CN108587986B

CN108587986B - Bacillus amyloliquefaciens with dual functions of preventing diseases and degrading organic phosphorus

Info

Publication number: CN108587986B
Application number: CN201810622393.8A
Authority: CN
Inventors: 马毅楠; 李博超; 孔令佳
Original assignee: Individual
Current assignee: Yunnan Sijiaotian Agricultural Technology Co.,Ltd.
Priority date: 2018-06-15
Filing date: 2018-06-15
Publication date: 2021-06-01
Anticipated expiration: 2038-06-15
Also published as: CN108587986A

Abstract

The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain WKBA-15, belonging to the field of agricultural microorganisms. The preservation number of the strain is CGMCC No. 14952. The invention also discloses a microbial agent containing the strain WKBA-15. The strain has good control effect on the tomato gray mold, and the average control effect is more than 80%; secondly, the bacterial strain has a wide antibacterial spectrum, and also has a good inhibitory effect on early blight of tomatoes, target spot of cucumbers and late blight of potatoes; meanwhile, the strain has strong capability of degrading organic phosphorus in soil, improves the utilization rate of the organic phosphorus in the soil, can promote the growth of crops, is a functional strain with dual functions, realizes multiple purposes of one strain and greatly reduces the production cost.

Description

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

Technical Field

The invention belongs to the field of agricultural microorganisms, and particularly relates to bacillus amyloliquefaciens with double functions of disease prevention and organic phosphorus degradation; also relates to a microbial agent containing the strain and application of the microbial agent in the aspect of preventing and treating plant diseases such as tomato gray mold and the like.

Background

Tomato gray mold is a more harmful and common disease on tomatoes caused by Botrytis cinerea (Botrytis cinerea). Besides tomato, the tomato plant also can damage more than 20 crops such as eggplant, hot pepper, cucumber, bottle gourd and the like. The annual hazard of low temperature and continuous rainy weather is serious. The tomato leaf, stem, flower and fruit are infected by the strain, and the yield loss caused by common years can reach 20-50%. At present, the method is developed and rises in various places in China, and great threat is brought to the production of tomatoes.

At present, the method for preventing and treating gray mold is mainly chemical prevention and treatment, but the chemical prevention and treatment has serious environmental pollution problem, especially gray mold has the characteristics of fast propagation, large genetic variation and strong adaptability, and is easy to generate drug resistance. Therefore, the search for microbial pesticides with high efficiency, low toxicity, low residue and environmental friendliness to control plant diseases becomes an important way. The bacillus sp has the characteristics of strong reproductive capacity, obvious stress resistance and the like, and is beneficial to industrial production. Thus, bacillus is a relatively ideal biocontrol microorganism.

Currently, Bacillus species for controlling tomato gray mold are mainly Bacillus amyloliquefaciens (Bacillus amyloliquefaciens, CN107338207A, CN107338202A, CN106811435A, CN107058180A, CN107043720A, CN106676047A CN106337032A, CN106434454A, CN105176894A, CN105199996A, Xinbei Zhao, etc., Chinese biological control bulletin 2016 (4): 485): Lihong Xiao, etc., Chinese biological control bulletin, 2016,32(4): 485): Paenibacillus polymyxa (Junbacteriaceae, CN107384836A, CN106085905A), Bacillus Licheniformis (Bacillus Licheniformis, CN107266539A, Von bud, university of northwest Master thesis, 2016), Bacillus subtilis (Bacillus subtilis, CN107164285A, CN106939290A, CN107043721A, CN 105028483A; Cynanchum Kimura, etc., Chinese biological control bulletin, 2017,33 (2); Shuichi university of Bacillus subtilis, CN104946567A, Hi-ji Hai, Hai-shi Hai-Si-Hai-Ha.

Phosphorus is an important nutrient element in the plant growth process, and is not only an important constituent of plant cells, but also essential in the composition of genetic material and the storage of energy. Phosphorus in soil mainly exists in the states of phosphorus-containing organic matters (lecithin, phytic acid and the like) and inorganic phosphorus compounds (calcium phosphate, sodium phosphate and the like), and the insoluble phosphorus is difficult to be absorbed and utilized by plants, so that the growth of the plants is influenced. The phosphate solubilizing bacteria can decompose and convert insoluble phosphorus in soil into soluble phosphorus which is easy to be absorbed and utilized by plants, and can improve the phosphorus supply level of the soil so as to promote the growth of the plants.

Currently, known Bacillus used for degrading organic phosphate fertilizer in soil are Bacillus subtilis (CN 106244504A; Wu-Dong et al, Jiangsu agri-Proc., 2017,33(4): 843-.

The disclosed strain has relatively single function, or only has the functions of bacteriostasis and disease prevention, or only has the function of degrading organic phosphorus; secondly, the biocontrol microorganism is a product of the evolution of pathogenic bacteria and is also an environmental product, and the function of the biocontrol microorganism can be lost along with the change of the pathogenic bacteria or the change of environmental conditions; meanwhile, the diversified biocontrol microorganisms can delay the time for generating resistance of pathogenic bacteria and enhance the durability of the drug effect, so that the continuous screening of new biocontrol microorganisms is the most economic and effective means for coping with the physiological race or/and the environmental change of the pathogenic bacteria.

Disclosure of Invention

The invention aims to provide a bacillus amyloliquefaciens strain which has high control effect on plant diseases such as tomato gray mold and the like and has strong organic phosphorus degradation capability.

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.

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 sixth purpose of the invention is to provide a genetically engineered bacterium.

The seventh purpose of the invention is to provide a molecular marker for identifying the bacillus amyloliquefaciens.

The invention is realized by the following technical scheme:

a Bacillus amyloliquefaciens strain WKBA-15 has a preservation number of CGMCC No. 14952.

The application of the strain WKBA-15 in preventing and treating plant diseases.

The plant diseases in the above application are tomato gray mold (Botrytis cinerea), tomato early blight (Alternari solani), cucumber target spot (Corynespora cassiicola), potato late blight (Phytophthora infestans) and the like. Preferably tomato gray mold (Botrytis cinerea).

The application of the strain WKBA-15 in degrading organic phosphorus.

The application of the strain WKBA-15 in degrading organic phosphorus in soil.

The strain WKBA-15 is applied to preventing and treating tomato gray mold and degrading organic phosphorus in soil.

The invention also provides a microbial agent which contains the strain WKBA-15.

The formulation of the microbial agent can be liquid or solid.

The microbial agent contains WKBA-15 strain with viable count of about 20.0 × 10⁷～20.0×10⁸cfu/mL or 20.0X 10⁷～20.0×10⁸cfu/g。

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

(1) activating strains: activating the WKBA-15 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 the temperature of 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 meal 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; sampling every 30 minutes for microscopic examination, and stopping fermentation culture when the spore rate reaches 90%; obtaining the liquid preparation of the bacillus amyloliquefaciens WKBA-15.

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 preventing and treating plant diseases.

The application of the microbial agent in degrading organic phosphorus.

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

The microbial agent is applied to preventing and treating tomato gray mold and degrading organic phosphorus.

The invention also provides a genetic engineering bacterium which takes the strain WKBA-15 as a receptor bacterium. The genetic engineering bacteria improve the control effect on plant diseases such as tomato gray mold and the like, or improve the capability of degrading organic phosphorus, or both.

The invention also provides a molecular marker for identifying the strain WKBA-15, wherein the molecular marker consists of a nucleotide sequence shown in SEQ ID No. 2.

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, and spraying the leaf surface before the tomato gray mold occurs. Or diluting the obtained microbial agent with water until viable bacteria count is 10 when tomato grows out 2-3 pieces of compound leaves⁷The cfu/mL is used for root irrigation treatment, and each seedling is irrigated with 200mL, so that the purposes of degrading organic phosphorus in soil and promoting the growth of tomatoes can be achieved.

The invention has the advantages and beneficial effects that: (1) the bacillus amyloliquefaciens WKBA-15 has good control effect on the tomato gray mold, the average control effect is more than 80.0 percent, and a new way is provided for the control of the tomato gray mold; (2) the strain WKBA-15 also has good capability of degrading organic phosphorus in soil, can obviously improve the utilization rate of the organic phosphorus in the soil, and has important effects of reducing the application amount of phosphate fertilizer, promoting the growth of crops and improving the yield and the quality; (3) the strain WKBA-15 has double functions of preventing and treating diseases and degrading organic phosphorus, can be used for multiple purposes, and greatly reduces the production cost; (4) the bacterial strain WKBA-15 has a wide antibacterial spectrum, and has a good inhibition effect on tomato gray mold, tomato early blight, cucumber target spot and potato late blight; (5) the bacterial strain WKBA-15 has strong specificity in preventing and treating plant diseases such as tomato gray mold and the like; the drug resistance is not easy to generate, and the drug effect durability is good; (6) the microbial agent is safe to people and livestock, and does not have the problem of environmental pollution; (7) the microbial preparation has the advantages of simple preparation method, low cost and simple use.

Biological preservation

The Bacillus amyloliquefaciens strain WKBA-15 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 at the preservation address of: 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. 14952.

Drawings

FIG. 1 is a phylogenetic tree of WKBA-15 strain obtained from the 16S rDNA sequence.

FIG. 2 is a phylogenetic tree diagram of WKBA-15 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.

The following examples tested a source of Botrytis cinerea strain BC-14: the strain is collected from tomato diseased fruits in white tower shop village in towns of high forests in Xushui county of Baoding city, Hebei province, and is identified as Botrytis cinerea through Hebei agricultural university, and the pathogenicity determination shows strong pathogenicity.

The tomato variety oxsert 968 tested in the examples below was purchased from beijing major source seed company. The variety has high resistance to leaf mold and tomato gray mold.

EXAMPLE 1 screening and isolation procedure and identification Classification of the Strain WKBA-15 of the present invention

Screening and separating process of strain WKBA-15

Soil samples are collected at five points in a tomato greenhouse with serious occurrence of tomato gray mold in Dong Niudou village county of Baoding City of Hebei province in 2015 9 months, and 5 parts of soil samples are collected, wherein each part is about 200 g. After even mixing, weighing 1g of the mixture and putting the mixture into a 250mL sterilized triangular flask, adding 100mL of sterile water, putting the mixture on a shaking table, oscillating the mixture for 30min at 180r/min, standing the mixture for 2h, taking 10mL of supernatant, adding the supernatant into a 50mL sterilized centrifuge tube, carrying out constant temperature water bath at 80 ℃ for 30min, taking 1mL of the supernatant, adding 9mL of sterile water, and obtaining 10mL10^-3Multiplying the soil microorganism suspension, and then diluting the soil suspension to 10^-4、10^-5、10^-6A double diluent; taking 100 mu L of each concentration of microorganism suspension to coat on an LB culture medium plate, repeating each concentration for 3 times, culturing for 1 d-3 d at the constant temperature of 30 ℃, and separating and purifying bacteria. Screening biocontrol bacteria by using tomato gray mold as a target through a flat plate confronting method, an in vitro leaf method and a pot test method; and then the organic phosphine degrading capability of the selected biocontrol bacteria is evaluated by a transparent ring method and a molybdenum-antimony colorimetric resistance method, and a strain with double functions of preventing and treating tomato gray mold and degrading organic phosphorus is screened out from the results, and is named as WKBA-15.

(II) classification and identification of WKBA-15 strain:

(1) morphological characterization

Culturing in LB culture medium to obtain rod-shaped thallus, culturing for 10 hr to produce spore, generating ellipse, preventing cyst from expanding, resisting acid staining, generating negative, having no parasitized crystal, enabling motion, and generating flagellum peritomatically. 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 (Dongxu bead et al, science Press, 2001), and it was preliminarily judged that the strain WKBA-15 belongs to Bacillus.

(2) Identification and classification using 16S rDNA sequences

Carrying out PCR amplification by taking the genome DNA of the WKBA-15 strain as a template and taking universal primers F27 and R1492 as primers, wherein the primer sequences are as follows: f27: 5'-AGAGTTTGATCATGGCTCAG-3', respectively; r1492: 5'-GGCTACCTTGTTACGACTT-3' are provided. 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/. mu.L) 1 μ L, F27(10 μmol/L)1 μ L, R1492(10 μmol/L)1 μ L, and WKBA-15 genomic DNA 50 ng; ddH₂O make up to 50. mu.L. The reaction conditions of PCR were: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.5 min; 10min at 72 ℃. Subjecting the obtained PCRAnd carrying out gel electrophoresis on the amplified product, and sending the gel to Shanghai biological engineering Co., Ltd for sequencing to obtain a 16S rDNA sequence (shown as SEQ ID No.1) of the WKBA-15 strain. Homology comparison is carried out on the 16S rDNA sequence of the WKBA-15 in Genbank, and the result shows that the homology of the strain WKBA-15 and the 16S rDNA of the bacillus reaches 98 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software, and the result (shown in figure 1) that WKBA-15 is polymerized with the Bacillus, indicates that WKBA-15 belongs to the Bacillus.

(3) Identification and classification by using gyrB gene sequence

Carrying out PCR amplification by taking WKBA-15 genome DNA as a template and taking degenerate primers gyrB-F and gyrB-R of a gyrB gene of bacillus as primers to obtain a PCR amplification product; wherein the sequence of the primer is as follows:

gyrB-F：5’-TTGRCGGHRGYGGHTATAAAGT-3’；

gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’。

wherein the PCR amplification reaction system (50 μ L) of gyrB is 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, WKBA-15 genomic DNA 50 ng; 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 ℃. And (3) delivering the amplified product to Shanghai biological engineering Co., Ltd for sequencing to obtain the gyrB gene sequence (shown as SEQ ID No.2) of the WKBA-15 strain. Homology comparison is carried out on the gyrB gene sequence of the WKBA-15 strain in Genbank, and the result shows that the homology of the WKBA-15 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 WKBA-15 strain and the Bacillus amyloliquefaciens are polymerized together indicates that the WKBA-15 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 WKBA-15 strain belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), is different from the existing Bacillus amyloliquefaciens strain, and is a novel Bacillus amyloliquefaciens strain.

Example 2 preparation of microbial Agents of the invention containing Strain WKBA-15

The method comprises the following steps:

(1) activating strains: activating a bacillus amyloliquefaciens strain WKBA-15 stored at the temperature of-80 ℃ on an LB plate culture medium (30 ℃), and selecting a single colony on an LB inclined culture medium to culture for 12 hours at the temperature of 30 ℃ 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-fermentation culturing for 48 hours to obtain WKBA-15 liquid preparation.

The viable bacteria content of the obtained WKBA-15 liquid preparation is 22 multiplied by 10 according to the determination result by adopting a plate colony counting method⁸cfu/mL。

EXAMPLE 3 antagonistic Effect of the inventive Strain WKBA-15 on Botrytis cinerea

The method comprises the following steps:

in 11-month-first ten days of 2015, Botrytis cinerea BC-14 was activated and cultured on a PDA plate for 4 days, and then a puncher was used

A bacterial plate is punched in the edge area of a bacterial colony, then a botrytis cinerea BC-14 bacterial plate is transferred to the center of another PDA plate, and the WKBA-15 strain activated in the step (1) of the example 2 is spotted at a position 2.0 cm away from an indicating bacterial plate, and a blank control is arranged (without spotting the WKBA-15 strain). Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculation of WKBA-15) of Botrytis cinerea when blank control is about to grow over the whole culture dish, 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.

Results (see table 1) the inhibition rate of the WKBA-15 strain of the invention on botrytis cinerea reaches 69.04%; the WKBA-15 strain is proved to have biocontrol potential for preventing and treating tomato gray mold.

TABLE 1 results of antagonistic test of WKBA-15 strain of the present invention against Botrytis cinerea

EXAMPLE 4 Effect test of WKBA-15 Strain of the present invention on controlling Botrytis cinerea

(I) test treatment:

(1) WKBA-15 liquid formulation: the liquid formulation of the strain WKBA-15 prepared in example 2 was diluted 50-fold with water.

(2) Chemical agents: azoxystrobin suspension (250 g/l) (mr. uk) was diluted 500-fold with water.

(3) Blank control: clean water

(II) test method:

culturing Oseit 968 tomato seedlings in a seedling culture plate, transplanting the seedlings into flowerpots with the diameter of 15cm when 2 compound leaves grow out, and planting 1 seedling in each flowerpot. When the plant grows to 5-6 compound leaves, selecting leaves with consistent leaf age and size, washing the surface of the leaves with sterile water, then sucking the water with sterile filter paper, and then soaking the leaves into 50 times of water dilution (containing 10 thalli) of the WKBA-15 microbial inoculum prepared in example 2⁷cfu/mL), the surfaces of the leaves are fully coated with the medicine, then the leaves are placed in a culture dish paved with double layers of sterilized filter paper, and meanwhile, a tomato botrytis cinerea BC-14 bacteria plate is connected to the center of the leaves

And (5) preserving moisture and culturing. The chemical treatment was performed by the same method. Leaves treated with clear water were used as a blank control. And (5) surveying the diameter of the disease spot after the blank control is fully diseased, and calculating the prevention and treatment effect.

The result (see table 2) shows that the liquid preparation of the strain WKBA-15 has the control effect of 70.54 percent on the gray mold of the tomato, and has no significant difference with 79.10 percent of the control effect of a chemical contrast medicament. The strain WKBA-15 and the liquid preparation thereof have outstanding prevention and treatment effects on tomato gray mold.

TABLE 2 test results of the prevention of tomato gray mold by the inventive strain WKBA-15

Treatment of	Diameter of lesion (mm)	Control effect (%)
			WKBA-15 liquid preparation	7.6b	70.54
Chemical agent	5.4b	79.10
			Blank control	25.8a	--

EXAMPLE 5 comparative test of controlling effect of WKBA-15 of the present invention on Botrytis cinerea

(I) test treatment:

(1) WKBA-15 liquid formulation: the WKBA-15 liquid formulation prepared in example 2 was diluted 50-fold with water.

(3) Blank control: clean water

(II) test method:

culturing Oselter 968 tomato seedling in sunlight greenhouse for 3 months 2016, transferring to artificial climate chamber after tomato grows out 8 pieces of compound leaves, selecting tomato seedling with consistent size, and diluting WKBA-15 liquid preparation prepared in example 2 with 50 times of water diluent (containing thallus 10)⁷cfu/mL) were sprayed evenly on tomato seedlings, incubated at 25 ℃ for 24 hours, and then a tomato Botrytis BC-14 bacterial plate was inoculated on each leaflet

And investigating the disease incidence result after the moisture preservation culture for 3d, measuring the diameter (L, W) of each lesion by adopting a cross method, and calculating the lesion area and the prevention and treatment effect. Clear water blank control and chemical agent control were set.

Area of lesion (mm)²)＝π×L×W/4；

The control effect (%) - (control lesion area-treatment lesion area)/control lesion area × 100.

The result (see table 3) shows that the lesion area treated by the strain WKBA-15 is obviously smaller than the lesion area treated by the chemical agent and the blank control, and the expansion of the tomato botrytis cinerea on tomato leaves can be obviously inhibited; the prevention effect of WKBA-15 on the gray mold of the tomato is 82.35 percent, which is obviously higher than that of 68.55 percent of chemical agent. The strain WKBA-15 and the liquid preparation thereof have outstanding prevention and treatment effects on tomato gray mold.

TABLE 3 comparative test results of the control effect of the inventive strain WKBA-15 on tomato gray mold

Treatment of	Area of lesion (mm)²)	Control effect (%)
			WKBA-15 liquid preparation	23.74c	82.35
Chemical agent	42.32b	68.55
			Blank control	134.58a	--

Example 6 comparative test of field control of tomato Gray mold by the inventive Strain WKBA-15

(I) test treatment:

(3) Blank control: clean water

(II) test method:

the experiment is carried out in a tomato greenhouse of Xianheng town Longhuacun in Baoding city of Hebei province from 3 months to 6 months in 2016. At the beginning of the test, the gray mold of the tomato leaf part in the whole shed is moderate, and the diseased leaves are removed before the application of the drug. 3 rows per cell, 4 repetitions, random block permutation. A50-fold aqueous dilution (containing cell 10) of the liquid preparation of the strain WKBA-15 prepared in example 2 was inoculated using an electric knapsack sprayer⁷cfu/mL); chemical treatment and blank control were also provided. Spraying the pesticide for 1 time after 7 days of the first pesticide application, investigating the number of single diseased leaves of each treatment cell on 7 days after the second pesticide application, and calculating the control effect.

The result (shown in table 4) shows that the number of the single plant disease leaves of the tomato gray mold after the liquid preparation treatment of the strain WKBA-15 is obviously lower than that of the single plant disease leaves of the tomato gray mold treated by a blank control and a chemical agent, the control effect reaches 82.13 percent and is obviously higher than that of the chemical agent by 63.98 percent, and the strain WKBA-15 and the liquid preparation thereof have the obvious control effect on the tomato gray mold.

TABLE 4 preventive effect test results of the inventive strain WKBA-15 on tomato gray mold

Treatment of	Leaf number of single plant disease	Control effect (%)
			WKBA-15 liquid preparation	1.22c	82.13
Chemical agent	2.46b	63.98
			Blank control	6.83a	--

Example 7 qualitative determination of the ability of the inventive Strain WKBA-15 to degrade organophosphorus

The method comprises the following steps:

the activated WKBA-15 strain obtained in step (1) of example 2 was inoculated with sterilized toothpicks onto an organophosphorus-decomposing plate medium (composed of 10.0g of glucose 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), then placing the mixture in a 30 ℃ constant temperature incubator to be cultured for 72 hours, and measuring the diameter of the transparent ring and the diameter of a bacterial colony.

The result shows that the strain WKBA-15 of the invention generates a transparent ring with the diameter of 15.8 mm on an organic phosphorus plate culture medium containing calcium phytate, and the strain WKBA-15 of the invention can well degrade the organic calcium phytate and has the potential of degrading organic phosphorus in soil.

EXAMPLE 8 quantitative determination of the ability of the inventive Strain WKBA-15 to degrade organophosphorus

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: the seed solution of the WKBA-15 strain obtained in the step (2) of example 2 and a blank control culture solution (LB liquid culture medium without the WKBA-15 strain) were inoculated into the fermentation medium prepared in the step (1) according to an inoculum size of 2% by weight, 3 replicates of each group were cultured at 30 ℃ and 180rpm for 6 days, and a fermentation broth was obtained.

(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 0.3792x-0.00232 (R)²0.99829, 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 0.3792x-0.00232), 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 5 quantitative determination test results of organophosphorus degrading ability of the strain WKBA-15 of the present invention

Strain numbering	OD720(nm)	Soluble phosphorus content (mg/L)
			WKBA-15	0.718	37.99
Blank control	0.157	8.29

The result (see Table 5) shows that the content of soluble phosphorus in the fermentation culture solution inoculated with the bacillus amyloliquefaciens WKBA-15 is increased to 37.99mg/L compared with that of a blank control, which indicates that the WKBA-15 strain has stronger capability of degrading the organic calcium phosphate.

Example 9 growth-promoting Effect of the inventive Strain WKBA-15 on tomato 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.

(II) test method:

firstly culturing Oseit 968 tomato seedlings, selecting tomato seedlings with consistent growth vigor after 3-4 compound leaves grow out, transplanting the tomato seedlings into flowerpots (the height is 20cm, the diameter of a pot opening is 22cm, and the diameter of a pot bottom is 15cm) filled with 3.5kg of sand, culturing 3 tomato seedlings in a phytotron, and starting a test after the seedlings are revived. Test set-up treatment and control, treatment was a dilution (concentration 1X 10) of the fermentation broth of the WKBA-15 strain prepared in example 2⁷CFU/mL)250mL/pot was irrigated to the roots of the crop, and the blank control was an equal volume of the original fermentation medium dilution prepared in step (3) of example 2.3 replicates, each replicate treating 1 pot. During the period, the tomato pest and disease control management is enhanced, and water is supplemented at the right time, wherein each time is 400 mL/pot. And 5d, pouring the phosphorus-deficient nutrient solution once (the components of the phosphorus-deficient nutrient solution are shown in patent application 201110107663X), and each time, 250mL per pot. And after 40 days, measuring the indexes of the tomato, such as the plant 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 tomato plant body and the like.

TABLE 6 test results of the effect of the WKBA-15 strain of the present invention on the plant height and fresh weight of potted tomatoes

As a result (see Table 6), compared with the blank control, the tomato plant height treated by the WKBA-15 fermentation liquid of the invention is increased by 3.92%; the growth rate of the fresh weight of the overground part is 31.34 percent, the growth rate of the fresh weight of the underground part is 62.52 percent, and the significant difference exists between the fresh weight of the overground part and the fresh weight of the underground part of the tomato plant and the blank control. The results show that the WKBA-15 strain of the invention has very obvious effect of promoting the growth of tomato plants.

TABLE 7 test results of the effect of WKBA-15 strain of the present invention on available phosphorus in substrate and tomato plants

As can be seen from Table 7, the increase rate of available phosphorus in soil is 7.03% after the strain WKBA-15 of the invention is used for treatment; the increase rate of available phosphorus in tomato plants was 127.55%. The strain WKBA-15 can effectively degrade the organic phosphorus in the soil and promote the tomato plant to absorb the degraded effective phosphorus, thereby promoting the growth of the tomato plant.

Example 10 test of inhibitory Effect of Bacillus amyloliquefaciens WKBA-15 on three disease pathogenic bacteria

Test pathogenic bacterial strain

(1) Tomato early blight AS-8: the tomato diseased fruit is separated from tomato diseased fruit in white tower village in township village in Gao forest of Xushui county of Baoding city, Hebei province and is identified as Alternaria solani (Alternari solani) by Hebei agricultural university.

(2) Cucumber leaf blight bacterium CC-6: the cucumber leaf was isolated from Cucumis sativus leaves of Codontobur village of eastern Hebao, Dingxing, Baoding City and identified as Corynespora spinosa (Corynespora cassicola) by Hebei agriculture university.

(3) Potato late blight bacterium PI-2: the potato tuber is separated from potato disease tubers in the Xiyanmukucun county, Jiahe, Shangyu county, Zhang Kokoku, and is identified as Phytophthora infestans (Phytophthora infestans) by Hebei agriculture university.

The three strains have strong pathogenicity through the pathogenicity measurement.

(II) test method:

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 formed by punching holes in 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 WKBA-15 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 WKBA-15 strain) is arranged. Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculation of WKBA-15) of Botrytis cinerea when blank control is about to grow over the whole culture dish, 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.

The results (see table 8) show that the inhibition rate of the bacillus amyloliquefaciens WKBA-15 on the early blight of tomato is 67.58%, the inhibition rate on the cucumber target spot bacteria is 65.93% and the inhibition rate on the potato late blight is 71.00%, which shows that the bacillus amyloliquefaciens WKBA-15 has obvious inhibition effect on the three pathogenic bacteria and has biological control potential for preventing and treating early blight of tomato, cucumber target spot disease and potato late blight.

TABLE 8 test results of inhibition of Bacillus amyloliquefaciens WKBA-15 on three pathogenic bacteria

Pathogenic bacteria	Normal growth (mm)	Inhibition of growth (mm)	Bacteriostatic ratio (%)
				Early blight of tomato (A. solani) AS-8	33	10.7	67.58
Cucumber target spot fungus (C. casilicola) CC-6	32	10.9	65.93
				Potato late blight (P.infestans) PI-2	40	11.6	71.00

Sequence listing

<110> Baoding micro-control Biotechnology Ltd

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

<130> 2018S1238INH

<141> 2018-06-15

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1364

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 1

gatgggagct tgctccctga tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc 60

ctgtaagact gggataactc cgggaaaccg gggctaatac cggatggttg tctgaaccgc 120

atggttcaga cataaaaggt ggcttcggct accacttaca gatggacccg cggcgcatta 180

gctagttggt gaggtaaggg ctcgccaagg cgacgatccg tagccgacct gagagggtga 240

tcggccacac tgggactgag acatggccca gactcatacg ggagacagca gtagggaatc 300

ttccgcaatg gacgaaagtc tgacggagca acaccgcgtg agtgatgaag gaattcggat 360

cgtaaagctc tgttgttagg gaagaacaag tgccgttcaa gtagggcggc accgtgacgg 420

tacctaacca gaaagctacg gctaactacg tgccagcagc cgcggtaata cgtaggcggg 480

aagcgttgtc cggaatgatt gggcgtaaag ggctcgcagg cggtttctta agtctgatgt 540

gaatgccccc ggctcaacag gggagggtca ttggaaactg gggaacttga gtgcagaaga 600

ggagagtgga attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg 660

cgaaggcgac tctctggtct gtaactgacg ctgagaagcg aaagcgtggg gagcgaacag 720

gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta atgggtttcc 780

gccccttagt gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac 840

tgtaactcaa acgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcgg 900

agcaacgcca agaaccttac caggtcttga catcctctga caatcctaga gataggacgt 960

ccccttcggg gacagagtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1020

ttgggttaag tcccgcaacg agcgcaaccc tagatcttag ttgccagcat tcagttgggc 1080

actctaaggt gactgccggt gacaaaccga aggaaggtgg ggatgacgtc aaatcatcat 1140

gccccttatg acctgggcta cacacgtgct acaatggaga gaacaaaggg cagcgaaacc 1200

gcgaggttaa gccaatccca caaatctgtt ctcagttcgg atcgcagtct gcaactcgtc 1260

tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg 1320

ggccttgtac acaccgcccg tcacaccacg agagtttgta acac 1364

<210> 2

<211> 813

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 2

acggaagaat ccactatcag gcgtacgagc gcggtgtacc cgtggctgat cttgaagtga 60

tcggtgatac tgataagacc ggaacgatta cgcacttcgt tccggatccg gaaatcttcg 120

aagaaacaat cgtatacgac tatgatctgc tttcaaaccg tgtccgggaa ctggccttcc 180

tgacaaaagg cgtaaacatc acgattgaag acaaacgtga aggacaagaa cggaaaaacg 240

agtaccacta cgaaggcgga atcaaaagct atgttgagta cttaaaccgt tccaaagaag 300

tcgttcatga agagccgatt tatatcgaag gcgagaaaga cggcataacg gttgaagttg 360

cgttgcaata caacgacagc tatacaagcc acatttattc tttcacaaat aacatcaaca 420

catacgaagg cgggacgcac gaagccggat ttaaaaccgg tctgacccgt gtcataaacg 480

actatgcaag aagaaaaggg attttcaaag taaatgatcc gaatttaagc ggggatgatg 540

tgagagaagg gctgactgcc attatttcaa ttaagcaccc tgatccgcaa ttcgaagggc 600

agacgaaatc gaagctcggc aactccgaag cgagaacgat cactgatacg ctgttttctt 660

ctgcgctgga aacattcctt cttgaaaatc cggactcagc ccgcaaaatc gttgaaaaag 720

gtttaatggc cgcaagagcg cggatagcag cgaaaaaagc gcgggaattg acccgccgca 780

aaagtgcgct tgagatttcc aatctgccgg gca 813

Claims

1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The strain WKBA-15 has the preservation number of CGMCC No. 14952.

2. The application of the strain WKBA-15 in preventing and controlling plant diseases and degrading organic phosphorus in soil according to claim 1; the plant diseases are tomato gray mold, tomato early blight, cucumber target spot and potato late blight.

3. A microbial inoculant comprising the strain WKBA-15 according to claim 1.

4. The use of the microbial agent of claim 3 for controlling plant diseases and degrading organic phosphorus in soil; the plant diseases are tomato gray mold, tomato early blight, cucumber target spot and potato late blight.

5. Molecular marker for identifying the strain WKBA-15 according to claim 1, characterized in that said molecular marker consists of the nucleotide sequence shown in SEQ ID No. 2.