suitable nitrogen source for strain P1 is potassium nitrate.

As can be seen from fig. 9, the different nitrogen sources have significant influence on the phosphorus-decomposing ability of 4 inorganic phosphorus-decomposing bacteria, and among the 5 different nitrogen sources, the phosphorus-decomposing ability of 4 inorganic phosphorus-decomposing bacteria is: 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 phosphate dissolving amount of the strain P30 is highest when ammonium sulfate is taken as a nitrogen source, is 598.89mg/L, the phosphate dissolving amount of the strain P30 is 556.82mg/L and is slightly lower than the phosphate dissolving amount of the strain P in ammonium sulfate, the phosphate dissolving amounts of the strain P12 and the strain P4 are respectively 461.45mg/L and 477.37mg/L when ammonium oxalate is taken as a nitrogen source, and the phosphate dissolving amount of the strain P8 is 559.78mg/L when ammonium sulfate is taken as a nitrogen source. As can be seen from the graph, the strains P12, P8 and P4 have better phosphate-dissolving effect by taking ammonium sulfate and ammonium oxalate as nitrogen sources, urea is inferior to the ammonium sulfate, the phosphate-dissolving amount is the lowest when potassium nitrate and sodium nitrate are taken as nitrogen sources, the strains P12, P8 and P4 have the best phosphate-dissolving effect when ammonium nitrogen is taken as nitrogen sources, the strain P30 has better phosphate-dissolving effect when ammonium sulfate and potassium nitrate are taken as nitrogen sources, the strain P30 can better utilize both nitrate nitrogen and ammonium nitrogen, but the phosphate-dissolving amount in ammonium sulfate is slightly higher than that in potassium nitrate, and the effective phosphorus content in urea is the lowest. In summary, the best nitrogen sources of the strains P30 and P8 are ammonium sulfate, the best nitrogen sources of the strains P12 and P4 are ammonium oxalate, and the 4 strains have better phosphate dissolving effect when the ammonium nitrogen is taken as the nitrogen source.

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

Glucose and ammonium sulfate in organic phosphorus and inorganic phosphorus liquid culture medium are used as carbon source and nitrogen source, the C/N ratio is adjusted to 40:1, 20:1 and 8:1, and the mixture is packaged in 150mL triangular bottles, 30mL of each bottle is sterilized at 121 ℃ for 20min. The inoculum size and shaking culture conditions in different C/N culture media were the same as those of 2.1, and the content of available phosphorus in the bacterial liquid was measured after 7d of culture, and the results are shown in FIG. 10 and FIG. 11.

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

As can be seen from FIG. 11, the strains P30, P12 and P4 all had the highest phosphorus-dissolving amounts at 20:1C/N, namely 581.89mg/L, 493.71mg/L and 501.44mg/L respectively, and were 40:1 secondly, the 3 strains had the lowest phosphorus-dissolving amounts at 8:1C/N, and the 3 strains had phosphorus-dissolving amounts at 20:1C/N were 2.07 times, 2.42 times and 2.28 times the phosphorus-dissolving amounts at 8:1C/N respectively. As can be seen from the graph, 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 capacity of the strain P8 is lowest when the C/N is 8:1, and the phosphorus-dissolving capacity of the strain P8 is not good for the strain to exert the phosphorus-dissolving capacity when the C/N is 40:1 as shown in the graph, wherein the phosphorus-dissolving capacity of the strain P8 is 532.61mg/L when the C/N is 40:1, and the phosphorus-dissolving capacity of the strain P8 is lowest when the C/N is 8:1.

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

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

As can be seen from fig. 12, the different salt concentrations have significant effects on the phosphate-solubilizing ability of 4 strains of organophosphorus bacteria, the phosphate-solubilizing ability of 4 strains is significantly reduced at salt concentrations of 5% and 10%, which means that the excessive salt concentration significantly inhibits the phosphate-solubilizing ability of the strains, and in different salt concentrations, the phosphate-solubilizing amounts of the strains P5, P8 and P1 are respectively 28.43mg/L,34.58mg/L and 27.27mg/L at 1.5% salt concentration, and significantly higher than the phosphate-solubilizing amounts of the strains under the treatment of other salt concentrations, respectively, the phosphate-solubilizing amounts of the strains are not significantly different at salt concentrations of 0.5% and 1.5%, and the phosphate-solubilizing amounts of the strains 3 at salt concentrations of 10% are respectively 1.16mg/L,1.02mg/L and 1.13mg/L, respectively. The strain P7 is different from other 3 strains, the phosphorus dissolution amount of the strain P7 is 40.63mg/L which is obviously higher than that of the strain under the treatment of other salt concentration when the salt concentration is 2.5%, and the phosphorus dissolution amount of the strain P7 is 1.25mg/L at the lowest 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 shown in FIG. 13, when the salt concentration reaches 2.5%, the 4-strain bacterial liquid still has higher effective phosphorus content, which indicates that the 4-strain bacterial liquid still has better salt tolerance at the salt concentration of 2.5%, when the salt concentration continues to rise to 5%, the bacterial liquid still has higher phosphorus dissolution capability, the effective phosphorus content in the bacterial liquid is 117.79mg/L, and the effective phosphorus content in the bacterial liquid of the bacterial strains P12, P8 and P4 is obviously reduced, namely 11.12mg/L, 16.76mg/L and 13.36mg/L, respectively, which indicates that the bacterial strain P30 has strong salt tolerance compared with other 3-strain bacterial liquid, and when the salt concentration reaches 10%, the effective phosphorus content of the 4-strain bacterial liquid is reduced to the minimum, thereby indicating that the bacterial liquid has a certain optimal salt concentration range, and the phosphorus dissolution capability of the bacterial liquid of the bacterial strain can be obviously inhibited by NaCl with too high concentration. As can be seen from the graph, the optimal salt concentration ranges of the strains P12, P8 and P4 are all 0-2.5%, wherein the optimal salt dissolving capacity of the strains P12 and P4 is strongest when the salt concentration is 0.5%, the effective phosphorus content in the bacterial liquid is 3235.35mg/L and 349.15mg/L respectively, the optimal salt concentration of the strains P12 and P4 is 0.5%, the phosphate dissolving capacity of the strains P8 is 79.06 times of the phosphate dissolving capacity of the strains P8 when the salt concentration is 10%, the phosphate dissolving capacity of the strains P8 is 400.70mg/L and 66.67 times of the phosphate dissolving capacity of the strains P8 when the salt concentration is 10%, the optimal salt concentration range of the strains P30 is 1.5%, the effective phosphorus content in the bacterial liquid is 450.33mg/L and the optimal salt concentration of the strains P30 is 47.40%, and the optimal salt concentration of the strains P30 is 47.40%. In summary, the optimal salt concentration was 1.5% for strain P30, 0.5% for strains P12 and P4, and 1.5% for strain P8.

Example 3 mechanism of phosphorus dissolution by rhizosphere phosphorus-dissolving bacteria of zenia

3.1 test methods

3.1.1 determination of the phosphate-solubilizing ability of the phosphate-solubilizing bacteria and the pH of the bacterial liquid

The strain to be tested is activated for 24 hours at 30 ℃ on a beef extract peptone solid culture medium, a loop of activated thalli is inoculated in 30mL of LB liquid culture medium, shaking culture is carried out for 24 hours, the rotating speed is 180r/min, and the temperature is 30 ℃. 1mL of seed solution of each strain cultured overnight is absorbed and inoculated into 30mL of organic phosphorus and inorganic phosphorus liquid culture medium respectively (the organic phosphorus bacteria are inoculated into the organic phosphorus liquid culture medium, the inorganic phosphorus bacteria are inoculated into the inorganic phosphorus liquid culture medium), 3 repeats are arranged for each treatment, the culture is carried out for 7 days under shaking at 30 ℃ and 180r/min, the soluble phosphorus content and the pH value in the bacterial liquid are detected at regular time every day, the effective phosphorus content is measured by adopting a molybdenum-antimony colorimetric method, the pH value is measured by adopting a pH meter, and the effective phosphorus content and the pH value of the bacterial liquid are recorded every day.

3.1.2 determination of the species and content of organic acids secreted by the phosphate-solubilizing bacteria

Inoculating inorganic phosphorus-decomposing bacteria into inorganic phosphorus liquid culture medium respectively, shaking culture for 7d and 7d, centrifuging the culture solution under 8000r/min for 15min, keeping supernatant after centrifuging, filtering the collected supernatant and each organic acid standard substance with a 0.22 μm filter membrane, removing impurities, ultrasonically degassing for 10min, and performing high performance liquid chromatography analysis. Setting chromatographic conditions identical to those of standard organic acid measurement, and comparing the peak time of each organic acid in the bacterial liquid with the standard organic acid according to the rule, so as to determine the types of the organic acids in the bacterial liquid, and comparing standard curves between the standard organic acid concentration and the peak area according to the peak area of each organic acid in the bacterial liquid, so as to obtain the content of each organic acid in the bacterial liquid.

3.1.3 determination of the Activity of the phospholyzing bacteria to secrete phosphatase

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

3.1.4 determination of the Phytophosphorus-degrading bacteria secretion phytase Activity

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

Sucking 1mL of organophosphorus bacteria bacterial liquid cultured for 7d, centrifuging at 4000r/min for 10min, diluting the centrifuged supernatant, sucking 1mL of sodium phytate solution, adding the diluted bacteria liquid into a 25mL volumetric flask, adding 1mL of trichloroacetic acid into the volumetric flask, reacting at 30 ℃ for 30min, adding TCA (ternary ammonium chloride) to terminate the reaction, measuring the absorbance at 710nm after volume fixing, and simultaneously calculating the soluble phosphorus content in the bacteria liquid, wherein the calculation formula of phytase activity is as follows:

/>

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

3.2 results

(1) As shown in FIGS. 14 to 19, in 4 strains of organophosphorus degrading bacteria, the strains P1 and P5 have the strongest phosphorus degrading ability in the culture for 72 to 96 hours, the strains P7 and P8 have the strongest phosphorus degrading ability in the culture for 48 to 72 hours, the 4 strains have the highest content of effective phosphorus in the bacterial liquid in the culture for 120 hours, the phosphorus degrading amount of the strain P7 is the highest in the whole culture process, and the phosphorus degrading amount of the 4 strains has no obvious correlation with the pH of the bacterial liquid. The 4 inorganic phosphorus-decomposing bacteria have the strongest phosphorus-decomposing capacity when being cultured for 48-72 hours, the bacterial strain P30 has the highest phosphorus-decomposing capacity when being cultured for 144 hours, the bacterial strains P12, P8 and P4 have the highest phosphorus-decomposing capacity when being cultured for 120 hours, the bacterial strain P30 has the highest phosphorus-decomposing capacity in the whole culture process, and the phosphorus-decomposing capacity of the 4 inorganic phosphorus-decomposing bacteria is extremely obviously inversely related to the pH of bacterial solution.

(2) As shown in FIG. 20, the phosphate solubilizing amounts of the strains P30, P8 and P4 in the three media were each represented by calcium phosphate > iron phosphate > aluminum phosphate, and the dissolving amount of the strain P12 was represented by iron phosphate > aluminum phosphate > calcium phosphate. Compared with the phosphate dissolving amount of 4 strains, the phosphate dissolving amount of 4 strains in the calcium phosphate and ferric phosphate culture medium is in the order of P30> P8> P4> P12, and the phosphate dissolving amount of 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 species and content of organic acid secretion 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	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 that: the data in the tables are all mean ± standard deviation, the different lower case letters indicate that the different strains differ significantly at 0.05 level (P < 0.05), and "-" indicates that no corresponding organic acid was detected in the bacterial liquid, and the same applies below.

TABLE 7 species and content of organic acid secretion by inorganic phosphate-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 acids	4855.36±19.19a	2266.93±14.38b	1496.03±14.49c	1195.63±18.53d

TABLE 8 species and content of organic acid secretion by inorganic phosphate-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	-	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 order of the organic acid secreted by the strain in the calcium phosphate medium is P30> P8> P4> P12, the total content order of the organic acid secreted by the strain in the aluminum phosphate medium is P30> P12> P8> P4, the total content order of the organic acid secreted by the strain in the iron phosphate medium is P30> P12> P8> P4, the phosphate-dissolving amount of the strain in other two mediums except for the iron phosphate corresponds to the total content of the organic acid secreted by the strain, the generation of the organic acid is an important factor for determining the phosphate-dissolving amount of the strain, and the total amount of the organic acid secreted by the strain P30 in the calcium phosphate, the aluminum phosphate and the iron phosphate is 9950.50mg/L,4855.36mg/L and 6556.17mg/L respectively. In the calcium phosphate culture medium, 9 organic acids are detected in bacterial liquid of the strain P30, wherein the lactic acid content is highest, 5 organic acids are detected by the strain P12, the glutaric acid content is highest, 7 organic acids are detected by the strain P8, the lactic acid content is highest, 6 organic acids are detected by the strain P4, and the succinic acid content is highest. In the aluminum phosphate culture medium, the strain P30 produces 6 organic acids, wherein the oxalic acid content is highest, the strain P12 produces 4 organic acids, the propionic acid content is highest, and the strains P8 and P4 secrete 5 organic acids and 4 organic acids respectively are all the highest in succinic acid content. In the ferric phosphate culture medium, the oxalic acid content in 4 organic acids produced by the strain P30 is highest, the glutaric acid content in 5 organic acids produced by the strain P12 is highest, the succinic acid content in 5 organic acids produced by the strain P8 is highest, and the tartaric acid content in 4 organic acids produced by the strain P4 is highest.

(4) As shown in FIGS. 21 to 23, it is clear from the graph that 4 strains of organophosphorus degrading bacteria can secrete phosphatase and phytase in the process of dissolving organophosphorus, wherein the activities of acid phosphatase and alkaline phosphatase secreted by the strains P7 and P8 are significantly higher than those of the strains P5 and P1, the activity of acid phosphatase secreted by the strain P7 is 16.61U/mL, the activity of alkaline phosphatase is 11.26U/mL, the activity of acid phosphatase secreted by the strain P8 is 15.08U/mL, the activity of alkaline phosphatase is 10.83U/mL, the activity of phytase secreted by the strain P7 is significantly higher than that of other strains, and the activity of phytase in the bacterial liquid is 0.80U/mL.

Example 4 study of the ability of the phosphorus-solubilizing rhizosphere bacteria of the Hospita tree to secrete phytohormones and iron carriers

4.1 test methods

Activating the strain to be tested on beef extract peptone agar slant culture medium at 30deg.C for 24 hr, picking part of strain with toothpick, inoculating into 30mL LB liquid culture medium, shake culturing for 24 hr at 180r/min, and preparing seed solution at 30deg.C.

1mL of seed solution for overnight culture of the bacteria for decomposing the organic phosphorus and the inorganic phosphorus is respectively taken and inoculated into the organic phosphorus liquid culture medium and cultured for 7d at the temperature of 30 ℃ under 180r/min in a shaking way. And centrifuging the bacterial liquid at 4000r/min for 20min, mixing the supernatant after centrifugation with Salkowsk colorimetric liquid in an equal volume, and measuring the absorbance value at 540nm after light-shielding reaction for 30min to obtain the IAA content.

1mL of seed solution for decomposing organophosphorus and inorganic phosphorus bacteria and culturing overnight is respectively taken and inoculated into an organophosphorus and inorganic phosphorus liquid culture medium, 7d is cultured at 30 ℃ under 180r/min in a shaking way, 7d is then centrifuged, 1mL of test solution is taken, the absorbance at 412nm is measured, and the gibberellin content is obtained.

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

Inoculating the strain into 30mL of LB liquid medium, oscillating overnight at the temperature of 30 ℃ at the rotating speed of 180r/min to ensure that bacterial liquid OD600 = 1, inoculating 1mL of bacterial liquid into MKB liquid medium, shake culturing for 48h at the temperature of 180r/min at 30 ℃, centrifuging for 10min at 5000r/min after 48h, absorbing and uniformly mixing the centrifuged bacterial liquid with 3mL of CAS detection liquid, measuring the absorbance at 630nm after 1h reaction, and taking the MKB liquid medium without bacterial liquid as a reference, wherein the calculation formula of the siderophore is as follows:

U(％)＝(Ar-As)/Ar×100

wherein, U: siderophore activity; as: absorbance of the mixed solution at 630 nm; ar: absorbance of MKB liquid medium without bacterial liquid.

4.2 results

The results are shown in FIGS. 24 to 26. As shown in FIG. 24, the 4 organophosphorus bacteria have the capability of secreting IAA, the IAA secretion amount is 61.57 mg/L-100.75 mg/L, and the content order of IAA secreted by the 4 organophosphorus bacteria is P7> P8> P5> P1. Wherein the secretion amount of the strain P7 IAA is 100.75mg/L at most, which is obviously higher than that of other strains, and is the strain P8, the IAA content secreted by the strains P5 and P1 is lower, the difference between the two 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 inorganic phosphorus bacteria also have the capability of secreting IAA, and the content order of the IAA secreted by the 4 inorganic phosphorus bacteria is P30> P8> P12> P4. Wherein the secretion amount of the strain P30 IAA is highest and is 103.27mg/L, the secretion amount of the strain P8 is 91.80mg/L, and the secretion amount of the strain P4 IAA is lowest and is 22.47mg/L. The secretion amount 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 IAA secretion among 4 strains is obvious.

As shown in FIG. 25, the 4 organophosphorus bacteria can secrete gibberellin, the gibberellin secretion amount is between 19.60mg/L and 57.20mg/L, the order of the gibberellin secretion content of the 4 strains is P8> P7> P5> P1, the gibberellin secretion amounts of the strains P7 and P8 are 52.99mg/L and 57.20mg/L respectively, the difference between the two is insignificant, the second is the strain P5, the gibberellin secretion amount is 32.86mg/L, the minimum gibberellin secretion amount of the strain P1 is 19.60mg/L, the gibberellin secretion amount of the strain P7 is higher than that of the strains P5 and P1 by 37.9% and 63.0%, the gibberellin secretion amount of the strain P8 is higher than that of the strains P5 and P1 by 42.6% and 65.7%, and the capacity of the strains P8 and P7 in the 4 organophosphorus bacteria is relatively strong. The secretion amount of 4 inorganic phosphorus bacteria gibberellin has obvious difference, the order of the content of 4 bacterial strains secreting gibberellin is P30> P8> P12> P4, the secretion amounts of bacterial strains P30 and P8 gibberellin are higher and are 57.65mg/L and 55.28mg/L respectively, the secretion amounts of bacterial strains P12 and P4 gibberellin are 23.17mg/L and 17.59mg/L respectively, the secretion amounts of bacterial strains P30 are 59.8% and 69.5% higher than those of the bacterial strains P12 and P4 respectively, and the secretion amounts of bacterial strains P8 are 58.1% and 68.2% higher than those of the bacterial strains P12 and P4 respectively. In conclusion, strains P30 and P8 in the 4 strains have stronger gibberellin secretion capacity.

From fig. 26, the order of the activities of 4 strains secreting siderophores is P7> P8> P5> P1, the activities of P7 and P8 siderophores are higher, 59.32% and 61.47% respectively, the activities of two siderophores are significantly higher than those of the strains P5 and P1, the activities of the strains P5 and P1 siderophores are lower, 24.14% and 24.04% respectively, and the differences of the activities of two siderophores are not significant, so that the strains P7 and P8 in the 4 strains for decomposing organophosphorus bacterial strains secrete siderophores are strong. The 4 inorganic phosphorus-decomposing bacteria have obvious difference of the activities of secreting the siderophores, the order of the activities of the siderophores is P8> P30> P4> P12, the highest siderophore activity in bacterial liquid of the strain P8 is 59.58%, the next time is P30, the siderophore activity is 48.28%, the lower siderophore activities of the strains P4 and P12 are 24.14% and 17.16%, and the highest siderophore secreting ability of the strain P8 in the inorganic phosphorus-decomposing bacteria is summarized.

Compared with 4 organophosphorus bacteria, IAA content and siderophore activity produced by the strain P7 are highest, respectively 100.75mg/L and 61.47%, and gibberellin content secreted by the strain P8 is highest, namely 57.20mg/L. IAA and gibberellin secreted by the strain P30 in the 4 inorganic phosphate-solubilizing bacteria are highest in content of 103.27mg/L and 57.65mg/L, and siderophore activity in bacterial liquid of the strain P8 is highest in content of 59.58%. The strain screened by the invention has better capability of secreting IAA, gibberellin and siderophores, and the microbial fertilizer prepared by the strain can well solve the problems of low content of available phosphorus in soil, slow root system development of the seedlings of the hopped tree and slow growth of the seedlings when the microbial fertilizer is applied to the soil.

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

Sequence listing

<110> university of guangxi; guangxi Zhuang autonomous region nan Ning Liang Fengjiang national forest park

<120> Bacillus cereus P30 strain and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1449

<212> DNA

<213> Bacillus cereus (Bacillus cereus)

<400> 1

actctgtccc ttaggcggct ggctccataa aggttacccc accgacttcg ggtgttacaa 60

actctcgtgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 120

tgatccgcga ttactagcga ttccagcttc atgtaggcga gttgcagcct acaatccgaa 180

ctgagaacgg ttttatgaga ttagctccac ctcgcggtct tgcagctctt tgtaccgtcc 240

attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc 300

ttcctccggt ttgtcaccgg cagtcacctt agagtgccca acttaatgat ggcaactaag 360

atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420

accatgcacc acctgtcact ctgctcccga aggagaagcc ctatctctag ggttttcaga 480

ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540

cttgtgcggg cccccgtcaa ttcctttgag tttcagcctt gcggccgtac tccccaggcg 600

gagtgcttaa tgcgttaact tcagcactaa agggcggaaa ccctctaaca cttagcactc 660

atcgtttacg gcgtggacta ccagggtatc taatcctgtt tgctccccac gctttcgcgc 720

ctcagtgtca gttacagacc agaaagtcgc cttcgccact ggtgttcctc catatctcta 780

cgcatttcac cgctacacat ggaattccac tttcctcttc tgcactcaag tctcccagtt 840

tccaatgacc ctccacggtt gagccgtggg ctttcacatc agacttaaga aaccacctgc 900

gcgcgcttta cgcccaataa ttccggataa cgcttgccac ctacgtatta ccgcggctgc 960

tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtgccag cttattcaac 1020

tagcacttgt tcttccctaa caacagagtt ttacgacccg aaagccttca tcactcacgc 1080

ggcgttgctc cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag 1140

gagtctgggc cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat 1200

cgttgccttg gtgagccgtt acctcaccaa ctagctaatg cgacgcgggt ccatccataa 1260

gtgacagccg aagccgcctt tcaatttcga accatgcggt tcaaaatgtt atccggtatt 1320

agccccggtt tcccggagtt atcccagtct tatgggcagg ttacccacgt gttactcacc 1380

cgtccgccgc taacttcttg agagcaagct ctcaatccat tcgctcgact gcatgtatag 1440

cccgccgcc 1449

Claims (5)

1. Bacillus cereus P30, which is deposited with China general microbiological culture Collection center with a date of 2021 and a date of 07 and 13 months and with a deposit accession number of CGMCC No.22827.

2. Use of bacillus cereus P30 of claim 1 for converting poorly soluble phosphorus in soil to available phosphorus that can be absorbed by plants, wherein the poorly soluble phosphorus is calcium phosphate, aluminum phosphate, and iron phosphate.

3. Use of bacillus cereus P30 of claim 1 for artificial forest cultivation of a zenith tree.

4. The use according to claim 3, for improving the seedling stage of the desired tree and the root system development of the young tree, and for promoting the growth.

5. A microbial agent comprising the Bacillus cereus P30 according to claim 1.