A strain of Bacillus amyloliquefaciens bred by space-induced mutation and its application

Abstract

The present invention discloses a strain of Bacillus amyloliquefaciens bred by space-induced mutagenesis and its application. The present invention provides Bacillus amyloliquefaciens HTYB‑MH71‑1, with a deposit registration number of CGMCC No.25355. Compared with the starting strain: the ability of Bacillus amyloliquefaciens HTYB‑MH71‑1 to produce siderophore, protease, cellulase, IAA, etc. has been significantly improved, the biofilm formation and growth rate have been significantly accelerated, and the fermentation time has been significantly shortened, thereby effectively reducing the production cost; and, the control effect of Bacillus amyloliquefaciens HTYB‑MH71‑1 on tomato gray mold has been greatly enhanced. Therefore, Bacillus amyloliquefaciens HTYB‑MH71‑1 is a more efficient biocontrol strain with great development value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a strain of Bacillus amyloliquefaciens bred by space-induced bacterial strain mutation and application thereof. Background Art

[0002] Gray mold caused by Botrytis cinera is a common fungal disease in protected tomato cultivation. The pathogen can infect the stems, leaves, flowers and fruits of tomatoes, often leading to large-scale production reductions and huge economic losses to growers. Chemical agents are currently the main measure for the prevention and control of tomato gray mold in production. Long-term and large-scale use will lead to a series of problems such as the emergence of pathogen resistance and ecological environmental pollution. Biological control has gradually become an effective alternative for green prevention and control of fruit and vegetable diseases such as tomato gray mold because it is not easy to develop resistance and is safe and friendly to the environment.

[0003] Bacillus can produce spores with excellent properties such as resistance to high temperature and ultraviolet radiation. It has the advantages of strong stress resistance, broad antibacterial spectrum, fast reproduction and high safety. It is an important biocontrol group widely used in agricultural production. Obtaining active strains with excellent biocontrol functions is the premise and key to the effective prevention and control of plant diseases. Some biological characteristics related to the healthy growth of microorganisms and plants are key factors in evaluating the biocontrol potential of strains. Some extracellular enzymes and antimicrobial protein active substances produced by Bacillus have great application value in the biological control of fungal diseases of fruit and vegetable crops.

[0004] With the continuous advancement of microbiological technology and the various needs of humans for functional strains, the relevant screening methods for microbial strains with special functions have also been diversified. In recent years, with the in-depth development and utilization of space resources by humans and the rapid development of the global aerospace industry, the biological effects of the space environment on life forms have also attracted the interest of many scholars. Space mutagenesis breeding has also achieved many results with these hot spots, especially with the rapid development of my country's aerospace industry. One of the research focuses at home and abroad is on the mutagenesis breeding of space microorganisms, which has specifically involved the research fields of morphology, cytology, physiology, biochemistry and molecular biology of microorganisms, and has begun to conduct ground simulated weightlessness tests. According to relevant information, the rapid development of space mutagenesis technology in recent years can provide a new and reliable way to screen excellent strains for biological control applications. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus amyloliquefaciens bred by spaceflight mutation and application thereof.

[0006] The present invention provides Bacillus amyloliquefaciens HTYB-MH71-1, whose full name is Bacillus amyloliquefaciens HTYB-MH71-1, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on July 20, 2022, and the deposit registration number is CGMCC No. 25355.

[0007] Bacillus amyloliquefaciens HTYB-MH71-1 has the performance of rapid growth, so its application in production can reduce the fermentation cost in production and greatly improve the cost performance of its products.

[0008] The present invention also protects the use of Bacillus amyloliquefaciens HTYB-MH71-1, which is as follows (a) or (b):

[0009] (a) inhibiting the pathogenic bacteria of gray mold;

[0010] (b) Prevent and control plant gray mold.

[0011] The present invention also protects the culture of Bacillus amyloliquefaciens HTYB-MH71-1, the fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, or the metabolite of Bacillus amyloliquefaciens HTYB-MH71-1.

[0012] The function of the culture of Bacillus amyloliquefaciens HTYB-MH71-1, the fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, or the metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 is as follows (a) or (b):

[0013] (a) inhibiting the pathogenic bacteria of gray mold;

[0014] (b) Prevent and control plant gray mold.

[0015] The present invention also protects a bacterial agent, characterized in that: the bacterial agent contains Bacillus amyloliquefaciens HTYB-MH71-1 or / and a culture of Bacillus amyloliquefaciens HTYB-MH71-1 or / and a fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1 or / and a metabolite of Bacillus amyloliquefaciens HTYB-MH71-1.

[0016] The function of the bacterial agent is as follows (a) or (b):

[0017] (a) inhibiting the pathogenic bacteria of gray mold;

[0018] (b) Prevent and control plant gray mold.

[0019] The bacterial agent may also contain other biological components or non-biological components. Those skilled in the art may determine other active components of the bacterial agent based on the effects of the bacterial agent.

[0020] The bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0021] The solid support may be a mineral material and / or a biological material.

[0022] The mineral material may be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth.

[0023] The biological material may be at least one of various crop stalks, pine husks, rice straw, peanut shells, corn flour, bean flour, starch, peat and animal feces.

[0024] The liquid carrier may be water or a liquid culture medium. In the bacterial agent, Bacillus amyloliquefaciens HTYB-MH71-1 may exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0025] The bacterial agent may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0026] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0027] The present invention also protects the culture of Bacillus amyloliquefaciens HTYB-MH71-1, the fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, the metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 or the use of the bacterial agent, which is as follows (a) or (b):

[0028] (a) inhibiting the pathogenic bacteria of gray mold;

[0029] (b) Prevent and control plant gray mold.

[0030] The present invention also protects Bacillus amyloliquefaciens HTYB-MH71-1, a culture of Bacillus amyloliquefaciens HTYB-MH71-1, a fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, a metabolite of Bacillus amyloliquefaciens HTYB-MH71-1, or a use of the bacterial agent in preparing a product;

[0031] The functions of the product are as follows (a) or (b):

[0032] (a) inhibiting the pathogenic bacteria of gray mold;

[0033] (b) Prevent and control plant gray mold.

[0034] The present invention also protects a product, which includes Bacillus amyloliquefaciens HTYB-MH71-1, a culture of Bacillus amyloliquefaciens HTYB-MH71-1, a fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, a metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 or the bacterial agent;

[0035] The functions of the product are as follows (a) or (b):

[0036] (a) inhibiting the pathogenic bacteria of gray mold;

[0037] (b) Prevent and control plant gray mold.

[0038] The present invention also protects the use of Bacillus amyloliquefaciens HTYB-MH71-1, the culture of Bacillus amyloliquefaciens HTYB-MH71-1, the fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, the metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 or the bacterial agent in the preparation of protease and / or cellulase and / or siderophore and / or indoleacetic acid.

[0039] The present invention also protects a product for preparing protease and / or cellulase and / or siderophile and / or indoleacetic acid, which comprises Bacillus amyloliquefaciens HTYB-MH71-1, a culture of Bacillus amyloliquefaciens HTYB-MH71-1, a fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1, a metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 or the bacterial agent.

[0040] The present invention also protects a method for preparing the bacterial agent, comprising the step of using Bacillus amyloliquefaciens HTYB-MH71-1 or / and a culture of Bacillus amyloliquefaciens HTYB-MH71-1 or / and a fermentation product of Bacillus amyloliquefaciens HTYB-MH71-1 or / and a metabolite of Bacillus amyloliquefaciens HTYB-MH71-1 as a component of the bacterial agent to obtain the bacterial agent.

[0041] The culture of any of the above-mentioned Bacillus amyloliquefaciens HTYB-MH71-1 is a substance obtained by culturing Bacillus amyloliquefaciens HTYB-MH71-1 in a microbial culture medium (such as a liquid culture containing Bacillus amyloliquefaciens HTYB-MH71-1 and a substance secreted into a liquid culture medium, or a solid culture containing Bacillus amyloliquefaciens HTYB-MH71-1 and a substance secreted into a solid culture medium).

[0042] The fermentation product of any of the above-mentioned Bacillus amyloliquefaciens HTYB-MH71-1 is a substance obtained by fermenting Bacillus amyloliquefaciens HTYB-MH71-1 in a microbial culture medium (such as a fermentation liquid containing Bacillus amyloliquefaciens HTYB-MH71-1 and a substance secreted into a liquid culture medium, or a solid fermentation product containing Bacillus amyloliquefaciens HTYB-MH71-1 and a substance secreted into a solid culture medium).

[0043] Any of the above metabolites of Bacillus amyloliquefaciens HTYB-MH71-1 can be obtained from the fermentation broth of Bacillus amyloliquefaciens HTYB-MH71-1. The metabolites of Bacillus amyloliquefaciens HTYB-MH71-1 can be sterile metabolites of Bacillus amyloliquefaciens HTYB-MH71-1 or bacteria-containing metabolites of Bacillus amyloliquefaciens HTYB-MH71-1. The sterile metabolites (sterile fermentation filtrate) of Bacillus amyloliquefaciens HTYB-MH71-1 can be prepared specifically according to the following method: culturing Bacillus amyloliquefaciens HTYB-MH71-1 in a liquid culture medium, filtering and removing Bacillus amyloliquefaciens HTYB-MH71-1 in the fermentation broth, and obtaining sterile metabolites of Bacillus amyloliquefaciens HTYB-MH71-1. The bacterial metabolites of Bacillus amyloliquefaciens HTYB-MH71-1 can be prepared specifically according to the following method: culturing Bacillus amyloliquefaciens HTYB-MH71-1 in a liquid fermentation medium, collecting the fermentation broth (containing Bacillus amyloliquefaciens HTYB-MH71-1 and substances secreted into the liquid medium), and the fermentation broth is the bacterial metabolites of Bacillus amyloliquefaciens HTYB-MH71-1.

[0044] Any of the above gray mold pathogens is Botrytis cinerea.

[0045] Any of the above-mentioned gray mold pathogens is the pathogen of leek gray mold or the pathogen of tomato gray mold.

[0046] Any of the above gray mold pathogens is Botrytis squamosa or Botrytis cinerea Pers.

[0047] Any of the above gray mold diseases is gray mold disease caused by Botrytis cinerea.

[0048] Any of the above gray mold diseases is leek gray mold disease or tomato gray mold disease.

[0049] Any of the above-mentioned gray mold diseases is gray mold disease caused by the pathogen of leek gray mold disease or the pathogen of tomato gray mold disease.

[0050] Any of the above gray mold diseases is gray mold disease caused by Botrytis squamosa or Botrytis cinerea Pers.

[0051] Specifically, any one of the above plants is a plant of the Liliaceae family.

[0052] Specifically, any one of the above plants is an Allium plant.

[0053] Specifically, any one of the above plants is a Solanaceae plant.

[0054] Specifically, any one of the above plants is a Solanum plant.

[0055] Specifically, any one of the above plants is leek or tomato.

[0056] The present invention discloses a strain of Bacillus amyloliquefaciens obtained by aerospace breeding technology, and the strain is named Bacillus amyloliquefaciens HTYB-MH71-1. Compared with Bacillus amyloliquefaciens MH71 as the starting strain: the ability of Bacillus amyloliquefaciens HTYB-MH71-1 to produce siderophore, protease, cellulase, IAA, etc. has been significantly improved, the biofilm formation and growth rate have been significantly accelerated, and the fermentation time has been significantly shortened, thereby effectively reducing the production cost; and the control effect of Bacillus amyloliquefaciens HTYB-MH71-1 on tomato gray mold has been greatly enhanced. Therefore, Bacillus amyloliquefaciens HTYB-MH71-1 is a more efficient biocontrol strain with great development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 These are the sample photos before spaceflight mutagenesis and the sample photos after returning in Example 1.

[0058] Figure 2 The following are photos of bacterial growth status for comparison of growth conditions in Example 1.

[0059] Figure 3 The electrophoresis diagram and sequencing results of the PCR amplification products in the bacterial species verification in Example 1 are shown.

[0060] Figure 4 These are photos of the culture process of the strains after mutagenesis selected according to morphological differences in Example 1.

[0061] Figure 5 This is a photograph of the morphological characteristics of Bacillus amyloliquefaciens HTYB-MH71-1 in Example 1.

[0062] Figure 6 This is a photograph of the confrontation culture of Bacillus amyloliquefaciens HTYB-MH71-1 in Example 1 for genetic stability.

[0063] Figure 7 This is the growth curve in Example 2.

[0064] Figure 8 These are photos of colony morphology and colony counts in Example 2.

[0065] Fig. 9 These are the photos and statistical results of the protease activity detection in Example 3.

[0066] Fig.10 These are the photos and statistical results of detecting cellulase activity in Example 3.

[0067] Fig.11 These are the photos and statistical results of detecting the siderophore secretion ability in Example 3.

[0068] Fig.12 These are photos and statistical results of detecting the IAA production capacity in Example 3.

[0069] Fig.13 This is a photo of the biofilm formation ability test in Example 4.

[0070] Fig.14 These are the results and photos of the plate antibacterial activity test on leek gray mold in Example 5.

[0071] Fig.15 These are the results and photos of the antibacterial activity test on tomato gray mold in Example 5.

[0072] Fig.16 These are the results and photos of the in vitro leaf protection efficacy test against tomato gray mold in Example 5. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the culture medium in the examples has been sterilized at 121°C for 20 minutes before use. Unless otherwise specified, the quantitative tests in the following examples are repeated three times, and the results are averaged.

[0075] PDA solid medium (natural pH): 20 g glucose, 200 g potato, 20 g agar powder, 1000 mL distilled water.

[0076] LB solid medium (pH 7.0-7.2): 10 g tryptone, 10 g NaCl, 5 g yeast extract, 20 g agar powder, and 1000 mL distilled water.

[0077] LB liquid culture medium (pH 7.0-7.2): 10 g tryptone, 10 g NaCl, 5 g yeast extract, and 1000 mL distilled water.

[0078] FB fermentation medium (pH 7.0): 36.6 g corn flour, 3.2 g beef extract, 1.33 g sodium chloride, 14 g glucose, 7 g peptone, 0.4 g magnesium sulfate heptahydrate, 0.8 g potassium hydrogen phosphate, 8 g calcium carbonate, and 1000 mL distilled water.

[0079] Example 1. Acquisition and preservation of Bacillus amyloliquefaciens HTYB-MH71-1

[0080] 1. Preparation before spaceflight mutagenesis

[0081] Starting strain: Bacillus amyloliquefaciens MH71. Bacillus amyloliquefaciens MH71, full name Bacillus amyloliquefaciens MH71, was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on December 12, 2012, with the deposit registration number of CGMCC No.6978. It has been authorized by the Chinese invention patent with the authorization announcement number of CN 103087951 B published on August 20, 2014.

[0082] 1. Activate the starting strain stored in ultra-low temperature glycerol by streaking on LB solid medium.

[0083] 2. After completing step 1, pick a single colony and streak it onto the PDA solid culture medium slant (made with 2mL CORNING cryopreservation tubes), and culture it in a 28℃ constant temperature incubator for 48h. Four parallel treatments were performed, and four bacteria-containing cryopreservation tubes were obtained accordingly, numbered JT-1, JT-2, JT-3, and JT-4, and the tubes were sealed with parafilm. JT-1 and JT-2 were used for step 2, and JT-3 and JT-4 were stored at 4℃ for subsequent simultaneous controls.

[0084] 3. After completing step 1, pick a single colony and inoculate it into LB liquid culture medium (100mL culture medium in a 500mL triangular bottle), shake and culture at 30℃ and 200rpm for 72h, then centrifuge at 4℃ and 6000rpm for 10min, discard the supernatant, vacuum freeze-dry the remaining bacteria, obtain bacterial powder, and put it into a 2mL CORNING cryopreservation tube. Four parallel treatments were performed, and four corresponding cryopreservation tubes containing bacterial powder were obtained, which were numbered JF-1, JF-2, JF-3, and JF-4, and the tube mouths were sealed with parafilm. JF-1 and JF-2 were used for step 2, and JF-3 and JF-4 were stored at 4℃ for subsequent simultaneous controls.

[0085] 2. Spaceflight Mutation

[0086] JT-1 and JT-2 as well as JF-1 and JF-2 were carried on the Long March 2F Yao 12 carrier rocket of the Shenzhou XII manned spacecraft. The returned samples were received by the inventor's work unit on September 22, 2021.

[0087] The main characteristics of the space environment are microgravity, strong cosmic rays, ultra-vacuum, alternating magnetic field, ultra-clean environment, etc. The flight parameters of Shenzhou XII are orbital altitude of 400 kilometers, inclination of 41 degrees, radiation dose of 0.4 millisievert (mSv), cabin temperature of 25℃, and humidity of about 30%. These space conditions are likely to cause genetic mutations in microorganisms.

[0088] See the sample photos before space mutagenesis Figure 1 The left picture of the sample after returning is shown in Figure 1 Right picture.

[0089] 3. Comparison of growth

[0090] JT-1, JT-2, JF-1, JF-2 that have completed step 2, and the starting strain activated in step 1 were streaked onto PDA solid culture medium and cultured in a constant temperature incubator at 28°C for 48 hours.

[0091] Observe the growth status of bacteria, see photos Figure 2 The results showed that the bacteria grew well after space-induced mutagenesis, and there was no death or weakening of bacterial growth. This result also shows that the bacterial strains induced by space-induced mutagenesis can be selected in the form of bacterial slant or vacuum-frozen freeze-dried powder before loading.

[0092] 4. Verification of bacterial species

[0093] Test samples: JT-1, JT-2, JF-1, JF-2 that have completed step 2, and JT-3, JT-4, JF-3, JF-4 that have been stored at 4°C in step 1.

[0094] The bacteria in the test sample were streaked onto LB solid medium for activation, and then genomic DNA was extracted using a bacterial genomic kit (Beijing Bomed Gene Technology Co., Ltd.). Using genomic DNA as a template, PCR amplification was performed using a primer pair consisting of 27F and 1492R, and the taxonomic status of the bacteria was determined by sequence comparison.

[0095] The target sequences of 27F and 1492R are located in the 16S rDNA gene. 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGTTACCTTGTTACGACTT-3'.

[0096] The electrophoresis diagram of PCR amplification products (each lane represents a sample) and the sequencing results (each line represents a sample) are shown in Figure 3 The results showed that the sequence similarity of all samples was 99.46%, with only a few base differences. All samples were identified as Bacillus amyloliquefaciens. This result also shows that the bacteria induced by space flight were not contaminated and no significant variation in species occurred.

[0097] 5. Select the mutagenized strain based on morphological differences

[0098] 1. Complete the JT-1, JT-2, JF-1, and JF-2 of step 2, and streak them onto PDA solid medium, then pick a single colony and inoculate it into LB liquid medium, shake and culture at 28°C and 180rpm for 24h, and then dilute them 10 times with sterile water. Take dilutions of 10 -6 Up to 10 -11 100 μL of the bacterial solution was spread on LB solid culture medium plates and cultured in a constant temperature incubator at 28°C for 48 h.

[0099] 2. After completing the previous step, observe the colony morphology and pick single colonies of different morphologies to inoculate into LB liquid culture medium, shake and culture at 28°C and 180rpm for 24h, and then dilute them 10 times with sterile water. -6 Up to 10 -11 100 μL of bacterial solution was spread on a PDA solid culture medium plate and cultured in a 28°C constant temperature incubator for 48 h. Repeat several times.

[0100] The starting strain was used as the control strain for morphological observation.

[0101] See the photos of the cultivation process. Figure 4(A is a photo of the starting strain, and B is a photo of an exemplary mutagenized strain). The colony diameter of the starting strain is small, nearly round, flat and dry, and the colony periphery is serrated. Some of the mutagenized strains are irregular and protruding in the shape of vacuolar water droplets (named Bacillus amyloliquefaciens HTYB-MH71-1), and some are smaller in diameter and round (named Bacillus amyloliquefaciens HTYB-MH71-2).

[0102] VI. Morphological characteristics of Bacillus amyloliquefaciens HTYB-MH71-1

[0103] On PDA solid medium plate: In the early stage of culture, the colonies are round or irregularly connected, with water drop-shaped protrusions on the surface; as the culture time increases, the protrusions gradually turn into nearly white or light brown, the protrusions gradually disappear and become flat, and the surface is wrinkled or the surrounding is jagged. See the morphological photos for details. Figure 5 .

[0104] Staining showed that it was Gram-positive bacteria.

[0105] VII. Genetic stability of Bacillus amyloliquefaciens HTYB-MH71-1

[0106] Bacillus amyloliquefaciens HTYB-MH71-1 was subcultured 15 times (using PDA solid culture medium plates), and the strain from each subculture was used as the test strain.

[0107] The test strain was inoculated with 1 loop of bacteria into LB liquid medium (100 mL medium in a 500 mL triangular bottle), cultured at 30°C and 200 rpm for 20 hours (this is called seed liquid), then diluted with sterile water, and then spread on a PDA solid medium plate, cultured in a 28°C constant temperature incubator for 72 hours, and then the number of colonies was counted to calculate the bacterial content in the seed liquid. The bacterial content results are shown in Table 1.

[0108] The seed liquid obtained in the above steps was inoculated into the FB fermentation medium at a 1% inoculation rate, and cultured at 30°C and 200rpm for 72 hours, then sampled and used as the test solution. Use a 7mm sterile puncher to punch holes on the PDA solid culture medium plate cultured with tomato gray mold to obtain the pathogenic bacteria cake. Take a new PDA solid culture medium plate, place the pathogenic bacteria cake with the mycelium side down in the center of the plate, then inoculate the test solution on both sides (take 1μL on the left and right sides to draw a line), and then culture in a constant temperature incubator at 28°C for 72 hours. The starting strain was tested in parallel as a control. See example photos. Figure 6 (CK represents the starting strain; A-1 and A-2 represent the 15th generation strains of Bacillus amyloliquefaciens HTYB-MH71-1; B-1, B-2 and B-3 represent the 1st generation strains of Bacillus amyloliquefaciens HTYB-MH71-1).

[0109] Table 1

[0110] <![CDATA[Bacterial content result (×10 10 CFU / mL)]]> Antibacterial activity results The strain of the 15th generation 5.2 +++ First generation strain 5.5 +++

[0111] The results showed that after 15 transfers, the colony morphology of Bacillus amyloliquefaciens HTYB-MH71-1 did not undergo abnormal changes, and the number of viable bacteria and its antibacterial activity against tomato gray mold were stable and maintained at a high level, indicating that this strain is a biocontrol strain with great potential for development and application.

[0112] 8. Storage of Bacillus amyloliquefaciens HTYB-MH71-1

[0113] Bacillus amyloliquefaciens HTYB-MH71-1, whose full name is Bacillus amyloliquefaciens HTYB-MH71-1, was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on July 20, 2022, with the deposit registration number CGMCC No.25355.

[0114] Example 2. Growth characteristics of three strains of Bacillus amyloliquefaciens

[0115] Test bacteria: Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, Bacillus amyloliquefaciens HTYB-MH71-2.

[0116] 1. Growth Curve

[0117] 1. Pick a single colony of the test bacteria and inoculate it into LB liquid culture medium (100 mL culture medium in a 500 mL Erlenmeyer flask), and culture it at 30°C and 200 rpm for 20 hours to obtain the seed solution.

[0118] 2. Inoculate the seed liquid into LB liquid medium (10 mL of liquid in a 50 mL triangular bottle) at a 1% inoculum, and culture at 30°C and 200 rpm. The culture time is 24 hours in total, and samples are taken at intervals of 1 hour, with the first sampling starting at 0 hours. Set up 3 parallel treatments for each culture time.

[0119] 3. After completing step 2, use the OD 600nm Detect the absorbance value. OD values ​​between 0.2-0.6 are valid data. OD values ​​exceeding 0.6 need to be appropriately diluted with LB liquid culture medium and re-tested.

[0120] Growth curves are shown in Figure 7The growth performance of Bacillus amyloliquefaciens HTYB-MH71-1 in the delayed phase was very stable, without any ups and downs. It entered the rapid growth phase after 10 hours, and entered the logarithmic phase about 5 hours earlier than Bacillus amyloliquefaciens MH71. The growth trend of Bacillus amyloliquefaciens HTYB-MH71-2 was basically the same as that of Bacillus amyloliquefaciens MH71.

[0121] 2. Colony morphology and colony count

[0122] Take the bacterial solution cultured for 17 hours in step 1, dilute it with sterile water and spread it on a PDA solid culture medium plate, culture it in a constant temperature incubator at 28°C for 48 hours, and then observe the colony morphology and count it.

[0123] Results Figure 8 .

[0124] The bacterial content of Bacillus amyloliquefaciens MH71 was 2.75×10 10 CFU / mL.

[0125] The bacterial content of Bacillus amyloliquefaciens HTYB-MH71-2 was 8.3×10 10 CFU / mL.

[0126] After the bacterial solution of Bacillus amyloliquefaciens HTYB-MH71-1 was inoculated and cultured, the colonies quickly grew and connected into pieces, making it impossible to count the exact number of colonies. Combined with visual observation, the bacterial content should be significantly higher than that of Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2.

[0127] The above results show that Bacillus amyloliquefaciens HTYB-MH71-1 has the performance of rapid growth, so its application in production can reduce the fermentation cost in production and greatly improve the cost performance of its products.

[0128] Example 3: Analysis of the characteristics of the production of phytohormones and enzymes

[0129] Test bacteria: Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, Bacillus amyloliquefaciens HTYB-MH71-2.

[0130] 1. Preparation of culture medium

[0131] 1. Protease detection plate: Mix the milk powder agar solution and LB solid medium in a volume ratio of 1:9, and then pour it into the plate. Milk powder agar solution: 10g imported skimmed milk powder, 15g agar, 1000mL distilled water; use after high pressure sterilization at 115℃ for 20min.

[0132] 2. Siderophore test plate: Pour solution 3 into solution 2, then mix with 30 mL of filtered and sterilized 10 g / 100 ml casamimo acid (acid hydrolyzed casein) solution, and finally add solution 1, mix slowly (avoid foaming), and then pour onto the plate.

[0133] Solution 1 (CAS / HDTMA solution): 50 mL of CAS solution and 10 mL of iron solution were mixed and added to 40 mL of HDTMA solution. The mixed blue-black liquid is the CAS / HDTMA solution; it is sterilized at 121°C before use. CAS solution: 60.5 mg of CAS (chrome azure) was dissolved in 50 mL of distilled water. Iron solution (pH 2.0): 0.216 g of FeCl3·6H2O was dissolved in 10 mL of 10 mmol / L HCl aqueous solution. HDTMA solution: 72.9 mg of HDTMA (trimethylhexadecyl ammonium bromide) was dissolved in 40 mL of distilled water.

[0134] Solution 2 (Salts / Buffer solution): Dissolve 30.24g Pipes (piperazine-1,4-diethanesulfonic acid) in 750mL Salts solution, then adjust pH to 6.8 with 50g / 100mL KOH aqueous solution, then add 15g agar, and then dilute to 800mL with distilled water; sterilize at 121℃ and cool to 50℃. Salts solution (750mL): KH2PO4 0.3g, NaCl 0.5g, NH4Cl1g, distilled water to 750mL.

[0135] Solution 3: glucose 2g, MgSO4·7H2O 493mg, mannitol 2g, CaCl2 11mg, ZnSO4·7H2O 1.2mg, H3BO3 1.4mg, MnSO4·2H2O 1.17mg, CuSO4 40μg, Na2MoO4·2H2O 1mg, make up to 750mL with distilled water; autoclave at 121℃ and then cool to 50℃.

[0136] 3. Cellulase detection plate (pH 7.0): MgSO4·7H2O 0.25 g, K2HPO4 0.5 g, cellulose 1.88 g, Congo red 0.2 g, agar 14 g, gelatin 2 g, distilled water to 1000 mL; autoclave at 121°C for 20 min before use.

[0137] 4. CCM liquid culture medium (pH 7.0): mannitol 5.0 g, sucrose 5.0 g, lactic acid 0.5 mL, MgSO4·7H2O 0.2 g, KH2PO4 0.2 g, K2HPO4 0.8 g, CaCl2·2H2O 0.06 g, NaCl 0.1 g, NaMo04·2H2O 2.5 mg, yeast powder 0.1 g, Na·Fe·EDTA solution 4 mL (1.64%), tryptophan 0.1 g, NH4NO3 1.0 g, distilled water to 1000 mL; autoclave at 121°C for 20 min before use.

[0138] 2. Detection of protease activity

[0139] 1. Inoculate a single colony of the test bacteria into LB liquid medium (100 mL medium in a 500 mL triangular bottle), shake and culture overnight at 30°C and 200 rpm, and then adjust the bacterial concentration with LB liquid medium to obtain 1×10 8 CFU / mL of bacterial suspension.

[0140] 2. Inoculate the bacterial suspension prepared in step 1 onto the protease detection plate (evenly set 3 spots, inoculate 1 μL at each spot), culture at 28°C for 72 h, take pictures and count the diameters of the transparent circles.

[0141] Set up three or more repeated treatments and take the average of the results.

[0142] See the sample photos Fig. 9 The left figure (numbers 1, 2, and 3 correspond to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively), the statistical results of the transparent circle diameter are shown in Fig. 9 The results showed that the protease activity of Bacillus amyloliquefaciens HTYB-MH71-1 and Bacillus amyloliquefaciens HTYB-MH71-2 was better than that of Bacillus amyloliquefaciens MH71; the protease activity of Bacillus amyloliquefaciens HTYB-MH71-1 was better than that of Bacillus amyloliquefaciens HTYB-MH71-2.

[0143] 3. Detection of cellulase activity

[0144] 1. Same as step 1 of step 2.

[0145] 2. Inoculate the bacterial suspension prepared in step 1 onto the cellulase detection plate (evenly set 3 spots, inoculate 1 μL at each spot), culture at 28°C for 72 hours, take pictures and count the diameters of the transparent circles.

[0146] Set up three or more repeated treatments and take the average of the results.

[0147] See the sample photos Fig.10The left figure (numbers 1, 2, and 3 correspond to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively), the statistical results of the transparent circle diameter are shown in Fig.10 The results showed that the cellulase activity of Bacillus amyloliquefaciens HTYB-MH71-1 and Bacillus amyloliquefaciens HTYB-MH71-2 were better than that of Bacillus amyloliquefaciens MH71; the cellulase activity of Bacillus amyloliquefaciens HTYB-MH71-1 was better than that of Bacillus amyloliquefaciens HTYB-MH71-2.

[0148] 4. Detection of siderophore

[0149] 1. Same as step 1 of step 2.

[0150] 2. Inoculate the bacterial suspension prepared in step 1 onto the siderophore detection plate (evenly set 3 spots, inoculate 1 μL at each spot), culture at 28°C for 72 h, take pictures and count the diameters of the transparent circles.

[0151] Set up three or more repeated treatments and take the average of the results.

[0152] Two exemplary photos are shown in Fig.11 The above figure (numbers 1, 2, and 3 correspond to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively), the statistical results of the transparent circle diameter are shown in Fig.11 The results showed that the siderophore secretion ability of Bacillus amyloliquefaciens HTYB-MH71-1 was better than that of Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2, and there was no significant difference in the siderophore production ability of Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2.

[0153] 5. Detection of indoleacetic acid

[0154] 1. Same as step 1 of step 2.

[0155] 2. Inoculate 1 mL of the bacterial suspension prepared in step 1 into 50 mL of CCM liquid culture medium (container is a 500 mL Erlenmeyer flask) and culture at 28°C and 130 rpm for 12 days.

[0156] 3. IAA qualitative detection

[0157] After completing step 2, take a sample of 100 μL of culture solution and drop it on a white colorimetric plate (the positive control well uses an equal volume of 10 mg / mL IAA aqueous solution instead of culture solution), and then add 100 μL of colorimetric solution (colorimetric solution: 0.5M FeCl3 1mL + H2SO4 30mL + distilled water 50mL; the same below). Place the white colorimetric plate at room temperature for 15 minutes and observe its color change. If the solution color is pink, it is positive, otherwise it is negative, so as to determine whether the strain has the ability to produce the auxin IAA. See photos for details. Fig.12 A (numbers 1, 2, and 3 correspond to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively).

[0158] 4. IAA quantitative detection

[0159] (1) Prepare IAA standard (Sigma) into a series of gradient concentration standard solutions of 10 mg / L, 5 mg / L, 2.5 mg / L, 1.25 mg / L, and 0.625 mg / L and store in a refrigerator at 4°C until use.

[0160] (2) After completing step 2, centrifuge at 4°C and 12,000 rpm for 5 min and collect the supernatant (test supernatant).

[0161] (3) Mix 1 mL of the supernatant obtained in step (2) with 1 mL of the colorimetric solution and let it stand in a light-proof environment for 30 min. Immediately measure the absorbance of each reaction solution and the IAA standard solution at 530 nm using a spectrophotometer, and create an IAA standard curve in Excel to calculate the IAA content of each test supernatant.

[0162] Set up three or more repeated treatments and take the average of the results.

[0163] Results Fig.12 B.

[0164] The results showed that Bacillus amyloliquefaciens HTYB-MH71-1 had the strongest IAA secretion ability, with a quantitative detection content of 1.19 mg / L, the quantitative detection content of Bacillus amyloliquefaciens MH71 was 0.82 mg / L, and the quantitative detection content of Bacillus amyloliquefaciens HTYB-MH71-2 was only 0.07 mg / L.

[0165] Example 4: Biofilm Formation Ability Detection

[0166] Test bacteria: Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, Bacillus amyloliquefaciens HTYB-MH71-2.

[0167] A single colony of the test bacteria was inoculated into LB liquid culture medium (100 mL culture medium in a 500 mL Erlenmeyer flask), cultured overnight at 30 °C and 200 rpm with shaking, then left at room temperature for 48 h to observe the biofilm formation on the surface of the culture medium.

[0168] See photos Fig.13 (No. 1, 2, 3 correspond to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1 and Bacillus amyloliquefaciens HTYB-MH71-2, respectively). The results showed that the biofilm formation ability of Bacillus amyloliquefaciens HTYB-MH71-1 was stronger than that of Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2. The biocontrol strain has a strong ability to form biofilms, indicating that it can form a strong colonization ability in the rhizosphere of crops, thereby effectively preventing and controlling pathogens from invading plants.

[0169] Example 5: Determination of antibacterial activity against Botrytis cinerea

[0170] Test biocontrol bacteria: Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, Bacillus amyloliquefaciens HTYB-MH71-2.

[0171] The pathogen of leek gray mold, Botrytis squamosa, is recorded in the following literature: Screening test of agents for the prevention and control of leek gray mold, Li Yameng, Bi Yang, Lu Caige, etc., China Plant Protection Guide, Issue 8, 2020, 63-68.

[0172] Tomato gray mold, namely the pathogen of tomato gray mold (Botrytis cinerea Pers.), is recorded in the following literature: Biocontrol activity of Bacillus amyloliquefaciens MH71 and detection of lipopeptide antibiotic genes, Lu Caige et al., Plant Protection, 2015, 41(3): 12-18.

[0173] Improved LB liquid medium (pH 7.2-7.4): glucose 10.0 g, peptone 10.0 g, sodium chloride 5.0 g, beef extract 3.0 g, manganese sulfate 5.0 mg, distilled water 1000 mL.

[0174] 1. Plate antibacterial activity test on leek gray mold

[0175] 1. Inoculate a single colony of the test biocontrol bacteria into LB liquid medium (100 mL medium in a 500 mL triangular bottle), shake and culture overnight at 30°C and 200 rpm, and then adjust the bacterial concentration with LB liquid medium to obtain 1×10 8 CFU / mL of bacterial suspension.

[0176] 2. Use a 7mm sterile hole puncher to punch holes on the PDA solid medium plate cultured with leek gray mold to obtain the pathogenic bacteria cake. Take a new PDA solid medium plate, stick the mycelium surface of the pathogenic bacteria cake on the center of the plate facing down, and then inoculate the bacterial suspension obtained in step (1) around the pathogenic bacteria cake (evenly set 3 points, inoculate the three kinds of test biocontrol bacteria suspensions respectively, and inoculate 1μL at each point), and culture in a 25°C constant temperature incubator for 72h. Set up a control with an equal volume of sterile water instead of the bacterial suspension.

[0177] See photos Fig.14 (Figure A is an exemplary photo of the three tested biocontrol bacteria inoculated, with numbers 1, 2, and 3 corresponding to Bacillus amyloliquefaciens HTYB-MH71-1, Bacillus amyloliquefaciens MH71, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively; Figure B is an exemplary photo of the control.) The results showed that the three strains had strong antibacterial activity against gray mold of leek, but Bacillus amyloliquefaciens HTYB-MH71-1 was able to grow faster and occupy the ecological niche in the confrontation with gray mold of leek, thereby inhibiting the expansion and growth of the pathogen.

[0178] 2. Antibacterial activity test on tomato gray mold

[0179] 1. Wash the spores on the freshly cultured tomato gray mold plate with sterile water, filter through four layers of sterile gauze, and prepare 1×10 8 CFU / mL spore suspension.

[0180] 2. Inoculate a single colony of the test biocontrol bacteria into modified LB liquid culture medium (100 mL culture medium in a 500 mL Erlenmeyer flask), shake and culture at 30°C and 180 rpm for 72 h, then centrifuge at 4°C and 10,000 rpm / min for 5 min, collect the supernatant, filter with a 0.45 μm pore size Millpore sterile filter membrane, and collect the filtrate.

[0181] 3. Take a culture dish with a diameter of 9 cm, pour 25 mL of melted PDA solid culture medium into it, add 100 μL of the spore suspension of tomato gray mold prepared in step 1 after the culture medium solidifies, spread it evenly with an applicator, then punch holes with a 7 mm diameter sterile puncher (evenly set 3 punching points), pick out the agar block with a sterile toothpick, add the filtrate prepared in step 2 into the hole (inject the filtrate prepared by the three test biocontrol bacteria respectively, and inject 100 μL into each hole), seal it with a parafilm sealing film, and culture it in a constant temperature incubator at 30°C for 72 h.

[0182] Set up three or more repeated treatments and take the average of the results.

[0183] See photos Fig.15(Figure A is an exemplary photo, with numbers 1, 2, and 3 corresponding to Bacillus amyloliquefaciens MH71, Bacillus amyloliquefaciens HTYB-MH71-1, and Bacillus amyloliquefaciens HTYB-MH71-2, respectively; Figure B is the statistical result of the diameter of the inhibition zone). The results showed that the fermentation broth of Bacillus amyloliquefaciens HTYB-MH71-1 had the strongest antibacterial activity against tomato gray mold, which was better than Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2, and there was no significant difference in the antibacterial activity between Bacillus amyloliquefaciens MH71 and Bacillus amyloliquefaciens HTYB-MH71-2.

[0184] 3. Detection of the efficacy of in vitro leaf protection against tomato gray mold

[0185] The tomato variety of the test tomato plants was Hard Powder Champion, and the seeds were purchased from Jingyan Yinong (Beijing) Seed Technology Co., Ltd.

[0186] 1. Obtain fresh and healthy leaves of the same age from the test tomato plants, wash the leaves with running water and let them dry naturally.

[0187] 2. Inoculate a single colony of the test biocontrol bacteria into a modified LB liquid medium (100 mL medium in a 500 mL triangular bottle), shake and culture at 30°C and 180 rpm for 72 hours to obtain the fermentation stock solution (i.e., the entire system containing bacteria and medium). Dilute the fermentation stock solution to 5 times its volume with sterile water to obtain the fermentation dilution solution.

[0188] 3. Use a 7mm sterile puncher to punch holes on the PDA solid culture medium plate cultured with tomato gray mold to obtain pathogenic bacteria cakes.

[0189] 4. Immerse the leaves obtained in step 1 in the fermentation solution or fermentation dilution solution prepared in step 2, soak at room temperature for 20 minutes, then take out and place on a tray covered with 3-5 layers of sterile gauze to dry naturally.

[0190] 5. Stick the pathogenic bacteria cake obtained in step 3 to the center of the leaf obtained in step 4. Soak the sterile gauze on the tray with water, seal the tray with sealing film, pierce holes at multiple locations on the film for ventilation, place it at room temperature for 72 hours, then observe and take pictures, measure the diameter of the lesions, and calculate the disease prevention effect.

[0191] As a positive control, sterile water was used instead of the fermentation stock solution.

[0192] A treatment in which sterile water was used instead of the fermentation stock solution and no pathogenic bacteria cake was added was set up as a blank control.

[0193] At least 5 replicates were performed and the results were averaged.

[0194] Results Fig.16 . Fig.16 In the figure, A corresponds to the fermentation stock solution and B corresponds to the fermentation dilution solution. Fig.16 In the leaf photos, from left to right, the first column corresponds to Bacillus amyloliquefaciens HTYB-MH71-2, the second column corresponds to Bacillus amyloliquefaciens HTYB-MH71-1, the third column corresponds to Bacillus amyloliquefaciens MH71, the fourth column corresponds to the positive control, and the fifth column corresponds to the blank control. The results showed that no lesions appeared on the tomato leaves treated with the fermentation broth of the three strains for 72 hours. Compared with the positive control, the control effect was calculated according to the lesion area, and the control effect was 100%. The results showed that no obvious lesions appeared on the tomato leaves treated with the fermentation dilution of Bacillus amyloliquefaciens HTYB-MH71-1 for 72 hours, and the pathogen used the nutrients of the mycelium block to grow, that is, a little bit of pathogen hyphae occasionally appeared on the mycelium block; after being treated with the fermentation dilution of Bacillus amyloliquefaciens MH71 or the fermentation dilution of Bacillus amyloliquefaciens HTYB-MH71-2 for 72 hours, lesions appeared on the leaves, but were smaller than the positive control; the protection effect was calculated based on the lesion area. The in vitro protection effect of Bacillus amyloliquefaciens HTYB-MH71-1 against tomato gray mold was 100%, while the protection effects of Bacillus amyloliquefaciens HTYB-MH71-2 and Bacillus amyloliquefaciens MH71 were 49.75% and 42.14%, respectively.

[0195] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Bacillus amyloliquefaciens HTYB-MH71-1, whose deposit registration number is CGMCC No.25355.

2. The use of Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1 is as follows (a) or (b): (a) inhibiting the pathogenic bacteria of gray mold; (b) Prevent and control plant gray mold.

3. The culture of Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1.

4. A microbial agent, characterized in that: The bacterial agent contains the Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1 and / or the culture according to claim 3.

5. The use of the culture according to claim 3 or the bacterial agent according to claim 4 is as follows (a) or (b): (a) inhibiting the pathogenic bacteria of gray mold; (b) Prevent and control plant gray mold.

6. Use of the Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1, the culture according to claim 3 or the bacterial agent according to claim 4 in preparing a product; The functions of the product are as follows (a) or (b): (a) inhibiting the pathogenic bacteria of gray mold; (b) Prevent and control plant gray mold.

7. A product comprising the Bacillus amyloliquefaciens HTYB-MH71-1 of claim 1, the culture of claim 3 or the bacterial agent of claim 4; The functions of the product are as follows (a) or (b): (a) inhibiting the pathogenic bacteria of gray mold; (b) Prevent and control plant gray mold.

8. Use of the Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1, the culture according to claim 3 or the bacterial agent according to claim 4 in the preparation of protease and / or cellulase and / or siderophore and / or indoleacetic acid.

9. A product for preparing protease and / or cellulase and / or siderophore and / or indoleacetic acid, comprising the Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1, the culture according to claim 3 or the bacterial agent according to claim 4.

10. A method for preparing the bacterial agent according to claim 4, comprising the step of using the Bacillus amyloliquefaciens HTYB-MH71-1 according to claim 1 or / and the culture according to claim 3 as components of the bacterial agent to obtain the bacterial agent.

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