CN115261267B - Application of brown alginate oligosaccharides in promoting bacillus movement and plant rhizosphere colonisation - Google Patents

Abstract

The invention provides an application of fucoidan oligosaccharide in promoting the movement of bacillus and promoting the colonization of bacillus in plant rhizosphere, belonging to the technical field of microorganisms. The bacillus includes one or more of Bacillus subtilis, Bacillus Veles and Bacillus amyloliquefaciens, and the fucoidan oligosaccharide significantly promotes the exercise ability of the bacillus. The present invention also provides a bacillus inoculum agent, which includes bacillus and fucoidan oligosaccharide, and the fucoidan oligosaccharide can significantly promote the motility of the bacillus, so that it can quickly and effectively colonize the crop rhizosphere, thereby The bacterium agent jointly prepared by the fucoidan oligosaccharide and the bacillus can significantly promote plant growth, thereby providing an effective way for reducing the application of chemical pesticides and fertilizers and promoting sustainable agricultural development.

Description

Application of fucoidan oligosaccharides in promoting Bacillus movement and colonization in plant rhizosphere

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to the application of fucoidan oligosaccharides in promoting the motility of bacillus.

Background technique

In agricultural production, plant rhizosphere growth-promoting bacteria play an important role in crop health and high yield. Its application is an effective way to reduce chemical pesticides and fertilizers in my country and promote sustainable agricultural development. At present, scholars at home and abroad have isolated a large number of biocontrol strains with disease prevention and growth promotion effects from the rhizosphere of important crops such as rice, wheat, corn, and cotton, and some strains have been used in commercial production. However, after biocontrol bacteria enter the soil, they face competition from indigenous microorganisms and complex soil environmental conditions, making it difficult to form dominant populations and exert effective biocontrol functions. Instability and other problems have hindered its agricultural application. Efficient rhizosphere colonization is an important prerequisite for microorganisms to exert various plant beneficial functions. Therefore, solving the problem of difficult colonization of microbial strains in the plant rhizosphere is the key to solving the limited agricultural application of biocontrol bacteria. The motility of the plant significantly affects its colonization in the crop rhizosphere.

Fucoidan oligosaccharide is a compound with strong solubility, easy absorption, safety and non-toxicity. Studies have found that fucoidan oligosaccharide has antioxidant, antibacterial and therapeutic effects on human diseases. At present, fucoidan oligosaccharides have broad development prospects in the fields of drug development and functional food development, but there is no report on the use of fucoidan oligosaccharides to promote bacterial motility.

Contents of the invention

In view of this, the object of the present invention is to provide the application of fucoidan oligosaccharides in improving the motility of bacillus and promoting the colonization of bacillus in the rhizosphere of plants, and effectively improve the motility of bacillus on the soil or plant surface by using fucoidan oligosaccharides , thereby promoting the colonization of the bacterial strain in the rhizosphere of the plant, and at the same time, the joint application of the fucoidan oligosaccharide and the bacillus of the present invention can also effectively promote the growth of the plant.

In order to achieve the purpose of the above invention, the present invention provides the application of fucoidan oligosaccharide in promoting the movement of bacillus.

Another object of the present invention is to provide the application of fucoidan oligosaccharide in promoting the colonization of bacillus in plant rhizosphere.

Optionally, the bacillus includes: one or more of Bacillus subtilis, Bacillus Veles and Bacillus amyloliquefaciens.

Optionally, the method for preparing fucoidan oligosaccharides includes the following steps: dissolving sodium alginate in water, adding enzymes for cleavage, centrifuging, and taking the supernatant to freeze-dry to obtain fucoidan oligosaccharides.

Preferably, in the preparation step of the fucoidan oligosaccharide, the enzyme is alginate lyase, the enzymolysis temperature is 25-32°C, and the enzymolysis time is 12-18h.

Optionally, the molecular weight of the fucoidan oligosaccharide is 1000-4000D.

Another object of the present invention is to provide a bacillus bacterial agent, which includes fucoidan and bacillus.

Optionally, the concentration of the fucoidan oligosaccharide is 1.0-7.0 g/L.

Optionally, the dosage form of the bacterial agent can be water, wettable powder, water dispersible granules or water suspension.

Another object of the present invention is to provide the application of the above-mentioned Bacillus inoculum in promoting plant growth.

Compared with the prior art, the present invention has the following beneficial effects:

The fucoidan oligosaccharide of the invention has low molecular weight, strong water solubility, high stability, safe and non-toxic physical and chemical properties. It can not only provide a carbon source for the growth of bacillus, but also effectively improve the motility of bacillus, and within a certain amount of fucoidan oligosaccharides, the promotion of the motility of the strain is positively correlated with the concentration of fucoidan oligosaccharides; The fucoidan oligosaccharide has broad development prospects in the fields of drug development and functional food development. The present invention adds the fucoidan oligosaccharide to the bacillus bacterial agent, providing a new application of the fucoidan oligosaccharide.

The bacillus bacterial agent provided by the present invention includes fucoidan oligosaccharides and bacillus. The fucoidan oligosaccharides in the bacterial agent can significantly promote the motility of bacillus, and then can promote the effective colonization of bacillus strains in the crop rhizosphere, thereby achieving further The purpose of improving biocontrol agent products. The bacillus added to the bacillus inoculum of the present invention is one of the soil and plant micro-ecological dominant microbial populations, which can play a role through nutrient competition, promote plant growth, induce plant resistance, and secrete antibacterial substances. Fucoidan oligosaccharides and The joint application of bacillus can promote the movement ability of the strain on the soil or plant surface, and can quickly and effectively colonize the bacillus in the rhizosphere of the plant to form dominant bacteria, which in turn can significantly promote the growth of the plant.

Biological Deposit Instructions

Bacillus velezensis EM-1 (Latin name Bacillus velezensis EM-1) described in the present invention, preservation unit: China Microbial Cultures Preservation Management Committee General Microbiology Center, preservation address: No. 1, Beichen West Road, Chaoyang District, Beijing, China Court No. 3, deposit number: CGMCCNO.21131, deposit date: November 09, 2020.

Description of drawings

Figure 1: Effects of sugars from different sources on the motility of Bacillus amyloliquefaciens Cas02, where CK is blank, COS is chitosan oligosaccharide, HSG is fucoidan oligosaccharide, HT is enteromorpha polysaccharide, KLG is carrageenan oligosaccharide, and KLJ is Carrageenan, SA is sodium alginate, YZ is fucoidan, and the added concentration of the above sugars is 5.0g/L;

Figure 2: Effects of different concentrations of fucoidan oligosaccharides on the motility of Bacillus amyloliquefaciens Cas02, the added concentrations were 1.0g/L, 2.5g/L, 5.0g/L, 10.0g/L;

Figure 3: Fucoidan oligosaccharides promote the effective colonization of Bacillus amyloliquefaciens Cas02 in the plant rhizosphere, where CK is the application of sterile water, Cas02 is the application of only Bacillus amyloliquefaciens Cas02 bacterial solution, and HSG+Cas02 is the application of fucoidan oligosaccharides The Bacillus amyloliquefaciens Cas02 bacterium liquid;

Figure 4: Agronomic traits of tobacco seedlings grown under different treatments, where CK is watering sterile water, HSG is watering fucoidan oligosaccharide solution, C is watering Bacillus amyloliquefaciens Cas02 bacterial solution, HSG+C is fucoidan oligosaccharides and amyloliquefaciens spores co-administration of bacteria.

Detailed ways

The invention provides the application of the fucoidan oligosaccharide in improving the motility of the bacillus and promoting the colonization of the bacillus in the plant rhizosphere. The fucoidan oligosaccharides of the present invention can provide a carbon source for the growth of bacillus, and can significantly improve the motility of the bacillus, thereby significantly promoting the effective colonization of the bacillus in the rhizosphere of plants; the bacillus in the present invention is a rhizosphere promoting Bacteria can play a role through nutrient competition, promote plant growth, induce plant resistance, and secrete antibacterial substances, and have biological activities of decomposing phosphorus, potassium, and nitrogen fixation.

The bacillus of the present invention includes one or more of Bacillus subtilis, Bacillus Veles and Bacillus amyloliquefaciens. The Bacillus is preferably Bacillus amyloliquefaciens, more preferably Bacillus amyloliquefaciens Cas02, which is a strain disclosed in Chinese patent CN108624543A, and has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 26, 2018 (Address: No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, China), and its biological deposit number is CGMCCNO.15514. In the present invention, Bacillus subtilis is preferably Bacillus subtilis Tpb55, which is a document Biocontrol potential of antagonist Bacillus subtilis Tpb55 against tobacco black shank (Han, T.et.al.BioControl, 2016, 61 (2), 195-205.) The strain disclosed in , and was preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms on December 29, 2008 (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China), and its biological deposit number is CGMCC NO .2853. The Bacillus Velez in the present invention is preferably Bacillus Velez EM-1, which was preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures (Address: Beichen, Chaoyang District, Beijing, China) on November 09, 2020. No. 3, No. 1 Yard, West Road), and its biological deposit number is CGMCC NO.21131. The bacillus added in the invention has strong stress resistance, can promote plant growth, and inhibit pathogenic bacteria from invading plants.

The fucoidan oligosaccharides in the present invention can effectively promote the motility of bacillus, and within the range of addition of fucoidan oligosaccharides, the promotion effect on the motility of bacillus is positively correlated with the concentration of fucoidan oligosaccharides; and fucoidan oligosaccharides can significantly promote the motility of bacillus Efficient colonization of crop rhizosphere by Bacillus strains.

As an implementable mode, the preparation method of the fucoidan oligosaccharides of the present invention comprises: dissolving sodium alginate in water, adding enzymes for cleavage, centrifuging, and vacuum freeze-drying the supernatant to obtain the fucoidan oligosaccharides.

In the present invention, sodium alginate is added to water according to a material-to-liquid ratio of 2-6:100g/mL, preferably a material-to-liquid ratio of 4-5.5:100g/mL; wherein, the present invention has no special limitation on the added water, and the method used in the art Conventional test water is sufficient, and as an implementable mode, distilled water is added in the present invention.

The enzyme added in the present invention is alginate lyase, the added concentration of alginate lyase is 0.1-2.5g/L, preferably 0.5-1.5g/L; the enzymolysis temperature is 25-32°C, preferably 28- 30°C; the enzymolysis time is 12-18h, preferably 15-16h; the enzymolysis solution is centrifuged to remove undegraded polysaccharides, the centrifugation parameters are: 5000-10000rpm, 3-10min, preferably 6000-8000rpm, 5-8min.

In the present invention, the fucoidan oligosaccharide solution obtained by centrifugation is dried. In the present invention, there is no special limitation on the drying method of the fucoidan oligosaccharide, and the conventional drying method in the field can be used. As an implementable method, the present invention adopts vacuum freeze-drying way, the drying time is 24-72h, preferably 36-48h;

The molecular weight of the fucoidan oligosaccharides added in the present invention is preferably 1000-4000D. Some studies have found that different molecular weights of fucoidan oligosaccharides have different effects on promoting plant growth. However, the present invention has found that brown algae with a molecular weight of 1000-4000D Oligosaccharides can effectively improve the biological activity of fucoidan oligosaccharides, facilitate the absorption and utilization of organisms, and improve the motility of Bacillus.

The present invention also provides a bacillus bacterial agent, which includes fucoidan oligosaccharide and bacillus.

The present invention has no special limitation on the method for cultivating the bacillus. As an implementable mode, the culturing of the bacillus of the present invention includes: cultivating the strain in LB liquid medium, and the culturing condition is aerobic culturing at 28° C. for 12 hours.

Wherein, the LB medium is a conventional medium in the field, and its components (g/L) are: tryptone 10.0, yeast extract 5.0, sodium chloride 10.0, pH7.0±0.2; In the manner of implementation, in the present invention, the liquid culture medium inoculated with the bacterial strain is placed on a shaker, and cultured by shaking at 120-160rpm, preferably at 130-150rpm.

In the present invention, the bacterium liquid obtained by culture is centrifuged to obtain the thallus, the centrifugation parameter is 6000rpm, 5min, and the thallus is resuspended to OD ₆₀₀ of 0.1-1.0 with sterile water, preferably the OD ₆₀₀ is adjusted to 0.2- 0.5: Add fucoidan oligosaccharides into the Bacillus bacteria liquid, preferably the concentration of the fucoidan oligosaccharides in the bacteria liquid is 1.0-7.0 g/L, more preferably 1.5-5.0 g/L.

The dosage form of the bacillus bacterial agent of the present invention can be water, wettable powder, water dispersible granule or water suspension.

Additives are also included in the bacillus inoculum of the present invention, and the additives include one or more of dispersants, wetting agents, disintegrants, binders, defoamers, antifreeze agents, thickeners, fillers and solvents. Various.

Wherein, the dispersant is an anionic dispersant and/or a nonionic dispersant, sodium lignosulfonate, sodium naphthalene sulfonate formaldehyde condensate, sodium methylene bis-naphthalene sulfonate, formaldehyde condensate sulfate , one or more of polycarboxylates, alkylphenol polyoxyethylene phosphates and fatty acid polyoxyethylene esters;

The wetting agent can be selected from one or more of sodium lauryl sulfate, sodium dodecylbenzene sulfonate, saponin powder, sapinberry powder, tea dry powder and pull-open powder BX;

The disintegrant can be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride and glucose;

The binder can be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose and carboxymethyl cellulose salt;

The defoamer can be selected from one or more of C8-C20 fatty alcohol compounds, C10-C20 saturated fatty acid compounds, epoxidized soybean oil, ethanol, silicone compounds and organic silicone oils;

The antifreezing agent can be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propylene glycol, glycerol, urea and sodium chloride;

The thickener can be selected from one or more of gelatin, xanthan gum, polyethylene glycol and polyvinyl alcohol;

The filler can be selected from one or more of light calcium carbonate, diatomaceous earth, bentonite, attapulgite and white carbon black;

The solvent may be selected from deionized water or methyl oleate.

Unless otherwise specified, according to the dosage form of the bacillus inoculum to be prepared in the present invention, the above-mentioned additives can be conventionally selected to make the desired dosage form.

Unless otherwise specified, the reagents and consumables involved in the present invention can be obtained from commercial sources. If the specific conditions of the experiment are not specified, it is usually carried out according to the conventional conditions or the conditions recommended by the reagent company.

The invention also provides the application of the bacillus bacterial agent in promoting plant growth. In the Bacillus inoculum provided by the present invention, fucoidan oligosaccharides and Bacillus are used in combination, and Fucoidan oligosaccharides can provide nutrition for Bacillus, so that after the application of the inoculum, the Bacillus maintains biological activity under the condition of sufficient nutrition, has high survival ability, and Fucoidan oligosaccharides can significantly promote the motility of Bacillus, significantly improve the motility of Bacillus on the soil or plant surface, and then can quickly and effectively colonize the plant rhizosphere, play a role, and achieve the effect of significantly promoting plant growth.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

Fucoidan oligosaccharides promote the motility of Bacillus

1. Preparation of fucoidan oligosaccharides:

(1) Take 5g of sodium alginate and add 100mL of distilled water;

(2) Add alginate lyase so that the concentration in the solution is 0.5g/L, the enzymolysis temperature is 30°C, and the enzymolysis time is 16h;

(3) Centrifuge the enzymatic solution (6000rpm, 5min) to remove undegraded polysaccharides, and the supernatant is the fucoidan oligosaccharide solution;

(4) Vacuum freeze-dry the fucoidan oligosaccharide solution for 48 hours, and grind to obtain a fucoidan oligosaccharide with a molecular weight of 1000-4000D.

2. Prepare a semi-solid medium containing different types of sugars. The components of the semi-solid medium are: 10g/L peptone, 5g/LNaCl, 0.7% agar, pH7.0±0.2, and the types and concentrations of added sugars are shown in Table 1, wherein The added fucoidan oligosaccharide is the fucoidan oligosaccharide in step 1. Pour 25 mL of the prepared semi-solid medium into a petri dish, dry it in an ultra-clean bench, and place it at room temperature for 12 hours for later use.

Table 1 Sugars from different sources and their concentrations added in semi-solid medium

3. Cultivate Bacillus amyloliquefaciens Cas02 in LB liquid medium under the conditions of 28°C and 150rpm shaking for 12 hours, adjust the cultured fresh bacterial suspension to OD ₆₀₀ =0.3 with sterile water, and absorb the above bacterial liquid Inoculate 1.0 µL to the semi-solid medium from step 2. Incubate at 37°C for 12 hours in a static state, take pictures during the cultivation and observe whether the strains have swimming phenomenon.

As can be seen from the results in Figure 1, when no strain was inoculated in the semi-solid medium, and 5 g/L of chitosan, enteromorpha polysaccharide, carrageenan oligosaccharide, carrageenan, sodium alginate, rock Algae polysaccharide, after cultivating strains for 12 hours, there was no obvious bacterial strain swimming phenomenon on the surface of the semi-solid medium; as can be seen from the results in Figure 2, when the concentration of 1.0g/L, 2.5g/L and 5.0 g/L were added to the semi-solid medium, respectively g/L fucoidan oligosaccharides, the culture medium surface of the cultured strains appeared obvious strain swimming phenomenon. When the concentration of fucoidan oligosaccharide was 10.0g/L, no strain grew after 12 hours of culture after inoculation, that is, the high concentration of fucoidan oligosaccharide inhibited the normal growth of the strain. It shows that in a certain range of addition amount, fucoidan oligosaccharide can significantly promote the exercise ability of the strain, and it is positively correlated with the concentration of fucoidan oligosaccharide.

Example 2

This embodiment provides a bacillus aqueous solution, the components of which are Bacillus amyloliquefaciens and fucoidan oligosaccharides.

The preparation method of described bacillus water preparation is:

1. Cultivate Bacillus amyloliquefaciens in LB liquid medium, the culture condition is 28°C, 150rpm shaking culture for 12h, centrifuge the bacteria solution (6000rpm, 5min) to obtain the bacteria, resuspend the bacteria in sterile water and Adjust OD ₆₀₀ =0.3, reserve;

2. Add the fucoidan oligosaccharide obtained in Example 1 to the bacterial liquid obtained in step 1, and adjust its concentration in the bacterial liquid to 5 g/L to obtain a bacillus water preparation.

Example 3

The difference from Example 2 is only that the concentration of fucoidan oligosaccharide in the bacterial liquid is 2.5g/L.

Example 4

The only difference from Example 2 is that the bacillus is bacillus subtilis.

Example 5

The only difference from Example 2 is that the bacillus is Bacillus velesi.

Example 6

Fucoidan oligosaccharides promote effective colonization of Bacillus in plant rhizosphere

1. Cultivate Bacillus amyloliquefaciens Cas02 in LB liquid medium under the conditions of 28°C, 150rpm shaking culture for 12h, centrifuge the bacterial liquid (6000rpm, 5min) to obtain bacterial cells, and resuspend the bacterial cells in sterile water And adjust OD ₆₀₀ =0.3, reserve;

2. Sterilize the tobacco seeds and place them in a sterilized nutrient matrix to grow seedlings. When the tobacco seedlings grow to 4-5 leaves, move them into the nutrient solution containing MS;

3. Divide the nutrient solution transplanted with tobacco seedlings into three groups:

Control group CK: Add 2 mL of sterile water to the nutrient solution;

Experimental group Cas02: apply 2 mL of Bacillus amyloliquefaciens (OD ₆₀₀ =0.3) to the nutrient solution;

Experimental group HSG+Cas02: 2 mL of the bacillus water solution in Example 2 was applied to the nutrient solution.

4. Tobacco seedlings are cultivated in the greenhouse (light conditions: 16h, 28°C; dark conditions: 8h, 20°C; relative humidity 60%), take out the tobacco seedlings after 4 days, wash the roots with sterilized water, and take the roots in the 1cm mature area Placed in a sterilized EP tube for laser confocal observation of the colonization of the strains in the rhizosphere. Among them, the laser confocal microscope model is Leica TCS SP8 (Mannheim, Germany), the excitation light wavelength is 488, and the acceptance light wavelength is 505-550.

The results are shown in Figure 3. It can be seen that the experimental group HSG+Cas02 has the largest number of bacillus colonized in the tobacco rhizosphere, indicating that the fucoidan oligosaccharides in the bacillus inoculum provided by the present invention can significantly promote the growth of bacillus in the plant rhizosphere. Colonize.

Example 7

Effects of joint application of fucoidan oligosaccharides and Bacillus amyloliquefaciens on plant growth

1. Sterilize the tobacco seeds and place them in a sterilized nutrient matrix to raise seedlings. When the tobacco seedlings grow to 4-5 leaves, move them into a flower pot (about 10 cm in diameter) containing about 180 g of natural soil. Greenhouse culture (light conditions: 16h, 28°C; dark conditions: 8h, 20°C; relative humidity 60%), watering 60mL/plant of sterilized water every week;

2. When the tobacco seedlings grow to 6-7 leaves, different treatments are carried out on the potted plants in which the tobacco seedlings are planted, and each treatment is repeated 5 times: 20 mL of sterile water (H ₂ O) and 20 mL of brown algae oligosaccharides are evenly poured around the plants respectively. Sugar (HSG), 20mL Bacillus amyloliquefaciens bacteria liquid (C), 20mL Bacillus water solution (HSG+C) in Example 2; wherein, the fucoidan oligosaccharide was prepared in Example 1, and the concentration was 5g/L; Bacillus amyloliquefaciens bacterium liquid is the fresh bacterium liquid cultivated with LB medium, the bacterium liquid is centrifuged (6000rpm, 5min), obtains the bacterium, resuspends the bacterium in sterile water and adjusts OD ₆₀₀ =0.3, and the cultivation method is the same as implementation Example 2;

Watering once every 7 days, twice in a row; 7 days after the second watering, the growth-promoting effect of Bacillus on tobacco seedlings was detected. The experimental results are shown in Figure 4 and Table 2.

Table 2 Agronomic traits of different experimental treatments

Note: The data is the average value of 5 repetitions, and different letters represent significant differences at the p﹤0.05 level among different treatments for the same trait.

As can be seen from Table 2, the joint application of fucoidan oligosaccharides and bacillus in the Bacillus inoculum agent provided by the present invention, compared with the application of only fucoidan oligosaccharides and only Bacillus amyloliquefaciens, the tobacco plant height, maximum leaf length, and maximum leaf width , the number of effective leaves, and the maximum leaf area all increased significantly, which promoted the growth of tobacco seedlings; compared with the control group irrigated with sterile water, the joint application of fucoidan and Bacillus increased tobacco plant height by 44.44%, and increased the maximum leaf length 66.23%, the maximum leaf width increased by 56.22%, the effective leaf number increased by 14.42%, and the maximum leaf area increased by 47.12%. It can be seen that the combined application of fucoidan oligosaccharides and Bacillus can significantly promote the growth of plants.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (3)

1. the application of fucoidan oligosaccharide in promoting the movement of bacillus, it is characterized in that, the preparation method of described fucoidan oligosaccharide comprises the following steps: take sodium alginate and dissolve in water, add enzyme cracking, centrifuge, take supernatant and freeze-dry , to obtain fucoidan oligosaccharide; the enzyme is alginate lyase, the enzymolysis temperature is 25-32°C, and the enzymolysis time is 12-18h; the molecular weight of the fucoidan oligosaccharide is 1000-4000D. 2. The application of fucoidan oligosaccharide in promoting the colonization of bacillus in plant rhizosphere, it is characterized in that, the preparation method of described fucoidan oligosaccharide comprises the following steps: take sodium alginate and dissolve in water, add enzyme cracking, centrifuge, take The supernatant is freeze-dried to obtain the alginate oligosaccharide; the enzyme is alginate lyase, the enzymolysis temperature is 25-32°C, and the enzymolysis time is 12-18h; the molecular weight of the alginate oligosaccharide is 1000-4000D. 3. The application according to claim 1 or 2, characterized in that, the bacillus is one or more of Bacillus subtilis, Bacillus Velez and Bacillus amyloliquefaciens.