CN113755358B - Bacillus bailii ZJ1 strain and fermentation broth preparation and use methods - Google Patents

Abstract

The invention discloses a bacillus bailii strainBacillus velezensis) The ZJ1 strain is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.20986 in the year 2020, 11. The preparation method of the strain fermentation broth comprises the steps of strain seed culture medium, seed liquid preparation, fermentation broth preparation and fermentation broth culture. The use method of the strain fermentation liquor is that the fermentation liquor is diluted by water for 10 times according to an external ratio method, tween 80 with the volume ratio of 10 percent of the dilution liquor is added as a surfactant to prepare the bacillus belicus ZJ1 field control microbial inoculum, the preparation is used for 1 time in the early 8-month quinoa grouting period, the preparation is used once every 10 days, the preparation is continuously used for 3 times, and the dosage is 50L/mu. The bacillus belgium ZJ1 fermentation liquor has good inhibition effect on pathogenic bacteria of quinoa black stem diseases, and laboratory inoculation shows that the inhibition rate of bacillus belgium ZJ1 on quinoa black stem diseases is 58.39 percent, and the field test control effect reaches 70.65 percent.

Description

Bacillus bailii ZJ1 strain and fermentation broth preparation and use methods

Technical Field

The invention belongs to the technical field of biological control of plant diseases, and particularly relates to bacillus bailii ZJ1 with antibacterial activity on pathogenic bacteria of quinoa black stem disease, namely, alternaria alternata, and a preparation method and a use method of bacillus bailii ZJ1 fermentation liquor.

Background

Quinoa is native to the andes mountain area in south america, is a high altitude crop, is cool and cool, has the characteristic of adapting to severe environments, and is planted in america, europe, asia, australia and the like. Because of its comprehensive nutritional ingredients, the federal grain and agricultural organization (FAO) recommends quinoa as a "full nutrient" food suitable for human use. The quinoa industry in China starts later, but along with the enlargement of planting area and continuous cropping cultivation, quinoa diseases become serious, and the development of the quinoa industry is seriously affected. Wherein, quinoa black stem disease is a new disease affecting quinoa stems, and is more easy to occur under the low temperature condition (15-25 ℃). Typical symptoms are black necrotic lesions formed on the stems, which can completely encapsulate the stems, cause lodging and blanking, and eventually develop into empty ears and sterile granules. Pathogenic bacteria of quinoa black stem diseaseAscochyta caulina) The germ infects mainly the stems of quinoa and produces a large number of conidia, occasionally the leaves of quinoa. At present, chemical control methods are mainly used for quinoa diseases in production, but the control is not timely, the loss is huge, and even the quinoa diseases are completely killed because the control is not clear.

Biological control makes up the shortages of chemical pesticides because of being friendly to human beings and environment, and becomes a very potential research direction at present. Endophytic bacteria, particularly bacillus, have strong stress resistance, and have good control effect on various plant diseases in recent years. Bacillus spBacillusIs a gram positive bacterium which can grow and propagate rapidly, can effectively colonise plant rhizosphere, can generate various active metabolic substances, has various biological functions, has wide application prospect in the aspects of industry, agriculture and medicine, and is a treasury for developing microorganism resources. The biocontrol bacillus is various, and the microbial pesticides currently used for pesticide registration in China relate to 9 kinds of bacillus, wherein the most registered pesticide products are developed by using bacillus subtilis, and the most registered pesticide products are bacillus polymyxa, bacillus amyloliquefaciens and pseudomonas fluorescens. Screening of endophytic bacteria for plants as biocontrol bacteria is usually carried out on the tissue of the target plant to be controlled or on the same habitatPlant-like tissue or plant tissue with antibacterial effect.

Disclosure of Invention

The invention aims to provide bacillus belicusBacillus velezensis) ZJ1 and a preparation and use method of the fermentation liquor, and bacillus belicus ZJ1 fermentation liquor has good antibacterial activity on the aschersonia aleyrodis and has remarkable control effect on quinoa black stem disease.

The technical scheme of the invention is as follows:

bacillus bailii strainBacillus velezensis) ZJ1 strain is preserved in China general microbiological culture Collection center (CGMCC) at 11/2/2020, and has a preservation number of CGMCC No.20986, address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.

The bacillus belgium ZJ1 is prepared from Bush plant Zostera marina of Zostera of loguaiaceaeBuddleja lindleyana Fortunes) were isolated and screened from stems of fortunes). After the strain is cultured by adopting a beef extract and peptone solid culture medium, the strain is off-white in color on the culture medium, has dry surface, is wrinkled and opaque, is moist in the interior, and has serrated and irregular colony edges; the surface of the picking plate is provided with a thin skin layer (figure 1); the ZJ1 cells were found to be rod-shaped by scanning electron microscopy, and the size of the periphyton was 0.5 to 1.5. Mu.m.times.1.5 to 2.0. Mu.m (FIG. 2).

Bacillus bailii ZJ1 can utilize glucose, lactose, sucrose, mannitol and maltose, can hydrolyze starch, can produce acid by utilizing glucose, has a growth temperature range of 10-65 ℃, has salt tolerance, and can normally grow at a salt concentration of 2-10%.

Homology alignment with Bacillus bailii using 16SrDNA sequenceBacillus velezensisBacillus amyloliquefaciensBacillus amyloliquefaciensThe highest homology of (3) cannot be identified to a species clearly (FIG. 3), and further homology alignment with Bacillus bailii is performed on the gyrA sequenceBacillus velezensisThe homology of the bacillus subtilis reaches more than 99 percent, and the bacillus subtilis is identifiedBacillus velezensis(FIG. 4).

The preparation method of bacillus belicus ZJ1 fermentation broth comprises the following steps:

(1) Strain seed medium: the culture medium of bacillus belicus ZJ1 strain seeds adopts NA culture medium;

(2) Preparing seed liquid: the ZJ1 strain of 48 h cultured on NA culture medium is inoculated into LB culture solution by picking a loop of single colony, and the temperature is 26-28 ℃ and 160-180 r.min ^-1 Shaking culture is carried out for 20-24 and h to obtain ZJ1 seed liquid;

(3) Preparing a fermentation liquid: mixing the seed solution prepared in the step (2) with the modified YSP culture solution according to the volume ratio of 1:100, mixing to prepare fermentation liquor;

(4) Culturing fermentation liquor: at 26-28deg.C and rotational speed of 180-190 r min ^-1 The fermentation time is 68-72 h, and the number of the bacillus bailii ZJ1 viable cells in the fermentation liquid is not less than 10 ⁹ cfu/mL。

The formulation of the NA culture medium is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, agar powder 15 g, distilled water 1000 mL, pH 7.0.

The formula of the LB culture solution is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, distilled water 1000 mL, pH 7.0.

The formula of the improved YSP culture solution is as follows: peptone 10.0 g, lactose 20.0 g, yeast powder 1.5 g, beef extract 1.5 g, peptone 2.0 g, distilled water 1000 ml, ph=7.0.

Bacillus bailii of the inventionBacillus velezensis) The fermentation liquor of ZJ1 strain is prepared by diluting bacillus belicus ZJ1 fermentation liquor with water by 10 times according to external ratio method, adding tween 80 with 10% of the dilution liquor as surfactant according to volume ratio, and making into bacillus belicus ZJ1 field control microbial inoculum, wherein the dosage is 1 time in the early quinoa grouting period of 8 months, once every 10 days, 3 times continuously, and 50L/mu.

Bacteriostasis test of bacillus bailii ZJ1 fermentation liquor

1. The test method comprises the following steps:

(1) Measuring the germ of the black stalk of quinoaAscochyta caulina) Is inhibited by (a) in the form of a suspension

After the quinoa black fungus is cultured by adopting a four-point plate confronting method to form 10 d, a fungus cake is manufactured at the edge by using a puncher with phi of 5 mm, the fungus cake is planted in the center of a solid PDA plate for culture, the separated and purified bacillus beijerinus ZJ1 strain is symmetrically inoculated on four sides 20 mm of the fungus cake, and only pathogenic fungi are inoculated as a control, and each test is repeated for 3 times. 25. After the incubator is inverted and cultured for 10 d under the condition of the temperature, the antibacterial effect is observed, the diameters of two groups of treated pathogenic bacteria are measured, and the antibacterial rate is calculated.

(2) Bacillus bailii ZJ1 fermentation broth stability test

A measurement of thermal stability

After 60 mL ZJ1 fermentation broth is equally placed in 6 sterile test tubes, the test tubes are treated in a water bath for 30 min, and the set temperatures are respectively the values required by the test: after cooling to room temperature at 20deg.C, 40deg.C, 60deg.C, 80deg.C, 100deg.C, and 121deg.C, mixing with PDA at a ratio of 1:9 (V: V) to obtain plate for detecting antibacterial activity, and repeating each test for 3 times without water bath treatment.

B acid-base stability determination

The 70 ml ZJ1 fermentation broth was equally placed in 7 sterile tubes and the pH of the fermentation filtrate was adjusted to the desired value for the test with HCl and NaOH standard solutions: 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0, and after standing for 2 h at room temperature, the natural pH value is adjusted back to prepare a flat panel detection antibacterial activity, and the control group is not subjected to pH adjustment treatment, and each test is repeated 3 times.

C ultraviolet radiation stability determination

The 60 ml ZJ1 fermentation broth is equally placed in 6 sterile test tubes, is vertically placed at a position 20 cm under an 18W ultraviolet lamp and irradiates 12 h, and samples are taken every 2 h in time to prepare a flat plate for detecting antibacterial activity, a control group is not subjected to ultraviolet irradiation treatment, and each test is repeated 3 times.

D determination of light stability

The 50 ml ZJ1 fermentation broth is equally placed in 5 sterile test tubes, vertically placed under an 18W incandescent lamp, irradiated with 20 cm for 48 h, sampled in time at 6 h, 12 h, 24 h, 36 h and 48 h respectively to prepare a flat-plate detection antibacterial activity, and the control group is not irradiated with the incandescent lamp, and each test is repeated for 3 times.

E determination of storage stability

After the 20 mLZJ1 fermentation broth is equally placed in 2 test tubes subjected to 90 mm aseptic treatment, the test tubes are respectively stored in a refrigerator at 4 ℃ and at room temperature in darkness, and the test samples are timely extracted and sampled at 7 d, 15 d, 30 d and 60 d to prepare a flat-plate detection antibacterial activity, and a control group is not subjected to darkness storage treatment and is repeated for 3 times per test.

(3) Bacillus bailii ZJ1 fermentation liquor field control effect test

Bacillus bailii ZJ1 fermentation broth (viable cell count 10) ⁹ cfu/mL) is diluted by water for 5 times, 10 times, 20 times, 50 times, 100 times and 200 times according to an external ratio method, 10 percent of Tween 80 (volume ratio) is added as a surfactant, and the bacillus belicus ZJ1 microbial inoculum is prepared for a field control effect test.

The test was conducted in Xin, shanxi province in Jinle county. Test agent bacillus beleiensis ZJ1 bacterial agent, 430 g/L tebuconazole suspension, clear water blank. The experiment had 8 treatments, 4 replicates each, for a total of 32 cells. Each cell 30 m ² A guard row is provided between cells. The trial was arranged to be administered 1 time at the early 8 month quinoa grouting period, once every 10 days, 3 times continuously, and completed before the 8 month end. The quinoa is sprayed manually or mechanically with water consumption of 50L/mu. The weather is clear and windless during the test period, and the medicine is 72-h free of precipitation. Investigation period: the field disease investigation was performed 10 d after the last application and completed the same day. The investigation method comprises the following steps: each cell was investigated, and the number of disease-causing plants was recorded. The incidence, i.e. the percentage of the number of diseased plants to the total number of all investigated plants, was calculated.

2. Test results:

(1) The inhibition rate of bacillus belgium ZJ1 on quinoa black stalk disease is 58.39%, and the antibacterial band width is 11.2+/-0.1 mm (figure 5).

(2) The results of the stability test of Bacillus bailii ZJ1 fermentation broth are shown in tables 1-5:

after the ZJ1 strain fermentation broth is treated for 30 min at 6 different temperatures, the antibacterial effect is slightly reduced along with the temperature rise, and the antibacterial effect is still kept above 55% after the treatment at 60 ℃, but is obviously weakened after the treatment at 80 ℃.

The ZJ1 strain fermentation liquor has certain antibacterial activity after being treated by different pH values, the antibacterial effect is optimal under the neutral (pH 7.0) condition, the antibacterial effect is respectively reduced to different degrees along with the increment of the acid-base property, but the antibacterial effect is still more than 30%, and the activity is kept in a wider acid-base range.

The ZJ1 strain fermentation liquor has certain antibacterial activity after being irradiated by ultraviolet lamps with different durations for 2-12 hours respectively, and the antibacterial activity is slightly reduced along with the increase of time, but is still maintained to be more than 50%, and the fermentation liquor has the antibacterial effect onA. caulinaAlso has better inhibition effect.

The ZJ1 strain fermentation liquor is respectively irradiated by incandescent lamps with different durations for 6-48 hoursA. caulinaAll have certain antibacterial activity, the antibacterial effect is not obviously reduced along with the increase of time, the antibacterial effect is very stable, and the antibacterial rate is kept at about 58%.

ZJ1 strain fermentation broth is respectively refrigerated at 4 ℃ and at room temperature of 25 ℃,after preservation for different lengthsA. caulinaAll have certain antibacterial activity, the antibacterial effect is kept above 57% along with the increase of the preservation time, the antibacterial effect is not obviously reduced, but the antibacterial effect at the room temperature is slightly reduced compared with the antibacterial effect at the condition of 4 ℃ at the same time point, and the refrigeration is beneficial to the preservation of ZJ1 strain fermentation liquor.

(3) Bacillus bailii ZJ1 fermentation broth field control effect test results (Table 6)

The optimal concentration of the bacillus belicus ZJ1 fermentation liquor is 10 times of diluent, the effect of preventing and treating quinoa black stem disease reaches 70.65 percent, no obvious difference is caused compared with the preventing and treating effect (68.46 percent) of 5 times of diluent, and the preventing and treating effect is higher than that of 5 times of diluent; significantly higher than the chemical tebuconazole (65.92%) and other fold dilutions. The bacillus belicus ZJ1 microbial agent has good prevention effect on quinoa black stem disease.

The invention has the advantages and beneficial effects that:

the bacillus belgium ZJ1 fermentation liquor has good inhibition effect on pathogenic bacteria of quinoa black stem disease, and laboratory inoculation shows that the inhibition rate of bacillus belgium ZJ1 on quinoa black stem disease is 58.39%, the field test control effect reaches 70.65%, and the effect is obviously higher than that of a chemical agent tebuconazole (65.92%). The bacillus bailii ZJ1 fermentation broth has good thermal stability, and the bacteriostasis rate is not obviously reduced under the condition of not exceeding 60 ℃; the acid-base adaptation range of the fermentation liquor is wider, and the bacteriostasis rate of the fermentation liquor is not obviously reduced under the condition of pH 5-9; can be stored at room temperature for a long time under the irradiation of visible light and ultraviolet light, is favorable for commercialization of the bacterial strain microbial inoculum, and has great application value in quinoa field production. The preparation process of the fermentation liquor is simple, the fermentation condition is not harsh, the raw materials used in the microbial inoculum formula are simple, the sources are wide, the cost is low, and the industrial production is easy to realize.

Drawings

FIG. 1 shows Bacillus bailii according to the present inventionBacillus velezensisColony morphology of ZJ1 strain on NA medium.

FIG. 2 shows Bacillus bailii according to the present inventionBacillus velezensisScanning electron microscope photographs of ZJ1 strain.

FIG. 3 shows Bacillus bailii according to the present inventionBacillus velezensis16SrDNA sequence phylogenetic tree of ZJ1 strain.

FIG. 4 shows Bacillus bailii according to the present inventionBacillus velezensisPhyra sequence phylogenetic tree of ZJ1 strain.

FIG. 5 shows Bacillus bailii according to the present inventionBacillus velezensisThe bacteriostasis effect of ZJ1 strain on four-point opposite culture of Alternaria alternata on PDA culture medium is shown schematically.

Detailed Description

The invention is further illustrated below with reference to examples.

Example 1

Bacillus bailii of the inventionBacillus velezensis) The ZJ1 strain is preserved in China general microbiological culture Collection center (CGMCC) in 11 months in 2020, and has a preservation number of CGMCC No.20986, address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.

The bacillus belicus ZJ1 strain is prepared from Bush plant Zostera marina of Zostera of loguaiaceaeBuddleja lindleyana Fortunes) were isolated and screened from stems of fortunes). After the strain is cultured by adopting a beef extract and peptone solid culture medium, the strain is off-white in color on the culture medium, has dry surface, is wrinkled and opaque, is moist in the interior, and has serrated and irregular colony edges; the surface of the picking plate is provided with a thin skin layer (figure 1); the ZJ1 cells were found to be rod-shaped by scanning electron microscopy, and the size of the periphyton was 0.5 to 1.5. Mu.m.times.1.5 to 2.0. Mu.m (FIG. 2). Bacillus bailii ZJ1 can utilize glucose, lactose, sucrose, mannitol and maltose, can hydrolyze starch, can produce acid by utilizing glucose, has a growth temperature range of 10-65 ℃, has salt tolerance, and can normally grow at a salt concentration of 2-10%. Homology alignment with Bacillus bailii using 16SrDNA sequenceBacillus velezensisBacillus amyloliquefaciensBacillus amyloliquefaciensThe highest homology of (3) cannot be identified to a species clearly (FIG. 3), and further homology alignment with Bacillus bailii is performed on the gyrA sequenceBacillus velezensisThe homology of the bacillus subtilis reaches more than 99 percent, and the bacillus subtilis is identifiedBacillus velezensis(FIG. 4).

As shown in FIG. 5, the inhibition rate of Bacillus belicus ZJ1 strain to quinoa black stalk disease is 58.39%, and the antibacterial band width is 11.2+ -0.1 mm.

Example 2

The preparation method of bacillus bailii ZJ1 fermentation broth comprises the following steps:

(1) Strain seed medium: the culture medium of bacillus belicus ZJ1 strain seeds adopts NA culture medium;

(2) Preparing seed liquid: the ZJ1 strain 48 h cultured on NA culture medium is inoculated into LB culture medium by picking a loop of single colony, and the temperature is 28 ℃ and 160 r min ^-1 Shaking culture is carried out for 24 h to obtain ZJ1 seed liquid;

(3) Preparing a fermentation liquid: mixing the seed solution prepared in the step (2) with the modified YSP culture solution according to the volume ratio of 1:100, mixing to prepare fermentation liquor;

(4) Culturing fermentation liquor: at 28 ℃, the rotation speed is 190 r min ^-1 The fermentation time is 72 and h, and the number of the bacillus bailii ZJ1 viable cells in the fermentation liquid is not less than 10 ⁹ cfu/mL。

The formulation of the NA culture medium is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, agar powder 15 g, distilled water 1000 mL, pH 7.0.

The formula of the LB culture solution is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, distilled water 1000 mL, pH 7.0.

The formula of the improved YSP culture solution is as follows: peptone 10.0 g, lactose 20.0 g, yeast powder 1.5 g, beef extract 1.5 g, peptone 2.0 g, distilled water 1000 ml, ph=7.0.

Experiments prove that the bacillus belicus ZJ1 fermentation liquor has good thermal stability, and the bacteriostasis rate is not obviously reduced under the condition of not exceeding 60 ℃; the acid-base adaptation range of the fermentation liquor is wider, and the bacteriostasis rate of the fermentation liquor is not obviously reduced under the condition of pH 5-9; can be stored at room temperature for a long time under the irradiation of visible light and ultraviolet light, is favorable for commercialization of the bacterial strain microbial inoculum, and has great application value in quinoa field production. The preparation process of the fermentation liquor is simple, the fermentation condition is not harsh, the raw materials used in the microbial inoculum formula are simple, the sources are wide, the cost is low, and the industrial production is easy to realize.

Example 3

Bacillus bailii of the inventionBacillus velezensis) The fermentation liquor of ZJ1 strain is diluted 10 times by water according to external ratio method, tween 80 with 10% of the dilution liquor is added as surfactant according to volume ratio to prepare the field control microbial inoculum of the bacillus belicus ZJ1, the preparation is used for 1 time in the early quinoa grouting period of 8 months, the preparation is used for 3 times every 10 days, the dosage is 50L/mu, and the control effect reaches 70.65%.

Claims (2)

1. Bacillus bailii strainBacillus velezensis) The ZJ1 strain is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.20986 in the year 2020, 11.

2. Bacillus bailii according to claim 1Bacillus velezensis) The preparation method of ZJ1 strain and fermentation broth thereof comprises the following steps:

(1) Strain seed medium: the culture medium of bacillus belicus ZJ1 strain seeds adopts an NA culture medium, and the formulation of the NA culture medium is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, agar powder 15 g, distilled water 1000 mL, pH 7.0;

(2) Preparing seed liquid: the ZJ1 strain of 48 h cultured on NA culture medium is inoculated into LB culture solution by picking a loop of single colony, and the temperature is 26-28 ℃ and 160-180 r.min ^-1 After shaking culture of 20-24 and h, ZJ1 seed liquid is obtained, and the formula of the LB culture liquid is as follows: beef extract 3 g, peptone 10 g, naCl 5 g, distilled water 1000 mL, pH 7.0;

(3) Preparing a fermentation liquid: mixing the seed solution prepared in the step (2) with the modified YSP culture solution according to the volume ratio of 1:100, and preparing a fermentation broth, wherein the formula of the improved YSP culture broth comprises the following components: peptone 10.0 g, lactose 20.0 g, yeast powder 1.5 g, beef extract 1.5 g, peptone 2.0 g, distilled water 1000 ml, ph=7.0;

(4) Culturing fermentation liquor: at 26-28deg.C and rotational speed of 180-190 r min ^-1 The fermentation time is 68-72 h, and the number of the bacillus bailii ZJ1 viable cells in the fermentation liquid is not less than 10 ⁹ cfu/mL。

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