Preparation and application of multifunctional microbial preparation for rhizosphere conservation

Abstract

The invention discloses preparation and application of a multifunctional microbial preparation for rhizosphere conservation, and relates to the technical field of microorganisms, and the technical key points are as follows: the microbial agent is prepared by mixing bacillus safensis S124, bacillus velezensis S719 and pseudomonas chlororaphis S1130 according to the proportion of 1: 1: 1, and the strain preservation numbers of the microbial agent are CGMCC (China General Microbiological Culture Collection Center) No.29817, CGMCC No.29818 and CGMCC No.29819 in sequence; the microbial preparation can inhibit pathogenic bacteria, plant pathogenic fungi and oomycetes, can promote crop growth, is free of drug resistance, safe and non-toxic, has the effects of preventing diseases and promoting growth, and can provide a biological control method for effective yield increase, weight reduction and pesticide reduction of crops.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to the preparation and application of a multifunctional microbial preparation for rhizosphere conservation. Background Art

[0002] Crop diseases caused by plant pathogens have had a serious impact on the production of food crops in my country and even around the world. For a long time, in order to increase crop yields and prevent and control diseases, excessive fertilizers and pesticides have often been applied during the planting process, which has led to a decline in the quality of cultivated soil, increased resistance of pathogens, and increased environmental pollution, causing great harm to the sustainable development of agriculture and the ecological environment. It is urgent for people to explore an environmentally friendly, efficient and safe production method and green prevention and control strategy.

[0003] Plant growth-promoting rhizobacteria (PGPR) are a type of beneficial microorganisms that exist in plant roots or rhizospheres and can promote plant growth, help plants absorb and utilize nutrients, improve soil physical and chemical properties, and antagonize plant pathogens. Plant growth-promoting rhizobacteria promote the utilization of nutrients such as minerals by plant roots through potassium and phosphorus solubilization, nitrogen fixation, and production of plant hormones; they directly or indirectly resist plant diseases and protect crops by producing antibiotics, lipopeptides, HCN and other antimicrobial substances, inducing plant immunity, occupying the ecological niche of the rhizosphere and competing with harmful bacteria for nutrients. Developing disease prevention and growth-promoting preparations using rhizosphere beneficial microbial resources has become one of the important ways to implement the green prevention and control strategy. The demand and supply of microbial preparations in my country maintain a rapid growth trend, and the market space and potential are huge. However, most of the commercialized microbial preparations currently still have problems such as homogeneity of production strains, lack of excellent strains, similar product combinations, and single mechanism of action.

[0004] To this end, the present invention aims to provide a preparation and application of a multifunctional microbial preparation for rhizosphere conservation to solve the above problems. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems and provide a preparation and application of a multifunctional microbial preparation for rhizosphere conservation. The microbial preparation of the present invention is prepared by mixing Bacillus shaforniensis S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 in a ratio of 1:1:1. The microbial preparation has a broad antibacterial spectrum, is suitable for a variety of crops, is non-resistant, safe and non-toxic, has disease prevention and growth promotion effects, and can provide a biological control method for effectively increasing crop yields and reducing fat and medication.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The invention provides a preparation method of a multifunctional microbial preparation for rhizosphere conservation. The microbial preparation is prepared from Bacillus sabourii S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130, the strains of which are deposited in CGMCC No. 29817, CGMCC No. 29818 and CGMCC No. 29819, respectively. Bacillus sabourii S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 are all deposited at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The preparation method comprises the following steps:

[0009] S1. Activation of bacterial strains and preparation of seed solution: Bacillus sabogensis S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130 stored in glycerol tubes were inoculated on LB plates for culture at 28° C. for 24 h, and single colonies were picked from the LB plates for inoculation into LB liquid culture medium, and shake-cultured overnight at 28° C. and 200 rpm to prepare fermentation seed solutions of Bacillus sabogensis S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130;

[0010] S2, scaled fermentation: the fermentation seed liquid of Bacillus sabdariffa S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130 were respectively transferred into a 1000L liquid fermentation tank at a ratio of 3% for scaled fermentation for 72 hours;

[0011] S3. Preparation of bacterial powder: After the fermentation is completed, the fermentation liquid is placed in a centrifuge, centrifuged at 10,000 rpm for 15 minutes, and the bottom bacterial mud is taken; the bacterial mud, glycerol, corn starch and sucrose are fully mixed in a ratio of 4:0.5:5:0.5, and finally dried to obtain the mother powder of Bacillus sabdariffa S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 (the mother powder standard is: effective viable bacteria count ≥ 50 billion CFU / g, miscellaneous bacteria rate ≤ 2.0%, water content ≤ 10.0%);

[0012] S4. Mixing auxiliary materials: add Bacillus sabdariffa S124 mother powder, Bacillus velezii S719 mother powder, Pseudomonas chlororaphis S1130 mother powder, potassium humate and maltodextrin into a mixer in a ratio of 5:5:5:5:80, mix thoroughly to obtain a microbial preparation.

[0013] The culture medium formula for the amplified fermentation of Bacillus sabovich S124 and Bacillus velezii S719 is: 30 g / L maltodextrin, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, and 1 g / L potassium dihydrogen phosphate;

[0014] The culture medium formula for the amplified fermentation of Pseudomonas chlororaphis S1130 is: 30 g / L corn flour, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, and 1 g / L potassium dihydrogen phosphate;

[0015] The fermentation temperature of the amplified fermentation of Bacillus saffron S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 is 30° C. and the pH is 7.0.

[0016] Application of a multifunctional microbial agent for rhizosphere conservation, strains S124, S719 and S1130 have different degrees of inhibitory effects on pathogenic bacteria such as rice Xanthomonas, tomato canker, Pseudomonas syringae, Pseudomonas aeruginosa, watermelon acidophilus, and plant pathogenic fungi and oomycetes such as rice blast, pseudograminearum Fusarium, tobacco moniliforme, Rhizoctonia solani, and tobacco phytopathogenic fungi and oomycetes; at the same time, it has growth-promoting properties such as solubilizing phosphate and potassium, producing iron carriers, proteases, amylases, cellulases, producing NH3, and secreting IAA. The results of the watermelon seedling disease prevention experiment showed that the bacterial liquid after the combination of strains S124, S719 and S1130 had a significantly higher control effect on watermelon wilt than the treatment of single strains and strains in pairs, with a control effect of up to 93.3%; in addition, the compound bacterial agent can effectively prevent and control tomato bacterial wilt, with a control efficiency of 91.3%. The compound bacterial agent can also significantly promote the growth and development of strawberries, and the weight of a single fruit increases by 15.2%.

[0017] In this scheme, the colonies of Bacillus sabogenes S124 on LB medium are white round colonies; the colonies of Bacillus velez S719 on LB medium are light yellow sticky round colonies; the colonies of Pseudomonas chlororaphis S1130 on LB medium are orange-red round colonies. Bacillus sabogenes S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 are mixed in a ratio of 1:1:1. The effective viable count is: Bacillus sabogenes S1243×10 8 CFU / g; Bacillus velezensis S7193×10 8 CFU / g; Pseudomonas chlororaphis S11303×10 8 CFU / g.

[0018] Compared with the prior art, this solution has the following beneficial effects:

[0019] The microbial preparation of the present invention is prepared by mixing Bacillus shaforniensis S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 in a ratio of 1:1:1. The microbial preparation can inhibit pathogenic bacteria, plant pathogenic fungi and oomycetes, and can promote crop growth, provide a biological control method for effectively increasing crop yields and reducing fertilizer and drug use, and provide an important reference for green prevention and control of plant diseases and improvement of soil and arable land quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 1 is a schematic diagram of a phylogenetic tree of S124 and related Pseudomonas constructed based on 16S rDNA sequences in an embodiment of the present invention;

[0021] Figure 2 16S rDNA sequence-based phylogenetic tree diagram of S719 and related Pseudomonas strains constructed in the present invention;

[0022] Figure 3 1 is a schematic diagram of a phylogenetic tree of S1130 and related Pseudomonas species constructed based on 16S rDNA sequences in an embodiment of the present invention;

[0023] Figure 4 Schematic diagram of the growth-promoting functional characteristics of strains S124, S719, and S1130 in the embodiments of the present invention;

[0024] Figure 5 Schematic diagram of the antagonistic effects of strains S124, S719, and S1130 on plant pathogenic bacteria in the embodiments of the present invention;

[0025] Figure 6 Schematic diagram of the antagonistic effects of strains S124, S719, and S1130 on plant pathogenic fungi in the embodiments of the present invention;

[0026] Figure 7 Schematic diagram of pairwise compatibility between strains S124, S719, and S1130 in the embodiment of the present invention;

[0027] Figure 8 Schematic diagram of the results of the control effect of the microbial preparation on watermelon wilt in the embodiment of the present invention;

[0028] Fig. 9 This is a graph showing the effect of the microbial preparation in the present invention on the prevention and treatment of tomato bacterial wilt in the field

[0029] Fig.10 This is a diagram showing the growth-promoting effect of the microbial preparation in an embodiment of the present invention on strawberries in the field. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0031] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0032] Example 1: Isolation of Bacillus saffron S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 (hereinafter referred to as isolation of strains S124, S719 and S1130)

[0033] Plant growth-promoting bacteria with antagonistic effects were initially isolated from tobacco rhizosphere soil in Yuxi City, Yunnan Province and wheat rhizosphere soil in Huantai County, Zibo City, Shandong Province. Weigh 1 g of rhizosphere soil sample, add 9 mL of sterile distilled water, and vortex for 2 min. After standing and stratifying, take 1 mL of the supernatant and dilute it to 10 -5 . Take 100 μL of each dilution gradient and spread it on NA medium plate, repeat 3 plates for each concentration gradient, and culture it in a 28°C incubator for 2 days. Pick single colonies with different morphology and re-streak them on LB plates to purify them, and obtain S124, S719, and S1130.

[0034] Example 2: Identification of strains S124, S719, and S1130

[0035] The strains S124, S719 and S1130 isolated above were subjected to morphological observation and genetic evolution analysis and identification. The specific methods of colony morphology, size, convexity and concavity, and edge were referred to the Manual of Identification of Common Bacterial Systems. The morphological characteristics showed that the colony morphology of S124 was milky white, opaque, round, with a raised center and neat edges; the colony morphology of S719 was light yellow, translucent, and moist and sticky on the surface; the colony morphology of S1130 was orange-red, opaque, round, with a raised center and neat edges.

[0036] The primers 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT were used to amplify the 16S rDNA fragment using the genomic DNA of strains S124, S719, and S1130 as templates using a PCR amplification reaction kit. The PCR reaction conditions were 95℃ pre-denaturation for 5min; 94℃30s, 53℃30s, 72℃10s for 30 cycles; 72℃ extension for 10min, and then stored at 4℃. Take 4μL of the PCR product, use DL2000 as a DNA molecule marker, methyl bromophenol blue as a dye, and 1% agarose gel electrophoresis to detect the PCR product. The products with the same amplification size were sent to Qingke Gene Company for sequencing analysis, and the 16S rDNA sequences of strains S124, S719, and S1130 were obtained, as shown in Seq ID No.1, No.2, and No.3, respectively. Based on the sequence alignment analysis in EzBioCloud and using MEGA11 software, the 16S rDNA phylogenetic tree was constructed using the Neighbor-Joining Tree method. The 16S rDNA identification results showed that strain S124 had the highest similarity with Bacillus safensis, with a homology of 99.93% ( Figure 1 ). Strain S719 has the highest similarity with Bacillus velezensis, 99.64% ( Figure 2 ), strain S1130 had the highest similarity with Pseudomonas chlororaphis, 100% ( Figure 3 The three strains were named B. safensis S124, B. velezensis S719, and P. chlororaphi S1130, respectively.

[0037] Seq ID No.1

[0038]

[0039] Seq ID No.2

[0040]

[0041] Seq ID No.3

[0042]

[0043] Example 3: Growth-promoting properties of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130

[0044] The activated B. safensis S124, B. velezensis S719 and P. chlororaphis S1130 were inoculated into LB liquid culture medium respectively, and cultured overnight at 28° C. and 200 rpm with shaking to obtain fermentation bacterial liquid.

[0045] (1) Determination of potassium-dissolving ability

[0046] Take 5 μL of bacterial liquid and inoculate it on potassium-dissolving medium (sucrose 5g, MgSO4·7H2O 0.5g, FeCl30.005g, CaCO30.1g, potassium feldspar powder 2g, agar 15g, ddH2O 1L), and inoculate 5 μL of liquid LB medium as control. Repeat each treatment 3 times. After drying the plate on the sterile operating table, transfer it to a 28℃ incubator and invert it for 3-5 days to observe the growth of the colony. If it can grow normally and the colony shows an oil droplet-like morphology, it proves that the strain can decompose potassium feldspar.

[0047] The results showed that there was no change in the control group and the plates inoculated with S124 and S1130 bacterial solutions, while colony growth was clearly observed on the plates inoculated with S719 bacterial solution ( Figure 4 a), indicating that B. velezensis S719 can decompose potassium feldspar, increase soil nutrient content, and improve the nutritional status of plant roots.

[0048] (2) Determination of phosphate solubilization capacity

[0049] Take 5 μL of culture solution and spot-spread it on a Montgena organophosphate medium plate (glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, FeSO4·7H2O 0.03g, MnSO4·4H2O 0.03g, egg yolk lecithin 0.2g, CaCO3 5g, yeast extract 0.4g, agar 15g, distilled water 1L, pH 7.0), and spot-spread 5 μL of LB liquid medium as a control. Each treatment was repeated 3 times. After drying the plate in a sterile operating table, transfer it to a 28°C incubator and invert it for 5 to 7 days to observe whether a hydrolysis halo appears. The results showed that there was no change in the control group and the plates inoculated with S124 and S719 bacterial solutions, while an obvious hydrolysis halo appeared around the colony inoculated with S1130 ( Figure 4 b), indicating that P.chlororaphis S1130 can hydrolyze organic phosphorus and can be used for soil improvement.

[0050] (3) Determination of protease production capacity

[0051] Take 5 μL of culture solution and spot-spread it on the protease detection medium plate (peptone 10.0g, NaCl5.0g, CaCl20.1g, skim milk 10.0mL, distilled water 990mL, 1.5% agar, sterilized at 115℃ for 30min, pH7.2-7.4), and spot-spread 5 μL of LB liquid culture medium as the control. Each treatment was repeated 3 times. After drying the plate in the sterile operating table, transfer it to a 28℃ incubator and invert it for 2 days to observe whether a transparent circle appears. The results showed that there was no change in the control group plate, while obvious hydrolysis transparent circles appeared on the plates inoculated with S124, S719 and S1130 bacterial solutions ( Figure 4 c), the average values ​​were 69.33, 49.00 and 31.00 mm respectively. This indicates that the protein in the plate was decomposed, and strains B. safensis S124, B. velezensis S719 and P. chlororaphis S1130 all have the ability to produce protease, among which B. safensis S124 has the strongest ability to decompose protein.

[0052] (4) Determination of amylase production capacity

[0053] Take 5 μL of culture solution and spot-spread it on the amylase detection medium plate (soluble starch 1g; peptone 5g; glucose 5g; NaCl 5g; beef extract 5g; agar 1.2%; distilled water 1L, 1.5% agar, sterilized at 115℃ for 30min, pH 7.2~7.4), and spot-spread 5 μL of LB liquid medium as control. Each treatment is repeated 3 times. After drying the plate in the sterile operating table, transfer it to a 28℃ incubator and invert it for 2~4d. After the culture is completed, pour Gram's iodine solution (I21g, KI 2g, distilled water 300mL) to cover the culture medium for 2min, discard the Gram's iodine solution and observe whether a transparent circle appears around the colony.

[0054] The results showed that there was no change in the control group and the plates inoculated with S124 and S1130 bacterial solutions. Only the plate inoculated with S719 bacterial solution showed an obvious transparent circle ( Figure 4 d), with a size of 37.67 mm. This indicates that B. velezensis S719 can produce amylase and has the ability to decompose starch, while strains B. safensis S124 and P. chlororaphis S1130 do not have the ability to decompose starch.

[0055] (5) Determination of cellulase production capacity

[0056] Take 5 μL of culture solution and spot-spread it on a sodium carboxymethyl cellulose medium plate (10 g of peptone; 10 g of yeast powder, 10 g of sodium carboxymethyl cellulose, 1 g of KH2PO4; 15 g of NaCl; 1.2% agar; 1 L of distilled water, 1.5% agar, sterilized at 115°C for 30 min), and spot-spread 5 μL of LB liquid medium as a control. Each treatment was repeated 3 times. After drying the plate in a sterile operating table, transfer it to a 28°C incubator and invert it for 2 to 4 days. After the culture is completed, pour a sufficient amount of 1 g / L Congo red solution into the culture dish for staining for 1 hour, then pour out the Congo red solution and rinse with 1 mol / L NaCl solution. After standing for 1 hour, pour out the NaCl solution and observe whether there is a transparent circle around the colony. If there is a transparent circle, it means that cellulase can be secreted. The results showed that there was no change in the control group and the plate inoculated with S1130 bacterial solution, while obvious hydrolysis transparent circles appeared on the plates inoculated with S124 and S719 bacterial solutions ( Figure 4 e), and their average values ​​were 58.67 and 51.00 mm, respectively. This indicates that the cellulose in the plate was decomposed, and strains B. safensis S124 and B. velezensis S719 have the ability to produce cellulase, among which B. safensis S124 has a stronger ability to decompose protein.

[0057] (6) Determination of IAA production capacity

[0058] The strains B.safensis S124, B.velezensis S719, and P.chlororaphis S1130 were inoculated into LB liquid culture medium containing 100 mg / L tryptophan, and cultured at 28°C and 200 rpm for 2 days, followed by centrifugation at 8000 r / min for 2 minutes, the supernatant was discarded, and the bacterial suspension was resuspended with an equal amount of physiological saline. Next, 100 μL of the bacterial suspension was dropped onto a white ceramic plate, and 100 μL of Salkowski colorimetric solution was added. The plate was placed at room temperature and away from light for 30 minutes before observation. If the color turns red, it means that IAA can be secreted, and the darker the color, the stronger the IAA production ability; if it does not change color, it means that IAA cannot be secreted. The results are as follows: Figure 4 As shown in f, strains B. safensis S124 and B. velezensis S719 did not change color after being tested in the dark, indicating that they could not produce IAA, but strain P. chlororaphis S1130 showed pink color in the dark, indicating that P. chlororaphis S1130 had the ability to secrete IAA, thereby promoting the germination of plant seeds and the growth of seedlings.

[0059] (7) Determination of ammonia production capacity

[0060] The strains B.safensis S124, B.velezensis S719, and P.chlororaphis S1130 were inoculated into peptone ammoniation medium (peptone 5g, Mg2SO4·7H2O 0.5g, NaCl 0.25g, FeSO40.01g, K2HPO40.5g, distilled water 1L), and cultured at 28℃ 200rpm for 2 days. Then, 3-5 drops of Nessler's reagent were added to the culture medium. If a yellow or brownish red precipitate appeared, it indicated that NH3 could be produced; if it did not change color, it indicated that it did not have the ability to produce ammonia. The results are as follows Figure 4 As shown in Figure h, after adding Nessler's reagent, the culture solutions of strains B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 all showed varying degrees of yellow or brown-red precipitation, indicating that B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 all have the ability to produce ammonia.

[0061] (8) Determination of siderophore production capacity

[0062] Preparation of CAS blue qualitative detection plate: Dissolve 0.012g CAS in 10mL deionized water and mix with 2mL 1mmol / L FeCl3 solution to obtain solution a. Dissolve 0.015g HDTMA in 8ml deionized water to obtain solution b.

[0063] Slowly add solution a to solution b, mix thoroughly to obtain dye solution c. Add 10 ml of 0.1 mol / L phosphate solution (2.427 g Na2HPO4·12H2O, 0.5905 g NaH2PO4·2H2O, 0.075 g KH2PO4, 0.125 g NaCl, 0.25 g NH4Cl, 100 mL deionized water, 10 times dilution when used) and 6.04 g piperazine diethanol sulfonic acid (pipes) to a clean conical flask containing 150 mL distilled water, mix well, and adjust the pH to 6.8 with 50% NaOH solution, and finally add 4 g agar powder to obtain culture medium d. Sterilize dye solution c, culture medium d, 1 mmol / L CaCl2, 1 mmol / LMgSO4·7H2O, 20% glucose, and 10% casamino acids (115°C, 20 minutes). Measure 0.2 mL of the above 1 mmol / L CaCl2, 4 mL of 1 mmol / LMgSO4·7H2O, 6 mL of 10% casamino acids, and 2 mL of 20% glucose solution and add them to the culture medium d. Then slowly add the dye solution c, shake well and pour the plate to obtain a blue siderophore qualitative detection plate.

[0064] The strains B.safensis S124, B.velezensis S719, and P.chlororaphis S1130 were inoculated on CAS blue qualitative detection plates and cultured at 28°C for 24, 48, 60, and 72 hours to observe whether a yellow halo was produced around the colonies. As the siderophores competed for the iron ions chelated by EDTA in the culture medium, the blue color in the culture medium faded, and a fading halo appeared around the colonies. The results showed that there were obvious fading halos around the colonies of B.velezensis S719 and P.chlororaphis S1130, proving that they have the ability to produce siderophores; however, there was no fading halo around the colonies of B.safensis S124, indicating that strain S124 does not produce siderophores ( Figure 4 i).

[0065] Table 1 Growth-promoting properties of strains S124, S719, and S1130

[0066] Potassium Phosphate solubilization Protease Amylase production Cellulase production Produced by IAA Ammonia production Siderophore production S124 - - + - + - + - S719 + - + + + - + + S1130 - + + - - + + +

[0067] Example 4: Antibacterial spectrum of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130

[0068] (1) Antibacterial spectrum of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 against pathogenic bacteria

[0069] Common plant pathogenic bacteria such as Xanthomonas oryzae, tomato canker bacteria, Pseudomonas syringae, Pseudomonas aeruginosa, and Acidophilus syringae were selected as the pathogenic bacteria to be tested. The pathogenic bacteria were activated for 16 hours under the conditions of their suitable temperature and culture medium to prepare pathogenic bacterial solution for use. Use a pipette to draw 1mL of the activated pathogenic bacterial solution into a 9cm diameter plate, and add 10mL of melted NA solid culture medium to it. After gently mixing, place it on a horizontal surface to make the agar layer form a uniform thickness. Open the plate cover and blow the plate near the alcohol lamp for half an hour. Then take 5μL of S124, S719 and S1130 bacterial solution and point it in the center of the solidified plate. After the bacterial solution is dried, take out the plate and put it into the incubator at 28℃ for 1d. Observe whether a transparent inhibition zone appears, and measure the diameter of the inhibition zone ( Figure 5 ).

[0070] As shown in Table 2, strain S719 has an inhibitory effect on the eight plant pathogenic bacteria tested, and the diameter of the inhibition zone for tomato ulcer pathogen, rice Xanthomonas, crucifer black pathogen, Pseudomonas syringae and avenaceae acidobacteria exceeds 30 mm, indicating that it has a strong inhibitory effect on these pathogenic bacteria. Strain S124 has an inhibitory effect on the four plant pathogens tested, including tomato ulcer pathogen, rice Xanthomonas, crucifer black pathogen and Staphylococcus aureus, among which the inhibitory effect on rice Xanthomonas is the strongest, and the diameter of the inhibition zone can reach 39.5 mm. Similarly, strain S719 has an inhibitory effect on the four plant pathogens tested, including tomato ulcer pathogen, rice Xanthomonas, crucifer black pathogen and Staphylococcus aureus, among which the inhibitory effect on rice Xanthomonas is the strongest, and the diameter of the inhibition zone can reach 32.5 mm.

[0071] Table 2 Antagonistic effects of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 on plant pathogenic bacteria

[0072]

[0073] (2) Antifungal spectrum of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 against pathogenic fungi

[0074] In view of the common diseases on food crops such as rice and wheat and cash crops such as watermelon, tomato and tobacco, rice blast, pseudofusarium graminearum, tobacco phytophthora, tobacco moniliforme and Rhizoctonia solani were selected as pathogenic fungi to be tested. The pathogenic fungi were inoculated on PDA plates in advance and cultured at a constant temperature of 25°C until the hyphae covered the entire plate. A sterile punch with a diameter of 5 mm was used to punch a fungus block at the edge of the pathogenic fungus and inoculated into the center of a new PDA plate. 5 μL of S124, S719 and S1130 bacterial solution were inoculated at two docking points 2.5 cm away from the pathogenic fungus block. Each treatment was repeated 3 times and cultured at 28°C for 3 to 5 days. The treatment in which the pathogenic fungus was inoculated only in the center of the PDA plate was used as the control. The mycelial growth diameter of the pathogenic fungus in the control group and the treatment group was measured, and the inhibition rate was calculated according to the inhibition rate = (control group colony diameter - treatment group colony diameter) / treatment group colony diameter × 100%. The distances from pathogenic fungi to colonies S124, S719, and S1130 were measured as the inhibition zone ( Figure 6 ).

[0075] As shown in Table 3, strains S124, S719 and S1130 have different antibacterial spectra and can complement each other to inhibit common pathogenic fungi. Among them, strain S719 has antibacterial activity against all tested fungi; strain S124 has antibacterial activity against Rhizoctonia solani, rice blast fungus, tobacco phytophthora, and tobacco thallus, among which the antibacterial activity against rice blast fungus is the strongest, with an inhibition rate of 65.69%; strain S1130 has antibacterial activity against all other tested fungi except Rhizoctonia solani. Although the inhibition rate of strain S1130 against rice blast fungus is only 11.36%, the inhibition rates of strains S124 and S719 against rice blast fungus are both over 60%, indicating that the three strains can form a complementary relationship and play a joint role in disease prevention.

[0076] Table 3 Antagonistic effects of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 on plant pathogenic fungi

[0077]

[0078] Example 5: Pairwise compatibility between B. safensis S124, B. velezensis S719, and P. chlororaphis S1130

[0079] The confrontation culture method was used to examine the pairwise compatibility between strains. One test bacterium was inoculated on one half of the LB solid culture medium, and another test bacterium was inoculated on the other half to form a V shape, ensuring that the bacterial liquids at the ends were close to each other. After the plate was placed in a 28°C incubator for 3 days, the growth of the colonies at the intersection was observed. If no inhibition band appeared, it was compatible, otherwise it was incompatible. The results are as follows Figure 7 As shown, B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 are compatible with each other and will not produce antagonism, making them suitable for development as compound microbial agents.

[0080] Example 6: Control effect of B. safensis S124, B. velezensis S719, and P. chlororaphis S1130 on watermelon wilt

[0081] Preparation of fermentation seed solution: Pick fresh single colonies of strains S124, S719, and S1130, transfer them to 5 mL of LB liquid culture medium with an inoculation needle, and culture them at 28°C and 200 rpm with shaking overnight to obtain seed solution.

[0082] Preparation of fermentation seed solution: The seed solution was inoculated into a conical flask containing 200 mL of LB liquid culture medium at an inoculation rate of 1%, and shaken and cultured at 28° C. and 200 rpm for 48 h to obtain a separate fermentation solution.

[0083] Culture of watermelon wilt pathogen: Pick the mycelium of watermelon wilt pathogen onto the PDA plate and culture it at 28℃ for 3-5 days. When the mycelium fills the culture dish, set it aside.

[0084] Watermelon wilt prevention test: Select watermelon seeds with full grains and uniform size, disinfect them with 2% sodium hypochlorite for 5 minutes, and rinse them with sterile water 2 to 3 times. Moisturize and germinate them under sterile conditions at 28°C for 5 days. Then soak the watermelon seeds with uniform germination in bacterial suspensions of S124, S719, S1130, S124+S719 (1:1), S124+S1130 (1:1:1) for 30 minutes. The concentration of the bacterial suspension is 1×10 8 CFU / mL, with immersion in sterile water for 30 minutes as the control. At the same time, use a 1cm diameter puncher to take the watermelon wilt pathogen cake, and place it with the mycelium side facing up in the soil layer in a 12mL centrifuge tube. The height of the soil layer is about 10mL. Finally, place the soaked watermelon seeds on the watermelon wilt pathogen cake, one seed per tube, cover with a thin layer of soil, pour an appropriate amount of sterile water, and culture at 25℃ for 10 days to observe the disease condition of the leaves and stem base ( Figure 8 The incidence rate, disease index and control effect were calculated (Table 4). The results showed that the control effect of S124+S719+S1130 was significantly higher than that of single strain treatment and two strains combination treatment, and the indoor control effect of S124+S719+S1130 on watermelon wilt reached 93.3%.

[0085] The calculation formula is as follows:

[0086] Incidence rate (%) = number of diseased plants / total number of treated plants × 100%;

[0087] Disease index = [∑(number of diseased plants at each level × corresponding disease level)] / (total number of plants surveyed × highest disease level) × 100;

[0088] Preventive and therapeutic effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0089] The grading standard for watermelon wilt disease: Grade 1 is when the wilting area of ​​leaves or stems accounts for 1 / 4 or less of the whole plant; Grade 2 is when the wilting area of ​​leaves or stems accounts for 1 / 2 of the whole plant; Grade 3 is when the wilting area of ​​leaves or stems accounts for 3 / 4 of the whole plant; Grade 4 is when the whole plant wilts and dies.

[0090] Table 4 Control of watermelon wilt by strains S124, S719 and S1130

[0091]

[0092]

[0093] Example 7: Preparation method of microbial agent

[0094] The strains S124, S719, and S1130 stored in glycerol tubes were inoculated on LB plates and cultured at 28°C for 24 h. Single colonies were picked and inoculated into LB liquid culture medium and shake-cultured overnight at 28°C and 200 rpm to prepare fermentation seed liquid.

[0095] The fermentation medium used by strains S124 and S719 (30 g / L maltodextrin, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, 1 g / L potassium dihydrogen phosphate) and the fermentation medium of strain S1130 (30 g / L corn flour, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, 1 g / L potassium dihydrogen phosphate) were sterilized at 121°C for 30 min for use. Then, the fermentation seed liquid of strains S124, S719, and S1130 was transferred to a 1000L liquid fermentation tank at a ratio of 3% for amplified fermentation for 72 hours. The fermentation temperature of strains S124, S719, and S1130 was 30°C and the pH was 7.0.

[0096] After the fermentation is completed, the fermentation liquid is separated by a centrifuge (10000rpm, 15min) to harvest the bacterial sludge. The bacterial sludge, glycerol, corn starch and sucrose are fully mixed in a ratio of 4:0.5:5:0.5, and finally dried to harvest the mother powders of Bacillus sabdariffa S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130. The mother powder of Bacillus sabdariffa S124, the mother powder of Bacillus velez S719, the mother powder of Pseudomonas chlororaphis S1130, potassium humate and maltodextrin are added to a mixer in a ratio of 5:5:5:5:80, and mixed thoroughly to obtain a composite microbial preparation.

[0097] Example 8: Field efficacy of microbial agents against tomato bacterial wilt

[0098] Field prevention experiments were conducted in a perennial tomato plantation in Shilibao Town, Miyun District, Beijing, which is a high-incidence plantation for tomato bacterial wilt. Tomato seedlings with consistent growth were selected for planting. After 7d and 40d of planting, compound microbial preparations (10kg / mu) were used for root irrigation, and a blank control group without added microbial preparations was set up. Three plots were set up for each treatment. About 30 days after the second root irrigation, the incidence of tomato bacterial wilt was counted. The results showed that the growth and disease prevention and control effects of the test group with microbial preparations were significantly better than those of the control group, with a prevention efficiency of 91.3% ( Fig. 9 ).

[0099] Example 9: Microbial agents significantly promote the growth and development of strawberries

[0100] Field trials were conducted in a strawberry farm in Xingshou Town, Changping District, Beijing. Strawberry seedlings with consistent growth were selected for planting. Fifteen days after the strawberries were planted, the roots were irrigated with a fungus (5 kg / mu). Thirty days after the fungus treatment, the growth indicators such as the height and canopy of the strawberries were counted. The same amount of clean water was applied as a control. Three plots were set up for each treatment. The results showed that the application of the fungus significantly promoted the growth of the strawberry plants. Compared with the treatment without the fungus, the height and canopy of the strawberry seedlings increased by 31.4% and 36.6% respectively ( Fig.10 ), and the growth-promoting effect is obvious. During the strawberry harvest period, the weight of single strawberry fruit was tested on site, and it was found that the weight of single strawberry fruit treated with the fungus agent was significantly higher than that of the control treatment, with an increase of 15.2%, which significantly increased the weight of single strawberry fruit. This shows that the fungus agent helps the growth and development of strawberries and increases yield.

[0101] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A preparation of a multifunctional microbial preparation for rhizosphere conservation, characterized in that: The microbial preparation is made of Bacillus saffron S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130; The preparation method comprises the following steps: S1. Activation of bacterial strains and preparation of seed solution: Bacillus sabogensis S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130 stored in glycerol tubes were inoculated on LB plates for culture at 28° C. for 24 h, and single colonies were picked from the LB plates for inoculation into LB liquid culture medium, and shake-cultured overnight at 28° C. and 200 rpm to prepare fermentation seed solutions of Bacillus sabogensis S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130; S2, scaled fermentation: the fermentation seed liquid of Bacillus sabdariffa S124, Bacillus velez S719, and Pseudomonas chlororaphis S1130 were respectively transferred into a 1000L liquid fermentation tank at a ratio of 3% for scaled fermentation for 72 hours; S3. Preparation of bacterial powder: After the fermentation is completed, the fermentation liquid is placed in a centrifuge, centrifuged at 10,000 rpm for 15 minutes, and the bottom bacterial mud is taken; the bacterial mud, glycerol, corn starch and sucrose are fully mixed in a ratio of 4:0.5:5:0.5, and finally dried to obtain the mother powder of Bacillus sabdariffa S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130; S4. Mixing auxiliary materials: add Bacillus sabdariffa S124 mother powder, Bacillus velezii S719 mother powder, Pseudomonas chlororaphis S1130 mother powder, potassium humate and maltodextrin into a mixer in a ratio of 5:5:5:5:80, mix thoroughly to obtain a microbial preparation.

2. The use of a multifunctional microbial preparation for rhizosphere conservation as claimed in claim 1, characterized in that: The microbial preparation can inhibit plant pathogenic bacteria, plant pathogenic fungi and oomycetes; The microbial preparation can promote the growth of crops.

3. The preparation of a multifunctional microbial preparation for rhizosphere conservation as claimed in claim 1, characterized in that: The culture medium formula for the amplified fermentation of Bacillus sabovich S124 and Bacillus velezii S719 is: 30 g / L maltodextrin, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, and 1 g / L potassium dihydrogen phosphate; The culture medium formula for the amplified fermentation of Pseudomonas chlororaphis S1130 is: 30 g / L corn flour, 5 g / L yeast powder, 5 g / L peptone, 1 g / L magnesium sulfate, 1 g / L calcium chloride, and 1 g / L potassium dihydrogen phosphate; The fermentation temperature of the amplified fermentation of Bacillus saffron S124, Bacillus velez S719 and Pseudomonas chlororaphis S1130 is 30° C. and the pH is 7.

0.

4. The use of a multifunctional microbial preparation for rhizosphere conservation as claimed in claim 2, characterized in that: The microbial preparation is used for preventing and controlling tomato bacterial wilt; The microbial preparation promotes the growth and development of strawberries.

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