Bacillus velezensis g-1 and application thereof in preventing and treating plant fusarium moniliforme

Abstract

The present application belongs to the technical field of agricultural microorganisms, and particularly relates to a bacillus velezensis G-1 and application thereof in prevention and treatment of plant smut fungus. The present application provides a bacillus velezensis G-1, and the preservation number of the bacillus velezensis G-1 is CGMCC No. 23750. The bacillus has the effect of preventing and treating plant smut fungus, and has remarkable effect in promoting seed germination and bud lengthening; after the bacillus velezensis G-1 and lipopeptide substances of the bacillus velezensis G-1 are simultaneously used to prepare a microbial suspension seed coating agent, the high efficient colonization ability of the bacillus velezensis G-1 can be improved in the process of inhibiting bacteria, and the competitive advantage between the microbial seed coating agent and indigenous microorganisms is improved, so that the effect of preventing smut fungus from invading plants can be achieved, and plant germination can be promoted.

Description

Technical Field

[0001] This invention relates to the technical field of agricultural microorganisms, specifically to a strain of Bacillus belye G-1 and its application in the control of plant smut. Background Technology

[0002] Sorghum is an important C4 crop in the global agricultural system, playing a vital role in medium- and low-grade and marginal farmland due to its drought resistance, tolerance to poor soil, and salt tolerance. However, sorghum smut disease occurs frequently in various producing areas, and it has become one of the major diseases restricting the increase of sorghum yield in my country.

[0003] To effectively control sorghum smut, scholars both domestically and internationally have conducted research on various aspects, including the breeding of resistant varieties, the selection of chemical control agents, the analysis of the pathogenicity of different physiological races, and infection characteristics. However, the resistance of resistant varieties to smut often degenerates with increasing planting years. Excessive concentration of chemical agents can cause phytotoxicity, while insufficient concentration leads to ineffective treatment. Furthermore, due to frequent pathogen mutations, the emergence of a newly differentiated physiological race often results in a rapid loss of resistance in a batch of resistant resources. Currently, there are no reports on the use of microorganisms to prepare suspension seed coatings for the control of sorghum smut. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus belye G-1 and its application in the control of plant smut fungus. It can not only control smut fungus but also promote plant growth, and has the characteristics of good control effect and high safety.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a strain of Bacillus belyssus G-1, the preservation number of which is CGMCC No. 23750.

[0007] This invention also provides a lipopeptide substance of Bacillus belyssus G-1, wherein the preparation method of the lipopeptide substance includes:

[0008] The activated Bacillus berleis G-1 was fermented, and the fermentation broth was centrifuged for the first time to obtain the first supernatant of the Bacillus berleis G-1 fermentation broth.

[0009] The first supernatant was mixed with an acidic solution and allowed to stand before being centrifuged a second time to obtain the first precipitate.

[0010] The first precipitate was resuspended, and the pH of the resuspended precipitate was adjusted to 7.0-8.0 before a third centrifugation was performed to obtain the second supernatant.

[0011] After adjusting the pH of the second supernatant to 2.0–3.0, a fourth centrifugation was performed to obtain the second precipitate.

[0012] The second precipitate was extracted with methanol, and the extract was concentrated to obtain the lipopeptide substance of Bacillus belyssus G-1.

[0013] Preferably, during the fermentation, the inoculum size of Bacillus belye G-1 is 0.5% to 2% of the total fermentation medium volume;

[0014] The fermentation time is 3 to 5 days, the temperature is 25 to 30°C, and the rotation speed is 120 to 180 r / min.

[0015] Preferably, the conditions for the first centrifugation, the second centrifugation, the third centrifugation and the fourth centrifugation are as follows: time is 5 to 10 minutes and speed is 7500 to 12000 rpm.

[0016] Preferably, the acidic solution includes a hydrochloric acid solution; the concentration of the hydrochloric acid solution is 1–6 mol / L.

[0017] The conditions for mixing and settling include: a time of 8 to 24 hours and a temperature of 0 to 10°C.

[0018] Preferably, during the extraction, the volume ratio of methanol to Bacillus vesiculosus G-1 fermentation broth is 0.5–1.5:0.5–2;

[0019] The extraction is performed 1 to 4 times.

[0020] This invention provides a microbial suspension seed coating agent, comprising the following raw materials in the following weight percentages: Bacillus belye G-1 as described in the above technical solution, 0.02%–0.06% of the lipopeptide substance as described in the above technical solution, 3%–4% of sodium carboxymethyl cellulose, 0.4%–0.6% of sodium alginate, 3%–4% of dispersant MF, 0.1%–0.2% of ferrous sulfate, 0.5%–2% of magnesium sulfate, 0.5%–4% of dipotassium hydrogen phosphate, 2%–6% of color paste, and the balance being water;

[0021] The concentration of Bacillus vesiculus G-1 in the microbial suspension seed coating agent is 5.0 × 10⁻⁶. 7 ~5.0×10 9 cfu / g.

[0022] Preferably, the preparation method of the lipopeptide substance of Bacillus belyssus G-1 includes:

[0023] This invention provides the application of the microbial suspension seed coating agent described in the above-mentioned technical solution in the control of plant smut fungus;

[0024] The plant mentioned includes sorghum.

[0025] This invention provides a method for controlling plant smut, comprising: coating plant seeds with the microbial suspension seed coating agent described in the above technical solution, and then sowing the coated plant seeds.

[0026] Preferably, the mass ratio of the microbial suspension seed coating agent to the plant seeds is 30-45:0.5-2.

[0027] Beneficial effects:

[0028] This invention provides a strain of Bacillus belyceae G-1, with the preservation number CGMCC No. 23750. This bacterium is effective in controlling plant smut fungus and is also significantly effective in promoting seed germination and shoot elongation.

[0029] Based on the above technical advantages, this invention provides a microbial suspension seed coating agent, comprising the following raw materials in the following mass percentages: *Bacillus belyssus* G-1 as described in the above technical solution, 0.02%–0.06% of lipopeptide of *Bacillus belyssus* G-1 as described in the above technical solution, 3%–4% sodium carboxymethyl cellulose, 0.4%–0.6% sodium alginate, 3%–4% dispersant MF, 0.1%–0.2% ferrous sulfate, 0.5%–2% magnesium sulfate, 0.5%–4% dipotassium hydrogen phosphate, 2%–6% color paste, and the balance being water; the concentration of *Bacillus belyssus* G-1 in the microbial suspension seed coating agent is 5.0 × 10⁻⁶. 7 ~5.0×10 9 cfu / g. This invention enhances the plant's ability to control diseases by adding an appropriate concentration of Bacillus berleis G-1; by adding the lipopeptide substances of Bacillus berleis G-1, it is beneficial to improve the efficient colonization ability of Bacillus berleis G-1 in the plant rhizosphere during the antibacterial process, thereby enhancing the competitive advantage between the microbial seed dressing agent and the indigenous microorganisms, and achieving the effect of preventing the infection of plants by the pathogen of Solanum smut.

[0030] This invention also provides the application of the microbial suspension seed dressing agent described in the above-mentioned technical solution in the control of plant smut pathogen. Experiments have shown that after adopting the technical solution provided by this invention, the infection prevention effect against smut pathogen in sorghum is as high as 87.74%, and it can significantly promote the growth of sorghum seedlings.

[0031] Biological Preservation Information

[0032] Bacillus velezensis G-1 was deposited on November 8, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23750. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0034] Figure 1 Cluster heatmap showing the effects of different treatments on germination rate and shoot length of sorghum seeds in Experiment Example 1;

[0035] Figure 2 The effects of different treatments on the infectivity of sorghum smut pathogen in Experiment Example 2;

[0036] Figure 3 This describes the formation of sorghum root tissue infected by *Sorghum smut* under different treatments in Experiment Example 2.

[0037] Figure 4 The effects of different film-forming agents on the physical properties, strain survival rate and seed germination of the biological suspension agent in Experiment Example 3;

[0038] Figure 5 This illustrates the effects of different nutrients on the physical properties, bacterial survival rate, and seed germination of the biological suspension in Experiment Example 4.

[0039] Figure 6 The effects of different dispersants MF on seeds in Experiment Example 6;

[0040] Figure 7 To estimate the marginal mean of different factors in the orthogonal experiment in Example 7 using analysis of variance;

[0041] Figure 8 To verify the effectiveness of the bio-suspension prepared with the optimal formulation in Experimental Example 7;

[0042] Figure 9 The effects of different treatments in Example 1 and Comparative Examples 1-4 on sorghum seed germination and sorghum smut infection;

[0043] Figure 10 To illustrate the effects of different treatments in Example 1 and Comparative Examples 5-6 on sorghum seed germination, seedling growth, and smut pathogen infection;

[0044] Figure 11 The results are the whole genome sequencing results of strain G-1 in Experiment Example 1. Detailed Implementation

[0045] Unless otherwise specified, all raw materials and reagents used in this invention are conventionally purchased.

[0046] This invention provides a strain of *Bacillus belyssus* G-1, with the preservation number CGMCC No. 23750. The *Bacillus belyssus* G-1 strain of this invention is preferably isolated from the fruit of *Catalpa bungei*. The G-1 strain exhibits good resistance to *Smut* disease and significantly promotes seed germination and shoot elongation. The *Bacillus belyssus* G-1 strain preferably comprises a suspension of *Bacillus belyssus* G-1.

[0047] The present invention also provides lipopeptide substances of Bacillus belyssus G-1. The preparation method of the lipopeptide substances includes: fermenting activated Bacillus belyssus G-1, and centrifuging the fermentation broth to obtain a first supernatant of the Bacillus belyssus G-1 fermentation broth; mixing the first supernatant with an acidic solution, allowing it to stand, and then centrifuging it a second time to obtain a first precipitate; resuspending the first precipitate, adjusting the pH of the resuspended first precipitate to 7.0-8.0, and then centrifuging it a third time to obtain a second supernatant; adjusting the pH of the second supernatant to 2.0-3.0, and then centrifuging it a fourth time to obtain a second precipitate; extracting the second precipitate with methanol, and concentrating the extract to obtain the lipopeptide substances of Bacillus belyssus G-1.

[0048] In this invention, *Bacillus belye* G-1 is preferably activated to obtain activated *Bacillus belye* G-1. In this invention, the activation method and reagents are not particularly required; techniques well-known in the art can be used.

[0049] After obtaining the activated Bacillus berleis G-1, the present invention preferably ferments the activated Bacillus berleis G-1 to obtain a Bacillus berleis G-1 fermentation broth. In the present invention, the fermentation medium is preferably BPY liquid medium; the inoculum amount of the activated Bacillus berleis G-1 is preferably 0.5% to 2% of the total volume of the BPY liquid medium, more preferably 1%; the fermentation method is preferably shaking culture; the shaking time is preferably 3 to 5 days, more preferably 4 days; the shaking temperature is preferably 25 to 30°C, more preferably 28°C; the shaking speed is preferably 120 to 180 r / min, more preferably 160 r / min, thereby obtaining the Bacillus berleis G-1 fermentation broth.

[0050] After obtaining the *Bacillus berberis* G-1 fermentation broth, the present invention preferably performs a first centrifugation on the *Bacillus berberis* G-1 fermentation broth to obtain a first supernatant of the *Bacillus berberis* G-1 fermentation broth. In the present invention, the speed of the first centrifugation is preferably 7500-12000 rpm, more preferably 12000 rpm; the time of the first centrifugation is preferably 5-10 min, more preferably 10 min; and the temperature of the first centrifugation is preferably 4-10℃, more preferably 4℃.

[0051] After obtaining the first supernatant, the present invention preferably mixes the first supernatant with an acidic solution and allows it to stand to obtain a mixture after standing. The acidic solution is preferably a hydrochloric acid solution; the concentration of the hydrochloric acid solution is preferably 1-6 mol / L, more preferably 1 mol / L; the amount of acidic solution added is not particularly required, but is based on the pH value of the supernatant of *Bacillus belyssiensis* G-1 fermentation broth reaching 2.0-3.0, with the pH value preferably being 2.0; the mixing and standing time is preferably 8-24 h, more preferably 12 h; the mixing and standing temperature is preferably 0-10℃, more preferably 4℃. After the mixing and standing, it is beneficial for the supernatant of the *Bacillus belyssiensis* G-1 fermentation broth to precipitate.

[0052] After obtaining the settled mixture, the present invention preferably performs a second centrifugation on the settled mixture to obtain a first precipitate. In the present invention, the method of the second centrifugation is the same as that of the first centrifugation, which has been described in detail above and will not be repeated here.

[0053] After obtaining the first precipitate, the present invention preferably resuspends the first precipitate and adjusts the pH value of the resuspended first precipitate. In the present invention, the first precipitate is resuspended with sterile water; the volume ratio of the sterile water to the volume of the Bacillus vesiculosus G-1 fermentation broth is preferably 50-150:1000, more preferably 100:1000, based on the volume of the Bacillus vesiculosus G-1 fermentation broth; the resuspension method has no special requirements and can be any technique well known in the art; the present invention preferably uses an alkaline solution to adjust the pH value of the resuspended first precipitate; the alkaline solution preferably includes sodium hydroxide solution; the concentration of the sodium hydroxide solution is preferably 1-6 mol / L, more preferably 1 mol / L; the pH value of the resuspended first precipitate is preferably 7.0-8.0, more preferably 7.0.

[0054] After adjusting the pH value of the first precipitate resuspension, the present invention preferably performs a third centrifugation on the pH-adjusted first precipitate resuspension to obtain a second supernatant. In the present invention, the third centrifugation method is the same as the first centrifugation method, which has been described in detail above and will not be repeated here.

[0055] After obtaining the second supernatant, the present invention preferably adjusts the pH value of the second supernatant. The present invention preferably uses an acidic solution to adjust the pH value of the second supernatant; the acidic solution is preferably a hydrochloric acid solution; the concentration of the hydrochloric acid solution is preferably 1-6 mol / L, more preferably 1 mol / L; the pH value of the second supernatant is preferably 2.0-3.0, more preferably 2.0.

[0056] After adjusting the pH value of the second supernatant, the present invention preferably performs a fourth centrifugation on the pH-adjusted second supernatant to obtain a second precipitate. In the present invention, the fourth centrifugation method is the same as the first centrifugation method, which has been described in detail above and will not be repeated here.

[0057] After obtaining the second precipitate, the present invention preferably extracts the second precipitate to obtain an extract. In the present invention, the extraction solvent is preferably a methanol solution; the volume percentage of the methanol solution is preferably 85%–100%, more preferably 100%; based on the volume of the *Bacillus vesiculosus* G-1 fermentation broth, the volume ratio of the methanol solution used to the volume of the *Bacillus vesiculosus* G-1 fermentation broth during extraction is preferably 0.5–1.5:0.5–2, more preferably 1:2; the number of extractions is preferably 1–4 times, more preferably 3 times; the extraction method has no special requirements and any technique well known in the art can be used. After extraction, it is beneficial to obtain an extract containing *Bacillus vesiculosus* G-1 lipopeptides.

[0058] After obtaining the extract, the present invention preferably concentrates the extract to obtain lipopeptide substances of Bacillus belyssus G-1. In the present invention, the concentration method is not particularly required, as long as no further liquid evaporation occurs. After the above operations, the resulting paste-like substance is the lipopeptide substance of Bacillus belyssus G-1. This lipopeptide substance of Bacillus belyssus G-1 can significantly enhance the efficient colonization ability of Bacillus belyssus G-1 in the plant rhizosphere.

[0059] This invention provides a microbial suspension seed coating agent, comprising the following raw materials in the following mass percentages: *Bacillus belye* G-1 as described in the above-mentioned technical solution, 0.02%–0.06% of lipopeptide of *Bacillus belye* G-1 as described in the above-mentioned technical solution, 3%–4% sodium carboxymethyl cellulose, 0.4%–0.6% sodium alginate, 3%–4% dispersant MF, 0.1%–0.2% ferrous sulfate, 0.5%–2% magnesium sulfate, 0.5%–4% dipotassium hydrogen phosphate, 2%–6% color paste, and the balance being water; the concentration of *Bacillus belye* G-1 in the microbial suspension seed coating agent is 5.0 × 10⁻⁶. 7 ~5.0×10 9 cfu / g.

[0060] In this invention, the concentration of Bacillus belyceae G-1 in the microbial suspension seed coating agent is 5.0 × 10⁻⁶. 7 ~5.0×10 9 cfu / g, preferably 5.0 × 10⁻⁶ 9 cfu / g.

[0061] In this invention, the microbial suspension seed coating agent comprises, by weight percentage, 0.02% to 0.06% of the lipopeptide substance of Bacillus belyssus G-1, preferably 0.04%. The preparation method of the lipopeptide substance of Bacillus belyssus G-1 has been described in detail above and will not be repeated here.

[0062] In this invention, the microbial suspension seed coating agent comprises 3% to 4% sodium carboxymethyl cellulose, preferably 4%, by weight percentage.

[0063] In this invention, the microbial suspension seed coating agent comprises 0.3% to 0.6% sodium alginate, preferably 0.6%, by mass percentage.

[0064] In this invention, the microbial suspension seed coating agent comprises 3% to 4% dispersant MF, preferably 4%, by mass percentage.

[0065] In this invention, the microbial suspension seed coating agent comprises 0.1% to 0.2% ferrous sulfate, preferably 0.2%, by mass percentage; the ferrous sulfate preferably comprises ferrous sulfate heptahydrate.

[0066] In this invention, the microbial suspension seed coating agent comprises 0.5% to 2% magnesium sulfate, preferably 1% by mass percentage; the magnesium sulfate preferably comprises magnesium sulfate heptahydrate.

[0067] In this invention, the microbial suspension seed coating agent comprises 0.5% to 4% dipotassium hydrogen phosphate, preferably 0.5%, by mass percentage; the dipotassium hydrogen phosphate preferably comprises dipotassium hydrogen phosphate trihydrate.

[0068] In this invention, the microbial suspension seed coating agent comprises 2% to 6% color paste, preferably 4%, by mass percentage; the color paste preferably includes apple green. Using the above method is beneficial for preparing the microbial suspension seed coating agent of this invention. There are no special requirements for the preparation method of the microbial suspension seed coating agent, as long as all raw materials are fully and evenly mixed.

[0069] This invention provides the application of the microbial suspension seed coating agent described above in the control of plant smut fungus; the plant preferably includes sorghum. The above-mentioned microbial suspension seed coating agent is beneficial in reducing the infection of sorghum by sorghum smut fungus.

[0070] The present invention also provides a method for controlling plant smut, comprising: coating plant seeds with the microbial suspension seed coating agent described in the above technical solution, and then sowing the coated plant seeds.

[0071] This invention preferably utilizes a microbial suspension seed coating agent to coat plant seeds. In this invention, the mass ratio of the microbial suspension seed coating agent to the plant seeds is preferably 30–45:0.5–2, more preferably 40:1; the plant seeds preferably include sorghum seeds; the coating method has no special requirements and any technique well-known in the art can be used.

[0072] After coating, the coated plant seeds are preferably sown according to the present invention. In this invention, there are no special requirements for the sowing method; techniques well known in the art can be used.

[0073] Experiments have shown that the technical solution provided by this invention can achieve an infection prevention effect of up to 87.74% against sorghum smut pathogen and can significantly promote the growth of sorghum seedlings.

[0074] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Bacillus belyes G-1 and its application in controlling plant smut, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0075] Experimental Example 1

[0076] Effects of bacterial suspensions of different concentrations and strains and their lipopeptides on sorghum seed germination

[0077] Preparation before the experiment:

[0078] Identification of Bacillus bereaves G-1: The whole genome of the isolated G-1 strain was sequenced by Shanghai Paisennong Biotechnology Co., Ltd. The sequencing results are shown below. Figure 11 .

[0079] Depend on Figure 11 It can be seen that G-1 is Bacillus belye, and its NCBI accession number is CP084485.

[0080] 1) The variety of sorghum seeds is: Jinza 2001;

[0081] 2) Preparation of Bacillus belysus G-1 bacterial suspension: The G-1 strain stored at -80℃ was activated, and the activated G-1 strain was inoculated into BPY liquid medium (the inoculation amount of G-1 strain was 1% of the volume of BPY liquid medium), and then cultured at 28℃ and 160r / min for 4 days to obtain G-1 bacterial suspension.

[0082] 3) Take the G-1 bacterial suspension from step 2) for concentration determination, and after dilution with sterile water, obtain a concentration of 1.0 × 10⁻⁶. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 CFU / mL G-1 bacterial suspension dilution;

[0083] 4) Preparation of Bacillus belyssus G-1 lipopeptide: Take 1000 mL of the G-1 bacterial suspension from step 2), centrifuge at 12000 rpm and 4℃ for 10 min to obtain the supernatant. Add 1 mol / L HCl solution to the supernatant to adjust the pH to 2.0. Let the supernatant with pH 2.0 stand at 4℃ for 12 h, then centrifuge (12000 rpm and 4℃ for 10 min). Collect the precipitate and dilute with 100 mL of sterile water. The protein was resuspended and its pH was adjusted to 7.0 using NaOH solution. After resolvation to remove denatured proteins, the supernatant was collected. The pH of the resuspended protein was then adjusted to 2.0 using HCl solution. The resuspended protein at pH 2.0 was centrifuged again (12000 rpm, 4℃ for 10 min). The precipitate was collected and extracted three times with 500 mL of methanol. The three extracts were mixed and evaporated to obtain a paste-like substance, which is the Bacillus belyssus G-1 lipopeptide.

[0084] 5) Determine the concentration of Bacillus belysin G-1 lipopeptide in step 4), and dilute with sterile water to obtain G-1 lipopeptide dilutions with concentrations of 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL and 1000 μg / mL.

[0085] 6) Preparation of Bacillus siamensis G-3 bacterial suspension dilution: Using the same method as in steps 2) and 3), a concentration of 1.0 × 10⁻⁶ was obtained. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 CFU / mL of G-3 bacterial suspension diluted; (Note: Bacillus siamensis G-3 is the same strain as described in the literature Controleffects of Bacillus siamensis G-3volatile compounds on raspberry postharvest disease caused by Botrytis cinerea and Rhizopus stolonifer[J].Biological Control, 2020, 141: 104-135.)

[0086] 7) Preparation of bacterial suspension dilution of strain M2: Using the same method as in steps 2) and 3), a concentration of 1.0 × 10⁻⁶ was prepared. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 CFU / mL M2 bacterial suspension dilution; wherein bacteria M2 were isolated from germinating sorghum seeds of Jinza 2001.

[0087] Experimental setup: 10 mL of different concentrations of G-1 bacterial suspension dilution from step 3), different concentrations of G-1 lipopeptide dilution from step 5), different concentrations of G-3 bacterial suspension dilution from step 6), and different concentrations of M2 bacterial suspension dilution from step 7) were added to petri dishes containing 30 grains of Jinza 2001 sorghum. A control (CK) with 10 mL of distilled water was added. After incubation at 28℃ for 7 days, the germination rate and sprout length of each treatment were recorded. Results are shown below. Figure 1 (exist Figure 1In the table, I represents the effect of different concentrations of bacterial suspension dilution on the germination rate of sorghum seeds; II represents the effect of different concentrations of bacterial suspension dilution on the sprout length of sorghum seeds; III represents the effect of different concentrations of G-1 lipopeptide on the germination rate of sorghum seeds; and IV represents the effect of different concentrations of G-1 lipopeptide on the sprout length of sorghum seeds. A1 to A5 represent bacterial suspension concentrations of different strains, respectively, at 1.0 × 10⁻⁶. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 cfu / mL; * indicates that the treatment is significantly different from the control and is higher than the control). Each concentration of dilution corresponds to one culture dish, each culture dish is one treatment, and each treatment is repeated three times.

[0088] Results and Analysis:

[0089] Combination Figure 1 It can be seen that with the increase of the concentration of strain G-1, the germination rate and shoot length of sorghum seeds initially increased and then decreased to varying degrees compared with the control. Among strains G-1, G-3, and M2, strain G-1 showed the best effect in promoting sorghum grain germination and shoot elongation, and its application concentration was 1.0 × 10⁻⁶. 7 ~1.0×10 9 At cfu / mL, the seed germination rate and shoot length at different time points were significantly higher than the control, while strain G-3 only showed a higher germination rate at 1.0 × 10⁻⁶ CFU / mL. 7 cfu / mL and 1.0×10 8 At CFU / mL, strain M2 showed a promoting effect on seed germination and shoot elongation; however, strain M2 did not show a promoting effect on seed germination at different treatment concentrations, and the effect was not observed at concentrations exceeding 1.0 × 10⁻⁶. 8 At a concentration of CFU / mL, it showed an inhibitory effect on germination. Compared with the control, the germination rate and shoot length of sorghum grains also showed a trend of first increasing and then decreasing with the increase of G-1 lipopeptide concentration, and the promoting effect was best when the concentration of G-1 lipopeptide was 400-600 μg / mL.

[0090] Experimental Example 2

[0091] Effects of different concentrations of dilution on the infection rate of sorghum smut

[0092] Preparation before the experiment:

[0093] 1) Preparation of sorghum sprout juice: When the seeds of Jinza 2001 germinate for 3 days, weigh 5g of seeds with only the roots and add 3mL of physiological saline to grind them.

[0094] 2) Spore powder suspension of sorghum smut: 40% formaldehyde (volume fraction) and 0.1% potassium permanganate solution were mixed at a volume ratio of 2:1 to obtain a mixed solution. Spore powder from sorghum smut, after fumigation with this mixed solution for 10 minutes, was counted using a hemocytometer. A spore concentration of 1.0 × 10⁻⁶ was prepared using sterile water. 7 A suspension of cfu / mL; sorghum smut spore powder is a mixed spore powder collected in the field when the disease occurs;

[0095] 3) In step 3) of Experiment Example 1, the concentration obtained was 1.0 × 10⁻⁶. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 CFU / mL G-1 bacterial suspension dilution;

[0096] 4) Step 5) of Experiment Example 1 yielded diluted solutions of G-1 lipopeptide substances with concentrations of 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL and 1000 μg / mL;

[0097] 5) Step 6) of Experiment Example 1 yielded a concentration of 1.0 × 10⁻⁶. 7 cfu / mL, 1.0×10 8 cfu / mL, 1.0×10 9 cfu / mL, 1.0×10 10 cfu / mL, 1.0×10 11 G-3 bacterial suspension dilution at cfu / mL.

[0098] Experimental setup:

[0099] Take 9 mL of the solution from step 3) with a concentration of 1.0 × 10⁻⁶. 7 ~1.0×10 11 A CFU / mL dilution of G-1 bacterial suspension was mixed with 100 μL of sorghum sprout extract from step 1) and 1 mL of sorghum smut spore powder suspension from step 2), wherein the concentration of the added solution was 1.0 × 10⁻⁶. 71 The CFU / mL G-1 bacterial suspension dilution is designated as Mixture 1, and then diluted sequentially to 1.0 × 10⁻⁶. 7 ~1.0×10 11 cfu / mL corresponds to mixtures 1–5 respectively;

[0100] Take 9 mL of the diluted G-1 lipopeptide solution with concentrations of 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL and 1000 μg / mL from step 4), respectively, and mix it with 100 μL of sorghum sprout juice from step 1) and 1 mL of sorghum smut spore powder suspension from step 2) to obtain mixed solutions, which are respectively recorded as mixed solution 6 to mixed solution 10;

[0101] Take 9 mL of the solution from step 5) with a concentration of 1.0 × 10⁻⁶. 7 ~1.0×10 11 The CFU / mL G-3 bacterial suspension dilution was mixed with 100 μL of sorghum sprout juice from step 1) and 1 mL of sorghum smut spore powder suspension from step 2) to obtain mixtures 11-15.

[0102] The control group (CK) was a treatment containing only water and no preferred bacterial suspension or lipopeptides.

[0103] Mixtures 1-15 and CK were added to sterile petri dishes containing 30 sorghum seeds of Jinza 2001, with one mixture added to each dish. Each dish was considered a treatment, and each treatment was repeated three times. The initial weight of each treatment was recorded. The plants were co-cultured at 28℃ for 7 days, during which water was added to the initial weight by weighing. After the culture was terminated, the roots of different treatments were rinsed with running water for 10 minutes to remove spores attached to the root surface. The hypocotyl of each sorghum seedling was then cut off with a scalpel. The smears were stained, prepared, and examined under a microscope according to the method for determining AM mycorrhizal fungal infection described in the literature (Shi Ning. Mechanism of interspecific and phosphorus-solubilizing biological interactions of arbuscular mycorrhizal fungi to promote phosphorus absorption in maize [D]. Beijing: China Agricultural University, 2016).

[0104] Results and Analysis:

[0105] Based on the mycelial infection rate of sorghum smut fungus in the root segment (divided into 6 levels, corresponding to N0(0), 0), ... <N1≤10%、10%<N2≤30%、30%<N3≤50%、50%<N4≤70%、70%<N5≤90%、N6> 90%) and spore abundance in root segments (spore count is divided into 6 levels, corresponding to B0(0), 0 <B1≤10、10<B2≤30、30<B3≤50、50<B4≤70、70<B5≤90、B6> 90. The unit of spore count is: spores. Determine the level of each root segment and calculate the infection rate and infection intensity of sorghum smut fungus under different treatments.

[0106] The effects of different concentrations of treatment on the infectivity of sorghum smut pathogen are shown in the figure. Figure 2 (exist Figure 2In the diagram, A represents the effect of different concentrations of bacterial strains on the infectivity of sorghum smut pathogen; B represents the effect of different concentrations of lipopeptide substances on the infectivity of sorghum smut pathogen. The formation of sorghum smut pathogen infection in sorghum root tissues under different concentrations is shown in [reference needed]. Figure 3 (exist Figure 3 In the study, the concentrations of bacterial suspensions of strains G-1, G-3, and M2 were all 1.0 × 10⁻⁶. 9 CFU / mL; lipopeptide concentration is 600 μg / mL (where 40× is the objective lens magnification and 10 μm is the microscopic observation scale).

[0107] Infection rate % = Number of infected root segments × 100 / Total number of root segments

[0108] Infection intensity = [(n6 + 0.90 × n5 + 0.70 × n4 + 0.50 × n3 + 0.30 × n2 + 0.10 × n1 + n0) + (b6 + 0.90 × b5 + 0.70 × b4 + 0.50 × b3 + 0.30 × b2 + 0.10 × b1 + b0)] / 2 × total number of infected segments

[0109] In the formula: n6 represents the number of root segments with an infection rate of >90% (N6 level) of sorghum smut pathogen; n5 represents the number of root segments with an infection rate of 70%–90% (N5 level), and so on. b6 represents the number of root segments with a rhizosphere spore abundance of >90 (N6 level); b5 represents the number of root segments with a rhizosphere spore abundance of 70–90 (B5 level), and so on.

[0110] Depend on Figure 2 and Figure 3 It can be seen that different strains and different concentrations of lipopeptides exhibit varying effects in controlling sorghum smut infection. Compared to the control, the inhibitory effects of different strains and lipopeptides on sorghum root tissue infected with sorghum smut increased with increasing concentration. Among the different antagonistic microorganisms at the same treatment concentration, strains G-1 and G-3 showed better effects, with an optimal treatment concentration of 1.0 × 10⁻⁶ for both. 9 ~1.0×10 11 The concentration of cfu / mL can control the infection rate and intensity of sorghum smut pathogen at around 15%, and the density of perispores and mycelia is significantly lower than that of the control. Figure 2 A in the middle; Figure 3 Compared to the control, the inhibitory effect of lipopeptide natural products also showed a gradual increasing trend with increasing concentration, and exhibited good inhibitory effect at concentrations higher than 600 μg / mL. Figure 2 B in the middle; Figure 3 Among them, strain G-1 (1.0 × 10⁻⁶) was the most common. 9The CFU / mL and G-1 lipopeptide (600 μg / mL) are the most effective in controlling sorghum smut.

[0111] Experimental Example 3

[0112] Screening experiment of film-forming agent and dosage

[0113] 1) Types of film-forming agents: sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, starch and xanthan gum. The amount of film-forming agent used is calculated as a percentage.

[0114] 2) Using the methods in steps 2) and 3) of Experimental Example 1, a concentration of 3.5 × 10⁻⁶ was prepared. 10 G-1 bacterial suspension at cfu / mL;

[0115] 3) Using the methods in steps 4) and 5) of Experiment Example 1, a G-1 lipopeptide substance with a concentration of 12 mg / mL was prepared;

[0116] 4) Preparation of suspension seed coating agent:

[0117] a. Weigh 0.5g (0.5g corresponds to 0.5%) of sodium carboxymethyl cellulose and mix it with 75mL of distilled water. Heat the mixture in a 70℃ water bath until dissolved. After cooling to room temperature, add 4g of color paste, 5mL of G-1 lipopeptide (12mg / mL from step 3), (final concentration 600μg / g), and 5mL of G-1 lipopeptide (3.5×10⁻⁶ from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 Add distilled water to make up to 100g, stir for 30min to mix evenly, and obtain suspension seed coating agent 1;

[0118] b. Following the method described in a above, weigh out 1%, 2%, 3%, 4%, 5%, and 6% sodium carboxymethyl cellulose to replace 0.5% sodium carboxymethyl cellulose in step a to prepare suspension seed coating agents, which are then referred to as suspension seed coating agents 2 to 7.

[0119] 0.5%, 1%, 2%, 3%, 4%, 5%, and 6% of starch were weighed to replace 0.5% of sodium carboxymethyl cellulose in step a to prepare suspension seed coating agents, which were then referred to as suspension seed coating agents 8 to 14.

[0120] 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 1% sodium alginate were weighed to replace 0.5% sodium carboxymethyl cellulose in step a to prepare suspension seed coating agents, which were then referred to as suspension seed coating agents 15 to 21.

[0121] 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 1% polyvinyl alcohol were weighed to replace 0.5% sodium carboxymethyl cellulose in step a to prepare suspension seed coating agents, which were respectively denoted as suspension seed coating agents 22 to 28;

[0122] 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% and 1% xanthan gum were weighed to replace 0.5% sodium carboxymethyl cellulose in step a to prepare suspension seed coating agents, which were then referred to as suspension seed coating agents 29 to 35.

[0123] c. Add 4g of color paste, 5mL of G-1 lipopeptide (12mg / mL, final concentration 600μg / g) from step 3), and 5mL of G-1 lipopeptide (3.5×10⁻⁶) from step 2) to 75mL of distilled water. 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 Add distilled water to make up to 100g, stir for 30min to mix evenly, and obtain suspension seed coating agent CK;

[0124] Experimental setup:

[0125] Weigh 1g each of suspension seed coating agents 1-35 and CK from step 4) and coat sorghum grains with them. Coat 150 sorghum grains with each suspension seed coating agent. Record the drying time of the seeds. After drying, randomly select 50 sorghum grains and place them in a petri dish containing 10mL of water. Incubate at 27℃ for 7 days. Count the number of germinations of seeds in each treatment and calculate the germination rate.

[0126] Thirty sorghum grains with different coating treatments were randomly selected, and the coating uniformity of different treatments was determined according to the method reported in the literature (Chen Fujia. Study on preparation and quality detection method of 15% gram-furo suspension seed coating agent [D]. Zhengzhou: Henan Agricultural University, 2011.).

[0127] The suspension rates of different treatments were determined according to the national standard GB / T 14825-2006, "Determination of Suspension Rate of Pesticides". Viscosity was measured using a viscometer. After 30 days of storage at room temperature in the dark, 1g of each of the biological suspension seed coating agents (1-35) and CK were diluted with sterile water at a concentration of 10 g / L. 4 The number of viable bacteria was then determined using the plate coating method on rifampicin resistant plates.

[0128] Results and Analysis:

[0129] The suspension rate, viscosity, coating uniformity, and film drying time of seeds under different treatments were analyzed. The results are shown in the figure. Figure 4 (exist Figure 4In this context, AF represents the concentration ranges of sodium carboxymethyl cellulose, polyvinyl alcohol, starch, sodium alginate, xanthan gum, and a preferred film-forming agent, respectively.

[0130] Depend on Figure 4 It can be seen that, compared with the control, the film-forming agents at different concentrations did not show a significant inhibitory effect on sorghum seed germination and strain G-1, but the physical properties of the bio-suspension seed coating agent prepared with starch as the film-forming agent were poor. Figure 4 (B) in the middle;

[0131] Regarding the effects of different film-forming agents on the germination rate of sorghum seeds, the optimal concentrations for sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, and xanthan gum were 0.5%–4%, 0.1%–0.6%, 0.1%, and 0.1%–1%, respectively. At these concentrations, the germination rates of sorghum seeds treated with different film-forming agents did not differ significantly from the control. Figure 4 (A, C~E in the original text);

[0132] Based on the survival rate of strain G-1, the optimal concentrations for sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, and xanthan gum were 0.5%–6%, 0.1%–1%, 0.1%–0.2%, and 0.1%–0.2%, respectively. Regarding the physical properties of the bio-suspension agent (suspension rate, viscosity, coating uniformity, and film-forming drying time), the optimal concentrations for sodium carboxymethyl cellulose, sodium alginate, and xanthan gum were 4%–6%, 0.6%, and 0.6%, respectively. While polyvinyl alcohol showed better suspension rate and viscosity at a concentration of 0.6%, its coating uniformity was poor. Figure 4 (A, C~E in the original text);

[0133] The criteria for selection are: germination rate ≥85%, suspension rate ≥90%, viscosity value 150–800 mPa·s, and viable count of strain G-1 ≥1.6 × 10⁻⁶. 9 Using CFU / g as the screening criterion, it can be seen that the film-forming agents that meet the criteria are sodium carboxymethyl cellulose and sodium alginate, and the preferred concentrations are 4% and 0.6% respectively. Figure 4 (F in the middle).

[0134] In addition, regarding the film-forming drying time ≤15 min, compared with the control, although sodium carboxymethyl cellulose and sodium alginate showed significantly reduced drying film-forming times when used at concentrations higher than 4% and 0.3% respectively, the film-forming drying times of all five film-forming agents at different test concentrations were >15 min, failing to meet the quality requirements. Figure 4 Further adjustments will require the addition of dispersants.

[0135] Experiment Example 4

[0136] Screening experiment of nutrients and dosage

[0137] 1) Types of nutrients: ferrous sulfate, magnesium sulfate, or potassium dihydrogen phosphate

[0138] 2) Same as step 2) of Experiment Example 3;

[0139] 3) Same as step 3) of Experiment Example 3;

[0140] 4) Preparation of suspension seed coating agent:

[0141] a. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 4g of color paste, 5mL of G-1 lipopeptide substance with a mass concentration of 12mg / mL (i.e., a final concentration of 600μg / g) from step 3), and 5mL of substance with a concentration of 3.5×10 from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 The mass of dipotassium hydrogen phosphate (cfu / g) and 0.5g (i.e. 0.5%) was added, and distilled water was added to make up to 100g. The mixture was stirred for 30 minutes to obtain suspension seed coating agent 36 (i.e. A2).

[0142] b. Following the preparation method described in a above, weigh out 1%, 2%, 3%, 4%, 5% and 6% by mass of dipotassium hydrogen phosphate to replace 0.5% of dipotassium hydrogen phosphate in step a, and prepare suspension seed coating agents 37-42 (i.e. A3-A8).

[0143] According to the preparation method of step a above, magnesium sulfate with a mass concentration of 0.5%, 1%, 2%, 3%, 4%, 5% and 6% was weighed to replace 0.5% of dipotassium hydrogen phosphate in step a, and suspension seed coating agents 43-49 (i.e. A9-A15) were prepared.

[0144] According to the preparation method of step a above, 0.1%, 0.2%, 0.3%, and 0.5% ferrous sulfate by mass concentration were weighed to replace 0.5% dipotassium hydrogen phosphate in step a, and suspension seed coating agents 50-54 (i.e., A16-A19) were prepared.

[0145] c. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 4g of color paste, 5mL of G-1 lipopeptide substance with a mass concentration of 12mg / mL (i.e., a final concentration of 600μg / g) from step 3), and 5mL of substance with a concentration of 3.5×10 from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 After adding distilled water to make up to 100g, stir for 30min to mix evenly to obtain suspension seed coating agent A1;

[0146] Experimental setup: Weigh 1g each of suspension seed coating agent 36-54 and A1 from step 4) and coat 150 sorghum grains. Record the drying time of the seeds. After drying, randomly select 50 sorghum grains and place them in a petri dish containing 10mL of water. Incubate at 27℃ for 7 days. Count the number of germinations of seeds in each treatment and calculate the germination rate.

[0147] Thirty sorghum grains with different coating treatments were randomly selected, and the coating uniformity of different treatments was determined according to the method reported in the literature (Chen Fujia. Study on preparation and quality detection method of 15% gram-furo suspension seed coating agent [D]. Zhengzhou: Henan Agricultural University, 2011.).

[0148] The suspension rates of different treatments were determined according to the national standard GB / T 14825-2006, "Determination of Suspension Rate of Pesticides". Viscosity was measured using a viscometer. After 30 days of storage at room temperature in the dark, 1g of each of the biological suspension seed coating agents 36-54 and A1 (with A1 as the control) were diluted with sterile water to a 10:10 concentration. 4 The number of viable bacteria was then determined using the plate coating method on rifampicin resistant plates.

[0149] Results and Analysis:

[0150] Germination rate, suspension rate, viscosity, and film-forming drying time of seeds under different treatments were statistically analyzed. The results are shown in [Table / Reference]. Figure 5 (exist Figure 5 In the diagram, A1 to A19 represent suspension seed coating agents with different nutrient concentrations, respectively; B1 to B6 represent germination rate, suspension rate, viscosity, film-forming drying time, coating uniformity, and survival rate of strain G-1, respectively; * indicates that the treatment is significantly different from the control and is lower than the control.

[0151] Depend on Figure 5 It can be seen that, compared with the control, different nutrients had no significant effect on the physical properties of the G-1 biological suspension. High concentrations of K2HPO4·3H2O, MgSO4·7H2O, and FeSO4·7H2O significantly inhibited sorghum seed germination, while only high concentrations of MgSO4·7H2O significantly reduced the survival rate of strain G-1. O4 ·7H2O and FeSO4·7H2O have significant inhibitory effects. Therefore, the optimal dosages of K2HPO4·3H2O, MgSO4·7H2O, and FeSO4·7H2O are 0.5%–4%, 0.5%–2%, and 0.1%–0.2%, respectively.

[0152] Experimental Example 5

[0153] Screening experiment on pigment dosage

[0154] 1) Apple green pulp, purchased regularly;

[0155] 2) Same as step 2) of Experiment Example 3;

[0156] 3) Same as step 3) of Experiment Example 3;

[0157] 4) Preparation of suspension seed coating agent:

[0158] a. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 2g (2%) of the color paste from step 1), 5mL of G-1 lipopeptide substance with a mass concentration of 12mg / mL (final concentration 600μg / g) from step 3), and 5mL of the substance with a concentration of 3.5×10 from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 The mixture consists of 0.1 g of ferrous sulfate (FeSO4·7H2O), 0.5 g of dipotassium hydrogen phosphate, and 0.5 g of magnesium sulfate. Distilled water is added to make up to 100 g of the mixture. The mixture is stirred for 30 min to obtain a 55 g suspension seed coating agent.

[0159] b. Following the preparation method described in a above, weigh out 4%, 6%, 8% and 10% mass concentration color pastes respectively to replace 2% of the color paste in step a, and prepare suspension seed coating agents 56-59 in sequence;

[0160] c. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 5mL of G-1 lipopeptide (12mg / mL from step 3), i.e., final concentration 600μg / g, and 5mL of G-1 lipopeptide (3.5×10⁻⁶ from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 The mixture consists of 0.1 g (i.e., 0.1%) of ferrous sulfate (FeSO4·7H2O), 0.1 g of ferrous sulfate (FeSO4·7H2O), 0.5 g of dipotassium hydrogen phosphate, and 0.5 g of magnesium sulfate. Distilled water is added to make up to 100 g of the mixture, and the mixture is stirred for 30 min to obtain the suspension seed coating agent CK.

[0161] Experimental setup: Weigh 1g each of suspension seed coating agent 55-59 and CK from step 4) and coat 150 sorghum grains. Record the drying time of the seeds. After drying, randomly select 50 sorghum grains and place them in a petri dish containing 10mL of water. Incubate at 27℃ for 7 days. Count the number of germinations of seeds in each treatment and calculate the germination rate.

[0162] Thirty sorghum grains with different coating treatments were randomly selected, and the coating uniformity of different treatments was determined according to the method reported in the literature (Chen Fujia. Study on preparation and quality detection method of 15% gram-furo suspension seed coating agent [D]. Zhengzhou: Henan Agricultural University, 2011.).

[0163] The suspension rates of different treatments were determined according to the national standard GB / T 14825-2006, "Determination of Suspension Rate of Pesticides". Viscosity was measured using a viscometer. After 30 days of storage at room temperature in the dark, 1g of the biological suspension seed coating agent (55-59 g) and the control (CK) were diluted with sterile water to a concentration of 10 g. 4 The number of viable bacteria was then determined using the plate coating method on rifampicin resistant plates.

[0164] Results and Analysis

[0165] The effects of different suspension seed coating agents on the physical properties, survival rate and seed germination of strain G-1 were statistically analyzed, and the results are shown in Table 1.

[0166] Table 1. Effects of different amounts of color paste on the physical properties, survival rate, and seed germination of strain G-1 biological suspension.

[0167]

[0168] Note: Different lowercase letters in the same column indicate significant differences at the P<0.05 level.

[0169] Table 1 shows that different concentrations of pigment paste had no significant effect on the suspension rate, drying film-forming time, and seed germination rate of the G-1 biological suspension seed coating agent. However, when the pigment paste concentration reached 8% or higher, it led to a significant decrease in the coating uniformity and the survival rate of strain G-1. Furthermore, the viscosity of the seed coating agent showed a continuous increasing trend with increasing pigment paste concentration. Therefore, the optimal dosage of pigment paste is 2%–6%.

[0170] Experimental Example 6

[0171] Screening experiment of dispersants

[0172] 1) Dispersant MF

[0173] 2) Same as step 2) of Experiment Example 3;

[0174] 3) Same as step 3) of Experiment Example 3;

[0175] 4) Preparation of suspension seed coating agent:

[0176] a. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 4g of apple green pulp from step 1), 5mL of G-1 lipopeptide with a mass concentration of 12mg / mL (i.e., a final concentration of 600μg / g) from step 3), and 5mL of G-1 lipopeptide with a mass concentration of 3.5×10 from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 The mixture consists of 0.1 g of FeSO4·7H2O, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 0.5 g of dispersant (i.e., 0.5%), and distilled water to a final volume of 100 g. After stirring for 30 minutes to ensure uniform mixing, a suspension seed coating agent 60 is obtained.

[0177] b. Following the preparation method described in a above, weigh out 1%, 2%, 3%, 4%, 5% and 6% by mass of dispersant to replace 0.5% of the dispersant in step a, and prepare suspension seed coating agents 61 to 66 in sequence.

[0178] c. Weigh 4g of sodium carboxymethyl cellulose and 0.6g of sodium alginate, mix them with 75mL of distilled water, and heat in a 70℃ water bath to dissolve. After cooling to room temperature, add 4g of apple green pulp from step 1), 5mL of G-1 lipopeptide with a mass concentration of 12mg / mL (i.e., a final concentration of 600μg / g) from step 3), and 5mL of G-1 lipopeptide with a mass concentration of 3.5×10 from step 2). 10 CFU / mL of Gram-1 bacterial suspension (final concentration 1.75 × 10⁻⁶) 9 Mix 0.1 g of FeSO4·7H2O, 0.5 g of dipotassium hydrogen phosphate, and 0.5 g of magnesium sulfate with distilled water to make up to 100 g, stir for 30 min to mix evenly, and obtain suspension seed coating agent CK.

[0179] Experimental setup: Weigh 1g each of suspension seed coating agent 61-66 and CK from step 4) and coat 100 sorghum grains. Record the drying time of the seeds. After drying, randomly select 50 sorghum grains and place them in a petri dish containing 10mL of water. Incubate at 27℃ for 7 days. Count the number of germinations of seeds in each treatment and calculate the germination rate.

[0180] Thirty sorghum grains with different coating treatments were randomly selected, and the coating uniformity of different treatments was determined according to the method reported in the literature (Chen Fujia. Study on preparation and quality detection method of 15% gram-furo suspension seed coating agent [D]. Zhengzhou: Henan Agricultural University, 2011.).

[0181] The suspension rates of different treatments were determined according to the national standard GB / T 14825-2006, "Determination of Suspension Rate of Pesticides". Viscosity was measured using a viscometer. After 30 days of storage at room temperature in the dark, 1g of each of the biological suspension seed coating agents 61-66 and CK were diluted with sterile water. 4 The number of viable bacteria was then determined using the plate coating method on rifampicin resistant plates.

[0182] Results and Analysis

[0183] The effects of seed coating agents with different dispersant dosages on the physical properties, survival rate, and seed germination of strain G-1 bio-suspension were statistically analyzed. The results are shown in Table 2 and 3. Figure 6 (exist Figure 6 In the experiment, A to H represent the control (CK) and the results of dispersant addition amounts of 0.5%, 1%, 2%, 3%, 4%, 5%, and 6%, respectively.

[0184] Table 2. Effects of different dispersant dosages on the physical properties, survival rate, and seed germination of strain G-1 biological suspension.

[0185]

[0186] Note: Different lowercase letters in the same column indicate significant differences at the P<0.05 level.

[0187] From Table 2 and Figure 6 It can be seen that different concentrations of dispersant MF have a significant impact on the film-forming drying time and viscosity of the G-1 biological suspension, both of which gradually decrease with increasing concentration. When the amount of dispersant MF increases to 3%, the film-forming drying time of the seed coating agent decreases to 11.00±1.00 min, meeting the quality requirement of ≤15 min for seed coating agents. When the amount of dispersant MF increases to 5%, the number of viable strains and the suspension rate of G-1 show a significant decrease. Figure 6 It can be seen that as the dosage of dispersant MF increases, the tendency for sorghum seeds to stick together during the coating process gradually decreases. Although the drying film-forming time is shortest at a dosage of 6%, the degree of seed surface coloring and coating uniformity decrease significantly. Therefore, the optimal dosage of dispersant MF is 3%–4%.

[0188] Experimental Example 7

[0189] Screening experiment of microbial suspension seed coating agent

[0190] 1) An orthogonal experiment was conducted based on the components of the microbial suspension seed coating agent. The factors and levels of the orthogonal experiment are shown in Table 3.

[0191] Table 3. Orthogonal Experiment Factors and Levels Design Table

[0192]

[0193]

[0194] Experimental setup: 1g of each of the suspension seed coating agents prepared at different levels (Table 3) was used to coat 100 sorghum grains. The drying time of the seeds was recorded. After drying, 50 sorghum grains were randomly selected and placed in a petri dish containing 10mL of water. The seeds were then cultured at a constant temperature of 27℃ for 7 days. The number of germinations of each treatment seeds was counted and the germination rate was calculated.

[0195] Thirty sorghum grains with different coating treatments were randomly selected, and the coating uniformity of different treatments was determined according to the method reported in the literature (Chen Fujia. Study on preparation and quality detection method of 15% gram-furo suspension seed coating agent [D]. Zhengzhou: Henan Agricultural University, 2011.).

[0196] The suspension rates of different treatments were determined according to the national standard GB / T 14825-2006, "Determination of Suspension Rate of Pesticides". Viscosity was measured using a viscometer. After 30 days of storage at room temperature in the dark, 1g of the biological suspension seed coating agent prepared at different levels was diluted with sterile water. 4 The number of viable bacteria was then determined using the plate coating method on rifampicin resistant plates.

[0197] Results and Analysis:

[0198] The G-1 biological suspension seed coating agent obtained from the orthogonal experiment in Table 3 was statistically analyzed, and the results are shown in Table 4 (in Table 4, the values ​​corresponding to different factors are the treatment levels of that factor).

[0199] Table 4. Optimization results of different treatment formulations in orthogonal experiments.

[0200]

[0201]

[0202] As shown in Table 4, the results of the orthogonal experiment indicate that different treatments have certain differences in their effects on the physical properties, seed germination rate, survival rate of strain G-1, and control of sorghum smut. However, further comprehensive evaluation of each indicator and determination of the effects of different factors on each indicator require range and variance analysis.

[0203] 2) Range and variance analysis were performed on the effects of the interactions of different factors in Table 4 on each indicator. The results are shown in Table 5.

[0204] Table 5. Significance analysis of the range and variance of each response index at different levels of each factor.

[0205]

[0206] Note: * indicates a significant difference at the p<0.05 level.

[0207] Table 5 shows that sodium carboxymethyl cellulose and sodium alginate are the main factors affecting sorghum seed germination in this formulation. Among the factors in the formulation, only factor A (the amount of strain G-1 added) has a significant impact on the number of viable strain G-1, with the largest range being 7.06. Dispersant MF is the main factor affecting the drying and film-forming time of the formulation, not only having the largest range but also showing a variance of P<0.05. This indicates that the amount of strain G-1 added, sodium carboxymethyl cellulose, sodium alginate, and dispersant MF are the main factors affecting the physical properties and application effects of the formulation.

[0208] 3) Estimate the marginal means of different factors based on the analysis of variance drawn in step 2). The results are shown in […]. Figure 7 .

[0209] Depend on( Figure 7 It can be seen that the optimal level of strain G-1 in this formulation is 5 (5.0 × 10⁻⁶). 9 The optimal levels of the following components were found to be: cfu / mL, lipopeptide 2 (400 μg / mL), sodium carboxymethyl cellulose 2 (4%), sodium alginate 2 (0.6%), dispersant MF 2 (4%), ferrous sulfate 2 (0.2%), magnesium sulfate 2 (1%), dipotassium hydrogen phosphate 2 (0.5%), and color paste 2 (4%). Therefore, the optimal preparation formula for strain G-1·lipopeptide bio-suspension seed coating agent is: strain G-1 (1.0 × 10⁻⁶ / mL). 11 5 mL of CFU / mL, 5 mL of lipopeptide (8 mg / mL), 4 g of sodium carboxymethyl cellulose, 0.6 g of sodium alginate, 4 g of dispersant MF, 0.2 g of ferrous sulfate, 1 g of magnesium sulfate, 0.5 g of dipotassium hydrogen phosphate, 4 g of color paste, and water to make up to 100 g.

[0210] strain G-1 (1.0 × 10⁻⁶) 11 A seed coating agent was prepared by mixing 5 mL of CFU / mL, 5 mL of G-1 lipopeptide (8 mg / mL), 4 g of sodium carboxymethyl cellulose, 0.6 g of sodium alginate, 4 g of dispersant MF, 0.2 g of ferrous sulfate, 1 g of magnesium sulfate, 0.5 g of dipotassium hydrogen phosphate, and 4 g of color paste with water to a final volume of 100 g. This seed coating agent was then used to coat Jinza 2001 sorghum seeds. Coated seeds and uncoated Jinza 2001 sorghum seeds were placed in petri dishes for verification. Each petri dish served as a control, and each control was tested in triplicate. After incubation at 28℃ for 7 days, the germination rate and anti-infection effect of each treatment were statistically analyzed. The results are shown in Table 4, which presents the statistical results of the optimal formulation. Figure 8 .

[0211] From Table 4 and Figure 8 It can be seen that the strain G-1 biological suspension seed coating agent prepared by this invention not only has good physical properties and does not reduce the germination rate of sorghum seeds, but also has an anti-infection effect of up to 89.63% against sorghum smut pathogen.

[0212] Example 1

[0213] Effects of microbial suspension seed coating agents on seed germination rate and resistance to smut pathogen infection

[0214] 1) Preparation before the experiment: Using the methods in steps 2) and 3) of Experiment Example 1, a concentration of 1.0 × 10⁻⁶ was prepared. 11 A suspension of Bacillus belyss G-1 bacteria at cfu / mL was prepared; using the methods in steps 4) and 5) of Experimental Example 1, Bacillus belyss G-1 lipopeptide at a concentration of 8 mg / mL was obtained.

[0215] 2) Preparation of microbial suspension seed coating agent: Take the Bacillus belyssus G-1 bacterial suspension from step 1) (1.0×10⁻⁶) 11 5 mL of CFU / mL Bacillus belysin G-1 lipopeptide (8 mg / mL) 5 mL, 4 g of sodium carboxymethyl cellulose, 0.6 g of sodium alginate, 4 g of dispersant MF, 0.2 g of ferrous sulfate, 1 g of magnesium sulfate, 0.5 g of dipotassium hydrogen phosphate, and 4 g of color paste were mixed with water to make up to 100 g to obtain a microbial suspension seed coating agent.

[0216] 3) Weigh 25g of the microbial suspension seed coating agent from step 2) and coat 1kg of sorghum seeds. After drying, randomly select 50 sorghum seeds and place them in a petri dish containing 10mL of water and incubate at 27℃. Each petri dish is one treatment, and each treatment is repeated three times in parallel.

[0217] Comparative Example 1

[0218] The difference from Example 1 is that the sorghum seeds are not coated in step 3).

[0219] Comparative Example 2

[0220] The difference from Example 1 is that in step 3), 15g of microbial suspension seed coating agent is used to coat 1kg of sorghum seeds.

[0221] Comparative Example 3

[0222] The difference from Example 1 is that in step 3), 35g of microbial suspension seed coating agent is used to coat 1kg of sorghum seeds.

[0223] Comparative Example 4

[0224] The difference from Example 1 is that in step 3), 45g of microbial suspension seed coating agent is used to coat 1kg of sorghum seeds.

[0225] Results and Analysis:

[0226] 1) Seed germination rate and sorghum smut pathogen infection rate were statistically analyzed for seeds cultured at 27℃ for 7 days in Example 1 and Comparative Examples 1-4, respectively. The results are shown in the table below. Figure 9 (exist Figure 9 In this text, 0g represents the result of Comparative Example 1, 15g represents the result of Comparative Example 2, 35g represents the result of Comparative Example 3, 25g represents the result of Example 1, and 45g represents the result of Comparative Example 4.

[0227] Depend on Figure 9 It can be seen that as the amount of seed dressing agent increased, the infection intensity of smut pathogen, the density of rhizosphere mycelium and spores gradually decreased, while the germination rate of sorghum seeds also gradually decreased. Although the amount of 15g had no significant effect on the germination of sorghum seeds, the infection intensity of smut pathogen was relatively high. The effect was best when 25g of seed dressing agent was used to coat 1kg of seeds.

[0228] 2) The microbial suspension seed coating agent prepared in Example 1 was stored at room temperature in the dark. The number of viable bacteria, lipopeptide content and the rate of decline in anti-infection effect of strain G-1 were measured and analyzed after 3, 6, 9 and 12 months of storage in accordance with the storage quality requirements of NY / T2293.2-2012 "Bacterial Microbial Pesticides Bacillus subtilis Part 2 Bacillus subtilis Wettable Powder". The results are shown in Table 6.

[0229] Table 6. Differences in the content and efficacy of effective components of microbial suspension seed coating agents under different storage times.

[0230]

[0231] As shown in Table 6, compared with the seed coating agent stored for 0 days (i.e., freshly prepared), the number of viable bacteria, the content of lipopeptides and the anti-infection effect in the microbial suspension seed coating agent all showed a downward trend with the extension of storage time. However, after 12 months of storage, the decrease rate of effective ingredient content was less than 10%, and the prevention effect only decreased by 1.63%, indicating that the microbial suspension seed coating agent prepared by the present invention has good storage stability.

[0232] Application Example 1

[0233] Pot experiment

[0234] 1) Take the coated and dried sorghum seeds (Jinza 2001) from step 3) of Example 1 for later use;

[0235] 2) Mix 1.5 kg of soil with 25 g of sorghum smut spore powder evenly and fill a plastic flowerpot with a bottom diameter of 14.6 cm, a height of 12.7 cm, and a top diameter of 18.0 cm. The sorghum smut spore powder was collected in the field, i.e., manually shaken off the diseased ears of grain. The viable count of the spore powder was 1.0 × 10⁻⁶. 6 cfu / g.

[0236] 3) Sow 6 coated sorghum seeds in step 1) in the flowerpots in step 2), cover the surface with 3cm of soil, and water to 70% of field capacity. During the entire experiment, the field capacity was controlled to 70% by weighing. Sow 5 pots.

[0237] Comparative Example 5

[0238] 1) Using 60 g / L tebuconazole suspension seed coating agent as a positive control, sorghum seeds were coated with tebuconazole suspension seed coating agent for later use. The mass ratio of seed coating agent to seeds was 0.3 g: 1 kg.

[0239] 2) Same as step 2) in Application Example 1;

[0240] 3) Same as step 3) in Application Example 1.

[0241] Comparative Example 6

[0242] 1) Mix 1.5 kg of soil with 25 g of sorghum smut spore powder evenly and fill a plastic flowerpot with a bottom diameter of 14.6 cm, a height of 12.7 cm, and a top diameter of 18.0 cm. The sorghum smut spore powder was collected in the field, i.e., manually shaken off the diseased ears. The viable count of the spore powder was 1.0 × 10⁻⁶. 6 cfu / g.

[0243] 2) Sow 6 sorghum seeds (Jinza 2001) in the flowerpots in step 1), cover the surface with 3cm of soil, and water to 70% of field capacity. During the entire experiment, the field capacity was controlled to 70% by weighing. Sow 5 pots.

[0244] Results and Analysis:

[0245] The following treatments were performed for each of the following examples: For each treatment, two pots were randomly selected at 2, 4, 6, and 8 days after sowing to harvest germinating sorghum seeds. After collecting the hypocotyl portion of the sorghum root system, the infection rate of sorghum smut pathogen under different treatments was measured. The results are shown in Figures 7 and 8. Figure 10 The remaining potted plants in each treatment were thinned at the 3-leaf stage, and the plant height, aboveground fresh weight, and underground weight were measured 15 days after thinning. The results are shown in Figures 7 and 8. Figure 10 .

[0246] Table 7. Effects of different seed dressing treatments on sorghum grain germination, seedling growth, and prevention of sorghum head smut pathogen infection.

[0247]

[0248] From Table 7 and Figure 10 It can be seen that, under in-situ soil conditions, after measuring the coating effect, the effect of sorghum smut pathogen prevention on sorghum root tissue and its influence on seed germination by pot inoculation experiment with sorghum smut pathogen, strain G-1 bio-suspension seed coating agent was found to still have a good inhibitory effect on sorghum root tissue infection by sorghum smut pathogen under in-situ soil conditions, with an infection intensity of 8.37, which was 87.74% lower than the control (68.27) and 50.85% lower than the control agent (17.03%). In terms of germination rate and growth promotion, strain G-1·lipopeptide bio-suspension seed coating agent not only had no inhibitory effect on sorghum seed germination, but also had a significant promoting effect on sorghum plant height and underground part development during the seedling stage. Compared with the control, strain G-1·lipopeptide bio-suspension seed coating agent significantly increased the plant height, aboveground fresh weight and underground fresh weight of sorghum seedlings (P<0.05).

[0249] In summary, the microbial suspension seed coating agent prepared by this invention can achieve an infection prevention effect of 87.74% against sorghum smut pathogen, and has a significant growth-promoting effect on sorghum seedlings, showing good application potential in the prevention and control of sorghum smut.

[0250] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis G-1, characterized in that, The preservation number of the Bacillus belyssus G-1 is CGMCC No. 23750.

2. A method for preparing lipopeptide substances from Bacillus belye G-1 as described in claim 1, characterized in that, The preparation method includes: The activated Bacillus berleis G-1 was fermented, and the fermentation broth was centrifuged for the first time to obtain the first supernatant of the Bacillus berleis G-1 fermentation broth. The first supernatant was mixed with an acidic solution and allowed to stand before being centrifuged a second time to obtain the first precipitate. The first precipitate was resuspended, and the pH of the resuspended precipitate was adjusted to 7.0-8.0 before a third centrifugation was performed to obtain the second supernatant. After adjusting the pH of the second supernatant to 2.0-3.0, a fourth centrifugation was performed to obtain the second precipitate; The second precipitate was extracted with methanol, and the extract was concentrated to obtain the lipopeptide substance of Bacillus belyssus G-1.

3. The preparation method according to claim 2, characterized in that, During the fermentation, the inoculum size of Bacillus belye G-1 is 0.5% to 2% of the total fermentation medium volume; The fermentation time is 3-5 days, the temperature is 25-30℃, and the rotation speed is 120-180 r / min.

4. The preparation method according to claim 2, characterized in that, The conditions for the first, second, third, and fourth centrifugations are as follows: time is 5-10 min, and speed is 7500-12000 rpm.

5. The preparation method according to claim 2, characterized in that, The acidic solution includes a hydrochloric acid solution; the concentration of the hydrochloric acid solution is 1~6 mol / L; The conditions for mixing and settling include: a time of 8 to 24 hours and a temperature of 0 to 10°C.

6. The preparation method according to claim 2, characterized in that, During the extraction, the volume ratio of methanol to Bacillus vesiculosus G-1 fermentation broth is 0.5~1.5:0.5~2; The extraction is performed 1 to 4 times.

7. A microbial suspension seed coating agent, characterized in that, The raw materials comprise the following components by weight percentage: Bacillus belyssus G-1 as described in claim 1, 0.02%~0.06% lipopeptide, 3%~4% sodium carboxymethyl cellulose, 0.4%~0.6% sodium alginate, 3%~4% dispersant MF, 0.1%~0.2% ferrous sulfate, 0.5%~2% magnesium sulfate, 0.5%~4% dipotassium hydrogen phosphate, 2%~6% color paste, and the balance being water; wherein the lipopeptide is a lipopeptide obtained by the preparation method described in any one of claims 2 to 6; The concentration of Bacillus vesiculus G-1 in the microbial suspension seed coating agent is 5.0 × 10⁻⁶. 7 ~5.0×10 9 cfu / g.

8. The application of the microbial suspension seed coating agent according to claim 7 in the control of plant smut fungus; wherein the plant is sorghum.

9. A method for controlling plant smut fungus, characterized in that, include: After coating plant seeds with the microbial suspension seed coating agent according to claim 7, the coated plant seeds are sown; the plant is sorghum.

10. The method according to claim 9, characterized in that, The mass ratio of the microbial suspension seed coating agent to the plant seeds is 30~45:0.5~2.

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