CN110079480B - Glyphosate-resistant paenibacillus mucilaginosus JD-07, microbial inoculum and application - Google Patents
Glyphosate-resistant paenibacillus mucilaginosus JD-07, microbial inoculum and application Download PDFInfo
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Abstract
The invention provides glyphosate-resistant paenibacillus mucilaginosus JD-07, a microbial inoculum and application, and relates to the field of microorganisms. The preservation number of the Paenibacillus mucilaginosus (Paenibacillus muciniginosus) JD-07 in the China general microbiological culture Collection center is as follows: CGMCC No.17201 has glyphosate resistant property, can grow on culture medium containing glyphosate isopropylamine salt in 5.0mmol/L, and has unchanged morphological characteristics and physiological and biochemical properties compared with wild strain of Paenibacillus mucilaginosus. The glyphosate-resistant paenibacillus mucilaginosus JD-07 and the microbial inoculum containing the same relieve the problem that the glyphosate causes the death of the paenibacillus mucilaginosus in a product but cannot normally exert the efficacy of the paenibacillus mucilaginosus.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to glyphosate-resistant paenibacillus mucilaginosus JD-07, a microbial inoculum and application.
Background
Glyphosate is a herbicide with high efficiency, low toxicity, broad spectrum, internal absorption and conduction, and non-selective foliar spraying, and is widely applied to agricultural production (old countries, etc., 2017). However, it is not limited toThe glyphosate is convenient for agricultural production and simultaneously remains in a large amount in soil. The domestic and foreign research on the glyphosate shows that the glyphosate is applied at a normal application rate of 1kg/hm2Calculating to obtain glyphosate residue of 0.45-2 mg/kg in soil with the surface layer of 13 cm; in areas with large glyphosate dosage, the residual quantity is as high as 10-20 mg/kg (Zhou et al, 2013). The residual glyphosate not only causes great pollution to soil, water and the like, but also seriously hinders the growth and the function of soil microorganisms. As proved by research of Wuxuening et al (2016), the glyphosate preparation has certain inhibition effect on soil microorganisms and is increased along with the increase of the concentration of the medicament dose. This inhibition renders many functional microorganisms applied to the soil ineffective. Therefore, the screening of functional microorganisms with certain glyphosate tolerance has important significance for the exertion of the functions of the microorganisms.
Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) is commonly called potassium bacteria, is one of silicate bacteria, and is a Plant Growth-Promoting Rhizobacteria (PGPR) (Lijun et al, 2011). The paenibacillus mucilaginosus is gram-negative bacteria, aerobic or facultative anaerobic, the optimal growth temperature is 28-30 ℃, and the optimal pH is 7-8.
The colony of the paenibacillus mucilaginosus on the silicate bacteria solid culture medium is characterized by being in a round and smooth semi-glass bead shape, is viscous, can be pulled into a filament and is not easy to break; the surface of the microscope is in a thick and long rod shape, the tail end of the microscope is in a circular shape, and polysaccharide covers the surface of the microscope without flagella and with capsules. Spores are generated in the later period, and the spores are oval.
The physiological and biochemical characteristics of the paenibacillus mucilaginosus are catalase activity positive, citric acid utilization test activity negative, glucose fermentation test negative, methyl red test positive, V.P test negative, starch hydrolysis test positive, indole test positive, gelatin liquefaction test negative and drug resistance negative.
The tolerance of the paenibacillus mucilaginosus to herbicide glyphosate is limited, and researches show that the tolerance of the paenibacillus mucilaginosus to the glyphosate is only 3mmol/L, and the paenibacillus mucilaginosus stops growing when the tolerance is higher than 3mmol/L, so that the application and the function exertion of the functional strain are seriously influenced. Therefore, screening of the glyphosate-tolerant paenibacillus mucilaginosus mutant has important significance for expanding the application range of the bacillus mucilaginosus and playing the functions of the bacillus mucilaginosus.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a glyphosate-resistant Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) JD-07 which is preserved in China general microbiological culture Collection center in 2019, 15.01 and with the preservation number: CGMCC No. 17201.
The second purpose of the invention is to provide a microbial inoculum, which comprises the paenibacillus mucilaginosus JD-07.
The third objective of the invention is to provide the paenibacillus mucilaginosus JD-07 or the application containing the paenibacillus mucilaginosus JD-07.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides glyphosate-resistant Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) JD-07 which is preserved in China general microbiological culture Collection center in 2019, 15.01 and with the preservation number: CGMCC No. 17201.
According to one aspect of the invention, the invention provides a microbial inoculum, which comprises the paenibacillus mucilaginosus JD-07.
Preferably, the microbial inoculum further comprises a carrier.
According to another aspect of the invention, the invention also provides application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in mineral degradation to release potassium elements and/or phosphorus elements.
According to another aspect of the invention, the invention also provides application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in nitrogen fixation.
According to another aspect of the invention, the invention also provides an application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in preparation of fertilizers.
Preferably, the fertilizer comprises a microbial fertilizer.
According to another aspect of the present invention, the present invention also provides a fertilizer comprising the above Paenibacillus mucilaginosus JD-07, or the above microbial agent.
According to another aspect of the invention, the invention also provides an application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in preparing a soil conditioner.
According to another aspect of the present invention, the present invention also provides a soil conditioner comprising the above paenibacillus mucilaginosus JD-07, or the above microbial agent.
Compared with the prior art, the invention has the following beneficial effects:
the Paenibacillus mucilaginosus JD-07 provided by the invention has the characteristic of glyphosate resistance and can grow on a culture medium containing 5.0mmol/L glyphosate isopropylamine salt.
The morphological characteristics and physiological and biochemical properties of the paenibacillus mucilaginosus JD-07 provided by the invention, such as the yield, the growth speed, the antibiotic resistance, the salt resistance and the antagonism to rhizoctonia solani, are not changed compared with the wild strain of the paenibacillus mucilaginosus.
The paenibacillus mucilaginosus JD-07 provided by the invention can play the same function as wild paenibacillus mucilaginosus, has the advantage of glyphosate resistance compared with wild paenibacillus mucilaginosus, and can be applied to actual agricultural production to solve the problem that the glyphosate causes death of the paenibacillus mucilaginosus in products, but the efficacy of the paenibacillus mucilaginosus cannot be normally played.
The invention also provides a fungicide containing the glyphosate-resistant paenibacillus mucilaginosus JD-07, which has all the beneficial effects of the paenibacillus mucilaginosus JD-07 and is not described herein again.
Compared with wild paenibacillus mucilaginosus, the paenibacillus mucilaginosus JD-07 and the microbial inoculum containing the same are applied to mineral degradation, nitrogen fixation, fertilizer preparation and soil conditioner preparation, and have the advantage of glyphosate tolerance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram showing the growth of wild type Paenibacillus mucilaginosus (JD-CK) and mutant Paenibacillus mucilaginosus JD-07 in the solid culture medium ACCC55 containing 5.0mmol/L glyphosate isopropylamine salt, wherein the left half of the culture dish is wild type Paenibacillus mucilaginosus (JD-CK) and the right half is mutant Paenibacillus mucilaginosus JD-07;
FIG. 2 shows the growth of wild type Paenibacillus mucilaginosus 3016 and mutant Paenibacillus mucilaginosus JD-07 on potassium-solubilizing medium.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The strain preservation date of the invention is 2019, 01, 15 and the preservation number is as follows: CGMCC No. 17201. The classification is named as: paenibacillus mucilaginosus (Paenibacillus muciniphunsus) JD-07, the name of the preservation unit is China general microbiological culture Collection center (CGMCC for short), No. 3 of Xilu No.1 of the Kyowa area of the Beijing city, China academy of sciences microbial research institute; the zip code 100101.
The morphological characteristics of the paenibacillus mucilaginosus JD-07 provided by the invention are the same as those of wild paenibacillus mucilaginosus, the bacterial colony is circular, convex, transparent, semi-glass strain-shaped, has larger viscoelasticity, is picked up and drawn, and the bacteria are rod-shaped, gram-negative and have a hypertrophic capsule when observed under an optical microscope. Paenibacillus mucilaginosus JD-07 has a 16S rRNA sequence shown in SEQ ID NO. 1.
The paenibacillus mucilaginosus can generate substances for promoting plant growth such as organic acid, hormone, polysaccharide and the like through secondary metabolism, and the paenibacillus mucilaginosus can also decompose potassium-containing minerals and release potassium and other nutrient elements to promote plant growth; the exopolysaccharide produced by the paenibacillus mucilaginosus can also enhance the nonspecific immunity of plants, reduce plant diseases and degrade the damage of toxic and harmful substances such as pesticides and the like to the plants. In conclusion, the paenibacillus mucilaginosus can be applied to promoting plant growth, improving plant immunity and improving soil fertility, and has wide application.
Furthermore, the Paenibacillus mucilaginosus JD-07 provided by the invention also has the characteristic of glyphosate resistance and can grow on a culture medium containing 5.0mmol/L glyphosate isopropylamine salt. Besides the advantage of being more tolerant to glyphosate compared with the wild strain, the morphological characteristics and the physiological and biochemical properties of the paenibacillus mucilaginosus JD-07 provided by the invention, such as the yield, the growth speed, the antibiotic resistance, the salt resistance and the antagonism to rhizoctonia solani, are not changed compared with the wild strain of the paenibacillus mucilaginosus.
The paenibacillus mucilaginosus JD-07 provided by the invention can play the same function as wild paenibacillus mucilaginosus, has the advantage of glyphosate resistance compared with wild paenibacillus mucilaginosus, and can be applied to actual agricultural production to solve the problem that the glyphosate causes death of the paenibacillus mucilaginosus in products, but the efficacy of the paenibacillus mucilaginosus cannot be normally played.
According to an aspect of the invention, the invention also provides a microbial inoculum, which comprises the paenibacillus mucilaginosus JD-07. The microbial inoculum can take paenibacillus mucilaginosus JD-07 as a main active ingredient; the Paenibacillus mucilaginosus JD-07 can also be used as an auxiliary active ingredient to be matched with other microorganisms or functional ingredients. The microbial inoculum contains the paenibacillus mucilaginosus JD-07, so that the microbial inoculum has all the beneficial effects of the paenibacillus mucilaginosus JD-07.
In some alternative embodiments, the microbial inoculum may also include other species of microorganisms; including but not limited to one or more of actinomycetes, azotobacteria, phosphorus solubilizing bacteria, phosphorus solubilizing fungi, silicate bacteria, rhizobia, growth promoting bacteria, growth promoting fungi, or photosynthetic bacteria.
In some alternative embodiments, the microbial inoculum may further comprise a carrier acceptable in the art, which refers to a solid material that is safe to human, animal, plant and environment for adsorbing the microorganism of interest and suitable for survival. In some specific embodiments, the carrier includes, but is not limited to, one or more of kaolin, montmorillonite, clay, talc, white carbon, sepiolite, diatomaceous earth, precipitated calcium carbonate, straw residue, wood flour, rice hulls, starch, polyvinyl alcohol, and polyethylene glycol.
In some alternative embodiments, the microbial inoculum may further include optional adjuvants acceptable in the art, including but not limited to one or more of solvents, dispersants, pH adjusters, emulsifiers, dispersants, wetting agents, stabilizers, humectants, and trace elements.
In some alternative embodiments, the formulation of the microbial inoculum may be, for example, but not limited to, a liquid, an emulsion, a microemulsion, a suspension, a powder, a granule, a wettable powder, or a water dispersible granule.
According to one aspect of the invention, the invention also provides the application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in mineral degradation.
The potassium element in the soil minerals can generally account for 90-98% of the total potassium in the soil, but potassium elements in the minerals cannot be directly utilized by plants, and the paenibacillus mucilaginosus can dissolve silicate minerals and apatite, release potassium elements, phosphorus elements and silicon elements in the soil minerals and improve soil fertility, so that the paenibacillus mucilaginosus JD-07 or a microbial inoculum containing the same can be applied to degrading the minerals in the soil to release the potassium elements and/or the phosphorus elements so as to improve soil labor and promote plant growth. Compared with wild paenibacillus mucilaginosus, the paenibacillus mucilaginosus JD-07 or the microbial inoculum can be applied to degrading the minerals in the soil containing glyphosate, so that the problem that the capability of degrading the minerals disappears due to death of the paenibacillus mucilaginosus caused by applying the glyphosate to the soil is solved. When the paenibacillus mucilaginosus JD-07 or the microbial inoculum is applied to degrading minerals, the paenibacillus mucilaginosus can degrade soil minerals and improve the content of potassium elements and/or phosphorus elements in soil while glyphosate is applied to plants for weeding.
According to one aspect of the invention, the invention also provides the application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in nitrogen fixation. The paenibacillus mucilaginosus can grow in a nitrogen-free culture medium and belongs to self-growing nitrogen-fixing bacteria. The paenibacillus mucilaginosus JD-07 or the microbial inoculum can fix nitrogen in the soil containing glyphosate.
According to one aspect of the invention, the invention also provides the application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in preparing fertilizers. The preparation of the fertilizer can be realized by firstly preparing the paenibacillus mucilaginosus JD-07 or the microbial inoculum into an additive of the fertilizer, and mixing the additive into the fertilizer prepared or purchased in advance when the additive is used; or the microbial inoculum is directly used as an additive of the fertilizer and is mixed into the fertilizer prepared or purchased in advance when in use; the paenibacillus mucilaginosus JD-07 or the microbial inoculum can also be used as a component in the fertilizer to be prepared into a finished fertilizer product together with other components. In some alternative embodiments, the fertilizer comprises a microbial fertilizer.
The microbial fertilizer refers to a product containing a living body of a specific microorganism, which is applied to agricultural products, and increases the supply of plant nutrients or promotes the growth of plants, increases the yield, and improves the quality of agricultural products and the agricultural ecological environment through the life activities of the microorganism contained therein. The microbial fertilizer has the advantages of improving soil fertility, improving soil structure and reducing the using amount of chemical fertilizer.
According to one aspect of the present invention, the present invention also provides a fertilizer comprising the above paenibacillus mucilaginosus JD-07, or the above microbial agent, which can be applied to soil containing glyphosate.
In some alternative embodiments, the fertilizer further comprises other ingredients acceptable in the fertilizer field, such as but not limited to inorganic materials, organic materials, additives and adjuvants acceptable in the fertilizer field. In some embodiments, the inorganic material includes, but is not limited to, one or more of ammonium bicarbonate, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, calcium nitrate, potassium nitrate, urea, calcium superphosphate, potassium chloride, potassium sulfate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; in some embodiments, the organic material includes, but is not limited to, one or more of kitchen waste, livestock manure, municipal sludge, biogas residue, humic acid, and crop waste; in some embodiments, the additive includes, but is not limited to, one or more of a trace element, a vitamin, an enzyme preparation, a microbial metabolite, or meat and bone meal; in some embodiments, the adjuvant includes, but is not limited to, one or more of a binder, a dispersant, a pH adjuster, a preservative, an anti-caking agent, a slow-release agent, a filler, a wet-spreading agent, an emulsifier, a synergist, a fixative, and a stabilizer.
According to one aspect of the invention, the invention also provides the application of the paenibacillus mucilaginosus JD-07 or the microbial inoculum in preparing a soil conditioner. Soil conditioners, also known as soil conditioners, refer to materials added to soil to improve the physical, chemical and/or biological shape of the soil. The soil conditioner can improve the soil structure, reduce the harm of soil salt and alkali, adjust the pH value of the soil, improve the soil moisture condition or repair the polluted soil and the like. The soil conditioner can be, for example but not limited to, a soil structure improving agent, a soil water retaining agent, a soil pH value adjusting agent, a saline-alkali soil improving agent or a contaminated soil repairing agent.
According to one aspect of the invention, the invention also provides a soil conditioner, which comprises the paenibacillus mucilaginosus JD-07 or the microbial inoculum. The soil conditioner can improve the soil containing glyphosate and increase the content of potassium element and phosphorus element in the soil.
In some alternative embodiments, the soil conditioner may also include, for example, may be, but is not limited to, natural minerals, solid waste, polymers, or biologics, and the like. In some embodiments, the natural minerals include, but are not limited to, one or more of peat, limestone, gypsum, bentonite, zeolite, silicates, and perlite; in some embodiments, the solid waste includes, but is not limited to, one or more of fly ash, phosphogypsum, blast furnace slag, municipal sludge, straw, wood chips, livestock manure, and pulp waste; in some embodiments, the polymer includes, but is not limited to, one or more of chitosan, humic acid, polymeric amino acids, hydrolyzed acrylic nitrile, polyacrylamide, polyvinyl alcohol, and polyethylene glycol.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
Example 1 determination of Glyphosate-resistant isopropylamine salt concentration of Paenibacillus mucilaginosus
The activated Paenibacillus mucilaginosus was inoculated to ACCC55 (sucrose sugar: 10.0g, yeast powder: 0.5g, K) containing glyphosate isopropylamine salts of various concentrations (1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L and 6mmol/L) by streaking2HPO4：0.5g，NaCl：0.2g，MgSO4：0.2g，CaCO3: 1.0 g; pH 7.0. + -. 0.2 to 1L), culturing in an incubator at 30 ℃, observing and recording the growth, wherein the result is shown in Table 1, wherein "+" represents growth and "-" represents no growth.
TABLE 1 growth of Paenibacillus mucilaginosus in Glyphosate of varying concentrations
| Glyphosate concentration (mM) | Growth of Paenibacillus mucilaginosus |
| 1 | + |
| 2 | + |
| 3 | + |
| 4 | － |
| 5 | － |
| 6 | － |
Example 2 determination of UV mutagen dose
Activated Paenibacillus mucilaginosus is inoculated into a 150ml conical flask containing 50ml of ACCC55 liquid medium for overnight culture, washed twice with an equal amount of 0.9% physiological saline and suspended with 0.9% physiological saline, 6ml of the suspension is taken out, irradiated for 0s, 6s, 12s, 18s, 24s and 30s respectively in an ultraviolet crosslinking instrument (energy: 1J/m2), and then a plate is coated and the colony number is recorded, and the experimental results are shown in Table 2.
TABLE 2 lethality of Paenibacillus mucilaginosus under different UV irradiation times
| Time/s | Number of bacteria/cfu | Mortality rate/% |
| 0 | 12100 | 0 |
| 6 | 1200 | 90.08 |
| 12 | 170 | 98.6 |
| 18 | 0 | 100 |
| 24 | 0 | 100 |
| 30 | 0 | 100 |
Example 3 ultraviolet screening of Glyphosate-resistant isopropylamine salt strains of Paenibacillus mucilaginosus
And (3) repeating the processes of activation, washing and ultraviolet mutagenesis at the time point with the lethality of 98.60%, inoculating 200 mu L of the mutagenized bacterial liquid into an ACCC55 solid culture medium containing 5.0mmol/L glyphosate isopropylamine salt, culturing in an incubator at 30 ℃, observing the growth condition of the bacterial strain, primarily screening 7 strains of Paenibacillus mucilaginosus with better growth, storing and carrying out subsequent tests. Inoculating 7 strains with glyphosate-resistant isopropyl amine salt of 5mmol/L into an ACCC55 liquid culture medium without glyphosate isopropyl amine salt, culturing for 12h, inoculating a bacterium solution into an ACCC55 solid culture medium with glyphosate isopropyl amine salt of 5.0mmol/L, culturing at 30 ℃, and observing the growth condition. The results show that only 1 Paenibacillus mucilaginosus strain of 7 strains can generate a single colony. As shown in table 3 and fig. 1, wherein "+" represents growth and "-" represents no growth. From FIG. 1, it can be seen that the wild type Paenibacillus mucilaginosus (JD-CK) did not grow, and that the Paenibacillus mucilaginosus JD-07 could grow well in the solid culture medium ACCC55 containing 5.0mmol/L glyphosate isopropylamine salt.
Growth of the strains of Table 37 in ACCC55 solid Medium containing 5.0mmol/L glyphosate isopropylamine salt
| Strain numbering | Growth conditions |
| Wild strain | － |
| JD-1 | － |
| JD-2 | － |
| JD-3 | － |
| JD-4 | － |
| JD-5 | － |
| JD-6 | － |
| JD-7(JD-07) | + |
EXAMPLE 4 determination of spore-forming Capacity of mutant Strain and wild Strain
The activated Paenibacillus mucilaginosus wild strain (JD-CK) and the screened Paenibacillus mucilaginosus (JD-07) resistant to glyphosate isopropylamine salt are inoculated into a 150ml conical flask containing 50ml of ACCC55 culture medium, the culture is carried out for 12h, OD600 is adjusted to be consistent, the culture is inoculated into a new ACCC55 culture medium for 48h, the difference of the germination rates of the two strains is measured by a microscope, and the experimental results are shown in Table 4.
TABLE 4 Blastogenicity of wild strains (JD-CK) and mutant strains (JD-07)
| Bacterial strains | Ratio of germinated spore (%) |
| JD-CK | 81 |
| JD-07 | 82 |
Example 5 comparison of growth rates of mutagenized strains with wild strains
Activated Paenibacillus mucilaginosus JD-CK and JD-07 were inoculated into 150ml Erlenmeyer flasks containing 50ml of ACCC55 liquid medium, the culture was carried out for 12 hours, OD600 was adjusted to be consistent, the Erlenmeyer flasks containing 50ml of ACCC55 liquid medium were inoculated into new 150ml Erlenmeyer flasks, and the OD600 of the bacterial liquid was measured every 3 hours by using an ultraviolet spectrophotometer, and the experimental results are shown in Table 5.
TABLE 5 growth rates of wild strain (JD-CK) and mutant strain (JD-07)
Example 6 antagonism of strains against Rhizoctonia solani by plate antagonism
After the rhizoctonia solani is activated by the PDA culture medium, a bacterium block is clamped by tweezers and placed in the center of the ACCC55 solid culture medium, activated paenibacillus mucilaginosus JD-CK and JD-07 are respectively inoculated to the periphery of the culture medium, and the culture is carried out for 72 hours at the temperature of 30 ℃. The size of the zone of inhibition was measured and the results recorded as shown in table 6.
TABLE 6 antagonistic action of wild strains (JD-CK) and mutant strains (JD-07) against Rhizoctonia solani
Example 7 resistance of strains to antibiotics by the dipstick method
Bacterial strain antibiotic susceptibility experiment, culturing Paenibacillus mucilaginosus JD-CK and JD-07 overnight to reach exponential phase, taking 100 mul of bacterial liquid to coat on ACCC55 solid culture medium, attaching sensitivity test paper (OXOID) on the surface of the culture medium, culturing for 24h and determining the size of a transparent ring, wherein the results are shown in Table 7.
TABLE 7 antibiotic resistance of wild strains (JD-CK) and mutant strains (JD-07)
EXAMPLE 8 Paenibacillus mucilaginosus salt tolerance test sample
After activated Paenibacillus mucilaginosus JD-CK and JD-07 were inoculated into R2A medium respectively and cultured overnight, 100. mu.l of the cultured Paenibacillus mucilaginosus JD-CK and the activated Paenibacillus mucilaginosus JD-07 were inoculated into R2A medium containing 1.0%, 2.0%, 3.0%, 4.0% and 5.0% of NaCl respectively and cultured overnight, and the results are shown in Table 8, wherein "+" represents growth and "-" represents no growth.
TABLE 8 salt tolerance of wild strains (JD-CK) and mutant strains (JD-07)
| Concentration of NaCl | JD-CK | JD-07 |
| 1％ | － | － |
| 2％ | － | － |
| 3％ | － | － |
| 4％ | － | － |
| 5％ | － | － |
In conclusion, the wild paenibacillus mucilaginosus is subjected to mutagenesis transformation, a mutant JD-07 with glyphosate-resistant paenibacillus mucilaginosus is screened, and compared with the wild strain of the mutant JD-07, the mutant JD-07 has no change in other properties such as yield, growth speed, antibiotic resistance, salt resistance and antagonism to rhizoctonia solani except for the glyphosate-resistant property.
Example 9 function study of Bacillusmucilaginosus on hydrolyzed potassium ores
Because the wild paenibacillus mucilaginosus has the potassium-decomposing function, the function is often used for improving the utilization efficiency of the potassium fertilizer of plants in the production of microbial fertilizers. Many minerals such as potassium feldspar contain many potassium elements, but the potassium elements present in the minerals are in a water-insoluble form that cannot be directly utilized by plants. The wild type paenibacillus mucilaginosus has the function of hydrolyzing potassium element from potassium mineral, improves the utilization of the potassium element by plants, and can further contribute to the improvement of the yield of the plants. Thus, Paenibacillus mucilaginosus is used as a raw material for microbial fertilizers. In order to ensure that the potassium-solubilizing function of the glyphosate-resistant mutant strain of the strain is not influenced by gene mutation, it is necessary to compare the potassium-solubilizing function of the mutant with that of a wild strain thereof. This example uses a comparison of glial wild-type 3016 strain and a mutant JD-07 from glyphosate-resistant glial spore rod to verify whether the potassium-solubilizing ability of mutant JD-07 is altered.
The formula of the culture medium for potassium-dissolving functional analysis is as follows: weighing 10g of glucose, 0.5g of yeast extract, 1g of ammonium sulfate, 2g of disodium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, 1g of calcium carbonate, 15g of agar powder and 1g of potassium feldspar by a ten-thousandth balance, weighing 1000ml of distilled water by a measuring cylinder, putting the distilled water into a reagent bottle, and sterilizing the reagent bottle in a high-temperature high-pressure sterilization pot at 121 ℃ for 20 min.
Pouring a potassium-decomposing culture medium cooled to 45-60 ℃ onto a sterile flat plate on an ultra-clean workbench under the ignited alcohol, burning a bacterium inoculating needle on the outer flame of an alcohol lamp to take bacteria after the flat plate is solidified, and respectively dipping the strain JD-07 and a wild type into the cooled bacterium inoculating needle to inoculate on the potassium-decomposing flat plate. The plates were inverted and incubated in a 30 ℃ incubator for 4 days and the results were observed. As a result, the mutant strain JD-07 showed the same potassium-solubilizing ability as the wild type strain, as shown in FIG. 2. Thus, the potassium-solubilizing function of the mutant strain is not affected by the gene mutation.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Beijing aerospace Hengfeng science and technology GmbH
<120> glyphosate-resistant paenibacillus mucilaginosus JD-07, microbial inoculum and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1418
<212> DNA
<213> （Paenibacillus mucilaginosus）
<400> 1
gtgctataca tgcagtcgag cggagcactt cggtgcttag cggcggacgg gtgagtaaca 60
cgtaggcaac ctgcctgtaa gatcgggata actaccggaa acggtagcta agaccggata 120
gctggtttcg gtgcatgccg gaatcatgaa acacggggca acctgtggct tacggatggg 180
cctgcggcgc attagctagt tggcggggta atggcccacc aaggcgacga tgcgtagccg 240
acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtaggg aatcttccgc aatgggcgca agcctgacgg agcaacgccg cgtgagtgat 360
gaaggttttc ggatcgtaaa gctctgttgc cagggaagaa tgtcgtggag agtaactgct 420
ctgcgaatga cggtacctga gaagaaagcc ccggctaact acgtgccagc agccgcggta 480
atacgtaggg ggcaagcgtt gtccggaatt attgggcgta aagcgcgcgc aggcggtctt 540
ttaagtctgg tgtttaagcc cggggctcaa ccccggttcg caccggaaac tggaagactt 600
gagtgcagga gaggaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660
gaacaccagt ggcgaaggcg gctttctgga ctgtaactga cgctgaggcg cgaaagcgtg 720
gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaggtgt 780
taggggtttc gatacccttg gtgccgaagt aaacacaata agcactccgc ctggggagta 840
cgctcgcaag agtgaaactc aaaggaattg acggggaccc gcacaagcag tggagtatgt 900
ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatccctc tgaaagccct 960
agagataggg ccctccttcg ggacagaggt gacaggtgga gcatggttgt cgtcagctcg 1020
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgacttt agttgccagc 1080
attgagttgg gcactctaga gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1140
tcaaatcatc atgcccctta tgacctgggc tacacacgta ctacaatggc cggtacaacg 1200
ggaagcgaag tcgcgagatg gagcgaatcc ttagaagccg gtctcagttc ggattgcagg 1260
ctgcaactcg cctgcatgaa gtcggaattg ctagtaatcg cggatcagca tgccgcggtg 1320
aatacgttcc cgggtcttgt acacaccgcc cgtcacacca cgagagttta caacacccga 1380
agccggtggg gtaacccgta agggagccag ccgtcgaa 1418
Claims (9)
1. A glyphosate-resistant Paenibacillus mucilaginosus (Paenibacillus mucilaginosus) JD-07 is preserved in China general microbiological culture Collection center in 2019, 01, 15 days, and the preservation number is as follows: CGMCC No. 17201.
2. A microbial agent comprising paenibacillus mucilaginosus JD-07 according to claim 1.
3. The microbial inoculum according to claim 2, further comprising a carrier.
4. Use of paenibacillus mucilaginosus JD-07 according to claim 1 or the microbial agent according to claim 2 or 3 for degrading potassium ore.
5. Use of paenibacillus mucilaginosus JD-07 of claim 1 or microbial inoculum of claim 2 or 3 in preparation of fertilizer.
6. Use according to claim 5, wherein the fertilizer comprises a microbial fertilizer.
7. A fertilizer comprising paenibacillus mucilaginosus JD-07 according to claim 1 or the microbial agent according to claim 2 or 3.
8. Use of paenibacillus mucilaginosus JD-07 according to claim 1 or the microbial agent according to claim 2 or 3 for preparing a soil conditioner.
9. A soil conditioner comprising paenibacillus mucilaginosus JD-07 according to claim 1 or a microbial agent according to claim 2 or 3.
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| CN102838417A (en) * | 2012-09-20 | 2012-12-26 | 江西省科学院微生物研究所 | Glyphosate microbiological degradation preparation and preparation method thereof |
| CN106434430A (en) * | 2016-09-07 | 2017-02-22 | 中山市润泽生物科技有限公司 | Compound microbial agent for degrading antibiotic and pesticide residues as well as preparation and application thereof |
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| CN102838417A (en) * | 2012-09-20 | 2012-12-26 | 江西省科学院微生物研究所 | Glyphosate microbiological degradation preparation and preparation method thereof |
| CN106434430A (en) * | 2016-09-07 | 2017-02-22 | 中山市润泽生物科技有限公司 | Compound microbial agent for degrading antibiotic and pesticide residues as well as preparation and application thereof |
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