Application of Exiguobacterium indicum and cyclo (leucine-proline) in plant tillering regulation
By using Microbial Indica R2567 and ring (leucine-proline) to regulate the tillering of grass plants such as rice, the problem of insufficient tillering regulation in plants in the prior art was solved, and effective control of tiller count and increase crop yield was achieved.
Patent Information
- Application Number
- CN202510418132.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, there are few researches on the application of cyclic dipeptides in plant tillering regulation, and it is difficult to effectively regulate the tiller number of grass plants such as rice, affecting crop yield and plant type structure.
Exiguobacterium indicum R2567 and its secretion ring (leucine-proline) were added to plant culture medium or planting soil to regulate plant tillering, and to optimize plant type and increase yield by inhibiting tillering number.
Without affecting plant growth, significantly adjust the number of tillers, reduce energy losses, improve crop quality and yield, reduce field operations, and improve economic benefits.
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Figure CN120349920A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to rhizosphere microorganisms of plants and their secretions, and specifically relates to the application of Exiguobacterium indicum and its cyclic dipeptide secretion cyclo(leucine-proline) in regulating tillering of plants, belonging to the technical field of agricultural production. Background Art
[0002] During the evolution of plants, the transition from an aquatic to a terrestrial lifestyle has prompted the formation of complex root systems to adapt to the terrestrial environment and obtain essential nutrients and water from the soil. This adaptive transition not only enhances the plant's adaptability to the terrestrial ecosystem but also promotes the formation and development of the rhizosphere microbiome. The rhizosphere microbiome refers to a large and diverse community of microorganisms enriched inside and around plant roots, which form a close symbiotic relationship with plant roots and accompany the entire growth cycle of plants. Rhizosphere microorganisms play a crucial role in the physiological processes of plant growth and development, nutrient absorption, disease resistance, and stress tolerance. They promote the acquisition and utilization of nutrients in the soil by plants through various mechanisms, such as converting nutrients that are difficult for plants to directly absorb into available forms through nitrogen fixation, phosphorus solubilization, and potassium solubilization. In addition, rhizosphere microorganisms can also regulate plant growth and development by secreting phytohormones (such as auxin, gibberellin, cytokinin, etc.) and enhance the stress resistance and adaptability of plants.
[0003] Tillering is an important biological process in which gramineous plants (such as rice, wheat, etc.) develop branches from axillary buds at the base during the vegetative growth stage, directly affecting the plant architecture, biomass accumulation, and yield formation of crops. The occurrence of tillering is synergistically regulated by multiple factors such as genetics, hormones, environment, and cultivation measures, and its molecular mechanism and agronomic application have become research hotspots in plant developmental biology and crop genetic improvement. Tillering originates from the activation and elongation of axillary buds at the basal nodes of the stem, and its occurrence process includes three stages: axillary bud primordium initiation, dormancy release, and tiller bud elongation. The tillering ability of gramineous plants is usually determined by the tillering angle and the number of tillers, among which the number of tillers is strongly inhibited by apical dominance. The classical model shows that the dynamic balance of phytohormones (such as auxin, cytokinin, and strigolactone) is the core factor regulating tillering: auxin inhibits axillary bud germination through polar transport, while cytokinin promotes the activation of tiller primordia; strigolactone, as a long-distance signaling molecule, indirectly inhibits tillering by inhibiting the expression of branching genes (such as TB1, FC1).
[0004] Cyclodipeptides (CDPs) are cyclic structures formed by the linkage of two amino acids through peptide bonds, with a stable 2,5-diketopiperazine (DKP) cyclic backbone. This structure endows cyclodipeptides with a wide range of biological activities, making them of important application value in drug research and development and the field of biotechnology. Cyclodipeptides have a wide range of sources, mainly from Gram-negative bacteria, and a small part comes from plants, animals, Gram-positive bacteria, and fungi. Research shows that cyclodipeptides have various biological activities such as antibacterial, antitumor, and antioxidant. For example, certain cyclodipeptides have been proven to have inhibitory effects on a variety of pathogenic fungi and pathogenic bacteria, which makes them have potential application value in antibiotic research and development. In addition, the role of cyclodipeptides in plant growth regulation has gradually been revealed. They can indirectly promote plant growth by regulating plant hormone levels or affecting the structure of the rhizosphere microbial community. For example, cyclodipeptides can enhance the disease resistance of Arabidopsis thaliana by manipulating the level of salicylic acid in Arabidopsis thaliana. However, the current research on the application of cyclodipeptides in plant tillering regulation is relatively scarce. Summary of the Invention
[0005] The present invention aims to explore microorganisms and compounds that affect plant tillering, providing a new regulatory means for agricultural production to increase crop yields and optimize plant type structures.
[0006] The present invention isolated and identified Exiguobacterium indicum R2567 (deposit center registration number: GDMCC No. 66084) from the rhizosphere of paddy rice in the field. This strain can significantly inhibit plant tillering in solar greenhouse and natural field environments. Further, a cyclo(leucine-proline) was extracted from the secretions of the tillering-inhibiting bacterium Exiguobacterium indicum R2567. It is a cyclic dipeptide formed by the condensation of one leucine and one proline, with the chemical formula C 11 H 18 N2O2, and the relative molecular mass is 210.2770. The structural formula of the cyclo(leucine-proline) is as follows:
[0007]
[0008] In the first aspect of the present invention, there is provided an Exiguobacterium indicum R2567 and its application in regulating plant tillering. By adding Exiguobacterium indicum R2567 to the culture solution or planting soil of plants during the vegetative growth period, plant tillering can be inhibited.
[0009] In some specific embodiments of the present invention, the Exiguobacterium indicum R2567 is added to the culture solution of plants, and the addition concentration is in the order of 10 6 ~10 7 cfu / mL, which is similar to the microbial abundance in the soil environment. In some other specific embodiments of the present invention, the Exiguobacterium indicum R2567 is added to the planting soil of plants. Preferably, the root system of the seedlings is first soaked in a bacterial suspension with a concentration of Exiguobacterium R2567 of 10 6 ~10 7 cfu / mL for a period of time, and then transplanted into the soil, so that the bacterial content in the soil around the root system is 10 6 ~10 7 cfu / g soil.
[0010] In the second aspect of the present invention, the application of cyclo(leucine-proline) in regulating plant tillering is provided. Plant tillering is inhibited by adding cyclo(leucine-proline) to the culture solution or planting soil of plants during the vegetative growth period.
[0011] In some specific embodiments of the present invention, the cyclo(leucine-proline) is added to the culture solution of plants. Preferably, the concentration of cyclo(leucine-proline) in the culture solution is 2-4 μM. In some other specific embodiments of the present invention, the cyclo(leucine-proline) is added to the planting soil of plants. Preferably, the root system of the seedlings is first soaked in a 2-4 μM cyclo(leucine-proline) solution for a period of time, and then transplanted into the soil. A cyclo(leucine-proline) solution is added to the soil so that the content of cyclo(leucine-proline) in the soil around the root system is 2-4 μmol / g soil.
[0012] In the third aspect of the present invention, a plant growth regulator is provided. The growth regulator contains Exiguobacterium indicum R2567 and / or the cyclo(leucine-proline). This growth regulator can regulate the number of plant tillers, improve the plant type, and thus improve the quality and yield of plants.
[0013] The plants in the present invention mainly refer to gramineous plants, especially gramineous crops, including but not limited to rice, wheat, corn, tobacco, sorghum, etc.
[0014] The present invention uses Exiguobacterium indicum R2567 and cyclo(leucine-proline) to inhibit the tillering of gramineous plants such as rice, and can effectively regulate the tillering number of different rice varieties without affecting plant growth, providing a theoretical basis and a new method for regulating plant architecture. By adjusting the tillering number of crops such as rice through the method of the present invention, the plant architecture can be controlled and optimized, the energy loss caused by ineffective tillering can be reduced, the quality and yield of crops can be improved, and field operations can be reduced, labor can be saved, and economic benefits can be increased.
[0015] Depositing of Biological Materials
[0016] Exiguobacterium indicum R2567 of the present invention was deposited at the Guangdong Provincial Microbial Culture Collection Center (abbreviated as GDMCC, address: Building 59, No. 100 Yard, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province) on April 1, 2025, and the accession number of the deposit center is GDMCC No. 66084. Description of the Drawings
[0017] Figure 1 It is the experimental result of regulating the tillering number of rice by Exiguobacterium R2567 in the solar greenhouse environment in Example 2.
[0018] Figure 2 It is the experimental result of regulating the tillering number of rice by Exiguobacterium R2567 in the field environment in Example 2, where (a) is a cross-sectional photo of tillers and (b) is a statistical chart of tiller numbers.
[0019] Figure 3 It is the mass spectrum of identifying cyclo(leucine-proline) in Example 3, where (a) is a 600M 1H NMR spectrum and (b) is a 150M 13C NMR spectrum of cyclo(leucine-proline) isolated from Exiguobacterium R2567 and the standard product.
[0020] Figure 4 It is the experimental result of the regulatory effect of cyclo(leucine-proline) on the tillering of japonica rice Nipponbare in the solar greenhouse environment in Example 4.
[0021] Figure 5 It is the experimental result of the regulatory effect of cyclo(leucine-proline) on the tillering of four rice varieties in the field environment in Example 4, which are Nipponbare, 9311, AUS NC1 / 536, and Karabaschak from left to right. Detailed Description of the Invention
[0022] The present invention will be further described in detail below with reference to the accompanying drawings through embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.
[0023] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, are all conventional biochemical reagents and can be obtained from commercial channels.
[0024] Example 1: Isolation and Identification of Exiguobacterium indicum R2567
[0025] 1. Isolation and Cultivation of Exiguobacterium indicum R2567
[0026] Exiguobacterium indicum R2567 (GDMCC No.66084) was isolated from a rice rhizosphere sample. The rice rhizosphere sample was collected from Changping District, Beijing, China in October 2016.
[0027] 1) Dig out rice plants that have grown for about 8 weeks from the field, cut off the root tissue about 15 cm from the rhizome connection, wash the roots with deionized water, and dry the surface moisture of the roots with filter paper.
[0028] 2) Cut the roots into about 2 mm segments, mix them evenly, take 0.02 g of root segments and put them into a 1.5 mL centrifuge tube, add 200 μL of sterile water, and grind the root tissue into a homogenate with a grinding rod.
[0029] 3) Dilute the homogenate with 1 / 10 TSB medium at gradients of 10-fold, 100-fold, 1000-fold, 10000-fold, and 100000-fold. Transfer 160 μL of the diluted solution into a bacterial culture plate with 45 culture plates for each gradient dilution. Cover the lid and seal it, and culture at room temperature.
[0030] 4) Place the bacterial culture plates for about 3 weeks, and retain the gradient dilution samples with 30 - 40% of the wells turbid for identification.
[0031] 5) Take out a part of the cultured bacteria in each well for bacterial identification, and add glycerol at a volume ratio of 1:1 to the remaining cultured bacteria, and store them at -80 °C for later use.
[0032] 2. Preliminary Identification of Exiguobacterium indicum R2567
[0033] 1) Take 6 μL of cultured bacteria into a 96-well PCR plate, add 10 μL of Buffer I (solute: 25 mM NaOH and 0.2 mM EDTA; solvent: water; pH 12), mix well, extract DNA at 95 °C for 30 min in a PCR instrument, and then add 10 μL of Buffer II (solute: 40 mM Tris-HCl; solvent: water; pH 7.5) to obtain the template DNA.
[0034] 2) Perform two-step PCR on the template DNA. In the first step, use the universal primers 799F and 1193R. In the second step, use 799F containing one of the 96-well barcodes and 1193R containing the plate barcode (Jingying Zhang, etal., NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nature Biotechnology, 2019, 37(6): 676 - 684) to amplify the variable regions V5 - V7 of the bacterial 16S rRNA gene and obtain the bacterial identification library.
[0035] 3) Sequence on the Hiseq 2500 platform. Blast align the bacterial 16S rRNA gene sequences in each well with those in NCBI to obtain the aligned bacterial species for each bacterium. The V5 - V7 sequence of the 16S rRNA gene of a bacterium with the strain number R2567 (hereinafter referred to as strain R2567) is SEQ ID No.1 in the sequence listing.
[0036] 3. Biological characteristics and morphological identification of Exiguobacterium indicum R2567
[0037] Identify the species of the isolated strain R2567 through biological characteristics and morphological observations as follows:
[0038] Observe the colony morphology, size, convexity, and edge, perform Gram staining, and refer to the "Manual for Systematic Identification of Common Bacteria" for the specific methods of physiological and biochemical characteristic tests. The results showed that the cell shape of strain R2567 was short rod-shaped, with an average cell size of 1-2 μm × 0.5-0.8 μm, Gram-positive staining, no endospores, no flagella, no fluorescence characteristics, and was a facultative anaerobe. When cultured on 1 / 2 TSB solid medium for 24 h, the colonies were moist and shiny, with a smooth and opaque surface, regular edges, initially light yellow in color, and gradually turning orange-red. The growth temperature range of this bacterium was 15-40 °C, the most suitable growth temperature was 25-35 °C, and the growth pH value was 7.0-7.6. The full-length 16S rRNA gene of strain R2567 was sequenced, and the result was SEQ ID No.2 in the sequence listing. The similarity of the 16S rRNA gene sequence of strain R2567 to Exiguobacterium indicum strains exceeded 99%.
[0039] Through the above morphological characteristics, cultural characteristics, physiological and biochemical characteristics, and 16S rDNA sequence, Blast alignment identified strain R2567 as Exiguobacterium indicum R2567. Exiguobacterium indicum R2567 was deposited in the Guangdong Provincial Microbial Culture Collection Center (abbreviated as GDMCC, address: Building 59, No. 100 Compound, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province) on April 1, 2025, and its deposit number in the collection center was GDMCC No. 66084. Hereinafter, it is referred to as Exiguobacterium indicum R2567 for short.
[0040] Example 2: Regulation of rice tillering by Exiguobacterium indicum R2567
[0041] 1. Activation of Exiguobacterium indicum R2567 and preparation of bacterial suspension
[0042] 1) Strain activation. Streak and activate Exiguobacterium indicum R2567 on a 1 / 2 TSB solid medium plate, seal it, and place it in an incubator at 28 °C for about 3 days.
[0043] 2) Strain identification. Add 17 μL of Buffer I (25 mM NaOH and 0.2 mM EDTA, pH = 12) to 10 μL of the bacterial solution, incubate at 95 °C for 30 min, and quickly add 17 μL of Buffer II (40 mM Tris HCl, pH = 7.5), mix well to obtain template DNA. Amplify the full length of the bacterial 16S rDNA using the universal primers 27F / 1492R and compare it with SEQ ID No.2.
[0044] 3) Bacterial liquid fermentation. Pick single colonies into 1 / 2 TSB liquid medium, place it on a shaker at 28 °C and 180 rpm for about 3 days to obtain the fermentation broth.
[0045] Among them, the 1 / 2 TSB solid medium was prepared as follows: 15 g of TSB powder, 20 g of agar, made up to 1 L with distilled water, adjusted the pH to 7.2 ± 0.2, and used after autoclaving at 121 °C for 15 min.
[0046] Among them, the 1 / 2 TSB liquid medium was prepared as follows: 15 g of TSB powder, made up to 1 L with distilled water, adjusted the pH to 7.2 ± 0.2, and used after autoclaving at 121 °C for 15 min.
[0047] 4) Prepare the bacterial suspension. Centrifuge the fermentation broth at 3000 rpm for 10 min in a high-speed centrifuge, discard the supernatant in a laminar flow hood, add 25 mL of sterile water, and vortex to mix evenly. Repeat the above steps 2 times. For the last resuspension, use 15 mL of sterile water. After vortexing, take out 1 mL and measure the bacterial OD 600nm value, record it and convert and dilute it to OD 600nm = 0.5 to obtain the Exiguobacterium indicum R2567 bacterial solution. The content of Exiguobacterium indicum R2567 in the Exiguobacterium indicum R2567 bacterial solution is 5 × 10 8 cfu / mL.
[0048] 2. Regulation of rice tillering by Exiguobacterium indicum R2567 under the solar greenhouse environment
[0049] 1) Seedling raising. Prepare the seeds of japonica rice Nipponbare, remove the seed coats, and select the seeds that are neat and consistent and have complete germ. Raise seedlings in an artificial climate chamber. The environmental conditions are 21% humidity, 25 °C temperature, a 16-hour light / 8-hour dark cycle mode, supplemented with white fluorescent lamps, and the light intensity is about 200 μM m-2s-1.
[0050] 2) Transplant the 7-day-old Nipponbare seedlings to the solar greenhouse. The environmental humidity is 50%, the light cycle is 16 hours of light / 8 hours of dark, and the temperature is 28 °C / 30 °C. Natural light and supplementary light ensure that the light intensity during the day is 250 - 300 μM m -2 s -1 . Use the modified Kimura culture solution: 91.17 μM KH2PO4, 273.46 μM MgSO4·7H2O, 182.38 μM (NH4)2SO4, 91.49 μM KNO3, 182.94 μM Ca(NO3)2·4H2O, 22.94 μM Fe-EDTA (C 10 H 12(FeN2NaO8), 0.20 μM CuSO4·5H2O, 2.99 μM H3BO3, 0.50 μM MnCl2·4H2O, 1.00 mM (NH4)6Mo7O 24 ·4H2O, 0.40 mM ZnSO4·7H2O (adjust the pH to 6.2 using 2 M NaOH solution).
[0051] 3) Transplant 12 seven-day-old seedlings into 14 L of the above 1 / 2 modified Kimura culture solution and let them acclimatize for 5 days. After acclimatization, the treatment group added the above-mentioned bacterial suspension with an OD 600nm = 0.5 to the modified Kimura culture solution at a volume ratio of 1:100 to obtain a culture solution with a content of 5×10 6 cfu / mL of Exiguobacterium indicum R2567, and this concentration is basically consistent with the microbial abundance in the soil environment. The control group was not added. Adjust the pH = 6.2 every 2 days, change the culture solution every 6 days, and count the tiller number after 50 days.
[0052] 4) The results are as Figure 1 shown. Through t-test, compared with the control group, the tillering of the Exiguobacterium indicum R2567 treatment group was significantly inhibited. The tiller number of the control group was 4 - 7, and the tiller number of the treatment group was 2 - 5. The average tiller number of the treatment group decreased by about 35%.
[0053] 3. Regulation of rice tillering by Exiguobacterium indicum R2567 in the field environment
[0054] 1) Deep plow the field soil to a depth of 15 - 20 cm. Set up spatially isolated plots for planting the control group and the treatment group. Before planting, no bacterial fertilizer or organic fertilizer was applied to the plots, and no pesticides or fertilizers were applied during the period. Pay attention to manual weeding.
[0055] 2) One day before transplanting, mix the bacterial suspension of Exiguobacterium indicum R2567 with a final concentration of 1.7×10 6 cfu / g into the field soil of the treatment group. According to the soil depth of 15 cm and density of 1.333 g / cm 3 , calculate the total mass of the soil in the plot used, and then calculate the dosage of the Exiguobacterium indicum R2567 bacterial suspension. The next day, soak the roots of 14-day-old rice seedlings in the Exiguobacterium indicum R2567 bacterial suspension with a concentration of 1.7×10 6 cfu / mL for 1 h, and then transplant them into the above field environment with a plant spacing of 20 cm. Calculation method for the dosage per seedling: Calculate the soil mass according to the soil volume of 20 cm × 20 cm × 15 cm and density of 1.333 g / cm 3 , and calculate the dosage per seedling according to 1.7×10 6 cfu / g. Measure the OD 600nmThe bacterial suspension with a concentration of 0.5 was diluted with water and applied once every 7 days. The statistics were carried out after 15 weeks.
[0056] 3) The results are as Figure 2 shown. Through t-test, compared with the control group, the tiller number of the treatment group was significantly reduced under field conditions, indicating that Exiguobacterium indicum R2567 could significantly inhibit tillering under field conditions. The tiller number of the control group was 7 - 14, while that of the treatment group was 6 - 13, and the average tiller number decreased by more than 15%.
[0057] Example 3: Isolation and Identification of Cyclo(leucine - proline)
[0058] 1. Extraction of the Crude Extract from the Fermentation Broth of Exiguobacterium indicum R2567
[0059] 1) The seed liquid of Exiguobacterium indicum R2567 was inoculated into 500 mL of medium at a volume ratio of 1:1000 and cultured in the dark at 28 °C and 200 rpm for 3 days. The OD was measured by a spectrophotometer 600nm >3 to obtain a fully fermented fermentation broth.
[0060] 2) The bacterial fermentation broth was mixed with an equal volume of ethyl acetate (Sigma - Aldrich, Cat#34858 - 4*4L) and broken by a 40 kHz ultrasonic crusher for 1 hour. During this period, attention should be paid to keeping the ultrasonic temperature not too high. The broken liquid was mixed evenly at 200 rpm in a shaker for 30 min, left standing for 15 min, and the upper organic phase was collected.
[0061] 3) The above extraction steps were repeated 3 times, and the organic phases collected three times were mixed evenly. The organic phase was concentrated using a rotary evaporator, and nitrogen blowing was used to obtain the crude extract of the secretion of Exiguobacterium indicum R2567.
[0062] 2. Step - by - step Separation and Identification of Cyclo(leucine - proline) in the Crude Extract
[0063] 1) The dried extract was separated by silica gel flash chromatography using gradient elution (petroleum ether - ethyl acetate). Combining with the activity experiment, a white solid active substance was separated from the F3 - 1 fraction and named S6. Through LCMS analysis using a Vanquish UPLC - Q - Exactive HRMS system (Thermo Fisher Scientific) equipped with a Kinetex EVO C18 column (1.7 μm, 100A, 100 mm×2.1 mm, Phenomenex), the chemical formula of S6 was observed to be C 11 H 19N2O2, with a mass-to-charge ratio of 211.1440 ([M+H]+, the molecular formula calculation is 211.1446). Mobile phases A and B are pure water containing 0.1% formic acid (A) and acetonitrile (B) respectively. The flow rate is set at 0.3 mL / min. For the analysis of each component, a method of 13.5 minutes is adopted: 5% B at 0 minute, increased to 30% B at 4 minutes, 80% B at 6 minutes, 100% B at 8 minutes and maintained for 4 minutes, then decreased to 5% B at 12.5 minutes and maintained for 1 minute. The source energy is 4.0 kV, using positive polarity, and the mass spectrometry scanning range is set at 100 - 1500 daltons for general analysis and 150 - 320 daltons for quantitative analysis.
[0064] 2) The structure of S6 was detected using nuclear magnetic resonance (NMR) spectroscopy. Through detailed analysis of the 1D and 2D NMR spectra of S6, this substance has the following characteristic signals: 2 carbonyl - C=O signals (δC 172.8 and 168.9 ppm), 1 - CH signal bound to the amide nitrogen (δH 4.25 and 4.12 ppm), and 1 - CH2 (δH 3.51 ppm) signal. Combining the 2D NMR correlation analysis of the remaining aliphatic signals (including 2 isopropyl - CH3 signals, 3 - CH2 signals, and 1 - CH signal), the structure of S6 was clearly determined to be a cyclic dipeptide of L - leucine and L - proline, namely cyclo(leucine - proline).
[0065] 3) Using the standard product (KKL Med, Cat#KCM13147) as a control, the structural characterization results using nuclear magnetic resonance proton spectroscopy (1H NMR) are as Figure 3 shown. The finally identified isolated compound is cyclo(leucine - proline), a cyclic dipeptide formed by the condensation of one leucine and one proline, with the exact chemical formula C 11 H 18 N2O2, with a relative molecular mass of 210.2770.
[0066] Example 4. Regulation of Rice Tillering by Cyclo(leucine - proline) under Solar Greenhouse and Field Environments
[0067] 1. Stability Test of Cyclo(leucine - proline)
[0068] 1) According to the identification results, commercially sourced cyclo(leucine - proline) (KKL Med, Cat#KCM13147) was purchased. Cyclo(leucine - proline) was dissolved in acetone to prepare a 50 mM stock solution and stored in a - 80 °C refrigerator for later use.
[0069] 2) Detection of the stability of cyclo(leucine-proline). Cyclo(leucine-proline) was dissolved in water at an initial concentration of 5 μg / L and placed at room temperature. Its content was detected daily. After detection, the content of cyclo(leucine-proline) remained basically unchanged in the first 6 days, decreased to 1 / 100 of the initial concentration on the 9th day, and was hardly detectable by the instrument on the 11th day.
[0070] 2. Regulation of rice tillering by cyclo(leucine-proline) under solar greenhouse conditions
[0071] 1) Prepare rice of four varieties: japonica rice Nipponbare, indica rice 9311, japonica rice Karabaschak, and AUS NC1 / 536. Remove the seed hulls and select seeds that are neat and consistent with intact germplasms. Raise seedlings in an artificial climate chamber with environmental conditions of 21% humidity, 25 °C temperature, a 16-hour light / 8-hour dark cycle mode, and white fluorescent lamps for supplementary lighting with a light intensity of approximately 200 μM m-2 s-1.
[0072] 2) Transplant 7-day-old rice seedlings to a solar greenhouse with an environmental humidity of 50%, a light cycle of 16 hours light / 8 hours dark, and a temperature of 28 °C / 30 °C. Natural light and supplementary lighting ensure a daytime light intensity of 250 - 300 μM m -2 s -1 . Use a modified Kimura nutrient solution: 91.17 μM KH2PO4, 273.46 μM MgSO4·7H2O, 182.38 μM (NH4)2SO4, 91.49 μM KNO3, 182.94 μM Ca(NO3)2·4H2O, 22.94 μM Fe-EDTA (C 10 H 12 FeN2NaO8), 0.20 μM CuSO4·5H2O, 2.99 μM H3BO3, 0.50 μM MnCl2·4H2O, 1.00 mM (NH4)6Mo7O 24 ·4H2O, 0.40 mM ZnSO4·7H2O (adjust the pH to 6.2 using a 2 M NaOH solution).
[0073] 3) Transplant 12 7-day-old seedlings into 14 L of the above 1 / 2 modified Kimura nutrient solution and let them acclimatize for 5 days. After acclimatization, the treatment group was cultured with a modified Kimura nutrient solution containing 2 μM of cyclo(leucine-proline), and the control group was added with the corresponding solvent. Replace the nutrient solution every 3 days and adjust the pH = 6.2. Culture for a total of 41 days. Count the tiller number every 4 days.
[0074] 4) Experimental results. Through t-test, starting from 12 days after inoculating cyclo(leucine-proline), compared with the control group, the tillering of rice in the cyclo(leucine-proline) treatment group began to be significantly inhibited, such asFigure 4 As shown, the average tiller number of japonica rice Nipponbare decreased by more than 50%.
[0075] 3. Regulation of rice tillering by cyclo(leucine-proline) under field conditions
[0076] 1) Compound fertilizer was applied at 40 kg / mu as basal fertilizer to the field plots, and the soil was deeply plowed by 15 - 20 cm. Plots with spatial isolation were set up for planting the control group and the treatment group. Before planting, no bacterial fertilizer or organic fertilizer was applied to the plots, and no pesticides and fertilizers were applied during the period. Manual weeding was noted.
[0077] 2) One day before transplanting, cyclo(leucine-proline) with a final concentration of 2 μmol / g was mixed into the soil of the treatment group fields. According to a soil depth of 15 cm and a density of 1.333 g / cm 3 , the total mass of the soil in the plots used was calculated, and then the dosage of cyclo(leucine-proline) was calculated. The next day, the roots of 14-day-old rice seedlings were immersed in a 2 μM cyclo(leucine-proline) working solution for 1 h, and then transplanted into the above-mentioned field environment with a plant spacing of 20 cm. The dosage calculation method for each seedling: The soil volume was calculated as 20 cm × 20 cm × 15 cm, and the density was 1.333 g / cm 3 to calculate the soil mass, and the dosage for each seedling was calculated according to the final concentration of 2 μmol / g. The cyclo(leucine-proline) stock solution was diluted 20 times with water and injected into the rice roots every 7 days for a total of 8 weeks of planting. The control group was applied with the corresponding solvent.
[0078] 3) The tiller number was counted in the 8th week. The results are as Figure 5 shown. Through t-test, for the 4 varieties of japonica rice Nipponbare, indica rice 9311, japonica rice Karabaschak, and AUS NC1 / 536, the tiller numbers of the treatment groups were significantly lower than those of the control groups. The average tiller numbers decreased by 27%, 30%, 38%, and 20% in sequence.
[0079] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, any change, application, or improvement of the present invention using conventional techniques in the art belongs to the protection scope of the present invention.
Claims
1. An Exiguobacterium indicum R2567, with the deposit center registration number GDMCC No. 66084.
2. The application of Exiguobacterium indicum R2567 with the deposit center registration number GDMCC No. 66084 in regulating plant tillering.
3. The application according to claim 2, wherein Adding the Exiguobacterium indicum R2567 to the culture solution or planting soil of plants during the vegetative growth period to inhibit plant tillering.
4. The application according to claim 3, wherein Add the Exiguobacterium indicum R2567 with a concentration of 10 6 ~10 7 cfu / mL to the culture solution of the plant; or, first soak the roots of the seedlings in a bacterial suspension with a concentration of 10 6 ~10 7 cfu / mL of Exiguobacterium indicum R2567 for a period of time, and then transplant them into the soil to which Exiguobacterium indicum R2567 has been applied, so that the bacterial content in the soil is 10 6 ~10 7 cfu / g soil.
5. Use of cyclo(leucine-proline) in regulating tillering of plants, wherein, The ring (leucine-proline) is a cyclic dipeptide formed by the condensation of one leucine and one proline, with the chemical formula C 11 H 18 N2O2.
6. The application according to claim 5, wherein The structural formula of the cyclic (leucine - proline) is as follows:
7. The application according to claim 5, characterized in that Adding the cyclic (leucine - proline) to the culture solution or planting soil of plants during the vegetative growth period to inhibit plant tillering.
8. The application according to claim 7, wherein Adding cyclic (leucine - proline) with a concentration of 2 - 4 μM to the culture solution of plants; or, first soaking the roots of the seedlings in a 2 - 4 μM cyclic (leucine - proline) solution for a period of time, then transplanting them into the soil and adding a cyclic (leucine - proline) solution to the soil so that the content of cyclic (leucine - proline) in the soil around the roots is 2 - 4 μmol / g of soil.
9. The application according to any one of claims 2 to 8, characterized in that, The plant is a gramineous plant, including but not limited to rice, wheat, corn, tobacco, sorghum.
10. A plant tillering regulator, wherein the growth regulator contains Exiguobacterium indicum R2567 and / or cyclic (leucine - proline), and the deposit center registration number of the Exiguobacterium indicum R2567 is GDMCC No. 66084.