CN108048360B

CN108048360B - Bacillus subtilis with dual functions of degrading organic phosphorus and preventing diseases

Info

Publication number: CN108048360B
Application number: CN201711455361.5A
Authority: CN
Inventors: 李博超; 马毅楠; 孔令佳
Original assignee: Individual
Current assignee: Yunnan Sijiaotian Agricultural Technology Co ltd
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2021-01-01
Anticipated expiration: 2037-12-28
Also published as: CN108048360A

Abstract

The invention discloses a Bacillus subtilis strain WKPHY-54 with the preservation number of CGMCC No. 14951. Also discloses a microbial agent containing the strain. The strain WKPHY-54 has dual functions of degrading organic phosphorus and preventing diseases, can improve the utilization rate of the organic phosphorus in phosphate fertilizer or soil, promote the growth of crops, reduce fertilizer investment and reduce production cost; meanwhile, the pesticide composition has higher control effect on plant diseases such as eggplant verticillium wilt and the like, and the average control effect is more than 70.0 percent; secondly, the bacterial strain WKPHY-54 has a wide antibacterial spectrum and has a good inhibition effect on eggplant verticillium wilt, cucumber fusarium wilt, cotton fusarium wilt, potato black nevus and the like; in addition, the WKPHY-54 strain has strong specificity on eggplant verticillium wilt and other plant diseases; the drug resistance is not easy to generate, and the drug effect durability is good.

Description

Bacillus subtilis with dual functions of degrading organic phosphorus and preventing diseases

Technical Field

The invention belongs to the field of agricultural microorganisms, and particularly relates to bacillus subtilis with dual functions of degrading organic phosphorus and preventing diseases, a microbial agent containing the bacillus subtilis, and applications of the bacillus subtilis and the microbial agent in degrading organic phosphorus and preventing and treating crop diseases.

Background

Phosphorus is one of mineral elements necessary for maintaining normal growth of plants, and is an important component of nucleic acid, various enzymes, coenzyme, ATP and the like in the plants. The phosphorus required for plant growth and development comes mainly from the supply of fertilizers and soil. Phosphate fertilizers applied to soil are easy to form insoluble phosphate and are difficult to be absorbed and utilized by crops, so that the key point for improving the phosphorus nutrition of plants is to convert and utilize the insoluble phosphorus in the soil. The phosphorus in the soil can not be transformed by the phosphorus-dissolving bacteria, and the phosphorus-dissolving bacteria are divided into inorganic phosphorus-dissolving bacteria and organic phosphorus-dissolving bacteria. The organic phosphorus degrading bacteria mainly decompose organic phosphorus compounds including phytate, phospholipid, organic phosphate and the like, and the action mechanism is mainly to degrade the organic phosphorus into an inorganic form by virtue of enzymes generated in the life activities of the bacteria, so that the organic phosphorus is absorbed and utilized by plants.

Currently, the bacteria that degrade organic phosphorus in soil mainly include Bacillus (Bacillus sp., CN106978347A), Pantoea (Pantoea sp., CN104498403A, CN104962500A), Acinetobacter (Acinetobacter sp., CN106544303A, CN104974962A), Pseudomonas (Pseudomonas sp., CN105112319A), and the like. Among the Bacillus, Bacillus subtilis (CN 106244504A), Bacillus amyloliquefaciens (CN106434455A, CN105420156A) and the like can be mentioned. The bacterial strains mainly degrade organic phosphorus and are not reported in the aspect of preventing and controlling plant diseases.

Eggplant Verticillium wilt is a soil-borne disease caused by Verticillium dahliae (Verticillium dahliae), mainly damages leaves, can be caused in all growing periods of the eggplant, and is the most serious in fruiting period. With the development of facility cultivation, continuous cropping cultivation of eggplants is more and more common, so that the verticillium wilt of the eggplants is increased year by year, and the yield and the quality of the eggplants are greatly reduced.

In production, the verticillium wilt of eggplants is mainly prevented and treated by using a large amount of chemical pesticides, so that the drug resistance of pathogenic bacteria is enhanced, the balance among soil microorganisms is destroyed, and pesticide residues on vegetables and environmental pollution are caused. Therefore, biological control has attracted much attention as an important measure for controlling plant diseases.

Currently, Bacillus for preventing and treating eggplant verticillium wilt mainly comprises Bacillus laterosporus (CN 107151641A), Bacillus amyloliquefaciens (CN105238723A), Bacillus subtilis (CN 102925394A; CN 102154186A; Luhong science, Hubei agricultural science, 2013, 52 (21): 5199-.

The known bacillus has relatively single function, or only has phosphate solubilizing effect, or only has disease control effect; secondly, with the change of environmental conditions or the evolution of pathogenic bacteria, the function of the bacillus can also evolve along with the evolution of the pathogenic bacteria, so that the bacillus with high-efficiency phosphorus dissolving function and wide antibacterial spectrum is continuously separated and screened from the soil, and the bacillus has important significance for adjusting the contradiction between supply and demand of soil phosphorus, improving the control effect on plant diseases, reducing the production cost, protecting the ecological environment and promoting the sustainable development of agriculture.

Disclosure of Invention

The invention aims to provide a bacillus subtilis strain which has double functions of degrading organic phosphorus and preventing and treating diseases.

The second purpose of the invention is to provide the application of the bacillus subtilis.

The third purpose of the invention is to provide a microbial agent containing the bacillus subtilis.

The fourth object of the present invention is to provide a method for producing the microbial agent.

The fifth purpose of the present invention is to provide the use of the microbial agent.

The invention is realized by the following technical scheme:

a Bacillus subtilis strain WKPHY-54 with a preservation number of CGMCC No. 14951.

The application of the strain WKPHY-54 in degrading organic phosphorus.

Application of strain WKPHY-54 in degrading organic phosphorus in soil

The application of the strain WKPHY-54 in promoting the growth of crops.

The application of the strain WKPHY-54 in preventing and treating plant diseases.

The plant diseases in the above application refer to eggplant Verticillium wilt (Verticillium dahliae), cucumber wilt (f.oxysporum f.sp.cucumerium), cotton wilt (f.oxysporum f.sp.vassifnfectium), potato black nevus (r.solani), and the like.

The strain WKPHY-54 is applied to degrading organic phosphorus and preventing and treating verticillium wilt of eggplants.

The invention also provides a microbial agent containing the strain WKPHY-54.

The microbial agent can be a liquid preparation or a solid preparation.

The microbial agent contains strain WKPHY-54 with viable count of 2.0 × 10⁶～2.0×10⁸cfu/mL or 2.0X 10⁶～2.0×10⁸cfu/g。

The preparation method of the liquid preparation of the microbial agent comprises the following steps:

(1) activating strains: activating the WKPHY-54 strain stored at low temperature on an LB plate culture medium, selecting a single strain, and culturing the single strain on an LB slant culture medium at 25-35 ℃ for 10-16 hours to obtain an activated strain;

(2) preparing a seed solution: scraping a ring of the activated strain in the step (1) by using a sterile inoculating ring, then inoculating the strain into 100mL of LB liquid culture medium, and culturing for 10-16 hours at the temperature of 25-35 ℃ and the rotating speed of a shaking table of 150-220 rpm to obtain seed liquid;

(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a corn flour and soybean flour culture medium (pH value is 7.2) according to the volume ratio of 1-3%, and fermenting and culturing for 36-40 h under the conditions that the temperature is 25-35 ℃ and the rotating speed of a shaking table is 150-220 rpm to obtain a fermentation liquid; detecting the quantity of the thalli and spores in the fermentation liquid every 30min, and stopping fermentation culture when mature spores in the fermentation liquid account for 90% of the total quantity of the spores and the thalli; the obtained product is a liquid preparation of WKPHY-54 strain.

The LB plate culture medium or LB inclined plane culture medium in the step (1) of the preparation method comprises the following components in percentage by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride, 12-18 g of agar powder and 1000mL of water.

The LB liquid culture medium in the step (2) of the preparation method comprises the following components in parts by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride and 1000mL of water.

The LB plate culture medium, the LB slant culture medium and the LB liquid culture medium are all prepared according to a conventional method.

The corn meal and soybean meal culture medium in the step (3) of the preparation method comprises the following components in percentage by weight: 1.0-3.0% of corn flour, 1.0-3.0% of soybean flour, 0.1-1.0% of NaCl, and MnSO₄·H₂0.5-1.0% of O and the balance of water.

The preparation method of the corn flour and soybean meal culture medium comprises the steps of mixing corn flour, soybean meal, NaCl and MnSO in percentage by weight₄·H₂And O, mixing, adding water, adjusting the pH value and stirring uniformly.

The microbial agent is applied to degrading organic phosphorus.

The microbial agent is applied to degrading organic phosphorus in soil.

The microbial agent is applied to promoting the growth of crops.

The microbial agent is applied to the prevention and treatment of plant diseases.

The microbial agent is applied to degrading organic phosphorus and preventing and treating verticillium wilt of eggplants.

The use method of the microbial agent comprises the following steps: diluting the obtained microbial agent with water until viable cell number is 10⁷cfu/mL, soaking seeds for half an hour before sowing eggplant or adsorbing the obtained microbial agent by using calcium carbonate to prepare bacillus amyloliquefaciens WKPHY-54 powder which can be applied in holes before sowing eggplant.

The invention also provides a genetic engineering bacterium which takes the strain WKPHY-54 as a receptor bacterium. The genetic engineering bacteria improve the capability of degrading organic phosphorus or improve the control effect of controlling plant diseases such as eggplant verticillium wilt and the like; or the dual functions of degrading organic phosphorus and preventing and treating plant diseases such as verticillium wilt of eggplants and the like are improved.

The invention has the advantages and beneficial effects that: (1) the strain WKPHY-54 is a microorganism with dual functions of degrading organic phosphorus and preventing and treating eggplant verticillium wilt; the strain WKPHY-54 has strong capacity of degrading organic phosphorus, and plays an important role in improving the utilization rate of fertilizer, promoting the growth of crops, reducing the fertilizer investment and reducing the production cost, and meanwhile, the strain WKPHY-54 has good effect of preventing and treating the verticillium wilt of eggplants, the average prevention effect is over 70.0 percent, and a high-efficiency microorganism is provided for preventing and treating the verticillium wilt of the eggplants; (2) the strain WKPHY-54 has a wide antibacterial spectrum, and has a good inhibition effect on eggplant verticillium wilt, cucumber wilt, cotton wilt, potato black nevus and the like. (3) The WKPHY-54 strain has strong specificity on eggplant verticillium wilt; the drug resistance is not easy to generate, and the drug effect durability is good; (4) the microbial agent is safe to people and livestock, and does not have the problem of environmental pollution; (5) the microbial preparation has the advantages of simple preparation method, low cost and simple use.

Biological preservation

The Bacillus subtilis strain WKPHY-54 is obtained by self screening of the inventor, is preserved in China general microbiological culture Collection center in 11 and 22 months in 2017, and has the preservation addresses as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and Beijing, the preservation number is: CGMCC No. 14951.

Drawings

FIG. 1 is a phylogenetic tree diagram of WKPHY-54 strain obtained based on 16S rDNA sequence.

FIG. 2 is a phylogenetic tree diagram of WKPHY-54 strain obtained based on the gyrB gene sequence.

Detailed Description

The invention is further illustrated and described by the following examples, which are not intended to be limiting in any way. The experimental methods in the following examples are all conventional methods unless otherwise specified; the percentages in the following examples are by weight unless otherwise specified.

Example 1 screening and isolation procedure and Classification of the Strain WKPHY-54 of the present invention

Process for collecting, separating and screening WKPHY-54 strain

9 months in 2015, 5 parts of soil sample per 200g is collected at five points by baoding micro-control biotechnology limited company in the alum-mountain phosphate region of the Hebei province, the Zhelu county of the Hebei province. Respectively weighing 1.0g of air-dried soil sample, adding into a triangular flask with sterilized glass beads, adding 99mL of sterile water, standing for 20min, and charging at 30 deg.C and 180r/min on a shaking tableOscillating for 30min, and performing gradient dilution by 10-fold dilution method to obtain 10 solutions^-3，10^-4，10^-5The diluted solution of (2) is 100 mu L, and is coated on an organophosphorus-decomposing culture medium, and each concentration is 3 times; standing in clean bench for 5-10min after coating, adsorbing the bacterial liquid into culture medium, and culturing at constant temperature of 35 deg.C for 5-7 d. And screening out the strains with the phosphate solubilizing function by a transparent ring method and a molybdenum-antimony colorimetric resistance method, screening the capability of the selected strains on controlling the verticillium wilt of the eggplants by a plate confronting method and a pot experiment method, and finally screening out the strains with the dual functions of solubilizing the phosphate and controlling the verticillium wilt of the eggplants, wherein the strains are named as WKPHY-54.

(II) classification and identification of WKPHY-54 strain:

(1) morphological characterization

The thallus is in a rod shape and cultured on an LB culture medium for 10 hours to generate spores which are Zhongsheng, oval, no cyst expansion, negative acid-fast staining, no parasporal crystal, motion and flagellum perigenesis. On a nutrient agar plate, the bacterial colony at the initial culture stage is light milk white, membranous and circular, and the bacterial colony at the later culture stage is light yellow and has irregular edge; carrying out streak culture on a nutrient agar inclined plane to form a straight line shape; the white mycoderm is formed on the surface of the culture medium by static culture in the liquid culture medium. These morphological characteristics were substantially identical to those of Bacillus described in the Manual of identification of common bacteria systems (Dongxu bead et al, science Press, 2001), and it was preliminarily judged that the strain WKPHY-54 belongs to Bacillus.

(2) Identification and classification using 16S rDNA sequences

Carrying out PCR amplification by taking genome DNA of WKPHY-54 as a template and universal primers F27 and R1492 as primers, wherein the primer sequence is as follows: f27: 5'-AGAGTTTGATCATGGCTCAG-3', respectively; r1492: 5'-GGCTACCTTGTTACGACTT-3', respectively; the PCR reaction system (50. mu.L) was 10 XPCR Buffer (Mg)²⁺)5 μ L of genomic DNA of 5. mu.L of dNTP mix (2.5mM)5 μ L, Taq (5U/. mu.L) 1 μ L, F27 (10. mu. mol/L)1 μ L, R1492 (10. mu. mol/L)1 μ L, WKPHY-54 50ng, ddH₂O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.5 min; 10min at 72 ℃. Subjecting the obtained PCR amplification product to gel electrophoresis, and delivering to Shanghai lifeThe 16S rDNA sequence of WKPHY-54 is obtained by sequencing of engineering bioengineering company, Ltd (see SEQ ID No: 1). Homology comparison is carried out on the 16S rDNA sequence of the WKPHY-54 in Genbank, and the result shows that the homology of the strain WKPHY-54 and the 16S rDNA of the bacillus reaches 99 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software, and the result (shown in figure 1) that WKPHY-54 is polymerized with the Bacillus together indicates that WKPHY-54 belongs to the Bacillus.

(3) Identification and classification using gyrB gene sequences

Carrying out PCR amplification by taking WKPHY-54 genome DNA as a template and taking degenerate primers gyrB-F and gyrB-R of a gyrB gene of bacillus as primers; wherein the primer sequence is as follows: gyrB-F: 5 '-TTGRCGGHRGYGGHTATAAAGT-3'; gyrB-R: 5 '-TCCDCCSTCAGARTCWCCCTC-3'. The PCR amplification reaction system (50. mu.L) of gyrB was 10 XPCR Buffer (Mg)²⁺)5 μ L, dNTP mix (2.5mM)5 μ L, Taq (5U/μ L)1 μ L, gyrB-F (10 μmol/L)1 μ L, gyrB-R (10 μmol/L)1 μ L, WKPHY-54 genomic DNA 50ng, ddH₂O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 45s, and 72 ℃ for 1 min; 10min at 72 ℃. The amplified product is sent to Shanghai biological engineering Co., Ltd for sequencing to obtain the gyrB gene sequence of the WKPHY-54 strain (see SEQ ID No: 2). Homology comparison is carried out on the obtained gyrB gene sequence in Genbank, and the result shows that the homology of WKPHY-54 and the gyrB gene sequence of the bacillus subtilis is the highest and reaches 99 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software, and the result (shown in figure 2) shows that the WKPHY-54 strain and the Bacillus subtilis are polymerized together, which indicates that the WKPHY-54 is the Bacillus subtilis and is a new strain.

By combining the morphological characteristics and the results of homology comparison analysis of 16S rDNA and gyrB gene sequences, the WKPHY-54 belongs to Bacillus subtilis, is different from the existing Bacillus strains, and is a new Bacillus subtilis strain.

Example 2 preparation of microbial Agents of the invention containing WKPHY-54 Strain

The method comprises the following steps:

(1) activating strains: activating a bacillus subtilis strain WKPHY-54 (preservation number is CGMCC No.14951) stored at-80 ℃ on an LB plate culture medium (30 ℃), and selecting a single colony to be cultured on an LB slant culture medium at 30 ℃ for 12 hours to obtain an activated strain; wherein the LB plate culture medium or LB inclined plane culture medium comprises the following components in percentage by weight: 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, 15g of agar powder and 1000mL of water;

(2) preparing a seed solution: 100mL of LB liquid culture medium (the components and the weight ratio of the components are 10g of tryptone, 5g of yeast extract, 5g of sodium chloride and 1000mL of water) is filled into a 250mL triangular flask, high-pressure damp-heat sterilization is carried out, after the temperature is reduced to room temperature, a strain activated in the inoculating loop step (1) is inoculated into each flask, and shaking culture is carried out for 12 hours under the conditions of 30 ℃ and the rotating speed of a shaking table of 180rpm, so as to obtain seed liquid;

(3) preparing a corn flour and soybean flour culture medium: according to the weight percentage, 2.5 percent of corn flour, 2.5 percent of soybean meal, 0.6 percent of NaCl and MnSO₄·H₂Adding 0.6% of O into water, and stirring and mixing uniformly to obtain a corn flour and soybean meal culture medium; subpackaging in 500mL triangular bottles, 200mL each; sterilizing the corn flour and soybean meal culture medium at 121 ℃ for 30 minutes, and then cooling to 30 ℃ for later use;

(4) fermentation culture: inoculating 2mL of the seed solution obtained in the step (2) into 200mL of the culture medium of each bottle of corn flour and soybean meal obtained in the step (3); performing fermentation culture at 30 ℃ and a shaking table rotation speed of 180rpm for 36 hours, sampling from a triangular flask every 30 minutes, performing microscopic examination, counting spores and total thallus in a visual field, and calculating the spore rate (%) -mature spore number/(mature spore number + thallus number) × 100); stopping fermentation culture when the spore rate reaches 90%; co-fermenting and culturing for 48 hours to obtain the liquid preparation of the bacillus subtilis WKPHY-54.

The viable bacteria content of the obtained Bacillus amyloliquefaciens WKPHO-12 liquid preparation is 2.6 multiplied by 10 according to the measurement result by adopting a flat plate colony counting method⁸cfu/mL。

Example 3 qualitative determination of the ability of the Strain WKPHY-54 of the present invention to degrade organophosphorus

The method comprises the following steps:

the activated WKPHY-54 strain obtained in step (1) of example 2 was inoculated with sterilized toothpick to organic phosphorus-decomposing plate medium (the composition and weight ratio of the strain are glucose 10.0g and (NH)₄)₂SO₄ 0.2g，MgSO₄·7H₂O 0.5g，KCl 0.1g，MgCl₂·6H₂O5.0 g, calcium phytate 2.0g, agar 20.0g, distilled water 1000mL, pH: 7.0-8.0), placing in a constant temperature incubator at 30 ℃ for culturing for 72 hours, and measuring the diameter of the transparent ring and the diameter of the bacterial colony.

The result shows that the WKPHY-54 strain of the invention generates a transparent ring with the diameter of 13.4 mm on an organic phosphorus plate culture medium containing calcium phytate, and the strain WKPHY-54 can well degrade the organic phosphorus calcium phytate and has the potential of degrading organic phosphorus in soil.

Example 4 quantitative determination of organophosphorus degrading ability of WKPHY-54 Strain of the invention

The method comprises the following steps:

(1) preparing a fermentation medium: 10.0g of glucose and (NH) are mixed according to the weight ratio₄)₂SO₄ 0.2g、MgSO₄·7H₂O 0.5g、KCl 0.1g、MgCl₂·6H₂Adding O5.0 g, calcium phytate 2.0g and agar 20.0g into 1000mL of distilled water, and mixing well to obtain fermentation culture medium (pH: 7.0-8.0); and (3) filling the fermentation medium into a conical flask according to the amount of 100mL/300mL, and sterilizing at high temperature and high pressure for later use.

(2) Preparing fermentation liquor: inoculating the strain WKPHY-54 seed solution obtained in the step (2) of the example 2 and a blank control culture solution (LB liquid culture medium without inoculating the WKPHY-54 strain) into the fermentation culture medium prepared in the step (1) according to the inoculation amount of 2 weight percent respectively, repeating the steps for 3 groups, and culturing at 30 ℃ and 180r/min for 6 days to obtain fermentation liquid.

(3) Plotting OD₇₂₀nm-phosphorus standard curve: respectively and accurately absorbing KH of 5mg/L₂PO₄Adding 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of standard solution into a 50mL colorimetric tube, adding 1-2 drops of 2, 4-dinitrophenol as an indicator, adjusting the pH value by using 10% NaOH and 5% dilute sulfuric acid solution, adding water to enable the total volume of each colorimetric tube to reach 30mL, and shaking up;finally, adding 5.0mL of molybdenum-antimony anti-reagent, mixing uniformly, developing, and fixing the volume. After 30min, the color is compared at the wavelength of 720nm, the phosphor concentration value is taken as the abscissa, the corresponding OD value is taken as the ordinate, and a standard curve is drawn. Obtaining the regression equation y of the phosphorus standard curve which is 0.3457x-0.00176 (R)²0.99927, y-OD value, x-phosphorus concentration).

(4) And (3) treating fermentation liquor: and (3) transferring the fermentation liquor obtained by culturing in the step (2) to a sterile centrifuge cup, and performing ultrasonic cell disruption by adopting a KQ5200DE numerical control ultrasonic wave cleaner (disruption condition: 200 and 240V, 2A, 50/60Hz, time 20min) to release available phosphorus in the cells. Centrifuging at 8000r/min for 10min, adding 2 drops of 2, 4-dinitrophenol as indicator into 2.5mL of supernatant in 50mL colorimetric tube, adjusting pH value with 10% NaOH and 5% dilute sulfuric acid solution until the solution is just yellowish, adding 5mL of molybdenum-antimony color-developing resisting agent, diluting to constant volume, and reacting for 30 min. The OD value of the supernatant at 720nm was measured with a T6 new century UV-Vis spectrophotometer. And obtaining the effective phosphorus content in the supernatant according to a standard curve.

(5) And (4) calculating a result: the sample solution is subjected to colorimetry to obtain an absorption value, then the phosphorus content (mg/L) of the corresponding colorimetric solution is calculated according to a working curve (y is 0.3457x-0.00176), and the effective phosphorus content in the fermentation liquor is calculated according to the following formula: available phosphorus (mg/L) is the phosphorus (mg/L) x dilution of the colorimetric solution.

TABLE 1 quantitative determination test result of organophosphorus degrading ability of WKPHY-54 strain of the invention

Strain numbering	OD720(nm)	Soluble phosphorus content (mg/L)
			WKPHY-54	0.693	40.19
Blank control	0.170	9.86

Compared with the blank control, the content of soluble phosphorus in the fermentation culture solution inoculated with the WKPHY-54 strain disclosed by the invention is increased to 40.19mg/L, and the obvious difference exists between the result (shown in Table 1) and the blank control, so that the WKPHY-54 strain disclosed by the invention has good capability of degrading organic calcium phytate.

Example 5 growth-promoting Effect of the Strain WKPHY-54 of the present invention on eggplant plants

Test materials (one):

(1) matrix: sand + substrate

Wherein: sand is washed for 3 times by water in advance, air-dried for standby, and the pH value is as follows: about 6.0;

substrate: calcium phytate, 1g/kg matrix.

(2) Eggplant variety: agricultural product 601

(II) test treatment:

(1) and (3) treatment: calcium phytate + WKPHY-54 fermentation liquor + phosphorus-deficient nutrient solution

(2) Comparison: calcium phytate, original fermentation medium and phosphorus-deficient nutrient solution

(III) test method: the test was carried out in a laboratory of Ningmu Biotech, Inc., in 1 month of 2016. Eggplant seedlings were cultivated in a nutrition pot, the seedlings were transplanted into a flowerpot (height: 20cm, pot mouth diameter: 21cm, pot bottom diameter: 14cm) containing 3.5kg of sand per pot when the first true leaf was unfolded, 2 plants per pot were placed in a greenhouse for cultivation, and the test was started after seedling revival. Two treatments were set up for the experiment, the treatment was watering 250mL of WKPHY-54 strain fermentation broth dilution (concentration 1X 10) prepared in example 4 step (2)⁷CFU/mL) in the roots of the crops, and the blank control was an equal volume of the original fermentation medium dilution prepared in step (1) of example 4. Each treatment was repeated 3 times, 3 pots each. Normal management in the middle period and timely supplementWater, 400mL per pot each time. And pouring the phosphorus-deficient nutrient solution once every 7 days (the components of the phosphorus-deficient nutrient solution are shown in patent application 201110107663X), and every time, 250mL is poured in each pot. And measuring the indexes of the height, the fresh weight of the overground part, the fresh weight of the underground part, the available phosphorus in the matrix, the phosphorus content in the eggplant plant body and the like after 50 days.

TABLE 2 test results of the effect of WKPHY-54 strain on eggplant growth

Compared with a blank control, the result (shown in table 2) shows that the height of the eggplant plant treated by the bacillus subtilis WKPHY-54 fermentation liquor is increased by 1.66 percent, and has no significant difference with the control; the fresh weight growth rates of the overground part and the underground part are respectively 27.98 percent and 42.23 percent, and the fresh weight growth rates are different from those of the blank control in significance. The results show that the WKPHY-54 strain can obviously promote the growth of eggplants.

TABLE 3 test results of the effect of the WKPHY-54 strain of the present invention on available phosphorus in substrate and eggplant plants

As can be seen from Table 3, the increase rate of available phosphorus in the substrate was 12.26% and the increase rate of available phosphorus in the eggplant plants was 105.36% after treatment with the WKPHY-54 strain of the present invention. The strain WKPHY-54 can effectively degrade the organic phosphorus in the matrix and promote the absorption of the degraded effective phosphorus by eggplant plants, thereby promoting the growth of the eggplant plants.

Example 6 test of inhibitory Effect of the inventive Strain WKPHY-54 on Verticillium dahliae

(one) test pathogenic bacterial strains: eggplant Verticillium wilt bacterium EVD-1: eggplant diseased fruits separated from Zhang Zhengchang shop village of Changxiang county of Qingyuan of Baoding city are separated and purified by Baoding micro-control biotechnology limited company and identified as Verticillium dahaliae by Hebei agricultural university, and the pathogenicity is measured to show strong pathogenicity.

(II) test method:

first, eggplant Verticillium wilt bacterium EVD-1 is activated and cultured on a PDA plate for 3-7 days, and then a hole puncher is used for

Punching holes on the edge area of the bacterial colony to prepare bacterial sheets; the cell piece was transferred to the center of another PDA plate, and the WKPHY-54 strain activated in step (1) of example 2 was spotted at a distance of 2.0 cm from the indicator cell piece, with a blank control (no spot inoculation of WKPHY-54 strain). Culturing at constant temperature of 25 deg.C for 3-10 days, observing the growth conditions of WKPHY-54 strain and pathogenic fungi to be tested day by day, and measuring the control growth amount (colony radius) of pathogenic bacteria and the growth amount (growth inhibiting radius after inoculation of WKPHY-54) treated by the strain of the invention when the blank control pathogenic bacteria grow to the edge of the culture dish, wherein the antagonistic action is expressed by bacteriostatic rate. The formula for calculating the bacteriostasis rate is as follows:

the bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100).

The result (shown in table 4) shows that the bacterial strain WKPHY-54 has an inhibitory rate of 63.12% on verticillium wilt of eggplant, and the WKPHY-54 has an obvious inhibitory effect on verticillium wilt of eggplant.

TABLE 4 test results of the bacteriostatic action of WKPHY-54 strain of the present invention on verticillium wilt of eggplant

Pathogenic bacteria	Normal growth (mm)	Inhibition of growth (mm)	Bacteriostatic ratio (%)
				Eggplant Verticillium (V.dahliae)	32.0	11.8	63.12

Example 7 Effect test of liquid preparation of the Strain WKPHY-54 of the present invention on controlling eggplant Verticillium wilt

Test materials (one):

(1) eggplant variety to be tested: farming land 601; is an eggplant variety bred by the university of Hebei agriculture.

(2) Test pathogenic bacteria strains: eggplant Verticillium dahliae EVD-1.

(II) test treatment:

(1) WKPHY-54 liquid preparation: the WKPHY-54 liquid formulation prepared in example 2 was diluted 100-fold with water.

(2) Medicament control: 10 hundred million viable spores/gram bacillus subtilis wettable powder (Baodin, Lufeng Biochemical technology, Inc.); diluted 100 times with water.

(3) Blank control: clean water

(III) test method:

soaking seeds in 100 times of water diluent of the WKPHY-54 liquid preparation prepared in example 2 for half an hour before sowing; soaking seeds in 100 times of water diluent of 10 hundred million live spores/gram of bacillus subtilis wettable powder for half an hour as medicament contrast; seeds are soaked in clear water for half an hour as a blank control. And (5) normally culturing after sowing. When 4 th to 5 th true leaves grow from the eggplant seedlings, adopting a root cutting method to inoculate spore suspension (10) of verticillium wilt of eggplant⁷spores/mL). And (5) performing conventional management, continuously culturing until a blank control is fully developed, investigating disease indexes, and calculating the prevention and treatment effect.

The result (table 5) shows that the WKPHY-54 strain has 72.60% of effect on controlling the verticillium wilt of eggplants, which is equivalent to the effect (70.29%) of the control of medicaments on the verticillium wilt of the eggplants. The WKPHY-54 and the liquid preparation thereof have good control effect on the verticillium wilt of eggplants.

TABLE 5 test results of the WKPHY-54 of the present invention on the verticillium wilt of eggplant

Example 8 Effect test of solid preparation of the Strain WKPHY-54 of the present invention on controlling verticillium wilt in eggplant

Test materials (one):

(1) eggplant variety: the farm is 601.

(2) Test pathogenic bacteria strains: eggplant Verticillium dahliae EVD-1.

(II) test treatment:

(1) WKPHY-54 powder: according to the following steps of 1: calcium carbonate was added to the WKPHY-54 liquid preparation prepared in example 2 at a ratio of 1, and stirred uniformly to obtain WKPHY-54 powder.

(2) Medicament control: 10 hundred million viable spores/gram bacillus subtilis wettable powder (Baodin, Lufeng Biochemical technology Co., Ltd.).

(3) Blank control: untreated

(III) test method:

sterilizing the seedling-raising soil by high-pressure steam for 2 hours, placing the seedling-raising soil to room temperature, paving a seedling-raising tray, planting 1 eggplant seedling in each hole of the seedling-raising tray, and culturing the eggplant seedlings until 4-5 true leaves are obtained for later use. Inoculating verticillium wilt fungus EVD-1 mycelium block in PDB culture solution, culturing at 25 deg.C and 180rpm/min for 5 days, filtering to remove mycelium to obtain conidia, mixing conidia into sterile seedling substrate to make the concentration of verticillium wilt fungus EVD-1 conidia 10⁶One per gram of soil. The mixed seedling substrate with the bacteria is put into a flowerpot (the diameter is 15cm), 2.0g of WKPHY-54 powder is applied to the position where the eggplant seedlings are planted, the eggplant seedlings are transplanted into the flowerpot (the seedling tray is lifted during transplanting to cut off the root tips of the eggplant seedlings so as to be beneficial to infection of pathogenic bacteria), the eggplant seedlings are covered with the soil with the bacteria, and one eggplant seedling is planted in each pot. 2.0g of bacillus subtilis wettable powder applied in a hole of 10 hundred million spores/g is taken as a medicament control, and eggplant seedlings directly transplanted without treatment are taken as a blank control. Each treatment was repeated 3 times, 5 pots each. Normal culture after transplantation to blank controlWhen the disease is fully developed, the disease index is investigated, and the prevention and treatment effect is calculated.

The result (table 6) shows that the prevention effect of the strain WKPHY-54 of the invention on the verticillium wilt of eggplants can reach 71.79 percent, which is equivalent to the effect (74.44 percent) of the control microbial bactericide on the verticillium wilt of eggplants. The strain WKPHY-54 and the solid preparation thereof have good control effect on the verticillium wilt of eggplants.

TABLE 6 test results of the present invention strain WKPHY-54 on controlling verticillium wilt in eggplant

Treatment of	Index of disease condition	Control effect (%)
			WKPHY-54 powder	22.69b	71.79
10 hundred million live spores/gram bacillus subtilis wettable powder	20.56b	74.44
			Blank control	80.43a	--

Example 9 test of inhibitory Effect of the Strain WKPHY-54 of the present invention on three pathogenic bacteria

Test pathogenic bacterial strain

(1): cucumber fusarium wilt bacterium FOC-1: cucumber plant isolated from Country Tokyo village of Dongdongbao county, dingxing, baoding City, and identified as Fusarium oxysporum cucumber transformant (Fusarium oxysporum f.sp. Cucumebrium) by Hebei agriculture university

(2): cotton wilt pathogen FOV-7: is separated from diseased cotton plant in Weixian county, from the Chachentai city, and is identified as Fusarium oxysporum wilting specialization type (Fusarium oxysporum f.sp.vasifectum) by the university of agriculture in Hebei

(3): potato black nevus RS-3: the potato diseased plant is separated from Shanghai city Shang Yi county, Jiahe county, Xiyanmukucun and identified as Rhizoctonia solani by Hebei agriculture university

The three strains have strong pathogenicity through the pathogenicity measurement.

(II) test method:

the test was carried out in a laboratory of Ann micro-controlled Biotechnology, Inc. in 6 am 2017. Firstly, the pathogenic bacteria to be tested are activated and cultured on a PDA plate for 4 days, and then a puncher is used

Punching holes on the edge area of the bacterial colony to prepare a bacterial plate, then transferring the bacterial plate to the center of another PDA flat plate, and then dotting the activated bacillus amyloliquefaciens WKPHY-54 obtained in the step (1) of the example 2 at a position 2.0 cm away from the bacterial plate of the indicator, and setting a blank control (not dotting the WKPHY-54 strain). Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculating WKPHY-54) of Botrytis cinerea when blank control is about to grow over the whole culture dish, and expressing antagonistic effect by antibacterial rate. The calculation formula of the bacteriostatic rate is as follows:

the bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100.

TABLE 7 test results of the inhibitory effect of the inventive strain WKPHY-54 on three pathogenic bacteria

The result (see table 7) shows that the inhibition rate of the strain WKPHY-54 of the invention on cucumber fusarium wilt is 74.55%, the inhibition rate on cotton fusarium wilt is 74.63%, and the inhibition rate on potato black nevus is 77.59%, which shows that the bacillus subtilis WKPHY-54 has obvious inhibition effect on the three pathogenic bacteria, has wide antibacterial spectrum, and has biological control potential for preventing and treating plant diseases such as cucumber fusarium wilt, cotton fusarium wilt, potato black nevus disease and the like.

Sequence listing

<110> Baoding micro-control Biotechnology Ltd

<120> bacillus subtilis with dual functions of degrading organic phosphorus and preventing diseases

<130> 2017S1138INH

<141> 2017-12-27

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1437

<212> DNA

<213> Bacillus subtilis

<400> 1

atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 60

gagtaacacg tgggtaacct gcctgtacga ctgggataac tccgggaaac cggggctaat 120

accggatggt tgtttgaacc gcatggttca aacataaaag gtggcttcgg ctaccactta 180

cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg 240

cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactcctt 300

cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 360

tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtaccgttc 420

gaatagggcg gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 480

gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttggccgtaa agggctcgca 540

ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 600

tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 660

agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 720

cgaaagcgtg gggtgcgaac aggattagat accctggtag tccacgccgt aaacgatgag 780

tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc 840

ctggggagta cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg 900

tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct 960

gacaatccta gagataggac gtccccttcg ggggcagagt gacaggtggt gcatggatgt 1020

cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt 1080

agttgccagc attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt 1140

ggggatgacg tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga 1200

cagaacaaag ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc 1260

ggatcgcagt ctgcaactcg actgcgtgaa gctggaatcc ctagtaatcg cggatcagca 1320

tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg 1380

taacacccga agtcggtgag gtaacctttt aggagccagc cgccgaaggt gggacag 1437

<210> 2

<211> 808

<212> DNA

<213> Bacillus subtilis

<400> 2

cgtgacggta gaattcaccg ccaaacctat aaacgcggag ttccggttac agaccttgaa 60

atcattggcg aaacggatca tacaggaacg acgacacaat ttgtcccgga ccctgaaatt 120

ttctcagaaa caaccgagta tgattacgat ctgcttgcca accgcgtgcg tgaattagcc 180

tttttaacaa agggcgtaaa catcacgatt gaagataaac gtgaaggaca agagcgcaca 240

aatgaatacc attacgaagg cggaattaaa agttatgtag agtatttaaa ccgctctaaa 300

gaggttgtcc atgaagagcc gatttacatt gaaggcgaaa aggacggcat tacggttgaa 360

gtggctttgc aatacaatga cagctacaca agcaacattt actcgtttac aaacaacatt 420

aacacgtacg aaggcggtac ccatgaagct cgcttcaaaa cgggcctgac tcgtgttatc 480

aacgattacg ccagaaaaaa agggcttatt aaagaaaatg atccaaacct aagcggagat 540

gacgtaaggg aagggctgac agcgattatt tcaatcaaac accctgatcc gcattttgag 600

ggccaaacaa aaacaaagct gggcaactca gaagcacgga cgatcaccga tacgttattt 660

tctacggcga tggaaacatt tatgctggaa aatccagatg cagccaaaaa aattgtcgat 720

aacggtttaa tggcggcaag agcaagaatg gctgcgaaaa aagcgcgtga actaacacgc 780

cgtaagagtg ctttggaaat ttcaaacc 808

Claims

1. A Bacillus subtilis strain WKPHY-54 with a preservation number of CGMCC No. 14951.

2. The application of the strain WKPHY-54 of claim 1 in degrading organic phosphorus and preventing and controlling plant diseases; wherein the plant diseases refer to eggplant verticillium wilt, cucumber wilt, cotton wilt and potato black nevus.

3. A microbial agent comprising the strain WKPHY-54 of claim 1.

4. The use of the microbial agent of claim 3 for degrading organophosphorus and controlling plant diseases; wherein the plant diseases refer to eggplant verticillium wilt, cucumber wilt, cotton wilt and potato black nevus.