Bacillus aerobacter, microbial agent and application of bacillus aerobacter and microbial agent

Abstract

The invention belongs to the technical field of microbiology and agricultural application thereof, and particularly relates to bacillus aerosus, a microbial agent and application of the microbial agent. According to the invention, a strain with good antagonism on tobacco black shank and fusarium root rot is separated and screened from tomato roots, and is identified as aerobacillus, and chitinase, cellulase and protease are secreted to dissolve the cell wall structure of pathogenic bacteria hyphae and inhibit the growth of pathogenic bacteria, so that the pathogenic bacteria lose the infection ability, and the pathogenic bacteria are effectively prevented from being infected. The biocontrol microbial inoculant has the antibacterial effect on more than 10 plant pathogenic fungi, the antibacterial rate is greater than 80%, especially the antibacterial rate on phytophthora nicotianae and fusarium oxysporum is greater than 90%, and pot experiments and field experiments prove that the biocontrol microbial inoculant has the control effect on tobacco black shank and fusarium root rot obviously superior to those of commercially available biocontrol microbial inoculants and chemical agents; meanwhile, the tobacco seed germination can be promoted, the growth and development of field tobacco plants are promoted, the subculture stability is better, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology and its agricultural application, and specifically relates to an air bacillus, a microbial agent and application thereof. Background Art

[0002] Tobacco black shank and Fusarium root rot are global soil-borne fungal diseases that restrict tobacco production. They have caused serious economic losses in tobacco-producing countries such as the United States, Australia, India, South Africa, South Korea, Japan, and Canada. They have also caused serious damage to my country's tobacco yield and quality. These two diseases often occur together. Due to their strong infectivity, short disease cycle, and rapid spread, they often become regional devastating diseases. At present, field production mainly adopts methods such as applying chemical pesticides, breeding disease-resistant varieties, and improving tillage and cultivation measures for prevention and control. However, the long-term and large-scale application of chemical agents can easily lead to the risk of resistance of pathogens, reduce the prevention effect, and harm the environment and human health. There are few tobacco varieties that are highly resistant to these two diseases in production, and in tobacco fields with limited arable land and increasing multiple cropping index, relying solely on tillage or cultivation measures to prevent and control diseases has very limited effects. Biological control has become an important means of comprehensive prevention and control of tobacco diseases at home and abroad due to its advantages such as high efficiency and environmental friendliness, and its position in the prevention and control of soil-borne diseases of tobacco rhizomes is becoming increasingly important.

[0003] The current primary task of tobacco biological control is to continuously discover new antagonistic bacteria resources and reveal their mechanism of action. Wang Yayue et al. isolated a strain of Streptomyces violascens from marine sponges, which had a field control effect of 83.5% on tobacco black shank disease. This strain can destroy the cell wall structure of pathogens by secreting proteases and cellulases, thereby achieving an inhibitory effect on pathogens. Liu Tianbo et al. screened out Acinetobacter pituitii. Acinetobater pittiiThe control efficiency against tobacco black shank reached 55.37%, and it had a significant effect on the growth and development of tobacco plant roots. Pseudomonas aeruginosa YZ66 and Pseudomonas chlororaphis YX33 screened from tobacco rhizosphere soil by Qiu Rui et al. can effectively inhibit Fusarium solani and Fusarium oxysporum. Previous studies have focused on the screening of antagonistic strains against tobacco black shank or Fusarium root rot, and there has been no report on the simultaneous prevention and control of these two diseases. Pseudomonas aeruginosa and Pseudomonas granadensis screened by Qian Huimin et al. have good disease prevention effects on tobacco black shank and root black rot; Song Yujuan et al. found that Trichoderma aculeatus T-6 has good control effects on tobacco black shank and root black rot. It shows that screening biocontrol bacteria that can simultaneously prevent and control multiple diseases has become a new strategy for biological control. Tobacco black shank and Fusarium root rot often infect tobacco plants together, and the symptoms are difficult to distinguish. Therefore, there is an urgent need to screen out biocontrol bacteria that can prevent and control these two diseases at the same time. Summary of the invention

[0004] In order to solve the problems existing in the prior art, one of the objects of the present invention is to provide an air Bacillus, which has an antibacterial effect on a variety of pathogens that can cause plant diseases, especially showing good prevention effect on tobacco black shank disease and Fusarium root rot, and can promote tobacco seed germination and field growth, and has both disease prevention and growth promotion effects.

[0005] The second object of the present invention is to provide a microbial agent, comprising the aerobic Bacillus provided by the present invention, which can be used for the dual prevention and treatment of tobacco black shank disease and Fusarium root rot, and promote tobacco seed germination and field growth.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The invention discloses an air bacillus, whose preservation number is CGMCC NO.21278.

[0007] A microbial agent containing the above-mentioned air bacillus.

[0008] The microbial agent is a freeze-dried powder or a liquid preparation; the liquid preparation can be the above-mentioned Bacillus aeruginosa fermentation liquid. In a specific embodiment of the present invention, the fermentation culture temperature of the Bacillus aeruginosa fermentation liquid is 30°C. Specifically, the concentration of Bacillus aeruginosa in the liquid preparation is 1.0×10 7 ~1.0×10 11 cfu / mL; preferably, the concentration of Bacillus aeruginosa in the liquid preparation is 1.0×10 8 ~1.0×10 10cfu / mL.

[0009] The above-mentioned airborne Bacillus and microbial agents have antibacterial effects on a variety of plant pathogenic fungi, and can be used to prevent and control plant diseases caused by plant pathogenic fungi, including Siamese Anthracnose, Fujikura Gibberellum, Pink Trichothecene, Tomato Alternaria, Verticillium, Solanum solani, Fujikura Fusarium, Fusarium solani, Cucumber Fusarium, Mutant Fusarium, Mango Anthracnose, Botrytis cinerea, Tobacco Phytophthora, Tobacco Oxysporum Fusarium, etc.

[0010] The above-mentioned air bacillus and microbial agent can be used to prevent and treat tobacco black stem disease and Fusarium root rot. As a preferred embodiment, the concentration of air bacillus in the microbial agent for preventing and treating tobacco black stem disease and Fusarium root rot is 1.0×10 8 cfu / mL; the above-mentioned air Bacillus and microbial agent can also be used to improve the disease resistance of tobacco plants, improve the germination rate of tobacco seeds and / or the field growth of plants. As a preferred embodiment, the concentration of air Bacillus in the microbial agent for improving the disease resistance of tobacco plants, improving the germination rate of tobacco seeds and / or the field growth of plants is 1.0×10 9 cfu / mL.

[0011] Beneficial effects of the present invention: (1) The aerium bacillus of the present invention is isolated from tomato rhizomes and identified as aerium bacillus (Bacillus aerius) by combining its morphological characteristics, physiological and biochemical properties and 16SrRNA homology analysis. It has a high efficiency and broad-spectrum antibacterial activity against plant pathogens, and the antibacterial rate against 17 plant pathogenic fungi is greater than 80%, among which the antibacterial rate against tobacco phytophthora, oxysporum, Siamese anthracnose, fujikura gibberellosis and pink trichothecene is greater than 90%; (2) The antimicrobial proteins (chitinase, cellulase and protease) derived from the aerobic Bacillus of the present invention can cause mycelial deformity, cell wall dissolution, cytoplasm leakage and loss of infection ability of tobacco Phytophthora and Fusarium oxysporum; pot tests showed that the relative control efficacy against tobacco black shank disease and Fusarium root rot was 63.6% and 82.8% respectively, and the relative control efficacy of field tests reached 72.4% and 75.1% respectively, which was significantly better than commercially available biocontrol agents and chemical agents; (3) The airborne Bacillus of the present invention can also induce the activity of defense enzymes (POD, PAL, PPO) of tobacco plants to increase in a short period of time (3 to 6 days), thereby improving the disease resistance of tobacco plants; (4) The airborne Bacillus of the present invention can promote the germination of tobacco seeds and the growth and development of tobacco plants in the field; (5) The aerobic Bacillus of the present invention has good subculture stability. After 50 subcultures, it still maintains a high antibacterial activity (>87%) against tobacco black shank disease and Fusarium root rot pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 The plate confrontation effect of strain C-1 on Phytophthora nicotianae and Fusarium oxysporum; Figure 2 The colony morphology (A and B) and microscopic morphology (C) of strain C-1; Figure 3 This is the electrophoresis of the 16SrDNA amplification product of strain C-1; Figure 4 This is the phylogenetic diagram of the 16S rDNA sequence of strain C-1 Figure 5 The effect of strain C-1 on pathogens under a scanning electron microscope; a. Normal Fusarium oxysporum hyphae; b. Fusarium oxysporum hyphae treated with the fermentation liquid of strain C-1; c. Normal Phytophthora nicotianae hyphae; d. Phytophthora nicotianae hyphae treated with the fermentation liquid of strain C-1; Figure 6 The effects of different concentrations of C-1 strain fermentation broth on the activities of POD, PAL and PPO enzymes in tobacco leaves; Figure 7 The effect of different concentrations of C-1 strain fermentation liquid on tobacco seed germination; Figure 8 The effects of different concentrations of C-1 strain fermentation liquid on the soluble protein content and total amylase activity during tobacco seed germination; Fig. 9 The inhibitory effect of strain C-1 on 12 pathogenic bacteria. DETAILED DESCRIPTION

[0014] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0015] The tobacco variety used in the following embodiments and test examples is "Zhongyan 100"; the pathogen of tobacco black shank disease (Phytophthora parasitica var. nicotianae) and the pathogen of tobacco fusarium root rot disease (Fusarium oxysporum) were isolated by the inventor's research group from tobacco black shank disease and fusarium root rot disease-infected tobacco plants in Pingdingshan tobacco fields.

[0016] Example 1 Screening and identification of target antagonistic bacteria 1.1. Isolation and screening of strains: The tissue homogenate method was used to isolate and purify the endophytic bacteria in tomato roots. Twenty strains were isolated from five tomato root samples. The purified single colonies were preserved by the slant method and the glycerol method for later use. 1.2. Antagonistic bacteria inhibition spectrum detection by plate confrontation method: 1 cm bacterial cakes of tobacco phytophthora and Fusarium oxysporum were inoculated in the center of PDA medium, and the fermentation broth of the isolated strains (cell concentration 1.0×10 8 cfu / mL) were inoculated around the pathogen in a triangle shape at an equal distance of 2 cm from the center of the plate. The plate inoculated with only pathogens was used as the control. The plates were cultured in a constant temperature incubator at 30°C for 5 days. Each treatment was repeated 3 times. When the control colony grew close to the edge of the plate, the diameter of the pathogen colony and the diameter of the inhibition zone were measured. Calculation formula: Antibacterial diameter = diameter of pathogen colonies in the control group - diameter of pathogen colonies in the treatment group Inhibition rate = [(colony diameter of control group - colony diameter of treatment group) ( / colony diameter of control group - diameter of bacterial cake)] × 100% A strain with good antagonistic effect on tobacco phytophthora and Fusarium oxysporum was screened and recorded as C-1, as shown in Table 1 and Figure 1 As shown in the figure, the antibacterial diameter of the bacteria against Phytophthora nicotianae and Fusarium oxysporum reached 72 mm, and the inhibition rate was greater than 90%, and the plate confrontation antibacterial effect was better.

[0017]

[0018] 1.3. Identification of strains: 1.3.1 Colony morphology and microscopic morphology observation Use NA solid medium to streak the C-1 colony and culture it at 30℃ constant temperature and humidity for 2-3 days to form a single colony. Observe its colony morphology, such as Figure 2 As shown, the colony of strain C-1 is beige in color, with pale edges that are milky white, flat and irregular in shape, with scattered petal-shaped edges, moist surface with wrinkled concave protrusions in the center, rough and opaque surface, with spores that are mesogenetic or eccentric, oval, and slightly swollen. Gram staining is positive, and microscopic examination revealed that the bacteria are rod-shaped.

[0019] 1.3.2 Physiological and biochemical characteristics were determined. The results are shown in Table 2:

[0020] The results shown in Table 2 indicate that the C-1 strain can liquefy gelatin, hydrolyze casein, starch, urea, cellulose, reduce nitrate, and is positive in oxidase, methyl red, and VP tests, but negative in lipase, tryptophan decomposition, and catalase tests, and can utilize carbon sources such as glucose, citric acid, and sucrose.

[0021] According to the Bergey's Manual of Bacterial Identification, combined with the morphological characteristics of C-1 colonies, it was preliminarily determined to be Bacillus ( Bacillus ).

[0022] 1.3.3 Molecular biological identification of strains: The genomic DNA of strain C-1 was extracted using a bacterial genome extraction kit, and PCR reaction was performed using bacterial 16S rDNA universal primers 27F and 1492R. The amplified products were subjected to agarose gel electrophoresis. Figure 3 As shown in the figure, the length of the 16S rDNA gene sequence of strain C-1 is 1434 bp; the 16S rDNA gene sequence of strain C-1 was sent to Tianjin Jinweizhi Biotechnology Co., Ltd. for sequence determination. The base pair sequences obtained after the determination were compared for similarity using BLAST software, and the eight sequences with the highest homology to strain C-1 were selected in turn. Then, the Neighbor-Joining method of MEGA7.0 was used to construct a phylogenetic tree, as shown in Figure 4 As shown in the figure, after 16S rDNA sequence comparison by Blast, it was found that strain C-1 was similar to Bacillus aeruginosa ( Bacillus aerius ) are in the same branch and have the highest homology.

[0023] Therefore, combined with the morphological characteristics, physiological and biochemical properties and 16SrDNA homology analysis, strain C-1 was identified as Bacillus aeruginosa ( Bacillus aerius ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 2, 2020, with the deposit number CGMCC NO.21278.

[0024] The strain C-1 was expanded and cultured to obtain the fermentation broth for subsequent experiments. The specific conditions for the expansion culture were as follows: the seed solution was inoculated into a 500 mL shake flask (200 mL liquid NA / flask) at a ratio of 1:100, and cultured at 30°C and 200 r / min for 3-5 days.

[0025] Example 2 Identification of the biocontrol effect and antibacterial mechanism of strain C-1 2.1 Analysis of the biocontrol potential of tobacco black shank and Fusarium root rot: C-1 bacterial solution was used to irrigate the roots of tobacco seedlings to determine its potted and field control effects: The potted plant experiment was conducted in four treatments: (1) CK (blank control): only the pathogen was inoculated; (2) C-1 treatment: the fermentation broth of the expanded C-1 strain was diluted to prepare a fermentation broth (cell concentration was about 1.0×10 8 cfu / mL), 20 mL was irrigated to each pot by root irrigation; (3) Wangtaibao treatment: Wangtaibao (Dajing Biological, effective bacteria: Trichoderma harzianum, commercially available) (biocontrol agent control) solution concentration was 1%, 65 mL was irrigated to each tobacco plant; (4) Metalaxyl-Mancozeb treatment: 58% Metalaxyl-Mancozeb wettable powder (Jiangsu Baoling Chemical Co., Ltd., metalaxyl content: 10%, mancozeb content: 48%, chemical agent control, commercially available) was diluted 600 times, 20 mL was irrigated to each plant. Each treatment was inoculated with 20 mL of tobacco oxysporum or tobacco phytophthora fermentation liquid 7 days after the first application of the fungicide or pesticide, and then the fungicide or pesticide of each treatment was irrigated once every 7 days, for a total of 3 times. Each treatment had 8 plants, 3 replicates, and the disease situation was investigated 14 days after inoculation.

[0026] The field test was conducted in Bailongmiao Village, Pingdingshan City. Except for CK, which was a natural disease, the other treatments were the same as potted plants. Each treatment had 50 plants and was repeated 3 times. The tobacco plants were transplanted on May 1, 2022. After the seedlings were returned, each treatment was applied at 10 times the volume of the potted plants. The roots were irrigated every 15 days for a total of 3 times. The disease situation was investigated 15 days after the last root irrigation. The rest of the measures were carried out according to the production of high-quality tobacco leaves.

[0027] The disease index grading standards for tobacco black shank and Fusarium root rot refer to the "Tobacco Industry Standard of the People's Republic of China" Tobacco Disease Grading and Investigation Methods (GB / T23222-2008, GB / T23224-2008).

[0028] Calculation formula: Incidence rate = (number of diseased plants / total number of plants investigated) × 100% Disease index = ∑ (number of diseased plants at each level × value of the disease level) / (total number of plants surveyed × highest level value) × 100 Relative prevention effect = (control disease index - treatment disease index) / control disease index × 100% The results of potted and field control tests are shown in Table 3:

[0029] The results of the pot test showed that the incidence of tobacco black shank in the control CK inoculated with pathogens was 91.7%, and the incidence of Fusarium root rot was 100%, indicating that the inoculation of pathogens was relatively successful, and the corresponding disease indexes were 57.3 and 89.3, respectively. The incidence of tobacco black shank and Fusarium root rot under the treatment of the commercially available biocontrol agent Wangtaibao was 75.0% and 100%, the disease indexes were 44.8 and 78.6, and the relative control efficiency was 21.7% and 11.9%, respectively; the incidence of chemical agent metaloxam mancozeb was 54.2% and 66.7%, the disease indexes were 33.1 and 42.9, and the relative control efficiency was 42.1% and 51.9%, respectively. The incidence of C-1 treatment was 29.2% and 20.8%, the disease indexes were 20.8, and 15.3, and the relative control efficiency was 63.6% and 82.8%, respectively. The incidence and disease index of the two diseases under the three treatments of commercially available biocontrol agents, chemical agents and C-1 were generally lower than those of the control, and all had certain relative prevention effects. The differences among the treatments were significant. The incidence and disease index of the two diseases treated with strain C-1 were the lowest, and the relative prevention effect was also significantly higher than that of other treatments.

[0030] The results of field tests showed that the incidence rates of tobacco black shank and Fusarium root rot in the naturally occurring CK treatment were 12.0% and 32.7% respectively; the incidence rates of the two diseases in the treatment of the commercially available biocontrol agent Wangtaibao were 7.3% and 16.7% respectively, the disease indexes were 6.2 and 11.0 respectively, and the relative control effects were 38.4% and 31.6% respectively; the incidence rates of the two diseases in the treatment of the chemical agent formosan frost were 6.7% and 10.0% respectively, the disease indexes were 6.0 and 7.3 respectively, and the relative control effects were 38.4% and 53.1% respectively; the incidence rates of the two diseases in the treatment of C-1 were 4.0% and 5.3% respectively, the disease indexes were 2.8 and 4.0 respectively, and the relative control effects reached 72.4% and 75.1% respectively; Compared with the control, commercially available biocontrol agents, chemical agents and C-1 treatments in field trials significantly reduced the incidence and disease index of tobacco black shank and Fusarium root rot, and all had a certain relative preventive effect, especially C-1, which had the best preventive effect. The relative preventive effects of the two diseases in field trials were both higher than 70%.

[0031] 2.2 Determination of antibacterial mechanism of antagonistic strains 2.2.1 Determination of cell wall degrading enzyme activity of antagonistic strains The supernatant filtrate of the fermentation broth of strain C-1 (the whole bacterial solution was centrifuged at 12000 rpm for 5 min, and the supernatant was filtered) and the fermentation broth bacterial cell fragments (100 mL of the whole bacterial solution was collected, centrifuged at 4°C, 6500 rpm for 10 min, 5 mL of 50 mmol / L pH7.5 KH2PO4-K2HPO4 buffer solution was added, and ultrasonic fragments were performed) were collected respectively. The activities of antimicrobial proteins (protease, chitinase and cellulase) in the supernatant filtrate of the fermentation broth of strain C-1 and the bacterial cell fragments were determined respectively: the cellulase content was determined by the cellulase activity detection kit (Shanghai Yuanxin Biotechnology, YX-SH-TQ22025), the chitinase content was determined by the chitinase activity detection kit (Shanghai Yuanxin Biotechnology, YX-W-B917), and the protease content was determined by the protease activity detection kit (Wuhan Merck Biological Company, KT59429).

[0032] The results are shown in Table 4: protease, chitinase and cellulase activities were detected in the supernatant filtrate and cell disrupted liquid of the fermentation broth of strain C-1, and there was no significant difference in the activity of the three cell wall degrading enzymes between the two treatments.

[0033]

[0034] 2.2.2 Scanning electron microscopy observation of inhibited pathogen hyphae Scanning electron microscope was used to observe Fusarium oxysporum, Phytophthora nicotianae and pathogens treated with C-1 fermentation broth. Figure 5 As shown: from Figure 5 Figure a shows that the normal hyphae of Fusarium oxysporum are tubular, with a smooth velvety surface and no branches. Figure 5 As shown in Figure b, after being treated with C-1 solution, its growth and spore production were significantly inhibited, and the mycelium branches of the inhibited pathogen increased, the mycelium was entangled, and the top was swollen. C-1 parasitized inside the mycelium of Fusarium oxysporum and secreted substances that inhibited the growth of the pathogen, causing it to shrivel and eventually die.

[0035] from Figure 5 As shown in Figure c, the mycelium of the control tobacco phytophthora grows normally, has a full shape, and has smooth cell walls. Figure 5 As can be seen from Figure d, after antagonistic treatment with the fermentation broth of strain C-1, the growth and spore production of Phytophthora were significantly inhibited. The surface of the mycelium of the inhibited pathogen shrank, became concave, shrunken, twisted and deformed, and part of the mycelium wall collapsed, eventually losing the ability to infect.

[0036] 2.2.3 Effects of strain C-1 on defense enzyme activities in tobacco seedlings In a potted experiment, 30 days after transplanting, the C-1 fermentation broth (cell concentration of about 1.0×108 cfu / mL), 10 times (cell concentration 1.0×10 9 cfu / mL), 100 times (cell concentration 1.0×10 10 cfu / mL) fermentation liquid, and water treatment was used as control (CK). Each treatment was repeated 10 times. The third leaf was taken at 0, 3, 6, and 9 days after inoculation to determine its defense enzyme activity.

[0037] Peroxidase (POD) was determined by the guaiacol method; polyphenoloxidase (PPO) was determined by the catechol method; phenylalanine ammonia lyase (PAL) was determined by the L-phenylalanine method.

[0038] The results are as follows Figure 6 As shown in Figure a, after inoculation with different concentrations of C-1 strain fermentation broth, the POD (peroxidase) activity in the tobacco plants showed a trend of first slightly decreasing on the 3rd day after inoculation, significantly increasing on the 6th day, and then decreasing again; Figures b and c show that the PAL (phenylalanine ammonia lyase) and PPO (polyphenol oxidase) activities showed a trend of steadily increasing from 0d to 6d after inoculation, and then significantly decreasing.

[0039] Compared with the control, after inoculation with different concentrations of fermentation broth of C-1 strain, its POD activity increased with the increase of C-1 fermentation broth concentration, especially on the 6th and 9th days after inoculation, the difference was significant; its PAL activity showed an overall trend of increasing with the increase of fermentation broth concentration, and on the 9th day, it was significantly lower than CK after inoculation with inoculation broth; while PPO activity was significantly higher than CK after inoculation with C-1 stock solution and 10 times concentration of fermentation broth, but lowered with 100 times concentration of fermentation broth. This indicates that inoculation with C-1 fermentation broth will induce the increase of defense enzymes (POD, PAL, PPO) of tobacco plants in a short period of time (3d to 6d), and the cell concentration is 1.0×10 9 cfu / mL is better.

[0040] Example 3 Evaluation of the feasibility of strain C-1 in promoting tobacco seed germination and field tobacco plant growth and development 3.1 Effects on tobacco seed germination Tobacco seeds were immersed in sterile culture dishes using clean water (CK), fermentation solution of strain C-1, 10-fold and 100-fold concentrated fermentation solutions, respectively, and cultured in a constant temperature incubator at 25°C. The germination rate was counted every 12 hours starting from the 3rd day (the germination standard was that the root length was equal to the seed length or the sprout length was equal to 1 / 2 of the seed length) and recorded until the 6th day.

[0041] The results are as follows Figure 7As shown in the figure, after soaking in water (CK), C-1 stock solution, 10-fold and 100-fold fermentation solution, the germination rates of tobacco seeds were 52.67%, 60.00%, 61.33% and 59.00% on the 4th day, respectively. Compared with the water control, different concentrations of C-1 fermentation solution had a significant promoting effect on tobacco seed germination. From the 4th to the 6th day after soaking, the 10-fold fermentation solution concentration (cell concentration 1.0×10 9 cfu / mL) had the highest germination rate, followed by the fermentation broth (cell concentration 1.0×10 8 cfu / mL).

[0042] 3.2 Effects on soluble protein content and total amylase activity during tobacco seed germination In the above seed germination test, 0.5 g of germinated seeds were taken on the 9th day after tobacco seeds germinated, 5 mL of phosphate buffer (pH 6.9) was added, and the mixture was thoroughly ground and allowed to stand for 30 min. The homogenate was then transferred to a centrifuge tube and centrifuged at 4 °C and 12000 r / min for 10 min. All supernatants were collected for later use. The soluble protein content was detected by the Coomassie brilliant blue method, and the amylase activity was determined by the 3,5-dinitrosalicylic acid method.

[0043] The results are as follows Figure 8 As shown in the figure, after tobacco seeds were soaked in water (CK), C-1 stock solution, 10 times and 100 times fermentation solution, the soluble protein content in the seeds was 7.32, 9.25, 10.70 and 8.01 mg / g respectively ( Figure 8 ). The soluble protein content of the treatments with different concentrations of C-1 strain fermentation liquid was higher than that of the control CK, and the difference between the 10-fold and original liquid treatments was significant. After soaking, the amylase activity in tobacco seeds increased with the increase of fermentation liquid concentration, and there was a significant difference between the treatments with different concentrations of fermentation liquid.

[0044] 3.3 Effects on tobacco agronomic traits At maturity, three tobacco plants were randomly selected from each treatment group in Example 2, and their plant height, stem girth, internode distance, number of effective leaves, maximum leaf length, leaf width, leaf area, fresh leaf weight, root weight and root length were measured. The results are shown in Table 5:

[0045] Compared with the control, the plant height, stem girth, internode distance, leaf length of the largest leaf, leaf width, leaf area, number of effective leaves, fresh leaf weight, root length, and root weight of C-1 treatment were significantly increased. Except for stem girth, the agronomic traits of C-1 treatment were also increased compared with those of microbial agent Wangtaibao and chemical agent mancozeb.

[0046] Example 4 Determination of the stability of subculture of strain C-1 Strain C-1 was subcultured on NA medium, transferred once every 48 hours, and subcultured 50 times. The plate confrontation test was used to determine the antibacterial effects of the primary strain and the subculture strain on tobacco phytophthora and Fusarium oxysporum. The effect of subculture on the antibacterial effect of antagonistic bacteria was analyzed based on the change in the width of the inhibition zone. The results are shown in Table 6:

[0047] As shown in Table 6, after 1, 5, 10, and 50 generations, the inhibition rates of strain C-1 against Phytophthora nicotianae were 90.55%, 87.97%, 89.45%, and 89.07%, respectively; and the inhibition rates against Fusarium oxysporum were 90.15%, 89.67%, 90.13%, and 89.24%, respectively. The inhibition rates of strain C-1 against the two pathogens were stable after different generations of culture, showing obvious differences.

[0048] Example 5 Determination of the antibacterial spectrum of strain C-1 The plate confrontation method was used to detect the antibacterial effect of strain C-1 on other plant pathogenic fungi. Five plates were used for each treatment and the experiment was repeated three times. The results are as follows: Fig. 9 As shown in Table 7 below:

[0049] The results showed that strain C-1 had a significant inhibitory effect on the growth of 12 pathogenic fungi ( Fig. 9 ), the inhibition rates of Colletotrichum siamese, Gibberella fujikura, Trichothecene pink, Alternaria tomato, Fusarium rotundifolia, Fusarium solani, Fusarium fujikura, Fusarium solani, Fusarium fujikura, Fusarium solani, Fusarium ovale, Fusarium variegata, Fusarium ovale, Fusarium variegata, Colletotrichum mangoes and Botrytis cinerea were 93.99%, 93.69%, 90.39%, 89.50%, 87.69%, 86.18%, 85.59%, 84.98%, 84.08%, 82.89%, 82.28% and 81.68% respectively (Table 7), and the antibacterial effect was obvious. The results show that the strain C-1 screened by the present invention is an efficient and broad-spectrum antagonistic bacterium, and has great potential application value in the prevention and control of plant diseases. In addition to having a dual preventive effect on tobacco black shank and Fusarium root rot, it also has potential application value in the prevention and control of strawberry stem base rot, anthracnose; strawberry root rot, corn ear rot and stalk ear disease, rice seedling blight, sugarcane tip rot; corn ear rot, wheat fusarium scurf, melon pink mildew / powdery mildew, tomato pink mildew, cucumber pink mildew, mushroom pink mildew, apple heart rot, mango fruit rot; tomato early blight; corn ear disease and stem rot, tomato fruit rot; tobacco Fusarium root rot, tomato Fusarium fruit rot, zucchini Fusarium fruit rot, sweet potato brown spot; strawberry root rot; eggplant blossom rot; cucumber Fusarium wilt; sweet potato stem rot; mango anthracnose and gray mold diseases of various crops.

[0050] The characterization results of the above-mentioned embodiment show that the air Bacillus C-1 separated from the tomato root of the present invention, and its derived antimicrobial proteins (chitinase, cellulase and protease) can cause the mycelium deformity of tobacco phytophthora and Fusarium oxysporum, cell wall dissolution, cytoplasm leakage, and loss of infection ability, and can show good prevention effect on both tobacco black shank disease and Fusarium root rot. Its field prevention effect is greater than 70%, which can increase the activity of defense enzymes such as PPO, POD and PAL in tobacco plants, induce tobacco plants to produce disease resistance, and promote tobacco seed germination and field growth, and has both disease prevention and growth promotion effects. In addition, the strain can effectively inhibit a variety of crop diseases, has good high-efficiency and broad-spectrum antibacterial properties, is a very potential disease prevention and growth promotion bacterium, and its good subculture stability can be widely used in biocontrol preparations.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An air bacillus, characterized in that Its accession number is CGMCC NO.21278.

2. A microbial agent containing the aerobic bacillus as claimed in claim 1.

3. The microbial agent according to claim 2, characterized in that The microbial agent is freeze-dried powder.

4. The microbial agent according to claim 2, characterized in that The microbial agent is a liquid preparation.

5. The microbial agent according to claim 4, characterized in that The microbial agent is the fermentation liquid of the aerobic Bacillus strain as claimed in claim 1.

6. The microbial agent according to claim 5, characterized in that The concentration of Bacillus aeruginosa in the microbial agent is 1.0×10 7 ~1.0×10 11 cfu / mL.

7. The microbial agent according to claim 6, characterized in that The concentration of Bacillus aeruginosa in the microbial agent is 1.0×10 8 ~1.0×10 10 cfu / mL.

8. Use of the air bacillus according to claim 1 or the microbial agent according to any one of claims 2 to 7 in preventing and controlling plant diseases caused by plant pathogenic fungi, characterized in that: The plant pathogenic fungi are selected from any one or more of Siamese Anthracnose, Fujikura Gibberella, Pink Trichothecene, Alternaria alternata, Verticillium fusariotii, Fusarium solani, Fujikura Fusarium, Fusarium solani, Cucumber Fusarium, Fusarium variegata, Mango Anthracnose, Botrytis cinerea, Phytophthora nicotianae, and Fusarium oxysporum.

9. The use according to claim 8, characterized in that The plant diseases are tobacco black stem disease and Fusarium root rot; Optionally, the concentration of Bacillus aeruginosa in the microbial agent is 1.0×10 8 cfu / mL.

10. Use of the air bacillus according to claim 1 or the microbial agent according to any one of claims 2 to 7 in improving tobacco plant disease resistance, tobacco seed germination rate and / or plant growth; Optionally, the concentration of Bacillus aeruginosa in the microbial agent is 1.0×10 9 cfu / mL.

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