CN108192838B

CN108192838B - Bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention

Info

Publication number: CN108192838B
Application number: CN201711454747.4A
Authority: CN
Inventors: 马毅楠; 李博超; 孔令佳
Original assignee: Individual
Current assignee: Qi Yongxin
Priority date: 2017-12-28
Filing date: 2017-12-28
Publication date: 2021-04-30
Anticipated expiration: 2037-12-28
Also published as: CN108192838A

Abstract

The invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain WKPHO-12, wherein the preservation number of the strain is CGMCC No. 14954. The invention also discloses a microbial agent containing the strain. The strain WKPHO-12 has dual functions of degrading inorganic phosphorus in soil and preventing diseases, namely, the strain can efficiently degrade the inorganic phosphorus in the soil, improve the utilization rate of the inorganic phosphorus in the soil and reduce the application amount of a phosphate fertilizer; the control effect on plant diseases such as eggplant verticillium wilt is high, and the average control effect is over 70 percent; secondly, the bacterial strain WKPHO-12 has wide antibacterial spectrum and has good inhibition effect on verticillium wilt of eggplants, fusarium wilt of cucumbers, fusarium wilt of cotton, potato black nevus and the like; in addition, the strain WKPHO-12 has strong specialization, good drug effect durability and environmental protection, and can also greatly reduce the production cost and promote the sustainable development of agriculture.

Description

Bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention

Technical Field

The invention belongs to the field of agricultural microorganisms, and particularly relates to bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention, a microbial agent containing the bacillus amyloliquefaciens, and application of the bacillus amyloliquefaciens in crop disease prevention and control and crop growth promotion.

Background

Phosphorus is one of three major elements in plant nutrition, is also an important component of organic compounds in plants, and plays an essential role in the growth and development of plants. Although the phosphorus content in the soil is high, about 95% of phosphorus in the soil is in an ineffective form, and the inorganic phosphorus which can be directly absorbed and utilized by plants only accounts for 2-3% of the total phosphorus. 74 percent of cultivated land in China is lack of phosphorus, and if chemical fertilizers are applied for a long time to supplement the phosphorus in the soil, the proportion of nutrients in the soil is disordered and the physical and chemical properties of the soil are changed. Improving the utilization rate of phosphorus in soil is the key to solve the phosphorus deficiency. The phosphate solubilizing bacteria can convert insoluble or insoluble phosphorus in soil into microorganisms which can be easily absorbed and utilized by plants, and in addition, the phosphate solubilizing bacteria can promote metabolic activity of beneficial microorganisms in the soil, improve root nutrition of the plants and improve crop yield.

Currently, known phosphate solubilizing bacteria are mostly concentrated in Bacillus (Bacillus), such as Bacillus amyloliquefaciens (CN105385638A, CN105420156A), Bacillus subtilis (CN 104263679A, CN103773709A), Bacillus megaterium (Wuhaiyan, etc., Gillen university Committee, 2014(2): 171-. None of the known phosphate solubilizing bacteria relate to the bacteriostatic and disease-preventing effects of the phosphate solubilizing bacteria.

Eggplant Verticillium wilt is a soil-borne disease caused by Verticillium dahliae (Verticillium dahliae). In recent years, with the rapid expansion of the planting area of eggplants in protected areas, the verticillium wilt of the eggplants is increased year by year under special conditions such as light temperature, water and fertilizer and continuous cropping. The eggplant has few fruit bearing after being infected by diseases, and the plant is premature senility and even dies. The general incidence rate is 15-20%, the incidence rate of serious cases is 30-50%, and the influence on the yield and quality of eggplants is great.

At present, the method for preventing and treating the verticillium wilt of eggplants in production mainly comprises grafting prevention and chemical prevention. But the grafting workload is large, and the popularization is difficult; the long-term use of a large amount of chemical pesticides results in the generation of drug residues and drug resistance. Therefore, the beneficial microorganisms are used for preventing and treating the verticillium wilt of the eggplant, and the method is a prevention and treatment measure with application prospect. Currently, Bacillus for preventing and treating eggplant verticillium wilt mainly comprises Bacillus amyloliquefaciens (CN105238723A), Bacillus subtilis (CN 102925394A; CN 102154186A; Luhong science, etc., 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 environmental conditions and change, so that the bacillus with high-efficiency phosphorus dissolving function and wide antibacterial spectrum is 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 amyloliquefaciens strain which has double functions of degrading inorganic phosphorus, preventing and treating verticillium wilt of eggplants and the like.

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

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

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.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a Bacillus amyloliquefaciens strain WKPHO-12 with a preservation number of CGMCC No. 14954.

The application of the strain WKPHO-12 in degrading inorganic phosphorus.

The strain WKPHO-12 is applied to degrading inorganic phosphorus in soil.

The application of the strain WKPHO-12 in promoting the growth of crops.

The application of the strain WKPHO-12 in preventing and treating plant diseases.

The plant diseases in the application include eggplant Verticillium wilt (Verticillium dahliae Kleb), cucumber wilt (F.oxysporum f.sp.cucumerium), cotton wilt (F.oxysporum f.sp.vassifnfectium), potato black nevus (R.solani) and the like.

The strain WKPHO-12 is applied to the prevention and treatment of verticillium wilt of eggplants and the degradation of inorganic phosphorus in soil.

The invention also provides a microbial agent containing the bacillus amyloliquefaciens strain WKPHO-12.

The microbial agent is in a liquid preparation or a solid preparation.

The viable count of the strain WKPHO-12 in the microbial agent is 2.0 × 10⁶～2.0×10⁸cfu/mL or 2.0X 10⁶～2.0×10⁸cfu/g。

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

(1) activating strains: activating the WKPHO-12 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 obtained in the step (1) by using a sterile inoculating ring, inoculating the ring 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; then detecting the quantity of the thalli and spores in the fermentation liquid every 30 minutes, 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 the liquid preparation of the WKPHO-12 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 or 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 inorganic phosphorus.

The microbial agent is applied to degrading inorganic 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 prevention and treatment of verticillium wilt of eggplants and degradation of inorganic phosphorus in soil.

The use method of the microbial agent comprises the following steps: diluting the microbial agent with water until viable bacteria count is 10⁷cfu/mL, soaking seeds for half an hour before sowing the eggplants; or adsorbing the microbial agent by using calcium carbonate to prepare WKPHO-12 powder, and mixing the powder according to the seed ratio of 1: 10 seed dressing.

The invention also provides a genetic engineering bacterium taking the strain WKPHO-12 as a receptor bacterium. The genetically engineered bacterium improves the capability of degrading inorganic phosphorus, or improves the control effect on pathogenic bacteria, or improves the dual functions of degrading inorganic phosphorus and controlling pathogenic bacteria.

The invention has the advantages and beneficial effects that: (1) the bacillus amyloliquefaciens WKPHO-12 is a microorganism which can degrade inorganic phosphorus and prevent plant diseases and has double functions, and can realize multiple purposes of one bacterium. The application of the strain can improve the utilization rate of inorganic phosphorus in soil, reduce the application amount of phosphate fertilizer, promote the growth of crops and reduce the production cost, and has higher control effect on various diseases such as verticillium wilt of eggplants, and the average control effect is more than 70.0 percent. (2) The strain WKPHO-12 has wide antibacterial spectrum, not only has the inhibition effect on verticillium wilt of eggplants, but also has better inhibition effect on fusarium wilt of cucumbers, fusarium wilt of cottons, potato black nevus and the like. (3) The strain WKPHO-12 has strong specificity to pathogenic bacteria, is not easy to generate drug resistance, has strong drug effect durability, and reduces the application amount and the application times. (4) The microbial agent is safe to people and livestock, and has no environmental pollution; (5) the microbial preparation has the advantages of simple preparation method, low cost and simple use.

Biological preservation

The Bacillus amyloliquefaciens strain WKPHO-12 is obtained by self screening of the inventor, is preserved in China general microbiological culture Collection center in 11 months and 22 days 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. 14954.

Drawings

FIG. 1 is a phylogenetic tree diagram of WKPHO-12 strain obtained based on 16S rDNA sequence.

FIG. 2 is a phylogenetic tree diagram of WKPHO-12 strain obtained based on the gyrB gene sequence.

Detailed Description

The invention is further clearly illustrated by the following specific examples, which are not to be construed as 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.

The pathogenic bacterium strain eggplant Verticillium wilt bacterium EVD-1 to be tested, which is referred to in the following examples, is separated from eggplant diseased fruits separated from Zhang Shandong village of Changxiang county, Qingyuan county, Baoding micro-control biotechnology limited company, is separated and purified, is identified as Verticillium dahaliae by Hebei agriculture university, and shows strong pathogenicity through pathogenicity determination.

The test eggplant variety Nongda 601 used in the following examples is a variety bred by Hebei agriculture university.

Example 1 isolation and screening Process and Classification of the Strain WKPHO-12 of the present invention

The process of separating and screening the bacterial strain of the invention comprises the following steps:

5 parts of soil sample, 200g each, are collected at five points in the area of alum mountain phosphate rock in Zhenju county, Zhang Kouzhou province in Hebei in 2015 9 months. 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, fully oscillating at 30 deg.C and 180r/min for 30min on a shaking table, performing gradient dilution according to 10-fold dilution method, respectively taking 10^-3、10^-4、10^-5Coating 100 mu L of the diluted solution on an inorganic phosphorus removal culture medium, wherein each concentration is 3 times; standing in a clean bench for 5-10min after coating, adsorbing the bacterial liquid into a culture medium, and culturing at constant temperature of 35 ℃ for 5-7 d. The strain with inorganic phosphorus degradation capability is screened by a transparent ring method and a molybdenum-antimony colorimetric resistance method, meanwhile, the verticillium wilt of the eggplant is taken as a target, screening is carried out by a flat plate confronting method and a pot experiment method, and finally, the strain with double functions of dissolving phosphorus and preventing and treating the verticillium wilt of the eggplant is screened out and named as WKPHO-12.

(II) classification and identification of WKPHO-12 strain:

(1) morphological characterization

The thallus is rod-shaped on LB culture medium, after 10h of culture, spores are generated, the spores are middle-grown and oval, cysts are not expanded, acid-fast staining is negative, no parasporal crystal exists, and the thallus can move and is periflagellar. On a nutrient agar plate, the colony is light milky white, purulent, round and neat in edge at the initial culture stage, the colony is raised into a steamed bun shape, and the surface is moist; the bacterial colony in the later culture period is light yellow, the edge is irregular, and the surface is dry and has wrinkles; 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 (east elegans bead et al, science Press, 2001), and it was preliminarily judged that the WKPHO-12 strain belongs to Bacillus.

(2) Identification and classification using 16S rDNA sequences

Carrying out PCR amplification by taking genome DNA of WKPHO-12 as a template and universal primers F27 and R1492 as primers, wherein the primer sequences are as follows: f27: 5'-AGAGTTTGATCATGGCTCAG-3', respectively; r1492: 5'-GGCTACCTTGTTACGACTT-3', respectively; wherein the reaction system (50. mu.L) of PCR is 10 XPCR Buffer (Mg)²⁺)5 μ L, dNTP mix (2.5mM)5 μ L, Taq (5U/μ L)1 μ L, F27(10 μmol/L)1 μ L, R1492(10 μmol/L)1 μ L, genomic DNA50ng of WKPHO-12; 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 ℃. And carrying out gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering Co., Ltd for sequencing to obtain a 16S rDNA sequence (shown as SEQ ID No:1) of WKPHO-12. Homology comparison is carried out on the 16S rDNA sequence of the WKPHO-12 in Genbank, and the result shows that the homology of the strain WKPHO-12 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 WKPHO-12 is polymerized with Bacillus, indicates that the WKPHO-12 belongs to the Bacillus.

(3) Identification and classification using gyrB gene sequences

Carrying out PCR amplification by taking WKPHO-12 genome DNA as a template and taking bacillus gyrB gene degenerate primers gyrB-F and gyrB-R as primers to obtain a PCR amplification product; wherein the sequences of the gyrB-F primers and the gyrB-R primers are as follows: gyrB-F: 5 '-TTGRCGGHRGYGGHTATAAAGT-3'; gyrB-R: 5 '-TCCDCCSTCAGARTCWCCCTC-3'; the PCR amplification reaction system of gyrB is 50 μ L, 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, WKPHO-12 genomic DNA50ng, 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 delivered to Shanghai biological engineering limited company for sequencing to obtainThe gyrB gene sequence of WKPHO-12 strain (see SEQ ID No: 2). Homology comparison is carried out on the obtained gyrB gene sequence of the WKPHO-12 strain in Genbank, and the result shows that the homology of the WKPHO-12 and the gyrB gene sequence of the bacillus amyloliquefaciens is the highest and reaches 99 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software, and the result (shown in figure 2) that the WKPHO-12 strain and the Bacillus amyloliquefaciens are polymerized together indicates that the WKPHO-12 strain is the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) 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 WKPHO-12 belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and is different from the existing Bacillus amyloliquefaciens strain and is a new Bacillus amyloliquefaciens strain.

Example 2 preparation of microbial Agents of the invention containing WKPHO-12 Strain

The method comprises the following steps:

(1) activating strains: activating a bacillus amyloliquefaciens strain WKPHO-12 (the preservation number is CGMCC No.14954) preserved at-80 ℃ on an LB plate culture medium (30 ℃), and selecting a single colony to be cultured on an LB inclined culture medium at 30 ℃ for 12 hours to obtain an activated strain; 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; at 121 ℃ toSterilizing a corn flour and soybean flour culture medium for 30 minutes, and 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%; and carrying out co-fermentation culture for 48 hours to obtain the liquid preparation of the bacillus amyloliquefaciens WKPHO-12.

The viable bacteria content of the WKPHO-12 liquid preparation is 2.3 multiplied by 10 measured by a flat plate colony counting method⁸cfu/mL。

Example 3 qualitative determination of the ability of the Strain WKPHO-12 of the present invention to degrade inorganic phosphorus

The method comprises the following steps:

the WKPHO-12 strain activated in step (1) of example 2 was inoculated with sterilized toothpicks in a medium (comprising 10.0g of glucose (NH4) for inorganic phosphorus removal)₂SO₄0.5g，MgSO₄·7H₂O 0.5g，NaCl 0.2g，Ca₃(PO₄)2 5.0g，KCl 0.2g，MnSO₄ 0.03g，FeSO₄0.003g, agar 20.0g, distilled water 1000mL, pH: 7.0-8.0), then placing in a constant temperature incubator at 30 ℃ for 5 days, and measuring the diameter of the transparent ring and the diameter of the bacterial colony.

As a result, the strain WKPHO-12 of the present invention contained Ca₃(PO₄)₂The transparent ring with the diameter of 13.6 mm is generated on the inorganic phosphorus plate culture medium, which shows that the WKPHO-12 strain can well degrade inorganic phosphorus Ca₃(PO₄)₂And has the potential of degrading inorganic phosphorus in soil.

Example 4 quantitative determination of inorganic phosphorus degrading ability of the inventive Strain WKPHO-12

The test was carried out in the climatic chamber of the fixed micro-controlled Biotechnology Limited in the last 2 months of 2016.

The method comprises the following steps:

(1) preparing a fermentation medium: mixing glucose 10.0g, (NH)₄)₂SO₄ 0.5g，MgSO₄·7H₂O 0.5g，NaCl 0.2g，Ca₃(PO₄)2 5.0g，KCl 0.2g，MnSO₄ 0.03g，FeSO₄Adding 0.003g of the fermentation medium into 1000mL of distilled water, and uniformly mixing to obtain the fermentation medium (pH: 7.0-8.0). 100mL of fermentation medium is filled into a 300mL conical flask and sterilized at high temperature and high pressure for later use.

(2) Preparing fermentation liquor: the WKPHO-12 strain seed solution obtained in the step (2) of the example 2 and a blank control culture solution (LB liquid culture medium without the WKPHO-12 strain) are respectively inoculated into the fermentation culture medium prepared in the step (1) according to the proportion of 2 percent by weight, each group is repeated for 3 times, and the culture is carried out for 6 days at the temperature of 30 ℃ and at the speed of 180r/min, thus obtaining the fermentation liquid.

(3) And (3) treating fermentation liquor: transferring the cultured fermentation liquor into a sterile centrifuge cup, and performing ultrasonic cell disruption by adopting a KQ5200DE numerical control ultrasonic cleaner under the following conditions: 200-240V, 2A, 50/60Hz, and 20 min. So as to release the available phosphorus in the cells. Centrifuging at 8000r/min for 10min, adding 2.5mL of supernatant into 50mL colorimetric tube, adding 2 drops of 2, 4-dinitrophenol as indicator, adjusting pH 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.

(4) And (4) calculating a result: calculating the phosphorus content (mg/L) of the corresponding colorimetric solution on a working curve according to the absorption value obtained by the sample solution colorimetry, and calculating the effective phosphorus content in the fermentation liquor according to the following formula:

available phosphorus (mg/L) is the phosphorus (mg/L) x dilution of the colorimetric solution.

Results (see Table 1) the soluble phosphorus content in the fermentation broth inoculated with the WKPHO-12 strain of the present invention was increased to 68.49mg/L compared to the blank control, indicating that the WKPHO-12 strain of the present invention has the ability to degrade inorganic tricalcium phosphate.

TABLE 1 quantitative determination test results of inorganic phosphorus degradation ability of WKPHO-12 strain of the present invention

Strain numbering	OD₇₂₀(nm)	Soluble phosphorus content (mg/L)
			WKBS-26	1.352	68.49
CK	0.203	10.27

Example 5 test of growth promoting effect of the inventive Strain WKPHO-12 on eggplant plants

Test materials (one):

(1) matrix: sand + substrate

Wherein: washing sand with water for 3 times in advance, and air-drying for later use, wherein the pH value is about 6.0;

substrate: tricalcium phosphate, 1g/kg of matrix.

(II) test treatment:

(1) and (3) treatment: tricalcium phosphate, WKPHO-12 fermentation liquor and phosphorus-deficient nutrient solution;

(2) comparison: tricalcium phosphate, original fermentation medium and phosphorus-deficient nutrient solution.

(III) test method: the test was carried out in a laboratory of Ningmu Biotech, Inc. in the first 6 th month of 2016. Culturing 601 eggplant seedlings in a nutrition pot, transplanting the seedlings into a flowerpot with the sand content of 3.5 kg/pot when the first true leaves are unfolded(height: 20cm, pot mouth diameter: 21cm, pot bottom diameter: 14cm), 2 plants per pot were cultured in a greenhouse, and after seedling rejuvenation, the test was started. And (3) experimental setting: the treatment was performed by pouring 250mL of a diluted solution (concentration: 1X 10) of the fermentation broth of WKPHO-12 strain prepared in the step (2) of example 4⁷CFU/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. Repeat 3 times, each 3 pots. During normal management, replenishing water at proper time, and every time, 400mL per pot; and pouring the phosphorus-deficient nutrient solution once at 7d (the components of the phosphorus-deficient nutrient solution are shown in patent application 201110107663X), and each time, 250mL per 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.

The result (shown in table 2) shows that the height of the eggplant plant treated by the fermentation liquor of the bacillus amyloliquefaciens WKPHO-12 is increased by 3.17 percent and has no significant difference with the control; the fresh weight growth rates of the overground part and the underground part are respectively 24.76 percent and 35.55 percent, and the fresh weight growth rates are different from those of the blank control in significance. The results show that the bacillus amyloliquefaciens WKPHO-12 strain can obviously promote the growth of eggplants.

Table 2 test results of the influence of the strain WKPHO-12 of the present invention on the plant height and fresh weight of potted eggplant

As can be seen from Table 3, the increase rate of available phosphorus in the substrate was 10.31% and the increase rate of available phosphorus in the eggplant plants was 107.05% after treatment with the Bacillus amyloliquefaciens WKPHO-12 of the present invention. The strain WKPHO-12 can effectively degrade inorganic phosphorus in the matrix and promote the eggplant plants to absorb the degraded effective phosphorus, thereby promoting the growth of the eggplant plants.

TABLE 3 test results of the effect of the inventive strain WKPHO-12 on the available phosphorus of substrate and eggplant plants

Example 6 test of inhibitory Effect of the inventive Strain WKPHO-12 on Verticillium dahliae

The method comprises the following steps:

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 pieces were transferred to the center of another PDA plate, and the activated Bacillus amyloliquefaciens WKPHO-12 in step (1) of example 2 was spotted at a distance of 2.0 cm from the indicator cell pieces, and a blank control (no spot inoculation of WKPHO-12 strain) was set. Culturing at 25 deg.C for 3-10 days, observing growth conditions of WKPHO-12 strain and pathogenic fungi to be tested day by day, when blank control pathogenic bacteria grow to the edge of the culture dish, measuring control growth amount (colony radius) of the pathogenic bacteria and growth amount (growth inhibiting radius after inoculation of WKPHO-12) treated by Bacillus amyloliquefaciens WKPHO-12, and expressing antagonistic action 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 (see table 4) shows that the bacteriostasis rate of the strain WKPHO-12 of the invention to the verticillium wilt of eggplant reaches 62.72 percent, which shows that the strain WKPHO-12 has obvious inhibition effect on the verticillium wilt of eggplant and has biocontrol potential on the verticillium wilt of eggplant.

TABLE 4 results of experiments on the bacteriostatic action of WKPHO-12 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)	33.8	12.6	62.72

Example 7 test of controlling Effect of the WKPHO-12 liquid preparation of the present invention on eggplant Verticillium wilt

(I) test treatment:

(1) WKPHO-12 liquid system: the WKPHO-12 liquid formulation prepared in example 2 was diluted 100-fold with water.

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

(3) Blank control: clean water

(II) test method:

the test was carried out in a laboratory of fixed micro-control biotechnology limited in 2017 and 3 months. Soaking seeds for half an hour by using 100 times of water diluent of the WKPHO-12 liquid preparation prepared in example 2 before sowing; soaking seeds in 100 times of water diluent of 10 hundred million viable spores/gram bacillus amyloliquefaciens 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 eggplant EVD-1⁷spores/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 control effect of the WKPHO-12 of the invention on the verticillium wilt of the eggplant is 72.48%, which is similar to the control effect (69.58%) of the medicament control on the verticillium wilt of the eggplant. The strain WKPHO-12 and the liquid preparation thereof have good control effect on the verticillium wilt of eggplants.

TABLE 5 test results of the prevention effect of the WKPHO-12 liquid preparation of the present invention on verticillium wilt of eggplant

Example 8 test of controlling Effect of the WKPHO-12 solid preparation of the present invention on verticillium wilt of eggplant

(I) test treatment:

(1) WKPHO-12 powder: according to the following steps of 1: 1 ratio calcium carbonate was added to the WKPHO-12 liquid preparation prepared in example 2 to prepare WKPHO-12 powder.

(2) Medicament control: 10 hundred million viable spores/gram bacillus amyloliquefaciens wettable powder (Baoding, Lufeng Biochemical technology, Inc., Baoding City).

(3) Blank control: untreated

(II) test method:

the test was carried out in a laboratory of fixed micro-control biotechnology limited in 2017 and 6 months. Sterilizing the seedling-raising soil by high-pressure steam for 2 hours, placing the seedling-raising soil to room temperature, paving a seedling-raising disk, fixedly planting 1 Kangda 601 eggplant seedling in each hole of the seedling-raising disk, and culturing the Kangda 601 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 conidium, mixing conidium with sterile seedling substrate to make the concentration of conidium of verticillium wilt fungus be 10⁶One per gram of soil. The mixed seedling substrate with the bacteria is filled into a flowerpot (the diameter is 15cm), 2.0 g of WKPHO-12 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 up 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. 2.0 g of bacillus amyloliquefaciens wettable powder applied in holes 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. Normally culturing after transplanting, investigating disease index when the blank control is fully developed, and calculating the prevention and treatment effect.

The result (table 6) shows that the prevention effect of the strain WKPHO-12 of the invention on the verticillium wilt of eggplants is 70.74 percent, which is equivalent to the control effect (73.30 percent) of medicaments on the verticillium wilt of the eggplants. The strain WKPHO-12 and the solid preparation thereof have good control effect on the verticillium wilt of eggplants.

TABLE 6 test results of the WKPHO-12 solid preparation of the present invention on the control of verticillium wilt of eggplant

Treatment of	Index of disease condition	Control effect (%)
			WKPHO-12 powder	24.94b	70.74
10 hundred million live spores/gram bacillus amyloliquefaciens wettable powder	22.76b	73.30
			Blank control	85.23a	--

Example 9 test of inhibitory Effect of the Strain WKPHO-12 of the present invention on three disease pathogenic bacteria

Test pathogenic bacterial strain

(1): cucumber fusarium wilt bacterium FOC-1: the cucumber plant was isolated from Cucumis sativus of Toyodo village, east-down fort, Dingxing, Baoding, 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 cotton diseased plants in Weixian county, the Chachentai city, and is identified as Fusarium oxysporum wilting specialization type (Fusarium oxysporum f.sp.vas.) by the university of Hebei agriculture.

(3): potato black nevus RS-3: the potato diseased strain is separated from potato diseased strain of Jiahe countryside, western poplar village in Shanghai prefecture of Zhang Kokou, and is identified as Rhizoctonia solani (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

A bacterial plate is made by punching holes on the edge area of a bacterial colony, then the bacterial plate is transferred to the center of another PDA plate, and the activated bacillus amyloliquefaciens WKPHO-12 in the step (1) of the example 2 is spotted at a position 2.0 cm away from the bacterial plate of an indicator, and a blank control (without spotting the WKPHO-12 strain) is arranged. Culturing at constant temperature of 25 deg.C, when blank control is about to grow over the whole culture dish, measuring control growth amount (colony radius) and treatment growth amount (growth inhibition radius after WKPHO-12 inoculation) of Botrytis cinerea, and expressing antagonistic action 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 WKPHO-12 on different pathogenic bacteria

The result (see table 7) shows that the bacterial strain WKPHO-12 of the invention has the inhibition rate of 74.05 percent on cucumber fusarium wilt, 75.38 percent on cotton fusarium wilt and 78.04 percent on potato black nevus germ, which indicates that the bacillus amyloliquefaciens WKPHO-12 has obvious inhibition effect on the three pathogenic bacteria and has biological prevention and control potential for cucumber fusarium wilt, cotton fusarium wilt and potato black nevus.

Sequence listing

<110> Baoding micro-control Biotechnology Ltd

<120> bacillus amyloliquefaciens with dual functions of inorganic phosphorus degradation and disease prevention

<130> 2017S1137INH

<141> 2017-12-27

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1348

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 1

agactgggat aactcaggga aaccggggct aataccggat ggttgtctga accgcatggt 60

tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcgtcg cattagctag 120

ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc 180

cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gactcttccg 240

caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa 300

agctctgttg ttagggcaga acaagtgccg ttcaaatagg gcggcacctt gacggtacct 360

aaccagaaag ccacggctaa gtacgtgcca gcagccgcgg taatacgtag gtggcaagcg 420

ttgtccggaa ttattgggcg taaagggctc gcaggcggta tcttaagtct gatgtgaaag 480

cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga 540

gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag 600

ccgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta 660

gaaaccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc 720

ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa 780

ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 840

cgcgaagaac cttaccaggt cttgacatcc tctgacaatg ctagagatag gacgtcccct 900

tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgacgtg agatgttggg 960

ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct 1020

aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1080

ttatgacctg ggctactcac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag 1140

gttaagccaa tcacacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt 1200

gaagctggaa tcgctagtaa tcgcgaatca gcatgccgcg gtgaatacgt tcccgggcct 1260

tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacca 1320

ttatggagcc agccgccgaa gtgaacag 1348

<210> 2

<211> 828

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 2

gacggaaaaa tccactatca ggcgtacgag tgcggtgtac ctgtggctga tcttgaagtg 60

atcggtgata ctgataagac cggaacgatt aagcacttcg ttccggatcc ggaaatcttc 120

aaagaaacaa tcgtatacga ctatgatctg ctttcaaacc gtgtccggga attggccttc 180

ctgactaaag gcgtaaacat cacgattgaa gacaaacgtg aaggacaaga acggaaaaac 240

gagtaccacg acgaaggcgg aatcaaaagc tatgttgagt acttaaaccg ttccaaagaa 300

gtcgttcatg aagagccgat ttatatcgaa ggcgagaaag acggcataac ggttgaagtt 360

gcgttgcaat acaacgacag ctatacaagc aacatttatt ctttcacaaa taacatcaac 420

acatacgaag gcgggacgca cgaagccgga tttgaaaccg gtctgacccg tgtcataaac 480

gactatgcaa gaagaaaagg gattttcaaa gaaaatgatc cgaatttaag cggagatgat 540

gtgagagaag ggctgactgc cattatttca attaagcacc ctgatccgca attcgaaggg 600

cagacgaaaa cgaagctcgg caactccgaa gcgagaacga tcactgatac gctgttttct 660

tctgcgctgg aaacatccct tcttgaaaat ccggactcag cccgcaaaat cgttgaaaaa 720

ggtttaatgg ccgcaagagc gcggatggca gcgaaaaaag cgcgggaatt gacccgccgc 780

aaaagtgcgc ttgagatttc caatctgccg gacaaactgg cggactgt 828

Claims

1. A Bacillus amyloliquefaciens strain WKPHO-12 with a preservation number of CGMCC No. 14954.

2. The use of the strain WKPHO-12 according to claim 1 for controlling plant diseases and degrading inorganic phosphorus; wherein the plant diseases refer to eggplant verticillium wilt, cucumber wilt, cotton wilt and potato black nevus.

3. A microbial agent comprising the strain WKPHO-12 of claim 1.

4. The microbial agent according to claim 3, wherein the viable count of WKPHO-12 is 2.0 x 10⁶～2.0×10⁸cfu/mL。

5. The use of the microbial inoculant according to claim 3 for controlling plant diseases and degrading inorganic phosphorus; wherein the plant diseases refer to eggplant verticillium wilt, cucumber wilt, cotton wilt and potato black nevus.