CN113881588B - Bacillus megaterium and application thereof - Google Patents
Bacillus megaterium and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a bacillus megaterium and application thereof, wherein the bacillus megaterium is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.20572. It has the effects of preventing and treating peanut rot, and has the capabilities of dissolving phosphorus, dissolving potassium and fixing nitrogen.
Description
Technical Field
The invention relates to bacillus megaterium.
Background
Peanuts are one of main oil crops and economic crops in China, peanut rot is a commonly occurring soil-borne disease in the world, and the peanut rot is reported in North America, australia, asia, africa and other places at present. In recent years, peanut rot is increasingly occurring in main peanut producing areas such as Hebei, shandong, henan and the like in China, and the trend that the peanut rot becomes increasingly serious becomes a great threat to peanut production. The disease has the characteristics of wide distribution range, serious harm, difficult control and the like, and the disease is characterized in that dark brown small disease spots appear on pod peels at the initial stage, then the disease spots gradually expand and spread to the whole pod, the endopleura becomes yellow, the seeds have poor development and are smaller than normal seeds. Most pod tips are infected first, the mild causes the whole pod or half pod to turn black, the severe whole pod is dark black, the peel and nuts are rotted, and the above-ground parts of the peanut plant are not different from the normal plant. In recent years, the disease has occurred in peanut main producing areas such as Hebei, henan, shandong, liaoning and the like, has a tendency of increasing weight year by year, and has become a main disease of peanut main producing areas in Hebei province and northern China. Peanut rot generally causes 30 to 100 percent of peanut pod rot, and the yield of serious disease plots can be reduced by more than 50 percent, even the peanut rot is completely harvested.
Examples of the peanut rot pathogens reported include fungi such as Fusarium neospora (Fusarium neocomosporillum), pythium neprilowii (Pythium myciorum), rhizoctonia solani (Rhizoctonia solani), and Fusarium solani (Fusarium solani). The main pathogenic bacterium of peanut rot in Hebei province is Fusarium neospora (F. Neocomosporium), which is called as Neososporia vasicinfecta (Neososporia vasicinfecta).
At present, the main cultivated species of peanuts in China are generally susceptible to fruit rot, and particularly, no species with high oleic acid exists. The method adopts a natural disease garden identification method to evaluate the peanut germplasm resources at home and abroad in the areas of He Meijing and the like to obtain 2 parts of high-resistance germplasm resources, but the distance for cultivating disease-resistant varieties has a longer path for production.
The chemical agent has the defects of short pesticide effect period, environment unfriendliness and the like in the aspect of preventing and treating soil-borne diseases, and the biological control can compensate the defects, so that the chemical agent has good performance in preventing and treating the soil-borne diseases.
Currently, although a certain number of excellent strains are provided in the biological control of peanut rot, the disease prevention effect of biocontrol microorganisms in the field control practice is unstable and nonuniform, so that no medicine is available for the disease in actual production. The root cause of the strain is that most of the biocontrol strains consider the inhibition effect on pathogenic bacteria, but do not consider the soil microenvironment where beneficial bacteria live and the interaction between the biocontrol strains and plants.
Therefore, a product or a method capable of effectively reducing the incidence rate of peanut rot, increasing the yield of peanuts and reducing pesticide residues in the peanuts is urgently needed, so that the effects of disease prevention, fruit preservation and yield increase are achieved.
Disclosure of Invention
One of the invention provides Bacillus megaterium (CGMCC), which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.20572.
The engineering bacteria obtained by genetic improvement of the strain can be endowed with more excellent and/or more performances, for example, the sterilization and/or bacteriostasis performances of the engineering bacteria can be increased and/or widened according to practical application by combining the characteristics of the strain, or the engineering bacteria have the insecticidal performance. That is, the strain of the present invention is genetically modified to have at least one of the above-mentioned properties. Because the engineering strain takes the bacillus megaterium as a modified object, namely, a specific gene and/or sequence and the like are transferred and/or knocked out, the genetically modified strain is still the bacillus megaterium.
Therefore, the second invention provides an engineering bacterium obtained by genetically modifying the bacillus megaterium of the first invention, wherein the engineering bacterium is obtained by transferring a functional gene into the bacillus megaterium of the first invention.
In a specific embodiment, the functional gene is at least one of a gene for controlling harmful plant pests, a gene for controlling pathogenic microorganisms of harmful plants, a gene for enhancing the effect of the bacillus megaterium on controlling peanut rot, a gene for enhancing the phosphorus dissolving capacity of the bacillus megaterium and a gene for enhancing the potassium dissolving capacity of the bacillus megaterium.
Although the transgenosis is suspected by partial population, the engineering bacteria obtained by genetically modifying the bacillus megaterium are not directly eaten by human beings or animals. And before it is put on the market for commercialization, it needs to be first evaluated for security by the relevant national departments to avoid the security problem. According to the safety conclusion of the engineering bacteria and the approval of relevant national departments, the engineering bacteria are reasonably used.
The third invention provides a composition, which comprises the bacillus megaterium according to the first invention and/or the engineering bacteria according to the second invention.
The fourth invention provides the application of one of the bacillus megaterium, the engineering bacteria and the composition, wherein the bacillus megaterium is used for preventing and treating at least one of peanut rot, dissolving phosphorus, dissolving potassium, fixing nitrogen and promoting growth, so that the yield of peanuts is increased.
In a specific embodiment, the application is peanut rot control, phosphorus dissolution, potassium dissolution, nitrogen fixation and growth promotion, so that the yield of peanuts suffering from the peanut rot is increased.
In a specific embodiment, during the growth period of peanuts, one of the bacillus megaterium according to one of the invention, the engineered bacterium according to the second of the invention and the composition according to the third of the invention is applied, together with a calcium fertilizer.
In a particular embodiment, the calcium fertilizer is calcium ammonium nitrate and/or calcium nitrate.
Unless otherwise defined, all terms used in the present invention are commonly used in the art.
The invention has the beneficial effects that:
the invention discovers for the first time that a strain has an inhibiting effect on pathogenic bacteria of peanut rot and the capacities of dissolving phosphorus, dissolving potassium and fixing nitrogen, and the strain is prepared into a bio-organic fertilizer applied to peanut planting, so that the strain has remarkable growth promoting, disease preventing and yield increasing effects compared with a strain which has a control effect on the peanut rot alone. Meanwhile, the biological organic fertilizer and the calcium fertilizer (such as calcium ammonium nitrate and calcium nitrate) prepared by the strain are applied according to a certain proportion to prevent and control the peanut rot, so that the yield can be increased by the growth promotion effect of the XJ-32 and the biological organic fertilizer thereof, and the yield can be increased by preventing and controlling the peanut rot through the synergistic effect of the XJ-32 biological organic fertilizer and the calcium fertilizer. Compared with chemical pesticides (suspension seed coating agents, the seed dressing agents of the chemical pesticides are set as blank controls) and microbial fertilizers (such as bacillus amyloliquefaciens), the bacterial strains have obvious, uniform and stable prevention and treatment effects and yield increasing effects, and the bacterial strains and the calcium fertilizers have synergistic effects.
The concrete test shows that:
(1) The strain XJ-32 can be colonized in rhizosphere soil in a pot experiment, and has the effect of remarkably promoting the growth of main roots compared with the bacillus amyloliquefaciens SWM-1 and GF-22 which have the effect of preventing and treating the fruit rot alone.
(2) The result of a plot experiment of the microbial organic fertilizer prepared by the strain XJ-32 shows that the XJ-32 microbial organic fertilizer prepared by the strain is better than Bacillus amyloliquefaciens SWM-1 and GF-22 which have the control effect on fruit rot and a blank control in the aspects of emergence rate and yield.
(3) The test results of field peanut rot prevention and control in 2018-2020 prove that when the effects of the XJ-32 biological organic fertilizer and calcium ammonium nitrate on prevention and control of peanut rot are respectively improved to 85.43% + -0.90%, 86.90% + -1.23% and 88.63% + -0.61%, the yield is improved to 27.08% + -1.39%, 26.64 + -0.66% and 27.49 + -0.64%, the prevention and control effects and the yield are remarkably better than those of blank control (2.5% metalaxyl-M +3.75% fludioxonil seed dressing or 3% fludioxonil +3% thifluzamide seed dressing) and conventional treatment (organic fertilizer, ammonium nitrate, organic fertilizer, calcium nitrate, bacillus amyloliquefaciens SWM-1 biological organic fertilizer and bacillus amyloliquefaciens) and than that when the XJ-32 biological organic fertilizer and the ammonium nitrate are independently applied, GF shows relatively uniform and stable prevention and control effects and yield increase, which show that the XJ-32 biological organic fertilizer and the calcium ammonium nitrate have synergistic effect. When the combined application of the XJ-32 bio-organic fertilizer and the calcium nitrate is used for preventing and treating peanut fruit rot and increasing the yield to 84.63% + -1.30%, 85.99% + -1.48% and 86.75% + -1.31% respectively, the yield is increased to 24.88% + -1.41%, 25.82% + -0.76% and 25.77% + -0.99%, and the prevention and treatment effect and the yield are equivalent to the combined application of the XJ-32 bio-organic fertilizer and the calcium nitrate, so that the prevention and treatment effect is remarkably better than blank control (the same as above) and conventional treatment (the same as above) and is also remarkably better than the independent application of the XJ-32 bio-organic fertilizer and the calcium nitrate, and relatively uniform and stable prevention and production increasing effects are also shown, which shows that the XJ-32 bio-organic fertilizer and the calcium nitrate have synergistic effect. The XJ-32 biological organic fertilizer and the calcium fertilizer are compounded for use, and the synergistic effect can be generated.
Drawings
FIG. 1 shows the colony morphology of the XJ-32 strain.
FIG. 2 shows the cell morphology of the XJ-32 strain.
FIG. 3 shows the inhibitory effect of XJ-32 strain on N.mairei.
FIG. 4 shows the preliminary verification results of dissolving phosphorus, dissolving potassium and fixing nitrogen of the XJ-32 strain.
FIG. 5 shows the soluble inorganic phosphorus content in the culture broth of strain XJ-32.
Biological preservation
The Bacillus megaterium (Bacillus megaterium) is named as XJ-32, and is preserved in China general microbiological culture Collection center (CGMCC) at 8-31.2020 with the preservation address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101 and CGMCC 20572.
Detailed Description
The invention is described in more detail below with reference to preferred embodiments, but the invention is not limited thereto.
The reagents in the examples of the present invention were all commercially available unless otherwise specified.
EXAMPLE 1 isolation and purification of the Strain
Collecting rhizosphere soil samples of healthy peanuts from farm peanut planting areas of North China scientific and technical teaching institute, changli county, qinhua, hebei province in 3 months in 2018, taking 10g of soil samples, placing the soil samples into a 250mL conical flask, adding 90mL of sterile water, placing three glass beads, shaking for 15min in a shaking table at 220rpm, standing for 30sec, and preparing to obtain 10 -1 A soil diluent; from 10 with a pipette -1 Sucking 1mL of soil diluent, adding into a large test tube containing 9mL of sterile water, and mixing to obtain 10 -2 A soil diluent; then from 10 -2 Sucking 1mL of soil diluent, adding into a large test tube containing 9mL of sterile water, and mixing to obtain 10 -3 A soil dilution solution ofBy analogy, respectively prepare 10 -4 ，10 -5 ，10 -6 The soil dilution was then pipetted 100. Mu.l each at 10 dilution -4 、10 -5 、10 -6 The soil dilution was passed through PDA medium (formulation: peeled potato 200.0g, glucose 20.0g, agar 20.0g, distilled water to a constant volume of 1000.0mL, natural pH), LB medium (formulation: yeast extract 5.0g, peptone 10.0g, naCl 10.0g, agar 20.0g, distilled water 1000.0mL, pH 7.0), gao's No. (formulation: soluble starch 20.0g, naCl 0.50g, KNO medium (formulation: soluble starch 20.0g, naCl 0.50g, KNO) 3 1.0g，K 2 HPO 4 ·3H 2 O 0.50g，MgSO 4 ·7H 2 O 0.50g，FeSO 4 ·7H 2 0.01g of O, 20.0g of agar, 1000.0mL of distilled water, pH 7.5) and a nitrogen-fixing medium (the formulation is as follows: 10.0g of mannitol, KH 2 PO 4 0.20g，MgSO 4 ·7H 2 O5.0 mg, sodium chloride 0.20g, caCO 3 5.0g，CaSO 4 ·2H 2 O0.10 g, distilled water 1000.0mL, agar 20.0g, pH 7.3) was spread using a dilution spread plate method, and samples at each concentration were repeated three times on each medium, and cultured in an inverted state at 28 ℃ for 2 days. And then performing separation, purification and culture on the cultured colonies by adopting a plate marking method, numbering, storing as a glycerol solution, and placing in a refrigerator at the temperature of-20 ℃ for later use.
The present invention was developed with respect to strain No. XJ-32.
EXAMPLE 2 identification of the strains
(1) The colony morphology of the XJ-32 strain on the LB culture medium is shown in figure 1, and the colony is circular, milky white, opaque, neat in edge, smooth in surface and slightly raised. Gram staining the cultured XJ-32 strain, and observing under an optical microscope, wherein the result is shown in FIG. 2, the strain is rod-shaped, produces spores, has continuous multi-double rods, is purple, and has a size of 1.18-1.54 × 2.84-4.71 μm; the spore size is 0.832-1.202X 1.527-2.364 μm. According to the manual of identifying common bacteria systems and gram staining results, the XJ-32 strain is preliminarily judged to be gram-positive bacillus.
(2) Physiological and biochemical identification: after the strain is activated on an LB culture medium, the 46 physiological and biochemical characteristics of the strain are analyzed and determined by a Meiriee VITEK-2 Compact full-automatic bacteria identification and analysis system and a BCL biochemical identification card, and the results are shown in Table 1. From the results of Table 1, it was shown that the probability of being Bacillus megaterium was 92%.
TABLE 1 XJ-32 physiological and biochemical identification
(3) 16S rDNA sequencing and sequence analysis
Extracting strain genome DNA by a boiling method, carrying out PCR amplification by taking universal primers 16SF (SEQ ID No. 1) and 16SR (SEQ ID No. 2) of 16S rDNA as templates, detecting an amplified product by using 1% agarose gel electrophoresis, cutting gel, recovering a target fragment, connecting the target fragment with a T-easy carrier, transforming the obtained product to E.coli JM109 competent cells after connection, sequentially adding 16 mu L of IPTG solution, 40 mu L of X-gal solution and 150 mu L of bacterial liquid into an LB solid plate, uniformly coating, and culturing at 37 ℃ overnight. Screening blue white spots to select positive clones for PCR and electrophoresis detection. And (4) sending the bacterial liquid of the positive clone which is successfully detected to Shanghai biological engineering GmbH for sequencing. The sequence result is spliced by software DNAMAN 6.0 to obtain the 16S rDNA sequence of 1516bp XJ-32 strain, which is shown as SEQ ID No. 3. BLAST comparison analysis was performed on the strain in GenBank, and the result showed that the similarity of the 16S rDNA sequence of the XJ-32 strain to Bacillus megaterium (Bacillus megaterium) having the gene accession number of CP032527 was 99.87%.
Based on the above results, XJ-32 was named Bacillus megaterium XJ-32 (hereinafter referred to as XJ-32).
Example 3 identification of endogenous control Effect
An indoor confrontation experiment is carried out on bacillus megaterium by using peanut fruit rot pathogen fusarium (F.neocomosum) as an indicator bacterium through a plate confrontation method, the indicator bacterium is inoculated in the center of a PDA (formula: peeled potato 200.0g, glucose 20.0g, agar 20.0g, distilled water to volume of 1000.0mL and natural pH) plate, three target bacteria are inoculated around the PDA plate, each treatment is set to be repeated for three times, and the average width of an XJ-32 inhibition ring is observed to be 0.39cm after 2 days of culture at 28 ℃ (figure 3). The results show that XJ-32 has an inhibitory effect on Fusarium neospora (F. Neocomosporium).
Example 4 preliminary verification of phosphorus solubilizing, potassium solubilizing and nitrogen fixing abilities
The bacterial liquid of the XJ-32 strain was activated on an LB medium (formulation: 5.0g yeast extract, 10.0g peptone, 10.0g NaCl, 10.0g agar, 1000.0mL distilled water, pH 7.0) by plating in a superclean bench, cake of uniform size was punched out on the activated culture with a 1mL pipette tip for a pipette, and then the cake was placed on a potassium bacteria-decomposing separation medium (formulation: na. RTM.) respectively 2 HPO 4 2.0g, sucrose 5.0g, mgSO 4 ·7H 2 O 5.0mg，FeCl 3 5.0mg, calcium carbonate 1.0g, potassium feldspar powder 10.0g, distilled water 1000.0mL, agar 2.0g, pH7.3), nitrogen-free medium (formula: mannitol 1.0g, KH 2 PO 4 0.20g，MgSO 4 ·7H 2 O5.0 mg, sodium chloride 0.20g, caCO 3 5.0g，CaSO 4 ·2H 2 0.10g of O, 1000.0mL of distilled water, 20.0g of agar, pH 7.3), and an organophosphorous bacteria isolation medium (the formula is as follows: (NH) 4 ) 2 SO 4 0.50g，MgSO 4 0.30g, 0.30g sodium chloride, 0.30g KCl, caCO 3 3.5g，FeSO 4 0.018g, lecithin 0.20g, agar 20.0g, glucose 10.0g, mnSO4.11g, distilled water 1000.0mL, pH 7.0) and an inorganic phosphate solubilizing medium (formulation: (NH) 4 ) 2 SO 4 0.50g，MgSO 4 0.30g, 0.30g sodium chloride 3 (PO 4 ) 2 10.0g, 10.0g of glucose, mnSO 4 0.123g，FeSO 4 0.018g agar 20.0g, distilled water 1000.0mL, pH 7.2), and after 2 days, it was observed that XJ-32 had a hydrolysis ring around the potassium decomposing bacteria separation medium, the organic phosphorus decomposing bacteria separation medium, and the inorganic phosphorus decomposing bacteria separation medium, the potassium decomposing hydrolysis ring had a diameter of 8mm, the organic phosphorus decomposing hydrolysis ring had a diameter of 10mm, and the inorganic phosphorus decomposing hydrolysis ring had a diameter of 11mm, and was able to continue to grow in the nitrogen-free medium (FIG. 4), indicating that XJ-32 had the functions of decomposing potassium and dissolving potassiumInorganic phosphorus dissolving, organic phosphorus dissolving and nitrogen fixing.
In addition, the same experiment was carried out using Bacillus amyloliquefaciens SWM-1 and Bacillus amyloliquefaciens GF-22 (hereinafter referred to as SWM-1 and GF-22), and the results showed that SWM-1 and GF-22 have only inorganic phosphorus-solubilizing ability and no organic phosphorus-solubilizing and potassium-solubilizing ability.
EXAMPLE 5 determination of phosphorus solubilizing ability
Quantitative determination of phosphate-solubilizing capacities of XJ-32, SWM-1 and GF-22 strains: inoculating activated strains into a PKO liquid culture medium (the formula is as follows: 0.3g of sodium chloride, 10g of glucose, 0.3g of potassium chloride, 5g of tricalcium phosphate, 0.5g of ammonium sulfate, 0.3g of magnesium sulfate heptahydrate, 0.03g of manganese sulfate, 0.03g of ferrous sulfate heptahydrate, 1000mL of distilled water and pH adjustment of 7.0), shaking and culturing at 30 ℃ for 3d, collecting centrifuged supernatant, firstly taking 5 50mL colorimetric tubes by a molybdenum blue colorimetric method, compiling into five numbers of 1, 2, 4, 6 and 8, respectively adding a phosphorus standard No.2 solution (the phosphorus standard solution: 0.4391g of anhydrous potassium dihydrogen phosphate is dissolved in 1000mL of water to serve as the No.1 solution, wherein 0.1mg/mL of phosphorus is contained), sucking 10mL of the No.1 solution, adding water to dilute to 100mL of 0.01mg/mL of phosphorus to serve as the No.2 solution) of 1, 2, 4, 6 and 8mL, and respectively adding water to 9, 8, 6 and 2mL of water in sequence. Then 8.0mL of 0.015% hydrazine sulfate aqueous solution and 2.0mL of 2.5% sodium molybdate dilute sulfuric acid aqueous solution were added to each of the 5 tubes, followed by shaking, removing the plugs, heating the 5 tubes in a boiling water bath for 10 minutes, taking out and cooling to room temperature, diluting with water to 50mL, shaking thoroughly, after 10 minutes, adjusting the zero point with a 1cm liquid bath and water using a spectrophotometer at a wavelength of 650nm, and measuring the extinction values. The extinction values were plotted on the ordinate and the phosphorus (0.01, 0.02, 0.04, 0.06, 0.08 mg) on the abscissa to obtain a standard curve. During color comparison, a pipette is used for sucking 10mL of a liquid to be detected, the liquid is injected into a 50mL colorimetric tube, 8.0mL of 0.015% hydrazine sulfate is added, 2.0mL of a sodium molybdate dilute sulfuric acid solution is added, a plug is added, the mixture is shaken up, the colorimetric tube is placed in a boiling water bath for heating for 10 minutes, the mixture is taken out and cooled to room temperature, the mixture is diluted to 50mL by water and shaken up fully, after 10 minutes, a spectrophotometer is used for 650nm, a 1cm liquid tank is used for adjusting the zero point by water, and the extinction value is measured. The determination of the available phosphorus content in the supernatant is shown in Table 2 and FIG. 5, which shows that the average values of the available phosphorus contents in the XJ-32, SWM-1 and GF-22 culture solutions are 0.160 mug/mL, 0.119 mug/mL and 0.127 mug/mL respectively, and the available phosphorus content in the XJ-32 culture solution is significantly higher than that in the SWM-1, GF-22 and blank control, which indicates that the ability of XJ-32 to dissolve inorganic phosphorus is stronger than that in the SWM-1 and GF-22.
TABLE 2 determination of the capability of dissolving inorganic phosphorus
| Strain of bacillus | Available phosphorus content (μ g/mL) |
| XJ-32 | 160.15±6.70a |
| SWM-1 | 120.02±7.83b |
| GF-22 | 127.07±7.95b |
| Blank control | 16.84±1.32c |
* The different letters (a-d) represent significant differences between groups (P < 0.05)
Example 6 Bacillus megaterium hemolysis assay
The purified XJ-32 strain is activated on an LB solid medium, after 24 hours of culture at 30 ℃, single colony is picked by using an inoculating loop, streaked on a blood agar plate (purchased from Qingdao Haibo biotechnology, co., ltd.), and cultured for 24 hours at 37 ℃, and the result of a hemolytic test shows that no hemolytic loop is generated. This indicates that the culture is not dangerous to human health or to animals, plants or to the environment.
Example 7 potted plant experiments to verify colonization and growth promotion effects
Peanut potting experiments are carried out in a greenhouse of research and development center of the institute of science and technology, north Hei in 2019, and 700g of sterilized soil is filled in each pot. Jihua No. 5 peanut seed with spore content of 10 8 After 12h of soaking the strain with CFU/mL XJ32 suspension, germination was accelerated in a petri dish lined with moist filter paper. After sprouting, the seedlings are sowed in sterilized sandy soil for pot experiment, cultured in a greenhouse and watered regularly. SWM-1 and GF-22 fermentation broth are used as conventional control, and culture medium is blank control. Each treatment was repeated 3 times, each for 40 pots, the main root length was counted as 0d, when the first pair of true leaves appeared after emergence of the peanut seedlings, 0d,7d,14d and 21d, and the growth promoting ability of each treatment was examined.
The results show that: the XJ-32 treatment produced significantly longer peanut taproots than SWM-1, GF-22 and the blank control in 21d (Table 3). This indicates that XJ-32 promotes the growth of the primary root.
TABLE 3 influence of XJ-32 Strain on peanut growth
* The different letters (a-c) represent significant differences between groups (P < 0.05)
EXAMPLE 8 preparation of XJ-32 seed liquid
Selecting XJ-32 strain glycerol bacteria, streaking on LB solid culture medium, and culturing at 30 deg.C for 48h to obtain activated bacteria; adding yeast extract powder 2.50g, tryptone 5.00g and sodium chloride 8.00g into water, stirring for dissolving, and diluting to a constant volume of 1L with water to obtain a seed liquid culture medium; the activated bacteria are transferred into a seed liquid culture medium and cultured for 12 hours at 30 ℃ and 200rpm/min to obtain a first culture which is used as seed liquid.
EXAMPLE 9 liquid fermentation of XJ-32 and preparation of Bio-organic Fertilizer
The specific process comprises the following steps: transferring the first culture as seed liquid of liquid shake flask fermentation to a liquid fermentation medium (formula: corn starch) containing 5% inoculation amount10g of bean flour, 10g of bean flour and (NH 4) 2 SO 4 5g、MgSO 4 ·7H 2 O 1g、FeSO 4 ·7H 2 O 0.3g、K 2 HPO 4 ·3H 2 O 0.2g、KH 2 PO 4 0.1 g) was cultured at 30 ℃ and 200rpm/min for 72 hours in a triangular flask to obtain a second culture. Coating the second culture on a flat plate for counting, adding sterile glass beads at 200rpm/min, shaking for 30min, bathing at 85 deg.C for 15min to kill the nutriment, and gradient diluting to obtain 10 concentration -5 、10 -6 、10 -7 The bacterial suspension of (4) is plated, each treatment is repeated for 3 times, after the inverted culture at 37 ℃ for 14 hours, the colony number of each plate is selected to be between 30 and 300 for plate counting, and the plate colony counting result is 2.81 +/-0.03 multiplied by 10 11 one/mL.
And centrifuging the second culture to collect bacterial sludge, uniformly mixing the bacterial sludge and the diatomite according to the proportion of 1.
The XJ-32 microbial inoculum is adsorbed on the surface of an organic fertilizer (the content of nitrogen, phosphorus and potassium is more than or equal to 5 percent, the content of organic matters is more than or equal to 45 percent, the water content is less than or equal to 30 percent and the pH is 6.5 to 7.5) according to the quantity of more than or equal to 0.5 hundred million/g, and the XJ-32 microbial inoculum is prepared for standby application, wherein the effective viable count is more than or equal to 0.5 hundred million/g.
The preparation methods of the SWM-1 biological organic fertilizer and the GF-22 biological organic fertilizer are the same as the above.
Example 10 greenhouse plot experiment verifies growth promoting and yield increasing effects of XJ-32 organic fertilizer
In 2019, a cell test is carried out in research and development center of Changli school of science and technology academy in Hebei to verify the growth promoting effect of the XJ-32 biological organic fertilizer.
The soil type is sandy loam, crops are not planted in previous crops, the treatment group applies XJ-32 bio-organic fertilizer in an amount of 50 kg/mu for broadcasting, after fertilization, the soil is turned over 20cm deep, each treatment is repeated for 3 times, and each treatment is repeated for 20m 2 . The SWM-1 biological organic fertilizer and the GF-22 biological organic fertilizer are used as conventional control, and the organic fertilizer is used as blank control. And (4) counting the emergence situation and the yield per mu, and calculating the emergence rate and the yield increase rate. The results are shown in Table 4, showing: the emergence rate of the XJ-32 biological organic fertilizer treatment is 93.06 percent, the yield per mu is 324.74kg, the yield increase rate is 6.29 percentThe emergence rate and the yield per mu are significantly higher than those of the conventional control. While the emergence rates of the treatment of the SWM-1 bio-organic fertilizer and the treatment of the GF-22 bio-organic fertilizer are 85.21 percent and 86.19 percent respectively, the yield per mu is 306.15kg and 306.34kg respectively, and the method has no significant difference with a blank control. The result shows that the XJ-32 bio-organic fertilizer has a remarkable promoting effect on the growth of peanuts.
TABLE 4 peanut emergence rate
| Treatment of | Rate of emergence (%) | Mu yield (kg/mu) | Yield increase (%) |
| XJ-32 biological organic fertilizer | 93.06±3.20a | 324.74±6.90a | 6.29±0.42a |
| SWM-1 biological organic fertilizer | 85.21±2.71b | 306.15±7.54b | 0.20±0.13b |
| GF-22 biological organic fertilizer | 86.19±2.46b | 306.34±7.44b | 0.26±0.14b |
| Blank control | 84.72±2.75b | 305.54±7.67b | 0.00b |
* The different letters (a-b) represent significant differences between groups (P < 0.05)
Example 11 field application method and disease prevention test
Disease prevention and growth promotion tests of the bio-organic fertilizer and the matching method thereof are respectively carried out in 2018, 2019 and 2020. The test place in 2018 is a peanut planting region of a farm of the northriver science and technology academy of education in Chanli county of Qinhua, hebei province. The soil type was sandy loam, the fertility level before sowing is shown in table 5, and no crop was planted in the previous crop. The peanut variety is Jihua No. 5, and all treated peanut seeds including blank control are dressed by using a suspension seed coating agent (2.5 percent of metalaxyl-M +3.75 percent of fludioxonil) and dried in the shade for later use.
The test site in 2019 and 2020 is a test field of new houses village of Duzhuang county in Qianzhuang city of Qianzhan province in Hebei province. The peanuts are planted in continuous cropping in 2019 and 2020. The soil type of the test field was sandy soil, and the fertility level before sowing was shown in table 6. The peanut variety is Jihua No. 16, and all treated peanut seeds including blank control are dressed by using a suspension seed coating agent (3% fludioxonil +3% thifluzamide) and dried in the shade for later use.
40kg of organic fertilizer per mu (the content of nitrogen, phosphorus and potassium is more than or equal to 10 percent, the content of organic matters is more than or equal to 45 percent, the water content is less than or equal to 30 percent, and the pH value is 5.5 to 8.5), 40kg of organic fertilizer per mu plus 25kg of ammonium nitrate per mu, 40kg of organic fertilizer per mu plus 25kg of calcium nitrate per mu, 40kg of SWM-1 biological organic fertilizer per mu and 40kg of GF-22 biological organic fertilizer per mu are respectively taken as routine treatment. 40 kg/mu of XJ-32 bio-organic fertilizer, 40 kg/mu of XJ-32 bio-organic fertilizer plus 25 kg/mu of calcium ammonium nitrate, 40 kg/mu of XJ-32 bio-organic fertilizer plus 25 kg/mu of calcium nitrate are treated as test groups. Water soluble potassium sulfate type compound fertilizer (total nutrient greater than or equal to 51%, N-P) is applied in each treatment 2 O 5 -K 2 The mass content of O is 17-17-17). Blank control seed dressing and application by using the suspension seed coating agent onlyWater-soluble potassium sulfate type compound fertilizer is added.
Before sowing, when the relative water content of sandy soil or sandy loam is 65-70% (water content is 15-20%), the treated fertilizers are scattered on the soil surface, turning is carried out for 25-30 cm, and then the water-soluble potassium sulfate type compound fertilizer is applied in a ditch. Each treatment is repeated for 3 times, and the peanut fruit rot disease is graded and investigated by a five-point sampling method 10 days before harvest, and the yield and the control effect on the peanut fruit rot are measured. Pod grading criteria were as follows: level 0: the pods are intact and have no rotting symptoms; level 1: the fruit peel has infection spots, and the fruit is intact; and 2, stage: 1/2 of pod rot; and 3, level: rot 1/2 of the pod. Disease index, prevention and treatment efficiency, dry fruit acre yield and yield increase rate are respectively expressed by formulas (1), (2), (3) and (4).
Disease index = ∑ (rotten fruit × rotten fruit level value)/(total fruit number × highest level value) × 100
Formula (1)
Control efficiency (%) = (blank control area disease index-treatment area disease index)/blank control area disease index is multiplied by 100
Formula (2)
Dry fruit per mu yield = wet fruit per mu yield × 50% × 85%
Formula (3)
Yield increase (%) = (treatment zone per mu yield-blank control zone per mu yield)/blank control zone per mu yield x 100
Formula (4)
2018-2020 field disease prevention tests show that (Table 7-9): when the suspension seed coating is used for dressing Jihua No. 5 (2.5% metalaxyl-M +3.75% fludioxonil) or Jihua No. 16 (3% fludioxonil +3% thifluzamide) (blank control), peanut rot still occurs, the disease index is 17.56 +/-4.31 when the disease is relatively mild in 2018, and the disease index is 26.99 +/-4.35 and 33.46 +/-4.15 when the disease is relatively severe in 2019 and 2020, so that the field control effect on the peanut rot by chemical agent treatment (blank control) is very limited.
When the disease is less in 2018 (Table 7), the prevention and treatment efficiencies of the XJ-32 bio-organic fertilizer, the SWM-1 bio-organic fertilizer and the GF-22 bio-organic fertilizer on peanut fruit rot are respectively 56.66%, 54.31% and 54.58%, the yield increasing rates are respectively 18.15%, 14.93% and 15.20%, and the prevention and treatment efficiencies and the yield increasing rates are both obviously higher than those of a blank control and an organic fertilizer. Although the control efficiency difference of the three biological organic fertilizers is not obvious, the yield increasing rate of the XJ-32 biological organic fertilizer is obviously higher than that of the SWM-1 and GF-22 biological organic fertilizers. When the disease incidence is serious in 2019 and 2020 (tables 8 and 9), the prevention and treatment efficiency and the yield increase rate of the XJ-32 bio-organic fertilizer are 55.95% + -3.09%, 17.56% + -2.25%, 51.01% + -4.27% and 16.23% + -2.66% in sequence, which are obviously better than those of the SWM-1 bio-organic fertilizer, the GF-22 bio-organic fertilizer, the organic fertilizer and a blank control (seed coating suspending agent). In a word, compared with a blank control and an organic fertilizer, the XJ-32 biological organic fertilizer has obvious prevention and treatment effects on peanut fruit rot and yield increase, and compared with an SWM-1 biological organic fertilizer and a GF-22 biological organic fertilizer, the XJ-32 biological organic fertilizer has more stable yield increase effect.
In 2018-2020 (tables 8 and 9), the prevention and control efficiency of the organic fertilizer, calcium ammonium nitrate and calcium nitrate which are respectively used as base fertilizers on peanut fruit rot is 54% -64% and 53% -62%, the yield is increased by 15% -18% and 14% -17%, and the prevention and control efficiency and the yield increase of the two treatments are obviously better than those of a blank control and an organic fertilizer. In 2018-2020 (tables 7-9), when the prevention and treatment effects of the XJ-32 bio-organic fertilizer and calcium ammonium nitrate on peanut fruit rot are respectively improved to 85.43% + -0.90%, 86.90% + -1.23% and 88.63% + -0.61%, the yield is improved to 27.08% + -1.39%, 26.64 + -0.66% and 27.49 + -0.64%, the prevention and treatment effects and the yield are remarkably better than blank control and conventional treatment and the XJ-32 bio-organic fertilizer and calcium ammonium nitrate are applied independently, and relatively uniform and stable prevention and treatment effects and yield increasing effects are also shown, which shows that the XJ-32 bio-organic fertilizer and calcium ammonium nitrate have a synergistic effect. When the combined application of the XJ-32 biological organic fertilizer and the calcium nitrate is used for improving the control effect and the yield increase rate of the peanut fruit rot to 84.63% + -1.30%, 85.99% + -1.48% and 86.75% + -1.31% respectively, the yield increase rate is improved to 24.88% + -1.41%, 25.82% + -0.76% and 25.77% + -0.99%, and the control effect and the yield increase rate are equivalent to the combined application of the XJ-32 biological organic fertilizer and the calcium ammonium nitrate, so that the control effect and the yield increase rate are obviously better than blank control and conventional treatment and the separate application of the XJ-32 biological organic fertilizer and the calcium nitrate, and the relatively uniform and stable control effect and the yield increase effect are also shown, which shows that the XJ-32 biological organic fertilizer and the calcium nitrate have the synergistic effect.
TABLE 5 soil fertility level tested at farm of Hubei institute of science and technology
| pH | Quick-acting nitrogen (mg, kg-1) | Quick-acting phosphorus (mg, kg-1) | Quick-acting potassium (mg, kg-1) |
| 7.1 | 126.56±6.328 | 17.65±0.88 | 94.05±4.70 |
TABLE 6 level of soil fertility tested in Zhanzhuang village, deng New House in Qian' an City
| pH | Quick-acting nitrogen (mg, kg-1) | Quick-acting phosphorus (mg, kg-1) | Quick-acting potassium (mg, kg-1) |
| 6.92±0.14 | 140.95±16.48 | 42.15±13.84 | 98.75±19.05 |
TABLE 7 disease-preventing and yield-increasing effects in 2018
* Different lower case letters represent significant differences between groups (P < 0.05).
TABLE 8 disease-preventing and yield-increasing effects in 2019
* Different lower case letters represent significant differences between groups (P < 0.05).
TABLE 9 disease-preventing and yield-increasing effects in 2020
* Different lower case letters represent significant differences between groups (P < 0.05).
Sequence listing
<110> Hubei institute of science and technology
<120> bacillus megaterium and application thereof
<130> LHA2160201
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 1516
<212> DNA
<213> Bacillus megaterium (Bacillus megaterium)
<400> 3
agagtttgat cctggctcag gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
gaactgatta gaagcttgct tctatgacgt tagcggcgga cgggtgagta acacgtgggc 120
aacctgcctg taagactggg ataacttcgg gaaaccgaag ctaataccgg ataggatctt 180
ctccttcatg ggagatgatt gaaagatggt ttcggctatc acttacagat gggcccgcgg 240
tgcattagcc agttggtgag gtaacggctc accaaggcaa cgatgcatag ccgacctgag 300
agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360
gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct 420
ttcgggtcgt aaaactctgt tgttagggaa gaacaagtac aagagtaact gcttgtacct 480
tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540
ggtggcaagc gttatccgga attattgggc gtaaagcgcg cgcaggcggt ttcttaagtc 600
tgatgtgaaa gcccacggct caaccgtgga gggtcattgg aaactgggga acttgagtgc 660
agaagagaaa agcggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 720
cagtggcgaa ggcggctttt tggtctgtaa ctgacgctga ggcgcgaaag cgtggggagc 780
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa gtgttagagg 840
gtttccgccc tttagtgctg cagctaacgc attaagcact ccgcctgggg agtacggtcg 900
caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960
attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaac tctagagata 1020
gagcgttccc cttcggggga cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1080
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc 1140
agttgggcac tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1200
atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt acaaagggct 1260
gcaagaccgc gaggtcaagc caatcccata aaaccattct cagttcggat tgtaggctgc 1320
aactcgccta catgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1380
cgttcccggg ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc 1440
ggtggagtaa ccgtaaggag ctagccgcct aaggtgggac agatgattgg ggtgaagtcg 1500
taacaaggta accgta 1516
Claims (6)
1. A kind of Bacillus megaterium (B.), (B.megaterium)Bacillus megaterium) It is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20572.
2. A composition comprising the bacillus megaterium of claim 1.
3. Use of a bacillus megaterium according to claim 1 or a composition according to claim 2 for the control of fusarium neospora (f: (f)), (ii) or (iii) aFusarium neocosmosporiellum) At least one of peanut rot, phosphorus dissolution, potassium dissolution, nitrogen fixation and peanut growth promotion, thereby increasing the yield of the peanut.
4. Use according to claim 3, for the control of Fusarium neospora (Fusarium neospora: (Fusarium neospora))Fusarium neocosmosporiellum) Caused by peanut rot, dissolving phosphorus, dissolving potassium, fixing nitrogen and promoting peanut growth, thereby causing the new fusarium (F) to suffer fromFusarium neocosmosporiellum) The yield of the peanuts with the peanut rot disease is increased.
5. Use according to claim 3 or 4, characterized in that the Bacillus megaterium according to claim 1 or the composition according to claim 2 is applied during the growth phase of peanuts, while a calcium fertilizer is used.
6. Use according to claim 5, wherein the calcium fertilizer is calcium ammonium nitrate and/or calcium nitrate.
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