CN108004185B - Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof - Google Patents
Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof Download PDFInfo
- Publication number
- CN108004185B CN108004185B CN201810017581.8A CN201810017581A CN108004185B CN 108004185 B CN108004185 B CN 108004185B CN 201810017581 A CN201810017581 A CN 201810017581A CN 108004185 B CN108004185 B CN 108004185B
- Authority
- CN
- China
- Prior art keywords
- bacillus
- microbial inoculum
- plant
- velezensis
- bacillus velezensis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention relates to a plant endophytic Bacillus belgii capable of preventing and treating plant diseases and having growth promoting and drought resisting functions and application thereof. The preservation number of the Bacillus velezensis E6 in the China general microbiological culture Collection center is CGMCC No. 9665. The strain is separated from plant roots, grows quickly, can be quickly colonized in a large amount at plant rhizosphere and has ecological niche advantages; the strain E6 has high-efficiency control effect on plant soil-borne fungal diseases, promotes the growth of plant root systems, improves the drought resistance of plants, can destroy the formation of pathogenic bacteria biofilm, is not easy to cause bacteria to generate drug resistance, and has more advantages than other bactericides; the nutritional requirement is simple, the production cost is low, and the application prospect is good.
Description
Technical Field
The invention belongs to the technical field of agricultural, forestry and horticultural crop protection, and particularly relates to a bacillus beijerinckii strain capable of preventing and treating plant soil-borne diseases and promoting growth and resisting drought and application thereof.
Background
The bacillus is a gram-positive rod-shaped aerobic bacterium which is widely distributed, and can generate endogenic spores, has strong heat resistance, radiation resistance, chemical resistance and other stress resistance, and is easy to be processed into preparations to be widely applied. Currently, commercial bacillus used for plant disease control is mainly a bacillus subtilis preparation. For example, Bacillus subtilis GB03, MBI600 and QST713 developed in the United states, Bacillus subtilis RB-14 and NB-22 developed by Tokyo technology research institute of Japan, Bacillus subtilis RC8, Bacillus subtilis RC9 and Bacillus subtilis RC11 developed in Argentina, Bacillus subtilis A213 developed in Australia, Bacillus subtilis B903, B908, B916, B1, B3 and BS208 developed in China, and the like. In recent years, studies have reported that Bacillus velezensis is applied to control plant diseases, but is mainly used for controlling leaf diseases such as Chinese cabbage black spot and soil-borne diseases such as sclerotinia rot of colza. The bacillus amyloliquefaciens which is close to the genetic relationship is mainly used for preventing and treating tomato gray mold, rice blast, rice bacterial streak, tomato wilt and bacterial wilt. The method is an important task in the future to obtain multifunctional high-efficiency biocontrol bacillus and cell-free preparations thereof which have independent intellectual property rights in China and can prevent and treat various soil-borne diseases.
The subject group is characterized in that a strain of Bacillus endophyticus E6 is separated from marigold roots of a corridor base of a Chinese agricultural academy of sciences in 2014 and is identified as Bacillus velezensis, and the strain has an inhibiting effect on various diseases such as cucumber fusarium wilt, tomato root rot, potato powdery scab, ginger blast, soft rot and the like, wherein the inhibiting effect on the cucumber fusarium wilt and the potato powdery scab is strong, and the control effect can reach more than 80 percent and is higher than that of other biocontrol bacteria. The strain not only has the function of preventing diseases, but also can improve the drought resistance of plants, and is a strain with very high application potential.
For plant disease biocontrol bacteria, the bacteria content in the preparation is improved, the production cost is reduced, and the method is a precondition for realizing large-scale production and application of the biocontrol bacteria. At present, few fermentation process researches related to Bacillus velezensis strains are carried out at home and abroad, only two reports (Wangshi et al, 2011; new Wu and Joe are frugal, 2013) are seen at home and abroad, the Bacillus velezensis strains used in the two studies are screened from pig manure, the required suitable fermentation temperature is 37 ℃, but the suitable growth temperature of the Bacillus E6 from marigold roots is 28-32 ℃, and other nutritional requirements are different due to different sources. Even different strains of the same species may have different nutritional requirements. Therefore, the research on the fermentation process of the biocontrol bacterium Bacillus velezensis E6 which has high-efficiency disease prevention effect on the plant soil-borne diseases is developed, and the development of the biocontrol bacterium agent with the independent intellectual property rights in China has important practical significance on the prevention and control of the plant soil-borne diseases.
Disclosure of Invention
The invention aims to provide a bacillus beijerinckii strain which can prevent and control plant soil-borne diseases and has the functions of growth promotion and drought resistance and application thereof.
The Bacillus belgii provided by the invention is specifically a Bacillus belgii (Bacillus velezensis) E6 strain which has been preserved in China general microbiological culture Collection center (code CGMCC, address: No. 3 of West Lu No. 1 of the sunward area of Beijing) in 9-15 days in 2014, and the preservation number is CGMCC No. 9665.
The invention also protects a microbial inoculum of which the active ingredient is the Bacillus velezensis E6.
The microbial inoculum can also comprise an adsorption carrier; the adsorption carrier can be at least one of diatomite, turf, vermiculite, rice hull powder, corncob powder, perlite and calcium carbonate.
More specifically, in one embodiment of the present invention, the adsorption carrier is specifically formed by mixing rice hull powder and diatomite according to a volume ratio of 2: 1.
Further, the microbial inoculum is prepared by mixing fermentation liquor of the Bacillus velezensis E6, or bacterial sludge obtained by centrifuging the fermentation liquor and the adsorption carrier according to the proportion of 1L: 1Kg to 1L: 3Kg (such as 1L: 1 Kg).
In an embodiment (example 2) of the present invention, a large amount of fermentation culture solution is used to prepare the fermentation liquid of bacillus belief (bacillus velezensis) E6, and the viable count of the bacillus belief E6 in the fermentation liquid of the bacillus belief 6 is 80-130 hundred million/mL (e.g., 80 hundred million/mL). Wherein, the solvent of the mass fermentation culture solution is water, and the solutes and the concentrations are as follows: 0.4-0.8% of cane sugar, 1.5-1.8% of corn starch, 1.2-1.5% of bean cake powder and 0.2-0.5% of calcium carbonate; wherein,% represents mass percentage content. Correspondingly, in the prepared microbial inoculum, the viable count of the Bacillus belgii E6 is 40-1000 hundred million/g (such as 1000 hundred million/g); the water content of the microbial inoculum is within 5-8%.
The preparation method of the microbial inoculum also belongs to the protection scope of the invention.
The preparation method of the microbial inoculum specifically comprises the following steps:
(1) culturing the Bacillus velezensis E6 to obtain a fermentation liquid of the Bacillus velezensis E6; in the fermentation liquor, the viable count of the Bacillus belgii is 80-130 hundred million/mL (such as 80 hundred million/mL);
(2) and (2) mixing the fermentation liquor obtained in the step (1) or the centrifuged bacterial sludge and the adsorption carrier according to the ratio of 1L: 1Kg to 1L: 4Kg (such as 1L: 1Kg) to obtain the microbial inoculum.
In the step (1), the solvent of the culture medium for culturing the Bacillus belief (Bacillus velezensis) E6 is water, and the solutes and the concentrations are as follows: 0.4-0.8% of cane sugar, 1.5-1.8% of corn starch, 1.2-1.5% of bean cake powder and 0.2-0.5% of calcium carbonate; wherein,% represents mass percentage content.
More specifically, the solvent of the culture medium is water, and the solutes and the concentrations are as follows: 0.5 percent of cane sugar, 1.7 percent of corn starch, 1.4 percent of bean cake powder and 0.45 percent of calcium carbonate; wherein,% represents mass percentage content.
The fermentation liquor of Bacillus velezensis E6 also belongs to the protection scope of the invention.
The invention also provides a product for preventing and controlling the soil-borne diseases of the plants, promoting the growth of the plants or improving the drought resistance of the plants.
The product provided by the invention is used for preventing and controlling plant soil-borne diseases.
The application of the Bacillus velezensis E6 or the microbial inoculum or the fermentation liquor in the following (1) or (2) also belongs to the protection scope of the invention:
(1) preventing and controlling plant soil-borne diseases, promoting plant growth or improving the drought resistance of plants;
(2) preparation of product for preventing and controlling plant soil-borne diseases
The plant soil-borne disease is specifically a soil-borne disease caused by pathogenic fungi and bacteria such as Fusarium oxysporum (Fusarium oxysporum), Fusarium solani (Fusarium solani), Rhizoctonia solani (Rhizoctonia solani), sphaera micrantha (spongospora), Ralstonia solani (Ralstonia solanacearum), and soft rot (petobacterium chrysogenum, Erwinia carofavora).
The plant can be fructus Cucumidis Sativi, fructus Lycopersici Esculenti, rhizoma Solani Tuber osi, rhizoma Zingiberis recens, rhizoma Amorphophalli, and semen Tritici Aestivi.
The invention also provides a method for preventing and controlling the soil-borne diseases of the plants.
The method for preventing and controlling the soil-borne diseases of the plants provided by the invention can be specifically as follows (A), (B), (C) or (D):
(A) the method comprises the following steps: adding the microbial inoculum diluent into plant seeds, and sowing after seed dressing. The concentration of the microbial inoculum diluent is 0.1 hundred million/mL;
(B) the method comprises the following steps: and (3) soaking the root system of the plant in the diluent of the microbial inoculum, planting the soaked plant, and irrigating the root in the growth period. The viable count of the microbial inoculum diluent is 0.1 hundred million/mL;
(C) the method comprises the following steps: the microbial inoculum is mixed with a seedling substrate, and then the plants are sown in the seedling substrate;
(D) any combination of two or three of the above methods (A), (B) and (C) is used.
In the (A), the viable count of the Bacillus velezensis E6 in the microbial inoculum is 40 hundred million/g; the mixing ratio of the plant seeds to the microbial inoculum (the viable count of the Bacillus velezensis E6 is 40 hundred million/g) can be 1: 1 (mass ratio). Further, the seed dressing specifically comprises: diluting the microbial inoculum (the viable count of the Bacillus velezensis E6 is 40 hundred million/g) and water into a thin paste according to the proportion of 1g to 400mL, and adding the plant seeds with the same mass as the microbial inoculum into the thin paste for stirring.
In the (B), the viable count of the Bacillus velezensis E6 in the diluted solution of the microbial inoculum is 0.1 hundred million/mL; the soaking time is 30 min.
In the (C), the viable count of the Bacillus velezensis E6 in the mixed matrix is 0.1 hundred million/g;
in the above (D), the concentration of the microbial inoculum is the same as the use concentration of each of the above (A), (B) and (C). .
The Bacillus velezensis (Bacillus velezensis) E6 CGMCC No.9665 provided by the invention has the following advantages:
1. the strain is separated from plant roots, can be more suitable for the plant rhizosphere environment, can grow rapidly, can be quickly colonized in a large amount at the plant rhizosphere, and has ecological niche advantages.
2. The current commonly used bacterial bactericides or copper ion bactericides are easy to cause bacteria to generate drug resistance after being used for a long time, thereby increasing the difficulty of disease control. The strain sterile fermentation filtrate can obviously inhibit the formation of biological membranes of pathogenic bacteria ralstonia solanacearum and soft rot bacteria, inhibits the bacteria by interfering signal transmission among the bacteria and a coating wrapped outside, is a new action mode, is not easy to cause the pathogenic bacteria to generate drug resistance, has more advantages than other sterilization action modes, and is a potential antagonistic bacterium.
3. Has high-efficiency prevention and control effect on various plant diseases, and can also promote the growth of plants and improve the drought resistance of the plants.
4. The strain has the advantages of simple nutritional requirement, short fermentation period, low cost, and easy large-scale production and commercial application.
The Bacillus belius (Bacillus velezensis) E6 CGMCC No.9665 provided by the invention is an endophytic bacterium separated from marigold root in the shelter base of the Chinese academy of agricultural sciences in 2014. The strain is characterized in that: when the strain is cultured on a nutrient medium plate, the bacterial colony protrudes to form wrinkles, and the bacterial colony is expanded rapidly, which is one of the preconditions for large-scale production and application of the biocontrol bacterium. Has inhibitory effect on cucumber fusarium wilt, tomato root rot, potato powdery scab and other diseases. The strain not only has the function of preventing diseases, but also can improve the drought resistance of plants, and is a strain with great potential. Through identification, the strain is Bacillus velezensis named as E6. The strain has strong inhibiting effect on cucumber fusarium wilt and potato powdery scab, and the prevention effect can reach more than 80 percent and is higher than that of other biocontrol bacteria. And can inhibit the growth of bacteria by destroying the biofilm formation of pathogenic bacteria, thereby avoiding the possibility that the pathogenic bacteria can generate drug resistance when being used in large quantity. When the E6 strain is fermented and cultured, the growth speed is high, the fermentation can be completed within 28 hours, the viable count can reach 80-130 hundred million/mL after the fermentation is finished, and the production cost is low. Therefore, E6 is a plant soil-borne disease biocontrol bacterium with very high potential, and the invention is proposed.
Deposit description
The strain name is as follows: bacillus belgii
Latin name: bacillus velezensis
The strain number is as follows: e6
The preservation organization: china general microbiological culture Collection center
The depository institution code: CGMCC (China general microbiological culture Collection center)
Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 9 and 15 days 2014
Registration number of the preservation center: CGMCC No.9665
Drawings
FIG. 1 is a diagram showing the growth conditions of wheat 1 day after drought stress.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 isolation and characterization of Bacillus velezensis E6
Isolation of Bacillus E6 Strain
The Bacillus velezensis E6 is obtained by diluting and coating tissue grinding fluid in marigold roots and then separating, and the separation method comprises the following steps:
(1) isolation of bacillus belgii: the roots of plants such as marigold are collected at the test base of agricultural academy in corridor city of Hebei province. Weighing 1g of root, washing with 1% NaClO sterile water, grinding in a sterile mortar, sucking 1mL of grinding fluid from the mortar by a fluid-moving gun, adding into another test tube containing 9mL of sterile water, mixing uniformly, and so on to obtain 10-3、10-4、10-5Solutions of different dilutions. And (3) coating 100 mu L of diluent on a TSA culture medium plate, coating three culture dishes on each gradient, and culturing for 2-4 days at 27-30 ℃. Selecting bacteria with different forms, inoculating to nutrient agar culture medium (NA solid culture medium), and streaking for purification.
Wherein the TSA culture medium comprises the following components in parts by weight: tryptone 15g, soybean peptone 5g, sodium chloride 5g, agar 15g, distilled water 1000mL, pH 7.0, 121 ℃ high pressure sterilization for 30 minutes.
The NA solid culture medium comprises the following components in percentage by weight: 10g of glucose, 10g of peptone, 3g of beef extract, 1g of yeast extract, 18g of agar and 1000mL of distilled water, adjusting the pH value to 7.0, and carrying out autoclaving at 121 ℃ for 30 minutes.
(2) Inhibition of pathogenic fungi by bacillus E6
Activated pathogenic fungi such as fusarium oxysporum, fusarium solani, rhizoctonia solani and the like are made into fungus cakes by a 5mm puncher and inoculated in the center of a PDA (personal digital assistant) flat plate, the bursiting of the bacillus beijerinckii E6 is performed at a position 2cm away from the fungus cakes, and the process is repeated for 3 times, and only inoculated pathogenic bacteria are used as blank control. And (4) culturing at 26 ℃ for 3-4 days, measuring the diameter of the colony, and calculating the colony growth inhibition rate. The results show that the bacillus E6 has the inhibiting effect on the pathogenic fungi.
TABLE 1 inhibitory Effect of Bacillus E6 on pathogenic fungi
(3) Inhibition of pathogenic bacteria by bacillus E6
Effect on the growth of pathogenic bacteria：
Make ralstonia solanacearum softInoculating the Cytophthora and Bacillus belgii E6 strains into 5mL beef extract peptone liquid culture solution, performing shake culture at 30 ℃ at 180 rpm, culturing Ralstonia solanacearum and Ralstonia solanacearum for 12-18 hours, counting by using a blood counting plate, and adjusting the concentration of pathogenic bacteria suspension to 108and/mL. Sucking 100 μ L of pathogenic bacteria culture solution, adding into beef extract peptone culture medium plate, coating, and air drying. After culturing the Bacillus belgii E6 for 48h, transferring the Bacillus belgii E6 into a centrifuge tube, centrifuging the Bacillus belgii E6 for 10 minutes at 4 ℃ at 10000 rpm, and filtering the supernatant with a 0.22 mu m filter membrane for later use. The Oxford cup was placed on a medium coated with pathogenic bacteria and 200. mu.L of sterile fermentation filtrate of Bacillus belgii E6 was added, each pathogen being replicated in 3 plates. The culture dish is placed in an incubator at 30 ℃ for culture, and after 24-48 hours, the diameter (mm) of the bacteriostatic circle is measured by a vernier caliper. The result shows that an obvious inhibition zone is generated around the oxford cup added with the bacillus E6 fermentation filtrate, and the diameter is more than 16 mm.
Inhibiting effect on pathogenic bacteria biofilm formation：
Respectively adding 150 mu L of pathogenic bacteria suspension into a flat-bottom 96-hole polystyrene cell culture plate, then adding 50 mu L of sterile filtrate of Bacillus beilaisi E6, placing the mixture into an incubator at 28-30 ℃ for standing culture for 2 hours, sucking out the culture solution and pathogenic bacteria which are not adhered to the culture plate by using a liquid transfer gun, gently rinsing the culture solution twice by using a phosphate buffer solution, finally, adding 200 mu L of beef extract peptone liquid culture solution, and standing culture for 24-36 hours until the biofilm is formed. The turbidity of the cells and the surface filming were observed. The result shows that the bacterial strain E6 sterile fermentation filtrate obviously inhibits the formation of the biofilm of pathogenic bacteria, and the inhibition rate is 43.0 percent and 45.8 percent respectively.
Identification of Bacillus E6 Strain
Genomic DNA from strain E6 was extracted, the 16S rDNA sequence was amplified with universal primers 27f and 1492r and sequenced.
27f：5′-AGA GTT TGA TCC TGG CTC AG-3′；1492r：5′-TAC GGC TAC CTT GTTACGACT T-3′。
The sequence of the 16S rDNA of strain E6 has been filed in GenBank under accession number MG 550967. In view of the above molecular biological characterization results, the strain E6 isolated and purified in the first step was identified as Bacillus belief (Bacillus velezensis). The Bacillus velezensis has been preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu No. 1 of Beijing Kogyo Beichen) in 9.15.2014, and the preservation number is CGMCC No. 9665.
Example 2:
preparation of Bacillus velezensis (Bacillus velezensis) E6 microbial inoculum
1. The lawn of Bacillus belgii (Bacillus velezensis) E6 CGMCC No.9665 obtained in example 1 is transplanted into Nutrient Broth (NB), and shaking culture is carried out on a shaker at 28-30 ℃ for 20h to obtain a seed solution.
Wherein, the formula of the Nutrient Broth (NB) is as follows: 10g of glucose, 10g of peptone, 3g of beef extract, 1g of yeast extract and 1000mL of distilled water, adjusting the pH value to 7.0-7.2, and carrying out autoclaving at 121 ℃ for 30 minutes.
2. Inoculating the seed solution obtained in the step (1) into a large amount of culture fermentation culture solution according to the proportion (volume ratio) of 1%, wherein the culture temperature is 30-32 ℃, the rotating speed is 150-180 r/min, the initial pH is 7.0-7.2, and the culture time is 28 h. And obtaining fermentation liquor after fermentation, wherein the viable count of Bacillus velezensis (Bacillus velezensis) E6 CGMCC No.9665 in the fermentation liquor can reach 120 hundred million/mL.
3. And (3) uniformly mixing bacterial sludge obtained by centrifuging the Bacillus beiLeisi fermentation liquor obtained in the step (2) with an adsorption carrier, namely rice hull powder and diatomite (the volume ratio is 2: 1) according to the volume-mass ratio of 1: 1 (namely 1L: 1Kg), so as to obtain the microbial inoculum. The centrifugal rotating speed is 11500 r/min, and the centrifugal time is 30 minutes; the viable count of Bacillus belgii (Bacillus velezensis) E6 CGMCC No.9665 in the microbial inoculum can reach 1000 hundred million/g, and the water content is within 5 percent.
Example 3: disease prevention effect of bacillus E6 microbial inoculum
Prevention and treatment effect on cucumber fusarium wilt: firstly, culturing fusarium by using Armstrong culture solution, performing microscopic examination counting after 3-4 days, and diluting the bacterial solution to 5 x 104cfu/mL for standby; diluting E6 microbial inoculum to 1 × 107cfu/mL. The spore suspension of fusarium is mixed with the E6 microbial inoculum in equal volume, and then the sterilized seeds are added into the mixed solution for soaking for 30 minutes. Cucumber seeds were sown 3 seeds per replicate per 10 replicates of treatment, and a treatment without application of microbial inoculum was used as a control. The cucumber growth was investigated 3 weeks after sowing.
The Armstrong culture solution comprises the following components in percentage by weight: glucose 20.0g, KCl 1.6g, MgSO4.7H2O0.4g、Ca(NO3)25.9g、KH2PO41.1g、FeSO40.2μg/mL、ZnSO40.2μg/mL、MnSO40.2. mu.g/mL, 1000mL of distilled water, and autoclaving at 121 ℃ for 30 min.
Wherein, FeSO4、ZnSO4、MnSO4The mother liquor can be prepared first. Namely, 0.02g of the three medicines are respectively weighed in 100mL of distilled water, and 1mL of the three medicines is taken in 999mL of mixed solution after being uniformly mixed.
The investigation result (table 2) shows that the control effect of the E6 microbial inoculum on cucumber fusarium wilt can reach 84.2%.
TABLE 2E 6 preventive effect of bacterial agents on cucumber fusarium wilt
Control effect on tomato root rot：
Collecting the diseased soil of the rhizoctonia, mixing the diseased soil with a sterile seedling culture substrate according to the mass ratio of 1: 1, and then putting the mixture into a square seedling culture box of 16.5cm multiplied by 11.5 cm. A small trench with the width of about 4cm and the depth of 6cm is dug in the diseased soil. Mixing the E6 bacterial agent with sterile matrix to give a final concentration of 106cfu/g. And then spreading the microbial inoculum into the ditches, transplanting the tomato seedlings into the ditches, and sealing the ditches with the diluted microbial inoculum, wherein 20g of the microbial inoculum needs to be spread in each ditch. Transplanting 10 seedlings in each pot after treating the repetition of 3 flowerpots, and putting the seedlings in a greenhouse after transplantingAnd (5) culturing. The temperature was 32 ℃ day and 22 ℃ night. The disease incidence is investigated after 9 days of transplantation, and the disease prevention effect is calculated. The result (table 3) shows that the control effect of the E6 microbial inoculum on the tomato root rot can reach 69.3%.
TABLE 3 soil disease method for determining the disease prevention effect of E6 microbial inoculum on tomato root rot
Prevention and treatment effect on potato powdery scab and early blight：
The seed potatoes are treated by the E6 microbial inoculum, the using amount is 1000 g/mu, the contrast is methyl thiophanate seed dressing, the using amount is 300 g/mu, potato pieces are dug out for investigation when the growing period is over, the surfaces of the potato pieces treated by the E6 microbial inoculum are smooth, the disease index of potato powdery scab is obviously reduced, and the control effect can reach 86.58%; the yield per mu increased by 17.2% (table 4).
TABLE 4E 6 prevention and treatment of powdery crust disease of potato by bacterial agent potato treatment
Example 4: growth promoting and drought resisting functions of bacillus E6
And taking field soil and filling the field soil into a flowerpot. Wheat seeds are soaked in the microbial inoculum and then sowed in soil. The concentration of the E6 microbial inoculum is 108cfu/g, 20 wheat grains per pot, 5 replicates per treatment. Wheat seeds without seed soaking were used as control CK. And (3) performing water shortage treatment for 2 days at 3 weeks after sowing, then watering, and measuring the plant height, root fresh weight and plant fresh weight of the wheat after 3 days.
The survey result shows that: the wheat seeds are treated by the E6 microbial inoculum, so that the growth of plants (shown in table 5) can be promoted, and the root system is developed; after drought stress, the growth was good, dark green in color, while the control leaves were yellow (fig. 1).
TABLE 5 Effect of E6 inoculum on wheat seed Germination and growth
Claims (8)
1. Bacillus velezensis E6, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 9665.
2. An agent, wherein the active ingredient of the agent is Bacillus velezensis E6 according to claim 1.
3. The microbial inoculum of claim 2, wherein: the microbial inoculum is a liquid microbial inoculum, and the number of live Bacillus belief (Bacillus velezensis) E6 in the liquid microbial inoculum is 80-130 hundred million/mL.
4. The microbial inoculum of claim 2, wherein: the microbial agent is a solid microbial agent, the solid microbial agent is formed by mixing fermentation liquor of Bacillus velezensis E6 and an adsorption carrier according to the proportion of 1L: 1Kg to 1L: 3Kg, or the bacterial sludge of the Bacillus velezensis E6 fermentation liquor after centrifugation and the adsorption carrier are mixed according to the proportion of 1L: 1Kg to 1L: 3 Kg.
5. The method for preparing the microbial inoculum according to claim 4, comprising the following steps:
(1) culturing the Bacillus subtilis E6 of claim 1 to obtain a fermentation broth of Bacillus subtilis E6;
(2) and (2) mixing the fermentation liquor obtained in the step (1) or bacterial sludge obtained after the fermentation liquor is centrifuged with an adsorption carrier according to the proportion of 1L: 1Kg to 1L: 3Kg to obtain the microbial inoculum.
6. The method for preparing the microbial inoculum according to claim 5, wherein the culture medium solvent for culturing the Bacillus subtilis E6 of claim 1 in the step (1) is water, and the culture medium solutes and concentrations are as follows: 0.4-0.8% of cane sugar, 1.5-1.8% of corn starch, 1.2-1.5% of bean cake powder and 0.2-0.5% of calcium carbonate; wherein,% represents mass percentage content.
7. The application of the Bacillus velezensis E6 according to claim 1, the microbial inoculum according to any one of claims 2 to 4 and the microbial inoculum prepared by the method according to any one of claims 5 to 6 in preventing and treating plant soil-borne diseases, promoting plant growth and improving plant drought resistance.
8. The use according to claim 7, wherein the plant soil-borne disease is a soil-borne disease caused by Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Spongospora uvarerana, Ralstonia solani, Pectobacterium chrysogenum, and Erwinia carofavora.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810017581.8A CN108004185B (en) | 2018-01-09 | 2018-01-09 | Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810017581.8A CN108004185B (en) | 2018-01-09 | 2018-01-09 | Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108004185A CN108004185A (en) | 2018-05-08 |
| CN108004185B true CN108004185B (en) | 2020-04-17 |
Family
ID=62050335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810017581.8A Active CN108004185B (en) | 2018-01-09 | 2018-01-09 | Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108004185B (en) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
| CN108587974B (en) * | 2018-05-15 | 2019-12-17 | 湖南农业大学 | Bacillus belgii, application and preparation method of fermentation liquor of bacillus belgii |
| CN108641981B (en) * | 2018-05-17 | 2020-10-02 | 十堰市农业科学院 | Biocontrol bacillus identification and application |
| CN108753664B (en) * | 2018-06-28 | 2021-03-26 | 广东省农业科学院农业资源与环境研究所 | Biological control agent, biological organic fertilizer and application thereof |
| CN109022305B (en) * | 2018-07-18 | 2020-11-13 | 浙江大学 | Polygonatum cyrtonema endophytic bacterium strain ZJU-C612-1 and application thereof |
| CN109077067B (en) * | 2018-08-16 | 2021-04-30 | 南京农业大学 | Biocontrol bacterium and application thereof in prevention and control of gray mold of crops |
| CN110982724A (en) * | 2018-09-29 | 2020-04-10 | 福建省农业科学院农业生物资源研究所 | Bacillus for antagonizing phytopathogen and promoting rooting and application thereof |
| CN111073825B (en) * | 2018-10-19 | 2022-07-12 | 沈阳药科大学 | Bacterium with plant soil-borne disease resistance effect and application thereof |
| CN109566656A (en) * | 2018-12-20 | 2019-04-05 | 江苏师范大学 | Bei Laisi bacillus is promoting the application in Garlic Growth |
| CN109652336A (en) * | 2019-01-22 | 2019-04-19 | 广西金穗生态科技股份有限公司 | One plant of Bei Laisi bacillus and its application |
| CN109749968B (en) * | 2019-03-04 | 2020-07-17 | 河北省科学院生物研究所 | Bacillus beiLeisi strain for biocatalytic synthesis of (R) -1, 3-butanediol and application thereof |
| CN110184221B (en) * | 2019-06-05 | 2021-03-05 | 中国农业科学院蔬菜花卉研究所 | Bacillus belgii and application thereof in prevention and treatment of phytophthora root rot of tomato |
| CN110283742B (en) * | 2019-06-17 | 2020-12-15 | 北京农业生物技术研究中心 | Broad-spectrum disease-resistant Bacillus belgii and application thereof |
| CN111234830A (en) * | 2019-12-20 | 2020-06-05 | 云南省微生物发酵工程研究中心有限公司 | Microbial modifier for improving soil aggregate structure and application thereof |
| CN110981610A (en) * | 2019-12-20 | 2020-04-10 | 云南省微生物发酵工程研究中心有限公司 | Microbial agent with disease prevention function and preparation method thereof |
| CN110982749B (en) * | 2019-12-26 | 2021-10-29 | 湖北省生物农药工程研究中心 | Bacillus belgii and application thereof in preparation of rice disease-resistant agent |
| CN113215010B (en) * | 2020-01-21 | 2022-08-30 | 中国农业科学院蔬菜花卉研究所 | Bacillus belgii ZF128 and application thereof in preventing and treating potato wilt |
| CN111172080B (en) * | 2020-02-24 | 2021-05-18 | 山东农业大学 | Bacillus belgii and application thereof |
| CN111518700B (en) * | 2020-03-13 | 2021-06-01 | 上海市农业科学院 | Carbon-based composite microbial inoculant and application thereof |
| CN112746033B (en) * | 2020-03-26 | 2022-10-14 | 湖南鑫业农牧科技有限公司 | Bacillus belgii, microbial inoculum and application of bacillus belgii in fermentation production of organic fertilizer |
| CN111304135B (en) * | 2020-04-10 | 2022-06-07 | 宁波市农业科学研究院 | Bacillus and application thereof in plant disease control |
| CN111961629B (en) * | 2020-08-28 | 2022-12-20 | 山东省果树研究所 | Cherry preservative and fresh-keeping microbial preparation and preparation method and application thereof |
| CN112167267A (en) * | 2020-11-04 | 2021-01-05 | 河南柏裕植物免疫科技有限公司 | Compound biological agent for preventing and treating tomato root rot and preparation method thereof |
| CN112586509A (en) * | 2020-11-13 | 2021-04-02 | 遵义医科大学 | Compound microorganism effervescent tablet for preventing tobacco bacterial wilt and promoting growth, and preparation method and application thereof |
| CN112920966A (en) * | 2020-12-31 | 2021-06-08 | 浙江省农业科学院 | Bacillus belgii, microbial agent thereof, preparation method of microbial agent and application of microbial agent |
| CN112940962B (en) * | 2021-01-07 | 2021-09-14 | 华南农业大学 | Bacillus belgii and application thereof in improving copper pollution in water body |
| CN112680380B (en) * | 2021-01-19 | 2022-10-04 | 深圳市芭田生态工程股份有限公司 | Preparation and application of biocontrol bacillus beleisi and microcapsule microbial inoculum |
| CN112899201B (en) * | 2021-03-16 | 2021-12-07 | 云南省农业科学院农业环境资源研究所 | Bacillus belgii, application thereof and method for preventing and treating banana wilt |
| CN113088469B (en) * | 2021-04-09 | 2022-02-22 | 中国科学院生态环境研究中心 | Bacillus belgii AM6, microbial inoculum and preparation method and application thereof |
| CN113403218B (en) * | 2021-05-13 | 2022-07-01 | 南京本源生态农业科技有限公司 | Biocontrol strain and application thereof in preventing and treating soft rot of thunberg fritillary bulb and promoting growth of thunberg fritillary bulb |
| CN114231437B (en) * | 2021-11-08 | 2023-06-09 | 南京农业大学 | Bacillus bailii DMW1 strain and application thereof |
| CN114058549B (en) * | 2021-11-29 | 2023-07-11 | 中国农业科学院农产品加工研究所 | Bacillus bailii Shu-6, production method and application |
| CN113969254B (en) * | 2021-12-07 | 2023-05-26 | 山东农业大学 | Sesbania endophytic bacillus ZH60, composite microbial inoculant and corn seed culture method |
| CN114276957B (en) * | 2021-12-20 | 2023-09-05 | 中农新科(苏州)有机循环研究院有限公司 | Biocontrol strain with effect of preventing and controlling various plant pathogenic fungi and application thereof |
| CN114317371A (en) * | 2022-01-12 | 2022-04-12 | 中国科学院东北地理与农业生态研究所 | Biocontrol growth-promoting bacterium for rice blast and application thereof |
| WO2023138678A1 (en) * | 2022-01-21 | 2023-07-27 | 慕恩（广州）生物科技有限公司 | Bacillus velezensis and use thereof |
| CN114672431B (en) * | 2022-03-25 | 2023-06-20 | 青岛农业大学 | Bacillus composite microbial inoculant for preventing and treating bacterial wilt and application thereof |
| CN114657097B (en) * | 2022-03-25 | 2023-04-07 | 青岛农业大学 | Bacillus belgii LGT-1 capable of efficiently antagonizing ralstonia solanacearum and application thereof |
| CN114836351B (en) * | 2022-05-19 | 2023-04-07 | 河北省科学院生物研究所 | Compound microbial agent for killing insects and preventing diseases and biological fertilizer containing microbial agent |
| CN115287214B (en) * | 2022-05-25 | 2023-04-14 | 甘肃省农业科学院植物保护研究所 | Compound microorganism, compound microbial agent and application |
| CN116445376B (en) * | 2023-06-15 | 2023-11-14 | 中国农业科学院农业资源与农业区划研究所 | Bacillus bailii and application thereof |
| CN116731928B (en) * | 2023-07-03 | 2024-03-26 | 宜春市科学院(江西富硒产业研究院) | Bacillus bailii YC5 and application thereof in preventing and treating black skin disease of Chinese yam |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101190650B1 (en) * | 2010-07-23 | 2012-10-17 | 대한민국 | A novel Bacillus velezensis B-42 for multi function |
| CN102286412B (en) * | 2011-08-30 | 2013-02-13 | 新疆农业科学院微生物应用研究所 | Bacillus velezensis and application thereof in tomato blight antagonism |
-
2018
- 2018-01-09 CN CN201810017581.8A patent/CN108004185B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108004185A (en) | 2018-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108004185B (en) | Plant endophytic Bacillus belgii with disease prevention, growth promotion and drought resistance functions and application thereof | |
| CN111172080B (en) | Bacillus belgii and application thereof | |
| CN105886428B (en) | One plant of Streptomycesalbidoflhaving and its application in microbial manure | |
| CN106591185B (en) | Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof | |
| CN101525587B (en) | Streptomyces strain and application thereof | |
| WO2004050861A1 (en) | The microbial preparation & method for preventing and curing the bacterial wilt the plant and its use | |
| CN109022320B (en) | Siamese bacillus and application thereof in preventing and treating root rot and southern blight | |
| CN109370955B (en) | Bacillus vallismortis HSB-2 and application thereof | |
| CN107325821B (en) | Biomass charcoal-based microbial inoculant for preventing and treating watermelon fusarium wilt and preparation method and application thereof | |
| CN102086444A (en) | Paenibacillus elgii strain and application thereof | |
| CN112795496B (en) | Paenibacillus polymyxa and application thereof in preventing and treating stem basal rot of Chinese cabbage | |
| CN105505836A (en) | Bacillus subtilis and methods for preparing and using bacillus subtilis microbial agent for controlling potato scab | |
| CN110295129B (en) | Biocontrol bacterium for preventing and treating gray mold and powdery mildew of cucumber and application thereof | |
| CN108893436B (en) | Saline-alkali-tolerant streptomyces flavochraceus and application thereof | |
| CN108220194B (en) | Streptomyces parvus with disease prevention and growth promotion functions and application thereof | |
| CN107287130A (en) | A kind of Streptomycesalbidoflhaving bacterial strain and its application in agricultural chemicals | |
| CN111808776B (en) | Saline-alkali-tolerant air bacillus and preparation method and application of viable bacteria preparation thereof | |
| CN104988098B (en) | One plant of prevention root rot of beets and the Bacillus strain for promoting Sugarbeet Growth | |
| CN102154178B (en) | Bacillus brevis for preventing and treating hot pepper epidemic disease as well as preparation method and application of biological agent | |
| CN116622547A (en) | Bacillus mojavensis YL-78 and application thereof | |
| CN116496922A (en) | Bacillus amyloliquefaciens and application thereof | |
| CN114921365A (en) | Streptomyces rochei and application thereof | |
| CN114456973A (en) | Streptomyces rochei strain in tobacco and application thereof in prevention and control of tobacco diseases | |
| CN114916563B (en) | Bacillus amyloliquefaciens and application of synergist thereof | |
| CN117384807B (en) | Bacillus mycoides HDMM 2 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220812 Address after: No. 888, south side of Xinle Xinwu Road, Shijiazhuang City, Hebei Province, 050799 Patentee after: Hebei Inuo Biochemical Co., Ltd. Address before: 100193 Institute of plant protection, Chinese Academy of Agricultural Sciences, 2 West Old Summer Palace Road, Beijing, Haidian District Patentee before: INSTITUTE OF PLANT PROTECTION CHINESE ACADEMY OF AGRICULTURAL SCIENCES |
|
| TR01 | Transfer of patent right |