CN115820477A - Plant growth promoting agrobacterium and application - Google Patents
Plant growth promoting agrobacterium and application Download PDFInfo
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- CN115820477A CN115820477A CN202211386202.5A CN202211386202A CN115820477A CN 115820477 A CN115820477 A CN 115820477A CN 202211386202 A CN202211386202 A CN 202211386202A CN 115820477 A CN115820477 A CN 115820477A
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Abstract
The invention relates to a plant growth-promoting Agrobacterium tumefaciens and application thereof, wherein the plant growth-promoting Agrobacterium tumefaciens is Agrobacterium larymoorei, which is preserved in China general microbiological culture Collection center at 5-9 months in 2022, and the preservation numbers are as follows: CGMCC No.24856. The plant growth promoting agrobacterium of the invention is separated from corn rhizosphere soil. The plant growth promoting agrobacterium can promote the growth of dicotyledonous leaf lettuce and monocotyledonous wheat. Compared with the prior art, the plant growth promoting Agrobacterium rhizogenes arrymoorei has a nitrogen fixation effect, can effectively improve nitrogen in wheat and leaf lettuce, has an obvious promotion effect on the growth of the wheat and the leaf lettuce, and is expected to be widely applied to the field of microbial fertilizers.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a plant growth promoting agrobacterium and application thereof.
Background
According to statistics, due to the fact that a large amount of chemical fertilizers are applied, the problems of large accumulation of chemical nutrients in farmland soil, soil acidification, secondary salinization and the like are more and more prominent, the micro-ecological balance of the soil is seriously damaged, a large amount of pathogenic bacteria are accumulated, the plants grow weakly, diseases are highly developed, and the serious threats are caused to the ecological environment safety and the food safety of farmlands. The method has the advantages that the production technical mode of excessive application of chemical fertilizers and pesticides in the current agricultural production is improved, the soil structure is improved, the quantity and activity of beneficial microorganisms in soil are increased, the plant growth is promoted, the utilization rate of fertilizers in the soil is improved, and the fertilizer supply capacity of the soil is improved, so that the method becomes an important direction of the current and future agricultural development in China, and is necessary for recovering good farmland ecological environment, realizing harmonious and harmonious co-development of agricultural production and nature, and promoting the sustainable development of agriculture ecology, economy and society.
The microbial fertilizer is a specific product containing a microorganism living body, and can obtain a specific fertilizer effect when being applied to agricultural production, wherein the living microorganisms in the product play a key role. Currently, microbial fertilizer products are generally divided into two broad categories: one is a microbial fertilizer in the narrow sense, which means that the supply of plant nutrient elements is increased by the life activities of microorganisms, including the total supply of plant nutrient elements in soil and production environment, resulting in improvement of plant nutrient status and further increase of yield, and the representative of the microbial fertilizer is rhizobia fertilizer; the other is a broad-sense microbial fertilizer, which not only can increase the supply of plant nutrient elements, but also can generate plant growth hormone through the life activities of microorganisms in the fertilizer, promote the absorption and utilization of the nutrient elements by plants or have the pathogenic effect of antagonizing certain pathogenic microorganisms, reduce crop diseases and insect pests and indirectly increase the crop yield. Compared with chemical fertilizers, the microbial fertilizer has the following advantages: the soil structure is not damaged; the ecological environment is protected, the environment is not polluted, and the fertilizer is non-toxic and harmless to people and livestock; the fertilizer efficiency is durable; the yield of the crops is improved, and the quality of the crops is improved; low cost, economy and effectiveness.
Plant growth-promoting rhizobacteria (PGPR) is a kind of microorganism capable of high-density colonizing in Plant rhizosphere, and has the functions of inhibiting Plant pathogenic bacteria and harmful microorganism in rhizosphere, promoting Plant growth and increasing crop yield. As an important resource library of biological fertilizers and biological pesticides, the research and application of PGPR plays a very important role, and the research and development of microbial fertilizers from the perspective of resource recycling has a realistic significance on resource comprehensive utilization and environmental protection.
Through research, the agrobacterium is also a plant growth-promoting rhizobacteria. Most agrobacteria can infect the root of dicotyledonous plants and promote the proliferation of roots or stems, but are not easy to infect monocotyledonous plants, and according to the related literature, the agrobacteria K1026 has no obvious effect on the growth of monocotyledonous wheat (DOI: 10.3976/j.issn.1002-4026.2017.04.005).
Disclosure of Invention
One of the purposes of the invention is to provide a plant growth promoting agrobacterium
The other purpose of the invention is to provide the application of the plant growth promoting agrobacterium.
In order to realize the purpose, the invention provides a plant growth promoting agrobacterium and application thereof.
The purpose of the invention can be realized by the following technical scheme:
one of the technical schemes of the invention is to provide a plant growth promoting agrobacterium. The plant growth-promoting agrobacterium is preserved in China general microbiological culture Collection center at 2022, 5 months and 9 days, and the preservation addresses are as follows: the preservation number of No.3 Xilu Beijing Hospital No.1, chaoyang area, beijing, is: CGMCC No.24856, named as Agrobacterium larymoorei.
Further, the colony characteristics of the plant growth promoting agrobacterium are as follows: after 24 hours of culture on an LB plate culture medium, the colony is round, smooth in surface, moist, raised, white and transparent, and regular in edge.
Furthermore, the 16S rDNA sequence of the plant growth promoting agrobacterium is shown as SEQ ID No.1.
Further, the technical scheme also provides a microbial inoculum containing the plant growth promoting agrobacterium.
Furthermore, the microbial inoculum comprises the following components of sterile water and plant growth promoting agrobacterium, wherein the ratio of the sterile water to the plant growth promoting agrobacterium is 100mL:1g of the total weight of the composition.
The second technical scheme of the invention is to provide the application of the plant growth promoting agrobacterium or the microbial inoculum according to the first technical scheme.
Further, the plant growth promoting agrobacterium or the microbial inoculum is applied to promoting plant growth.
Still further, the plant is an angiosperm.
Further, the angiosperm is leaf of Ottelia tetragonolobus and wheat.
Further, the plant growth promoting agrobacterium or the microbial agent acts on the roots of the plant.
Further, the plant growth promoting agrobacterium or the microbial inoculum is applied to preparation of biological fertilizers.
Compared with the prior art, the invention has the beneficial effects that: the Agrobacterium arrymobacter larymoorei has a nitrogen fixation effect, can effectively improve nitrogen in monocotyledon wheat and dicotyledon leaf lettuce, has an obvious promotion effect on growth of the monocotyledon wheat and the dicotyledon leaf lettuce, and is expected to be widely applied to the field of microbial fertilizers.
Drawings
FIG. 1 is a colony morphology of Agrobacterium rasimulus on LB solid medium.
FIG. 2 is a graph of an experimental group and a control group of leaf lettuce in example 2.
FIG. 3 is a diagram of experimental group and control group of potting wheat in example 3.
FIG. 4 is a graph of the experimental group and the control group of wheat in example 3.
Where Ck is the control group and 23002 is the experimental group.
Biological material protectionHiding information
The plant growth-promoting bacteria of the invention are preserved in the China general microbiological culture Collection center in 2022, 5 months and 9 days, and the preservation addresses are as follows: the preservation number of No.3 Xilu Beijing Hospital No.1, chaoyang area, beijing, is: CGMCC No.24856, named as Agrobacterium larymoorei.
Detailed Description
The invention is described in detail below with reference to the figures and the specific embodiments.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1:
separation and identification of the Larimorella agrobacterium:
the specific separation method is as follows:
(1) Collecting corn rhizosphere soil of a corn planting base in Suizhiya, heilongjiang province by adopting a five-point sampling method, and pretreating the collected corn rhizosphere soil by a sieve with the aperture of 2 mm;
(2) Adding 90mL of sterile water into 10g of pretreated corn rhizosphere soil, adding about 20 glass beads, shaking for 10min on a shaking table at 150r/min, and standing to obtain the soil with the concentration of 10 -1 g/mL of mother liquor;
(3) Adding 1mL of supernatant of the mother liquor obtained in the step (2) into 9mL of sterile water to obtain a soil concentration of 10 -2 g/mL of diluent, and the like, and then carrying out gradient dilution by 3-9 times to obtain the soil with the concentration of 10 -3 -10 -10 g/mL of diluent;
(4) Respectively coating each diluent in the step (3) on an ACCC55 medium plate for culture, coating 3 ACCC55 medium plates on each diluent for parallel experiment, selecting growing bacterial colonies, re-inoculating the bacterial colonies on the ACCC55 medium plate for culture for 4 days at 28 ℃, repeating for multiple times, and screening to obtain nitrogen-fixing single bacterial colonies;
(5) And (3) inoculating the single nitrogen-fixing colony in the step (4) on an LB solid culture medium, performing purification culture at 30 ℃ for 24h, and performing 16S rDNA sequencing on the purified colony, wherein a primer F (SEQ ID No. 2) is AGAGAGTTTGATCCTGGCTCAG, a primer R (SEQ ID No. 3) is TACGGYTACCTTACGACTT, and the 16S rDNA sequence is shown in SEQ ID No.1. As shown in FIG. 1, after 24 hours of cultivation on LB plate medium, the colony was round, smooth, moist, raised, white, transparent, and regular-edged.
The formula of the ACCC55 culture medium is as follows: 10.0g of sucrose, 0.2g of sodium chloride, 1.0g of calcium carbonate, 0.5g of monopotassium phosphate trihydrate, 0.2g of magnesium sulfate heptahydrate, 18.0g of agar and 1000mL of distilled water, wherein the pH value is adjusted to 7.4-7.6. Sterilizing at 121 deg.C for 15min, and pouring into flat plate.
The LB solid medium formulation is as follows: 5g of yeast extract, 10g of tryptone, 10g of NaCl, 18.0g of agar and 1000mL of distilled water, and the pH value is adjusted to be about 7.0. Sterilizing at 121 deg.C for 15min, and pouring into flat plate.
Example 2:
potting experiment of leaf lettuce:
culturing the seeds of the leaf lettuce in a seedling culture plate, culturing the seeds for 1 week under natural conditions, and selecting the leaf lettuce seedlings with the same growth vigor as experimental objects of a pot culture experiment. Transplanting the seedlings of the leaf lettuce into a cup filled with equal amount of soil, fixing for two days every week, adding equal amount of water (a control group) and bacterial suspension (an experimental group) into the leaf lettuce, selecting Tuesday and Friday in the embodiment, adding 100mL of water or bacterial suspension into the leaf lettuce, and continuously culturing the leaf lettuce for 1 month. And after the culture is finished, harvesting the leaf lettuce, weighing, calculating the growth rate, and detecting the total nitrogen and total phosphorus indexes in the leaf lettuce.
The bacterial suspension was prepared as follows:
the formula of the LB liquid culture medium is as follows: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 1000mL of distilled water, and adjusting the pH value to about 7.0. Sterilizing at 121 deg.C for 15 min.
Inoculating Agrobacterium larymoorei to LB liquid culture medium, performing shake culture at 30 deg.C and 220rpm for 2d, and centrifuging to obtain the strain block. 1g of the bacterial block is suspended by 100mL of sterile water to obtain bacterial suspension.
The lettuce in the experimental group grew better than the control group, as shown in fig. 2. As shown in Table 1, the growth rate of the leaf lettuce in the experimental group was 2 times higher than that in the control group.
TABLE 1 growth of leaf lettuce
Grinding and drying the leaf lettuce, sieving the leaf lettuce by a 0.25-0.5mm sieve, weighing 0.1-0.2g of sieved leaf lettuce, placing the leaf lettuce in a 100mL digestion tube, wetting the leaf lettuce with water, adding 5mL of concentrated sulfuric acid, slowly heating, gradually raising the temperature by white smoke when the concentrated sulfuric acid decomposes, dropwise adding 300g/L hydrogen peroxide when the solution is completely brownish black, heating to slightly boiling for 10-20min, slightly cooling, then adding 300g/L hydrogen peroxide, and repeating the steps for 2-3 times until the solution is colorless or clear. And (4) adding deionized water to make the volume of the solution be 100mL, filtering, and taking the filtrate to measure the N and P elements. The results are shown in table 2, where the total nitrogen content and the total phosphorus content in the experimental group are higher than those in the control group, where the total nitrogen content in the experimental group is more than 2 times that in the control group.
TABLE 2 Total Nitrogen and Total phosphorus determination in leaf of Ottelia
Example 3:
potting experiments with wheat:
culturing wheat seeds in a seedling culture plate, culturing the seedlings for 1 week under natural conditions, and selecting the wheat seedlings with the same growth vigor as experimental objects of a pot culture experiment. Transplanting the wheat seedlings into a cup filled with equal amount of soil, fixing for two days every week, adding equal amount of water and bacterial suspension into the wheat, selecting Tuesday and Friday in the embodiment, adding 100mL of water or bacterial suspension into the wheat, and continuously culturing the wheat for 3 months. After the culture is finished, harvesting the wheat, and detecting the total nitrogen and total phosphorus indexes in the wheat.
The bacterial suspension was prepared as follows:
the formula of the LB liquid culture medium is as follows: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 1000mL of distilled water, and adjusting the pH value to about 7.0. Sterilizing at 121 deg.C for 15 min.
Inoculating the Agrobacterium larymoorei into an LB liquid culture medium, carrying out shaking culture at 30 ℃ and 220rpm for 2 days, and centrifuging to obtain a bacterial block. 1g of the bacterial block is suspended by 100mL of sterile water to obtain bacterial suspension.
In the case of the wheat potted plant shown in fig. 3, compared with the control group, the growth vigor of the wheat in the experimental group was significantly better than that in the control group. As shown in fig. 4, the root system of wheat in the experimental group was significantly more developed than that in the control group. As shown in Table 3, the growth rate of the wheat in the experimental group was significantly higher than that in the control group, which was 3 times higher than that in the control group.
TABLE 3 growth of wheat
Grinding and drying wheat, sieving the wheat by a 0.25-0.5mm sieve, weighing 0.1-0.2g of the sieved wheat, placing the wheat in a 100mL digestion tube, wetting the wheat by water, adding 5mL of concentrated sulfuric acid, slowly heating, gradually raising the temperature by white smoke when the concentrated sulfuric acid decomposes, dropwise adding hydrogen peroxide with the mass fraction of 300g/L when the solution is completely brownish black, heating to slightly boiling for 10-20min, slightly cooling, then adding the hydrogen peroxide with the mass fraction of 300g/L, and repeating the steps for 2-3 times until the solution is colorless or clear. And (4) adding deionized water to make the volume of the solution be 100mL, filtering, and taking the filtrate to measure the N and P elements. The results are shown in table 4, where the total nitrogen content and the total phosphorus content in the experimental group are higher than those in the control group, where the total nitrogen content in the experimental group is more than 2 times that in the control group.
TABLE 4 Total Nitrogen and Total phosphorus determination in wheat
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.
SEQ ID No.1
CGCATCGCAAGATGAGTGGCAGACGGGTGAGTAACGCGTGGGAACATACCCTTTTCTACGGAATAGCTCTGGGAAACTGGAATTAATACCGTATACGCCCTACGGGGGAAAGATTTATCGGGGAAGGATTGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGATGAAGATAATGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGATATTTAAGTCAGGGGTGAAATCCCAGAGCTCAACTCTGGAACTGCCTTTGATACTGGGTATCTTGAGTATGGAAGAGGTAAGTGGAATTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCTCTTGACATTCGGGGTATGGTCATTGGAGACGATGACCTTCAGTTCGGCTGGCCCTAGAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACAGCGATGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGCTGCGCTAACCGCAA
Claims (10)
1. The plant growth promoting agrobacterium is characterized by being preserved in China general microbiological culture Collection center on 9 th 5 th 2022 at the preservation address of: xilu No.1 Hospital No.3, beijing, chaoyang, with the deposit number: CGMCC No.24856, named as Agrobacterium larymoorei.
2. The plant growth promoting agrobacterium of claim 1, wherein the colony characteristic of the plant growth promoting agrobacterium is: after 24 hours of culture on an LB plate culture medium, the colony is round, smooth in surface, moist, raised, white and transparent, and regular in edge.
3. The sequence of the 16S rDNA of the plant growth promoting agrobacterium is shown as SEQ ID No.1.
4. A microbial agent comprising the plant growth promoting Agrobacterium of claim 1.
5. The microbial inoculum according to claim 4, wherein the composition of the microbial inoculum comprises sterile water and plant growth promoting agrobacterium in a ratio of 100mL:1g of the total weight of the composition.
6. A plant growth promoting Agrobacterium according to claim 1 or use of a bacterial formulation according to claim 4 for promoting plant growth.
7. The use according to claim 6, wherein the plant is an angiosperm.
8. The use as claimed in claim 7, wherein the angiosperm is leaf of Ottelia tetragonoloba and wheat.
9. The use according to claim 6, wherein said plant growth-promoting Agrobacterium or said microbial inoculum is applied to the roots of said plant.
10. Use of the plant growth promoting agrobacterium of claim 1 or the microbial inoculum of claim 4 in the preparation of biofertilizer.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121147A (en) * | 2023-03-14 | 2023-05-16 | 昆明理工大学 | Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof |
| PL444472A1 (en) * | 2023-04-18 | 2024-03-25 | Uniwersytet Mikołaja Kopernika W Toruniu | New strain of Agrobacterium sp. Azo12 bacteria, biomass for growing wheat and method of obtaining it, a biopreparation for growing wheat and method of obtaining it with the participation of the Agrobacterium sp. Azo12 strain, intended for growing wheat under normal and salt stress conditions |
| PL444444A1 (en) * | 2023-04-18 | 2024-10-21 | Uniwersytet Mikołaja Kopernika W Toruniu | Bacterial strain Agrobacterium sp. Azo12 |
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2022
- 2022-11-07 CN CN202211386202.5A patent/CN115820477A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121147A (en) * | 2023-03-14 | 2023-05-16 | 昆明理工大学 | Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof |
| CN116121147B (en) * | 2023-03-14 | 2023-12-01 | 昆明理工大学 | Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof |
| PL444472A1 (en) * | 2023-04-18 | 2024-03-25 | Uniwersytet Mikołaja Kopernika W Toruniu | New strain of Agrobacterium sp. Azo12 bacteria, biomass for growing wheat and method of obtaining it, a biopreparation for growing wheat and method of obtaining it with the participation of the Agrobacterium sp. Azo12 strain, intended for growing wheat under normal and salt stress conditions |
| PL444444A1 (en) * | 2023-04-18 | 2024-10-21 | Uniwersytet Mikołaja Kopernika W Toruniu | Bacterial strain Agrobacterium sp. Azo12 |
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