CN117165498A - Bacillus belicus and application thereof - Google Patents

Abstract

The invention provides bacillus beleiensis which is named asBacillus velezensis QN ₃ NO-3, wherein the preservation number is CCTCC NO: m2021304. The bacillus belicus and the fermentation liquor thereof and the strain volatile matters generated in the culture process have broad-spectrum antibacterial activity and have good antagonism on tomato fusarium wilt and the like. The bacterial strain has antibacterial and other biological properties, can reduce the dependence on pesticides, improve the yield and quality of crops, reduce the negative influence on the environment, and has wide application in the fields of agriculture and environmentIs used for the application of the composition.

Description

Bacillus belicus and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to bacillus beijerinus and application thereof.

Background

Nitrogen (N) in NPK fertilizers is one of the vital nutrients in agricultural production, which is a basic chemical element necessary for vital activities of plants and animals. However, farmland soil is generally deficient in nitrogen. Increasing the nitrogen content in organic and chemical fertilizers to increase the productivity of the ecosystem is an effective way to achieve yield-increasing benefits. However, excessive nitrogen application can cause environmental pollution, not only can reduce the utilization efficiency of nitrogen, but also can negatively affect the quality of water and atmospheric environment, and threaten the balance of animal and plant ecological systems and human health. China is one of countries with the largest consumption of chemical fertilizer nitrogen, and the use of a large amount of nitrogen fertilizer promotes the development of agriculture in China, but also causes the problems of water eutrophication and the like. As the population grows and demand for agricultural products increases, fertilizer nitrogen consumption will continue to increase. Therefore, the utilization efficiency of nitrogen is improved, excessive use of fertilizer nitrogen is avoided, adverse effects on environmental quality (such as soil hardening and the like) are prevented, economic benefit and environmental benefit are taken into consideration, and the method has become the basic principle and important task of agricultural nitrogen management. The method for cultivating the nitrogen fertility of the soil is actively explored while ensuring the yield of crops so as to realize sustainable development of agriculture.

Microorganisms are the oldest organisms on the earth, have the history of at least 41 hundred million years, exist in soil, water bodies, air, animal and plant bodies and other environments in different forms, physiological characteristics and metabolic capacities, and the diversity of the earth microorganisms such as bacteria, fungi, viruses and the like enriches a microorganism resource library and provides precious resources for scientific research and application in the fields of agriculture, medicine, environment and the like. Nitrogen-fixing bacteria, which are a special class of bacteria, play an important role in microorganisms on earth as a group of bacteria capable of converting nitrogen (N ₂ ) Conversion to plant-available nitrogen compounds, e.g. ammonia (NH) ₃ ) And Nitrate (NO) ₃ ^- ) Is critical to the nitrogen circulation of the earth's ecosystem. Nitrogen is an important element of living essential substances such as proteins and nucleic acids synthesized by living organisms, but most living organisms cannot directly utilize nitrogen in the atmosphere. The nitrogen fixing bacteria convert nitrogen into a form available for other organisms through nitrogen fixing effect, so that the healthy development of biodiversity and an ecological system is promoted. Nitrogen-fixing bacteria widely exist in soil, water, plant rhizosphere and other environments, and form symbiotic relation with plants, for example, leguminous plant rhizobium is combined with root systems of leguminous plants to jointly promote nitrogen fixation and plant growth. In addition, the nitrogen fixing bacteria can also live together with other microorganisms to form a complex microbial community, and participate in the key ecological processes of soil organic matter decomposition, nutrition circulation and the like. Classifying and physiology of nitrogen-fixing bacteriaThe researches on characteristics, nitrogen fixation mechanisms, interaction with plants and other microorganisms and the like are helpful for revealing the nitrogen circulation mechanism of the earth ecological system, improving the agricultural production efficiency and promoting sustainable development. However, research on nitrogen-fixing bacteria has also faced challenges such as excavation and rational utilization of nitrogen-fixing bacteria resources, development and utilization of nitrogen-fixing bacteria, improvement of nitrogen-fixing efficiency, and influence of environmental factors on nitrogen-fixing effect. Accordingly, further research is still required to make continuous efforts to fully exert an important role of nitrogen-fixing bacteria in microorganisms of the earth.

Endophytes are considered to be microorganisms which at least live in healthy plants at a certain stage or even in the whole life cycle and do not cause obvious diseases to the plants, and are attracting attention since the first mention of K1eopper et al in 1992, the variety diversity determines wide application in the fields of biological control, medicine, forestry and the like, and under the double impact that the strengthening of agriculture sustainable development concept and chemical control are difficult to work, the microorganisms have the advantages of wide sources, large quantity, low cost and the like, so that the development space is wider and wider, including soil fertility improvement, soil restoration, crop yield and quality improvement, crop stress resistance improvement and the like are obtained. After entering soil, the microorganisms with antagonistic function establish a competition mechanism with plant pathogenic bacteria, occupy ecological sites, compete for nutrients, oxygen and the like, or inhibit the pathogenic bacteria in a parasitic and dissolved mode, induce the crops to generate resistance, and reduce the incidence rate. The previous reports that antagonizing the metabolites produced by the strain during the metabolic process can improve the disease resistance of plants or through destroying the pathogenic bacterial cell structure, the mycelium is fissionally distorted and the infection ability is lost. Zeng Yibo through research on development of growth-promoting and disease-resistant functions of photosynthetic bacteria PSB06, it is found that extracellular proteins produced by bacterial strain metabolism can effectively inhibit rice blast, anming et al isolate a biocontrol strain Y-1 resistant to Petasites in dendrobium stem, and determine the action mechanism of polypeptide metabolites thereof for inhibiting energy production and amino acid biosynthesis in pathogenic bacteria respiratory chain through proteomics research. Bacillus bailii has certain advantages in controlling crop diseases as a biological pesticide, can produce a series of antibacterial substances such as antibiotics, enzymes, antibacterial peptides and the like, and has an inhibiting effect on various plant pathogenic bacteria. The bacillus belicus biological pesticide product can be excavated and developed, so that the use amount of chemical pesticides can be effectively reduced, the pollution to the environment and the harm to the human health can be reduced, and the crop yield and quality can be improved. However, for specific applications, it is also necessary to select suitable Bacillus belicus strains and modes of application according to different crops and diseases.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides bacillus belicus and application thereof, and the bacillus belicus and the application thereof have broad-spectrum antibacterial activity, have good antagonism on various germs, have high-efficiency nitrogen fixation, IAA (IAA) production and cellulose decomposition capabilities, can improve seed germination rate, remarkably and early dew Bai Qi and the like, and have wide development space and application prospect.

In a first aspect, the present invention provides Bacillus bailii, designated asBacillus velezensisQN ₃ NO-3 (hereinafter also referred to as Bacillus bailii QN) ₃ NO-3), registration and preservation in China center for type culture collection (CCTCC NO): m2021304.

In a second aspect the present invention provides a fermentation broth of Bacillus belicus or a supernatant of a fermentation broth or a crude ammonium sulfate protein of a fermentation broth according to the first aspect of the present invention.

In a third aspect, the invention provides a strain volatiles produced by sealed culture of Bacillus bailii according to the first aspect of the invention.

In a fourth aspect the invention provides the use of Bacillus belicus according to the first aspect of the invention, or of Bacillus clarkii according to the second aspect of the invention, or of ammonium sulphate crude protein of the fermentation broth or of the supernatant of the fermentation broth or of the volatiles of the strain according to the third aspect of the invention, for antagonising tomato fusarium wilt and/or banana fusarium wilt No. 4 physiological race and/or banana fusarium wilt No. 1 physiological race and/or cucumber fusarium wilt and/or pepper fusarium anthracnose and/or strawberry fusarium anthracnose and/or litchi fusarium anthracnose and/or banana fusarium anthracnose and/or vegetable heart anthracnose and/or colletotrichum gloeosporum and/or corn curvularia leaf spot and/or corn alternaria leaf spot and/or wheat scab and/or wheat fusarium, and/or rice blast and/or mango fusarium graminearum.

In a fifth aspect, the invention provides the use of bacillus besii according to the first aspect of the invention, or of a crude ammonium sulphate protein of a fermentation broth or a supernatant of a fermentation broth or a crude ammonium sulphate protein of a fermentation broth according to the second aspect of the invention, or of a strain volatiles according to the third aspect of the invention, for controlling tomato fusarium wilt and/or banana fusarium wilt No. 4 physiological race and/or banana fusarium wilt No. 1 physiological race and/or cucumber fusarium wilt and/or pepper fusarium anthracnose and/or strawberry fusarium anthracnose and/or litchi fusarium anthracnose and/or banana fusarium anthracnose and/or vegetable heart anthracnose and/or gelatin spore anthracnose and/or corn curvularia leaf spot and/or corn alternaria leaf blight and/or wheat scab and/or wheat fusarium, and/or rice blast and/or mango fusarium pseudoalternifolium.

The cell wall component of pathogenic fungi mainly consists of chitin and glucan, and bacillus bailii QN of the invention ₃ NO-3 can produce chitinase and beta-1, 3-glucanase in different culture periods, and can damage fungal cell walls. Accordingly, in a sixth aspect the invention provides the use of Bacillus belicus according to the first aspect of the invention, or a fermentation broth or supernatant of a fermentation broth according to the second aspect of the invention, for disrupting fungal cell walls.

The bacillus besii and the fermentation liquor thereof and the strain volatiles generated by fermentation have broad-spectrum antibacterial activity, have good inhibition effects on tomato fusarium wilt, banana fusarium wilt No. 4 physiological micro-species, banana fusarium wilt No. 1 physiological micro-species, cucumber fusarium wilt, pepper anthracnose pathogen, strawberry anthracnose pathogen, litchi anthracnose pathogen, banana anthracnose pathogen, colletotrichum gloeosporioides, corn curvularia leaf spot, corn alternaria leaf spot, wheat gibberella, trichoderma mangiferum, amaranthus spinosus leaf spot, rice blast, mango fusarium wilt and the like, have high nitrogen fixation and IAA production capacity, and can promote plant growth, such as effectively promote seed germination, promote seed to enter a white period in advance, promote germ radicle growth, increase plant fresh weight, promote plant crown and root system growth, plant lateral root number and drought resistance of plants. The strain also has activities of producing cellulase (endoglucanase, exoglucanase, beta-glucosidase, filter paper cellulase and the like), chitinase, beta-1, 3-glucanase and the like, and can efficiently degrade cellulose and destroy fungal cell walls. The strain provided by the invention has special biological characteristics, exerts antibacterial and nitrogen fixation capabilities and other biological characteristics, can reduce the dependence on pesticides and fertilizers, improves the yield and quality of crops, reduces the negative influence on the environment, and has wide application potential in the fields of agriculture and environment.

Drawings

FIG. 1 is a QN ₃ NO-3 cell scanning electron microscope results and gram staining results.

FIG. 2 shows strain QN ₃ NO-3 partial physiological biochemical experiments.

FIG. 3 shows strain QN ₃ NO-3 IAA production ability measurement results.

FIG. 4 is an IAA content calibration curve.

FIG. 5 shows strain QN ₃ NO-3 cellulolytic ability assay (Image J screen shot) results.

FIG. 6 shows strain QN ₃ And (5) measuring the cellulase activity in the NO-3 fermentation liquid.

FIG. 7 shows strain QN ₃ Germination of corn seeds after NO-3 treatment. Left diagram: germination conditions of 1-3 d; right figure: and sprouting condition on the third day.

FIG. 8 shows the bacteriostatic activity of volatiles from the strain measured by the double dish-on-button method.

FIG. 9 shows strain QN ₃ Antagonism of tomato fusarium wilt by NO-3 fermentation supernatant and ammonium sulfate crude protein.

FIG. 10 shows strain QN ₃ NO-3 fermentation supernatant was assayed for chitinase and beta-1, 3-glucanase activity.

FIG. 11 shows strain QN ₃ Results of NO-3 broad-spectrum antibacterial activity study.

Detailed Description

The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1. Material

1.1 sample Source and handling

The fruits of the Nornii living plant in the bridgehead Zhennoni plantation (N19 DEG 58 '9', E109 DEG 55 '19') in Chengmai county, hainan province were subjected to multipoint sampling, bagging and numbering, and were stored at 4 ℃.

1.2 Culture medium

LB medium: yeast powder 5g, tryptone 10g, sodium chloride l 0g, agar 20g, distilled water 1L and pH 7.2-7.5;

ababetes medium: glucose 10g, potassium dihydrogen phosphate 0.2 g, sodium chloride 0.2 g, magnesium sulfate 0.2 g, calcium sulfate 0.1 g, calcium carbonate 5g, agar 20g, distilled water 1L, and pH 7.0-7.2;

cellulosic congo red medium: microcrystalline cellulose 1.88 g, congo red 1 g, gelatin 2.0 g, agar 20g, distilled water 1L, pH 7.0.

1.3 Main reagent

DNS reagent: weighing 10g of 3, 5-dinitrosalicylic acid, dissolving in 500ml of distilled water, adding 20g of sodium hydroxide and 200g of potassium sodium tartrate, heating and dissolving the solution, adding 2g of phenol and 0.5 g of anhydrous sodium sulfite, cooling to room temperature after the solute is dissolved, and fixing the volume to 1000ml. The reagent is stored in a brown volumetric flask, and is filtered for use after being placed for one week;

CMC substrate solution: 0.625 g of carboxymethyl cellulose sodium salt is weighed and dissolved in 100ml of sodium acetate buffer solution, and the solution is heated and stirred until the sodium salt is dissolved;

microcrystalline cellulose substrate solution: 0.5 g of microcrystalline cellulose is weighed and dissolved in 100ml of sodium acetate buffer solution;

filter paper substrate solution: 0.5 g of filter paper is weighed and immersed in 100ml of sodium acetate buffer solution;

standard glucose solution: 27.0 g of glucose was dissolved in 25ml of distilled water to prepare a glucose solution of 6 mol/L. A glucose solution standard curve was prepared according to table 1;

1.10 mol/L sodium hydroxide: 400 g analytical grade sodium hydroxide is weighed and dissolved in 500ml distilled water, and then the volume is fixed to 1L;

2% boric acid-indicator solution: weighing 20g boric acid solution 1000mL distilled water, then adding methyl red-bromocresol green mixed indicator 5mL, and adjusting to reddish purple by dilute acid or dilute alkali, wherein the reagent is ready to be prepared;

gao Xiaokai a nitrogen-fixing catalyst tablet.

1.4 Test crop

The test material is corn, the variety is Meiyu No. 16, and the sweet glutinous variety is provided by Hainan LvChuan seedling limited company.

1.5 Data processing

Gray scale area is measured and calculated by Image J (Version 1.38) software, microsoft Excel 2007 software and single factor analysis of variance experimental data are adopted, and the Duncan new complex polar difference test method (DMRT method) is adopted for difference significance analysis.

2 experimental methods and results

2.1 endophytic bacteria isolation and screening

Sample sterilization: and (5) cleaning and air-drying the noni fruits for later use. Sterilizing with 75% ethanol for 5min, treating with sodium hypochlorite in dark place for 20 min, taking out, soaking in 10% sodium bicarbonate for 10min, washing with sterile water for 5 times, air drying on sterilized filter paper, and labeling.

Sample blotting experiments: samples were placed in LB medium for 5min by tissue blotting and incubated at 28℃for 3d for sterility testing.

Sample grinding: the sample was sliced and ground to paste in a sterile mortar, each juice 1 mL was taken in a centrifuge tube, 4mL of LB medium was added, and 1 h was incubated at room temperature 180 r/min, vortexing.

And (3) separating and screening: gradient dilution with sterile Water (10 ^-1 、10 ^-2 、10 ^-3 ) Making into suspension, shaking, uniformly inoculating 200 μl of the diluted solution on Abbe's disease culture medium by plate coating method, repeating for 3 times, inverting dark box at 28deg.C, culturing for 5d, selecting different bacterial colonies with good morphology and growth vigor and rapid growth speed, and purifying on new solid Abbe's disease culture medium by inoculating loop plate streaking method for multiple times to pure culture.

The method comprises the steps of sterilizing a noni fruit sample to prepare a suspension, primarily separating and purifying the suspension to 71 endophytic bacteria by a dilution plate method, selecting 5 dominant bacteria for nitrogen fixation and growth promoting function rescreening (nitrogen fixation function rescreening refers to '2.2 nitrogen fixation capacity identification and activity determination', growth promoting function rescreening refers to '2.6 biological activity influence of the strain on corn growth'), selecting 1 strain with the best effect as a research object, and naming QN ₃ NO-3. The colony was irregularly round, huang Huise, opaque, smooth-surfaced with wrinkles, edge waving curves, central ridges, oval or short bars of cells, gram-stained purplish-red positive bacteria on LB medium (fig. 1). The colonies tend to dry flat and have irregular edge folds due to prolonged incubation time.

2.2 Physiological and biochemical identification of strains

And carrying out physiological and biochemical identification on the screened antagonistic strains according to the Berger's bacteria identification manual and the common bacteria system identification manual.

(1) Single carbon source utilization experiment: in the physiological and biochemical identification of bacteria, the utilization condition of the strain on a carbon source is an important index. The carbon source is utilized differently by different bacteria. The single carbon source utilization experiment is to add a single carbon source into a basic culture medium of Prague at a concentration of 1%, then add the strain to be identified, culture for one to two weeks at 28 ℃, add the strain on the basic culture medium without adding any carbon source as a blank control, and observe the growth condition of the strain on each culture medium. If the strain can grow, the strain is positive, which indicates that the strain has the ability to utilize the carbon source, and if the strain can not grow, the strain is negative, which indicates that the strain does not have the ability to utilize the carbon source. The carbon source includes: alpha lactose, D-fructose, D-galactose, D-mannitol, glucose, mannitol, anhydrous lactose, soluble starch, trehalose, sorbitol, inositol, melezitose, rhamnose, xylose, maltose, sucrose, melibiose, and the like.

(2) Single nitrogen source utilization experiment: the nitrogen source utilization conditions of different bacteria are different from the carbon source utilization conditions. Adding a single nitrogen source into a nitrogen source basic culture medium according to the concentration of 0.5%, preparing a flat plate, then inoculating the strain, inoculating the strain onto the basic culture medium without adding any nitrogen source as a blank control, culturing at 28 ℃ for one to two weeks, and observing the growth condition of the strain. If the strain can grow, the strain is positive, which indicates that the strain has the energy of utilizing the nitrogen source; if growth is not possible, it is negative, indicating that the ability to utilize the nitrogen source is not available. The nitrogen sources added include: tryptone, asparagine, valine, histidine, oxalic acid, ammonium sulfate, ammonium acetate, ammonium chloride, ammonium molybdate tetrahydrate, nitric acid, phenylalanine, glycine, cysteine, amino acid, arginine, serine, tryptophan, methionine and tyrosine.

(3) Starch hydrolysis experiments: the nutrient agar medium is taken as a basic medium, and 1% of soluble starch is added on the basis. Antagonistic strains were inoculated onto formulated plates by spot grafting (i.e., inoculation diameter < 0.5. 0.5 cm), and after incubation at 28℃for one week, iodine was added dropwise around the colonies. If a transparent circle exists around the colony, the result is positive, the strain is capable of producing amylase, and the diameter of the transparent circle indicates the activity of the produced amylase; if amylase is not produced, the whole plate will display blue color.

(4) Gelatin liquefaction experiments: the strain to be identified was inoculated into a tube containing gelatin medium, then cultured at 28℃and observed for gelatin liquefaction at 5, 10, 20, 30 d. If the strain has the liquefaction phenomenon, the strain is positive, which indicates that the strain has the ability of liquefying gelatin, otherwise, the strain is negative.

(5) Cellulose decomposition experiment: preparing a cellulose decomposition culture medium according to a formula, immersing a section of the filter paper strip in a liquid culture medium, sterilizing the culture medium, inoculating a strain in the culture medium, standing for culturing, observing whether the filter paper strip is decomposed or not after one month, if so, determining that the cellulose decomposition enzyme is generated, otherwise, performing negative.

(6) Nitrate reduction: nitrate reduction culture medium is prepared according to the formula, strains are inoculated into the culture medium, and are subjected to static culture at 28 ℃ for 7 d and 14 d, and the culture medium without the strains is used as a blank control. Taking blank test tubes, adding a small amount of 7 d culture solution and 14 culture solution d culture solution into different test tubes respectively, and simultaneously dripping the solution A and the solution B prepared before. If the solution appears pink, rose red, brown or orange, the nitrate is reduced to positive; if no color appears, 1 or 2 drops of diphenylamine reagent are added dropwise, and the solution is blue, the solution is still negative, otherwise, the solution is treated positively.

(7) Urease experiment: preparing a urease culture medium according to a formula, inoculating a strain to be tested on the urease culture medium, culturing for 4 days in an inverted mode at 28 ℃, and observing the color change of the culture medium. If the strain to be tested has the capability of producing urease, the culture medium becomes pink, positive, does not change color, and negative.

(8) Lipase (tween 20, 40, 80) experiment: after the lipase medium was formulated, the individually encapsulated tween 20, 40, 80 was sterilized together and the tween was mixed with the medium to plate. The strain was inoculated on a plate, cultured for one to two weeks, and the plate was observed, and if a faint halo around the colony was generated, it was positive, whereas the other was negative.

(9) Salt tolerance experiments: media with different NaCl concentrations (1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%) were prepared, the other components of the media were the same, strains to be identified were inoculated on these media, cultured at 28℃and observed once a week for four weeks, and finally whether the strains were grown on the media was recorded to determine the upper and lower limit concentrations of the tolerance of the strains to be identified to NaCl.

(10) pH tolerance experiment: the pH values of the liquid culture medium are respectively adjusted to 4, 5, 6, 7, 8, 9 and 10, strains to be detected are respectively inoculated into the culture mediums with different pH values, other culture conditions are consistent, the culture is carried out under shaking at 28 ℃, the culture is observed once a week for four weeks, and finally the growth condition of the strains on each culture medium is determined. And determining the upper limit and the lower limit of the pH value at which the strain can grow and the optimal pH value.

The experimental measurement results show that QN ₃ NO-3 reacts positively in starch hydrolysis, gelatin liquefaction, urease assay, enzymatic assay; only sensitive to tween 40 in the enzyme-lipid experiments; no reaction occurs in the test of methyl red, malonic acid, phosphate reduction and hydrogen sulfide, and the test is negative. Can grow in the pH range of 4-10, and the optimal growth environment is pH9; the NaCl can grow in the concentration of 1% -9% in the salt tolerance experiment, and 11% -15% has no growth.

According to the experimental result of carbon and nitrogen source utilization, QN ₃ The NO-3 can be carbon sources such as alpha-lactose, D-fructose, D-galactose, D-mannitol, glucose, mannitol, anhydrous lactose, soluble starch, trehalose, sorbitol, inositol, melezitose, rhamnose, xylose, maltose, sucrose, and melibiose; less nitrogen sources such as tryptone, asparagine, valine, histidine, and oxalic acid are available, and ammonium sulfate, ammonium acetate, ammonium chloride, ammonium molybdate tetrahydrate, nitric acid, phenylalanine, glycine, cysteine, amino acid, arginine, serine, tryptophan, methionine, and tyrosine are not available.

Preliminary identification of the bacteria asBacillus velezensisQN ₃ NO-3 (China center for type culture Collection, wuhan, CCTCC NO: M2021304, 2021, 3 months and 30 days).

Table 1 QN ₃ Physiological and biochemical characterization of NO-3 strain

2.3 Nitrogen fixation capacity identification and activity determination

The strain to be tested is selected into an Ashby nitrogen-free solid culture medium by using a sterilized toothpick, and the strain is passaged for 6 generations, so that the strain can grow well to show that the strain has nitrogen fixation capability and can not grow to show that the strain does not have nitrogen fixation capability.

The method comprises the steps of determining the nitrogen fixation activity of a bacterial strain by using a Kjeldahl nitrogen fixation method, selecting dominant nitrogen fixation bacteria by using an inoculating loop, fermenting and culturing in LB culture solution for 2 days, centrifuging the fermentation solution at 4 ℃ for 10min at 10000rmp, removing supernatant, placing the bacterial strain in a mortar, quickly grinding the bacterial strain by using liquid nitrogen, weighing 1 g at the bottom of a dry digestion tube, adding a small amount of water for wetting, and adding 5mL concentrated sulfuric acid and 2 efficient Kjeldahl nitrogen fixation catalyst tablets. And (3) performing digestion on the digestion tube until the solution in the digestion tube turns grey and white and green, and then continuing digestion for a period of time. The digestion tube is taken down. And measuring the nitrogen content in each digested culture solution by using a full-automatic Kjeldahl apparatus.

The nitrogen content in the bacterial suspension is measured by using a Kjeltec 8200 Kjeltec azotometer, under the action of a catalyst, organic nitrogen is converted into inorganic ammonium salt by using concentrated sulfuric acid to digest a sample, then the ammonium salt is converted into ammonia under an alkaline condition, the ammonia is distilled out along with water vapor and absorbed by excessive boric acid liquid, and then standard hydrochloric acid is used for titration, so that the nitrogen content in the sample is 152.75 mg/L and the protein content is 954.69 mg (protein content=nitrogen content/16%), which indicates that the bacterial strain has high-efficiency nitrogen fixation capability.

2.4 IAA-producing Capacity identification and Activity determination

Preparing liquid LB containing 100 mg/L alpha-aminoindolyl propionic acid, inoculating to strain to be tested, culturing at room temperature 180 r/min for 2d, sucking 50 μl of cultured bacterial suspension on a white drip plate, and setting 2 groups in comparison: 50 mu L of non-inoculated LB is used as a negative control, 50 mu L of IAA liquid with the concentration of 50 mu g/mL is used as a positive control, 50 mu L of Salkowski colorimetric liquid is added simultaneously, and the mixture is kept stand for 15min at normal temperature in a dark place, so that the color is not changed to be negative, and IAA is not produced; when the color turns pink, the IAA bacteria can be used for measuring the IAA content.

Preparing IAA mother solution (100 μg/mL), respectively preparing IAA solutions with constant volume concentrations of 0, 10, 20, 30, 40, 50, 60 μg/mL in 25mL constant volume bottle, and measuring OD with ultraviolet spectrophotometer _530nm And drawing an IAA standard curve. Placing the bacterial suspension in a centrifuge tube, centrifuging at an initial rotation speed of 12000 r/min for 10min, removing precipitate, collecting 4mL supernatant, mixing with Salkowski colorimetric solution of equal volume, and determining by the same method without adding supernatant as blankThe IAA yield of the strain was calculated on the basis of the IAA standard curve.

According to the Salkowski colorimetric assay (FIG. 3), strain QN ₃ The NO-3 mixed solution and the IAA positive control group are pink, and the non-inoculated negative control group has NO color change, which indicates the strain QN ₃ NO-3 can secrete IAA. IAA (FIG. 4) was plotted using the measurement results of the ultraviolet spectrophotometer to calculate QN ₃ NO-3 secreted IAA in the LB medium containing L-tryptophan in an amount of 40.61.+ -. 0.43. Mu.g/mL.

2.5 identification of cellulolytic Capacity and measurement of cellulase Activity

Bacterial samples were streaked onto cellulose Congo red medium with an inoculating loop, incubated at 28℃for 2-3d, and after colony growth, three replicates were set by observing whether a transparent loop was produced around the colonies of the medium. The generation of transparent circles indicates that the strain can produce the cellulase, and the cellulase activity is primarily judged according to the size of the transparent circles. After photographing and acquisition, using Image J software to calculate the gray scale area (pix) and colony Area (AR) of the irregular transparent ring on the flat plate, solving the ratio of the gray scale area (pix) and the colony Area (AR), calculating the AR/AR ratio, and preliminarily reflecting the activity of the strain for producing the fibror cellulose enzyme. And performing experimental data conversion and analysis by using Excel 2007 and analysis of variance software.

Calculating a ratio formula:

ratio (AR/AR) =colony area (pix)/transparent circle area (pix).

Based on the plate streaking observations (FIG. 5), a large area transparent circle was created on the cellulosic Congo red medium, demonstrating QN ₃ NO-3 can produce the hydrolytic cellulase. The average value of irregular transparent ring Area (AR) on the plate was 12690.33 pix, the average value of colony Area (AR) was 823.33 pix, and the ratio AR/AR was 15.41, as determined by Image J (Version 1.38) software. The strain has stronger cellulase activity and can be identified and subjected to subsequent experiments.

The bacterial suspension (10) was cultured for 2d ⁸ CFU/mL) was inoculated into 2mL of a 250mL flask containing 100mL of CMC liquid medium, and cultured at 28℃with shaking at 150r/min for 2 days. The fermentation broth was centrifuged at 10000rmp for 5 minutes at 4℃and the supernatant was used as enzyme extract. By measuringDetermining the amount of reducing sugar released from microcrystalline cellulose (MCC), carboxymethylcellulose (CMC) and Salicylic Acid (SAL), and determining the activities of exoglucanase, endoglucanase and beta-glucosidase. The total cellulase activity was expressed as filter paper cellulase activity (FPA) and determined by the filter paper assay described in Ghose (1987) using Whatman No. 1 filter paper as substrate. Cellulase activity was determined according to the dinitrosalicylic acid (DNS) method at 540nm and monosaccharide analysis was performed. D-glucose solutions of different concentrations were used as calibration curves. All samples were measured at 540nm with an ultraviolet-visible spectrophotometer. The unit of enzyme activity (U) is defined as the amount of enzyme that releases 1. Mu. Mol (. Beta. -glucosidase to 2. Mu. Mol) of reducing sugar (in terms of glucose) per ml of fermentation supernatant per minute.

The result of the cellulase activity measurement is shown in FIG. 6, after 2d of culture, the exoglucanase activity is 61.81 IU/mL, the endoglucanase activity is 47.11 IU/mL, the beta-glucosidase activity is 53.31 IU/mL, and the filter paper cellulase activity is 29.47 IU/mL, which indicates QN ₃ The NO-3 strain has good enzyme activities of endoglucanase, exoglucanase, beta-glucosidase and the like, which shows that the strain can efficiently degrade cellulose.

2.6 Biological Activity Effect of strains on maize growth

The corn seeds are placed in a refrigerator for vernalization at 4 ℃ for 3 days, taken out and placed in 10% (v/v) sodium hypochlorite for surface sterilization for 15min, and repeatedly washed with sterile water for 5 times. Sterilized corn seeds were suspended in a bacterial suspension (. Times.10) ^-1 ) Soaking for 60 min, placing in a culture dish with sterilized filter paper, culturing in dark at 28deg.C for 3 days, standing for germination, and counting germination rate. Transferring into a transparent incubator until 7 days, collecting the whole seedling, slowly washing filter paper attachments in clear water, recording the lengths and fresh weights of roots and overground parts, and calculating seedling growth indexes (overground fresh weight g, underground fresh weight g, germ length cm, radicle length cm, lateral root quantity pcs and root crown ratio). The control group was culture broth without inoculated strain, and each treatment was repeated 3 times with 10 corn seeds each.

Germination rate= (number of germinated seeds/total number of seeds) ×100%; root cap ratio = root fresh weight/aerial fresh weight.

Corn seeds are subjected to bacterial suspension (10) ^-1 ) Treatment, using culture medium as control, after 3 days of culture, the germination rate of seeds is 93.99% and 77.78%, respectively, and 93.99% of seeds are exposed in the first-day fungus suspension culture treatment group, and the control group is only 66.67%; QN the next day ₃ The NO-3 bacterial suspension promotes the growth and development of corn seed radicle, the embryo is also rapidly expanded, and 80% of seeds are exposed and white in the treatment of the culture medium. The two treatments were significantly different, indicating that the strain can bring the corn seeds into the white-out period in advance, and the stability is good, and the germination accelerating effect is achieved on the corn (figure 7).

The quality of maize seedlings greatly influences the later growth and yield, and the biological activity of the robust seedlings is generally stronger. Corn seed inoculation bacillus beleiensis QN ₃ After NO-3 grows to 7 days, the biological activity index of corn seedlings is measured, and the result shows that QN inoculation is carried out ₃ Compared with the unvaccinated control group, the corn seedlings of NO-3 have remarkable promotion effect on the length and fresh weight of germs and radicle, and have a certain increment effect on the quantity of contralateral roots. QN (quality control N) ₃ The NO-3 strain promotes the fresh weight of the corn seedlings to be increased by 35.29 percent and 80 percent respectively compared with the fresh weight of the non-inoculated seedlings on the ground and the fresh weight of the corn seedlings on the underground, the root cap ratio is also increased from 0.60 to 0.80, the root growth of the underground part of the corn seedlings is obviously promoted, and the growth quantity of the overground part is further promoted. Strain treated group germ 6.04 cm was 2.22 cm longer than the media treatment; the average length of radicle is 11.45 and cm, which is 1.56 times of the culture medium group, and the functional activity of root system is obviously enhanced. Lateral roots play an important role in drought tolerance of corn, the number of lateral roots in the seedling stage culture medium group is 8.07, and the number of lateral roots after strain treatment is 8.3. The above results indicate that QN ₃ NO-3 can promote the growth and development of corn seedlings and plays an important role in root system growth.

2.7 Method for measuring broad-spectrum antibacterial activity of strain by plate counter culture

Test pathogenic bacteria: tomato fusarium wiltF. oxysporum f.sp.lycopersicNo. 4 physiological race of banana fusarium wiltF. oxysporum f. sp. cubenseRace 4 (ATCC 76255), no. 1 physiological Race of banana fusarium wiltFusarium oxysporum SchlechtF.sp.cube Race 1 (ATCC MYA-3244), cucumberFusarium wilt germFusarium oxysporum sp. Cucumebrium(ACCC 30220), pepper anthracnose germColletotrichum capsici (ATCC 96157), strawberry anthracnoseColletotrichum fragariae(ATCC 58718) lychee anthracnose pathogenColletotrichum gloeosporioides(ATCC 58691), banana anthracnoseColletotrichum musae(ATCC 96167), colletotrichum gloeosporioidesColletotrichum higginsianum(KACC 40807), colletotrichum gloeosporioidesColletotrichum gloeosporioides(ATCC 58222), curvularia lunata leaf spotCurvularia lunata(ATCC 60937), alternaria zeaeAlternaria tenuissima(ATCC 26276), wheat scabFusarium graminearumSchwabe (ATCC MYA-4620), chestnut blight germCryphonectraa paraiticaAprio cercospora amabilis (Linnaeus) LinnaeusPestalotiopsis mangiferae(MTCC 3412), pyricularia oryzaeMagnaporthe grisea(ATCC 208987) and mango anthracnose pathogenColletotrichum gloeosporioides（ATCC MYA-4131）。

Preparing a PDA culture medium plate, taking down a pathogenic bacteria cake (phi= mm) which is cultured for 5d and has good growth vigor by using a puncher, placing the plate in the center of the PDA culture medium plate, drawing a cross by taking the center of the plate as the center, inoculating a tested strain at a position 2.5 cm away from the center on 4 sides of the cross, inoculating 3 repeats at each 4-dish, taking only pathogenic bacteria as a control, inversely culturing at 28 ℃ for 5-7 d, and calculating the bacteriostasis rate, wherein the bacteriostasis rate is = (control colony diameter-treated colony diameter)/control colony diameter multiplied by 100%. The results are shown in Table 2 and FIG. 11, strain QN ₃ NO-3 has broad-spectrum resistance to 17 host crops/plant pathogenic bacteria such as banana, tomato, mango, rubber, rice and the like.

Table 2 results of broad-spectrum antibacterial Activity study of strains

Sequence number	Chinese name of germ	Latin name	Antibacterial rate
				1	Tomato fusarium wilt	Fusarium oxysporum (Schl. ) f.sp.lycopersici(Sacc. ) Snyder et Hansen.	61.18%
2	No. 4 physiological race of banana vascular wilt	Fusarium oxysporum Schlecht.f.sp.cubense(E.F.Sm.) Snyd.et Hans,Foc 4	64.12%
				3	No. 1 physiological race of banana vascular wilt	Fusarium oxysporum Schlecht.f.sp.cubense(E.F.Sm.) Snyd.et Hans,Foc 1	55.29%
4	Cucumber fusarium wilt	Fusarium oxysporum(Schl.)F.sp.cucumerinum Owen.	61.76%
				5	Chilli anthracnose germ	Colletotrichum capsici	82.35%
6	Strawberry anthracnose pathogen	Colletotrichum fragariaeBrooks	62.94%
				7	Colletotrichum gloeosporioides (L.) DC	Colletotrichum gloeosporioidesPenz.	68.82%
8	Bacillus anthracis (Roxb.) kurz	Colletotrichum musaeBerk.et Curt.	72.35%
				9	Colletotrichum glomeratum (L.) Kuntze	Colletotrichum higginsianumSacc.	63.53%
10	Colletotrichum gloeosporioides	Colletotrichum gloeosporioidesPenz.	64.12%
				11	Curvularia lunata leaf spot of corn	Curvularia lunata(Wakker) Boed	70.59%
12	Alternaria alternata leaf blight of corn	Alternaria tenuissima	65.88%
				13	Wheat scab fungus	FusaHum graminearum Sehw	65.88%
14	Chestnut epidemic disease germ	Cryphonectraa paraitica	77.65%
				15	Aprio cercospora mandshurica of mangiferum	Pestalotiopsis mangiferae(P.Henn.) Steyaert	74.71%
16	Rice blast fungus	Magnaporthe grisea(Hebert) Barr	81.76%
				17	Mango anthracnose pathogen	Colletotrichum gloeosporioidesPenz.	72.35%

2.8 Double dish buckling method for measuring antibacterial activity of bacterial strain volatile matter

The method for measuring the bacteriostatic activity of the volatile bacteriostatic substances of the microbial sources uses a double-dish buckling method. Will QN ₃ The NO-3 strain was streaked on LB medium and incubated 2d in an incubator at 28 ℃. Then respectively inoculating the tomato fusarium wilt and the banana fusarium wilt No. four physiological small seed cakes (phi=5 mm) to the central positions of the PDA plates, and respectively growing with the bacterial strain QN for 2 days ₃ NO-3 is buckled, two flat plates are sealed by sealing films, and the flat plates buckled with an empty LB culture dish are used as a comparison. Incubation was continued in an incubator at 28℃for 7 d. Repeated 3 times. Measurement of QN ₃ The NO-3 strain can release volatile substances to inhibit growth of tomato fusarium wilt and banana fusarium wilt, and the diameter of pathogenic bacteria is measured by using imageJ software.

Antibacterial ratio = (control colony diameter-treated colony diameter)/control colony diameter x 100%

The bacteriostasis rates are 49.22% and 42.16% respectively.

2.9 Investigation of supernatant and crude proteins of Strain fermentation broths

（1）QN ₃ Antagonism determination of NO-3 fermentation liquor supernatant and ammonium sulfate crude protein on tomato fusarium wilt

The bacterial suspension (10) was cultured for 2d ⁸ CFU/mL) was inoculated into 2mL of a 250mL flask containing 100mL of CMC liquid medium, and cultured at 28℃with shaking at 150r/min for 2 days. Centrifuging the fermentation broth at 4deg.C with 10000rmp for 5min, and collecting supernatant as QN ₃ And (3) determining the bacteriostasis rate of the supernatant of the NO-3 fermentation liquor.

And (3) utilizing an ammonium sulfate fractional precipitation method, centrifuging 1L of fermentation liquor stock solution in batches at a high speed in a centrifuge with a centrifugation condition of 4 ℃ and 12000 r/min and 30min, discarding the precipitate, slowly adding corresponding amounts of ammonium sulfate to the final concentration of 20%, 30%, 50%, 70% and 80% of solution in batches to obtain supernatant, stirring until the supernatant is clear, and standing the supernatant in a refrigerator with the temperature of 4 ℃ to precipitate 24 h. After being split-packed by a 50mL centrifuge tube, the mixture is placed in a centrifuge for centrifugation at 12000 r/min for 30min at 4 ℃ and the precipitate is collected. Adding a small amount of 10 mmol/L PBS buffer solution into each stage of precipitate in a beaker until the precipitate is dissolved, placing the solution into a dialysis bag added with the PBS buffer solution with the same concentration, standing overnight at 4 ℃, sterilizing the solution after dialysis and desalination by using a filter to obtain a fermentation broth crude protein solution, and measuring the bacteriostasis rate.

And (3) antibacterial rate measurement: respectively measuring QN by using sterile water as a reference and adopting a plate perforation bacteriostasis test ₃ Antibacterial activity of NO-3 fermentation broth supernatant and fermentation broth crude protein liquid. The PDA plates were punched with a punch having a hole diameter of 7 mm at the center distance of the plate from the edge 25 mm, 100. Mu.L of each treatment liquid was added to each hole, each treatment was repeated 3 times, and pathogenic fungi (tomato blight bacteria) were added at the center position of the plate. After the control plate is cultivated at normal temperature and is full of fungi, QN is measured by using imageJ software ₃ Pathogen diameter of NO-3 broth supernatant and crude protein solution of different concentration gradients, antibacterial rate = (control colony diameter-treated colony diameter)/control colony diameter x 100%.

The results are shown in FIG. 9. QN (quality control N) ₃ The NO-3 supernatant can antagonize tomato fusarium wilt, the bacteriostasis rate is 49.11 percent, and the ammonium sulfate crude proteins with different concentrations have obvious inhibition effects on the tomato fusarium wilt, and the bacteriostasis rate reaches more than 46.39 percent.

(2) Strain fermentation supernatant chitinase and beta-1, 3-glucanase activity assay

QN ₃ The NO-3 strain is incubated in a sterile LB liquid medium at 28 ℃ and 180 rpm for 5 days to collect bacterial suspension, and centrifuged at 8000 rpm for 15 min. Chitinase activity and beta-1, 3-glucanase activity were determined from the supernatants collected daily, respectively.

The results are shown in FIG. 10. The cell wall component of pathogenic fungi mainly consists of chitin and glucan, and experimental data show that bacillus bailii QN ₃ NO-3 can produce chitinase and beta-1, 3-glucanase in different culture periods. Incubation to 5d, chitinase and beta-1, 3-glucanase activities were highest at 19.79.+ -. 0.52U/mL and 15.19.+ -. 0, respectively38U/g. By combining with bacteriostasis experiments, the chitinase can weaken or destroy the structural integrity of fungal cell walls by degrading chitin, and can directly inhibit the growth and development of various plant pathogenic fungi. In addition, beta-1, 3-glucanases hydrolyze another key component of the fungal cell wall by degrading beta-1, 3-glucans, both enzymes leading to osmotic imbalance, cell lysis and fungal growth inhibition. Chitinase and beta-1, 3-glucanase activities provide basis for the bacteriostatic mechanism of biological control agents, and understanding the existence and activity of these enzymes can further support the development and optimization of plant pathogen management biological control strategies.

2.10 Pesticide sensitization test

The QN3NO-3 strain drug sensitivity experiment is carried out by an agar dilution method: agar dilution plates containing various bactericides (3 concentrations, 3 replicates each) were prepared and inoculated with QN3NO-3 streaks and strain growth was observed and the results are shown in Table 3. According to the result, the QN3NO-3 strain can grow in 7 alternative broad-spectrum bactericides of thiophanate methyl, zhongshengmycin, mancozeb, carbendazim, dixone, metalaxyl and hymexazol, can inhibit growth and grow poorly when the mancozeb concentration is too high, and can grow well when the carbendazim concentration reaches 500%. The QN3NO-3 strain has a certain development potential in the agriculture industry with serious pesticide residue problems nowadays when being used as a microbial source fertilizer, can replace bactericides to inhibit bacteria and fungus pathogenic bacteria, and can also normally reproduce in farmlands polluted by pesticide residues to promote plant growth.

TABLE 3 results of studies on pesticide resistance of strains

Note that: +: slightly long, ++: the growth is carried out normally, the growth is carried out, ++, and: mass growth, -: is not long.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (6)

1. Bacillus belicus, characterized by being named asBacillus velezensis QN ₃ NO-3, wherein the preservation number is CCTCC NO: m2021304.

2. The crude ammonium sulfate protein of bacillus belicus fermentation broth or supernatant of the fermentation broth or fermentation broth according to claim 1.

3. The strain volatiles produced by the sealed culture of bacillus belicus according to claim 1.

4. Use of bacillus besii according to claim 1, or of a fermentation broth or of a supernatant of a fermentation broth or of an ammonium sulphate crude protein of a fermentation broth according to claim 2, or of a strain volatiles according to claim 3, for antagonizing tomato fusarium wilt, and/or of a physiological race No. 4 of banana fusarium wilt, and/or of a physiological race No. 1 of banana fusarium wilt, and/or of cucumber fusarium wilt, and/or of pepper fusarium anthracnose, and/or of strawberry fusarium anthracnose, and/or of banana fusarium anthracnose, and/or of colletotrichum zeae, and/or of alternaria leaf spot, and/or of alternaria zeae, and/or of wheat fusarium gibberella, and/or of trichoderma pseudoginseng leaf blight, and/or of rice blast, and/or of mango anthracnose.

5. Use of bacillus besii according to claim 1, or of the fermentation broth or of the supernatant of the fermentation broth or of the ammonium sulphate crude protein of the fermentation broth according to claim 2, or of the strain volatiles according to claim 3, for controlling diseases caused by tomato fusarium wilt, and/or of the physiological race No. 4 of banana fusarium wilt, and/or of the physiological race No. 1 of banana fusarium wilt, and/or of cucumber fusarium wilt, and/or of pepper anthracnose, and/or of strawberry anthracnose, and/or of litchi anthracnose, and/or of banana anthracnose, and/or of colletotrichum gloeosporioides, and/or of alternaria corn, and/or of wheat gibberella, and/or of trichoderma pseudolaris, and/or of rice blast, and/or of mango anthracnose.

6. Use of bacillus belicus according to claim 1, or of a fermentation broth or supernatant of a fermentation broth according to claim 2, for disrupting a fungal cell wall.