A rice phosphate-dissolving bacteria and its application

Abstract

The invention provides a rice phosphate-dissolving bacteria and its application, belonging to the technical field of biological microbial agents. The strain of the invention: rice phosphate-dissolving bacteria Acinetobacter sp.X2 can increase the germination rate of rice by 31.8%-38.1%; there are significant differences in rice plant height, root dry weight, total root length, aboveground dry weight and aboveground fresh weight among treatments. It has broad application prospects in increasing rice yield and the like, and helps to stabilize or increase rice yield and ensure food security.

Description

Technical Field

[0001] The invention relates to the technical field of biological microbial agents, and in particular to rice phosphate-dissolving bacteria and applications thereof. Background Art

[0002] Rice is one of the most important grains in the world. More than half of the world's population relies on rice as their staple food, so ensuring rice production is crucial to ensuring food security.

[0003] Phosphorus is one of the three major nutrients for plants and an essential nutrient for the formation of crop yields. It needs to be supplemented by applying phosphorus fertilizers during crop cultivation. The phosphorus absorbed and utilized by plants mainly comes from the soil, and 95% of the phosphorus in the soil exists in a form that is difficult for plants to absorb, resulting in phosphorus deficiency in about 74% of my country's arable land. In order to pursue high yields, the input of phosphorus fertilizers often exceeds the actual demand of crops, resulting in increased accumulation of soil phosphorus. Phosphorus in the soil is easily reacted with Fe 3+ , Ca 2+ 、Al 3+ Combined with water, it forms insoluble compounds and reduces its effectiveness, resulting in a utilization rate of only 5% to 25% of the applied phosphate fertilizer in the season. Rice production has a large demand for phosphate fertilizer, but the utilization rate of phosphate fertilizer is low. Some phosphorus is lost through surface runoff, causing agricultural non-point source pollution. Therefore, improving the effectiveness of soil phosphorus is the key to increasing rice production and technological breakthroughs.

[0004] Phosphate solubilizing bacteria (PSB) are a type of microorganism that can solubilize phosphorus. They can secrete organic acids, phosphatases and other substances to activate soil phosphorus, change its form, and convert the poorly soluble or insoluble phosphorus that is difficult for plants to absorb and utilize into a usable form, thereby improving the effectiveness of phosphorus in the soil.

[0005] Microorganisms are the driving force of soil phosphorus cycle. They are of great significance in regulating the composition of soil phosphorus pool and promoting the absorption and utilization of phosphorus by plants by affecting the dissolution, mineralization, absorption and distribution of phosphorus. Studies have shown that phosphate-dissolving microorganisms are widely present in soil. Using these microorganisms to promote the dissolution of mineral-bound phosphorus can improve the utilization efficiency of phosphorus fertilizer. In recent years, the isolation of phosphate-dissolving bacteria has received more and more attention, but few have been confirmed to promote rice seed germination, plant rice seedlings, promote rice absorption of phosphorus, and increase rice dry weight and other indicators. Microorganisms are regarded as the second genome of plants, and they are closely related to plant growth. Therefore, it is particularly necessary to isolate, identify and apply phosphate-dissolving bacteria suitable for rice growth environment. Summary of the invention

[0006] The object of the present invention is to provide a rice phosphate-dissolving bacteria having excellent effects.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a rice phosphate-dissolving bacterium. The rice phosphate-dissolving bacterium is rice phosphate-dissolving bacterium Acinetobacter sp.X2, and the preservation number is CCTCC NO.M 2024472.

[0009] Preferably, the 16S rDNA gene sequence of the rice phosphate-solubilizing bacteria is as shown in SEQ ID NO.1.

[0010] The present invention also provides a fermentation medium for culturing the rice phosphate-dissolving bacteria, wherein the fermentation medium is a PVK medium or an LB medium.

[0011] The present invention also provides a method for culturing the rice phosphate-dissolving bacteria, comprising the following steps:

[0012] The rice phosphate-solubilizing bacteria Acinetobacter sp.X2 is inoculated into the fermentation medium and cultured to obtain a rice phosphate-solubilizing bacteria fermentation liquid.

[0013] Preferably, the culture temperature is 25-35°C.

[0014] Preferably, the culturing time is more than 12 hours.

[0015] The present invention also provides a rice phosphate-dissolving bacteria fermentation liquid obtained by the above-mentioned culture method.

[0016] The present invention also provides the use of the rice phosphate-dissolving bacteria or the rice phosphate-dissolving bacteria fermentation liquid in preparing a soil conditioner.

[0017] The present invention also provides the use of the rice phosphate-dissolving bacteria or the rice phosphate-dissolving bacteria fermentation liquid in the preparation of an inorganic insoluble phosphorus hydrolyzing agent or a plant phosphorus absorption accelerator.

[0018] The present invention also provides a soil conditioner, which contains the rice phosphate-solubilizing bacteria or the rice phosphate-solubilizing bacteria fermentation liquid.

[0019] Beneficial effects of the present invention:

[0020] The strain of the present invention: Acinetobacter sp.X2, a rice phosphate-dissolving bacterium, can increase the germination rate of rice by 31.8%-38.1%. There are significant differences in the plant height, root dry weight, total root length, aboveground dry weight and aboveground fresh weight of rice between the treatments. It has broad application prospects in increasing rice yield and the like, and is helpful to stabilize or increase rice yield and ensure food security. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Qualitative detection of phosphate solubilizing ability of phosphate-solubilizing bacteria X2;

[0022] Figure 2 The bacterial morphology of strain X2;

[0023] Figure 3 The content of dissolved inorganic phosphorus by phosphate-dissolving bacteria X2;

[0024] Figure 4 Rice plant height after inoculation with phosphate-solubilizing bacteria X2;

[0025] Figure 5 Fresh weight of rice roots after inoculation with phosphate-solubilizing bacteria X2;

[0026] Figure 6 Fresh weight of aboveground part of rice after inoculation with phosphate-solubilizing bacteria X2;

[0027] Figure 7 Dry weight of rice roots after inoculation with phosphate-solubilizing bacteria X2;

[0028] Figure 8 Dry weight of aboveground part of rice after inoculation with phosphate-solubilizing bacteria X2;

[0029] Fig. 9 Total root length of rice after inoculation with phosphate-solubilizing bacteria X2;

[0030] Fig.10 Total phosphorus content in the aboveground and root systems of rice after inoculation with phosphate-solubilizing bacteria X2.

[0031] Biological Deposit Description

[0032] Rice phosphate-solubilizing bacteria Acinetobacter sp.X2 is deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, China, the deposit date is March 21, 2024, and the deposit number is CCTCC NO.M2024472. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example

[0035] Isolation and identification of phosphate-solubilizing bacteria X2

[0036] Preparation of LB medium: Weigh 10 g of tryptone (Oxoid LTD LP0042, England), 5 g of yeast extract (Oxoid LTD LP0021, England), and 10 g of sodium chloride (NaCl, Sinopharm Chemical Reagent Co., Ltd., 10019318), add 1000 mL of water and stir evenly. When preparing solid culture medium, add 15 g of agar, sterilize under high pressure steam, and package it. Sterilize it at 121°C for 20 min and store it for future use.

[0037] Isolation and identification of phosphate-solubilizing bacteria X2: The rice rhizosphere soil used for strain isolation was taken from the Science and Education Base of Fujian Agriculture and Forestry University.

[0038] Collect rhizosphere soil during the jointing stage of rice, place it in a sterilized conical flask, and add 95 ml of sterile water. Shake and mix at 30°C and 180 rpm for 1 hour, then let it stand for 10 minutes. Pipette 10 ml of the supernatant on the liquid surface into a 15 ml sterile centrifuge tube in the clean bench, and set the concentration gradient to 10. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Take 100 μl of the soil resuspension and spread it on the LB medium plate. Set three replicates for each concentration gradient. Put the medium plate in a 30℃ incubator for incubation. After three days, observe the growth of the colonies on the plate and pick a single colony. Streak it on the LB medium for purification three times to obtain the purified colony, which is stored at 4℃ for later use.

[0039] Phosphate-dissolving bacteria X2 dissolve inorganic phosphorus, such as Figure 1 shown.

[0040] Molecular identification of phosphate-solubilizing bacteria X2: The 16S rDNA sequence of bacterial universal primers was used as the design primers 16s-r RNA-F / 16s-r RNA-R, and the 16S rDNA sequence of strain X2 was amplified by PCR. The amplified sequence (SEQ ID NO.1:

[0041] GCAGTCGAGCGGGGGAAGGTAGCTTGCTACCGGACCTAGCGCGGGAC

[0042] GGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCG

[0043] AAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATC

[0044] TTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGT

[0045] GGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGA

[0046] TGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG

[0047] GCAGCAGTGGGGAATATTGGACAATGGGCGGCAAGCCTGATCCAGCCA

[0048] TGCCGCGTGTTGGAAGAAGCCTTATGGTTGTAAAGCACTTTAAGCGAG

[0049] AGAGCTACTTTAGATATACCTAGAGATAGTGGACGTTACTCGCAGAATA

[0050] AGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCA

[0051] AGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTA

[0052] AGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACT

[0053] GGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGT

[0054] GAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCT

[0055] GGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGA

[0056] TTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGG

[0057] GGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCT

[0058] GGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCC

[0059] CGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAC

[0060] CTTACCTGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCC

[0061] TTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCG

[0062] TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTT

[0063] GCCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAAACT

[0064] GGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAG

[0065] GGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACACAGCG

[0066] ATGTGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGC

[0067] AACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATG

[0068] CCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCA

[0069] TGGGAGTTTGTTGCACCAGAAGTAGCTAGCCTAACTGCAAAGAGGGCGGTAC) was transformed into competent E. coli cells, and positive clones were selected for sequencing. Comparative analysis was performed at NCBI to determine its taxonomic status. Combining the colony morphology of the bacterium on LB medium plates with related physiological and biochemical characteristics, it was named Acinetobacter sp.X2. The sequences of primers 16s r RNAF / 16s r RNAR are as follows:

[0070] 16s r RNAF: 5'AGAGTTTGATCCTGGCTCAG 3', as shown in SEQ ID NO.2

[0071] 16s r RNAR: 5'TACGGCTACCTTGTTACGACTT 3', as shown in SEQ ID NO.3

[0072] The phosphate-soluble bacteria X2 was sent to the China Center for Type Culture Collection for preservation and was classified and named Acinetobacter sp. X2, which is the strain provided by the present invention.

[0073] Determination of phosphate-solubilizing ability of phosphate-solubilizing bacteria X2, qualitative detection of phosphate-solubilizing ability: inoculate a single colony of the purified strain on PVK solid culture medium, invert it in a constant temperature incubator at 30°C and culture it for 7 days. Select the strain with phosphate-solubilizing ability and the ability to produce phosphate-solubilizing ring, measure the ratio of the phosphate-solubilizing ring diameter (D) / colony diameter (d), and make a preliminary judgment on the strength of the strain's phosphate-solubilizing ability based on the ratio.

[0074] Gram staining of bacteria: Use the Gram staining kit G1060-10 of Solarbio for the experiment, and the steps are as follows: 1) Prepare a smear of the bacterial culture on a glass slide, and fix it by flame drying. 2) For primary staining, add solution A (crystal violet) to cover the bacterial film, stain for 1-2 minutes, and rinse off the stain with clean water. 3) For mordant staining, add solution B (iodine solution) to cover the bacterial film, stain for 1 minute, and wash with water. 4) For decolorization, use filter paper to absorb the residual water on the glass slide, add solution C (95% ethanol), shake the glass slide, and decolorize for about 30-60 seconds depending on the thickness of the smear until the ethanol flowing out is no longer purple, then wash with water and absorb the water. 5) For re-staining, add solution D (safranine), re-stain for 1 minute, and wash with water. 6) For microscopic examination, use filter paper to absorb the water or air dry, and then examine with an oil microscope. Figure 2 shown.

[0075] Quantitative detection of phosphate-solubilizing ability of phosphate-solubilizing bacteria X2: Pick a single colony from the strains screened out during the qualitative detection experiment of phosphate-solubilizing ability and inoculate it into LB liquid culture medium. After culturing for 18 hours, take 1 ml of the bacterial solution and inoculate it into a 250 ml conical flask containing 100 ml of NBRIP liquid culture medium. The treatment without bacteria inoculation is used as a blank control, and each treatment is repeated 3 times. The conical flask is placed in a shaker at 30°C and cultured at 180 rpm for 5 days. Take 5 ml of the mixed bacterial solution at regular intervals every day, break the cells with ultrasound, and centrifuge at 10,000 rpm for 5 minutes. Collect the supernatant after centrifugation, and use the molybdenum antimony colorimetric method to determine the water-soluble phosphorus (PO4 3- ) content, such as Figure 3 shown.

[0076] Effects of Phosphate-Solubilizing Bacteria X2 on the Germination Rate of Rice

[0077] Comparison of germination rate between phosphate-dissolving bacteria X2 and control (CK): Select complete and full rice seeds. After seed selection, disinfect with 1% NaClO for 3-5 minutes, wash with sterile water for 3-5 times, and air-dry for use. Bacterial suspension volume: seed mass = 10:1 (ml:g), activate bacterial suspension, 24h, transfer it to a sterile 50ml centrifuge tube, centrifuge at 7000r / min for 10min, resuspend the cells in sterile water, mix well, and adjust the OD value to 2.0. Add the seeds to the bacterial suspension and shake immediately to make the seeds evenly contact with the initiation liquid. Initiate for 24 hours, shake gently every 4 hours to ensure that the seeds are fully in contact with the initiation liquid and can absorb water evenly. After the initiation is completed, rinse the seeds quickly with tap water, wipe off the surface moisture and return to the original moisture, and then conduct a germination test. The seeds are placed in a germination pot (12cm×12cm×6cm) padded with three layers of moist filter paper for paper germination. After the seeds germinate, 12 seedlings of the same growth potential are selected for hydroponic culture. After 25 days of culture, samples are taken to measure plant height, total root length, plant fresh and dry weight, and plant total phosphorus for analysis. The results are as follows: Figures 4 to 10 shown.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rice phosphate-dissolving bacteria, characterized in that: The rice phosphate-dissolving bacteria is rice phosphate-dissolving bacteria Acinetobacter sp.X2, the deposit number is CCTCC NO.M 2024472.

2. A method for culturing rice phosphate-dissolving bacteria according to claim 1, characterized in that: The following steps are involved: The rice phosphate-dissolving bacteria Acinetobacter sp.X2 is inoculated into a fermentation medium and cultured to obtain a rice phosphate-dissolving bacteria fermentation liquid; The fermentation medium is PVK medium or LB medium.

3. The method for cultivating rice phosphate-dissolving bacteria according to claim 2, characterized in that: The culture temperature is 25-35°C.

4. The method for cultivating rice phosphate-dissolving bacteria according to claim 2, characterized in that: The culture time is more than 12 hours.

5. A rice phosphate-solubilizing bacteria fermentation liquid obtained by the culturing method according to any one of claims 2 to 4.

6. Use of the rice phosphate-dissolving bacteria according to claim 1 or the rice phosphate-dissolving bacteria fermentation liquid according to claim 5 in preparing a soil conditioner.

7. Use of the rice phosphate-dissolving bacteria according to claim 1 or the rice phosphate-dissolving bacteria fermentation liquid according to claim 5 in the preparation of an inorganic insoluble phosphorus hydrolyzing agent or a plant phosphorus absorption accelerator.

8. A soil conditioner, characterized in that: The soil conditioner comprises the rice phosphate-solubilizing bacteria according to claim 1 or the rice phosphate-solubilizing bacteria fermentation liquid according to claim 5.

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