Rice growth-promoting rhizobacteria for activating insoluble phosphorus in red soil and application of rice growth-promoting rhizobacteria

Abstract

The invention relates to a rice growth-promoting rhizobacteria for activating insoluble phosphorus in red soil and application of the rice growth-promoting rhizobacteria. The invention discloses a Pandoraea sp. PanRPS strain with phosphorus dissolving capacity in acid red soil and application of the Pandoraea sp. PanRPS strain. The strain PanRPS can take insoluble iron phosphate as a substrate to activate phosphorus in the insoluble iron phosphate, so that the growth of rice under low phosphorus stress is promoted. The functional strain provided by the invention is of great significance in relieving low-phosphorus stress in a rice planting system in an acid red soil medium-low yield field, promoting rice growth and increasing the rice yield in a red soil area.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural biological product production, in particular to a rhizosphere phosphorus-solubilizing bacterium Pandora Pan_R_PS for promoting the activation of difficult-to-dissolve phosphorus in southern acidic red soil and application thereof. BACKGROUND

[0002] The low availability of phosphorus in red soil is one of the most important factors limiting agricultural activities in red soil. Red soil is rich in iron and aluminum minerals, and phosphorus is mainly in the form of difficult-to-dissolve Fe-P, Al-P and occluded phosphorus, which has very low availability. At present, biological organic fertilizer and microbial fertilizer are commonly used to improve red soil and increase the availability of phosphorus.

[0003] Microbial fertilizer is a special fertilizer containing active microorganisms. Through the life activities of microorganisms in the fertilizer, the content of various mineral nutrients in the soil can be increased, thereby increasing soil fertility and promoting plant growth. Phosphorus bacterial fertilizer mainly contains phosphorus-solubilizing strains, which can activate difficult-to-dissolve phosphorus in red soil and has important significance for improving the phosphorus deficiency status of red soil.

[0004] Therefore, it is necessary to screen efficient phosphorus-solubilizing strains from the rhizosphere of plants under natural conditions and prepare microbial fertilizer to fully exert the potential of promoting plant phosphorus absorption, enhancing soil health and reducing dependence on chemical fertilizers. SUMMARY

[0005] The purpose of the present application is to solve the problem of low biological availability of phosphorus in low-yield fields in southern red soil, and to screen rhizosphere growth-promoting bacteria that can activate difficult-to-dissolve phosphate in red soil from the rhizosphere of red soil rice.

[0006] To solve the above technical problems, the present application first provides Pandora Pan_R_PS, which is isolated from the rhizosphere of red soil rice.

[0007] The strain provided by the present application has been preserved in the China General Microbiological Culture Collection Center (CGMCC; address: No. 1, Yihuangyuan, Beichen West Road, Chaoyang District, Beijing, China; postcode: 100101). The Pandora Pan_R_PS has a preservation number of CGMCC NO. 27802.

[0008] The rhizosphere growth-promoting bacteria for activating difficult-to-dissolve phosphate in red soil described herein are prepared by the following method: inoculating the activated strain Pan_R_PS into LB liquid medium for fermentation.

[0009] The LB culture medium contains 5g of yeast extract, 10g of protein peptone, 3g of sodium chloride per liter, 1000ml of distilled water, and is sterilized at 115 DEG C for 15 minutes. 600 The fermentation temperature is specifically 30 DEG C, and the rotation speed is specifically 170 rpm.

[0010] The fermentation product is the bacteria liquid, and after centrifugation, the fermentation product is resuspended with sterile water to obtain a bacteria suspension. 7 The bacteria suspension is used for root immersion treatment for 6-12 hours, and then the rice is transplanted into the field.

[0011] The phosphorus-dissolving bacteria strain activates the water-soluble phosphorus iron in the rhizosphere of the rice, improves the content of the effective phosphorus in the rhizosphere soil, and thus relieves the phosphorus stress of the rice and promotes the growth of the crops.

[0012] Beneficial effects:

[0013] The application provides a phosphorus-dissolving bacteria strain which can activate the water-soluble phosphorus iron in the rhizosphere of the rice in red soil. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Phosphorus-dissolving ability identification of Pandora Pan_R_PS Figure 2 Colony morphology of the strain Pan_R_PS on a solid plate Figure 3 Phylogenetic tree of the strain Pan_R_PS Figure 4 Effect of Pandora Pan_R_PS on the plant height of rice seedlings under low-phosphorus stress Figure 5 Effect of Pandora Pan_R_PS on the root configuration of rice seedlings under low-phosphorus stress Figure 6 Effect of Pandora Pan_R_PS on the root length of rice seedlings under low-phosphorus stress Figure 7 Effect of Pandora Pan_R_PS on the total root length of rice seedlings under low-phosphorus stress Figure 8 Effect of Pandora Pan_R_PS on the total root surface area of rice seedlings under low-phosphorus stress Figure 9 Effect of the strain Pan_R_PS on the content of the available phosphorus in the rhizosphere soil of the rice under field conditions Figure 10 Effect of strain Pan_R_PS on phosphorus content of rice plants under field conditions Figure 11 Effect of strain Pan_R_PS on yield of rice under field conditions Biological material preservation information

[0015] Pan_R_PS, classified as Pandora sp. Pandoraea sp. , was preserved in the China General Microbiological Culture Collection Center on July 5, 2023, at the address of No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences; and the preservation number is CGMCC NO. 27802. DETAILED DESCRIPTION

[0016] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified. The rice variety is Nipponbare.

[0017] Example 1: Screening and phosphorus solubilizing ability identification of Pandora sp. strain Pan_R_PS Strain isolation and initial screening of phosphorus solubilizing ability: The tissue culture bottle planted with rice was inverted, and the seedlings were carefully separated from the soil body. The roots were washed with running water to remove the larger soil clumps. At this time, the soil attached to the surface of the rice roots that could not be directly washed off was the rhizosphere soil of the rice. The rhizosphere soil was extracted according to the following steps: the rice roots were cut with sterile scissors, and the part 2-6 cm away from the root tip was placed in 30 ml PBS-S buffer solution, and the mixture was placed in a shaking bed at 30°C and shaken at 200 rpm for 30 min to separate the rhizosphere soil from the plant roots. The rhizosphere soil suspension was passed through a 100 μm nylon cell filter to remove larger soil clumps and plant residues, and the filtrate was placed in a new 50 ml centrifuge tube. The filtrate was centrifuged at 3500 g for 15 min, and the supernatant was removed. Then, 1 ml of PBS-S was added to resuspend the precipitate, which was transferred to a new sterile 2 ml centrifuge tube and centrifuged at 10000 g for 5 min. The precipitate was the rhizosphere soil of the rice.

[0018] The above PBS-S buffer solution is prepared as follows: NaH2PO4·H2O 6.33 g, Na2HPO4·7H2O 16.5 g, Silwet L-77 200 μL, and distilled water to 1000 mL.

[0019] The collected rice rhizosphere samples were gradient diluted with PBS-S buffer solution, and the dilution gradient was selected as 10 -5The sample was inoculated on LB solid medium plate with 200 μL suspension.

[0020] The LB medium formula is as follows: yeast extract 5 g, peptone 10 g, sodium chloride 3 g, agar powder 20 g, distilled water to 1000 mL, sterilized at 115°C for 15 min. The solid medium of the medium is prepared by adding 1% agar to the liquid medium.

[0021] Different single colonies were picked from the LB plate and purified by streaking. The colony morphology was observed. The purified strain was cultured in LB liquid medium at 30°C and 170 rpm for 12 h. When the OD600 was 1.0, the culture was centrifuged at 2000 g for 5 min, and then washed twice with sterile water to completely remove the LB medium. The resuspended bacterial suspension was inoculated into a test tube containing MS-Fe liquid medium at a 1% inoculation amount, and cultured at 30°C and 170 rpm for 7 days. The effective phosphorus content in the supernatant was determined, with the medium without inoculation as a control (also cultured at 30°C and 170 rpm for 7 days). The strain that could significantly increase the effective phosphorus content in the fermentation broth was selected for re-screening.

[0022] The phosphorus-solubilizing effect of the strain was detected in MS-Fe medium, which was prepared by replacing the soluble phosphorus component with insoluble iron phosphate in the MS medium. The medium formula is as follows: glucose 10 g, FePO4 5 g, MgCl2·6H2O 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, pH adjusted to 6.0, distilled water to 1000 mL. The solid medium of the medium is prepared by adding 1% agar to the liquid medium.

[0023] Solid plate preparation and re-screening of strain phosphorus-solubilizing capacity: the strain was streaked on LB solid medium and cultured at 30°C. After the strain grew into single colonies on the plate, a single colony was picked and inoculated into 3 mL of LB liquid medium. The culture was shaken at 170 rpm and 30°C until the OD600 of the bacterial solution was 1.0. Then, 1.5 mL of the bacterial solution was inoculated into a 500 mL triangular flask containing 150 mL of LB liquid medium. The culture was shaken at 170 rpm and 30°C until the OD600 of the bacterial solution was 1.0. The bacterial fermentation broth was centrifuged, and the supernatant was discarded. The bacterial pellet was resuspended and washed twice with sterile water to remove the LB liquid medium in the bacterial pellet. Finally, the bacterial pellet was resuspended in sterile water to an OD600 of 1.0. 150 μL of the bacterial suspension was added to a 13*13 square culture dish, with a blank control of adding the same amount of sterile water without adding the bacterial suspension. Then, 1 / 2 MS-Fe medium was poured into the culture dish, mixed well, and left to solidify. 600 =1.0, and then 1.5 mL of the bacterial solution was inoculated into a 500 mL triangular flask containing 150 mL of LB liquid medium. The culture was shaken at 170 rpm and 30°C until the OD600 of the bacterial solution was 1.0. The bacterial fermentation broth was centrifuged, and the supernatant was discarded. The bacterial pellet was resuspended and washed twice with sterile water to remove the LB liquid medium in the bacterial pellet. Finally, the bacterial pellet was resuspended in sterile water to an OD600 of 1.0. 150 μL of the bacterial suspension was added to a 13*13 square culture dish, with a blank control of adding the same amount of sterile water without adding the bacterial suspension. Then, 1 / 2 MS-Fe medium was poured into the culture dish, mixed well, and left to solidify. 600 =1.0, and then 1.5 mL of the bacterial solution was inoculated into a 500 mL triangular flask containing 150 mL of LB liquid medium. The culture was shaken at 170 rpm and 30°C until the OD600 of the bacterial solution was 1.0. The bacterial fermentation broth was centrifuged, and the supernatant was discarded. The bacterial pellet was resuspended and washed twice with sterile water to remove the LB liquid medium in the bacterial pellet. Finally, the bacterial pellet was resuspended in sterile water to an OD600 of 1.0. 150 μL of the bacterial suspension was added to a 13*13 square culture dish, with a blank control of adding the same amount of sterile water without adding the bacterial suspension. Then, 1 / 2 MS-Fe medium was poured into the culture dish, mixed well, and left to solidify. 600 =1.0, and then 1.5 mL of the bacterial solution was inoculated into a 500 mL triangular flask containing 150 mL of LB liquid medium. The culture was shaken at 170 rpm and 30°C until the OD600 of the bacterial solution was 1.0. The bacterial fermentation broth was centrifuged, and the supernatant was discarded. The bacterial pellet was resuspended and washed twice with sterile water to remove the LB liquid medium in the bacterial pellet. Finally, the bacterial pellet was resuspended in sterile water to an OD600 of 1.0. 150 μL of the bacterial suspension was added to a 13*13 square culture dish, with a blank control of adding the same amount of sterile water without adding the bacterial suspension. Then, 1 / 2 MS-Fe medium was poured into the culture dish, mixed well, and left to solidify.

[0024] The 1 / 2MS-Fe medium is a half of MS-Fe full component medium, and the medium formula is: glucose 5 g, FePO4 2.5 g, MgCl2·6H2O 2.5 g, MgSO4·7H2O 0.125 g, KCl 0.1 g, (NH4)2SO4 0.05 g, pH is adjusted to 6.0, and distilled water is added to 1000 mL. The solid medium of the medium is prepared by adding 1% agar to the liquid medium.

[0025] After the above solid plate is cultured in a 30°C constant temperature incubator for 3 days, the effective phosphorus content of the solid plate is measured. In a clean bench, a sterilized plate puncher is used to take samples from the four corners of the solid plate and transfer them to 2 mL centrifuge tubes, with three replicates for each treatment. After adding 1 mL of sterile water to the centrifuge tubes, vortex for 10 min until the sample is fully mixed with the sterile water, centrifuge at 6000g for 5 min, and measure the effective phosphorus content of the supernatant.

[0026] 1.5 mL of the above supernatant is added to a 2 mL sterile centrifuge tube, centrifuged at 6000g for 5 min, 1 mL of the supernatant is transferred to a 50 mL centrifuge tube, 30 mL of deionized water is added, 5 mL of molybdenum antimony anti-color developing solution is added, and then the volume is adjusted to 50 mL with deionized water. After standing at room temperature for 20 min, the colorimetric determination is carried out at a wavelength of 700 nm and the value is recorded, and then the phosphorus content standard curve is calculated to calculate the phosphorus solubilizing capacity of the strain.

[0027] The method for drawing the phosphorus standard curve is as follows: take 25 mL of P standard solution, transfer it to a 500 mL volumetric flask, add deionized water to make up the volume, and prepare a P standard solution of 5 mg / L. Take 0, 2, 4, 6, 8, 10, 12 mL of the initial phosphorus solution into 7 50 mL volumetric flasks, add 200 mL of deionized water, add 5 mL of molybdenum antimony anti-color developing agent, and then add deionized water to make up the volume and shake well. Thus, 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / L of P standard solution is obtained. After standing for 30 min, the absorbance value is recorded at a wavelength of 700 nm with 0 g / mL P standard solution as the control, and the standard curve is drawn. As shown in Figure 1 The results show that a small amount of soluble phosphate is still produced on the plate after the addition of iron phosphate, with a concentration of about 0.357 mg / L; the water-soluble phosphorus content is significantly improved after inoculation of Pandora Pan_R_PS, about 3.43 times that of CK, 1.223 mg / L (*** indicates significant test P<0.001).

[0028] Figure 2 is its morphology after being cultured in LB solid medium for 48 h. As shown in Figure 2As shown, Pan_R_PS colonies were transparent, slightly yellow, about 2 mm in diameter, with a convex surface and irregular edges.

[0029] Strain classification and identification: 16S rRNA gene was selected for amplification, Illumina Miseq PE 2000 platform was used, and the primers were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'. DNA library construction, high-throughput sequencing and raw data output were completed by Nanjing Qikexing Biological Technology Co., Ltd. The DNA sequences obtained by sequencing were uploaded to NCBI (https: / / www.ncbi.nlm.nih.gov / ) for BLAST comparison, and strain identification was performed according to the comparison results. The DNA sequences of the top ten strains with high genetic similarity to the sequenced strain were downloaded for constructing a phylogenetic tree. MEGA10.0 was used for phylogenetic analysis to construct a phylogenetic tree. According to the top 10 similar strains, a phylogenetic tree was constructed as shown in Figure 3 According to the results of the phylogenetic tree, it was shown that strain Pan_R_PS was most closely related to strains P. pnomenusa LMG 18087 and P. pnomenusa CCUG 38742. Combined with morphological characteristics and molecular biology, the strain was identified as P. pnomenusa , and named P. pnomenusa Pan_R_PS.

[0030] Example 2: Role of Pandora strain Pan_R_PS in promoting the growth of rice seedlings under low phosphorus stress Nipponbare rice was selected as the experimental variety, and three treatments were set up, namely (1) normal phosphorus level: phosphorus MS (1 / 2 conventional MS medium, in which phosphorus element is provided in the form of KH2PO4); (2) low phosphorus stress: no phosphorus MS + iron phosphate (1 / 2 MS-Fe medium); (3) low phosphorus stress with functional strain: Pan_R_PS (1 / 2 MS-Fe medium, inoculated with Pan_R_PS strain).

[0031] Rice seed germination: in the clean bench, select full and size consistent rice seeds, use 75% alcohol solution to sterilize rice seeds for 1 min, then use 2% sodium hypochlorite solution to soak rice seeds for 30 min, rinse with sterile ultrapure water for 6 times, wash the sodium hypochlorite solution attached to the surface of the seed, and then take the last washing liquid to coat on the LB solid medium. The complete sterilization of the seed surface is represented by the generation of sterile colonies. The sterilized rice seeds are soaked in sterile ultrapure water for 1 day. The soaked rice seeds are placed in the sterilized MS medium (1 / 2MS+3‰ sigma phytagel plant gel) and placed in a 22℃ light incubator for 4 days.

[0032] Solid culture plate preparation: (1) phosphorus MS treatment: add 30 mL of sterilized 1 / 2 MS medium in a 13 cm*13 cm square culture dish; (2) no phosphorus MS+iron phosphate treatment: add 1 / 2 MS-Fe medium in a 13*13 square culture dish; (3) low phosphorus stress added functional strain treatment: add 150 μL of OD 600 =1.0 bacterial suspension to 1 / 2 MS-Fe medium.

[0033] The germinated rice seeds are transplanted to the solid plate in the clean bench, and the rice seedlings with consistent growth are transplanted to the plate. Three rice seedlings are transplanted to each plate. After transplantation, the plate is placed in a light incubator for culture, and the culture conditions are 25℃, light for 16 h and dark for 8 h, and humidity of 50%. After 10 days of culture, the plate is rinsed with tap water, and the physiological indexes of the rice plants are measured.

[0034] The detection indexes include plant height, root length, total root length, root surface area and root architecture. The plant height and root length are measured with a tape measure, and the total root length, root surface area and root architecture are measured by a scanner (EPSON PERFECTION V700 PHOTO).

[0035] Results and analysis As shown in Figure 4 , under low phosphorus stress, the addition of Pandora Pan_R_PS has obvious differences in rape root architecture compared with the no phosphorus MS+iron phosphate control.

[0036] As shown in Figure 5 , under low phosphorus stress, the addition of Pandora Pan_R_PS can significantly increase the plant height of rice under low phosphorus stress compared with the no phosphorus MS+iron phosphate control. The average increase of rice plant height is 5.6 cm, reaching 15.9 cm, which has no significant difference with the plant height under normal phosphorus supply level.

[0037] As shown in Figure 6As shown, under low phosphorus stress, compared with the phosphorus-free MS + iron phosphate control, the addition of Pandora bacteria Pan_R_PS significantly increased the root length of rice under low phosphorus stress. The average root length of rice increased by 6.9 cm, reaching 8.6 cm, which was not significantly different from the root length under normal phosphorus supply levels.

[0038] like Figure 7 As shown, under low phosphorus stress, compared with the phosphorus-free MS + iron phosphate control, the addition of Pandora bacteria Pan_R_PS significantly increased the total root length of rice under low phosphorus stress. The average total root length of rice increased by 40.13 cm, reaching 43.45 cm, which was not significantly different from the total root length under normal phosphorus supply levels.

[0039] like Figure 8 As shown, under low phosphorus stress, compared with the phosphorus-free MS + iron phosphate control, the addition of Pandora bacteria Pan_R_PS significantly increased the total root area of ​​rice under low phosphorus stress, and the average total root length of rice increased by 4.07 cm, reaching 6.31 cm.

[0040] Example 3: Field effects of Pandora bacteria (Pan_R_PS) on promoting rice growth Nipponbare was selected as the experimental variety, and red soil was used as the test soil. Two treatments were set up in the field experiment: (1) a blank control, and (2) treatment with Pan_R_PS strain alone. Each treatment was replicated in six plots, with each plot measuring 10m². 2 .

[0041] Rice seedlings are raised in a nursery. Before transplanting them to the field, the roots are first treated with a bacterial solution for 6 hours, with a total bacterial count of approximately 1×10⁻⁶. 7 CFU / plant, and then transplant the rice seedlings into the community.

[0042] Rhizosphere soil samples were collected at the rice maturity stage to determine the available phosphorus content using the sodium bicarbonate extraction-ultraviolet spectrophotometry method. Rice yield was also measured simultaneously.

[0043] Results and Analysis like Figure 9 As shown, the available phosphorus content in the rhizosphere of rice treated with CK was 4.5 mg / kg. Compared with CK, the available phosphorus content in the rhizosphere of rice after inoculation with Pandora bacteria Pan_R_PS was 2.36 times that of CK, reaching 10.6 mg / kg (*** indicates significance test P <0.001).

[0044] like Figure 10 As shown, the phosphorus content of rice plants treated with CK was 2.5 g / kg. Compared with CK, the phosphorus content of rice plants after inoculation with Pandora bacteria Pan_R_PS was 1.44 times that of CK, reaching 3.6 mg / kg (*** indicates significance test P <0.001).

[0045] As Figure 11 shown, the selected soil in field test is relatively low in phosphorus and low in fertility, which is the main limiting factor of rice yield, and the rice yield is also low. The rice yield of CK treatment is only 453 kg / acre. Compared with CK, the rice yield is increased by 19.6% after inoculation with Pandora Pan_R_PS, reaching 542 kg / acre (*** indicates significant test P <0.001).

[0046] Therefore, the use of phosphate-solubilizing functional strains to develop growth-promoting preparations for rice field planting can significantly improve soil fertility, alleviate phosphorus deficiency stress of rice, and increase rice yield.

Claims

1. A rhizosphere phosphate-solubilizing Pandora bacterium ( Pandoraea sp. Pan_R_PS, Classification and Naming: Pandora ( Pandoraea sp. (), deposited at the China General Microbiological Culture Collection Center, China General Microbiological Culture Collection Center, on July 5, 2023, with accession number CGMCC NO.27802.

2. The bacterial agent prepared from Pan_R_PS, a rhizosphere phosphate-solubilizing bacterium, as described in claim 1.

3. The microbial agent according to claim 2, characterized in that... The bacterial agent is a bacterial solution, suspension, or powder prepared from rhizosphere phosphate-solubilizing Pandora bacteria (Pan_R_PS).

4. The microbial agent according to claim 3, characterized in that... The bacterial agent is prepared by the following method: (1) The activated strain Pan_R_PS was inoculated into LB liquid medium and cultured at 30℃ and 170 rpm until OD. 600 =1.0, which is the phosphate-solubilizing bacterial solution; (2) The bacterial suspension is obtained by centrifuging the bacterial solution, washing the bacterial cells twice with sterile water, and then resuspending the bacterial cells with an equal volume of sterile water. (3) The bacterial powder is obtained by centrifuging the bacterial liquid, washing the bacterial cells twice with sterile water, sealing the opening with sterile gauze, placing it in a 37°C constant temperature incubator to dry the bacterial cells completely, and grinding it into powder.

5. The application of the rhizosphere phosphate-solubilizing Pan_R_PS as described in claim 1 or the inoculum agent as described in any one of claims 2 to 4 in activating insoluble iron phosphate in red soil and increasing phosphorus availability.

6. The application of the rhizosphere phosphate-solubilizing Pan_R_PS as described in claim 1 or the bacterial agent as described in any one of claims 2 to 4 in activating insoluble iron phosphate in the rice rhizosphere, increasing the available phosphorus content in the rhizosphere soil, thereby alleviating phosphorus deficiency stress in rice and promoting rice growth.

7. The application according to claim 6, characterized in that, Promoting rice growth manifests as increasing rice plant height, root length, dry and fresh weight, plant phosphorus content, and rice yield.

8. The application according to claim 6, characterized in that, When transplanting rice seedlings, apply the inoculant at a total strain concentration of not less than 1×10⁻⁶. 7 After the CFU plants were soaked in water to remove roots, they were transplanted to the field.

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