CN116574649A - Lignin degrading bacterium M15H2 with growth promoting, disease resisting and phosphorus dissolving capabilities and application thereof - Google Patents
Lignin degrading bacterium M15H2 with growth promoting, disease resisting and phosphorus dissolving capabilities and application thereof Download PDFInfo
- Publication number
- CN116574649A CN116574649A CN202310545364.7A CN202310545364A CN116574649A CN 116574649 A CN116574649 A CN 116574649A CN 202310545364 A CN202310545364 A CN 202310545364A CN 116574649 A CN116574649 A CN 116574649A
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- China
- Prior art keywords
- straw
- fermentation broth
- seeds
- strain
- raoultella planticola
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention provides lignin degrading bacteria M15H2 with the capabilities of promoting growth, resisting diseases and dissolving phosphorus and application thereof, and belongs to the technical field of agricultural microorganisms. The invention provides a Raoult plant (Raoultella planticola) M15H2, wherein the preservation number of the Raoult plant M15H2 is CGMCC NO.26627. The Raoultella planticola M15H2 provided by the invention can improve lignin degradation rate and promote straw decomposition, namely promote straw decomposition in returning to the field and improve straw returning efficiency; the Raoultella planticola M15H2 can generate growth-promoting hormone, so that seed germination and crop growth are promoted, and crop yield is increased; the plant-growing Raoult bacteria M15H2 can also inhibit the growth of rice leaf spot disease germs, can effectively solve the problem of soil-borne diseases such as leaf spot disease and the like, and has important significance for guaranteeing the sustainable development of the rice industry.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to lignin degrading bacteria M15H2 with the capabilities of promoting growth, resisting diseases and dissolving phosphorus and application thereof.
Background
As a renewable resource, the straw can not only relieve the energy problem to a certain extent, but also protect the environment and reduce the potential safety hazard if being reasonably utilized. As grain yields increase year by year, straw yields also increase year by year. Therefore, the efficient utilization of crop straws becomes a subject which must be paid attention to and solved in China. At present, the most widely-used straw returning mode is still direct returning, however, many problems exist in the direct returning process of the straw, for example, the straw is slowly decomposed in a natural state, the growth of aftercrop crops is adversely affected, for example, the germination of crop seeds and the growth of seedlings are affected, meanwhile, the straw is not subjected to high-temperature decomposition treatment, and the accumulation of pathogenic bacteria in soil can be increased by directly returning the straw.
Therefore, the screening of the straw degradation functional strain with the functions of promoting growth and inhibiting diseases has positive significance for realizing straw resource utilization, soil fertility and crop yield increase.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a Raoultella planticola (Raoultella planticola) M15H2 strain, wherein the strain M15H2 can promote straw degradation and has the functions of promoting plant growth and preventing and controlling diseases.
The invention aims at realizing the following technical scheme:
the invention provides a Raoultella planticola M15H2, wherein the preservation number of the Raoultella planticola M15H2 is CGMCC NO.26627.
Preferably, the nucleotide sequence of the 16S rDNA of the Raoultella planticola M15H2 is shown as SEQ ID NO. 1.
The invention provides the fermentation broth of the Raoultella planticola M15H 2.
The invention provides a preparation method of the fermentation broth, which comprises the following steps:
culturing the Raoultella planticola M15H2 in a culture medium to obtain a fermentation broth.
The invention provides application of the Raoultella planticola M15H2 in the technical scheme, the fermentation broth in the technical scheme or the fermentation broth prepared by the preparation method in the technical scheme in straw degradation, plant growth promotion and/or disease control.
Preferably, the disease comprises a leaf streak disease.
The invention provides application of the Raoultella planticola M15H2 in the technical scheme, the fermentation broth in the technical scheme or the fermentation broth prepared by the preparation method in the technical scheme in preparation of a preparation for inhibiting rice stripe disease germ.
The invention provides a straw degradation method, which comprises the following steps:
mixing the crushed straw with water and sodium nitrate to obtain a straw material;
inoculating the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method of the technical scheme into straw materials for degrading the straw.
The invention also provides a plant growth promoting method, which comprises the following steps:
sequentially soaking seeds, sterilizing and accelerating germination to obtain pretreated seeds;
soaking seeds with the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method prepared by the technical scheme to obtain seeds treated by a microbial inoculum;
culturing the seed treated by the microbial inoculum.
The invention also provides a method for controlling diseases by plants, which comprises the following steps:
seed soaking is carried out on seeds by using the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method prepared by the technical scheme, so as to obtain seeds treated by a microbial inoculum;
and accelerating germination of the seeds treated by the microbial inoculum, and sowing.
The invention has the beneficial effects that:
the invention provides a Raoultella planticola M15H2, wherein the preservation number of the Raoultella planticola M15H2 is CGMCC NO.26627. The Raoultella planticola M15H2 provided by the invention can generate lignin enzyme, so that lignin degradation rate is improved, and straw decomposition is promoted, namely straw returning efficiency is improved; the strain M15H2 can generate growth-promoting hormone IAA and the like, promote seed germination and crop growth, and realize crop yield increase; in addition, the strain M15H2 can inhibit the growth of rice leaf spot disease germs, can effectively solve the problem of leaf spot disease and other diseases, and has important significance in guaranteeing the sustainable development of the rice industry.
Description of biological preservation
Raoultella planticola (Raoultella planticola) M15H2 is preserved in China general microbiological culture Collection center, the unit is called China academy of sciences microbiological culture Collection center for short, and the preservation number is CGMCC No.26627, the institute of China academy of sciences No. 1, no. 3, the Beijing area, the Chaetomium, at 2023.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of strain M15H 2;
FIG. 2 is a phylogenetic tree of a strain of Raoultella planticola M15H2 constructed from a 16S rDNA sequence;
FIG. 3 shows lignin enzyme-producing profiles of different strains of M15H2, M60H3 and M120H 2;
FIG. 4 is a standard graph of IAA;
FIG. 5 is a map of the phosphate solubilizing circle of strain M15H 2;
FIG. 6 is a circle chart of the inhibition of strain M15H 2;
FIG. 7 is a graph showing growth of strain M15H2 in LB medium;
FIG. 8 is a graph showing the effect of different carbon sources on the growth status of strain M15H 2;
FIG. 9 is a graph showing the effect of different nitrogen sources on the growth status of strain M15H 2;
FIG. 10 is a graph showing the effect of different pH on the growth status of strain M15H 2;
FIG. 11 is a graph showing the effect of different temperatures on the growth of strain M15H 2.
Detailed Description
The invention provides a Raoultella planticola M15H2, wherein the preservation number of the Raoultella planticola M15H2 is CGMCC NO.26627.
The Raoultella planticola M15H2 is separated from wheat straw. The Raoult fungus M15H2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 26627 in 2023 and 2-21 days.
In the present invention, the colony morphology of Raoultella planticola M15H2 is shown in FIG. 1. The colony of the Raoult fungus M15H2 on the LB culture medium is yellow, round, opaque, neat in edge, moist and smooth in surface and easy to pick. The Raoultella planticola M15H2 is a gram-negative bacterium; the Raoult bacteria M15H2 is aerobic bacteria, can generate catalase (positive contact enzyme), can hydrolyze starch, can utilize citrate, is positive in methyl red test, cannot generate gelatinase, is negative in V-P test, can generate IAA, can generate lignin enzyme, and has phosphate dissolving capacity.
The 16S rDNA sequence of the Raoultella planticola M15H2 is shown as SEQ ID NO. 1.
SEQ ID NO.1：
ACACACAAAGCCCCCGTTGACCTTTCACTGATGGTGCTAGGCAATATGGTGACTAACCTGATCAATAGTAGCGTCGCGCCGGCGCAGCGTCAGGCGATCGCCCGTTCTTTTGCCCAGGCTCTGCAGTCTTCAATCAACGACGACCCGGCGCACTAAGGATAACCAACAACTGTATATGGTGACTCTTCGTCAGCCCTACCGAGAAAAAATCTCCCAGATGGTCAGCTGGGGGCACTGGTTCGCCCTGTTCAACATGTTGTTGGCCATGGTGCTCGGCAGCCGCTATCTTTTTGTCGCCGACTGGCCCACGACTCTGGGCGGACGTATTTTCTCGTATGTCAGCCTCGTGGGTCACTTCAGTTTTCTGGTGTTTACCAGCTATGTCCTGATTCTCTTCCCGCTGACCTTTATCGTCATATCGCAGCGATTGATGCGCTTCCTGTCGGTCATTCTGGCGACCGCGGGCATGACGCTTCTGCTGATCGATAGCGAAGTCTTTACCCGTTTCCACCTGCATCTTAACCCGATTGTCTGGGAACTGGTCATCAACCCTGACCAGAATGAGATGGCGCGCGACTGGCAGCTGATGTTTATCAGCGTGCCGATTATCTTCCTGCTGGAAATGCTGTTTGCGACCTGGAGCTGGCAGAAGCTGCGCAGCCTGACGCGTCGCCGCCACTACGCCCGGCCTGTCGCCTGGTTTTTCTTTCTCTCTTTTATCAGCAGCCACCTGATTTATATCTGGGCGGATGCGAATTTCTATCGCCCGATCACCATGCAACGGGCGAATCTGCCGCTCTCCTATCCGATGACGGCCCGCCGTTTCCTTGAGAAACATGGCCTGCTCGATGCCCAGGATTACCAGCGTCGGCTGGTAGAACAGGGGGCGCCGGAAGCCGTCTCCGTTGAGTATCCGCTGAGCAACCTGCGCTATCGCGATCTGGGGGCTGGCTACAACGTTCTGCTCATTACCGTCGATAACCTGAACTATTCACGGTTTGAGAAAAATATGCCGGAACTGGCCCGTTTCGCACAAGAGAACGTCAACTTTACCCAGCATATGAGCTCCGGCAACACTACCGATAGCGGTATGTTTGGCCTGTTCTACGGGATTTCGCCGGGCTATATGGATGGCGTGCTGTCGGCCCGCATTCCGGCCGCGTTGATTACGGCCTTTAACCAGCAGGGATATCAGCTGGGGCTGTTCTCCTCCGATGGCTTCAGCAGCCCGCTCTATCGCCAGGCGCTGCTGTCCGATTTCTCGCTGCCAAATGAGAAAACGCAAAGCGATGAGCAAACGGCGGACCAGTGGATTGGCTGGCTCAATCGCTATGCGCAGGATGAAAACCGCTGGTTCTCATGGATTTCGTTAAACGGCACCACGCTGGACAACAGTCAGCAGCAAAACTTCGCGCGTCGCTACAGTCGTGCGGCGGGAGATGTTGATAGTCAAATCAGCCGGGTACTGAGCGCCCTGAGCGATGCCGGAAAACTGGATAACACCGTGGTGATTATCACCGCCGCTCACGGTATGCCGTTGAATCCGCAGCGCGGTAAGTTTGACTGGTCCCGGGAGCAGCTGCAGGTGCCGCTGGTTATTCACTGGCCAGGAATTCCGGCGCAGAACATCGCGACGCTGACCAGCAACAAAGATGTGATGACCACGCTGATGCAGCGCCTGCTGCACGTCTCCACGCCTGCCAACGAATATTCACAGGGTGAAGACCTGTTCAGCCCCGCGCGTCGCCGGGCCTGGGTGACGGCAGCCAACGGCGATACGCTGGCGGTCACGTCGCCAGAGGTGACCGTGGTGCTGAACCACAACGGCACCTACAGCACCTGGAACAGCGATGGCAAAAAGATTGATAACCGCAAACCTCAGCTCAGTCTGCTGCTGCAGATCCTGACTGACGAAAAACGCTTTATCGCTAACTGATTGATTAATTATGAATCAATCAGCCGTTCCCCGGCTTGCATTCGGTCGGGGAACGGGTAATATTGTCCTCCATACGTCGGCACGTAGCGCAGCCTGGTAGCGCACCGTCATGGGGTGTCGGGGG。
The 16S rDNA sequence of the strain M15H2 is obtained and is subjected to BLAST analysis and comparison with sequences in a GenBank database, and a phylogenetic tree of the M15H2 strain is constructed, as shown in FIG. 2. And (3) identifying the strain M15H2 as Raoult plantlet (Raoultella planticola) by combining phylogenetic tree analysis, morphological analysis and physiological and biochemical result characteristics of the strain.
The invention provides the fermentation broth of the Raoultella planticola M15H 2.
The invention provides a preparation method of the fermentation broth, which comprises the following steps:
culturing the Raoultella planticola M15H2 in a culture medium to obtain a fermentation broth.
The Raoultella planticola M15H2 is cultured in a culture medium to obtain fermentation broth. The invention is not particularly limited in the kind of the culture medium, and the strain M15H2 can be grown normally. In the present invention, the medium is preferably a TSB medium, LB medium, liquid fermentation medium, aniline blue medium, phosphate solubilizing fermentation medium or lignin enrichment medium, more preferably an LB medium. In the present invention, the pH of the medium is preferably 4 to 10, more preferably 5 to 9, and even more preferably 5. In the present invention, when the medium is LB medium, the medium preferably further comprises the following components in percentage by weight0.1% of a carbon source and 1% by weight of a nitrogen source. In the present invention, the carbon source preferably includes one or more of glucose, mannitol, sucrose, maltose, xylose and lactose, more preferably sucrose; the nitrogen source preferably includes one or more of potassium nitrate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, more preferably yeast powder. The temperature of the culture according to the invention is preferably 20 to 40 ℃, more preferably 24 to 36 ℃, still more preferably 28 to 32 ℃, still more preferably 28 ℃; the time for the culture is preferably 0 to 24 hours, more preferably 0 to 18 hours, and still more preferably 18 hours. In the present invention, the cultivation is preferably carried out in a shaking table, and the rotation speed of the shaking table is preferably 140 to 180rpm, more preferably 180rpm. In the present invention, the concentration of the fermentation broth is preferably 1X 10 6 ～1×10 8 CFU/mL, more preferably 1X 10 7 ～1×10 8 CFU/mL, more preferably 1X 10 8 CFU/mL. In the present invention, the concentration of the bacterial liquid obtained by the culture is preferably higher than the concentration of the fermentation bacterial liquid. The invention preferably makes the concentration of the bacterial liquid reach the concentration required by the fermentation bacterial liquid by adding water for dilution.
The invention provides application of the Raoultella planticola M15H2 in the technical scheme, the fermentation broth in the technical scheme or the fermentation broth prepared by the preparation method in the technical scheme in preparation of a preparation for inhibiting rice leaf spot disease germ. In the invention, the Raoultella planticola M15H2 can inhibit the growth of rice leaf spot bacteria with high efficiency.
The invention provides application of the Raoultella planticola M15H2 in the technical scheme, the zymotic fluid in the technical scheme or the zymotic fluid prepared by the preparation method in the technical scheme in straw degradation, plant growth promotion and/or disease control.
The invention provides application of the Raoultella planticola M15H2 and fermentation broth thereof in straw degradation.
The Raoultella planticola M15H2 provided by the invention can generate lignin enzyme, so that the degradation of straw is efficiently promoted, and the straw returning efficiency is further improved. In the present invention, the straw is preferably wheat straw. The wheat straw is degraded by using the Raoultella planticola M15H2 provided by the invention, so that the degradation efficiency of the wheat straw can be effectively promoted. The wheat straw degradation rate of the wheat straw degradation method is 34.32% higher than that of the wheat straw degradation control group when the wheat straw degradation method is used for degrading wheat straw by 30 days, and is 32.59% higher than that of the wheat straw degradation control group when the wheat straw degradation control group is degraded by 55 days.
The invention provides application of the Raoultella planticola M15H2 and fermentation broth thereof in plant growth promotion.
The Raoultella plantlet M15H2 provided by the invention can generate IAA and has phosphate dissolving capacity, and the Raoultella plantlet M15H2 is used for treating plants, so that the growth of the plants can be efficiently promoted. The Raoultella planticola M15H2 provided by the invention can be used for treating rice to promote the growth of the rice. The Raoult plantlet M15H2 is used for treating rice seeds, so that the germination rate of the rice seeds can be improved efficiently, and the growth of rice buds and roots can be promoted. The invention adopts the Raoult fungus M15H2 to soak the rice seeds, which obviously improves the germination rate of the seeds relative to the control group and promotes the growth of the bud length and root length of the rice seeds. According to the invention, after seeds are soaked by using the Raoult plantlet M15H2, the germination rate of rice seeds is improved by 5.67% relative to a control group, the bud length is improved by 31% relative to the control group, and the root length is improved by 40% relative to the control group.
The invention provides application of the Raoultella planticola M15H2 and fermentation broth thereof in disease control.
The Raoultella planticola M15H2 can prevent and treat diseases. In the present invention, the disease preferably includes a leaf streak disease. The invention treats rice seeds by using the plant-growing Raoult bacteria M15H2, can efficiently prevent and treat rice leaf spot, and improves the preventing and treating effect of rice on leaf spot. The invention adopts the plant-growing Raoult bacteria M15H2 to soak the rice seeds, and compared with a control group, the control effect of the rice on the rice stripe disease is obviously improved. The invention adopts the Raoult fungus M15H2 to soak the seeds, and the prevention and treatment effect of the rice stripe spot is up to 15.70%.
The invention provides a straw degradation method, which comprises the following steps:
mixing the crushed straw with water and sodium nitrate to obtain a straw material;
inoculating the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method of the technical scheme into straw materials for degrading the straw.
The invention mixes crushed straw with water and sodium nitrate to obtain straw materials.
In the present invention, the straw is preferably wheat straw. The invention preferably pulverizes the straw before degrading the straw to obtain pulverized straw. The method for crushing the straw is not particularly limited, and the conventional crushing method in the field can be adopted. After the straw is crushed, the invention preferably screens the crushed straw, and the crushed straw is obtained by taking the screen lower matters. The pore size of the screen according to the invention is preferably 20 mesh. After the crushed straw is obtained, the crushed straw is mixed with water and sodium nitrate to obtain a straw material. In the invention, the mass volume ratio of the crushed straw to the water is preferably (5-20) g: (10-30) mL, more preferably 5g:30mL. In the invention, the mass ratio of the crushed straw to the sodium nitrate is preferably (5-20): (2-5), more preferably 5:2. The method for mixing the crushed straw with water and sodium nitrate is not particularly limited, and the conventional mixing method in the field can be adopted. The purpose of adding sodium nitrate into crushed straw is to provide nitrogen source for microorganisms and accelerate decomposition.
After straw materials are obtained, the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method of the technical scheme are inoculated into the straw materials for degrading the straw.
When the plant-grown Raoult bacteria M15H2 is used for degrading straw materials, the bacterial activity of the fermentation bacterial liquid is preferably 1 multiplied by 10 6 ～1×10 8 CFU/mL, more preferably 1X 10 8 CFU/mL. When the invention is used for inoculation, the mass volume ratio of the inoculum size of the Raoultella planticola M15H2 fermentation broth to the straws is preferably (5-10) mL: (5-20) g, more preferably 10mL:5g. The temperature for carrying out straw degradation in the invention is preferably 28-36 ℃, more preferably 28 ℃. The straw degradation is preferably accompanied by vibration, and the rotating speed of the vibration is preferably 140-180 r/min, and more preferably 180r/min. The invention has no special limit to the time of straw degradation, and can completely degrade the straw. The time for degrading the straw is preferably more than or equal to 55d.
The straw degradation method provided by the invention can be used for effectively improving the degradation rate of the straw. The wheat straw is degraded by using the straw degradation method provided by the invention, and the degradation rate of the wheat straw reaches 14.95% after 30 days of degradation, and is improved by 34.32% compared with a control group; after 55 days of degradation, the degradation rate of the wheat straw reaches 15.50%, and is improved by 32.59% compared with a control group.
The invention also provides a plant growth promoting method, which comprises the following steps:
sequentially soaking seeds, sterilizing and accelerating germination to obtain pretreated seeds;
soaking the pretreated seeds by using the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method prepared by the technical scheme to obtain the seeds treated by the microbial inoculum;
culturing the seed treated by the microbial inoculum.
The invention sequentially carries out seed soaking, disinfection and germination acceleration on the seeds to obtain pretreated seeds.
In the present invention, the seeds are preferably uniformly sized, mature, full, mildew-free seeds. After the seeds are selected, the seeds are soaked in the seed soaking agent. The seed soaking method is not particularly limited, and the conventional seed soaking method in the field can be adopted. The seed soaking mode is preferably to mix seeds with water for seed soaking. The amount of water used in the present invention is not particularly limited, and the amount of water used is not limited to the seed. The seed soaking time of the present invention is preferably 30 to 45 minutes, more preferably 30 minutes. After seed soaking is completed, the seeds are preferably sterilized. The method of sterilization according to the present invention is not particularly limited, and a conventional sterilization method in the art may be employed. The method of disinfection according to the invention preferably comprises in sequence disinfection with an aqueous ethanol solution and then with an aqueous sodium hypochlorite solution. In the invention, the volume percentage of the ethanol in the ethanol water solution is preferably 70-75%, more preferably 75%; the volume percentage of sodium hypochlorite in the sodium hypochlorite aqueous solution is preferably 0.5-1%, more preferably 1%. The time for sterilization using the aqueous ethanol solution of the present invention is preferably 5 to 10 minutes, more preferably 5 minutes. The time for sterilization with the aqueous sodium hypochlorite solution of the present invention is preferably 5 to 10 minutes, more preferably 5 minutes. After the sterilization is completed, the seeds after sterilization are preferably washed with sterile water. The washing according to the present invention is preferably performed 3 to 5 times, more preferably 5 times. After the cleaning is finished, the invention preferably carries out germination acceleration on the seeds to obtain pretreated seeds. The germination accelerating method of the invention is not particularly limited, and the conventional germination accelerating method in the field can be adopted. The germination accelerating method of the invention is preferably to soak the cleaned seeds in a small beaker with sterile water for culture. The temperature of the culture according to the invention is preferably 28-32 ℃, more preferably 28 ℃; the time of the culture is preferably 18 to 24 hours, more preferably 24 hours.
After the pretreated seeds are obtained, the pretreated seeds are soaked by the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method of the technical scheme, so as to obtain the seeds treated by the microbial inoculum.
When seeds are soaked by using the Raoultella planticola M15H2, the bacterial activity of the fermentation bacterial liquid is preferably 1 multiplied by 10 6 ～1×10 8 CFU/mL, more preferably 1X 10 7 ～1×10 8 CFU/mL, more preferably 1X 10 8 CFU/mL. The soaking time is preferably 25-30 min, more preferably 30min; the soaking temperature is preferably 28 to 32 ℃, more preferably 28 ℃.
After the seed treated by the microbial inoculum is obtained, the seed treated by the microbial inoculum is cultivated.
The method of the present invention is not particularly limited, and any conventional culture method in the art may be used. In the present invention, the method of culturing is preferably performed in a culture dish; the culture dish is preferably laid with sterile filter paper. The temperature of the culture according to the invention is preferably 25 to 28 ℃, more preferably 28 ℃; the ratio of the light time to the dark time of the culture is preferably (12-16): (12-8), more preferably 16:8; the humidity of the culture is preferably 60% to 70%, more preferably 70%. The culture humidity of the present invention is preferably achieved by supplementing water in filter paper. In the culture process, whether the filter paper is dried or not is checked preferably every 2 hours, and water is timely supplied.
The plant growth promoting method provided by the invention can obviously improve the germination rate of plant seeds and promote the growth of buds and roots. According to the plant growth promoting method disclosed by the invention, the rice seeds are treated by adopting the plant growth promoting method, so that the germination rate of the rice seeds is remarkably improved, and the bud length and root length of the rice are improved, wherein when the rice seeds are cultured for 5 days, the germination rate, the rice bud length and the root length of the rice seeds are respectively increased by 5.67%, 31% and 40% compared with that of the control treatment.
The invention also provides a method for controlling diseases by plants, which comprises the following steps:
seed soaking is carried out on seeds by using the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method prepared by the technical scheme, so as to obtain seeds treated by a microbial inoculum;
and accelerating germination of the seeds treated by the microbial inoculum, and sowing.
According to the invention, seed soaking is carried out on seeds by using the Raoult fungus M15H2, the fermentation broth or the fermentation broth prepared by the preparation method according to the technical scheme, so as to obtain seeds treated by the microbial inoculum.
In the present invention, the seeds are preferably uniformly sized, mature, full, mildew-free seeds. After the seed selection is completed, the seeds are preferably sterilized. The method of sterilization according to the present invention is not particularly limited, and a conventional sterilization method in the art may be employed. The method of disinfection according to the invention preferably comprises in sequence disinfection with an aqueous ethanol solution and then with an aqueous sodium hypochlorite solution. In the invention, the volume percentage of the ethanol in the ethanol water solution is preferably 70-75%, more preferably 75%; the volume percentage of sodium hypochlorite in the sodium hypochlorite aqueous solution is preferably 0.5-1%, more preferably 1%. The time for sterilization using the aqueous ethanol solution of the present invention is preferably 5 to 10 minutes, more preferably 5 minutes. The time for sterilization with the aqueous sodium hypochlorite solution of the present invention is preferably 5 to 10 minutes, more preferably 5 minutes. After the sterilization is completed, the seeds after sterilization are preferably washed with sterile water. The washing according to the present invention is preferably performed 3 to 5 times, more preferably 5 times. After the cleaning is finished, seeds are soaked by the Raoultella planticola M15H2 prepared by the technical scheme, the fermentation broth prepared by the technical scheme or the fermentation broth prepared by the preparation method of the technical scheme. The bacterial activity of the fermentation broth of the Raoultella planticola M15H2 used for seed soaking is preferably 1 multiplied by 10 6 ～1×10 8 CFU/mL, more preferably 1X 10 7 ～1×10 8 CFU/mL, more preferably 1X 10 8 CFU/mL. The seed soaking time is preferably 24-48 hours, more preferably 48 hours; the seed soaking temperature is preferably 28 to 30 ℃, more preferably 28 ℃.
After the seed treated by the microbial inoculum is obtained, the seed treated by the microbial inoculum is germinated and then sowed.
The germination accelerating method of the invention is not particularly limited, and the conventional germination accelerating method in the field can be adopted. The germination accelerating method of the invention is preferably to soak the cleaned seeds in a small beaker with sterile water for culture. The temperature of the culture according to the invention is preferably 28-32 ℃, more preferably 28 ℃; the time of the culture is preferably 18 to 24 hours, more preferably 24 hours. After germination accelerating seeds are obtained, seeds are sown. The invention is not particularly limited to the sowing method, and the conventional sowing method in the field can be adopted. In the present invention, the sowing is preferably greenhouse potting sowing. After sowing seeds, the method for cultivating the plants is not particularly limited, and the conventional management method in the field can be adopted.
The method for preventing and controlling the diseases can obviously improve the disease control effect of plants. The method for preventing and controlling diseases provided by the invention is used for treating rice seeds, so that the preventing and controlling effect of rice on leaf streaks can be obviously improved, and the preventing and controlling effect is up to 15.70%.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Culture medium composition and formulation instructions
LB medium: 25g of LB broth powder, 1000mL of distilled water and sterilization at 121℃for 20min (agar 20g was added to the solid medium in this formulation).
Liquid fermentation medium: 2.5g of sodium nitrate, 1.0g of monopotassium phosphate, 1.0g of magnesium sulfate heptahydrate, 1.0g of sodium chloride, 0.5g of calcium chloride, 70mL of microelement solution, 20g of straw, 1000mL of distilled water and sterilizing for 20min at 121 ℃.
Microelement solution in liquid fermentation medium: ferric chloride hexahydrate: 0.16g, zinc sulfate heptahydrate: 1.5g, cobalt chloride hexahydrate: 0.16g, copper sulfate pentahydrate: 0.15g of manganese sulfate monohydrate: 1.5g, boric acid: 0.3g of sodium molybdate crystal: 0.1g, 1000mL of distilled water.
Lignin enrichment medium: 3g of sodium lignin sulfonate, 2g of ammonium sulfate, 1g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.1g of anhydrous calcium chloride, 0.02g of manganese sulfate, 1g of monopotassium phosphate, 0.05g of ferrous sulfate and 1000mL of distilled water, and sterilizing for 20min at 121 ℃.
Aniline blue screening medium: 10g of yeast powder, 10g of glucose, 0.1g of aniline blue, 18g of agar and 1000mL of distilled water, and sterilizing for 20min at 121 ℃.
Phosphate-solubilizing fermentation medium: 10g of glucose, 5g of tricalcium phosphate, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate heptahydrate, 0.03g of ferrous sulfate heptahydrate, 0.03g of manganese sulfate monohydrate, 1000mL of distilled water and sterilization at 121 ℃ for 20min (agar 20g is added to the solid culture medium in the formula).
NA medium: beef extract 3g, peptone 10g, sodium chloride 5g, distilled water 1000mL, and sterilizing at 121deg.C for 20min (agar 20g is added to the solid culture medium).
Example 1
1. Strain screening
(1) The place is located in a comprehensive test station in Anhui province's Hefei city, lu Jiang county, guo He Zhenhui agricultural university, and the wheat straw collected from the comprehensive test station in Anhui province's Hefei province, lu Jiang county, guo He Zhenhui agricultural university is subjected to strain screening.
The specific operation is as follows: intercepting wheat straw with the length of about 10cm, spreading the wheat straw on a paddy field, and ploughing and uniformly mixing; drying the straw synchronously, cutting into pieces with a length of about 3cm, and sieving with 200 mesh sieve of 15×20cm 2 20g of straw is packed in nylon net bags, and the nylon net bags are vertically placed in soil with the depth of about 15cm in a paddy field. The nylon mesh bags were removed at test nos. 7d, 15d, 60d, 120 d. Taking a nylon net bag filled with wheat straw back to a laboratory, aseptically weighing 0.5g of fresh wheat straw sample, adding the fresh wheat straw sample into 100mL of lignin enrichment medium, and placing the mixture into a constant-temperature shake incubator at 28 ℃ and 180rpm for culturing for 3d to obtain bacterial suspension.
In a sterile operation table, 1mL of prepared bacterial suspension is added with 9mL of sterile water to prepare the bacterial suspension with the concentration of 10 -1 Is a raw liquid of (a) a liquid. Separating and purifying the strain by adopting a dilution coating flat plate method and a flat line drawing method, and storing the pure cultured strain in a refrigerator at 4 ℃.
And then respectively inoculating the separated and purified strains on an aniline blue screening culture medium to screen strains with lignin degradation capability. Placing the screened lignin strain in LB culture solution at 28 ℃ and 180r/min for overnight shake flask culture, inoculating the cultured bacterial solution to an aniline blue solid culture medium, culturing for 3d at 28 ℃, and judging the lignin degradation capability of the strain according to the fading condition of an aniline blue flat plate.
The discoloration of the screened strain is shown in Table 1.
Table 1 fading of the selected Strain on aniline blue Medium
| Bacterial numbering | Whether or not to fade |
| M7H1 | Whether or not |
| M7H2 | Whether or not |
| M7H3 | Whether or not |
| M15H1 | Whether or not |
| M15H2 | Is that |
| M60H2 | Whether or not |
| M60H3 | Is that |
| M120H2 | Is that |
| M120H3 | Whether or not |
As can be seen from Table 1, the results from the discoloration indicate that the M15H2, M60H3 and M120H2 strains have lignin degrading ability.
(2) Lignin enzyme activity determination of M15H2, M60H3 and M120H2 strains
Separately inoculating M15H2, M60H3 and M120H2 strains into a liquid fermentation culture medium, placing the liquid fermentation culture medium into a constant-temperature shake incubator with the temperature of 28 ℃ and 180rmp for shake culture for 48 hours to prepare bacterial suspension, placing the bacterial suspension into a 10mL centrifuge tube, placing the bacterial suspension into a freeze-drying centrifuge with the temperature of 10000r/min,10min and 4 ℃ for centrifugation, collecting bacterial strain precipitate, and sending a sample to Suzhou Ming biotechnology Co Ltd for measuring lignin enzyme activity.
The results of the enzyme activity measurement are shown in FIG. 3.
As can be obtained from FIG. 3, the screened 3 strains have strong lignin degrading capability of M15H2, and lignin peroxidase activity can reach 129.49 U.mL -1 Manganese peroxidase can reach 23.91 U.mL -1 The enzyme activity of laccase can reach 8.91 U.mL -1 。
2. Strain M15H2 Performance assay
(1) Determination of IAA secretion Capacity
IAA standard curve drawing
Weighing 50mg IAA on a ten-thousandth balance, dissolving in a 500mL volumetric flask with three-level water, adding water to a fixed volume to a scale mark, and uniformly mixing, wherein the concentration of IAA in the solution is 100mg/L. Then respectively sucking 0.00mL, 0.15mL, 0.30mL, 0.45mL, 0.60mL, 0.75mL, 0.90mL, 1.05mL, 1.25mL, 1.35mL and 1.50mL of IAA standard solution into a clean test tube, adding distilled water to complement 2mL, adding 4mL of Salkawski's reaction solution, mixing uniformly to obtain IAA standard series solution with final concentration of 0.0 mug/mL, 2.5 mug/mL, 5.0 mug/mL, 7.5 mug/mL, 10.0 mug/mL, 12.5 mug/mL, 15.0 mug/mL, 17.5 mug/mL, 20.0 mug/mL, 22.5 mug/mL and 25.0 mug/mL, and carrying out light-shielding reaction at room temperature for 30min, and measuring the light absorption value at 530 nm. IAA standard curves were plotted with the measured absorbance on the ordinate and IAA concentration (mg/L) on the abscissa. The standard curve related data are shown in table 2, and the drawn standard curve is shown in fig. 4.
TABLE 2 IAA Standard Curve correlation data
| Concentration (mg/L) | 0.0 | 2.5 | 5.0 | 7.5 | 10.0 | 12.5 | 15.0 | 17.5 | 20.0 | 22.5 | 25.0 |
| OD 530 | 0.000 | 0.130 | 0.278 | 0.453 | 0.622 | 0.870 | 0.968 | 1.257 | 1.465 | 1.659 | 1.822 |
As can be seen from table 2 and fig. 4, the standard curve equation for IAA is y= 0.0754x-0.077, r 2 ＝0.9945。
IAA production assay for Strain M15H2
Inoculating strain M15H2 into a strain containing 5.0mmol L -1 Culturing in TSB medium containing L-tryptophan at 28deg.C under 180rmp for 48 hr, centrifuging the bacterial suspension to remove bacterial cells, collecting 1.0mL sterile filtrate, and adding 2.0mL Salkawski's color development agent (50 mL 35% HClO) 4 ，1mL 0.5mol·L -1 FeCl 3 Light-shielding preservation), and after being kept under light shielding for 30min, the glass fiber reinforced plastic film is observed.
After the substrate was kept for 30 minutes under light shielding, the substrate was observed and the absorbance at 530nm was measured. Blank medium was also used as a control. IAA secretion was calculated by IAA standard curve.
The measured OD530 value was 0.218, and the IAA secretion amount of strain M15H2 was 3.90mg/L, which was obtained by substituting it into the IAA standard curve equation.
(2) Determination of phosphate-solubilizing ability
The purified strain M15H2 is selected by an inoculating loop and inoculated in LB culture medium, and shake culture is carried out for 24 hours at 28 ℃ and 180r/min, so as to be used as seed liquid for the test.
And qualitatively detecting, namely dipping seed liquid by using an inoculating loop, inoculating the seed liquid to the central position of the phosphate solubilizing and fermenting solid plate, placing the plate in a constant temperature incubator at 28 ℃ for culturing after drying, observing the phosphate solubilizing and fermenting solid plate after culturing for 5-7 d, and measuring phosphate solubilizing rings on the plate. The bacterial strain is provided with three repeats, and the size of the diameter of the phosphate solubilizing ring on the flat plate is measured to preliminarily compare the phosphate solubilizing capability of the bacterial strain.
The qualitative detection results are shown in FIG. 5.
Quantitative detection: taking 1% of seed liquid, inoculating the seed liquid into 100mL of phosphate-dissolving fermentation medium, culturing for 5d in a constant-temperature shake incubator at 28 ℃ and 180r/min by using a 250mL volumetric flask, and measuring the content of soluble phosphorus in the supernatant by using a molybdenum-antimony colorimetric method by using an inactivated strain as a control, wherein each group is provided with 3 repetitions.
The quantitative determination results of the phosphorus dissolving capacity of the experimental group and the control group are shown in table 3.
TABLE 3 quantitative determination of the phosphate solubilizing ability of the experimental and control groups
As can be seen from FIG. 5, strain M15H2 was found to be capable of producing P-turn by qualitative determination of the phosphorus-resolving power of the strain, and the ratio of transparent-turn diameter to colony diameter was 1.08.+ -. 0.02. As can be seen from Table 3, the dynamic monitoring of the P-decomposing ability of the strain M15H2 shows that the M15H2 has the strongest inorganic phosphorus-decomposing ability, up to 4.97mg.L on day 5 -1 。
(3) Antagonistic pathogen capability assay
Inoculating rice leaf spot pathogen into NA culture solution, shake culturing at 28deg.C under 180r/min to obtain pathogen fermentation broth, taking out after 48 hr, adding 9mL of sterile water into 1mL of prepared pathogen fermentation broth, and making into gradient of 10 -1 Is repeatedly diluted to 10 -3 . Bacterial strain M15H2 is inoculated into LB culture solution, shake culture is carried out at 28 ℃ and 180r/min to prepare antagonistic bacteria solution, and the antagonistic bacteria solution is taken out after 24 hours. 0.1mL gradient was taken as 10 -3 Uniformly coating the bacterial fermentation liquor on an NA solid culture medium, standing and cooling, sucking 1.0 mu L of antagonistic bacteria, dripping the antagonistic bacteria on a flat plate containing the bacterial fermentation liquor, taking sterile water as a reference, air-drying for 10min, culturing the bacterial fermentation liquor in a 28 ℃ incubator for 48h, recording the radius of a bacteriostasis ring, and setting 3 repetitions for each group. Observing whether a bacteriostasis ring appears on the culture medium, and recording the size of the bacteriostasis ring by adopting a crisscross method.
By measuring the disease-suppressing ability of the strain, the strain M15H2 was found to be capable of producing a zone of inhibition (FIG. 6), and the ratio of the diameter of the zone of inhibition to the diameter of the colony was recorded to be 1.20.+ -. 0.01. The ratio of the diameter of the inhibition zone to the diameter of the inhibition zone is more than 1, and the inhibition effect is certain.
3. Identification of strains
(1) Colony morphology observation
The strain M15H2 was streaked on LB solid medium, cultured at 28℃for 48 hours, and the morphology of single colonies was observed and recorded as shown in FIG. 1.
As can be seen from FIG. 1, the colony of strain M15H2 is yellow, round, opaque, clean in edge, moist and smooth in surface and easy to pick.
(2) Gram staining
The strain M15H2 is observed under an oil lens after being dyed by a gram-type dyeing method, and the strain is gram-negative bacteria.
(3) Physiological and biochemical characteristics
The physiological and biochemical characteristics of strain M15H2 were determined. The test results are shown in Table 4.
TABLE 4 physiological and biochemical characterization of M15H2
| Project | Results | Project | Results |
| Starch hydrolysis | + | Citrate utilization test | + |
| V-P test | - | Gelatin liquefaction test | - |
| Methyl Red test | + | Gram typeDyeing | - |
| Contact enzyme test | + | Aerobic test | + |
Note that: + indicates positive reaction, -indicates negative reaction
As shown in Table 4, the strain M15H2 was an aerobic bacterium, was able to produce catalase (contact enzyme positive), was able to hydrolyze starch, was able to use citrate, was positive in the methyl red test, was unable to produce gelatinase, and was negative in the V-P test.
(4) Molecular characterization
The strain M15H2 was extracted for DNA, subjected to PCR, sequenced by Shanghai Meiji biomedical technologies, and aligned in GenBank database according to the obtained 16S rDNA sequence result, blast searched for homologous sequences, and a phylogenetic tree was constructed by a neighbor-Joining method using MEGA-X software, as shown in FIG. 2.
The strain is identified as Raoult planticola (Raoultella planticola) by combining morphological analysis and physiological and biochemical result characteristics of the strain. Raoult bacteria (Raoultella planticola) M15H2 are preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 26627 in the year 2023, 2 and 21. Deposit center address: the national academy of sciences of China, national institute of sciences, no. 1, 3, north Chen West Lu, the Korean region of Beijing, city.
Example 2
1. Determination of growth curves of Strain M15H2 in LB Medium
Inoculating the strain M15H2 into LB liquid medium at 28deg.C for 180r/min overnight to obtain seed solution, inoculating 100 μl of seed solution into 10mL of LB culture solution, shake culturing in shake box at 28deg.C for 180r/min, taking out 3mL of bacterial solution respectively at 0H, 3H, 6H, 9H, 12H … … H, placing in refrigerator,blank culture solution is used as reference control, and OD is measured by ultraviolet spectrophotometer 600 Numerical values, and drawing a growth curve.
The growth curve plotted is shown in fig. 7.
The results are shown in FIG. 7: the growth speed of the strain is faster, 0 to 18 hours after inoculation is the logarithmic growth phase, and the OD of the 18 th logarithmic growth phase 600 The value reached the maximum and the growth of the strain reached the maximum.
2. Influence of different carbon sources on strain growth
And (3) taking ammonium nitrate as an optimal nitrogen source, and researching the influence of maltose, sucrose, lactose, xylose, glucose and mannitol serving as different carbon sources on the growth of the strain M15H 2.
The strain M15H2 is inoculated into LB liquid medium for culturing for 18H at 28 ℃ and 180r/min, and seed liquid is prepared.
Preparing LB culture solution, adding different carbon sources with weight fractions of 0.1% into the LB culture solution, inoculating 0.5mL of seed solution, culturing at 28deg.C and 180r/min for 48h, and setting 3 replicates for each treatment. Taking the fermentation liquor at OD 600 Determination of UV absorbance at nm wavelength, OD 600 The higher the value, the higher the concentration of bacteria, and the better the growth state.
The growth of the strain cultured for 48 hours under different carbon source conditions is shown in FIG. 8.
The results are shown in FIG. 8: when sucrose is taken as a carbon source, the absorbance of the strain M15H2 reaches the maximum, and OD 600 1.08.
3. Influence of different nitrogen sources on strain growth
And (3) taking sucrose as an optimal carbon source, and researching the influence of ammonium sulfate, potassium nitrate, peptone, yeast powder, urea and glutamic acid as different nitrogen sources on the growth of the strain M15H 2.
The strain M15H2 is inoculated into LB liquid medium for culturing for 18H at 28 ℃ and 180r/min, and seed liquid is prepared.
Preparing LB culture solution, adding different 1% nitrogen sources into LB culture solution, inoculating 0.5mL seed solution, culturing at 28deg.C at 180r/min for 48 hr, and setting 3 weight per treatmentAnd (5) repeating. Taking the fermentation liquor at OD 600 Ultraviolet absorbance was measured at nm wavelength.
The growth of the strain cultured for 48h under different carbon source conditions is shown in FIG. 9.
The results are shown in FIG. 9: when yeast powder is taken as a nitrogen source, the absorbance of the strain M15H2 reaches the maximum, and OD 600 3.95.
4. Influence of different pH on strain growth
Sucrose is used as an optimal carbon source, yeast powder is used as an optimal nitrogen source, and the influence of different pH values (4, 5, 6, 7, 8, 9 and 10) on the growth of the strain M15H2 is studied.
The strain M15H2 is inoculated into LB liquid medium for culturing for 18H at 28 ℃ and 180r/min, and seed liquid is prepared.
Preparing LB culture solution, taking sucrose as an optimal carbon source, taking yeast powder as an optimal nitrogen source, adjusting the pH value of the LB culture solution to be 4, 5, 6, 7, 8, 9 and 10, and inoculating 0.5mL of seed solution, culturing for 48h in a shaking table at 28 ℃ and 180r/min, taking the LB culture solution without bacteria as a reference, and setting 3 repetitions for each treatment. Measuring bacterial liquid OD 600 Values.
The results are shown in FIG. 10: at pH 5, the absorbance of the strain M15H2 reaches the maximum and OD 600 5.04.
5. Influence of different temperatures on the growth of strains
Sucrose was used as an optimal carbon source, yeast powder as an optimal nitrogen source, and ph=5 as an optimal pH, and the effect of different temperatures (20 ℃, 24 ℃, 28 ℃, 32 ℃, 36 ℃, 40 ℃) on the growth of the strain M15H2 was studied.
The strain M15H2 is inoculated into LB liquid medium for culturing for 18H at 28 ℃ and 180r/min, and seed liquid is prepared.
Preparing LB culture solution, taking sucrose as an optimal carbon source, yeast powder as an optimal nitrogen source, adjusting the pH value of the LB culture solution to 5, inoculating 0.5mL of seed solution, respectively culturing in a shaking table at 20 ℃, 24 ℃, 28 ℃, 32 ℃, 36 ℃, 40 ℃ and 180r/min for 48 hours, taking the LB culture solution without inoculating bacteria as a reference, and setting 3 repetitions for each treatment. Measuring bacterial liquid OD 600 Values.
The results are shown in FIG. 11: at a temperature of 28-32 DEG CThe absorbance of the strain M15H2 reaches the maximum and OD 600 8.57.
Example 3
1. Straw corrosion promotion dynamic monitoring test using bacterial strain M15H2 as microbial inoculum
M15H2 bacterial liquid preparation: inoculating M15H2 into LB culture solution, culturing in a constant temperature shake incubator at 28deg.C and 180r/min for 18 hr, taking out shake cultured bacterial liquid, adding sterile water, and diluting to bacterial liquid concentration of 1×10 8 CFU·mL -1 。
The test process comprises the following steps: weighing 5g of crushed wheat straw powder passing through a 20-mesh sieve, putting the crushed wheat straw powder into a 250mL triangular flask, adding 30mL of water and 2g of sodium nitrate, and adding 10mL of water with the concentration of 10 8 CFU·mL -1 Culturing the left and right bacterial solutions in a constant temperature shake incubator at 28 ℃ and 160r/min, taking out the triangular flask after 30d and 55d culture, putting the straw in the triangular flask into a funnel of three layers of gauze for filtering, discarding the filtrate, and taking the straw on the gauze. The filtered straw on the gauze is placed in an oven at 80 ℃ to be dried to constant weight, sterile water is additionally arranged to replace bacterial liquid, other steps are consistent, and the treatment is used as control treatment, and each treatment is repeated three times.
The formula of the straw decomposition rate is a weight loss rate method, and the formula is as follows.
Formula of straw degradation rate
Wherein: w (W) 0 Represents the dry weight of wheat straw in the liquid culture medium before inoculation, W 1 The dry weight of the wheat straw after the completion of the cultivation is shown.
The dynamic detection result of the bacterial strain M15H2 as a microbial inoculum on the degradation of the wheat straw is shown in table 5.
TABLE 5 dynamic detection results of M15H2 degradation of straw by strain
| Treatment of | Shaking bacteria 30d | Shaking bacteria 55d |
| M15H2 | 14.95±2.78* | 15.50±0.47* |
| Sterile water | 11.13±2.98 | 11.69±2.24 |
Note that the table shows significant differences, P <0.05.
As can be obtained from table 5, after 30 days of degradation, the degradation rate of the wheat straw treated by the microbial inoculum reaches 14.95%, and the degradation rate of the wheat straw treated by the microbial inoculum is improved by 34.32% relative to that of the control group; after 55 days of degradation, the degradation rate of the wheat straw treated by the microbial inoculum reaches 15.50%, and the degradation rate of the wheat straw treated by the microbial inoculum is improved by 32.59% compared with that of a control group.
2. Growth promotion test of bacterial strain M15H2 as microbial inoculum on rice
Test soil: taken from Lujiang county Guo Hezhen, a Hefei city, anhui province
Seed used in the test: variety is Lin-Han No. 1
M15H2 bacterial liquid preparation: inoculating M15H2 into LB culture solution, culturing in a constant temperature shake incubator at 28deg.C and 180r/min for 18 hr, taking out shake cultured bacterial liquid, adding sterile water, and diluting to bacterial liquid concentration of 1×10 8 CFU/mL。
Selecting rice seeds which are uniform in size, mature, full and free from mildew, and soaking the rice seeds.
Seed soaking: pouring a proper amount of rice seeds into a small beaker, adding pure water (the water quantity is less than the seeds) into the small beaker, soaking for 30min, and removing the seeds floating on the upper side.
And (3) disinfection: sterilizing the surface of 75% alcohol for 5min, soaking in 1% sodium hypochlorite water solution for 5min, and cleaning with sterile water for 5 times.
Accelerating germination: the washed seeds were immersed in a small beaker containing sterile water and incubated in an incubator at 28℃for 24 hours.
Bacterial liquid soaking: the bacterial activity of the seeds after germination acceleration is 1 multiplied by 10 8 The CFU/mL bacterial liquid is soaked for 30min at 28 ℃.
The rice seeds are uniformly paved in a culture dish of sterile filter paper after the steps of seed soaking/disinfection/germination accelerating/bacterial liquid soaking and the like, and 3 repetitions are set for each group by taking sterile water soaking as a control. 55 seeds were placed in each dish and the dishes were placed at the following temperature: culturing at 28deg.C in a light incubator with light and darkness ratio of 16:8 and humidity of 70%, checking whether the filter paper is dry every 2 hours, supplementing water in time, and observing germination rate, bud length and root length after culturing for 5 days.
The effects of the microbial inoculum M15H2 on the germination rate, shoot length and root length of rice are shown in Table 6.
TABLE 6 Effect of microbial inoculum M15H2 on germination Rate, bud Length and root Length of Rice
| Strain | Germination percentage (%) | Bud length (cm) | Root length (cm) |
| M15H2 | 67.88±4.20 | 0.38±0.07 | 0.70±0.03 |
| Sterile water | 64.24±8.20 | 0.29±0.03 | 0.50±0.17 |
As shown in Table 6, the M15H2 strain has the effect of promoting plant growth, and in this test, the germination rate, the bud length and the root length of rice plants are improved after the inoculation treatment. Wherein the germination rate, bud length and root length are respectively increased by 5.67%, 31% and 40% compared with the control treatment. Strain M15H2 was able to promote plant growth.
3. Test of bacterial strain for preventing and controlling rice stripe rot germ
Test soil: taken from Lujiang county Guo Hezhen, a Hefei city, anhui province
Seed used in the test: variety is Lin-Han No. 1
M15H2 bacterial liquid preparation: inoculating M15H2 into LB culture solution, culturing in a constant temperature shake incubator at 28deg.C and 180r/min for 18 hr, taking out shake cultured bacterial liquid, adding sterile water, and diluting to bacterial liquid concentration of 1×10 8 CFU/mL。
Preparing bacterial suspension of leaf spot disease bacteria: inoculating the strain to NA culture solution, culturing in a constant temperature shake incubator at 28deg.C and 180r/min for 48 hr, taking out the shake cultured bacterial solution, adding sterile water, and diluting to bacterial solution concentration of 1×10 8 CFU/mL。
After rice seeds with uniform size, full maturity and no mildew are selected, the seeds are sterilized in the following modes: sterilizing the surface of 75% alcohol for 5min, soaking in 1% sodium hypochlorite water solution for 5min, and cleaning with sterile water for 5 times.
After the cleaning is completed, the rice seeds are put into M15H2 bacterial suspension and soaked for 48 hours at 28 ℃. After seed soaking, accelerating germination of seeds and accelerating germination methodIn order to cultivate the seeds soaked by the bacterial liquid in a small beaker filled with sterile water in a 28 ℃ incubator for 24 hours, and then placing the seeds in a greenhouse for potting and sowing. The seed soaking with clean water is used as a control, and each group is repeated for 3 times. When the rice seedlings grow to 1 heart 2 leaf stage, the spray inoculation concentration is about 1.0X10 8 About 10mL of CFU/mL of the strip spot pathogenic bacteria suspension is covered with the film for moisturizing for 24 hours. And measuring the size of each group of strip spot after spraying the pathogenic bacteria liquid for 14d, and calculating the control effect according to the control effect formula.
The formula of the germ prevention and control effect is as follows:
the control effect of the bacterial agent M15H2 on rice leaf spot is shown in Table 7.
Surface 7 prevention and treatment effect of bacterial agent M15H2 on rice leaf spot
| Seed soaking treatment | Disease spot (cm) | Preventing effect (%) |
| Clean water | 6.74±0.81 | 0.00 |
| M15H2 | 5.68±0.61 | 15.7 |
According to the test result, the prevention effect of the bacterial suspension M15H2 on the leaf spot is 0.00% after the seed soaking in clear water, and is 15.70% obviously higher than the prevention effect after the seed soaking in clear water. Thus: the strain M15H2 has the inhibiting function on rice leaf spot.
In conclusion, the plant-growing Raoult bacteria M15H2 provided by the invention can promote the decomposition of straw returning to the field, improve the straw returning efficiency, promote the germination of seeds and the growth of crops, realize the yield increase of crops, inhibit the growth of rice leaf spot bacteria and effectively solve the problems of leaf spot diseases and other diseases.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. The Raoulella plantarum M15H2 strain is characterized in that the preservation number of the Raoulella plantarum M15H2 strain is CGMCC NO.26627.
2. The Raoultella planticola M15H2 of claim 1, wherein the nucleotide sequence of the 16SrDNA of the Raoultella planticola M15H2 is shown as SEQ ID NO. 1.
3. The fermentation broth of Raoultella planticola M15H2 according to claim 1 or 2.
4. A method for preparing a fermentation broth according to claim 3, comprising the steps of:
culturing the Raoultella planticola M15H2 in a culture medium to obtain a fermentation broth.
5. The application of the Raoultella planticola M15H2 according to claim 1 or 2, the zymophyte liquid according to claim 3 or the zymophyte liquid prepared by the preparation method according to claim 4 in straw degradation, plant growth promotion and/or disease control.
6. The use according to claim 5, wherein the disease comprises a leaf streak disease.
7. Application of Raoult bacteria M15H2 produced by plants in claim 1 or 2, fermentation broth produced by the preparation method in claim 3 or fermentation broth produced by the preparation method in claim 4 in preparation of preparation for inhibiting rice stripe disease bacteria.
8. The straw degradation method is characterized by comprising the following steps of:
mixing the crushed straw with water and sodium nitrate to obtain a straw material;
inoculating the Raoultella planticola M15H2 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4 into straw materials for degrading straw.
9. A method for plant growth promotion comprising the steps of:
sequentially soaking seeds, sterilizing and accelerating germination to obtain pretreated seeds;
soaking the pretreated seeds with the Raoultella planticola M15H2 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4 to obtain the seeds treated by the microbial inoculum;
culturing the seed treated by the microbial inoculum.
10. A method for controlling diseases of plants, which is characterized by comprising the following steps:
seed soaking is carried out on seeds by using the Raoultella planticola M15H2 according to claim 1 or 2, the fermentation broth according to claim 3 or the fermentation broth prepared by the preparation method according to claim 4, so as to obtain seeds treated by the microbial inoculum;
And accelerating germination of the seeds treated by the microbial inoculum, and sowing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117402796A (en) * | 2023-12-14 | 2024-01-16 | 中国农业大学 | Degradable microplastic and straw as well as Raoultella planticola with growth promoting and stress resisting functions and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117402796A (en) * | 2023-12-14 | 2024-01-16 | 中国农业大学 | Degradable microplastic and straw as well as Raoultella planticola with growth promoting and stress resisting functions and application thereof |
| CN117402796B (en) * | 2023-12-14 | 2024-03-08 | 中国农业大学 | Degradable microplastic and straw as well as Raoultella planticola with growth promoting and stress resisting functions and application thereof |
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