CN116790426B - Flavobacterium strain for reducing application of plant phosphate fertilizer and application thereof - Google Patents

Abstract

The invention discloses a Flavobacterium strain for reducing the application of plant phosphate fertilizer and application thereof, namely Flavobacterium (Flavobacterium sp.) C2, which is preserved in China general microbiological culture collection center (CGMCC) No.26904 at 24 days of 2023 and 3. C2 has a good capacity to dissolve poorly soluble inorganic phosphorus and has IAA secreting properties. The C2 is beneficial to promoting the high-efficiency utilization of phosphorus by plants (particularly rape) while dissolving inorganic phosphorus, so that the investment of phosphate fertilizer is reduced, the yield of crops is ensured, and the microorganism can be used for particularly and obviously promoting the growth of lateral roots of phosphorus-sensitive rape and enhancing the adaptability of the rape to low-phosphorus environment. The invention increases the quality microorganism germplasm resource of China, and has important significance for reducing the application of chemical fertilizer and promoting the green development of agriculture.

Description

Flavobacterium strain for reducing application of plant phosphate fertilizer and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and relates to a flavobacterium strain for reducing application of plant phosphate fertilizer and application thereof.

Background

Phosphorus is involved in the synthesis of important compounds in plants, such as nucleic acid, phospholipid, protein, high-energy phosphate compounds, etc., and is required by plants in large quantities, which is one of three essential nutrient elements of plants, and accounts for about 0.2% of dry weight of the plants. However, the soil often cannot supply enough available phosphorus to plants, so in agricultural production, phosphorus is often applied to the soil by processing phosphorus ores into phosphate fertilizers to meet the requirements of high yield of crops for phosphorus. On the one hand, as the global population increases further, the consumption rate of phosphate ores will increase further in order to keep up with more population. After the phosphate fertilizer is applied into the soil, phosphorus is easily adsorbed and fixed by clay minerals in the soil to become an invalid state. It is estimated that when the phosphate fertilizer utilization rate in the season is only 10 to 20%, most of it is fixed by the soil, phosphorus accumulated in the soil between 1980 and 2007 (P ₂ O ₅ ) The cumulative amount exceeds 8500 ten thousand tons. Excessive phosphorus in the soil can enter underground water along with the leaching effect of rainwater, or directly enter rivers and lakes along with runoff in rainy seasons to cause non-point source pollution such as water enrichment. The contribution rate of phosphorus to agricultural non-point source pollution is reported in literature to be even as high as 67%.

Cabbage type rape is a heterotetraploid crop formed by distant hybridization of cabbage and cabbage in nature, and is originally originated in Europe at the earliest, introduced into China in the thirties of the last century, and becomes the first large oil crop in China, and the oil yield is nearly 50% of that of the domestic oil crop. The Yangtze river basin is a main production area of rape in China, and because of the desilication and aluminizing effects of the soil, the soil in the area is rich in Fe and Al, and free phosphorus in the soil is often fixed into Fe-P and Al-P which cannot be absorbed and utilized by plants, so that the lack of effective phosphorus is caused. Rape is sensitive to phosphorus nutrient deficiency, and when rape lacks phosphorus slightly, bolting and flowering of rape are affected, branches and fruits at corners are reduced, and seeds are not full and blighted. When serious phosphorus deficiency happens, the growth of the rape root system is inhibited, the leaves become dark purple, shrinkage cannot be flattened, and part of plants even die. In summary, the lack of available phosphorus in the soil becomes a major limiting factor in rape production.

How to reduce the investment of phosphate fertilizer and increase the effectiveness of indissolvable phosphorus in soil is an urgent problem to be solved in the current agricultural production. It has been found that microorganisms in soil can colonize the rhizosphere and plant interiors and promote plant growth when applied to seeds, plant surfaces or soil. They can not only improve soil fertility and crop productivity by adding nutrients to the soil or improving the availability of nutrients, but also protect plants from diseases and insect pests. In addition, the biological fertilizer is environment-friendly, economical, efficient and nontoxic, and after the biological fertilizer is continuously used for 3-4 years, the biological fertilizer does not need to be continuously applied due to the self-growth and propagation effects of microorganisms, and enough microorganisms exist in soil. Therefore, the method for solving the problems has the highest potential and prospect that the phosphorus-dissolving microorganism in the soil is searched or has the function of promoting the efficient utilization of phosphorus by plants, and the microorganism is processed into a microorganism preparation for improving the utilization efficiency of the phosphorus in agricultural production.

Although various microorganisms are reported at present, the main development and application objects are melon, fruit and grain crops, few microorganisms which are suitable for oil crops, especially for reducing phosphate fertilizer of rape and do not reduce or even increase efficiency are reported. The general microbial preparation is difficult to play a stable effect on different crops, so the general microbial preparation is difficult to popularize to farmers. Therefore, screening microorganisms for rape production and developing corresponding microbial preparations have great significance for promoting green development of rape agriculture and guaranteeing national edible oil safety.

Disclosure of Invention

Therefore, the invention aims to provide the flavobacterium strain for reducing the application of the plant phosphate fertilizer and the application thereof, which can play a role in stabilizing and promoting growth, improve the phosphorus availability in soil and promote plant growth.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. a strain of Flavobacterium (Flavobacterium sp.) C2 for reducing phosphorus fertilizer application is preserved in China general microbiological culture Collection center (CGMCC) No.26904 at 24-month of 2023.

Preferably, the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.

2. A composition comprising the aforementioned flavobacterium strain.

3. A preparation contains the above Flavobacterium strain as effective component.

4. The application of the flavobacterium strain, composition or preparation in dissolving indissolvable phosphorus and increasing the content of available phosphorus.

Preferably, the poorly soluble phosphorus includes, but is not limited to: calcium phosphate, aluminum phosphate, iron phosphate.

5. Use of the foregoing flavobacterium strain, composition or formulation for promoting plant growth.

Preferably, promoting plant growth includes some or all of the following:

(1) Increasing biomass of the aerial part or the underground part of the plant in different growth and development periods;

(2) Promoting the plant height of the plant in different growth and development periods;

(3) Promoting the growth of plant root systems;

preferably, the plant is rape, and the promotion of plant growth further comprises:

(4) Promoting the growth of the root system of the rape under the low-phosphorus condition, in particular to the growth of the root system of the phosphorus-sensitive rape:

(5) Promoting the phosphorus content of the rapeseed grains;

(6) Promoting the oil content of the rapeseed grains.

6. The method for promoting plant growth by using the flavobacterium strain comprises the following specific steps:

grinding and air-drying soil, sieving with a 2mm sieve, mixing the soil with vermiculite and perlite, and sterilizing to obtain sterilized soil;

step two, sub-packaging sterilized soil into a seedling raising basin, sterilizing plant seeds, then sprouting the plant seeds in a 1/2MS culture medium, transplanting the plant seeds into the sterilized soil, inoculating bacterial liquid prepared by the flavobacterium strains into the root of the seeds after the seeds bud, and culturing for 14 days.

Preferably, in the first step, the soil is red soil, and the soil is from Yunnan stone forest.

Preferably, in the first step, the volume ratio of soil, vermiculite and perlite is 6:3:1.

preferably, in the first step, sterilization is performed by using an autoclave, and the conditions are as follows: at 121℃for 1 hour, and after 24 hours, sterilization was again performed under the same conditions.

Preferably, in the second step, the number of holes of the seedling raising basin is as follows: 5X 10, wherein the height and caliber of a single hole are respectively 12cm and 5cm, one hole is planted in each hole, and the process is repeated for 10 times; the amount of bacterial liquid in each inoculation is 5ml, and OD=0.5.

Preferably, the method for sterilizing the plant seeds comprises the following steps:

adding about 1/4 volume of plant seeds into a 2ml centrifuge tube, adding 1ml of 70% alcohol with volume concentration, reversing the solution for 2min to enable the alcohol to be fully contacted with the plant seeds, and sucking the alcohol out by a liquid-transferring gun;

secondly, adding 1ml of sterile water into the centrifuge tube after sucking out the alcohol, sucking the sterile water by a pipetting gun, and repeating the steps for three times to clean the residual alcohol;

thirdly, adding 10% sodium hypochlorite aqueous solution with volume concentration into the centrifuge tube, reversing the centrifuge tube for 5min to enable the sodium hypochlorite aqueous solution to fully contact with plant seeds, and sucking the added sodium hypochlorite aqueous solution by using a pipette;

(IV) adding 1ml of sterile water into the centrifuge tube, sucking the sterile water by a pipetting gun, and finally sucking out the sterile water, and repeating the steps for six times to clean the residual sodium hypochlorite.

Preferably, in the second step, the bacterial liquid is prepared by the following method: culturing the flavobacterium strain at 28deg.C, collecting single colony, inoculating into TSB liquid culture medium, shaking overnight culturing, centrifuging to obtain precipitate, re-suspending the precipitate with sterile water, centrifuging again to obtain precipitate, and adjusting the precipitate to OD=0.5+ -0.02 with sterile water.

Preferably, in the second step, the plant seeds are rape seeds, and the specific variety is ZS11 or R4474.

The invention has the beneficial effects that:

the invention screens and obtains a new Flavobacterium strain, namely Flavobacterium sp C2, which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 26904 in the 3 rd month of 2023 and 24 days. The strain is obtained by high-flux separation and culture of bacteria of rape root system, and is identified as Flavobacterium sp by a method of measuring 16S rDNA. C2 has a good capacity to dissolve poorly soluble inorganic phosphorus and has IAA secreting properties. The C2 is beneficial to promoting the high-efficiency utilization of phosphorus by plants (particularly rape) while dissolving inorganic phosphorus, so that the investment of phosphate fertilizer is reduced, the yield of crops is ensured, and the microorganism can be used for particularly and obviously promoting the growth of lateral roots of phosphorus-sensitive rape and enhancing the adaptability of the rape to low-phosphorus environment. The invention increases the quality microorganism germplasm resource of China, and has important significance for reducing the application of chemical fertilizer and promoting the green development of agriculture.

The experiment of plate and soil cultivation is carried out on C2 under three phosphorus levels (100% P;50% P;2% P), and the growth promotion benefit of C2 on two main push rape varieties (ZS 11, normal varieties; R4474, P sensitive varieties) is verified. The experimental results show that under different phosphorus levels, compared with the control group, the fresh weight of the overground part or the underground part of rape is obviously increased after C2 inoculation, the difference shown by the P-sensitive variety R4474 is more obvious, and the plate culture experiment is consistent with the soil culture experiment result. The invention provides a targeted and stable microbial germplasm resource for first large oil crop rape, in particular P-sensitive rape in China. The special function of the bacteria is beneficial to reducing the investment of phosphorus fertilizer in agriculture and reducing the risk of phosphorus loss to the environment; and the effectiveness of phosphorus in the soil is improved, and the absorption of the phosphorus by plants is promoted. The microbial preparation is applied to agricultural production, meets the sustainable requirements of low carbon and environmental protection, and is beneficial to the green development of agriculture in China. The invention expands the seed quality resources of the growth promoting bacteria, particularly expands the seed quality resources of the growth promoting bacteria aiming at oil plants and rape, and lays a bacterial foundation for developing efficient and stable special microbial fertilizers in the future.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention is illustrated in the following drawings.

FIG. 1 is a colony morphology of C2.

FIG. 2 shows that C2 dissolves calcium phosphate in PKO inorganic phosphorus medium to produce a transparent ring.

FIG. 3 is a phylogenetic tree of C2.

FIG. 4 is a graph of a qualitative analysis of IAA produced by C2.

FIG. 5 shows the phenotype of phosphorus-sensitive rape R4474 plated to 7d at low phosphorus (2%P) levels, wherein A is Mock (sterile treatment) and B is C2.

FIG. 6 is a graph showing the biomass statistics of C2 inoculated into R4474 and ZS11 of rape and cultivated in soil to 14d, wherein A is the fresh weight statistics of the upper part and B is the fresh weight statistics of the lower part at three phosphorus levels (100%, 50%, 2%).

FIG. 7 is a root phenotype plot of R4474 after inoculation with C2 to 14d under low phosphorus (2%P) conditions, wherein A is Mock (sterile treatment) and B is C2 treatment.

FIG. 8 is a graph showing the biomass statistics of the inoculated C2 and another Flavobacterium L2P42D4 (D4) cultivated in soil to 14D, wherein A is the fresh weight statistics of the upper part under the condition of low phosphorus (2%P), and B is the fresh weight statistics of the lower part.

Preservation information

Classification naming: flavobacterium (Flavobacterium)

Latin Wen Xueming: flavobacterium sp.

Preservation unit name: china general microbiological culture collection center (CGMCC)

Deposit unit address: beijing city, chaoyang area, north Chenxi Lu No.1 and 3

Preservation date: 2023, 3, 24

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1:

separation and purification of rape root endophyte

The main reference is the text of "high-throughput isolated culture identification of plant root bacteria" issued by the Baiyang group of the institute of Chinese academy of science, genetics, on Nature Protocols.

1. Experimental materials

(1) Biological material

The samples were derived from Brassica napus root lines (Brassica napus l.).

(2) Reagent(s)

Tryptone Soy Broth (TSB) medium, phosphate Buffer (PBS), sterile water, 10mM magnesium chloride solution, 80% glycerol aqueous solution by volume, agar.

(3) Instrument and equipment

Ultra-clean bench, electronic balance, constant temperature table, high-speed centrifuge, -80 ℃ ultra-low temperature refrigerator, -20 ℃ refrigerator.

(4) Others

Tweezers, scissors, a 50mL sterile centrifuge tube, a plastic grinding rod, a 13X 13cm sterile square dish, a 60mm bacterial culture dish, a 96-hole bacterial culture plate, a 96-hole ELISA plate, a 2.0mL frozen storage tube, microbank bacteria preservation, a channel pipette, a single-channel pipette, a pipette gun head and a Parafilm sealing film.

2. Experimental method

Pulling out the rape roots on natural soil, collecting about 3g of a 10cm root system sample of the rape, and placing the rape roots into a 50mL sterile centrifuge tube. The macroscopic soil particles on the root system were washed away with sterile water. Then, 5g of the root system was transferred to a new 50mL centrifuge tube, 30mL of sterilized phosphate buffer was added to the tube, and the tube was placed on a plate shaker at room temperature and shaken at 180rpm for 15min, and repeated 3 times. And adding 30ml of sterile water, cleaning for 4 times, uniformly coating sterile water disinfected for the last time on the surface of the TSB solid culture medium, placing the culture medium in a 28 ℃ environment for 3 days, and checking whether the surface of a root system is sterilized.

The root tissues after washing and water absorption are sheared into small sections of 2mm by sterile scissors and are uniformly mixed. 0.02g of root tissue was weighed and placed into a 1.5mL sterile centrifuge tube. In a biosafety cabinet 200. Mu.L of sterilized 10mM magnesium chloride solution was added to the centrifuge tube and the roots were ground with a sterile grinding rod until homogenized. And preserving 0.1g of the uniformly mixed root system tissue in a refrigerator at-20 ℃ for later detection of root system microbiome.

The root homogenate was transferred to a 50mL tube containing 25mL of 10mM magnesium chloride, mixed well and allowed to stand at room temperature for 15min.

The dilution gradient is prepared. 500. Mu.L, 167. Mu.L, 56. Mu.L and root homogenates were added to 3 reagent bottles containing 1L of 10% TSB solution by mass and diluted to 2000X, 6000X and 18000X gradient dilutions, respectively.

The dilutions were dispensed into 96-well cell culture plates. In a biosafety cabinet, shake well each bottle of diluent, pour 50mL of diluent into a 13 x 13cm sterile square dish, draw the liquid with a lance into each well of a 96 well cell culture plate, transfer 160 μl per well, transfer 3 cell culture plates per bottle of diluent.

Each cell culture plate was sealed with Parafilm. The plates were incubated at room temperature for 2 weeks.

After 2 weeks the growth of bacteria in 96-well cell culture plates was observed, leaving about 30% of the wells in the plate in a visually hazy state.

140. Mu.L of 80% sterilized glycerol aqueous solution was added to each well of the 96-well cell culture plate and stored in a-80℃refrigerator.

Bacterial cultures were picked from the frozen surface with a sterile gun head and transferred to 1/2TSB solid plates.

Single colonies were picked and transferred to new 1/2TSB solid plates after 3-5 days incubation at room temperature. This step was repeated 2 times.

After 3 times of purification on the plate, single colonies on the 3 rd plate are picked, and the plates are subjected to shaking culture by using 1/2TSB liquid medium, and the plates are subjected to shaking culture for 5-7 days at the temperature of 28 ℃ at the rotating speed of 180 rpm.

At room temperature, 30mL of bacterial liquid is centrifuged for 10min at 5000r/min, the supernatant is discarded until 1mL remains, bacterial cells are resuspended, 800 mu L of the resuspended bacterial liquid is absorbed and transferred into a freezing tube, an equal volume of sterile glycerol aqueous solution with the volume concentration of 80% is added, and the mixture is uniformly mixed and stored to the temperature of minus 80 ℃.

3. Experimental results

By the high-flux bacterial separation method, about 300 strain resources are separated from rape root system.

Example 2:

identification of isolated endophytes

In addition to the sequencing effort, the amplification and purification of the isolated strain 16S DNA, and subsequent screening effort, was accomplished in the laboratory.

1. Experimental materials

(1) Biological material

The rape root endophytic bacteria and the escherichia coli are separated by the method.

(2) Reagent(s)

NaOH alkaline lysate, HCl neutralization solution, various reagents required by a PCR reaction system shown in the following table, nuclease-free water, agarose, TAE buffer solution, nucleic acid dye solution and DNAmarker.

(3) Instrument and equipment

PCR instrument, -20deg.C refrigerator, gel electrophoresis apparatus, microwave oven, and gel imager.

(4) Others

96-well PCR plate, channel pipettor, single channel pipettor and pipette gun head

2. Experimental method

Double-side label two-step PCR amplification method for amplifying bacterial 16S rDNA sequence

10ul of bacterial solution was added to each well of a 96-well PCR plate, followed by 16. Mu.L of alkaline lysis solution.

Blowing and mixing the above system with a row gun, and sealing with a sealing plate film. The PCR plate was placed in a PCR apparatus and the cells were lysed at high temperature in an alkaline environment, with the procedure set at 95℃for 30min.

After cooling down, 16. Mu.L of neutralization buffer was added to each well, mixed well and centrifuged, and stored in a-20℃refrigerator.

First round PCR System, primer 799F (AACMGGATTAGATACCCKG)

/1193R (ACGTCATCCCCACCTTCC). 96-well PCR plates a12 and B12 wells were added nuclease-free water and DNA of e.coli, respectively, as negative and positive controls on each plate. (Table 1, table 2)

TABLE 1 first round PCR reaction System

Composition of the components	Volume (mu L)
		10×Buffer	3
dNTPs(2.5mM each)	2.4
		799F(10mM)	0.3
1193R(10mM)	0.3
		HS taq(5U/μL)	0.15
gDNA	3
		Double distilled water	20.85
Total volume of	30

TABLE 2 first round PCR reaction procedure

Cycle number	Denaturation (denaturation)	Annealing	Extension
				1	94℃,2min
2–31	94℃,30s	55℃,30s	72℃,1min
				32			72℃,5min

The first round PCR amplification product and two controls were diluted 40-fold with nuclease-free water as templates for the second round PCR amplification.

The second round of PCR amplification uses 799F and 1193R primers with a tag (Barcode) and a linker required for sequencing to achieve the addition of a tag at the 3 'and 5' ends of the fragment of interest and the sequence required for Illumina sequencing. (Table 3, table 4)

TABLE 3 second round PCR reaction System

TABLE 4 second round PCR reaction procedure

Cycle number	Denaturation (denaturation)	Annealing	Extension
				1	94℃,2min
2–26	94℃,30s	55℃,30s	72℃,1min
				27			72℃,5min

After the second round of PCR amplification, 5. Mu.L of the negative and positive control PCR products were aspirated, and mixed with 5. Mu.L of DNA loading buffer, respectively, and detected by 1.0% agarose gel electrophoresis. The positive control produced a band of about 500bp, and the negative control was not banded, indicating that the second round of PCR amplification was acceptable.

After quality inspection, the products are sent to a company for sequencing, and bacteria isolated and cultured are identified by a bioinformatics method.

3. Experimental results

As a result of sequencing, it was revealed that the obtained strains were Pseudomonas, bacillus, klebsiella, leifer, paenibacillus, serratia, oligotrophic monad, sphingomonas, proprietar, mycobacterium, polyphagia, sphingomonas, flavobacterium, pseudomonas fluorescens, etc.

Example 3:

screening bacterial strain for separation, purification and identification

1. Experimental materials

(1) Biological material

The separated rape root system bacteria

(2) Reagent(s)

TSB medium, sterile water, PKO inorganic phosphorus medium

(3) Instrument and equipment

High pressure steam sterilizing pot, shaking table, spectrophotometer, centrifuge and super clean bench.

(4) Others

Bacteria culture dish, 50ml sterile centrifuge tube.

2. Experimental method

Preparing TSB solid culture medium: 15g/L of pancreatic protein soybean broth culture medium and 15g/L of agar; sterilizing the culture medium in a high-pressure steam sterilizing pot at 121deg.C for 20min, cooling to 50deg.C, pouring into a plate on a super clean bench, and cooling for use.

A proper amount of bacterial liquid is selected from a sterilized inoculating loop in bacterial preservation, streaked in the TSB solid culture medium, and bacteria are cultured at 28 ℃.

The TSB broth was prepared as described above, except that agar was not added.

Pouring 20ml of the cooled TSB liquid culture medium into a sterilized 50ml centrifuge tube, selecting single colonies separated by plate streaking, inoculating the liquid culture medium into the centrifuge tube, and placing the centrifuge tube into a shaking table at 28 ℃ and the rotating speed of 180r/min for expansion culture.

Taking out the culture medium in the centrifuge tube after the culture medium is turbid to a certain degree, centrifuging in the centrifuge at 6000r/min for 6min, pouring out the supernatant, adding sterile water for resuspension, centrifuging again, discarding the supernatant, repeating the steps for two times, and adding the sterile water again for resuspension bacteria liquid.

The OD value of the bacterial liquid is regulated to 0.5, 5 mu L of the bacterial liquid is absorbed and coated on PKO inorganic phosphorus culture medium, and the bacterial liquid is cultured for 5 days, and the bacterial strain C2 with the phosphorus-dissolving ring is selected.

3. Experimental results

The 15 strains were found to have phosphorus dissolving ability and were classified into 8 genera including 5 flavobacterium, 3 flavomonas, 2 oligotrophic monads, 1 levator, 1 praecox, 1 mycobacterium, 1 polyphagia, 1 sphingosine. One of the strains of flavobacterium C2 (fig. 1, 3) exhibited a prominent phosphorus-dissolving capacity and was therefore selected for the subsequent experiments. After C2 was plated on PKO inorganic phosphorus medium, the medium developed a transparent loop as shown in fig. 2. The whole genome of C2 was determined and functionally annotated. Genomic information predicts that this microorganism has IAA-producing function, and later this was verified by Salkowski staining, demonstrating that C2 can not only solubilize inorganic poorly soluble phosphorus, but also produce IAA.

Example 4:

IAA determination

1. Experimental materials

(1) Biological material

The separated endophyte C2

(2) Reagent(s)

L-tryptophan, salkowski reagent and IAA standard solution.

(3) Instrument and equipment

A microorganism constant temperature incubator, a high-pressure steam sterilization pot, a shaking table, a centrifuge, an ultra-clean bench and a spectrophotometer.

(4) Others

50mL centrifuge tube, 2mL centrifuge tube, pipette.

2. Experimental method

Single colonies separated by plate streaking are selected in an ultra clean bench and inoculated into a sterilized 50mL centrifuge tube filled with liquid LB medium, 1mL of culture solution is taken and centrifuged at 12000r/min for 15min after adding L-tryptophan with the concentration of 0.5g/L and about 2.5mmol/L for 7d, 0.5mL of supernatant is sucked into the 2mL sterilized centrifuge tube, 1mL of Salkowski reagent is added, the reaction is carried out in the dark for 30min at room temperature, and then the OD value is measured at the wavelength of 530nm by using a spectrophotometer. And simultaneously, preparing IAA standard solution with gradient concentration by taking pure IAA as a material, drawing a standard curve, and calculating the concentration of IAA generated by C2 after 7d culture according to the drawn standard curve and OD value.

3. Experimental results

After 7d of incubation at 28℃the concentration of IAA produced from C2 in LB medium supplemented with L-tryptophan was:

1.40+/-0.20 mg/L. (FIG. 4)

Example 5:

c2 sterile Petri dish plate experiments

1. Experimental materials

(1) Biological material

The separated rape root system bacteria C2, R4474 and ZS11 rape seeds

(2) Reagent(s)

TSB culture medium, sterile water, 1/2 phosphorus-deficient MS culture medium, agar, HCl solution and KOH solution

(3) Instrument and equipment

High-pressure steam sterilizing pot, shaking table, spectrophotometer, centrifuge, super clean bench, microorganism incubator and PH meter.

(4) Others

Bacterial culture dishes, 50ml centrifuge tubes, 25×25 sterile square dishes, inoculating loop, sealing film.

2. Experimental method

Pure culture of C2: taking out a proper amount of bacterial liquid from bacterial preservation by using an inoculating loop, streaking and separating on a plate, placing into an incubator at 28 ℃ for culturing, picking up single bacterial colony and TSB liquid culture medium after single bacterial colony grows, and placing into a shaking table for overnight culture.

Sterilizing rape seeds: the method comprises the following steps: adding about 1/4 volume of rape seeds into a 2ml centrifuge tube, adding 1ml of 70% alcohol, reversing for 2min to make the alcohol fully contact with the rape seeds, and sucking out the alcohol by using a pipette; adding 1ml of sterile water into the centrifuge tube after sucking out the alcohol, sucking out the sterile water finally by using a pipetting gun, and repeating for three times to clean the residual alcohol; adding 10% sodium hypochlorite into the centrifuge tube, reversing the centrifuge tube for 5min to enable the sodium hypochlorite to fully contact with rape seeds, and sucking out the added 10% sodium hypochlorite by using a pipetting gun; after the above sodium hypochlorite solution was sucked out, 1ml of sterile water was added to the centrifuge tube, and finally the sucked out sterile water was sucked out by a pipette, and repeated six times to clean the remaining sodium hypochlorite.

Three phosphorus level 1/2MS medium was prepared: according to the medicine specification and the solution dosage, adding proper amount of 1/2MS phosphorus deficiency culture medium and potassium dihydrogen phosphate with different contents (100%, 50%, 2%P level phosphorus concentration are 625 μm, 312.5 μm and 12.5 μm respectively) into an conical flask, adjusting pH to 5.85, adding 15g/L agar, adding proper amount of sterile water, and sterilizing at 121deg.C for 20min.

Centrifuging the bacterial liquid, removing the supernatant, re-suspending with sterile water, centrifuging, removing the supernatant, and regulating the OD of the bacterial liquid with sterile water, wherein the OD=0.5+/-0.02 meets the requirements. Taking out the sterilized culture mediums with different phosphorus contents, cooling to about 50 ℃, adding bacterial liquid into the culture medium, uniformly mixing, and pouring the mixture into a flat plate, wherein the volume ratio of the bacterial liquid to the culture medium is 1:200. and (3) placing sterilized rape seeds on a sterile square dish of 25 multiplied by 25cm at certain intervals on the same horizontal line, sealing the square dish by using a sealing film, horizontally placing the square dish in a culture room for dark culture for 1d, enabling the radicle of the rape seeds to be pricked into a culture medium, vertically placing the square dish, illuminating for 16h, carrying out dark culture for 8h, and culturing until 7d harvesting.

3. Experimental results

Inoculating C2 at different phosphorus levels can promote rape growth compared with control, and it is especially notable that C2 significantly promotes growth of phosphorus sensitive R4474 rape lateral roots under low phosphorus conditions (as shown in FIG. 5). The increase in plant lateral roots in nature can help plants to take up more nutrients to enhance the plants' ability to resist stress, and thus the applicant believes this is an advantageous phenotype. In order to enable the rape inoculated with increased C2 lateral roots to show the growth advantages in the later period, potting soil culture experiments are carried out.

Example 6:

c2 potting test

1. Experimental materials

(1) Biological material

ZS11, R4474 rape seed, C2.

(2) Reagent(s)

70% alcohol, 10% sodium hypochlorite, sterile water, TSB medium, 1/2MS medium, agar.

(3) Instrument and equipment

An ultra-clean bench, a centrifuge, a high-pressure steam sterilizing pot and a spectrophotometer.

(4) Others

50mL centrifuge tube, 20mL centrifuge tube, 13X 13cm sterile culture dish, pipette

2. Experimental method

Pure culture of C2: taking out a proper amount of bacterial liquid from bacterial storage by an inoculating loop, streaking and separating on a flat plate, and culturing in an incubator at 28 ℃.

Sterilizing rape seeds: adding about 1/4 volume of rape seed into 2ml centrifuge tube, adding 1ml of 70% alcohol by volume concentration, shaking for 2min, and sucking out alcohol with a pipette; adding 1ml of sterile water into the centrifuge tube after sucking out the alcohol, sucking out the sterile water finally by using a pipetting gun, and repeating for three times to clean the residual alcohol; adding 10% sodium hypochlorite aqueous solution with mass concentration into the centrifuge tube, shaking for 5min, and sucking out the added residual sodium hypochlorite aqueous solution by using a pipette; after the above aqueous sodium hypochlorite solution was sucked out, 1ml of sterile water was added to the centrifuge tube, and finally the sterile water was sucked out by pipetting gun and repeated six times to clean the remaining sodium hypochlorite.

13X 13cm square seedling: selecting single colony of C2, inoculating to 1/2TSB (beef extract soybean broth) liquid culture medium, culturing in shaking table until logarithmic growth later stage, centrifuging, pouring supernatant to obtain thallus, adding sterile water for resuspension, and regulating OD value of microbial agent to 0.5; then adding the microbial inoculum into a 1/2MS culture medium cooled to 50 ℃, uniformly mixing, pouring the mixture into a flat plate, and spotting the sterilized rape seeds on the flat plate for dark culture for one day to sprout.

Soil cultivation test: grinding air-dried soil, collecting a 0.5mm sieve soil sample, and mixing the soil with vermiculite perlite according to a ratio of 6:3:1, filling the mixed soil into two layers of biosafety bags, sterilizing in a high-pressure steam sterilizing pot at 121 ℃ for 1h, and then killing the soil again after 24h, wherein the sterilized soil is ready for use; subpackaging the sterilized soil sample into a seedling raising basin, selecting the rape seeds which are uniformly germinated in a flat plate, transplanting the rape seeds into soil, inoculating 1mL of bacterial liquid with OD=0.5+/-0.02 into roots after the seeds are germinated from the soil, and culturing for 14 days to obtain rape.

3. Experimental results

Inoculating C2 can promote the growth of two rape varieties R4474 and ZS11 under each phosphorus level. In the case of potted plant experiments to reduce the application of phosphate by 50% and to apply phosphate by 2% (100% P is 1kg soil plus 200mg P) ₂ O ₅ 50% p:100mg/kg,2% P:4mg/kg; the phosphorus source is calcium phosphate), and compared with a control group without inoculating the strain, the rape plant height, the fresh weight, the dry weight, the leaf area, the main root length, the lateral root density and the like of the rape are obviously increased. And after inoculation of both rapes, the biomass is even higher at the applied 50% phosphate level than that of the non-inoculated rapes normally applied with phosphate. Especially, the promotion effect of C2 on the growth of the phosphorus-sensitive rape lateral root under the low phosphorus condition is more remarkable than that of the normal ZS11 lateral root, so that the C2 has great significance on adapting to the low phosphorus environmental stress of the phosphorus-sensitive rape. (Table 5, table 6, FIG. 7)

TABLE 5 fresh weight of ZS11 above and below ground at various phosphorus levels (14 d)

TABLE 6 fresh weight of R4474 above and below ground at various phosphorus levels (14 d)

The experiment uses Flavobacterium L2P42D4 (D4 for short) with growth promoting effect, which is purchased from China general microbiological culture Collection center (Beijing.) through electric commerce, and rape inoculation experiment is performed. The experimental results are shown in fig. 8, which show that compared with flavobacterium D4, C2 has more remarkable effect on the rape promotion and has greater application potential.

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (8)

1. A strain of Flavobacterium for reducing application of phosphate fertilizer to plants is characterized in that the strain is FlavobacteriumFlavobacterium sp.) C2 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of No.26904 in 2023 and 24 days.

2. A composition comprising the flavobacterium strain of claim 1.

3. A preparation comprising the flavobacterium strain according to claim 1 as an active ingredient.

4. Use of the flavobacterium strain of claim 1, the composition of claim 2 or the formulation of claim 3 for dissolving poorly soluble phosphorus and increasing the available phosphorus content.

5. Use of the flavobacterium strain of claim 1, the composition of claim 2 or the formulation of claim 3 for promoting plant growth.

6. The use according to claim 5, wherein promoting plant growth comprises some or all of:

(1) Increasing biomass of the aerial part or the underground part of the plant in different growth and development periods;

(2) Promoting the plant height of the plant in different growth and development periods;

(3) Promote the growth of plant root system.

7. The use of claim 6, wherein the plant is canola, and the promoting plant growth further comprises:

(4) Promoting the growth of the root system of the rape under the low-phosphorus condition.

8. A method for promoting plant growth using the flavobacterium strain of claim 1, comprising the specific steps of:

grinding and air-drying soil, sieving with a 2mm sieve, mixing the soil with vermiculite and perlite, and sterilizing to obtain sterilized soil;

step two, sub-packaging sterilized soil into a seedling raising basin, sterilizing plant seeds, then sprouting the plant seeds in a 1/2MS culture medium, transplanting the plant seeds into the sterilized soil, inoculating bacterial liquid prepared by the flavobacterium strains into the root of the seeds after the seeds bud, and culturing for 14 days.