Comamonas aquatica and use thereof
By adding Agromonta R780 or its metabolites to rice culture medium, the problem of unclear rice tillering regulation was solved, and the number of rice tillers was significantly increased, resulting in a yield increase of 11% to 75%.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-16
- Publication Date
- 2026-03-31
AI Technical Summary
In existing technologies, the regulatory mechanism of rice tiller formation is unclear, the influence of soil microorganisms on rice tiller growth has not been fully explored, and there is a lack of effective biological means for artificial regulation.
Pelomonas sp. R780 (CGMCC No. 25842) was used to add the strain or its metabolites to the rice culture medium, and the culture conditions were optimized to increase the number of rice tillers.
The number of rice tillers was significantly increased under both laboratory and field conditions, achieving effective artificial control of rice tillering, with the number of tillers increasing by 11% to 75%.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and more specifically, to a rice rhizosphere sphaeromonas and its applications. Background Technology
[0002] The tillering process in rice mainly involves two steps: the formation of tiller buds and their elongation. Typically, one axillary bud, or tiller bud, forms in the leaf axil of each leaf position in rice. However, the axillary buds at the node where the flag leaf rests often degenerate during the growth and development of most mid-season rice varieties, while they generally survive in early-season rice varieties. Tillering is a complex regulated process, and the known mechanisms regulating tillering are only a small part. Regarding the formation of a single tiller, the process from the appearance of the tiller primordium to the formation of the tiller bud is almost unaffected by internal or external conditions, proceeding sequentially according to the leaf emergence process of the mother stem. However, whether the tiller bud can develop into a tiller after formation is subject to various internal and external factors. Environmental factors, plant nutrition, plant hormones, and genetics all influence rice tillering growth.
[0003] Soil microorganisms, as important members of environmental factors, have been recognized by researchers as an indispensable influencing factor in plant growth and development. The soil is home to a large number and diverse range of microorganisms, which are selected and enriched by plant roots to form the rhizosphere microbiome. These microorganisms accompany the plant throughout its entire growth cycle, participating in important physiological processes such as growth and development, nutrient absorption, and disease and stress resistance. The interaction between plants and the rhizosphere microbiome is related to important agricultural issues such as efficient nutrient utilization, continuous cropping, and crop rotation. Existing research shows that rice rhizosphere microorganisms change with the growth and development stages, and that rice can synergistically utilize soil organic nitrogen with rhizosphere microorganisms. All these indications suggest the enormous potential of rhizosphere microorganisms in aiding rice growth. Currently, it is unclear whether rhizosphere microorganisms affect rice tillering. Exploring beneficial rhizosphere bacteria in rice not only plays a leading role in exploring the molecular mechanisms of plant branching but also provides important microbial resources for increasing rice yield. Summary of the Invention
[0004] The technical problem to be solved by this invention is how to achieve artificial regulation (increase) of [something] through biological means.
[0005] To solve the above-mentioned technical problems, the present invention provides a *Azotobacter* species, wherein the *Azotobacter* species (… Pelomonas sp. The strain number is R780, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 25842.
[0006] Mucor ( Pelomonas spR780 bacteria are short rod-shaped, with an average size of approximately 1.0 μm × 1.5–2.0 μm. They are Gram-negative, non-spore-forming, and capsule-containing. When cultured on TSB solid medium for 48–72 hours, the colonies are relatively large, round, flat, with a bright, smooth surface, slightly raised in the center, and regular edges. The colonies are initially milky white, gradually turning pale yellow. The optimal growth temperature is 28℃, and the optimal pH range is 6.8–7.6, with an optimum pH of 7.0. (Cryptospira) Pelomonas sp R780 has the 16S rDNA shown in sequence 1 of the sequence listing.
[0007] The present invention also provides a method for increasing the number of tillers in rice, characterized by the step of adding the above-mentioned *Agromonae R780* or / and metabolites of *Agromonae R780* or the aforementioned inoculant to the rice culture medium.
[0008] The amount of *Sphaeromonas hydrophila* added to the culture medium was 10. 5 -10 7 cfu / mL.
[0009] Any of the following applications of *Azoobacterium* or / and its metabolites should also be within the scope of protection of this invention:
[0010] 1) Application in the preparation of products for increasing the number of tillers in rice;
[0011] 2) Application in increasing the number of rice tillers.
[0012] The present invention also provides a microbial agent containing *Staphylococcus aureus* R780 and / or metabolites of *Staphylococcus aureus* R780.
[0013] Any of the following applications of the bacterial agent should also be within the scope of protection of this invention:
[0014] 1) Application in the preparation of products for increasing the number of tillers in rice;
[0015] 2) Application in increasing the number of rice tillers.
[0016] The present invention also provides a product for increasing the number of tillers in rice, wherein the active ingredients of the product include the above-mentioned *Azotomyces r780* or / and metabolites of *Azotomyces r780* or the above-mentioned inoculant.
[0017] The present invention also provides a method for preparing the above-mentioned product, comprising the step of using the above-mentioned *Azotomyces r780* and / or the metabolites of *Azotomyces r780* or the bacterial agent as the active ingredient of the product to obtain the product.
[0018] The product may be a liquid or solid dosage form.
[0019] The product may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material or a biological material; the mineral material may be at least one of peat moss, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material may be at least one of various crop straws, pine shells, rice straw, peanut shells, corn flour, soybean flour, starch, peat moss, and animal manure; the liquid carrier may be water; in the product, *Azoobacterium sarcodactylon* R780 and / or its metabolites may exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate. The product may be in various dosage forms, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.
[0020] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the product.
[0021] In the above text, the metabolites of *Aeromonas R780* can be the fermentation broth of *Aeromonas R780*. The fermentation broth of *Aeromonas R780* can be prepared by the following method: culturing *Aeromonas R780* in a liquid fermentation medium, collecting the fermentation broth (containing *Aeromonas R780* and substances secreted into the liquid culture medium), which is the metabolite of *Aeromonas R780*.
[0022] Adding Sphingomonas sp. R780 provided by this invention to the rice culture medium can increase the number of tillers in rice under both laboratory and field culture conditions, providing a possibility for artificially controlling the tillering of rice.
[0023] Preservation Instructions
[0024] Classification and nomenclature of biological materials: *Sphaeromonas* ( Pelomonas sp. )
[0025] Strain number of the biological material: R780
[0026] Name of the institution that deposits biological materials: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee
[0027] The abbreviation for the depository of biological materials is CGMCC.
[0028] Address of the depository for biological materials: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.
[0029] Preservation date of biological material: September 28, 2022
[0030] Biological materials depository registration number: CGMCC No. 25842 Attached Figure Description
[0031] Figure 1 The diagram shows the cultivation status of the laboratory system, the planting pattern (left), the aseptic seedlings transferred into the blue box after 6 days of cultivation (middle), and the growth of rice after 50 days of cultivation in the greenhouse (right).
[0032] Figure 2 In Example 2, the tillering of rice cultured with added bacterial solution and rice cultured without added bacterial solution were tested under laboratory conditions.
[0033] Figure 3 This is a growth chart of rice 40 days after transplanting into the field, representing a field planting system.
[0034] Figure 4 In Example 3, the tillering of rice cultured with added bacterial solution and rice cultured without added bacterial solution were tested under field conditions. Detailed Implementation
[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0037] The sequences involved in the following embodiments are shown in Table 1.
[0038] Table 1. Relevant sequences of monoclonal antibodies
[0039]
[0040] Example 1
[0041] In this embodiment, *Azotobacter oryzae* R780 CGMCC No. 25842 was isolated from the rhizosphere of rice. The rice rhizosphere sample was collected in Changping District, Beijing, China, in October 2016.
[0042] Isolation and culture of rice rhizosphere microorganisms
[0043] Fresh rice plants grown to about 8 weeks old were dug up from the field. Root tissue about 15cm from the rootstock was cut off. Large soil particles on the surface were washed away with deionized water. The surface moisture of the roots was blotted dry with filter paper. The roots were cut into small segments of about 2mm, mixed evenly, and 0.02g of root segments were placed in a 1.5mL centrifuge tube. 200uL of sterile water was added, and the root tissue was homogenized with a grinder. The homogenate was serially diluted 10, 100, 1000, 10000, and 100000 times. 160uL of the diluted solution was transferred into bacterial culture plates using a pipette. 45 culture plates were prepared for each serial dilution. The plates were sealed and incubated at room temperature.
[0044] After the bacterial culture plates have been stored for about 3 weeks, a gradient of turbidity with 30-40% of the wells remaining on each plate is maintained for identification. A portion of the cultured bacteria from each well is taken out for bacterial identification, and the remaining cultured bacteria are added to 80% (v / v) glycerol at a volume ratio of 1:1 and stored at -80℃ for later use.
[0045] Identification and strain comparison of isolated bacteria
[0046] Take 6 μL of cultured bacteria into a 96-well PCR plate, add 10 μL of buffer I (solutes: 25 mM NaOH and 0.2 mM EDTA; solvent: water; pH 12), mix well, and extract DNA in a PCR instrument at 95℃ for 30 min. Add 10 μL of buffer II (solutes: 40 mM Tris-HCl; solvent: water; pH 7.5) to extract template DNA.
[0047] Two-step PCR was performed on the template DNA. The first step used 799F and 1193R as primers, and the second step used 799F containing one of the 96 well bars and 1193R containing the plate barcode as primers (Jingying Zhang, Yong-XinLiu, Na Zhang, Bin Hu, Tao Jin, Haoran Xu, Yuan Qin, Pengxu Yan, XiaoningZhang, Xiaoxuan Guo, Jing Hui, Shouyun Cao, Xin Wang, Chao Wang, Hui Wang, Baoyuan Qu, Guangyi Fan, Lixing Yuan, Ruben Garrido-Oter, Chengcai Chu, Yang Bai., NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nature Biotechnology) to amplify the variable regions V5-V7 of the bacterial 16S rRNA gene, thus obtaining a bacterial identification library.
[0048] Sequencing was performed on the HiSeq 2500 platform. The 16S rRNA gene sequence of bacteria in each well was sequenced and aligned using NCBI BLAST to obtain the aligned bacterial species for each bacterium. The 16S rRNA gene sequence of one of the bacteria, numbered R780 (hereinafter referred to as strain R780), is sequence 1 in the sequence listing.
[0049] The *Sphaeromonas* provided by this invention ( Pelomonas sp Strain R780, with the registration number CGMCC No. 25842 at the China General Microbiological Culture Collection Center (CGMCC), was deposited on September 28, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It will be referred to as *C. 780* below.
[0050] Mucor ( Pelomonas spR780 cells are short rod-shaped, with an average size of approximately 1.0 μm × 1.5–2.0 μm. They are Gram-negative, non-spore-forming, and capsule-containing. When cultured on TSB solid medium for 48–72 hours, the colonies are relatively large, round, flat, with a bright, smooth surface, a slightly raised center, and regular edges. The colonies are initially milky white, gradually turning pale yellow. The optimal growth temperature is 28℃, and the optimal pH range is 6.8–7.6, with an optimum pH of 7.0.
[0051] Example 2
[0052] Under laboratory conditions, the tillering of rice cultured with and without bacterial culture was tested. Specific experimental methods included: ① Sterile seedling germination: Plump rice seeds were selected, and the seed coat was removed. 70% alcohol was added for 30 seconds for disinfection, and the alcohol was discarded. A 2.5% sodium hypochlorite solution with available chlorine concentration was added for 15 minutes for disinfection, and the sodium hypochlorite was discarded. This step was repeated 3 times. Sterile deionized water was added for 10 minutes for rinsing, and the water was discarded. This step was repeated 3 times. Using sterile forceps, the seeds were neatly and evenly spread on the surface of 1 / 2 MS solid medium, with the embryo facing upwards at the bottom 1 / 3 of the plate. The plate was sealed with Parafilm sealing film. The culture dish was placed vertically and cultured for 6 days in a culture room at 25℃, 16 hours of light, and 21% humidity. ② Transplanting greenhouse: Rice seedlings with uniform growth were selected and transplanted into blue boxes containing 15L of nutrient solution in the greenhouse, with one seedling per hole wrapped with a sponge. ③ Inoculation: For treatments requiring inoculation, add 150mL of bacterial solution with an OD600 of 0.5 to each box and stir well. Change the nutrient solution every 6 days, adding bacteria at the same time, and adjust the pH to 6.3-6.8. The planting conditions in the above experiment were: greenhouse, natural light, hydroponics. Figure 1 As shown in Table 2.
[0053] After rice seedlings have been cultivated in a greenhouse for 50 days and tillering has stabilized, the number of rice tillers is measured. Figure 2 As shown, the experimental group with added bacterial solution had a significantly higher number of tillers than the control group without added bacterial solution. The average number of tillers in the experimental group was 11% higher than that in the control group, with a maximum increase of 3 tillers and a maximum increase of 75%.
[0054] Table 2 Nutrient solution composition
[0055]
[0056] Example 3
[0057] Under field conditions, the tillering of rice cultured with and without bacterial culture was tested. Specific experimental methods included: ① Setting up paddy field plots, each 1.8 meters wide × 2.2 meters long. The control plot received no additional bacterial culture, while the experimental plots received 4.5 L of bacterial culture with an OD600 of 1, and were thoroughly mixed. Bacterial culture was added every 7 days after transplanting. ② Seed disinfection, germination, and transplanting: Plump rice seeds were selected, the husks were removed, and 70% alcohol was added for disinfection for 30 seconds, with continuous shaking to ensure each seed was in full contact with the alcohol. The alcohol was discarded. A 2.5% sodium hypochlorite solution was added for disinfection for 15 minutes, and the solution was discarded. This step was repeated 3 times. The seeds were then rinsed with sterile deionized water for 10 minutes, and this step was repeated 3 times. Using sterile forceps, the seeds were neatly and evenly spread on half of the MS solid culture medium surface, with the embryo facing upwards at the bottom 1 / 3 of the plate. The plate was then sealed with Parafilm sealing film. Place the petri dishes vertically and incubate for 7 days in a culture room at 25℃, 16 hours of light, and 21% humidity. Select sterile seedlings with uniform growth and wash away any residual culture medium from the roots with sterile deionized water. Transplant the cleaned seedlings into the field in 6×6 squares, spacing each rice plant 20 cm apart. The cultivation process is as follows. Figure 3 As shown.
[0058] Experimental group: The soil in the field was watered until the water level was about 1-2 cm below the soil surface, and 5% of the soil surface was added. 10 6 5 L of bacterial culture with cfu / g was stirred thoroughly with a spatula. After 7 days of sterile seedling culture, the roots were cleaned and then treated with 5... 10 6 After soaking the roots in a CFU / mL bacterial solution for 1 hour, transplant them into field-treated soil. Add the bacterial solution every 7 days (add 5% CFU / mL when watering). 10 6 5L of bacterial culture with cfu / g).
[0059] Control group: All steps of adding bacterial solution in the experimental group were replaced with watering only, while other steps remained unchanged.
[0060] Forty days after transplanting, when the rice tillering was stable, the number of tillers in the control group and the experimental group was measured. Figure 4 As shown, the addition of R780 bacterial solution to the experimental group significantly increased the number of rice tillers. The experimental group had an average increase of about 2 tillers compared to the control group, which increased the average number of rice tillers by 16%, with a maximum increase of 8 tillers and a maximum increase rate of 73%.
[0061] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A method for increasing the number of tillers in rice, characterized by, The present application relates to a method for improving the growth of rice, comprising adding a strain of the genus Comamonas, in particular Comamonas sp. R780, or / and metabolites of the strain of the genus Comamonas, in particular Comamonas sp. R780, to a culture solution of rice, wherein the strain of the genus Comamonas, in particular Comamonas sp. R780, has a strain number R780, and the strain of the genus Comamonas, in particular Comamonas sp. R780, is registered in the China General Microbiological Culture Collection Center (CGMCC) with a registration number CGMCC No. 25842 Pelomonas sp. .
2. The method of claim 1, wherein, The amount of the added H. fluvialis R780 in the culture solution is 10 5 -10 7 cfu / mL.
3. Use of any one of the following: a) S. limax R780, or / and b) metabolites of S. limax R780, Pelomonas sp. having a strain number of R780, and having a registration number of CGMCC No. 25842 at China General Microbiological Culture Collection Center. 1) Use in the manufacture of a product for increasing the number of tillers in rice plants; 2) Use for increasing the number of tillers in rice plants.
4. Use of any one of the following: a microbial agent containing a Hymenobacter R780 and / or metabolites of the Hymenobacter R780, the Hymenobacter R780 (Hymenobacter sp. R780) Pelomonas sp. ), which has a strain number of R780 and a registration number of CGMCC No. 25842 in the China General Microbiological Culture Collection Center, in the preparation of a plant growth promoting agent. 1) Use in the manufacture of a product for increasing the number of tillers in rice plants; 2) Use for increasing the number of tillers in rice plants.
5. A product for increasing the number of tillers of rice, characterized by, The active ingredient of the product comprises the Pelomonas sp. R780 or / and metabolites of Pelomonas sp. R780 as described in claim 3.
6. Process for the preparation of the product according to claim 5, characterized in that, The steps for obtaining the product comprise the Pelomonas sp. R780 and / or metabolites of Pelomonas sp. R780 as described in claim 3 as active ingredient of the product.
7. The method of claim 6, wherein, The product is a liquid formulation or a solid formulation.