Saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium and application thereof

Abstract

The invention is applicable to the technical field of microorganisms, and provides a saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium and application thereof.The phosphate-solubilizing algal-inhibiting bacterium is bacillus subtilis (Bacillus sp.) JL-10 and is preserved in the China General Microbiological Culture Collection Center on October 17, 2025, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.36241. The saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium has the advantages that the saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium can be applied to saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium can be applied to saline-alkaline tolerant phosphate-solubilizing algal-inhibiting bacterium; according to the invention, a novel bacterial strain with phosphorus-dissolving and algae-inhibiting functions is obtained through separation, the bacterial strain has relatively strong phosphorus-dissolving capacity, and the highest phosphorus-dissolving amount of the bacterial strain is as high as 112.65 mg / L measured by a molybdenum-antimony resistance colorimetric method; the compound has a remarkable inhibition effect on algae such as spirogyra, and the inhibition rate can reach 74.89% or above; meanwhile, the strain has strong colonization ability and good soil affinity in a saline-alkali environment, and can effectively promote the growth of rice in saline-alkali soil, which is found for the first time that the bacillus subtilis has the comprehensive effects of efficiently dissolving phosphorus, inhibiting the growth of spirogyra and promoting the growth of rice in the saline-alkali soil environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a salt and alkali-tolerant phosphorus-solubilizing algae-inhibiting bacterium and application thereof. BACKGROUND

[0002] About 70% of the soil in the world is under low phosphorus stress. At present, the conventional method to solve the problem of insufficient available phosphorus content in soil is to apply phosphorus fertilizer. However, after the phosphorus fertilizer enters the soil, it is often adsorbed and fixed, and cannot be directly absorbed and utilized by plants. Moreover, the concentration of phosphate gradually decreases from the surface or near-surface to the underground.

[0003] As an important reserve arable land resource, the soil of saline-alkali land has the characteristics of high salt and high alkali, which leads to extremely low bioavailability of phosphorus elements, and seriously restricts the growth of crops. The phosphorus-solubilizing bacteria can convert the insoluble phosphorus in the saline-alkali land into available phosphorus through the metabolites secreted in the process of growth and reproduction, or through synergistic action with other bacteria, so as to improve the dissolution degree of insoluble phosphate in the soil, facilitate the direct absorption and utilization of crops, and further improve the phosphorus nutrition status of crops. Therefore, the phosphorus-solubilizing bacteria used alone or in combination with phosphorus fertilizer can effectively improve the utilization rate of phosphorus elements in the soil of saline-alkali land by crops, reduce the application amount of phosphorus fertilizer, and reduce the pollution risk of soil secondary salinization caused by excessive application of phosphorus fertilizer. It has important significance for improving the yield of crops in saline-alkali land and promoting the sustainable development of agriculture in saline-alkali land.

[0004] In addition to the problem of soil phosphorus utilization, in aquatic or humid environments such as rice fields, water weeds such as water moss are also prone to breeding. When water moss proliferates in large quantities, it will compete with crops for nutrients, light and growth space, leading to inhibited growth of crops, and may also cause problems such as water body oxygen deficiency, affecting the balance of farmland ecological system.

[0005] At present, the application of phosphorus-solubilizing bacteria is mostly concentrated in conventional farmland to improve the yield of economic crops. The research on phosphorus-solubilizing bacteria for this special environment of saline-alkali land is relatively limited, and strains that can adapt to high salt and high alkali conditions in saline-alkali land and efficiently solubilize phosphorus are relatively scarce. More importantly, the functions of existing phosphorus-solubilizing bacteria are mostly limited to phosphorus solubilization and growth promotion, and strains with both phosphorus solubilization and algae inhibition functions are extremely rare. The application of phosphorus-solubilizing bacteria to inhibit the growth of water moss and other water weeds in rice fields and other scenarios is rarely reported, and it is difficult to simultaneously address the dual problems of phosphorus deficiency in saline-alkali land and water moss proliferation in rice fields. Therefore, screening strains that can adapt to the saline-alkali land environment, have high phosphorus solubilization capacity, and can inhibit the growth of water moss and other water weeds will provide important microbial resources for saline-alkali land rice planting, and has broad application prospects. SUMMARY

[0006] The purpose of the embodiments of the present application is to provide a salt and alkali-tolerant phosphorus-solubilizing algae-inhibiting bacterium, which aims to solve the problems raised in the background.

[0007] The application embodiment is implemented in the following manner: a salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium is Bacillus sp. JL-10, which was preserved in the China General Microbiological Culture Collection Center on October 17, 2025, at 1st Yard, No. 3, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC NO. 36241.

[0008] Another object of the application embodiment is to provide application of the salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium in preparation of a phosphorus-solubilizing preparation.

[0009] Another object of the application embodiment is to provide application of the salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium in preparation of an algae-inhibiting preparation.

[0010] Another object of the application embodiment is to provide application of the salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium in preparation of a phosphorus-solubilizing and algae-inhibiting preparation.

[0011] Another object of the application embodiment is to provide application of the salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium in preparation of a rice growth promoter.

[0012] Another object of the application embodiment is to provide a phosphorus-solubilizing preparation, which comprises the above-mentioned salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium.

[0013] Another object of the application embodiment is to provide an algae-inhibiting preparation, which comprises the above-mentioned salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium, and the water algae targeted by the preparation is Spirogyra.

[0014] Another object of the application embodiment is to provide a phosphorus-solubilizing and algae-inhibiting preparation, which comprises the above-mentioned salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium.

[0015] Another object of the application embodiment is to provide a rice growth promoter, which comprises the above-mentioned salt-tolerant and alkali-tolerant phosphorus-solubilizing and algae-inhibiting bacterium.

[0016] The application embodiment separates a new strain with both phosphorus-solubilizing and algae-inhibiting functions, which is identified as Bacillus sp. and has strong phosphorus-solubilizing capacity, with a maximum phosphorus-solubilizing amount of 112.65 mg / L determined by molybdenum-antimony resistance colorimetry; and has a significant inhibiting effect on water algae such as Spirogyra, with an inhibiting rate of more than 74.89%; meanwhile, the strain has strong colonization capacity in a saline-alkali environment and good soil affinity, and can effectively promote rice growth in a saline-alkali soil, which is also the first time to find that the Bacillus sp. has the comprehensive effects of efficient phosphorus solubilization, Spirogyra growth inhibition and rice growth promotion in a saline-alkali soil environment. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A colony morphology diagram of the strain provided for the application embodiment 1 cultured for 3 days; Figure 2A colony morphology chart of the strain provided for the present embodiment 1 cultured for 7 days; Figure 3 A standard curve diagram of the strain provided for the present embodiment 2 for phosphorus release; Figure 4 A control diagram of the inhibitory effect of the strain provided for the present embodiment 6 on waterweeds, A is an untreated rice field, and B is a treated rice field. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0019] The specific implementation of the present application is described in detail below in combination with specific embodiments.

[0020] Embodiment 1, screening and identification of strains: 1. Screening and isolation of strains: The target strain is isolated from the saline-alkali soil of a rice field, and the specific method is as follows: 10 g of soil sample is weighed and added to a 250 mL triangular flask containing 100 mL of sterile water, and cultured at 37℃ and 180 r / min for 30 min. 100 µL of the mixed and uniform soil suspension is sucked with a pipette and added to an EP tube containing 900 µL of sterile water to prepare a 10 -1 gradient bacterial suspension; the same method is used for subsequent dilutions to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 gradient bacterial suspensions; 100 µL of bacterial suspensions of different gradients are sucked and spread on a saline-alkali inorganic phosphorus solid medium (medium composition: glucose 10.0 g, ammonium sulfate 0.5 g, yeast extract powder 0.5 g, sodium chloride 5.0 g, potassium chloride 0.3 g, magnesium sulfate 0.3 g, ferrous sulfate 0.03 g, manganese sulfate 0.03 g, tricalcium phosphate 5.0 g, agar 20 g, pH adjusted to 8.0~9.0, deionized water added to 1000 mL, 121℃ sterilized for 20 min), 3 repeats for each gradient, and cultured in a 37℃ incubator for 3~7 days; The colony with obvious phosphorus-dissolving ring was selected, streak inoculated on LB solid medium (containing 5% sodium chloride, pH 8.5), and cultured at 37°C until the colony grew out, and then a single colony was picked and inoculated on the solid medium containing saline-alkaline inorganic phosphorus, and cultured at 37°C for 7 days. The purification and screening were repeated for 3-5 times, and finally a strain with stable growth and large phosphorus-dissolving ring was obtained, named JL-10, which was streaked and preserved on LB slant medium (containing 5% sodium chloride, pH 8.5) and stored in a refrigerator at 4°C for standby use.

[0021] 2. Strain identification: (1) Morphological identification: The strain JL-10 was inoculated on the solid medium containing saline-alkaline inorganic phosphorus and cultured at 37°C for 3 days and 7 days, and the colony morphology was observed. The colony cultured for 3 days was round, milky white, with neat edges, rough surface and no luster, as shown in FIG. 1; the colony cultured for 7 days was increased in volume, darkened in color, and satellite colonies appeared, as shown in FIG. 2. Figure 1 Figure 2 (2) Physiological and biochemical identification: The strain JL-10 was subjected to physiological and biochemical tests such as Gram staining, glucose fermentation and starch hydrolysis, and the results are shown in Table 1 (“+” indicates positive or has the relevant ability, and “-” indicates negative or does not have the relevant ability); Table 1 Physiological and biochemical identification results

[0022] (3) Molecular biology identification: The genomic DNA of the strain JL-10 was extracted, and PCR amplification was performed using bacterial 16S rDNA universal primers. The 16S rDNA sequence was obtained after sequencing the amplification product, as shown in SEQ NO. 1. The sequencing results were submitted to the NCBI database for BLAST comparison, and the MEGA software was used to construct a phylogenetic tree. Combined with morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain was identified as Bacillus sp. (4) Strain preservation: The strain was preserved at the China General Microbiological Culture Collection Center (CGMCC) on October 17, 2025, the address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation number is CGMCC NO. 36241.

[0023] Example 2, determination of the phosphorus-dissolving ability of the strain: 1. Verification by phosphorus-dissolving ring method: The seed liquid of the strain JL-10 was inoculated on the solid medium containing saline-alkaline inorganic phosphorus and cultured at 37°C for 7 days. The diameter (D) of the phosphorus-dissolving ring and the diameter (d) of the colony were measured, and the D / d value was calculated. The results are shown in Table 2: Table 2 Variation of the diameters (D) of the phosphorus-dissolving ring, the diameters (d) of the colonies and the D / d values with culture time ​​

[0024] 2. Molybdenum-antimony anti-colorimetric method for determining the amount of phosphorus: (1) Standard curve preparation: 0, 2, 4, 6, 8, 10, and 12 mL of 5 mg / L phosphorus standard solution were taken into 50 mL volumetric flasks, diluted with water to 20 mL, 2 drops of 2, 4-dinitrophenol indicator was added, 5 mL of molybdenum-antimony anti-color developing agent was added, shaken well, and diluted to 50 mL. The phosphorus standard series solution of 0-1.2 mg / L was obtained. Colorimetric analysis was performed at a wavelength of 700 nm, and the standard curve was drawn with the phosphorus concentration as the horizontal coordinate and the absorbance as the vertical coordinate. The regression equation was y=0.0405x-0.0398 (R 2 =0.9996), and the specific standard curve is shown in Figure 3 ; (2) Determination of phosphorus release amount: 1% of the strain JL-10 seed liquid was inoculated into the saline-alkaline NBRIP liquid medium (initial phosphorus content of 4.92 mg / L), and cultured at 37°C and 180 r / min for 7 days. The effective phosphorus content was determined every day, and the results are shown in Table 3: Table 3 Determination results of phosphorus release amount

[0025] Example 3, determination of the salt-alkali tolerance of the strain: Different salt concentrations (0.5%, 1%, 3%, 5%, 7%, and 10% NaCl) and different pH values (7.0, 8.0, 9.0, 10.0, and 11.0) of LB liquid medium were used, and the strain JL-10 seed liquid (inoculation amount 1%) was inoculated, and the OD600 value was determined after 24 h of culture at 37°C and 180 r / min. The results are shown in Table 4: Table 4 Growth of the strain under different salt concentrations and pH conditions (OD600)

[0026] The results show that the strain JL-10 can grow well in the range of NaCl concentration ≤7% and pH value 8.0-10.0, indicating that it has strong salt-alkali tolerance and can adapt to the saline-alkaline environment.

[0027] Example 4, determination of the algae inhibition ability of the strain: The log phase of the water moss algae was taken and inoculated into BG11 medium, and sterile fermentation liquid of the strain JL-10 (final concentration 10%) was added. The water moss culture liquid with the same amount of sterile medium was used as a control, and the water moss was cultured at 25°C for 7 days with 12 h light / 12 h darkness. The fresh weight and inhibition rate of water moss were determined, and the results are shown in Table 5: Table 5 Determination results of the algae inhibition ability of the strain

[0028] Example 5, application effect of the strain in saline-alkali soil rice pot experiment: The saline-alkali soil (containing salt 3.5%, pH 9.0) was selected and loaded into a flowerpot, and rice seedlings were planted. A control group (irrigated with sterile water) and a treatment group (irrigated with strain JL-10 bacterial suspension, concentration 10 8 CFU / mL, 10 mL per pot) were set up, each group had 3 repeats, and related indexes were measured after 30 days of culture. The results are shown in Table 6: Table 6: Results of saline-alkali soil rice simulation pot experiment

[0029] The results show that compared with the control group, the soil available phosphorus content of the treatment group increased by 173.43%; the dry weight of rice increased by 75.00%; and the coverage of water moss decreased by 72.31%, indicating that the strain JL-10 can effectively improve the soil fertility of saline-alkali land, promote the growth of rice, and inhibit the spread of water moss.

[0030] Comprehensive examples 1-5 show that the strain JL-10 provided by the embodiments of the present application has the functions of high-efficiency phosphorus solubilization, salt-alkali tolerance, algal inhibition and growth promotion, and has important application value in saline-alkali soil rice fields.

[0031] Example 6, influence of the strain on the growth of rice in saline-alkali land and the inhibitory effect on water moss: To verify the promotion effect of the strain JL-10 on the growth of rice in the actual saline-alkali land rice field environment, a field experiment was carried out in a typical saline-alkali land rice planting area, focusing on the influence on the height of rice and the development of root system. The experimental design is as follows: The test plot is located in the saline-alkali land rice planting area in the west of Jilin Province (soil salt content 3.2~4.0%, pH 8.5~9.2, initial available phosphorus content 9.1 mg / kg), with a total area of 1 mu, divided into a control group and a treatment group, each group has 3 plots (each plot area is about 167 m 2 ), 50 cm isolation ridge is set between plots (to prevent water and fertilizer from interpenetrating), the test rice variety is the local main saline-alkali tolerant variety "Baijing No. 1", transplanted on May 20, 2025, with a transplanting density of 30 cm x 13 cm (3 plants per hole); The treatment group was sprayed with strain JL-10 bacterial suspension (concentration 10 8 CFU / mL) one day before transplanting, with a dosage of 100 mL / m 2 (diluted with water 50 times and uniformly sprayed on the transplanting hole); the control group was sprayed with the same amount of sterile water; other field management (irrigation, weeding, disease and pest control, etc.) was carried out according to the local conventional measures, and the two groups were consistent; At the grain filling stage of rice (60 days after transplanting), 10 rice plants were randomly selected from each plot, and the following indexes were measured: Plant height: vertical distance from the ground surface to the top of the ear (without awn); Effective tiller number: the number of tillers per plant with full grains; Maximum root width: the maximum diameter of the lateral distribution of the root system; Root dry weight per plant: after washing the roots, the roots were blanched at 105°C for 30 minutes, dried at 80°C to a constant weight, and then weighed; The results are shown in Table 7: Table 7: Results of rice field test in saline-alkali soil

[0032] The results show that the plant height, effective tiller number, root distribution range, and root dry weight of the treatment group rice are significantly higher than those of the control group, with the effective tiller number increasing by 67.3% and the root dry weight per plant increasing by more than 70%. This indicates that under actual field planting conditions, strain JL-10 can significantly improve the nutrient absorption capacity and growth potential of rice by improving root growth (expanding the absorption range and increasing the biomass) and promoting vertical plant growth, further verifying its growth-promoting effect on saline-alkali soil rice.

[0033] To verify the algal inhibition effect of strain JL-10 in the actual rice field environment, the growth of water moss was observed simultaneously in the field test provided in Example 6, and the results are shown in Table 8. Figure 4 As shown in Table 8, the water moss coverage rate of the treatment group is less than 5%, while about 60% of the area in the control group rice field is covered by water moss. This result fully demonstrates that the inhibition effect of strain JL-10 on water moss in the actual field environment is stable and significant, further confirming that it can effectively control the growth of water moss in rice fields.

[0034] In summary, the strain JL-10 isolated in the examples of the present application has the characteristics of phosphorus solubilization, improvement of soil fertility in saline-alkali soil, promotion of growth of saline-alkali soil rice, and inhibition of growth of water moss. When used, an effective amount of the strain, a bacterial preparation thereof, or a fertilizer containing the strain can be applied to the soil or water body of a saline-alkali soil rice field. When used, the strain can be prepared into a bacterial preparation alone according to the need, or used in combination with other microorganisms having a synergistic effect, or used together with other fertilizers / fertilizer additives / adjuvants, etc. that have no inhibitory effect on the growth and metabolism of the strain. The strain can be used in the form of a solution, a freeze-dried powder, a granule, etc.

[0035] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A salt-tolerant and alkaline-tolerant phosphorus-solubilizing anti-algal bacterium, characterized in that, The phosphorus-dephosphorizing and algae-inhibiting bacteria is Bacillus sp. JL-10, which was preserved in China General Microbiological Culture Collection Center on October 17, 2025, at No. 1, Xibahe Road, Haidian District, Beijing, with a preservation number of CGMCC NO. 36241.

2. Use of the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1 in the preparation of a phosphorus-dephosphorizing preparation.

3. Use of the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1 in the preparation of an algae-inhibiting preparation.

4. The use of the salt-tolerant and alkaline-tolerant phosphorus-degrading and algae-inhibiting bacteria according to claim 3 in the preparation of an algae-inhibiting preparation, characterized in that, The algae targeted by the algae-inhibiting preparation is water moss.

5. Use of the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1 in the preparation of a phosphorus-dephosphorizing and algae-inhibiting preparation.

6. Use of the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1 in the preparation of a rice growth promoter.

7. A phosphorus-unblocking preparation, characterized by The preparation comprises the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1.

8. An algaecidal formulation, characterized by comprising: The preparation comprises the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1, and the algae targeted by the preparation is water moss.

9. A phosphorus-releasing and algae-inhibiting preparation, characterized in that, The preparation comprises the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1.

10. A rice growth accelerator, characterized by, The promoter comprises the salt-tolerant and alkali-tolerant phosphorus-dephosphorizing and algae-inhibiting bacteria in claim 1.

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