CN110079484B - Bacillus subtilis and application thereof in agricultural production - Google Patents
Bacillus subtilis and application thereof in agricultural production Download PDFInfo
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Abstract
The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus subtilis and application thereof in agricultural production. The bacillus subtilis sieve is selected from calcareous soil, and the preservation number is CCTCC NO of M2019402. The strain has strong enzyme production capability, can effectively improve the content of available phosphorus in soil, increase the crop yield, and can effectively prevent and treat common plant diseases such as root rot, scab and the like, and the effect is obvious.
Description
Technical Field
The invention relates to the technical field of screening and application of functional microorganisms, in particular to novel bacillus subtilis and application thereof in agricultural production.
Background
Phosphorus is a necessary nutrient element for plant growth, but phosphorus in soil is easy to be fixed to cause effective phosphorus deficiency, so how to convert insoluble phosphorus in soil into soluble effective phosphorus is the key to solve the problem of phosphorus deficiency. The phosphorus-solubilizing microorganisms can activate soil insoluble phosphorus, and have become one of the hot spots of the current research.
Phosphate-Solubilizing Microorganisms (PSM) can convert insoluble Phosphate in soil into Phosphate which can be absorbed and utilized by plants by utilizing self metabolites or synergistic action with other organisms. The types of phosphate solubilizing microorganisms in soil are various, including bacteria, fungi, actinomycetes and the like. The most varied bacteria include bacteria of 16 genera, such as Bacillus (Bacillus), Enterobacter (Enterobacter), Salmonella (Salmonella), Erwinia (Erwinia), Alcaligenes (Alcaligenes), Azotobacter (Azotobacter), Pseudomonas (Pseudomonas), Agrobacterium (Agrobacterium), Serratia (Serratia), Flavobacterium (Flavobacterium), Micrococcus (Micrococcus), Rhizobium (Bradyrhizobium), Chromobacter (Clomobacterium), Thiobacillus (Thiobacillus), Escherichia (Escherichia), Arthrobacter (Arthrobacter), and the like. The phosphate-solubilizing fungi are also an important component of phosphate-solubilizing microorganisms in soil, but the number of the phosphate-solubilizing fungi is far less than that of the phosphate-solubilizing bacteria, and the types of the phosphate-solubilizing bacteria are relatively few. The phosphate solubilizing fungi reported so far mainly include Aspergillus (Aspergillus), Rhizopus (Rhizopus), Penicillium (Penicillium), Fusarium (Fusarium), Sclerotium (Sclerotium), and the like, and among them, Aspergillus and Penicillium are most studied. In addition, researches show that mycorrhizal fungi (Arbuscular mycorrhiza) can also promote the host to absorb and utilize insoluble phosphorus in soil, and the content of available phosphorus in the soil can be obviously improved by inoculating the Arbuscular mycorrhizal fungi and the phosphate solubilizing bacteria simultaneously.
The most important application of phosphate-solubilizing microorganisms is to use the phosphate-solubilizing microorganisms as microbial fertilizers in agricultural production. After the phosphate solubilizing microorganisms are applied to soil, the yield and the quality of crops can be remarkably improved, the utilization efficiency of phosphorus in the soil is further improved, and plants can be helped to adsorb microelements such as calcium, magnesium and iron at the rhizosphere. Some phosphate solubilizing bacteria are rapidly propagated around the rhizosphere of the crops, can inhibit the growth of other pathogenic microorganisms, play the role of reducing the diseases of the crops and effectively improve the yield of the crops. The Zhaoyangqiong combines the screened high-efficiency phosphate-solubilizing bacterial strain pseudomonas Y2 with bacillus amyloliquefaciens T-5 and NJN-6, and is matched with a certain proportion of inorganic fertilizer to develop a compound microbial fertilizer by a disc granulation method, and the result shows that the Zhaoyiqiong compound microbial fertilizer has better growth promotion effect on tomatoes, eggplants, potatoes, corns, tobaccos and the like. The von Ruiz et al can make phosphate-solubilizing microorganisms into microbial inoculum, and field test results show that the biomass of oat can be remarkably improved by applying the microbial inoculum and half amount of phosphate fertilizer in combination. In addition, in the process of developing the phosphate-solubilizing microbial fertilizer, the recycling of agricultural wastes such as crop straws, livestock and poultry manure and the like can be realized.
The research on the phosphate solubilizing microorganisms at home and abroad has been carried out for a long time, but many researches are still not mature due to the various types of the phosphate solubilizing microorganisms. At present, the development of phosphate-solubilizing microbial products in China is still in the initial stage, the types of the products sold in the market are relatively few, the using effect is unstable, and the popularization difficulty is high. Therefore, screening of microbial strain resources for efficient phosphate solubilizing is still the primary task of the development of the phosphate solubilizing microbial products at present.
Disclosure of Invention
The invention provides a new bacillus subtilis and application thereof in agricultural production for solving the problems of the prior art. The bacillus subtilis screen is selected from calcareous soil, has strong enzyme production capacity, can effectively improve the content of available phosphorus in the soil, increase the crop yield, and can effectively prevent and treat common plant diseases such as root rot, scab and the like, and has remarkable effect.
The invention provides a Bacillus subtilis LLH-3(Bacillus subtilis LLH-3) which is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan, China in 2019, 5 and 27 days, and the preservation number is CCTCC NO: and M2019402.
The application of the bacillus subtilis in biological fertilizer.
The application of the bacillus subtilis in the prevention and treatment of plant diseases.
In another aspect, the present invention provides a microbial preparation comprising the Bacillus subtilis LLH-3 described above.
The microbial preparation also comprises any one or a combination of two or more of bacillus, pseudomonas, agrobacterium, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces.
The viable bacteria content of Bacillus subtilis LLH-3 in the microbial preparation is at least 109CFU/g。
The invention also provides application of the microbial preparation in a biological fertilizer.
The invention also provides application of the microbial preparation in plant disease control.
The plant diseases comprise any one of root rot, southern blight, powdery mildew, scab, gray mold, blight, brown spot and fruit rot.
Advantageous effects
The bacillus subtilis LLH-3 screened by the invention can convert insoluble phosphorus (Ca)3(PO4)2) Decomposing into soluble effective phosphorus with phosphorus dissolving efficiency up to 85%, and the strain has strong enzyme production capability, fermenting and culturing for 28-32h, the phytase activity in the supernatant is up to 13.5U/mL, and the cellulase activity is up to 4.04U/mL. The bacillus subtilis LLH-3 can also obviously improve the seedling quality of rice, and the plant height, stem thickness, root number, leaf age and second leaf length of rice seedlings and the chlorophyll content are respectively improved by 24.1%, 27.3%, 29.3%, 30.9%, 10.3% and 10.2%. In addition, the bacillus subtilis LLH-3 has obvious control effects on root rot, southern blight, scab and brown spot, wherein the control efficiency on the root rot and the scab is over 80 percent, the control efficiency on the southern rot and the brown spot is over 60 percent, and the control efficiency on the southern rot and the brown spot is over 60 percentThe treatment effect is obviously better than that of the traditional chemical bactericide.
The bacillus subtilis LLH-3 provided by the invention can be used alone or combined with other microorganisms with a phosphate solubilizing effect or a biocontrol effect to serve as a biological fertilizer and a biocontrol microbial inoculum, so that the soil environment is improved, the soil fertility is improved, common plant diseases are prevented and treated, the bacillus subtilis LLH-3 is environment-friendly, the quality of crops is improved, the conversion of traditional agriculture to ecological agriculture and green agriculture is promoted, and the healthy and sustainable development of agriculture is realized.
Detailed Description
The invention is further illustrated by the following specific examples. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention. The equipment and reagents used in the present invention may be selected from any commercially available ones.
Example 1 screening of microorganisms having phosphate solubilizing function
1. Soil sample: calcareous soil in the southwest mountain area of Qingzhou city, Shandong province.
2. Preparing a soil diluent: 0.5g of a soil sample was weighed and dissolved in 4.5ml of sterile water to make 1: 10, then sucking 0.5ml of the soil solution from the soil solution and putting the soil solution into 4.5ml of sterile water to prepare a mixture of 1: 100, by analogy with this method, 1: 106-107The soil dilution solution of (1).
0.1ml of the diluted soil solution was applied to a hardly soluble inorganic phosphorus solid medium (10 g of glucose, (NH)4)2SO40.5g,NaCl 0.3g,KC1 0.3g,MgSO4·7H20 0.3g,FeSO4.7H20 0.03g,MnSO4.4H20 0.03g,Ca3(PO4)25.0g, 1000ml of distilled water, pH 7.0-7.5, agar 20g, sterilized at 115 ℃ for 30min), inversely culturing in an incubator at 30 ℃ for 3 days, observing colonies growing on the culture medium, wherein 12 colonies among them produce obvious color changes around and have transparent circles, and are named LLH-1, LLH-2, LLH-3, … … and LLH-12 respectively.
EXAMPLE 2 rescreening of phosphate solubilizing microorganisms
12 strains of the bacteria preliminarily screened in example 1 were inoculated on a solid culture medium of hardly soluble inorganic phosphorus, and after culturing at 30 ℃ for 3 days, the size of a transparent circle around the colony was observed, and as a result, it was found that the three strains having the largest transparent circles were LLH-2, LLH-3, and LLH-10, respectively.
The three strains with the largest transparent circle were inoculated into 50mL of a hardly soluble inorganic phosphorus liquid medium (glucose 10g, (NH)4)2SO4 0.5g,NaCl 0.3g,KC1 0.3g,MgSO4.7H20 0.3g,FeSO4.7H20 0.03g,MnSO4.4H20 0.03g，Ca3(PO4)25.0g, distilled water 1000ml, pH 7.0-7.5, sterilized at 115 ℃ for 30min), cultured at 30 ℃ and 200rpm for 6 days while using a liquid phosphate solubilizing medium without any bacteria as a control group; respectively detecting the number of viable bacteria in the culture solution.
2.1 drawing of phosphorus Standard Curve
Sequentially sucking 0.0, 0.2, 0.4, 0.8, 1.6, 2.0, 3.2 and 4.0ml of phosphorus standard solution of 5mg/l into a test tube, then respectively adding 2ml of molybdenum-antimony color-resisting agent, fixing the volume to 20ml with distilled water, shaking up and standing for 20min, and measuring the absorbance at the wavelength of 700 nm. The phosphorus concentration in each tube was then divided into: 0.00, 0.05, 0.10, 0.20, 0.40, 0.50, 0.80, 1.00 mg/l. And drawing a phosphorus standard curve by taking the phosphorus concentration as an abscissa and the absorbance as an ordinate.
2.2 determination of the effective phosphorus content of the culture solution
Taking 5ml of each culture solution of the three strains under aseptic conditions, centrifuging at 8000rpm for 5min, taking supernatant, diluting to a proper concentration, sucking 0.5ml of diluent into a test tube, adding 5ml of distilled water, adding 2 drops of 2, 4-dinitrophenol indicator, adding 2ml of molybdenum-antimony anti-color-developing agent, then using distilled water to fix the volume to 20ml, shaking uniformly and standing for 20min, carrying out color comparison at 700nm wavelength, substituting the absorbance value into a standard curve to calculate the effective phosphorus content in the supernatant, and obtaining the specific result shown in table 1.
TABLE 1 phosphate solubilizing Effect of different strains
| Sample (I) | Viable count (CFU/mL) | Effective phosphorus content (mg/L) |
| Blank control group | 0 | 0 |
| LLH--2 | 106-107 | 332 |
| LLH--3 | 107-108 | 856 |
| LLH--10 | 107-108 | 562 |
As can be seen from the data in Table 1, the three strains of bacteria screened from the soil sample by the applicant all have strong phosphate solubilizing ability and can convert the insoluble phosphate (Ca) in the culture medium3(PO4)2) Decompose into soluble available phosphorus with remarkable effect. Wherein, the phosphorus dissolving efficiency of the LLH-3 strain can reach 85 percent at most, the effective phosphorus content reaches 856mg/L, and unexpected effects are achieved.
Example 3 identification of LLH-3 Strain
1) Colony morphology of LLH-3 Strain:
the colony of the LLH-3 strain is flat, rough in surface, opaque, rough after the initial smooth edge, white to dirty white, with spore size of about 0.5-0.9 × 0.8-1.3 μm, oval or columnar, mesogenic or near-mesogenic; gram reaction positive, catalase reaction positive, V.P reaction positive, starch hydrolysis test positive, indole test positive, can utilize glucose, arabinose, xylose and mannitol, the growth temperature range is 20-45 ℃, and the pH range is 5-10.
2) Molecular biological characterization of LLH-3 strains:
the LLH-3 strain obtained by screening is identified by adopting a molecular biology method, the 16s rDNA sequence SEQ ID NO:1 is measured, and blast comparison is carried out in a GenBank nucleic acid database.
SEQ ID NO 1 is shown below:
the applicant confirms that the LLH-3 strain is Bacillus subtilis (Bacillus subtilis) and is named as Bacillus subtilis LLH-3(Bacillus subtilis LLH-3) according to the colony morphology of the LLH-3 strain and the comparison result of 16 srDNA.
The applicant has already preserved the Bacillus subtilis LLH-3(Bacillus subtilis LLH-3) in 27.5.2019 in China, China center for type culture collection, Wuhan university, Wuhan, China, with the preservation number of CCTCC NO: M2019402.
Example 4 determination of enzyme-producing Activity of Bacillus subtilis LLH-3
1. Preparation of bacterial liquid
Selecting a fresh circle of Bacillus subtilis LLH-3 bacterial mud, inoculating into 100mL LB culture medium (1000mL water, 10.0 peptone, 5.0 yeast extract powder, 5.0gNaCl, pH7.0-7.2), culturing at 30 deg.C and 200rpm for 12-16h to obtain seed solution; inoculating the seed solution into sterilized nutrient broth culture medium at an inoculum size of 5%, culturing at 30 deg.C and 200rpm for 28-32 hr, stopping fermentation culture when the microscopic spore yield is above 95%, to obtain viable bacteria content of 108-109cfu/ml fermentation broth.
2. Enzyme activity detection
Centrifuging the fermentation liquor at 4 deg.C and 8000rpm for 5min, collecting supernatant, and determining phytase and cellulase activity in the supernatant according to the following method. The result shows that the bacillus subtilis LLH-3 obtained by screening has stronger enzyme production capability, the enzyme activity of the phytase in the fermentation supernatant is as high as 13.5U/mL, the enzyme activity of the cellulase is as high as 4.04U/mL, and unexpected technical effects are obtained.
(1) Method for measuring enzyme activity of phytase
Definition of enzyme activity unit: the enzyme quantity required for releasing 1 mu mol of inorganic phosphorus from the sodium phytate solution with the concentration of 5mg/ml per minute is an enzyme activity unit U under the conditions of 30 ℃ and pH value of 5.0.
The determination method comprises the following steps: taking 4ml of sodium phytate solution with the concentration of 7.5mmol/L (prepared by acetic acid buffer solution with the pH value of 5.00.25mol/L), adding the sodium phytate solution into a colorimetric tube, balancing for 5min at the temperature of 30 ℃, adding 2ml of phytase enzyme solution which is properly diluted by the acetic acid buffer solution with the pH value of 5.00.25mol/L and well balanced at the temperature of 30 ℃, uniformly mixing, and accurately preserving the temperature and reacting for 30min at the temperature of 30 ℃. After the reaction was completed, 4ml of a terminating solution (2 parts nitric acid solution (nitric acid: water: 1:2), 1 part of 100g/L ammonium molybdate solution and 1 part of 2.35g/L ammonium vanadate solution) were added thereto and mixed to terminate the reaction. Then, the mixture is placed at room temperature for 10min for color development, and the absorbance at 415nm of a spectrophotometer is measured.
The enzyme activity calculation formula is as follows:
U＝(A-A0-0.0016)×F/(0.0415×30)
in the formula: a is the light absorption value of the sample; a. the0The light absorption value of a blank sample is obtained; f is the total dilution multiple of the actual sample solution before reaction; and 30 is the enzymolysis reaction time, min.
(2) Method for determining enzyme activity of cellulase
Definition of enzyme activity unit: under the conditions of 50 ℃ and pH value of 6.0, the enzyme amount required by degrading and releasing 1 mu mol of reducing sugar from 5mg/ml of carboxymethyl cellulose sodium solution per minute is one enzyme activity unit U, and the reducing sugar is equal to glucose.
The determination method comprises the following steps: adding 0.5mL CMC substrate into each of the three test tubes, and preheating with the enzyme solution to be tested in 50 deg.C water bath for 5 min. 0.5mL of the solution to be detected was added to each of the first and second test tubes, and the mixture was reacted in a water bath at 50 ℃ for 15min while counting the time. After the reaction, 1.5mL of DNS reagent is added into each of the three test tubes, and 0.5mL of enzyme solution to be detected is added into the third test tube. Taking out and shaking three test tubes evenly, and reacting for 5min in a boiling water bath. It was rapidly cooled to room temperature and adjusted to 5.0mL with water. And (3) measuring the absorbance of the first and second test tube solutions at 540nm wavelength by using the third test tube solution as a control, preferably the absorbance is 0.25-0.35. The absolute value of the difference between the absorbance of the enzyme liquid reaction solution to be detected and the absorbance of the enzyme liquid reaction solution for horizontal control is not more than 0.015.
Enzyme activity X ═ (glucose equivalent/180/15/0.5). times.n
Wherein X is enzyme activity unit, IU/g (mL);
180-conversion of glucose from micrograms to micromoles;
15-reaction time of the solution to be tested and the substrate;
0.5-the amount of enzyme solution to be measured added;
n is the dilution multiple.
Example 5 application of Bacillus subtilis LLH-3 in spinach field cultivation experiment
1. A place: the greenhouse for planting the Laixi spinach in Qingdao city has uniform soil integral condition.
2. Procedure of experiment
30 experimental zones, each of which is a square area of 2m × 2m, were provided with a 1m interval maintained therebetween.
The experiment was carried out in 3 groups: blank control group: no substance is added; ② culture medium treatment group: in each experimental zone at 40mL/m2The sterilized nutrient broth culture medium is uniformly sprayed according to the proportion, and then soil with the thickness of 5-10cm on the surface layer is effectively and uniformly mixed; ③ treatment group of Bacillus subtilis LLH-3: in each experimental zone at 60mL/m2The Bacillus subtilis LLH-3 zymogen liquid (10) described in example 4 is evenly sprayed in the proportion8-109cfu/ml), and then effectively mixing the soil with the thickness of 5-10cm on the surface layer. 10 experimental regions were randomly selected for each group.
1) Seed treatment: sterilizing spinach seed with 5% sodium hypochlorite for 10min, washing with distilled water for 3-4 times to remove sodium hypochlorite, standing at room temperature for 30min, and naturally drying.
2) Sowing and harvesting: 25g of spinach seeds were uniformly sown in each experimental area, watered and managed at regular time without fertilizer application. 50 days after sowing, all spinach were harvested, and the fresh weight and dry weight of spinach in each experimental area were measured, respectively, and the average fresh weight and average dry weight of spinach in each treatment group were calculated and compared.
3) And (3) respectively collecting soil samples of each experimental area while harvesting spinach, detecting the content of available phosphorus in the soil samples by adopting an Olsen method, and comparing.
The test result shows that: compared with a blank control group, the average fresh weight and dry weight of the spinach of the culture medium treatment group are respectively increased by 4.8 percent and 4.1 percent, and the effective phosphorus content is increased by 3.2 percent; the average fresh weight and dry weight of spinach in the bacillus subtilis LLH-3 treated group are respectively improved by 295.7% and 228.2% compared with those in the blank control group, 290.9% and 224.1% compared with those in the culture medium treated group, the effective phosphorus content is improved by 78.5% compared with that in the blank treated group, and is improved by 75.3% compared with that in the culture medium treated group.
The results show that the bacillus subtilis LLH-3 screened by the method can greatly improve the soil fertility, thereby greatly improving the yield of planted crops, and can be widely applied to the field of agricultural production as a biological fertilizer.
Example 6 application of Bacillus subtilis LLH-3 in Rice seedling raising
1. Preparation of fungal powder
Selecting a fresh circle of Bacillus subtilis LLH-3 bacterial mud, inoculating into 100mL LB culture medium, culturing at 30 deg.C and 200rpm for 12-16h to obtain seed liquid; inoculating the seed solution into a sterilized nutrient broth culture medium with an inoculation amount of 5%, culturing at 30 ℃ and 200rpm for 28-32h, and stopping fermentation culture when the microscopic spore yield is more than 95%; centrifuging the fermentation liquid (8000rpm, 5min), collecting thallus, adding dextrin, stirring, vacuum freeze drying to obtain product with bacterial load of 109-1010CFU/g Bacillus subtilis LLH-3 powder.
2. Procedure of experiment
(1) The experimental site: a seedling raising greenhouse in Dandong City of Liaoning province.
(2) Experiment design:
blank control group: only adopting a seedling substrate (purchased from Harbin seedling and strong agriculture science and technology development Co., Ltd.);
② a bacillus subtilis CGMCC1.8886 contrast group: 1.5kg of Bacillus subtilis CGMCC1.8886 bacterial powder (10)9-1010CFU/g) and 1 ton of seedling raising matrix are mixed evenly;
(iii) Bacillus subtilis LLH-3 treatment group: 1.5kg Bacillus subtilis LLH-3 powder (10)9-1010CFU/g) and 1 ton of seedling raising matrix are mixed evenly;
the two treatment groups have the same management of sowing, irrigation, fertilization and the like, and the whole seedling period is about 40 days.
3. Results and analysis of the experiments
(1) Evaluation of Rice seedling morphology and chlorophyll content
Randomly extracting 100 seedlings from the center of each treatment group seedling raising plate, washing the seedlings with clear water, testing the seedlings, and detecting the chlorophyll content of the second leaf. The results are shown in Table 3.
TABLE 2 influence of Bacillus subtilis LLH-3 on growth of rice seedlings
As can be seen from the data in Table 2, compared with the blank control group, the plant height, stem thickness, root number, leaf age, second leaf length and chlorophyll content of the rice seedlings of the Bacillus subtilis LLH-3 treated group provided by the invention are respectively increased by 24.1%, 27.3%, 29.3%, 30.9%, 10.3% and 10.2%, and the effect is very obvious. The rice seedlings treated by the commercial bacillus subtilis CGMCC1.8886 only have the plant height and stem thickness respectively improved by 10.2 percent and 8.9 percent, and the root number, leaf age, second leaf length and chlorophyll content are not obviously increased. Therefore, the bacillus subtilis LLH-3 provided by the invention can obviously improve the seedling raising quality of rice, and the effect is obviously better than that of the bacillus subtilis sold in the market.
(2) Evaluation of rice seedling quality
The dry weight of hundreds of plants and the strong seedling index are important indexes of the quality of seedlings.
The strong seedling index is (stem thickness x 100 dry weight of the whole seedling) per plant height.
The dry weight and seedling raising index of the rice seedlings in the bacillus subtilis LLH-3 treatment group provided by the invention are respectively increased by 38.56% and 58.07% compared with the blank control group, and are respectively increased by 30.48% and 48.76% compared with the bacillus subtilis CGMCC1.8886 control group, so that unexpected technical effects are achieved.
Example 7 determination of bacteriostatic ability of Bacillus subtilis LLH-3
1. Culturing pathogenic fungi:
in a sterile operating table, small blocks of peanut root rot, sclerotium rolfsii, scab and brown spot of about 0.5cm × 0.5cm were inoculated into PDA medium with tweezers, and cultured in an inverted state at 28 deg.C for 3 days.
2. Inoculating the confronting bacterium:
when the pathogenic fungi grow to be about in the culture dish 1/3, inoculating bacillus subtilis LLH-3 at a position 2cm away from the fungi respectively, taking the fungi which are not inoculated with bacillus subtilis LLH-3 as a control, continuously culturing for 3 days at 28 ℃, and then respectively measuring the colony radius of each pathogenic fungus and calculating the bacteriostasis rate.
The inhibition rate is [ (control fungus growth radius-treatment fungus growth radius)/control fungus growth radius ] × 100%.
The results show that the bacillus subtilis LLH-3 has obvious antagonistic action on the four peanut pathogenic fungi. Wherein, LLH-3 has the strongest inhibition effect on peanut root rot and scab, and the inhibition rates are respectively up to 87.4% and 81.6%; the inhibition effect on the southern blight and brown spot is relatively weak, and the inhibition rates are 63.3% and 59.8% respectively.
Example 8 application of Bacillus subtilis LLH-3 in prevention and treatment of peanut diseases
1. The experimental site:
in the peanut continuous cropping field in ShaLingcun after Qingdao flatness, the root rot, southern blight, scab and brown spot of peanuts are serious.
2. Experiment design:
experimental zones are randomly arranged, each experimental zone is a rectangular area of 6m multiplied by 10m, and the interval of more than 3 m is kept between each experimental zone. The row spacing of the peanuts is 40cm, and the plant spacing is 20 cm. Three parallel experimental zones were set up for each experimental group.
(1) Blank control group: clear water;
(2) and (3) a bactericide treatment group: 800 times of 50% carbendazim solution;
(3) b, treatment group of Bacillus subtilis CGMCC 1.8886: respectively using Bacillus subtilis LLH-3 fermentation liquor (10) at the sowing time and 15d, 30d and 45d after sowing8-109cfu/mL) and each plant is watered to dilute about 30mL of fermentation liquor with the amount of 100 times each time;
(4) bacillus subtilis LLH-3 treatment group: respectively using Bacillus subtilis LLH-3 fermentation liquor (10) at the sowing time and 15d, 30d and 45d after sowing8-109cfu/mL) and about 30mL of 100-fold diluted fermentation broth per plant.
Other field management was performed in the same manner as normal production, and the disease was investigated 75 days later, and the results are shown in tables 3 to 6.
Grading standard of root rot:
no disease spots on the stem base and the main fibrous root at the 0 level;
stage 1, a small amount of scabs exist on the stem base and the main root;
in grade 3, the disease spots on the stem base and the main root are more, and the area of the disease spots accounts for 1/4-1/2 of the total area of the stem base and the root;
in stage 5, the disease spots on the stem base and the main root are many and large, and the area of the disease spots accounts for 1/2-3/4 of the total area of the stem base and the root;
7, the stem winding phenomenon is formed by connecting the lesion spots on the stem base and the main root, but the root system is not dead;
grade 9, root necrosis, wilting or death of the overground part of the plant.
Grading standard of southern blight:
stage 0: the plant is asymptomatic;
level 1: lesions are produced only at the base of the stem;
and 2, stage: stem base produces constriction symptoms, and less than one third of the whole plant shows systemic symptoms (withering, death, wilting, etc.);
and 3, level: less than two thirds of the whole plant presents with systemic symptoms;
4, level: more than two thirds of the whole plant showed systemic symptoms.
Scab grading criteria:
level 0: healthy plant
Level 1: small lesions on the top tender leaves and stalks
And 2, stage: small spots appear on tender leaves, fruit stalks and stems
And 3, level: the tender leaf has upward curled edge and scab-like appearance on the stem and stalk of peanut
4, level: the stalks and stems of the fruits are seriously bent, and the plants are burnt
Brown spot grading standard:
level 0: no disease symptom;
level 1: the victim blade area accounts for less than 1/10 of the survey blade area;
and 2, stage: the victim blade area accounts for less than 1/4 of the survey blade area;
and 3, level: the victim blade area accounts for less than 1/2 of the survey blade area;
4, level: the victim blade area accounts for more than 1/2 of the survey blade area, fallen leaves.
The disease rate is the number of diseased plants/total plants multiplied by 100%
Disease index ═ Σ (number of disease-level representative values × number of disease-level plants) × 100/(number of survey-level plants × number of highest-level disease-level representative values)
The prevention and treatment efficiency is [ (control disease index-treatment disease index)/control disease index ] × 100%
TABLE 3 comparison of control effects on peanut root rot
TABLE 4 peanut southern blight control effect comparison
TABLE 5 peanut scab control Effect comparison
TABLE 6 comparison of the control effects of cercospora brown spot of peanut
As can be seen from the field experimental data in tables 3-6, the Bacillus subtilis LLH-3 provided by the invention has obvious control effects on flower root rot, southern blight, scab and brown spot, wherein the control efficiency on the root rot and the scab is over 80 percent, the control efficiency on the southern rot and the brown spot is over 60 percent, and the control effect is obviously higher than that of the medicament treatment carbendazim. The commercial bacillus subtilis CGMCC1.8886 has certain control effect on flower root rot, southern blight, scab and brown spot, but the control efficiency is only 11.7-13.6%, which is far lower than that of the bacillus subtilis LLH-3.
The results show that the prevention and treatment effect of the bacillus subtilis LLH-3 on common peanut diseases is obviously superior to that of the traditional chemical bactericide, and the bacillus subtilis LLH-3 is environment-friendly, is beneficial to improving the quality of crops, and can be widely applied to green agricultural production.
Sequence listing
<110> Qingdao Lihui Biotechnology GmbH
<120> bacillus subtilis and application thereof in agricultural production
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 960
<212> DNA
<213> Bacillus subtilis
<400> 1
ctctagggtt ttcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa 60
accacatgct ccaccgcttt tgcgggcctc cgtcaattcc tttgagtttt agccttgcgg 120
ccgtactccc caggcggagt ggttaatgcg ttaacttcag cactaaaggg cggaaaccct 180
ctaacactta gaactcatcc tttacggcgt ggactaccag ggtatctaat cctgtttgct 240
ccccacgctt tcgcgcctca gtgtcagtta cagaccagaa agtcgccttc gccactggtg 300
ttcctccata tctctacgca tttcaccgct acacatggaa ttccactttc ctcttctgca 360
ctcaagtctc ccagtttcca atgaccctcc acggttgagc cgtgggcttt cacatcagac 420
ttaagaaacc acctgcgcgc gctttacgcc caataattcc ggataacgct tgccacctac 480
gtattaccgc ggctgctggc acgtagttag ccgtggcttt ctggttaggt accgtcaagg 540
tgccagctta ttcaactagc acttgttctt ccctaacaac agagttttac gacccgaaag 600
ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcggg ggattccgtc 660
ctgctgcctc ccctaggagg ctgggccgtg tctcagtccc agtgtggccg atcaccctct 720
caggtcggct acgcatcgtt gccttggtga gccgttacct caccaactag ctaatgcgac 780
gcgggtccat ccataagtga cagccgaagc cgcctttcaa tttcgaacca tgcagttcaa 840
aatgttatcc ggtattagcc ccggtttccc ggagttaccc cagtcttatg ggcaggttac 900
ccacgtgtta ctcacccgtc cgccgctaac tcactcgagc atgctactag cttttgcccc 960
Claims (3)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) The bacillus subtilis is characterized in that the preservation number of the bacillus subtilis is CCTCC NO: m2019402.
2. Use of the bacillus subtilis of claim 1 in a biological fertilizer.
3. The use of the bacillus subtilis of claim 1 for the control of peanut root rot, peanut southern blight, peanut scab or peanut brown spot.
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