CN111808778B

CN111808778B - Bacillus wegener for preventing and treating verticillium wilt and culture method thereof, microbial inoculum and preparation method and application thereof

Info

Publication number: CN111808778B
Application number: CN202010744196.0A
Authority: CN
Inventors: 李淑敏; 李威; 马辰; 孟令波; 桑平; 刘晓丹
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-07-29
Filing date: 2020-07-29
Publication date: 2021-05-14
Anticipated expiration: 2040-07-29
Also published as: CN111808778A

Abstract

The invention relates to the field of disease control, in particular to a bacillus wegener for controlling verticillium wilt and a culture method thereof, a microbial inoculum and a preparation method and application thereof. The invention provides a Bacillus westernella for preventing and treating verticillium wilt, which is Bacillus westerntephanensis (Bacillus westerntephanensis) SM19 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation address is No. 3 of No.1 Siro-Chen of the Hongyang area in Beijing, the preservation date is 2020, 01 and 15 days, and the preservation number is CGMCC No. 19355. The strain provided by the invention can inhibit the verticillium wilt of eggplants and increase the yield of the eggplants.

Description

Bacillus wegener for preventing and treating verticillium wilt and culture method thereof, microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the field of disease control, in particular to a bacillus wegener for controlling verticillium wilt and a culture method thereof, a microbial inoculum and a preparation method and application thereof.

Background

Eggplant Verticillium wilt is a soil-borne disease caused by Verticillium dahliae (Verticillium dahliae), also called as hemibarbus, melanosis and wilting disease, is a main soil-borne disease harmful to eggplant, pathogenic bacteria of the eggplant can survive in soil for a long time in the form of microsclerotia, and no effective medicament for preventing and treating the disease strain infected by the fungi exists at present. In recent years, the verticillium wilt of eggplants is increased year by year, the incidence rate in severe years is more than 70 percent, and the incidence rate in general years is 40 to 50 percent. In order to pursue economic benefits, farmers often adopt a method of fumigating soil with chemical agents to disinfect the soil and control the occurrence of eggplant verticillium wilt, but because the chemical agents are all biocidal agents, harmful and beneficial microorganisms in the soil are killed after the application, and the ecological environment is destroyed, the method for controlling diseases by using chemical pesticides brings huge threats to the agricultural ecological environment and the health of people and livestock, the soil ecological environment is continuously deteriorated, the two agents mutually promote, secondary salinization and acidification degree are aggravated along with soil nutrient unbalance, and the occurrence of soil-borne diseases is aggravated, and as a result, the yield and quality of the eggplant are seriously affected. Therefore, how to effectively control the verticillium wilt of eggplants in vegetable production is an important problem to be solved urgently in current horticultural vegetable production.

Disclosure of Invention

In view of the above, the invention provides a bacillus wegener for preventing and treating verticillium wilt, a culture method thereof, a microbial inoculum, a preparation method thereof and application thereof.

The invention provides a Bacillus westernella for preventing and treating verticillium wilt, which is Bacillus westerntephanensis (Bacillus westerntephanensis) SM19 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation address is No. 3 of No.1 Siro-Chen of the Hongyang area in Beijing, the preservation date is 2020, 01 and 15 days, and the preservation number is CGMCC No. 19355.

The invention provides a culture method of the bacillus westermani in the technical scheme, which comprises the steps of inoculating the bacillus westermani in a culture medium, and culturing under a constant temperature condition to obtain the expanded and propagated bacillus westermani.

Preferably, the medium comprises LB medium; the constant temperature is 28-30 ℃; the concentration of the cultured salt is 4.5-5.5%; the initial pH value of the culture is 5.0-7.0; the culture time is 48-72 h.

The invention provides a microbial inoculum for preventing and treating verticillium wilt, which comprises the bacillus wegener SM19 and solid auxiliary materials in the technical scheme.

Preferably, the solid auxiliary materials are activated carbon, biochar and diatomite; the mass percentage of the activated carbon in the solid auxiliary material is 60-70%, the mass percentage of the biological carbon in the solid auxiliary material is 10-20%, and the mass percentage of the diatomite in the solid auxiliary material is 10-20%.

The invention provides a preparation method of the microbial inoculum in the technical scheme, which comprises the following steps: mixing the fermentation liquor of the bacillus westernii SM19 with solid auxiliary materials, and then drying and crushing the mixture in sequence to obtain the microbial inoculum.

Preferably, the mixing mass ratio of the bacillus westernii SM19 fermentation liquid to the solid auxiliary material is 1: 3-1: 4; the drying temperature is 30-35 ℃; and the crushing is to pass through a 40-60-mesh sieve.

The invention provides application of the bacillus wegener in the technical scheme, the microbial inoculum in the technical scheme or the microbial inoculum prepared by the preparation method in the technical scheme in preventing and treating verticillium wilt.

The invention also provides application of the bacillus wegener in the technical scheme, the microbial inoculum in the technical scheme or the microbial inoculum prepared by the preparation method in the technical scheme in controlling the verticillium wilt of eggplants.

Preferably, the number of live bacillus wegener in the microbial inoculum for preventing and treating the verticillium wilt of eggplants is 5-10 hundred million/g.

The invention provides a Bacillus westerntephanensis strain for preventing and treating verticillium wilt, wherein the strain is Bacillus westernstephanensis (Bacillus westerntephanensis) SM19, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of No.1 Siro-West Luo of the area facing the sun in Beijing, the preservation date is 2020, 01-15 days, and the preservation number is CGMCC No. 19355. The strain provided by the invention can adjust the microbial community structure around the plant rhizosphere, has a remarkable prevention and control effect on the verticillium wilt of eggplants, and promotes the growth of plants. And has no pollution to the environment and no pesticide residue. The embodiment shows that the strain provided by the invention can obviously promote the growth of eggplants, and the weight of the eggplants is increased by 28 percent compared with that of the dry matters in the prior art; the disease incidence and disease index of the eggplant verticillium wilt can be reduced, and the relative control effect is improved by 12.3 percent compared with the prior art; meanwhile, the strain provided by the invention can transform soil from a fungal type to a bacterial type; and the yield of the eggplants can be improved by 52.1 percent compared with a control group.

The invention also provides a fungicide for preventing and controlling verticillium wilt, and the fungicide provided by the invention can be used for remarkably preventing and controlling verticillium wilt of eggplants and ensuring normal growth of the eggplants.

The invention also provides a preparation method of the verticillium wilt prevention and treatment microbial inoculum, and the method is simple and feasible and is suitable for industrial production.

Biological preservation Instructions

The Bacillus westerntephanensis SM19 is preserved in China general microbiological culture Collection center (CGMCC) in 1 month and 15 days of 2020, the preservation address is No. 3 of the Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No.19355 of the institute of microbiology of the Chinese academy of sciences.

Drawings

FIG. 1 is a graph of the control and antagonist pair test in example 2; wherein, the left figure is the test result of a control group which only inoculates the verticillium dahliae agar block and does not inoculate the bacillus weckeri SM19 strain, and the right figure is the test result of the antagonism of the bacillus weckeri SM19 strain and the verticillium dahliae;

FIG. 2 is an electron microscope scanning picture of Bacillus wegener SM19 in example 2;

FIG. 3 is a phylogenetic tree of Bacillus wegener SM 19;

FIG. 4 is a graph of the effect of different salt concentrations on the growth of Bacillus wegener SM19 in example 5;

FIG. 5 is a graph showing the effect of the growth of Bacillus wegener SM19 in example 6 at various pH values.

Detailed Description

The Bacillus wegener (Bacillus weihenstephanensis) SM19 is derived from rhizosphere soil of healthy eggplant plants in gardening and separate houses of Heilongjiang province.

The invention provides a culture method of bacillus westermani, which comprises the steps of inoculating the bacillus westermani into a culture medium, and culturing under a constant temperature condition to obtain the propagated bacillus westermani.

The invention inoculates the bacillus Welshi in the culture medium, and cultures under the condition of constant temperature, thus obtaining the bacillus Welshi. In the present invention, the medium preferably includes an LB medium; the LB culture medium is preferably a liquid culture medium; the preferable LB culture medium comprises 10g/L of peptone, 5g/L of yeast extract and 5g/L of NaCl; the pH value of the LB culture medium is preferably 6.0; the constant temperature is preferably 28-30 ℃, more preferably 28.5-30 ℃ and most preferably 29 ℃; the concentration of the salt for culture is preferably 4.5-5.5%, more preferably 4.8-5.2%, and most preferably 5%; the initial pH value of the culture is preferably 5.0-7.0, more preferably 5.5-6.5, more preferably 5.8-6.3, and most preferably 6.0; the culture time is preferably 48-72 h, more preferably 60-70 h, and most preferably 60 h. In the present invention, the LB medium is not limited at all, and any product conventionally purchased by those skilled in the art may be used. Under the parameter conditions provided by the invention, the bacillus westermani with the highest activity can be obtained by culturing.

In the invention, the solid auxiliary materials are preferably activated carbon, biochar and diatomite; the mass content of the activated carbon in the solid auxiliary material is preferably 60-70%, more preferably 62-68%, and most preferably 65%; the mass content of the biochar in the solid auxiliary material is preferably 10-20%, more preferably 13-20%, and most preferably 20%; the mass content of the diatomite in the solid auxiliary material is preferably 10-20%, more preferably 11-17%, and most preferably 15%. The activity of the bacillus weckerii SM19 in soil can be improved by adding biological carbon into the solid auxiliary material.

The invention mixes the fermentation liquor of the bacillus westernii SM19 with solid auxiliary materials, and then sequentially dries and crushes the mixture to obtain the microbial inoculum. In the present invention, the mixing mass ratio of the bacillus westernii SM19 fermentation liquid to the solid excipient is preferably (1:3) to (1:4), and more preferably (1:3.2) to (1: 3.7); the activity of the bacillus wecker SM19 in the bacillus wecker SM19 fermentation liquid is preferably 15-35 hundred million/ml, and more preferably 20-35 hundred million/ml; the drying temperature is 30-35 ℃, and more preferably 32-35 ℃; the crushing is preferably carried out by sieving with a 40-60 mesh sieve, and more preferably 60 mesh sieve. In the invention, the microbial activity can be maintained by adopting low-temperature drying, the bacillus westernii SM19 can be uniformly mixed with the solid auxiliary material by sieving, and the inoculation effect can be improved.

In the present invention, the obtaining method of the bacillus wegener SM19 fermentation liquid preferably includes:

(1) inoculating a bacillus wecker SM19 strain into a triangular flask liquid culture medium, and performing shake culture under constant temperature and shaking table conditions to obtain a first fermentation solution;

(2) inoculating the first fermentation liquid into a seed tank for fermentation culture to obtain a second fermentation liquid;

(3) and (3) inoculating the second fermentation liquid into a fermentation tank for fermentation culture to obtain the bacillus wegener SM19 fermentation liquid.

The invention inoculates the bacillus westernii SM19 strain in a culture medium, and performs shake culture under the conditions of constant temperature and shaking table to obtain a first fermentation solution. The first-level strain of the bacillus westernii SM19 strain is preferably inoculated in a culture medium and shake-cultured under the conditions of constant temperature and shaking table to obtain a first fermentation solution. In the invention, the first-class species of the Bacillus westernii SM19 strain is preferably obtained by culture in a test tube slant culture medium; the test tube slant culture medium is preferably LB culture medium; the LB culture medium is preferably a solid culture medium; the LB culture medium preferably comprises 10g/L of peptone, 5g/L, NaCl 5g/L of yeast extract and 20g/L of agar; the pH value of the LB culture medium is preferably 6.0. The inoculation mode of the bacillus wewinii SM19 strain is preferably inoculation by using an inoculating loop; in the present invention, the medium preferably includes an LB medium; the LB culture medium is preferably a liquid culture medium; the preferable LB culture medium comprises 10g/L of peptone, 5g/L of yeast extract and 5g/L of NaCl; the pH value of the LB culture medium is preferably 6.0; the constant temperature is preferably 28-30 ℃, more preferably 28.5-29.5 ℃ and most preferably 29 ℃; the rotating speed of the shaking table is preferably 150-180 r/min, more preferably 155-175 r/min, and most preferably 170 r/min; the culture time is preferably 48-72 h, more preferably 60-70 h, and most preferably 70 h; the culture medium is preferably LB culture medium; the LB culture medium is preferably a liquid culture medium; the LB culture medium preferably comprises peptone 10g/L and yeast extract 5g/L, NaCl 5.0.0 g/L.

The first fermentation liquid is inoculated into a seed tank for fermentation culture to obtain a second fermentation liquid. In the present invention, the seed tank preferably has a capacity of 10L. The culture medium used by the seed fermentation tank is LB liquid culture medium. In this step, the culture conditions are the same as those for obtaining the first fermentation solution, and are not described herein.

The second fermentation liquid is inoculated into a fermentation tank for fermentation culture to obtain the fermentation liquid of the bacillus westernii SM 19. In the invention, the culture medium used by the seed fermentation tank is LB culture medium. In this step, the culture conditions are the same as those for obtaining the first fermentation solution, and are not described herein.

The invention provides application of the bacillus wegener in the technical scheme, the microbial inoculum in the technical scheme or the microbial inoculum prepared by the preparation method in the technical scheme in preventing and treating verticillium wilt. In the application, the viable count of the bacillus westernii in the microbial inoculum is preferably 5-10 hundred million/g, more preferably 8-10 hundred million/g, even more preferably 9-10 hundred million/g, and most preferably 10 hundred million/g.

The invention also provides application of the bacillus wegener in the technical scheme, the microbial inoculum in the technical scheme or the microbial inoculum prepared by the preparation method in the technical scheme in controlling the verticillium wilt of eggplants. In the application, the number of viable bacteria of the bacillus westernii in the microbial inoculum is preferably 5-10 hundred million/g, more preferably 8-10 hundred million/g, and most preferably 10 hundred million/g.

For further illustration of the present invention, the following detailed description of a bacillus wegener for preventing and treating verticillium wilt, its culture method, microbial inoculum, and its preparation method and application are provided in the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Isolation of Bacillus wegener SM19 Strain

Collecting rhizosphere soil of healthy eggplant plants continuously grown in gardening and courtyard greenhouse of Heilongjiang province, removing plant residues and other impurities, sieving with a 2mm sieve, returning to a laboratory, and storing in a refrigerator at-4 ℃ for later use.

Weighing 10g of rhizosphere soil sample, adding 90ml of sterile water, standing for 20min, and oscillating for 30min in a shaking table at 200r/min to obtain a soil suspension. Sucking 5ml of soil suspension by using a sterile pipette, adding the soil suspension into 45ml of sterile water, and sequentially diluting to obtain 10^-1，10^-2，10^-3，10^-4，10^-5The gradient soil dilution is sucked by 100 mul of the final dilution and evenly spread on LB (lysis broth) culture medium, and the culture is repeated for 3 times, and the constant temperature culture is carried out for 2 days at 28 ℃. The LB culture medium formula is: 10.0g/L of peptone, 5.0g/L of yeast extract, 10.0g/L of NaCl, 20g/L of agar and 7.0-7.5 of pH.

Sterilizing a micro-sprayer at 120 ℃ for 20min, pouring about 1.5mL of Verticillium dahliae spore solution, uniformly spraying the spore suspension on the plate with the grown bacteria, airing the spore solution in a clean bench, sealing a culture dish, and culturing at the constant temperature of 28 ℃ for 3 d.

And (3) picking out bacteria in the inhibition zone, and streaking, purifying and culturing in a new LB plate until a single-character bacterial colony is obtained in the plate. Namely the strain of bacillus westernii SM 19.

Example 2

The test of the confronting of the verticillium dahliae comprises the following specific steps:

the agar blocks of 5mm in diameter for growing Verticillium dahliae were inoculated into the center of a potato dextrose PDA medium plate, incubated at 28 ℃ for 4 days, and then the purified antagonistic strain (Bacillus westermanii SM19 strain obtained in example 1) was inoculated into 4 spots on a circumference of 2.5cm from the agar blocks, and the treatment was repeated 3 times with respect to the plate inoculated with only the agar blocks of Verticillium dahliae without the antagonistic strain. Culturing at 28 deg.C for 10 days, measuring the diameter of Verticillium dahliae colony in each treatment (figure 1), and testing the bacteriostatic ability, wherein each treatment tests three groups of data. Wherein, the formula of the bacteriostasis rate is as follows: bacteriostatic activity (%) - (diameter of colony of control verticillium dahliae-diameter of colony of treated verticillium dahliae (cm))/(diameter of colony of control verticillium dahliae (cm)) × 100%. The results of the tests are shown in fig. 1 and table 1, and the average inhibitory potency of the isolated antagonistic bacteria was calculated to be 44.18%.

TABLE 1 antagonistic pair test replicate colony diameter (cm) and inhibitory potency (%)

As can be seen from the data described in Table 1 and FIG. 1, the bacterial strain provided by the invention can obviously inhibit verticillium dahliae, and the bacteriostatic ability reaches 44.18%.

Meanwhile, the Bacillus westernii SM19 strain was observed on NA (beef extract peptone agar: 5g, peptone 10g, NaCl 5.0g/L, agar 20g/L) medium, and the growth was observed.

The observation results show that: the growth condition of the bacillus wewinii SM19 strain is better, a single colony is circular and is opalescent and opaque, and the surface of the bacillus wewinii SM19 strain is slightly wrinkled when observed under an electron microscope (shown in figure 2), so that the growth of the bacillus wewinii SM19 strain is uniform and full. Through measurement and calculation, the length of the bacillus westernii SM19 strain is 1.528 mu m, the diameter of the strain is 0.68 mu m, and the volume of the strain is 1.039 mu m²。

Example 3

Physiological and biochemical measurements were performed on the strain Bacillus wegener SM 19. The physiological and biochemical indexes are shown in Table 2.

TABLE 2 physiological and biochemical indexes of Bacillus wegener SM19 strain

Note: "+" indicates that the test reaction was positive; "-" indicates that the test reaction was negative

The results are shown in Table 2, in which Bacillus wechsleri SM19 strain is gram-positive, catalase test is positive, V-P test is positive, methylred test is negative, indole test is positive, starch hydrolysis test is positive, H₂The gas production test of S is a positive reaction, the citrate test is a negative reaction, the nitrate reduction test is a positive reaction, the gelatin liquefaction test is a positive reaction, L-arabinose in the saccharide fermentation test is a negative reaction, D-glucose is a positive reaction, D-lactose is a negative reaction, D-sucrose is a negative reaction, D-xylose is a negative reaction, and the surface of the Bacillus westernii SM19 strain is wet, viscous and easy to pick, irregular in edge and slightly raised.

Example 4

Performing PCR amplification and sequencing on the separated antagonistic Bacillus westernii SM19 strain, submitting a PCR sequencing sequence to NCBI, performing BLAST similarity analysis in GenBank, performing clustering analysis by adopting MEGA7.0(Molecular evolution analysis) software Neighbor-join, constructing a phylogenetic tree from the first 15 bacteria with higher similarity to the strain gene sequence in GenBank, and combining morphological observation, physiological and biochemical index analysis and 16S rDNA gene sequence comparison result comprehensive analysis of the strain. The results are shown in FIG. 3.

As can be seen from FIG. 3, the strain Bacillus wechenstetphanensis SM19 was identified as Bacillus wechenstevensis.

Example 5

Effect of different salt concentrations on growth of Bacillus wegener SM19 Strain

Adding NaCl (0%, 0.5%, 2%, 5%, 6%, 7%, 10%) with different concentrations into LB culture medium, inoculating strain, culturing at 29 deg.C for 24 hr, and determining OD of culture solution₆₀₀The value is obtained. The results are shown in table 3 and fig. 4.

TABLE 3 Effect of different salt concentrations on the growth of Bacillus wegener SM19 Strain

Salt concentration (%)	0	0.5	2	4	5	6	7	10
									Absorbance at 600nm	0.184	0.189	0.193	0.200	0.203	0.198	0.181	0.165

As can be seen from table 3 and fig. 4. With the continuous increase of the initial culture salt concentration, the growth rate of the bacillus westernii SM19 strain shows a trend of increasing and then decreasing. When the culture salt concentration is 5%, the growth condition of the Bacillus wegener SM19 strain is best, and the growth condition of SM19 is linearly reduced along with the increase of the salt concentration. The optimal growth salt concentration of the bacillus wewinii SM19 strain is 5%, which shows that the bacillus wewinii SM19 strain is not tolerant to growth under the culture condition with higher salt concentration and can normally grow under the salt concentration lower than 5%.

Example 6

Effect of different initial pH values on growth of Bacillus wegener SM19 Strain

Setting LB liquid culture medium with different initial pH (3, 5, 6, 7, 8, 9, 11), inoculating strain, culturing at constant temperature of 30 deg.C for 24 hr, and determining OD of culture solution₆₀₀The value is obtained. The effect of different initial culture pH values on the growth of bacillus wegener SM19 strain is shown in table 4 and fig. 5.

TABLE 4 Effect of different initial pH values on the growth of the Bacillus wegener SM19 strain

pH	3	5	6	7	8	9	11
								Absorbance value	0.202	0.247	0.284	0.214	0.253	0.24	0.173

As can be seen from Table 4 and FIG. 5, the strain Bacillus wegener SM19 shows a tendency to increase and then decrease and turn back with increasing culture pH. When the culture condition is pH 6.0, the growth condition of the Bacillus weckerensis strain SM19 is best, and the growth of the Bacillus weckerensis strain SM19 shows a gradual decline and then a slow rise trend along with the increase of the pH value. It can be seen that when the culture pH is more than 8, the growth of the Bacillus wegener SM19 strain is not favored. The pH value of the optimum growth of the bacillus westernii SM19 strain is 6.0, the bacillus westernii strain is suitable for neutral growth, and the culture condition of over-acidity or over-alkalinity is not beneficial to the growth.

Example 7

(1) A loop strain is picked from the inclined plane of the strain of the bacillus wegener SM19 by using an inoculating loop, the loop strain is inoculated into a conical flask filled with 100mL of LB liquid culture medium, and shaking culture is carried out on a shaking table at the constant temperature of 29 ℃ and at 170r/min for 70h to obtain a first fermentation solution. The LB liquid culture medium comprises the following components in percentage by weight: 10g/L of peptone, 5g/L of yeast extract and 5g/L of NaCl, and the pH value is 6.0.

(2) Fermenting in a seeding tank: inoculating the first fermentation liquid into a 10L seed tank, fermenting and culturing at 29 deg.C and 170rpm for 70h to obtain a second fermentation liquid. The formula of the fermentation medium is the LB liquid medium.

(3) Fermentation in a fermentation tank: and (4) inoculating the second fermentation broth into a fermentation tank, and carrying out fermentation culture at 29 ℃ and 170rpm for 70h to obtain the fermentation broth of the bacillus westernii SM 19. The formula of the fermentation medium is the LB liquid medium.

(4) Preparation of a microbial inoculum: adding the fermentation liquor of the bacillus westernii SM19 into a solid auxiliary material, wherein the mass ratio of the solid auxiliary material to the fermentation liquor is 3.5: 1, uniformly stirring, drying at 30 ℃, crushing, sieving by a 40-mesh sieve, packaging and bagging to obtain the microbial inoculum with the total bacterial count activity of 10.0 hundred million/g. The solid auxiliary material accounts for 77.8 percent of the mixed mass content of the bacillus westermanii SM19 fermentation liquid and the solid auxiliary material, wherein the mass percent of the activated carbon in the solid auxiliary material is 65.0 percent, the mass percent of the biochar in the solid auxiliary material is 20.0 percent, and the mass percent of the diatomite in the solid auxiliary material is 15.0 percent.

Application example 1

Preparation of a verticillium dahliae spore suspension: activating Verticillium dahliae on a PDA culture medium, taking out the bacterial blocks, performing shake culture in the PDA liquid culture medium in the dark for about 7 days, filtering with double-layer medical gauze, and removing the mycelium pellet. The filtrate was used in a hemocytometer to count the spore concentration of the fungus, and the spore suspension was adjusted to 1X 10 with sterile water before inoculation⁷CFU·mL^-1。

The test soil is taken from a continuous cropping eggplant greenhouse of a gardening and division hospital of an agricultural academy of Heilongjiang province, sieved by a 2mm sieve and sterilized for 2h at 121 ℃ for later use. Setting a control group, a comparison group and an experimental group, wherein the control group is not treated by adding a fungicide and adopts the sterilized soil; the experimental group is that 20g of the microbial inoculum prepared in the example 3 is added into each pot, and the sterilized soil is adopted; the control group was a treatment with a chemical agent, and the amount of dazomet was 45 g.times.m according to the specification^-2Fumigating soil collected by the greenhouse and not sterilized for 15 days.

Taking culture pots with the specification of 20cm multiplied by 10cm multiplied by 20cm, filling 2.5kg of sterilized soil, adding 20ml of verticillium dahliae spore suspension into each pot, transplanting eggplant seedlings with 4 leaves and 1 heart into each treatment culture pot, repeating each treatment for 8 times, and managing the eggplants according to a conventional mode. And measuring the growth index of the eggplant and the incidence rate of the verticillium wilt after 45 days. The results are shown in Table 5.

TABLE 5 Effect of the biocontrol agent SM19 on eggplant growth and verticillium wilt disease incidence

Note: different letters in the same column indicate significant level of inter-treatment difference (P < 0.05).

As can be seen from Table 5, the experimental group significantly promoted the growth of eggplant, the dry matter weight was increased by 30.7% and 28.0% respectively compared with the control group and the control group, the plant height and stem thickness were also significantly higher than those of the control group and the control group, the incidence rate and disease index of verticillium wilt were significantly lower than those of the control group and the control group, and the relative control effect of the experimental group was 56.4%. Therefore, the bacterial strain and the microbial inoculum provided by the invention can obviously inhibit the verticillium wilt of eggplants and promote the growth of the eggplants.

Meanwhile, bacterial and fungal diversity indexes (Shannon and Simpson) of eggplant rhizosphere soil samples treated differently are measured by adopting a high-throughput sequencing technology for investigation, wherein the larger the Shannon index value is, the larger the species diversity is represented, and the more uniform the individual distribution is; simpson index is reversed. The results are shown in Table 6.

TABLE 6 Effect of the biological agent SM19 treatment on the alpha diversity index of eggplant rhizosphere soil bacteria and fungi

Note: different letters in the same row of bacterial or fungal diversity index indicate significant level of inter-treatment variation (P < 0.05).

As can be seen from table 6, the bacterial alpha diversity index of the rhizosphere soil of the eggplant shows significant difference among 3 treatments, wherein the experimental group significantly improves the Shannon index of the rhizosphere soil, which is increased by 14.4% and 13.8% respectively compared with the control group and the comparison group, and the Simpson index of the experimental group is minimum, which indicates that the bacterial diversity of the rhizosphere soil of the eggplant is significantly increased after the soil is treated by the biological agent; the alpha diversity index of the eggplant rhizosphere fungi shows obvious difference among 3 treatments, wherein an experimental group obviously reduces the Shannon index of rhizosphere soil and 31.44 percent and 9.29 percent respectively compared with a control group and a comparison group, and simultaneously the Simpson index of the experimental group is the largest, so that the eggplant rhizosphere fungi diversity of the experimental group is obviously lower than that of the control group and the comparison group, the rhizosphere soil fungi diversity of the control group is the highest, and the fungi diversity of the rhizosphere soil of the eggplant is obviously reduced after the soil is treated by the biological agent; therefore, the strain and the microbial inoculum provided by the invention convert soil from a fungal type to a bacterial type, so that the soil is healthier.

Application example 2

The test is carried out in a continuous cropping greenhouse of gardening and separated eggplant of agricultural institute of Heilongjiang province, the test is carried out in 2018, 4 and 30 days, eggplant seedlings with four leaves and one center are transplanted and planted, the eggplant seedlings are planted on ridge platforms in an inverted triangle mode, the ridge platforms are 5m long and 1m wide, the row spacing is 30 x 50cm, and the planting density is 2.5 ten thousand plants hm^-2Before planting, each treatment is carried out at 1200kg hm^-2The compound fertilizer (12-11-18) is applied in the amount of the fertilizer, the compound fertilizer is completely applied at one time, the additional fertilizer is not applied at the later stage, and the compound fertilizer and the additional fertilizer are uniformly managed in a film covering and drip irrigation mode.

In the experiment, the eggplant continuous cropping without adding the anti-biological agent is treated as a control group, the anti-biological agent (the agent obtained in example 7) is applied to the eggplant in a hole mode during transplanting according to the dosage of 4 kg/mu to be used as an experimental group, each treatment is repeated for 3 times, and the experimental cell is managed in a conventional mode by adopting a random block group design.

The disease incidence, disease index and prevention and treatment effect index are investigated respectively at 15 days 6 months, 27 days 6 months and 12 days 7 months at the early stage of verticillium wilt of eggplant at the beginning of 6 months, and the investigation is continuously carried out for 3 times. The yield was the cumulative eggplant yield from the time when the ripe eggplant fruits were picked at 25 days 6 months to the time when the picking was completed at 5 days 8 months, and the results of the investigation are shown in table 7.

TABLE 7 prevention and cure effect of biological control agent SM19 on verticillium wilt of continuous cropping eggplant

Note: different letters in the same column indicate significant differences between different treatments (P < 0.05).

As can be seen from the experimental data recorded in Table 7, the verticillium wilt starts to sporadically develop at the beginning of 6 months, the disease index of the verticillium wilt of the eggplant in the experimental group is obviously lower than that of the verticillium wilt of the eggplant in the control group, and the relative control effects of the biocontrol microbial inoculum in three investigation periods are respectively 61.0%, 51.5% and 51.3%; the incidence of the eggplant verticillium wilt in the experimental group is obviously lower than that of the eggplant verticillium wilt in the control group; therefore, the experiment group has obvious prevention and control effects on the verticillium wilt of the greenhouse continuous cropping eggplant. The yield of the eggplants in the soil treated by the experimental group is increased by 52.1 percent compared with that of the control group. Therefore, the treatment group can obviously inhibit the verticillium wilt of the eggplants, reduce the number of verticillium dahliae in the soil and improve the yield of the eggplants.

Meanwhile, the number of verticillium wilt pathogens in the soil is investigated by adopting the method in the flowering stage, the initial fruit stage and the full fruit stage of the eggplant, and the result is shown in table 8.

TABLE 8 influence of the biological agents on the number of verticillium wilt pathogens in continuous cropping eggplant soil (× 10)⁴Mean copies Verticillum dahliae·g^-1Dry soil)

Treatment of	Flowering phase	Early fruit stage	Full fruit period
				Experimental group	1.2±0.2	2.2±0.1	4.5±0.2
Control group	3.5±0.1	7.1±0.5	7.3±0.2

As shown in Table 8, the number of verticillium wilt pathogens in the soil treated by the control group was 2.91, 3.23 and 6.17 times that in the experimental group in the flowering, early fruiting and full fruiting period, respectively. Therefore, the experimental group can obviously inhibit the verticillium wilt of the eggplant and reduce the number of verticillium dahliae in the soil.

The above-mentioned examples show that the strain provided by the invention can obviously inhibit the verticillium wilt of eggplants, reduce the number of verticillium dahliae in soil and improve the yield of the eggplants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The bacillus wegener for preventing and treating verticillium wilt has the preservation number of CGMCC No. 19355.

2. The method for culturing Bacillus weckerii strain of claim 1, wherein the method comprises inoculating Bacillus weckerii strain into a culture medium, and culturing under a constant temperature condition to obtain expanded Bacillus weckerii strain.

3. The culture method according to claim 2, wherein the medium comprises an LB medium; the constant temperature is 28-30 ℃; the concentration of the cultured salt is 4.5-5.5%; the initial pH value of the culture is 5.0-7.0; the culture time is 48-72 h.

4. A bacterial agent for preventing and treating verticillium wilt, which is characterized by comprising the bacillus wegener of claim 1 and solid auxiliary materials.

5. The microbial inoculum according to claim 4, wherein the solid auxiliary materials are activated carbon, biochar and diatomite; the mass percentage of the activated carbon in the solid auxiliary material is 60-70%, the mass percentage of the biological carbon in the solid auxiliary material is 10-20%, and the mass percentage of the diatomite in the solid auxiliary material is 10-20%.

6. The method for preparing the microbial agent according to claim 4 or 5, comprising the steps of: and mixing the bacillus westernii fermentation liquor with solid auxiliary materials, and then sequentially drying and crushing to obtain the microbial inoculum.

7. The preparation method of claim 6, wherein the mixing mass ratio of the bacillus westernii fermentation liquid to the solid auxiliary material is 1: 3-1: 4; the drying temperature is 30-35 ℃; and the crushing is to pass through a 40-60-mesh sieve.

8. The use of the bacillus wegener of claim 1 for controlling eggplant verticillium wilt.

9. The application of claim 8, wherein the viable count of the bacillus westernii in the microbial inoculum for controlling the verticillium wilt of eggplants is 5-10 hundred million/g.