Fermentation method of Myxococcus fulvus WCH05

Abstract

The invention relates to the technical field of a microbial fermentation process, in particular to a fermentation method of Myxococcus fulvus WCH05 (Myxococcus fulvus WCH05) and a preparation method of the Myxococcus fulvus WCH05. Preparing a fermentation seed solution, namely scraping myxococcus citrinus WC H05 pellicle, inoculating the myxococcus citrinus WC H05 pellicle into the seed culture solution, and carrying out shake culture at 28-32 DEG C and 180-220 r / min for 48-56 hours to obtain the fermentation seed solution; the seed culture solution is an MD1 culture medium. And fermenting: inoculating the fermentation seed solution into a fermentation culture medium, and culturing for 68-80 hours under the conditions that the temperature is 28-32 DEG C and the rotating speed is 180-220 r / min to obtain the myxococcus citrinus WCH05 fermentation liquor. The volume of the inoculated fermentation seed solution accounts for 5-10% of the volume of the fermentation culture medium; the fermentation culture medium comprises the following components: 8.0 g / L-10.0 g / L of soluble starch, 3.0 g / L-5. 0 g / L of yeast extract, 1.0 g / L-1. 6 g / L of MgSO4 and the balance of water, and the volume is fixed to 1 L; the pH (Potential of Hydrogen) is 6.8 to 7.2. According to the invention, the defect that the biomass in the existing myxococcus citrinus strain WCH05 culture medium is relatively low is overcome, and technical support and theoretical guidance are provided for industrial production of the myxococcus citrinus strain WCH05 and obtaining of a high-yield bacterial agent.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation technology, and more specifically to a fermentation method of Myxococcus fulvus WCH05. Background Art

[0002] Myxobacteria, Latin name myxobacteria, is a kind of advanced prokaryotes with multicellular group behavior and complex life history. They have a wide range of predation capabilities on bacteria and fungi and are recognized as a generalist microbial predator. Related reports have shown that myxobacteria can prey on and antagonize pathogens by secreting lytic enzymes, synthesizing antibacterial metabolites, and regulating the structure of soil microbial communities. They are regarded as a new type of biocontrol microorganism. As a new type of biocontrol strain, myxobacteria have characteristics that are superior to those of reported biocontrol bacteria, including rich and diverse predation strategies, a wide range of prey spectra, good colonization ability, the ability to produce abundant new secondary metabolites, the ability to produce myxospores with strong stress resistance, and participation in soil organic matter metabolism. These characteristics make myxobacteria have good advantages as biocontrol bacteria. They have shown potential application value in the biological control of plant diseases such as apricot fire blight, pear rot, cucumber wilt, and sugarcane tip rot.

[0003] Chinese invention patent 202211078553.X applied for on September 5, 2022 discloses a strain of Myxococcus fulvus and its application in biological control of plant bacterial diseases, and discloses the preparation and application of Myxococcus fulvus WCH05 (Myxococcus fulvusWCH05) strain, with a preservation number of CGMCC NO.24981. Its application is as follows: Myxococcus fulvusWCH05 exhibits good antibacterial activity against a variety of plant pathogenic bacteria, showing broad-spectrum resistance to plant pathogenic bacteria. In particular, it exhibits strong antibacterial activity against fire blight pathogen, and through in vitro inflorescence and potted pear seedling tests, it is shown that Myxococcus fulvus WCH05 has a good control effect on fire blight (Reference 1: HAN, Jian, et al. From predator to protector: Myxococcus fulvus WCH05 emerges as a potent biocontrol agent for fire blight. Frontiers in Microbiology, 2024, 15: 1378288; Reference 2: Dong Zhiming, et al. Screening of myxobacterial strains that prey on fire blight pathogen and their biocontrol potential [J]. Journal of Fruit Science, 2024, 41(01): 143-154.).

[0004] The above application has no problem in biological control, but the existing culture method has the problem of low biomass in the culture medium of Myxococcus fulvus WCH05, which seriously restricts the industrial production and application of Myxococcus fulvus WCH05. Summary of the invention

[0005] To solve the above problems, the present invention provides a fermentation method of Myxococcus fulvus WCH05, which is used to increase the biomass of Myxococcus fulvus WCH05 during fermentation.

[0006] The present invention is achieved through the following technical solutions:

[0007] A fermentation method of Myxococcus fulvus WCH05 comprises the following steps:

[0008] Prepare fermentation seed liquid: scrape the bacterial film of Myxococcus aurantiacus WCH05 and inoculate it into the seed culture liquid, culture at 28℃~32℃, 180r / min~220r / min and shaking for 48h~56h to obtain fermentation seed liquid, wherein the seed culture liquid is MD1 medium.

[0009] Fermentation: Inoculate the fermentation seed liquid into the fermentation medium, and culture for 68h to 80h at a temperature of 28°C to 32°C and a rotation speed of 180r / min to 220r / min to obtain the orange Myxococcus WCH05 fermentation liquid. The volume of the inoculated fermentation seed liquid accounts for 5% to 10% of the volume of the fermentation medium.

[0010] The fermentation medium comprises: 8.0 g / L to 10.0 g / L soluble starch, 3.0 g / L to 5.0 g / L yeast extract, 1.0 g / L to 1.6 g / L MgSO4, and water to a volume of 1 L; the pH value is 6.8 to 7.2.

[0011] Preferably, the volume of the inoculated fermentation seed liquid accounts for 7% of the volume of the fermentation medium.

[0012] Preferably, the fermentation medium comprises: 8.0 g / L soluble starch, 4.0 g / L yeast extract, 1.2 g / L MgSO4, and water to 1 L; the pH is 7.0.

[0013] Preferably, the fermentation temperature is 30°C.

[0014] Preferably, the fermentation rotation speed is 200r / min.

[0015] Preferably, the fermentation culture time is 72h.

[0016] The fermentation method is used to obtain the orange Myxococcus WCH05 fermentation liquid.

[0017] The application of the Myxococcus aurantiacus WCH05 fermentation liquid in improving the biological control effect of amylovora aurantifolia.

[0018] A bacterial agent for preventing and treating fire blight of amylopectin, wherein the active ingredient of the agent comprises the fermentation liquid of Myxococcus aurantiacus WCH05.

[0019] Preferably, the bacterial agent further comprises one or more of a carrier, a stabilizer, a protective agent and a wetting agent.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a fermentation method of Myxococcus fulvus WCH05, comprising the following steps: preparing fermentation seed liquid: scraping the bacterial film of Myxococcus fulvus WCH05 and inoculating it into the seed culture liquid, 28°C to 32°C, 180r / min to 220r / min shaking culture for 48h to 56h, and obtaining the fermentation seed liquid; the seed culture liquid is MD1 culture medium; such culture conditions can effectively promote the growth of the bacteria, ensure the bacterial concentration and vitality of the seed liquid, and provide high-quality seed liquid for subsequent fermentation. Fermentation: inoculating the fermentation seed liquid into the fermentation medium, under the conditions of a temperature of 28°C to 32°C and a rotation speed of 180r / min to 220r / min, culturing for 68h to 80h to obtain the fermentation liquid of Myxococcus fulvus WCH05. The volume of the inoculated fermentation seed liquid accounts for 5% to 10% of the volume of the fermentation medium; such fermentation culture conditions can significantly increase the biomass of the bacteria, while ensuring the growth consistency and stability of the bacteria, and improving the fermentation efficiency. The fermentation medium comprises: 8.0 g / L to 10.0 g / L of soluble starch, 3.0 g / L to 5.0 g / L of yeast extract, 1.0 g / L to 1.6 g / L of MgSO4, and water to 1 L; the pH is 6.8 to 7.2. As a carbon source, soluble starch provides energy and carbon skeleton required for bacterial growth, and promotes rapid proliferation of bacteria. As a nitrogen source and growth factor, yeast extract provides nutrients such as amino acids and vitamins required for bacterial growth, and enhances the metabolic activity of bacteria. As a trace element, MgSO4 participates in the active center of multiple enzymes, and promotes the normal metabolism and growth of bacteria. pH: maintained in the range of 6.8 to 7.2, provides a suitable growth environment for bacteria, and avoids the adverse effects of acidity and alkalinity on bacterial growth. Experiments have shown that the fermentation method of Myxococcus fulvus provided by the method increases the biomass of the bacteria by 2.49 times compared with the basic fermentation process, and can also improve its effect in biological control of apricot fire blight. The invention solves the problem of low biomass in the existing culture medium of orange myxococcus strain WCH05, and the method has low cost and is easy to operate, thus providing technical support and theoretical guidance for the industrial production of orange myxococcus strain WCH05 and obtaining a high-yield bacterial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1The figure shows the effects of different seed culture solutions of the present invention on the growth of Myxococcus fulvus WCH05.

[0024] Figure 2 This is the growth curve of Myxococcus fulvus WCH05 of the present invention.

[0025] Figure 3 The present invention shows the effects of the carbon source and carbon source concentration on the growth of Myxococcus fulvus WCH05.

[0026] Figure 3 In the figure, A shows the effect of different carbon sources on the growth of Myxococcus fulvus; B shows the effect of different concentrations of soluble starch on the growth of Myxococcus fulvus WCH05.

[0027] Figure 4 The present invention shows the influence of the nitrogen source and nitrogen source concentration on the growth of Myxococcus fulvus WCH05.

[0028] Figure 4 In the figure, A shows the effect of different nitrogen sources on the growth of Myxococcus fulvus WCH05; B shows the effect of different concentrations of yeast extract on the growth of Myxococcus fulvus WCH05.

[0029] Figure 5 The present invention shows the effects of the inorganic salt and the inorganic salt content on the growth of Myxococcus fulvus WCH05.

[0030] Figure 5 In the figure, A shows the effect of different inorganic salts on the growth of Myxococcus fulvus WCH05; B shows the effect of different contents of MgSO4 on the growth of Myxococcus fulvus WCH05.

[0031] Figure 6 The figure shows the effects of different culture conditions on the growth of Myxococcus fulvus WCH05 in the present invention.

[0032] Figure 6In the figure, A is the effect of culture medium pH on the growth of Myxococcus fulvus WCH05; B is the effect of culture temperature on the growth of Myxococcus fulvus WCH05; C is the effect of culture rotation speed on the growth of Myxococcus fulvus WCH05; D is the effect of seed liquid inoculation amount on the growth of Myxococcus fulvus WCH05; E is the effect of conical flask liquid volume on the growth of Myxococcus fulvus WCH05.

[0033] Figure 7 This is the response surface of the influence of the four main effect factors of the present invention on the growth of Myxococcus fulvus WCH05.

[0034] Figure 7 In the figure, a is the interaction between yeast extract and soluble starch; b is the interaction between liquid filling volume and soluble starch; c is the interaction between seed liquid inoculation volume and soluble starch; d is the interaction between liquid filling volume and yeast extract; e is the interaction between seed liquid inoculation volume and yeast extract; f is the interaction between seed liquid inoculation volume and liquid filling volume.

[0035] Figure 8 Comparison of the dry weight of Myxococcusfulvus WCH05 before and after the response surface test confirmation of the fermentation method of the present invention. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The culture medium used in the following examples is specifically described as follows:

[0039] VY / 4 medium: Angel Yeast 5.0 g / L, CaCl2·2H2O 1.0 g / L, water to 1 L; pH 7.2.

[0040] LBS medium: soluble starch 7.0 g / L, yeast extract powder 5.0 g / L, casein peptone 1.0 g / L, MgSO4·7H2O 1.0 g / L, water to 1 L; pH 7.2.

[0041] TPM medium: Tris-HCl 10mM / L, KH2PO4 1mM / L, MgSO4 8mM / L, water to 1L; pH 7.6.

[0042] MD1 medium: casein peptone 6.0 g / L, soluble starch 2.0 g / L, CaCl2·2H2O 0.4 g / L, MgSO4·7H2O 2.0 g / L, water to 1 L; pH 7.2 g / L.

[0043] CYE medium: casein peptone 10.0 g / L, yeast extract 5.0 g / L, MgSO4·7H2O 1.0 g / L, water to 1 L; pH 7.6.

[0044] NA medium: peptone 10.0 g / L, NaCl 5.0 g / L, sucrose 5.0 g / L, beef extract 3.0 g / L, agar powder 15.0 g / L, water to 1 L; pH 7.2.

[0045] VY / 2 medium: Angel Yeast 5.0g / L, CaCl2·2H2O 1.0g / L, water to 1L; pH 7.2, after sterilization, add VB to a final concentration of 50μg / mL 12 .

[0046] The above culture media were sterilized by high pressure steam at 121°C for 25 min.

[0047] In the following examples, Myxococcus fulvus WCH05 is the Myxococcus fulvus WCH05 in the document "Dong Zhiming, Bai Xinhe, Dou Xinyu, Luo Ming, Lv Wen, Han Jian. Screening of myxobacterial strains that prey on apricot fire blight and their biocontrol potential [J]. Journal of Fruit Science, 2024, 41(01): 143-154", which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a strain collection number of CGMCC No. 24981. Erwinia amylovora E.a001 is an isolate of fragrant pear from Korla City, Xinjiang. The public can obtain the biological material from the applicant, which is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0048] The beneficial effects of the present invention are described below by means of specific embodiments:

[0049] Example 1: Fermentation method of Myxococcus fulvus WCH05

[0050] Preparation of fermentation seed liquid: scrape an appropriate amount of activated orange Myxococcus WCH05 biofilm and inoculate it into seed culture liquid, and culture it at 28°C and 180r / min for 48h to obtain fermentation seed liquid; the seed culture liquid is MD1 medium, and its components are: casein peptone 6.0g / L, soluble starch 2.0g / L, CaCl2·2H2O 0.4g / L, MgSO4·7H2O 2.0g / L, and water is fixed to 1L; pH is 7.2.

[0051] Fermentation: The fermentation seed liquid was inoculated into the fermentation medium, and cultured for 68 hours at a temperature of 28° C. and a rotation speed of 180 r / min to obtain the orange Myxococcus WCH05 fermentation liquid. The volume of the inoculated fermentation seed liquid accounted for 5% of the volume of the fermentation medium.

[0052] The fermentation medium comprises: 8.0 g / L soluble starch, 3.0 g / L yeast extract, 1.0 g / L MgSO4, and water to a volume of 1 L. The pH of the medium may be 6.8.

[0053] Example 2: A fermentation method of Myxococcus fulvus WCH05

[0054] Preparation of fermentation seed liquid: scrape an appropriate amount of activated orange sticky coccus WCH05 biofilm and inoculate it into the seed culture liquid, and culture it at 30°C and 200r / min with shaking for 52h to obtain the fermentation seed liquid; the seed culture liquid is MD1 medium, and its components are: casein peptone 6.0g / L, soluble starch 2.0g / L, CaCl2·2H2O0.4g / L, MgSO4·7H2O 2.0g / L, and water is fixed to 1L; the pH is 7.2.

[0055] Fermentation: The fermentation seed liquid was inoculated into the fermentation medium, and cultured for 72 hours at a temperature of 30° C. and a rotation speed of 200 r / min to obtain the orange Myxococcus WCH05 fermentation liquid. The volume of the inoculated fermentation seed liquid accounted for 7% of the volume of the fermentation medium.

[0056] The fermentation medium comprises: 8.0 g / L soluble starch, 4.0 g / L yeast extract, 1.2 g / L MgSO4, and water to a volume of 1 L. The pH of the medium may be 7.0.

[0057] Example 3: Fermentation method of Myxococcus fulvus WCH05

[0058] Preparation of fermentation seed liquid: scrape an appropriate amount of activated orange Myxococcus WCH05 biofilm and inoculate it into seed culture liquid, and culture at 32°C and 220r / min with shaking for 56h to obtain fermentation seed liquid; the seed culture liquid is MD1 medium, and its components are: casein peptone 6.0g / L, soluble starch 2.0g / L, CaCl2·2H2O 0.4g / L, MgSO4·7H2O 2.0g / L, and water is fixed to 1L; pH is 7.2.

[0059] Fermentation: The fermentation seed liquid was inoculated into the fermentation medium, and cultured for 80 hours at a temperature of 32° C. and a rotation speed of 220 r / min to obtain the orange Myxococcus WCH05 fermentation liquid. The volume of the inoculated fermentation seed liquid accounted for 10% of the volume of the fermentation medium.

[0060] The fermentation medium comprises: 10.0 g / L soluble starch, 5.0 g / L yeast extract, 1.6 g / L MgSO4, and water to a volume of 1 L. The pH of the medium may be 7.2.

[0061] In Comparative Examples 1, 2, 3 and 4, VY / 4, VY / 2, LBS and CYE were used as seed culture media respectively, and the rest of the fermentation method was exactly the same as that in Example 2.

[0062] The carbon sources in the fermentation medium components in Comparative Examples 5, 6, 7, 8, 9 and 10 are pea starch, glucose, dextrin, sweet potato starch, sucrose and galactose, respectively, and the rest of the fermentation method is exactly the same as that in Example 2.

[0063] The nitrogen sources in the fermentation medium components in Comparative Examples 11, 12, 13, 14, 15, 16 and 17 are soy peptone, trypsin, beef extract, peptone, casein peptone, KNO3 and urea, respectively, and the rest of the fermentation method is exactly the same as in Example 2.

[0064] The inorganic salts in the fermentation medium components in Comparative Examples 18, 19, 20, 21, 22 and 23 are CuSO4, MnSO4, CaSO4, FeSO4, K2SO4 and ZnSO4 respectively, and the rest of the fermentation method is exactly the same as in Example 2.

[0065] The pH of the fermentation medium in Comparative Example 24, Comparative Example 25, Comparative Example 26, Comparative Example 27, Comparative Example 28, Comparative Example 29, Comparative Example 30 and Comparative Example 31 were 6.0, 6.2, 6.4, 6.6, 7.4, 7.6, 7.8 and 8.0, respectively, and the rest of the fermentation method was exactly the same as in Example 2.

[0066] The fermentation culture temperatures of the fermentation medium in Comparative Examples 32 and 33 were 26° C. and 34° C., respectively, and the rest of the fermentation method was exactly the same as in Example 2.

[0067] The volume of the inoculated fermentation seed liquid in Comparative Examples 34, 35, 36 and 37 accounted for 1%, 2%, 3% and 4% of the volume of the fermentation medium, respectively. The rest of the fermentation method is exactly the same as that in Example 2.

[0068] The fermentation culture speeds of the fermentation medium in Comparative Examples 38 and 39 were 160 r / min and 240 r / min, respectively, and the rest of the fermentation method was exactly the same as in Example 2.

[0069] Experimental Example 1: Effects of different seed culture media on the growth of strain WCH05 and determination of its growth curve

[0070] The growth curve of the fermented seed solution of strain WCH05 prepared from different seed culture solutions in Example 2 and Comparative Examples 1 to 4 was measured, the bacteria were dried and weighed, and a total of 25 time points were set, with 3 repetitions at each time point. The growth curve was drawn with the dry weight of strain WCH05 bacteria as the ordinate and the culture time as the abscissa.

[0071] The results are as follows Figure 1 As shown, there are significant differences in the growth of strain WCH05 in the five seed culture solutions in Example 2 and Comparative Examples 1 to 4. Among them, the dry weight of the bacteria obtained by culturing in MD1, VY / 4 and CYE medium is significantly higher than that in VY / 2 and LBS medium, and the dry weight of the bacteria obtained in MD1 medium is the highest, reaching 130.43 mg / 100 mL, so MD1 medium is selected as the seed culture solution.

[0072] The growth curve of strain WCH05 was measured using MD1 medium as the seed culture medium. The results are as follows Figure 2 As shown in the figure, strain WCH05 enters the logarithmic phase after about 16 hours of growth in MD1 medium, and the dry weight of the bacteria increases exponentially at 16 hours. It enters the stable phase at 68 hours, when the dry weight of the bacteria reaches the maximum. After 80 hours, the growth enters the decay phase. Therefore, the optimal culture time of the seed solution for subsequent experiments is selected at 48 hours, and the dry weight of the bacteria is measured at 72 hours, i.e., the stable phase, as the response value for further confirmation of the evaluation of the fermentation conditions.

[0073] Experimental Example 2: Effect of Carbon Source in Fermentation Medium on Biomass Obtained by Fermentation of Strain WCH05

[0074] The WCH05 fermentation broth obtained in Example 2 and Comparative Examples 5 to 10 was respectively collected and the dry weight was measured. The results are as follows: Figure 3As shown in A. The strain WCH05 can grow in the culture medium with 7 different carbon sources. When soluble starch is used as the carbon source, the dry weight of the strain WCH05 is significantly higher than that of other carbon sources, so soluble starch is selected as the best carbon source. The effects of different concentrations of soluble starch on the growth of strain WCH05 are further determined. The results are shown in Figure 3 As shown in Figure B, the dry weight of strain WCH05 increased with the increase of soluble starch concentration. When the soluble starch concentration reached 0.8%, the dry weight of strain WCH05 was the highest, reaching 267.63 mg / 100 mL. Therefore, 0.8% soluble starch was selected as the optimal carbon source concentration for fermentation culture of strain WCH05.

[0075] Experimental Example 3: Effect of nitrogen source in fermentation medium composition on biomass obtained by fermentation of strain WCH05

[0076] The WCH05 fermentation broth obtained in Example 2 and Comparative Examples 11 to 17 was respectively collected and the dry weight was measured. The results are as follows: Figure 4 As shown in A, strain WCH05 can grow in 8 different nitrogen source culture media. When yeast extract is used as the nitrogen source, the dry weight of strain WCH05 is significantly higher than that of other nitrogen source treatment groups. Therefore, yeast extract is selected as the best nitrogen source. The effects of different concentrations of yeast extract on the growth of strain WCH05 are further determined. The results are shown in Figure 4 As shown in B, when the concentration of yeast extract was 0.4%, the dry weight of the bacteria reached the highest, which was 290.7 mg / 100 mL. Therefore, 0.4% yeast extract was selected as the optimal nitrogen source concentration for the fermentation culture of strain WCH05.

[0077] Experimental Example 4: Effect of Inorganic Salts in Fermentation Medium on the Biomass Obtained by Fermentation of Strain WCH05

[0078] The WCH05 fermentation broth obtained in Example 2 and Comparative Examples 18 to 23 was respectively collected and the dry weight was measured. The results are as follows: Figure 5 As shown in A, the addition of MgSO4 can significantly promote the growth of strain WCH05 compared with other inorganic salts, so MgSO4 was selected as the optimal inorganic salt for the culture medium. The effects of different concentrations of MgSO4 on the growth of strain WCH05 were measured, and the results are shown in Figure 5 As shown in B, when the MgSO4 concentration is 0.12%, the dry weight of the bacteria is significantly higher than that of other inorganic salts. Therefore, 0.12% MgSO4 is selected as the optimal inorganic salt concentration for fermentation culture of strain WCH05.

[0079] Experimental Example 5: Effect of pH of fermentation medium on biomass obtained by fermentation of strain WCH05

[0080] The WCH05 fermentation broth obtained in Examples 1 to 3 and Comparative Examples 24 to 31 was centrifuged to collect the bacterial cells, dried and weighed. The results are as follows: Figure 6 As shown in A, the dry weight of the bacteria was higher when the pH of the fermentation medium was between 6.8 and 7.2, and the dry weight of the bacteria was the highest when the pH was 7.0.

[0081] Experimental Example 6: Effect of the temperature of the fermentation medium on the biomass obtained by fermentation of strain WCH05

[0082] The WCH05 fermentation broth obtained in Examples 1 to 3 and Comparative Examples 32 to 33 was centrifuged to collect the bacterial cells, dried and weighed. The results are as follows: Figure 6 As shown in B, the dry weight of the bacteria is higher when the temperature is 28℃~32℃, and the dry weight of the bacteria is the highest when the temperature is 30℃.

[0083] Experimental Example 7: Effect of the volume ratio of the inoculated fermentation seed solution in the fermentation medium on the biomass obtained by the fermentation of strain WCH05

[0084] The WCH05 fermentation broth obtained in Examples 1 to 3 and Comparative Examples 34 to 37 was centrifuged to collect the bacterial cells, dried and weighed. The results are as follows: Figure 6 As shown in C, the dry weight of the bacteria is higher when the volume of the inoculated fermentation seed liquid accounts for 5% to 10% of the volume of the fermentation medium, and the dry weight of the bacteria is highest when the volume of the inoculated fermentation seed liquid accounts for 7% of the volume of the fermentation medium.

[0085] Experimental Example 8: Effect of fermentation culture speed on the biomass obtained by fermentation of strain WCH05

[0086] The WCH05 fermentation broth obtained in Examples 1 to 3 and Comparative Examples 38 to 39 was centrifuged to collect the bacterial cells, dried and weighed. The results are as follows: Figure 6 As shown in D, the dry weight of the bacteria is higher when the fermentation culture speed is 180r / min~220r / min, and the dry weight of the bacteria is the highest when the speed is 200r / min.

[0087] The liquid volume was set to 30 mL / 250 mL to 150 mL / 250 mL, the gradient was 10 mL / 250 mL, and there were 13 gradients in total. The other conditions were the same as those in Example 2. After the culture was completed, the cells were collected by centrifugation, dried and weighed. The results were as follows: Figure 6 The liquid volume shown in E is 100mL / 250mL, which has the highest dry weight of bacteria.

[0088] Experimental Example 9: Response Surface Methodology to Determine the Effects of Fermentation Medium Composition and Fermentation Conditions on Fermentation Biomass of Myxococcus fulvus WCH05

[0089] 1. Plackett-Burman, also known as PB test

[0090] Based on the results of the single-factor experiment, each factor was selected at two levels, high and low, and the Design-expert 13.0 software was used to design a PB experiment with N=12 times. The dry weight of the bacteria after fermentation was taken as the response value, and the main effect analysis was performed on 8 factors, namely carbon source, nitrogen source, inorganic salt, fermentation temperature, pH, inoculation size, liquid volume and rotation speed, to identify the main factors affecting the growth of strain WCH05.

[0091] The results of the main effect factor analysis are shown in Table 1. The significance of the eight factors on the growth of strain WCH05 is as follows: soluble starch > yeast extract > liquid volume > seed liquid inoculation volume > MgSO4 > pH > rotation speed > temperature. The P values ​​of soluble starch, yeast extract, liquid volume, and inoculation volume were all less than 0.05, so these four factors were selected as the main factors for constructing the model for response surface method.

[0092] Table 1 Significance analysis of Plackett-Burman test results

[0093]

[0094]

[0095] Note: *Significant difference (P<0.05); **Extremely significant difference (P<0.01); NSNo significant difference (P>0.05).

[0096] 2. Response Surface Methodology Confirmation of Fermentation Conditions of Strain WCH05

[0097] In order to maximize the biomass of strain WCH05, based on the PB test results, the four main effect factors of soluble starch, yeast extract, liquid volume, and seed liquid inoculation volume were used as independent variables, and the optimal value point of the single factor test was used as the central point data, that is, the concentration range of soluble starch was 0.6% to 1.0%, the concentration range of yeast extract was 0.2% to 0.6%, the liquid volume range was 80mL / 250mL-120mL / 250mL, and the seed liquid inoculation range was 5% to 9%. The dry weight of the bacteria was used as the response value, and the response surface optimization was performed using Box-Behnken design, as shown in Table 2. The experimental data were analyzed and statistically analyzed to obtain the regression equation and determine the best combination of factors.

[0098] The test results were solved by quadratic regression equation and variance analysis, and the regression equation was obtained: Y1 = +312.71-8.16A-15.19B-16.83C-9.76D-9.83AB-3.98AC+3.39AD-0.9619BC-6.62BD-27.20CD-10.85A2 -21.37B 2 -22.53C 2 -22.67D 2 .

[0099] From the variance analysis, as shown in Table 3, the regression equation P < 0.05, the lack of fit term is not significant P = 0.4766 > 0.05, R 2 The value is 0.9417, indicating that the model is significant, the model prediction value has a high degree of fit with the actual value, and can better reflect the change of the response value. The analysis results of the F value show that the influence of each factor on the dry weight of strain WCH05 is as follows: yeast extract > liquid volume > soluble starch > seed liquid inoculation volume. The response surface of the interaction on the dry weight of the bacteria obtained by the regression equation shows that Figure 7 As shown, the graphs are all three-dimensional space surface graphs with openings downward, indicating that the response surface value has extremely high values, and the optimal value exists in the designed factor level. Among them, the interaction between soluble starch and inoculation amount is more significant than other factors, P>0.05. Solving the regression equation, the optimal concentration of soluble starch is 0.8%, the optimal concentration of yeast extract is 0.4%, the optimal liquid volume is 100mL / 250mL, and the optimal seed liquid inoculation amount is 7%.

[0100] Combining the single factor and response surface results, the optimal liquid fermentation medium formula for strain KS01 was finally determined to be: soluble starch 8g / L, yeast extract 4g / L, MgSO4 1.2g / L; the optimal fermentation conditions were: medium pH 7.0, shake bottle volume 100mL / 250mL, seed liquid inoculation 7.0%, rotation speed 200r / min, and culture at 30℃ for 72h. Under these conditions, the model predicted that the maximum dry weight of strain WCH05 was 320.81mg / 100mL.

[0101] Table 2 Response surface methodology experimental design and results

[0102]

[0103]

[0104] Table 3 Analysis of variance of fitted regression equation

[0105] parameter sum of squares Degrees of Freedom Mean Square F-number P-value Significance Model 49938.75 14 3567.05 16.16 <0.0001 ** A-Soluble starch 965.05 1 965.05 4.37 0.0552 NS B-Yeast Extract 1412.46 1 1412.46 6.40 0.0240 * C-Liquid volume 1068.01 1 1068.01 4.84 0.0451 * D-seed liquid inoculation amount 358.34 1 358.34 1.62 0.2233 NS AB 2751.00 1 2751.00 12.46 0.0033 ** AC 451.56 1 451.56 2.05 0.1745 NS AD 183.60 1 183.60 0.8319 0.3772 NS BC 4.87 1 4.87 0.0221 0.8840 NS BD 311.52 1 311.52 1.41 0.2546 NS CD 2192.52 1 2192.52 9.93 0.0071 ** <![CDATA[A 2 ]]> 11681.14 1 11681.14 52.93 <0.0001 ** <![CDATA[B 2 ]]> 8961.73 1 8961.73 40.61 <0.0001 ** <![CDATA[C 2 ]]> 6170.03 1 6170.03 27.96 0.0001 ** <![CDATA[D 2 ]]> 3190.62 1 3190.62 14.46 0.0019 ** Residual 3089.78 14 220.70 / / / Lack of Fit 2303.62 10 230.36 1.17 0.4766 NS Pure Error 786.16 4 196.54 / / / Total error 53028.53 28 / / / / <![CDATA[R 2 =0.9417]]> / / / / / /

[0106] Note: *Significant difference (P<0.05); **Extremely significant difference (P<0.01); NSNot significant difference (P>0.05); “ / ” means no such item.

[0107] 3. Optimal Model Verification of Fermentation Medium and Culture Conditions

[0108] According to the optimal culture parameters obtained by response surface experiment, shake flask fermentation was carried out to obtain the actual dry weight of bacteria after fermentation. It was compared with the predicted value and the dry weight of fermentation before response surface experiment to verify the reliability and effectiveness of the model and obtain the best fermentation result.

[0109] The results are as follows Figure 8 As shown in Figure 1, the actual dry weight of strain WCH05 was 325.0 mg / 100 mL, with a relative error of 1.30% with the predicted value. The results confirmed that the model was reliable, indicating that the actual fermentation conditions under the conditions confirmed by the response surface test had a good fit with the prediction. Before the response surface test was confirmed, the dry weight of the bacteria obtained using MD1 medium was only 130.43 mg / 100 mL, as shown in Experimental Example 1. Compared with the response surface test confirmation, the biomass obtained by fermentation after confirmation increased by 2.49 times.

[0110] Experimental Example 10: Comparison of the efficacy of Myxococcus fulvus WCH05 fermentation liquid against fire blight before and after confirmation of fermentation conditions by response surface experiment

[0111] The two-year-old healthy potted Pyrus betula seedlings were used as inoculation materials to determine the efficacy of the fermentation liquid of strain WCH05 against amylopectin. There were four treatments in the experiment: A. The fermentation liquid of strain WCH05 obtained before the response surface test, that is, the fermentation liquid of strain WCH05 obtained by shaking culture in MD1 medium. Liquid volume 100mL / 250mL, seed liquid inoculation amount 3%, pH 7.2, 30℃, 180r / min culture for 72h; B. The fermentation broth of strain WCH05 obtained after confirmation by response surface test, that is, the fermentation broth of strain WCH05 obtained under the optimal culture conditions determined by the above test: soluble starch 8g / L, yeast extract 4g / L, MgSO4 1.2g / L, liquid volume 100mL / 250mL, seed liquid inoculation amount 7.0%, pH 7.0, rotation speed 200r / min, 30℃ culture for 72h; C. 4000 times liquid of agricultural streptomycin, purchased from North China Pharmaceutical Factory, with an active ingredient of 72%; D. Sterile water control.

[0112] Use a handheld sprayer to spray the fermentation liquid of strain WCH05, agricultural streptomycin, and sterile water until the leaves and branches are completely wet, 30mL / pot, and spray and inoculate 10 7 cfu / mL suspension of fire blight pathogen, 30mL / pot, 3 pots were sprayed for each treatment, and repeated 3 times. The inoculated pear seedlings were placed in a solar greenhouse at 30℃ and 70% relative humidity. During the period, the disease situation was observed regularly, the number of diseased branches was recorded, the length of branch death and the disease level were measured, and the incidence rate, disease index and prevention effect were calculated. After the experiment, the diseased plant materials were sterilized by dry heat and destroyed.

[0113] The disease grading standard for potted Pyrus betula seedlings is as follows: Grade 0, no lesions on the branches; Grade I, the length of the lesions on the branches is 1 / 3 of the length of the inoculated branches; Grade III, the length of the lesions on the branches is 2 / 3 of the length of the inoculated branches; Grade V, the length of the lesions on the branches is greater than 2 / 3 of the length of the inoculated branches.

[0114] Incidence rate (%) = (number of diseased branches / total number of inoculated branches) × 100.

[0115] Disease index (%) = ∑ (number of diseased branches at each level × representative value of disease level) / (total number of inoculated branches × highest level value) × 100.

[0116] Preventive effect (%) = (control disease index - treatment disease index) / control disease index × 100.

[0117] The results of the control efficacy test showed that, as shown in Table 4, the fermentation broth of strain WCH05 confirmed by the response surface test could significantly reduce the incidence rate and disease index of Pyrus betula seedlings, P < 0.05. The 7-day control efficacy of the fermentation broth confirmed by the response surface test was 86.22%, which was significantly higher than the 80.25% control efficacy of the fermentation broth confirmed by the response surface test, and the 14-day control efficacy was 82.57%, which was still higher than the 79.64% control efficacy of the fermentation broth before confirmation by the response surface test. The average control efficacy of the fermentation broth confirmed by the response surface test from 7 to 14 days was 84.40%, which was 4.45% higher than that of the fermentation broth confirmed by the response surface test.

[0118] Table 4 Indoor control efficacy of Myxococcus fulvus WCH05 against fire blight

[0119]

[0120] Note: Different lowercase letters in the same column indicate significant differences among treatments at P < 0.05 level; “ / ” indicates no such difference.

[0121] The indoor control effect results of amylopectin disease show that the fermentation liquid of strain WCH05 obtained after the response surface test confirmation selection of the present invention can significantly reduce the incidence of amylopectin disease, and the control effect of the fermentation liquid obtained after the response surface test confirmation of the present invention on amylopectin disease is significantly better than that of the myxobacterium fermentation liquid obtained before the response surface test confirmation. This shows that the fermentation process adopted by the present invention has significantly improved the control effect of strain WCH05.

[0122] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0123] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A fermentation method of Myxococcus fulvus WCH05, characterized in that: The steps include: Prepare fermentation seed solution: take the biofilm of Myxococcus aurantiacus WCH05 and inoculate it into the seed culture solution, culture it at 28℃~32℃, 180r / min~220r / min shaking for 48h~56h, and obtain fermentation seed solution; the seed culture solution is MD1 medium; Fermentation: inoculating the fermentation seed liquid into the fermentation medium, culturing for 68h to 80h at a temperature of 28°C to 32°C and a rotation speed of 180r / min to 220r / min to obtain a fermentation liquid of Myxococcus aurantiacus WCH05, wherein the volume of the inoculated fermentation seed liquid accounts for 5% to 10% of the volume of the fermentation medium; The fermentation medium comprises: 8.0 g / L to 10.0 g / L soluble starch, 3.0 g / L to 5.0 g / L yeast extract, 1.0 g / L to 1.6 g / L MgSO4, and water to a volume of 1 L; the pH value is 6.8 to 7.

2.

2. The fermentation method of Myxococcus aurantiacus WCH05 as claimed in claim 1, characterized in that: The volume of the inoculated fermentation seed liquid accounts for 7% of the volume of the fermentation medium.

3. The fermentation method of Myxococcus aurantiacus WCHH05 as claimed in claim 1, characterized in that: The fermentation medium comprises the following components: 8.0 g / L soluble starch, 4.0 g / L yeast extract, 1.2 g / L MgSO4, and water to a volume of 1 L; the pH value is 7.

0.

4. The fermentation method of Myxococcus aurantiacus WCH05 as claimed in claim 1, characterized in that: The fermentation temperature was 30°C.

5. The fermentation method of Myxococcus aurantiacus WCH05 as claimed in claim 1, characterized in that: The fermentation speed is 200r / min.

6. The fermentation method of Myxococcus aurantiacus WCH05 as claimed in claim 1, characterized in that: The fermentation culture time is 72h.

7. The Myxococcus aurantiacus WCH05 fermentation liquid obtained by the fermentation method as claimed in claim 1.

8. Use of the fermentation liquid of Myxococcus aurantiacus WCH05 as claimed in claim 7 in improving the biological control effect of fire blight.

9. A fungal agent for preventing and treating fire blight of amylopectin, characterized in that: The active ingredient comprises the fermentation broth of Myxococcus aurantiacus WCH05 according to claim 6.

10. The bacterial agent according to claim 9, characterized in that The bacterial agent also includes one or more of a carrier, a stabilizer, a protective agent and a wetting agent.

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