CN105624056B - The production method of one plant of plant growth-promoting bacteria for being isolated from phyllosphere and its microbial inoculum - Google Patents

Abstract

The invention relates to a high-efficiency plant growth-promoting bacterium-Bacillus subtilis, and a low-cost production method and application method of the bacterial agent, belonging to the technical field of microbial fertilizer and energy saving and emission reduction. The bacterial strain that the present invention adopts is a Bacillus subtilis (Bacillus subtilis) SZX11 bacterial strain that isolates and screens out from pine leaves and has the function of promoting plant growth. CGMCC No.8188. The present invention utilizes sweet potato starch wastewater as a liquid fermentation medium to cultivate Bacillus subtilis on a large scale and prepare agricultural microbial agent. The process has low energy consumption, low production cost, simple operation, and resourceful utilization of sweet potato starch wastewater. Reduce pollution emissions. The bacterial agent produced by the invention is used in the production of tea leaves and vegetables, not only can significantly increase the output, but also can obviously improve the quality of agricultural products, showing broad application prospects.

Description

A plant growth-promoting bacterium isolated from the phyllosphere and production method thereof

Technical field:

The invention relates to a high-efficiency plant growth-promoting bacterium (Bacillus subtilis SZX11 strain) isolated from pine leaves, and a low-cost production method of the bacterial agent, belonging to the technical field of microbial fertilizer and energy saving and emission reduction.

Background technique:

Bacillus subtilis (Bacillus subtilis) is a type of aerobic, endophytic rod-shaped bacteria resistant to reverse spores, with a size of (0.8-1.2) μm×(1.5-4.0) μm, Gram staining is positive, and can produce capsules. Mesogenic or mesogenic spores, coarse colonies, capable of liquefying gelatin, peptonizing milk, reducing nitrate, hydrolyzing starch, typical aerobic bacteria widely present in soil, lakes, oceans, and the surfaces of animals and plants, and they are not pathogenic sex. Bacillus subtilis can successfully colonize the rhizosphere, body surface or body of plants, compete with pathogenic bacteria for nutrients around plants, secrete antibacterial substances to inhibit the growth of pathogenic bacteria, and induce plant defense systems to resist pathogenic bacteria invasion, thus having a biocontrol effect. Bacillus subtilis can also produce substances similar to cytokinins and plant growth hormones to promote plant growth. For example: the U.S. AgraQuest company has developed the live bacteria preparation fungicide SerenadeTM with the Bacillus subtilis QST713 bacterial strain, and passed the registration of the U.S. Environmental Protection Agency (EPA) in 2000, and is used for preventing and treating powdery mildew, frost and dew disease, blight, Botrytis and other diseases. Bacillus subtilis MBI600 has been registered in MicroBioGroup Ltd. in the UK, IdemistuKosanCo., Ltd. in Japan, etc., and has been industrialized for the prevention and treatment of leaf diseases (grey mold, powdery mildew) and root diseases (wilt disease, root rot, black spot, etc.). Bacillus subtilis FZB24 has been registered in Germany, the United States and other countries, and put into production at Bayer Company in Germany. It is used to prevent and control tomato late blight, gray mold and wheat powdery mildew, and can also be used as a production increase accelerator. With the continuous deepening of the research on Bacillus subtilis, it is found that it has a wide application value in industry, agriculture, medicine and health, food health care, aquaculture and other aspects.

A Chinese invention patent (application number: 201210328716.5) discloses a Bacillus subtilis T-500 strain, the preservation number is CGMCC No.6058, which is effective against rice blast fungus, rice sheath blight fungus, rice bacterial blight fungus, rice smut fungus , rice bacterial streak, Fusarium graminearum, Aspergillus flavus and Aspergillus niger and other pathogenic bacteria, exhibit strong antibacterial activity, and have a good control effect on the diseases caused by it. The composition of the fermentation medium includes : Carbon source (sucrose or starch), nitrogen source (soybean powder, bean cake powder, corn steeple, or a combination of several), trace elements (beef extract, yeast extract, or fish meal, or a combination of two) . A Chinese invention patent (application number: 201210229066.9) discloses a Bacillus subtilis CX31 strain with antibacterial activity, the preservation number is CCTCC No. M2012138, and the cultivation of the strain uses beef extract peptone medium. A Chinese invention patent (application number: 201110205587.6) discloses an endophytic Bacillus subtilis strain with a preservation number of CGMCC No.4844, which has a good control effect on pepper blast. The culture medium used for cultivating the strain is: PDA medium.

A Chinese invention patent (application number: 201210215134.6) discloses a strain of Bacillus subtilis RB, the preservation number is CGMCC No.6268, and discloses the application of the strain in the treatment of sludge, poultry manure or crop straw. The culture medium of the bacterial strain is prepared by using cornstarch, brown sugar, wheat bran, fish meal, sucrose and inorganic salts. A Chinese invention patent (application number: 201210127519.7) discloses a strain of Bacillus subtilis) KW119, the preservation number of which is CGMCC No.5789. as the substrate. A Chinese invention patent (application number: 201110443948.0) discloses a strain of Bacillus subtilis CH-001, the preservation number is CCTCCNo. Animal protein feed additive, the main components of its culture medium are feather meal and inorganic salt. A Chinese invention patent (application number: 201010129783.5) discloses a strain of Bacillus subtilis ANSB060, the preservation number is CGMCC No.3440, and discloses its application in animal feed. The main components of its culture medium are peptone, glucose and inorganic Salt. Disclosed in the Chinese invention patent (application number: 200410101021.9) is a bacillus subtilis B115 bacterial strain, and preservation number is CGMCCNo.1210, and discloses its method for preparing microecological water quality improver for fishery, and its culture medium main component is Meal flour, bran and cornmeal.

In summary, there are many strains of Bacillus subtilis that can promote plant growth and antagonize plant diseases, and the strains used for animal feed are also relatively common, and their cultivation methods are also different. However, no strains isolated from the phyllosphere of plants have been seen. Bacterial strains, and there is no report on the cultivation of Bacillus subtilis using high-concentration sweet potato starch wastewater.

my country is the largest sweet potato producer in the world, with a stable total output of more than 100 million tons, accounting for about 80% of the world's total. Among the main crops in the country, sweet potatoes rank fourth after rice, wheat and corn. Sweet potato planting requires less investment and more output, and the edible dry matter per unit area ranks first among all kinds of crops. Sweet potato has strong disaster resistance, drought tolerance, and barrenness tolerance. It can also be planted in hilly areas, and it can also obtain better yields in places where other crops are difficult to grow. Sweet potatoes are perishable and should not be stored for a long time. The deep processing of sweet potatoes can solve the phenomenon of a large number of rotten potatoes caused by improper storage of fresh potatoes. The refined starch of sweet potatoes can be processed at different depths to produce hundreds of valuable products, and the value-added is about 10-30 times. However, the current processing technology produces about 10 tons of high-concentration organic wastewater for every 1 ton of sweet potato starch produced. Among them, the sweet potato cell fluid after starch separation has the highest organic content, and the COD can reach more than 20,000 mg/L, mainly containing soluble starch. , protein, pectin, organic acid and a small amount of oil, if not handled properly, they are easily spoiled and fermented, making the water black and smelly. However, using general sewage treatment processes to treat such high-concentration wastewater up to the standard discharge will consume a lot of energy. From another point of view, organic matter (including protein, pectin, sugar, etc.) in sweet potato starch wastewater is a good nutrient substrate for microbial growth, which can be made into microbial culture medium for resource utilization.

Invention content:

The purpose of the present invention is to provide a strain of Bacillus subtilis which can antagonize plant pathogenic bacteria and promote plant growth, and provide a method capable of resource utilization of high-concentration sweet potato starch wastewater to produce its bacterial agent.

The bacterial strain used in the present invention is a Bacillus subtilis (Bacillus subtilis) SZX11 bacterial strain that has the function of promoting plant growth and antagonizing plant pathogenic bacteria isolated and screened by the inventor from the leaves of pine trees. This bacterial strain was released on September 16, 2013. Preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the deposit number is: CGMCC No.8188, the address of the depository unit: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode: 100101.

The production method of Bacillus subtilis agricultural microbial bacterial agent of the present invention is as follows:

1, sweet potato starch wastewater pretreatment: add 0.1%～1% plant ash (from the solid waste of biomass thermal power plant, its main component is potassium carbonate) according to weight ratio in high-concentration sweet potato starch wastewater, repeat mixing, and then Adjust the pH of the waste water to 7.0-8.5 with lime water, and heat it with microwave at 95-110°C for 3-15 minutes.

2. Preparation of liquid seeds: Inoculate the Bacillus subtilis preserved on the slant in the triangular flask equipped with pretreated sweet potato starch wastewater, and culture at 45-55°C and 120-200 rpm for 24-36 hours to obtain liquid seeds .

3. Liquid fermentation: inoculate liquid seeds with a volume ratio of 2% to 15% in a fermentation tank equipped with pretreated sweet potato starch wastewater, and cultivate them in sterile air for 12 to 30 hours at 35 to 50°C; The fermentation liquid is counted by plate bacteria, and the number of viable bacteria reaches more than 2×10 ⁹ CFU/ml, and the obtained fermentation liquid can be directly used as a liquid bacterial agent.

4. Preparation of solid microbial agent: use biochar (the remaining solid matter after pyrolysis of biomass, the main component is carbon) passing through a 30-mesh sieve as the carrier, according to the weight of fermentation broth: biochar of 1:1～1:3 For example, add the fermented liquid to the biochar, mix thoroughly to obtain a solid microbial agent, ventilate and dry it at 55-60°C until the water content of the bacterial agent is 15%-25% by weight, pack it, and store it in a cool and dry place. place, spare.

The using method of Bacillus subtilis microbial bacterial agent of the present invention is as follows:

1. Dilute the fermented liquid or solid microbial agent with irrigation water until the number of viable bacteria is 3×10 ⁶ CFU/ml to 3×10 ⁷ CFU/ml, mix well, and use 0.3 to 3 cubic meters of dilution per mu for the Spray the whole crop evenly or furrow into the soil near the root of the crop;

2. Add the fermented liquid or solid bacterial agent to the organic fertilizer, and mix well, so that the number of viable Bacillus subtilis in the organic fertilizer is 2×10 ⁷ CFU/g~2×10 ⁸ CFU/g, according to 1~ The amount of 3 tons is applied to the soil as a base fertilizer.

Detailed ways:

The Bacillus subtilis provided by the invention can promote the absorption and utilization of nutrients by crops, and can effectively prevent and treat various plant diseases. The microbial bacterial agent produced by the technical solution provided by the invention can be used in agricultural production to reduce the use of chemical fertilizers and pesticides. Therefore, the environmental quality of farmland is improved; and the production cost is very low, high-concentration wastewater can be utilized as resources, and pollutant discharge can be reduced. The specific implementation is as follows:

Embodiment 1: sweet potato starch waste water produces Bacillus subtilis microbial bacterial agent

(1) sweet potato starch wastewater pretreatment: in high-concentration sweet potato starch wastewater, add 0.5% plant ash (from the solid waste of biomass thermal power plant, its main component is potassium carbonate) according to weight ratio, repeat mixing, and then use lime The pH of the wastewater was adjusted to 8.0 with water and microwaved at 100°C for 5 minutes.

(2) Liquid seed preparation: inoculate the Bacillus subtilis preserved on the slant into a triangular flask containing pretreated sweet potato starch wastewater, and vibrate at 50° C. at 150 rpm for 28 hours to obtain liquid seeds.

3. Liquid fermentation: inoculate the liquid seeds with a volume ratio of 5% in the pretreated sweet potato starch waste water fermenter tank, at 45°C, cultivate in sterile air for 16 hours; The number of viable bacteria reaches more than 2×10 ⁹ CFU/ml, and the obtained fermentation broth can be directly used as a liquid microbial agent.

4. Preparation of solid microbial agent: take the biochar (the remaining solid matter after pyrolysis of biomass, the main component is carbon) of passing through the 30 mesh sieve as the carrier, according to the weight ratio of fermentation broth: biochar of 1: 2, ferment The liquid is added to the biochar, mixed thoroughly to obtain a solid bacterial agent, ventilated and dried at 55-60°C until the water content of the bacterial agent is 15%-25% by weight, packaged, stored in a cool and dry place, and used for later use.

Embodiment 2: Bacillus subtilis microbial inoculum is applied to the production of organic tea

In the organic tea garden, choose about 2 acres of experimental land with uniform growth of tea trees. The management of the experimental tea trees is the same as that of other tea trees in the base, but the fertilization is different. The test site is divided into 5 blocks, corresponding to 5 treatments respectively. There is an isolation row of about 2 meters between each treatment. The bacterial agent is diluted with tap water or the fermentation broth is directly used. The 5 treatments are respectively clean water control (CK), starch wastewater control (FCK), high concentration (2×10 ⁷ CFU/ml) spray (H), medium concentration (1×10 ⁷ CFU/ml) spray (M), low concentration (5× 10 ⁶ CFU/ml) spraying (L), according to the amount of 2 liters of bacterial agent dilution per square meter. In the same way, each treatment was applied 3 times inoculum in winter, and the tea leaves were harvested in spring. The results showed that the yield of spring tea in the control treated with wastewater dilution was not significantly different from that in clean water, while the yield of spring tea in the application of inoculum increased significantly, among which , the yield of spring tea with medium concentration and high concentration of bacterial agent is equal, and the yield of spring tea with low concentration of bacterial agent is also increased by about 20% compared with that of clean water control. Moreover, the contents of tea polyphenols and amino acids in the tea leaves treated with bacterial agents were significantly higher than those in the clean water control.

Example 3: Organic fertilizer rich in Bacillus subtilis is applied to tea production

In the tea garden, a test site with uniform growth of tea trees was selected, and it was evenly divided into 6 plots, each with an area of about 200 square meters. They were respectively set as the control without fertilization, the application of organic fertilizer, and the application of Bacillus subtilis (6×10 ⁷ CFU/ gram) of organic fertilizer 3 kinds of treatment, every kind of treatment 2 parallels, each treatment is provided with the isolation line of about 2 meters, organic fertilizer and the organic fertilizer that is rich in Bacillus subtilis are according to the consumption of 1 ton per mu, in Furrow fertilization was applied once before winter, and tea leaves were harvested in spring. The results showed that: the yield of spring tea with organic fertilizer was about 15% higher than that of the control, and the yield of spring tea with bio-organic fertilizer was more than 35% higher than that of the control. The contents of water extracts, tea polyphenols and amino acids in fertilized tea were significantly higher than those in the control.

Embodiment 4: Bacillus subtilis microbial inoculant is applied to rape production

Using the vegetable greenhouse plot experiment, set up 8 experimental plots with exactly the same conditions, each plot 100m ² , 2 plots are a group of parallel experiments, divided into 4 groups, planting rapeseed, in the rapeseed seedling stage, the first group has 2 plots No fertilization was used as a control; the 2 plots in the 2nd group were sprayed with the bacterial agent dilution (2×10 ⁷ CFU/ml) once after the rape completely germinated and 3 weeks later according to the dosage of 100 kg/mu; the 2 plots in the 3rd group According to the fertilization rate of 15 kg/mu, the urea was applied once after the rape fully germinated and 3 weeks later; the 2 groups of the fourth group were respectively applied urea once after the rape fully germinated and 3 weeks later according to the fertilization rate of 15 kg/mu. And one day after each application of urea, the bacterial agent dilution (2×10 ⁷ CFU/ml) was sprayed once at 100 kg/mu, and the irrigation and management methods were the same for each plot. The yield and quality of rapeseed were investigated after 5 weeks of growth. The yield of rapeseed with the application of bacterial agent and urea alone is basically the same, and the yield of rapeseed is about 50% higher than that of the control plot without fertilization; The content of urea was significantly lower than that of the plots applied with urea alone.

Claims (3)

1. a kind of production method of bacillus subtilis agricultural microbial agent, includes the following steps：

(1) Sweet Potato Starch Processing Waste Water pre-processes：According to the grass of weight ratio addition 0.1%~1% in high concentration Sweet Potato Starch Processing Waste Water Wood ash repeats to be uniformly mixed, then adjusts the pH to 7.0~8.5 of waste water with limewash, in 95~110 DEG C of 3~15 points of microwave heatings Clock；The plant ash comes from the solid-state castoff of biomass thermal power plant, and main component is potassium carbonate；

(2) prepared by liquid seeds：The withered grass of slant preservation is inoculated with into the triangular flask equipped with the Sweet Potato Starch Processing Waste Water pre-processed Bacillus, in 45~55 DEG C, 120~200 revs/min of shaken cultivations 24~36 hours obtain liquid seeds；

(3) liquid state fermentation：To equipped with the liquid that inoculation volume ratio is 2%~15% in the Sweet Potato Starch Processing Waste Water fermentation tank pre-processed Body seed leads to filtrated air culture 12~30 hours in 35~50 DEG C；Cultured zymotic fluid is through count plate, viable bacteria Number reaches 2 × 10⁹CFU/ milliliters or more, gained zymotic fluid can directly be used as liquid bacterial agent；

(4) prepared by solid fungicide：Using the biological carbon of the sieve of 30 mesh excessively as carrier, according to zymotic fluid：Biological carbon is 1：1~1：3 weight Ratio is measured, zymotic fluid is added in biological carbon, is sufficiently mixed uniformly, obtains solid-state microbial inoculum, at 55~60 DEG C, aeration-drying is extremely By weight being 15%~25%, packaging is stored at shady and cool drying microbial inoculum water content, spare；The biological carbon is biomass Remaining solid matter after pyrolysis, main component are carbon；

Wherein, the bacillus subtilis is with the bacillus subtilis for promoting plant growth, antagonistic phytopathogen function (Bacillus subtilis) SZX11 bacterial strains, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life Object center, deposit number：CGMCC No.8188.

2. the application method of agricultural microbial agent described in claim 1, it is characterised in that：Zymotic fluid or solid fungicide are used It is 3 × 10 that irrigation water, which is diluted to viable count,⁶CFU/ milliliters~3 × 10⁷It CFU/ milliliters, is uniformly mixed, it is vertical according to per acre 0.3~3 The dosage of square rice dilution to the whole strain of crop uniformly spray or ditch apply crop root near soil in.

3. the application method of agricultural microbial agent described in claim 1, it is characterised in that：Zymotic fluid or solid fungicide are added Enter in organic fertilizer, be uniformly mixed, it is 2 × 10 to make the bacillus subtilis viable count in organic fertilizer⁷CFU/ grams~2 × 10⁸CFU/ Gram, in being manured into soil as base manure according to 1~3 ton per acre of dosage.