A strain of Bacillus amyloliquefaciens with biological control and nitrogen fixation functions and its application

Abstract

The present invention relates to the technical field of biological control of plant diseases, and specifically to a strain of Bacillus amyloliquefaciens with biological control and nitrogen fixation functions and its application. Through in vitro plate antibacterial experiments, it was found that Bacillus amyloliquefaciens NBL-AP73 had a good antibacterial effect on the specialized type of tomato neck rot and root rot of Fusarium oxysporum. The control effect of Bacillus amyloliquefaciens NBL-AP73 on tomato neck rot and root rot and the growth-promoting effect on tomato seedlings were determined by potted plant experiments. The results showed that Bacillus amyloliquefaciens NBL-AP73 can effectively prevent and control tomato neck rot and root rot, with a disease index of 10.50 and a control effect of up to 80.26%; at the same time, experiments found that Bacillus amyloliquefaciens NBL-AP73 has a good growth-promoting effect on tomato seedlings. Furthermore, Bacillus amyloliquefaciens NBL-AP73 has a growth-promoting effect on wheat. In addition, Bacillus amyloliquefaciens NBL-AP73 has an excellent nitrogen fixation effect and can be used to prepare products with nitrogen fixation ability.

Description

Technical Field

[0001] The invention relates to the technical field of biological control of plant diseases, and in particular to a Bacillus amyloliquefaciens strain with biological control and nitrogen fixation functions and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Tomato (Solanum esculentum L.) is an important vegetable crop. It is rich in nutrients, can be grown all year round, has high yield, a variety of varieties, and a wide range of uses. It is deeply loved by growers and consumers. However, during the long-term and large-scale production of tomatoes, it will be affected by different pathogens such as viruses, viroids, fungi, oomycetes, bacteria and nematodes, thereby reducing tomato yields.

[0004] Among them, Fusarium crown and root rot (FCRR) is a devastating soil-borne disease caused by Fusarium oxysporum f.sp.radicis-lycopersici (FORL), with a mortality rate of up to 100% in severe cases. The disease was first discovered in Japan in the 1970s and then spread to many countries. It has now become one of the important diseases affecting tomato cultivation, seriously affecting tomato yield and quality.

[0005] At present, the prevention and control of tomato neck rot and root rot mainly rely on biological control and chemical control. Although chemical control is simple, fast and effective, it has poor degradability and is easy to cause environmental pollution. In addition, the long-term use of the same type of chemical agents has made the problem of drug resistance increasingly prominent. Therefore, a green prevention and control measure is urgently needed to ensure the sustainable development of the vegetable industry. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides a Bacillus amyloliquefaciens strain with biological control and nitrogen fixation functions and application thereof.

[0007] The first aspect of the present invention provides a strain of Bacillus amyloliquefaciens with biocontrol and nitrogen fixation functions, named Bacillus amyloliquefaciens NBL-AP73, which was deposited in the China Center for Type Culture Collection on June 19, 2024, with a deposit number of CCTCC NO: M 20241295.

[0008] The 16S rDNA sequence of Bacillus amyloliquefaciens NBL-AP73 is shown in SEQ ID NO.1.

[0009] In the present invention, the morphological characteristics of Bacillus amyloliquefaciens NBL-AP73 are: milky white, smooth, shiny, opaque, raised, irregular and wavy edges, and sticky texture.

[0010] The second aspect of the present invention provides a microbial preparation, wherein the microbial preparation comprises the Bacillus amyloliquefaciens NBL-AP73 described in the first aspect.

[0011] The third aspect of the present invention provides a bacterial suspension, which is the bacterial suspension of Bacillus amyloliquefaciens NBL-AP73 described in the first aspect, wherein the content of Bacillus amyloliquefaciens NBL-AP73 is 1.0×10 5 ~1.0×10 8 CFU / mL; preferably 1.0×10 6 CFU / mL.

[0012] The fourth aspect of the present invention provides the use of Bacillus amyloliquefaciens NBL-AP73 described in the first aspect and / or the microbial preparation described in the second aspect and / or the bacterial suspension described in the third aspect in any of the following:

[0013] (A1) Control tomato crown rot and root rot;

[0014] (A2) Prepare a product for preventing and controlling tomato crown rot and root rot.

[0015] The fifth aspect of the present invention provides the use of Bacillus amyloliquefaciens NBL-AP73 described in the first aspect and / or the microbial preparation described in the second aspect and / or the bacterial suspension described in the third aspect in any of the following:

[0016] (B1) Promote plant growth and development;

[0017] (B2) Preparing products for promoting plant growth and development.

[0018] In one or more embodiments, the plant is a vegetable, fruit or grain, preferably tomato and wheat.

[0019] In one or more embodiments, the promoting plant growth and development includes but is not limited to promoting root development and increasing at least one selected from plant height, fresh weight, root length, root weight, main stem length, main stem thickness and main stem weight.

[0020] The sixth aspect of the present invention provides the use of Bacillus amyloliquefaciens NBL-AP73 described in the first aspect and / or the microbial preparation described in the second aspect and / or the bacterial suspension described in the third aspect in any of the following:

[0021] (C1) inhibiting plant pathogenic fungi;

[0022] (C2) preparing products for inhibiting plant pathogenic fungi;

[0023] (C3) Control of diseases caused by plant pathogenic fungi;

[0024] (C4) Preparation of products for controlling diseases caused by phytopathogenic fungi.

[0025] In one or more embodiments, the plant pathogenic fungus comprises Fusarium oxysporum.

[0026] The seventh aspect of the present invention provides a method for preventing and controlling tomato crown rot and root rot, comprising: applying the Bacillus amyloliquefaciens NBL-AP73 described in the first aspect and / or the microbial preparation described in the second aspect and / or the bacterial suspension described in the third aspect to tomato plants or seeds.

[0027] The application can be carried out in multiple growth stages of tomato seedlings, and the use method is simple. It can be carried out by flushing, pouring or drip irrigation. The application concentration is low, and a single application can maintain a long effective time. The stress resistance and growth promotion effect after application lasts for a long time.

[0028] The eighth aspect of the present invention provides the use of Bacillus amyloliquefaciens NBL-AP73 described in the first aspect and / or the microbial preparation described in the second aspect and / or the bacterial suspension described in the third aspect in any of the following:

[0029] (D1) Nitrogen fixation;

[0030] (D2) Prepare products with nitrogen fixation ability.

[0031] In one or more embodiments, the product having nitrogen fixation ability includes biological fertilizer, soil conditioner and bioremediation agent.

[0032] The beneficial effects of the present invention are:

[0033] Through in vitro plate antibacterial experiments, it was found that the antibacterial ability of Bacillus amyloliquefaciens NBL-AP73 against Fusarium oxysporum tomato neck rot root rot type (FORL) reached 93.38%, thus proving that it has a good antibacterial effect. The pot experiment was used to determine the control effect of Bacillus amyloliquefaciens NBL-AP73 on tomato neck rot and root rot and the growth-promoting effect on tomato seedlings. The results showed that Bacillus amyloliquefaciens NBL-AP73 can effectively control tomato neck rot and root rot, with a disease index of 10.50 and a control effect of 80.26%. At the same time, the experiment found that Bacillus amyloliquefaciens NBL-AP73 has a good growth-promoting effect on tomato seedlings, which can effectively promote plant height increase, fresh weight increase, root length increase, root weight increase, main stem length increase, main stem thickness increase and main stem weight increase. At the same time, through field experiments, it was found that in multiple growth stages of tomato seedlings, flushing or drip irrigation of 1.0×10 6 CFU / mL concentration of bacterial suspension can achieve better results, and the stress resistance and growth promotion effect lasts for a long time after application. Bacillus amyloliquefaciens NBL-AP73 can have a growth-promoting effect on tomato seedlings. Therefore, Bacillus amyloliquefaciens NBL-AP73 has great development potential in the prevention and treatment of tomato neck rot and root rot and the promotion of tomato growth. Furthermore, Bacillus amyloliquefaciens NBL-AP73 has a growth-promoting effect on wheat, and the plant height and root system of wheat treated with Bacillus amyloliquefaciens NBL-AP73 are significantly improved compared with the control group. In addition, Bacillus amyloliquefaciens NBL-AP73 has an excellent nitrogen fixation effect and can be used to prepare products with nitrogen fixation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 This is a colony diagram of the specialized type of tomato neck rot and root rot of Fusarium oxysporum screened in Example 1;

[0036] Figure 2 The detection result of PCR in Example 1;

[0037] Figure 3 This is the colony image of Bacillus amyloliquefaciens NBL-AP73 screened in Example 2;

[0038] Figure 4 The results of the effects of different treatments on tomato neck rot in Example 3 are shown, and each treatment includes an overall picture of the plant and an enlarged picture of the neck and root;

[0039] Figure 5 The results of the wheat growth promotion test of Bacillus amyloliquefaciens NBL-AP73 in Example 4 are shown;

[0040] Figure 6 The results of drug sensitivity test of Bacillus amyloliquefaciens NBL-AP73 and NBL-BQ39 in Example 5, wherein 1 is dimethomorph, 2 is carbendazim, 3 is bromothiocarb·prochloraz, 4 is mancozeb, 5 is zhongshengmycin, 6 is thiophanate-methyl, 7 is oligosaccharide·tetramoxine, 8 is tetramycin·sinophil, 9 is benzyl·prochloraz, 10 is oxamectin, and 11 is flutolan·tebuconazole;

[0041] Figure 7 The results of the effects of the control group and the bacterial agent treatment group on tomato plants in Example 7 are shown. DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0043] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0045] The various culture media involved in the following embodiments include:

[0046] Potato dextrose agar medium (PDA): 6 g potato powder, 20 g agar, 20 g glucose, 1 L distilled water, pH 5.6±0.2, sterilize at 121℃ for 20 min.

[0047] YPDA medium: 10 g yeast extract, 20 g peptone, 20 g glucose, 0.03 g adenine sulfate, 1 L distilled water, natural pH, sterilized at 121°C for 20 min.

[0048] Nutrient broth (NB): peptone 10g, beef extract 3g, sodium chloride 5g, distilled water 1L, pH natural, sterilized at 121℃ for 20min.

[0049] NA medium: peptone 10g, beef extract 3g, sodium chloride 5g, agar 20g, distilled water 1L, pH natural, sterilize at 121℃ for 20min.

[0050] Sodium carboxymethyl cellulose medium: sodium carboxymethyl cellulose 15g, ammonium nitrate 1g, yeast extract 1g, MgSO4 0.5g, KH2PO4 1g, distilled water 1L, pH natural, sterilize at 121℃ for 20min.

[0051] Physiological saline: NaCl 0.9 g, distilled water 100 mL, sterilized at 121°C for 20 min.

[0052] Instruments and equipment:

[0053] Clean bench, constant temperature shaker, oscillator, electric constant temperature incubator, autoclave, electron microscope.

[0054] Test strains:

[0055] Bacillus amyloliquefaciens NBL-BQ13, Bacillus amyloliquefaciens NBL-BQ39, and Bacillus amyloliquefaciens NBL-BQ62 are all from the Culture Collection Center of Shandong Bilan Biotechnology Co., Ltd.

[0056] Fusarium oxysporum was obtained from the strain collection center of Shandong Bilan Biotechnology Co., Ltd.

[0057] Pesticides: dimethomorph, carbendazim, bromothiocarb·prochloraz, mancozeb, zhongshengmycin, thiophanate-methyl, oligosaccharide·streptomycin, cinnamyl·tetramycin, benzyl·prochloraz, oxazolidinone, fluopicolide·tebuconazole, Chinese herbal medicine extracts: oregano extract and spearmint extract, all of which are commercially available products;

[0058] Example 1 Screening of Tomato Neck and Root Rot Specialized Type (FORL) of Fusarium oxysporum

[0059] 1.1 Select tomato plants that have recently developed the disease (tomato neck rot and root rot) as the isolation material, and use the tissue separation method to isolate the pathogens from the tomato neck rot root disease. Wash the surface of the pathogen with sterile water, cut off the epidermal tissue with a sterilized scalpel, take samples at the junction of the disease, cut a small piece of discolored vascular tissue, cut it into small cubes, first soak it in 75% alcohol for 30 seconds, then soak it in 10% sodium hypochlorite for 2 minutes, and finally wash it with sterile water 3 times, the washing time is 4 minutes, 3 minutes, and 2 minutes respectively. After washing, put it on filter paper to dry, take four pieces and evenly stick them on the PDA culture medium, four pieces on each culture medium, and culture at 25℃ for 7 days to grow colonies. Observe the morphology, size and color of the colonies.

[0060] 1.2 Purification: Pick the colonies with lavender morphology for purification until a purified colony with a single morphology is obtained, and store the single colony on PDA slant medium at 4°C for later use.

[0061] 1.3 Morphological characteristics of pathogens: A single colony was streaked onto a PDA solid plate medium, and the plate was inverted in a constant temperature incubator and cultured at 25°C for 7 days. The hyphae were luxuriant, grayish white to white, velvety, and a lavender pigment was produced on the back of the medium. Figure 1 .

[0062] 1.4 The DNA of the pathogen was extracted and amplified by PCR using universal primers ITS1 / ITS4 for fungi and sequenced. The nucleotide sequence was compared and analyzed in the NCBI blast (https: / / blast.Ncbi.Nlm.Nih.gov / Blast.cgi) and LPSN (https: / / bacterio.net / ) databases, and it was confirmed to be Fusarium oxysporum.

[0063] Table 1 Sequencing results

[0064]

[0065]

[0066] 1.5 PCR detection of DNA of seven pathogenic strains using the specific primers FrlDel30F / R of Fusarium oxysporum for tomato neck rot and root rot ( Figure 2 ). A specific fragment can be amplified from the genome of pathogen No. 2, and the sequence is consistent with the genome sequence of Fusarium oxysporum tomato neck rot and root rot specialized type (JH651385.1), which indicates that strain No. 2 belongs to Fusarium oxysporum tomato neck rot and root rot specialized type.

[0067] Example 2 Isolation and Screening of Bacillus amyloliquefaciens NBL-AP73

[0068] 2.1 Soil sample collection: Soil samples were collected from the area where tomato crown and root rot occurred all year round in Dongping County, Tai'an City, Shandong Province, China, stored at 4°C, and transported back to the laboratory.

[0069] Separation and purification: Weigh 10 g of soil sample and add it into a 250 mL Erlenmeyer flask containing 90 mL of sterile water (with glass beads), shake it at 30 °C and 180 r / min for 30 min, and dilute the bacterial suspension to 10 with sterile water. -1 ~10 -7 Each gradient was replicated three times and spread on NA solid agar medium and cultured at 30°C until single colonies grew out. Different single colonies were picked and purified by streaking on the plate. After single colonies with good growth and stable passage appeared on the plate, they were stored on NA slants at 4°C.

[0070] 2.2 Plate confrontation: The purified multiple strains were targeted with the tomato neck rot and root rot specialized type of Fusarium oxysporum (screened in Example 1), and the strains were screened using the plate confrontation method. A bacterial cake of tomato neck rot and root rot specialized type of Fusarium oxysporum with a diameter of 8 mm was inoculated into the center of the PDA plate. After the bacterial cake was placed in the middle of the plate of the treatment group, the bacterial liquid was streaked around 2 cm from the center of the plate. The plate was transferred to a 28°C constant temperature incubator for 7 days. The plate without biocontrol liquid was used as the control. After the colony of the control group covered the plate, the inhibition rate was calculated. Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / control group colony diameter × 100%. After calculation, NBL-AP73 had the highest inhibition rate for tomato neck rot and root rot specialized type of Fusarium oxysporum, with an inhibition capacity of 93.38%.

[0071] 2.3 Morphological characteristics of strains:

[0072] The selected strain (NBL-AP73) is as follows Figure 3 As shown, the morphological characteristics of the colonies are: milky white, smooth, shiny, opaque, raised, with irregular and wavy edges and a viscous texture.

[0073] 2.4 The 16S rDNA gene of strain NBL-AP73 was amplified by PCR and sequenced. According to the comparison analysis of the 16S rDNA nucleotide sequence in the NCBI blast (https: / / blast.Ncbi.Nlm.Nih.gov / Blast.cgi) and LPSN (https: / / bacterio.net / ) databases, the similarity between strain NBL-AP73 and Bacillus amyloliquefaciens was 100%.

[0074] The 16S rDNA sequence of Bacillus amyloliquefaciens NBL-AP73 is shown as SEQ ID NO.1.

[0075] The 16S rDNA sequence of Bacillus amyloliquefaciens NBL-AP73 is:

[0076]

[0077] 2.5 Comparison of antibacterial activity of Bacillus amyloliquefaciens:

[0078] The antibacterial properties of Bacillus amyloliquefaciens NBL-AP73 were compared with NBL-BQ13, NBL-BQ39, and NBL-BQ62. Fusarium oxysporum and Fusarium oxysporum tomato neck rot and root rot specialized type (screened in Example 1) were used as targets, and the antibacterial rates were compared using the plate confrontation method. The antibacterial rates of the three tested Bacillus amyloliquefaciens against Fusarium oxysporum were higher than those against Fusarium oxysporum tomato neck rot and root rot specialized type, and NBL-AP73 had the highest antibacterial rate against Fusarium oxysporum tomato neck rot and root rot specialized type. The results are shown in Table 2.

[0079] Table 2 Antibacterial activity of different strains against Fusarium oxysporum and Fusarium oxysporum tomato neck rot and root rot specialized type

[0080]

[0081] Example 3 Experiment on Biocontrol of Bacillus amyloliquefaciens NBL-AP73 in Potted Plants

[0082] 3.1 Test materials

[0083] The tomato variety used for the test was “Opel Red Lady Tomato”, which is the main local variety in Dongping County, Tai’an City, Shandong Province, China.

[0084] 3.2 Pathogen treatment

[0085] The tomato neck rot and root rot specialized type of Fusarium oxysporum obtained by screening in Example 1 of the present invention was cultured on a PDA plate medium, and then inoculated into a conical flask containing 800 mL of sodium carboxymethyl cellulose medium, and cultured at 25° C. with shaking at 140 r / min for 5 days. The spore production was examined under a microscope. When the spore production in the field of view reached more than 80%, the culture solution was filtered to remove the mycelium, and then centrifuged at 4000 r / min for 10 minutes, the supernatant was discarded, and sterilized distilled water was added to dilute it to 10 7 CFU / mL of inoculum suspension.

[0086] 3.3 Potted plant experiment

[0087] Tomato seedlings were inoculated with Fusarium oxysporum tomato neck rot type using the root immersion method. The specific operation is: after the tomato seedlings grow to 2-3 true leaves, select tomato seedlings with the same growth from the plug tray, gently pull them out, wash the soil on the roots with water, cut off the tip of the main root by about 0.5 cm, and place the roots of the seedlings in a 10-meter-high hole. 7 The seeds were soaked in a FORL spore suspension of CFU / mL for 30 minutes and then transplanted into a sterilized seedling medium.

[0088] After the above-mentioned FORL infection, the seedlings were allowed to grow for 5 days, and 180 tomato seedlings with consistent growth were selected and randomly divided into 6 groups (4 treatment groups and 2 control groups), with 3 parallels in each group and 10 tomato seedlings in each parallel. 6 CFU / mL Bacillus amyloliquefaciens NBL-AP73, NBL-BQ13, NBL-BQ39, NBL-BQ62 bacterial solution was used for irrigation, and each seedling was irrigated with 50mL of bacterial solution; the control group 1 (CK-H2O) was irrigated with water, and the control group 2 (CK-NB) was irrigated with nutrient broth (NB culture medium solution), and each seedling was irrigated with 50mL. After inoculation, they were placed in an indoor environment at 18-22℃ for 16h·d -1 The disease severity scale (DSS) was as follows: Level 0: normal root system; Level 1: slight browning at the base of the main root, normal lateral roots, and normal vascular bundles in longitudinal section of the stem; Level 2: browning at the base of the main root, slight browning at the lateral roots, and browning at the longitudinal section of the stem; Level 3: obvious browning of the main root and lateral roots, lateral roots falling off, and obvious browning of the vascular bundles in longitudinal section of the stem; Level 4: the plant wilts and dies, or the base and root of the stem are severely browned or even rotten, the lateral roots fall off, and the vascular bundles in longitudinal section of the stem are severely browned.

[0089] Disease index (DI) = [Σ(disease level value × number of plants with that disease level)] / (highest disease level value × number of plants surveyed) × 100.

[0090] The control effect = [(disease index of the control group - disease index of the treatment group) / (disease index of the control group)] × 100%.

[0091] The control effects were calculated with control group 1 as the reference.

[0092] 3.4 Determination indicators and methods

[0093] After 4 weeks, the tomato seedlings were pulled out, the disease situation was investigated, and the disease index and incidence were counted. The incidence of the CK-H2O and CK-NB groups was 100%. The control effect of the three strains of Bacillus amyloliquefaciens (NBL-BQ13, NBL-BQ39, NBL-BQ62) was less than 50% and the incidence was slightly higher. The incidence of Bacillus amyloliquefaciens NBL-AP73 was low after treatment, which was 8.33%, and the control effect on Fusarium oxysporum tomato neck rot and root rot reached 80.26%, with significant control effect. See Table 3 and Figure 4 .

[0094] Table 3 Effects of different bacterial agents on tomato neck rot and root rot

[0095]

[0096] Example 4 Experiment on promoting wheat growth by Bacillus amyloliquefaciens NBL-AP73

[0097] In order to verify the growth-promoting effect of Bacillus amyloliquefaciens NBL-AP73 on wheat, a control group and an experimental group were set up, with 3 parallels in each group and 2 pots in each parallel. Fifty wheat seeds were sown in each pot. The control group (CK-H2O) was irrigated with plain water, and the experimental group was irrigated with 10 6 CFU / mL of Bacillus amyloliquefaciens NBL-AP73 fermentation liquid was used for irrigation, and 50 mL was poured into each pot, and normal management was performed in the later stage. Observation was performed after two weeks. The plant height (aboveground part) and root system (underground part) of wheat treated with Bacillus amyloliquefaciens NBL-AP73 increased by 16.34% and 1.66% respectively compared with the control group, indicating that Bacillus amyloliquefaciens NBL-AP73 has obvious plant growth promotion function, see Figure 5 and Table 4.

[0098] Table 4 Effect of bacterial solution on wheat growth

[0099]

[0100]

[0101] Example 5 Antimicrobial Susceptibility Test of Bacillus amyloliquefaciens NBL-AP73 and NBL-BQ39

[0102] Filter paper method: Bacillus amyloliquefaciens NBL-AP73 and NBL-BQ39 were tested for drug sensitivity against 11 pesticides (dimethomorph, carbendazim, bromothiocarb·prochloraz, mancozeb, zhongshengmycin, thiophanate-methyl, oligosaccharide·streptomycin, cinnamyl·tetramycin, benzyl·prochloraz, oxadiazine, flutolanil·tebuconazole) and 2 Chinese herbal medicine extracts (oregano extract and spearmint extract). 100 μL of 10 6 CFU / mL bacterial solution was applied to the plate, a 6 mm diameter filter paper was attached to the NA medium, 20 μL of pesticide and Chinese medicine extract were respectively dripped onto the filter paper, the plate was transferred to a 28°C incubator for 1 day, and the plate was observed for transparent circles. Figure 6 .

[0103] The results showed that Bacillus amyloliquefaciens NBL-AP73 could not be used simultaneously with sinocycline and thiophanate-methyl, but could be used simultaneously with other pesticides it was insensitive to; NBL-BQ39 could not be used simultaneously with mancozeb, sinocycline, thiophanate-methyl and flutolanil, but could be used simultaneously with other pesticides it was insensitive to; Bacillus amyloliquefaciens NBL-AP73 and NBL-BQ9 could be mixed with Chinese herbal medicine extracts; compared with NBL-AP73, NBL-BQ39 was more sensitive to commonly used pesticides, as shown by a larger transparent zone.

[0104] Example 6 Nitrogen fixation experiment of Bacillus amyloliquefaciens NBL-AP73

[0105] Bacillus amyloliquefaciens NBL-BQ13, NBL-BQ39, NBL-BQ62, and NBL-AP73 were added to fresh NB culture medium, respectively, and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The fermentation broth was assayed for enzyme activity using microbial nitrogenase (NITS) ELISA kits (purchased from Baoruyi (Beijing) Biotechnology Co., Ltd., model RE8798-96T). The results are shown in Table 5. The results show that Bacillus amyloliquefaciens NBL-AP73 has the highest nitrogenase activity, reaching 347.78 ng / L. Its application to field crops can promote plants to increase total nitrogen content.

[0106] Table 5 Effects of different strains on plant nitrogenase activity

[0107]

[0108] Example 7 Field Trial of Bacillus amyloliquefaciens NBL-AP73

[0109] Tomato seedlings were inoculated 10 6CFU / mL concentration of Bacillus amyloliquefaciens NBL-AP73 bacterial solution, each seedling was irrigated with 50mL of bacterial solution, and the control group was irrigated with 50mL of water. Three parallels were set up for each group, and each parallel had 60 trees. Watering was done once after the seedlings were grown and once 30 days after transplanting. The growth of tomato seedlings in the field was investigated 40 days after transplanting, and samples were taken to measure the growth indexes and defense indexes (POD enzyme activity, SOD enzyme activity, MDA content) of the seedlings. The malondialdehyde (MDA) content detection kit (colorimetric method) (purchased from Sangon Biotech (Shanghai) Co., Ltd., model D799761-0050); total superoxide dismutase (T-SOD) test kit (hydroxylamine method) (purchased from Nanjing Jiancheng Bioengineering Research Institute, model A001-1-2); peroxidase (POD) determination kit (plant detection) (colorimetric method) (purchased from Nanjing Jiancheng Bioengineering Research Institute, model A084-3-1) were used to determine the enzyme activity of tomato leaves. The results are as follows Figure 7 As shown in Table 6, the results showed that the leaves of tomato seedlings in the control group (CK-H2O) turned yellow and dried, and the defense indicators POD and SOD enzyme activities were low and the MDA content was high; the leaves of tomato seedlings in the treatment group (applied with Bacillus amyloliquefaciens NBL-AP73) were normal, and the defense indicators POD (peroxidase) and SOD (superoxide dismutase) enzyme activities were high, and the MDA (malondialdehyde) content was low. The tomato seedlings applied with the powder containing Bacillus amyloliquefaciens NBL-AP73 were almost not affected by growth stress, and their plant height and stem diameter were significantly increased compared with the control group. Compared with the control group, the tomato seedlings applied with Bacillus amyloliquefaciens NBL-AP73 showed significant differences, indicating that the strain improved the stress resistance and growth promotion ability of tomato seedlings.

[0110] Table 6 Effect of application of fungi on tomato growth

[0111]

[0112] The stress rate is defined as the proportion of tomatoes that show adverse symptoms due to environmental or biological factors. In this experiment, the stress rate specifically refers to the proportion of tomato seedlings with dead leaves to the total number of tomato seedlings. A higher stress rate usually indicates that the plant is more seriously affected.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens, characterized in that It was named Bacillus amyloliquefaciens NBL-AP73 and was deposited in the China Center for Type Culture Collection on June 19, 2024, with the deposit number CCTCC NO: M 20241295.

2. A microbial preparation, characterized in that: The microbial preparation comprises the Bacillus amyloliquefaciens NBL-AP73 according to claim 1.

3. A bacterial suspension, characterized in that: The bacterial suspension is the bacterial suspension of Bacillus amyloliquefaciens NBL-AP73 according to claim 1, wherein the content of Bacillus amyloliquefaciens NBL-AP73 is 1.0×10 5 ~1.0×10 8 CFU / mL.

4. The bacterial suspension as claimed in claim 3, characterized in that The content of Bacillus amyloliquefaciens NBL-AP73 was 1.0×10 6 CFU / mL.

5. Use of the Bacillus amyloliquefaciens NBL-AP73 according to claim 1 and / or the microbial preparation according to claim 2 and / or the bacterial suspension according to claim 3 in any of the following: (A1) Control tomato crown rot and root rot; (A2) Prepare a product for preventing and controlling tomato crown rot and root rot.

6. Use of the Bacillus amyloliquefaciens NBL-AP73 according to claim 1 and / or the microbial preparation according to claim 2 and / or the bacterial suspension according to claim 3 in any of the following: (B1) Promote plant growth and development; (B2) preparing products for promoting plant growth and development; in, The plants are tomato and wheat.

7. Use of the Bacillus amyloliquefaciens NBL-AP73 according to claim 1 and / or the microbial preparation according to claim 2 and / or the bacterial suspension according to claim 3 in any of the following: (C1) inhibiting plant pathogenic fungi; (C2) preparing products for inhibiting plant pathogenic fungi; (C3) Control of diseases caused by plant pathogenic fungi; (C4) Preparation of products for controlling diseases caused by phytopathogenic fungi; in, The plants are tomatoes and wheat; the plant pathogenic fungus is Fusarium oxysporum.

8. A method for preventing and treating tomato crown rot and root rot, characterized in that: include: Apply the Bacillus amyloliquefaciens NBL-AP73 described in claim 1 and / or the microbial preparation described in claim 2 and / or the bacterial suspension described in claim 3 to tomato plants or seeds.

9. Use of the Bacillus amyloliquefaciens NBL-AP73 according to claim 1 and / or the microbial preparation according to claim 2 and / or the bacterial suspension according to claim 3 in any of the following: (D1) Nitrogen fixation; (D2) Prepare products with nitrogen fixation ability.

10. The use according to claim 9, characterized in that: The products with nitrogen fixation ability include biological fertilizers, soil conditioners and bioremediation agents.

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