Phosphorus-solubilizing bacteria screened from straw biochar and eurytopic application of phosphate-solubilizing bacteria

Abstract

The invention discloses a phosphate solubilizing bacterium with eurytopic characteristic and application thereof, the bacterium is separated from corn straw biochar in which rice is continuously planted for 3 years, is identified as Pseudomonas paaversuta and is named as MPP2401, the preservation unit of the bacterium is China Center for Type Culture Collection (CCTCC), the address is Wuhan University, Wuchang District, Wuhan, Hubei province, the preservation date is November 6, 2024, and the preservation number is CCTCC NO: M20242439. The strain can keep stable phosphate solubilizing capacity under the environmental conditions of 15-30 DEG C, pH of 7-10 and NaCl 0-0.8 mol.L <-1 >, and still has functional phosphate solubilizing capacity even under the condition of extreme low temperature of 5 DEG C, the composite stress of pH of 7-8 and NaCl 0-0.8 mol.L <-1 > and the composite stress of pH of 9-10 and NaCl 0-0.6 mol.L <-1 >. Meanwhile, the bacterium has the broad-spectrum antibacterial characteristic, and the antibacterial rate on fusarium graminearum, fusarium verticillioides and magnaporthe oryzae reaches 41.0-77.6%. Due to the unique wide environment adaptability and the phosphate solubilizing-bacteriostasis dual-function synergistic characteristic, a microbial germplasm resource with production-ecology dual values is provided for biochar bacterial fertilizer development, saline-alkali phosphorus activation and biological prevention and control.

Description

Technical Field

[0001] The present invention relates to the field of applied microbiology technology, and specifically relates to screening phosphate-solubilizing bacteria from straw biochar and the application of the bacteria in wide-temperature and saline-alkali-tolerant environments and their biological control potential. Background Art

[0002] In agricultural ecosystems, most of the soil phosphorus is in an inert state, and only a small part exists in a soluble form, restricting the soil-plant nutrient cycle, especially in saline-alkali soil environments. Phosphate-solubilizing bacteria can convert insoluble phosphates into forms that can be directly utilized by organisms, which is regarded as an effective way to improve the bioaccessibility of recalcitrant phosphorus in the soil. Among them, some phosphate-solubilizing bacteria also exhibit biological control potential, which can reduce and inhibit the activity of pathogenic bacteria, and improve the nutritional status and health level of plants. This dual function further broadens the application prospect of phosphate-solubilizing bacteria in agricultural production.

[0003] Existing studies have shown that problems such as low survival rate and limited function are likely to occur when phosphate-solubilizing bacteria are used alone to improve the soil. Therefore, the method of combining phosphate-solubilizing bacteria with protective carriers such as biochar has been applied in soil improvement. However, it is difficult for the existing technology to solve the compatibility problem between exogenous phosphate-solubilizing bacteria and biochar, which is a key factor restricting the biological activity of phosphate-solubilizing bacteria. To address this problem, this patent innovatively proposes to screen dominant phosphate-solubilizing bacteria from the corn straw biochar of rice continuously planted for 3 years. After long-term domestication in the biochar microenvironment, the strain forms a high degree of compatibility with it, can well adapt to the alkaline environment created by biochar, and fully utilizes its porous structure to achieve stable loading and continuous proliferation, extend its metabolic activity cycle, and enhance its survival ability and phosphate-solubilizing efficiency in the soil. There are no relevant literature and patent reports on the method of screening strains from biochar.

[0004] In addition, it can be known from the publicly available information that current research mainly focuses on the saline-alkali adaptability of phosphate-solubilizing bacteria, but often limits the evaluation of phosphate-solubilizing ability at a single temperature. In real natural environments (such as Northeast China), the temperature fluctuates greatly and the soil properties vary greatly, which may significantly affect the functional stability of the strain and lead to poor actual application effects. Therefore, phosphate-solubilizing bacteria with wide environmental condition (including temperature, pH value, salinity, etc.) adaptability have more important value in actual production applications. Chinese patents 202210777205.5 and 202210190459.9 disclose that under the conditions of 15 °C and pH 8, Mixta theicola ) and Acinetobacter calcoaceticus Acinetobacter calcoaceticus -1 respectively achieve the highest phosphate-solubilizing ability of 384.4 mg·L -1 and 444.7 mg·L -1, but its phosphate solubilization ability is obviously inferior to that of the strain in this patent. At the same time, the existing Chinese patent only emphasizes the phosphate solubilization effect based on constant temperature conditions (15 ℃), which cannot fully reflect the application performance of phosphate solubilizing bacteria under real environmental conditions. In order to be closer to practical applications, it is urgent to screen strains that can play a high-performance phosphate solubilization role under the coupling conditions of a wide temperature range (5-30 ℃) and a wide range of salt and alkali, which has application value in the Northeast (large temperature difference, high salt and alkali) region. Summary of the invention

[0005] In order to solve the compatibility problem between phosphate-solubilizing functional strains and biochar, the present invention screened phosphate-solubilizing bacteria from corn straw biochar grown with rice for three consecutive years. The saline-alkali microenvironment of biochar itself was used as the selection pressure to screen phosphate-solubilizing bacteria that are adapted to the physical and chemical properties of biochar.

[0006] In order to solve the problem of temperature adaptability of phosphate-solubilizing bacteria, the present invention further provides a method for enriching and separating phosphate-solubilizing bacteria, which is characterized in that the strain is separated from pure corn straw biochar grown from rice for three consecutive years, and the specific steps are as follows:

[0007] (1) Acclimation and enrichment: 10 g of biochar was placed in 90 mL of LB culture medium at 30 °C and 150-220 r / min. -1 Shake for 3-5 days; inoculate 1-5% of the culture medium into LB culture medium and shake and culture again for 3-5 days under the same conditions. Then lower the temperature to 15 and 5 °C and repeat the above steps to ensure that it can grow and reproduce normally at different temperatures.

[0008] (2) Isolation and purification: The domesticated and enriched target bacterial solution is gradually diluted 10 -3 -10 -8 Afterwards, 0.1 mL of each was evenly spread on inorganic phosphorus solid culture medium and inverted and cultured at 30 °C for 3-7 days. The single colonies with good growth were qualitatively and quantitatively analyzed to obtain the strain MPP2401 with strong phosphate solubilization ability.

[0009] The phosphate-solubilizing bacteria provided by the present invention are Pseudomonas paraversuta MPP2401, strain deposited in China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, deposit date: November 6, 2024, deposit number: CCTCC NO: M20242439.

[0010] The phosphate-solubilizing bacteria MPP2401 provided by the present invention has the wide adaptability of efficiently converting insoluble phosphate into soluble phosphate under different temperatures and saline-alkali environments. The strain MPP2401 can convert insoluble phosphate into soluble phosphate under the conditions of pH 7-10 and NaCl 0-0.8 mol·L -1All showed significant soluble phosphorus release ability in the environment. Specifically, when the culture temperature was 30 °C, its optimal phosphorus solubilization conditions were pH 7 and NaCl 0.2 mol·L -1 , and the soluble phosphorus release amount reached 594.40 mg·L -1 ; at 15 °C, the optimal parameters were adjusted to pH 7 and NaCl 0.4 mol·L -1 , and the release amount was 536.06 mg·L -1 . This strain still maintained significant activity in the extremely low temperature (5 °C) environment, and the highest phosphorus solubilization amount reached 496.98 mg·L -1 , and its phosphorus solubilization efficiency was outstanding in the concentration ranges of pH 7-8, NaCl 0-0.8 mol·L -1 and pH 9-10, NaCl 0-0.6 mol·L -1 , showing the metabolic adaptation ability to wide-range temperature and environmental stress.

[0011] The phosphorus-solubilizing bacterium MPP2401 provided by the present invention has both disease-resistant functions. The diseases are Fusarium graminearum, Verticillium tricorpus, and Magnaporthe oryzae, and its antibacterial rate is 41.0-77.6%.

[0012] The Pseudomonas paraversuta MPP2401 of the present invention was screened from pure corn straw biochar with three consecutive years of rice planting. This strain not only provides an ideal candidate for biochar-loaded phosphorus-solubilizing bacteria, effectively solving key problems such as strain colonization and survival time, but also has significant disease resistance, can effectively prevent crop diseases and promote the healthy growth of crops. In addition, the phosphorus-solubilizing bacterium MPP2401 has stable and efficient phosphorus-solubilizing performance under different temperature and saline-alkali conditions, filling the blank of the limited application of traditional phosphorus-solubilizing bacteria in the face of complex and changeable environments, and enhancing its applicability in various soil environments. The present invention provides a new method for solving the problem of the compatibility between phosphorus-solubilizing bacteria and biochar, a new strain with wide temperature and saline-alkali adaptability for the development of biochar-based fertilizers, saline-alkali soil improvement, and efficient utilization of phosphorus, etc., and has practical application value. Description of the Drawings

[0013] Figure 1 is Pseudomonas paraversuta the phosphorus solubilization circle diagram of MPP2401

[0014] Figure 2 is Pseudomonas paraversuta the streak plate diagram of MPP2401

[0015] Figure 3 is Pseudomonas paraversuta the morphological identification diagram of MPP2401; A spore staining, B Gram staining

[0016] Figure 4 Yes Pseudomonas paraversuta Phosphate-solubilizing effect of MPP2401 under different temperature conditions

[0017] Figure 5 Yes Pseudomonas paraversuta Schematic diagram of the phylogenetic tree of MPP2401

[0018] Figure 6 It is under the condition of 30 °C, Pseudomonas paraversuta Dynamic characteristics of the phosphate-solubilizing ability of MPP2401 with the change of saline-alkali concentration gradient

[0019] Figure 7 It is under the condition of 15 °C, Pseudomonas paraversuta Dynamic characteristics of the phosphate-solubilizing ability of MPP2401 with the change of saline-alkali concentration gradient

[0020] Figure 8 It is under the condition of 5 °C, Pseudomonas paraversuta Dynamic characteristics of the phosphate-solubilizing ability of MPP2401 with the change of saline-alkali concentration gradient

[0021] Figure 9 Yes Pseudomonas paraversuta Inhibitory effect of MPP2401 on pathogenic bacteria of diseases; A Fusarium graminearum, B Verticillium dahliae, C Magnaporthe oryzae Specific implementation manner

[0022] The present invention will be further described in detail below through embodiments in conjunction with the accompanying drawings, but the present invention is not limited to the following listed embodiments.

[0023] Example 1: Enrichment and isolation of phosphate-solubilizing bacteria with wide adaptability to temperature

[0024] The phosphate-solubilizing bacteria provided by the present invention are Pseudomonas paraversuta MPP2401, the strain is preserved in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, preservation date: November 6, 2024, preservation number: CCTCC NO: M20242439.

[0025] The present invention provides Pseudomonas paraversuta A cultivation method of MPP2401 phosphate-solubilizing bacteria, including the enrichment and isolation of the bacterial strain, characterized in that the strain is isolated from pure corn straw biochar with continuous rice cultivation for 3 years, and the specific steps are as follows:

[0026] (1) Culture medium: (a) LB medium (g·L -1 ) : Tryptone 10 g, NaCl 10 g, yeast extract 5 g, agar 18 - 20 g (not added to the liquid medium), distilled water 1 L, pH 7.0 - 7.2, sterilized at 121 °C for 20 min; (b) Inorganic phosphorus medium (g·L -1): 10 g of glucose, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.5 g of (NH4)2SO4, 0.03 g of MnSO4·4H2O, 0.03 g of FeSO4·7H2O, 5 g of Ca3(PO4)2, 18 - 20 g of agar (not added to liquid medium), 1 L of distilled water, pH 7.0 - 7.5, sterilized at 115 °C for 30 min.

[0027] (2) Acclimation and enrichment: Take 10 g of biochar and place it in 90 mL of LB culture medium, shake it at 30 °C and 180 r·min -1 for 3 d; inoculate it into the LB culture medium at an inoculation amount of 5%, and shake and culture it again under the same conditions for 3 d. Subsequently, lower the temperature to 15 and 5 °C respectively and repeat the above operations.

[0028] (3) Isolation and purification: Gradually dilute the acclimated and enriched target bacterial liquid by 10 -3 -10 -8 times. Then, take 0.1 mL of each dilution and spread it evenly on the inorganic phosphorus solid medium, and incubate it upside down at 30 °C for 3 - 7 d. Conduct qualitative analysis on the single colonies with better growth, measure the colony diameter (d) and the diameter of the transparent circle (D), calculate the phosphorus solubilization coefficient (D / d), and preliminarily judge the phosphorus solubilization ability of the strain ( Figure 1 , Table 1).

[0029] Strain Colony diameter (d) / cm Diameter of phosphorus solubilizing circle (D) / cm Phosphorus solubilizing index D / d MPP2401 0.71±0.07 1.54±0.08 2.18±0.10

[0030] (4) Observation of strain morphology: On the LB plate, the colonies are yellow, opaque, with a smooth and raised surface and neat edges, as Figure 2 shown. Staining analysis reveals that it is a Gram-negative bacillus without spores, as Figure 3 shown.

[0031] (5) Quantitative determination of the strain: After activating the strain preserved in the cryotube, inoculate it into the inorganic phosphorus culture medium at an inoculation amount of 5%. Under three temperature gradients of 30, 15, and 5 °C, shake and culture it at a rotation speed of 180 r·min -1 for 7 d. Take 1 mL of the culture medium every day and place it in a centrifuge tube, centrifuge it at 4000 r·min -1 for 20 min, take the supernatant, and use the molybdenum antimony anti-colorimetric method to determine the soluble phosphorus content. Use the inorganic phosphorus culture medium without inoculating the phosphorus-solubilizing bacteria as the blank control (CK). The results show that ( Figure 4 ) Pseudomonas paraversutaThe phosphorus-solubilizing rate of MPP2401 is different under different temperature conditions. At 30 and 15 °C, the strain MPP2401 showed high growth activity at the initial stage of cultivation (the 1st day), and its phosphorus-solubilizing effect was significant. As the cultivation time extended to 3 days, the phosphorus-solubilizing efficiency showed a downward trend. Under the extreme temperature condition of 5 °C, the strain MPP2401 grew relatively slowly compared to other temperatures, but still had functional phosphorus-solubilizing ability.

[0032] (6) Strain identification: The strain MPP2401 was amplified with primers 27F and 1492R and its 16S rDNA sequence was obtained, with a sequence length of 1094 bp. The specific sequence is shown as Seq ID No.1. By comparing with the sequences in the Blast and GenBank databases, the result showed that the homology with Pseudomonas paraversuta was 99.93%. The isolated strain was Pseudomonas paraversuta , named MPP2401, as shown in Figure 5 .

[0033] Example 2: Pseudomonas paraversuta Application of MPP2401 in phosphorus solubilization under different temperature and saline-alkali conditions

[0034] (1) Culture medium: On the basic inorganic phosphorus liquid medium, gradients of pH 7, 8, 9, and 10 were set, and at each pH gradient, the NaCl concentration was set to 0, 0.2, 0.4, 0.6, 0.8 mol·L -1 , forming 20 different saline-alkali gradient inorganic phosphorus culture solutions.

[0035] (2) Determination of phosphorus-solubilizing ability: Take the activated and enriched bacterial solution of the above example, inoculate it at 5% into the inorganic phosphorus culture solution, and under three temperature gradients of 30, 15, and 5 °C, shake culture at a rotation speed of 180 r·min -1 for 3 days, and use the molybdenum antimony anti-colorimetric method to determine the phosphorus-solubilizing amount of the strain.

[0036] (3) Determination results: The strain Pseudomonas paraversuta MPP2401 showed significant phosphorus-solubilizing ability under different temperature and saline-alkali conditions. After shaking culture at 30 °C and 180 r·min -1 for 3 days, the soluble phosphorus content in the environment of pH 7 - 10 and NaCl 0 - 0.8 mol·L -1 reached 113.10 - 594.40 mg·L -1 , among which the phosphorus-solubilizing amount was the highest at pH 7 and NaCl 0.2 mol·L -1 ( Figure 6); At 15 °C, the phosphorus solubilization amount of strain MPP2401 was 136.84 - 536.06 mg·L -1 , and the optimal conditions were pH 7 and NaCl 0.4 mol·L -1 ( Figure 7 ); At 5 °C, the highest phosphorus solubilization amount under normal conditions was 496.98 mg·L -1 , among which, in the range of pH 7 - 8 and NaCl 0 - 0.8 mol·L -1 , and in the range of pH 9 - 10 and NaCl 0 - 0.6 mol·L -1 environment, insoluble phosphorus ( Figure 8 ).

[0037] Example 3: Pseudomonas paraversuta Inhibitory effect test of MPP2401 on three pathogenic bacteria

[0038] (1) Tested pathogenic bacteria: Fusarium graminearum, Verticillium tricorpus, Magnaporthe oryzae

[0039] (2) Conduct a plate confrontation test of Pseudomonas paraversuta MPP2401 and the three pathogenic bacteria. Take 0.1 mL of the bacterial solution and spread it evenly on the PDA plate. Use a sterilized punch (6 mm) to punch holes in the PDA plate covered with the pathogenic bacteria. Use a sterile toothpick to pick up the bacterial cake and invert it with the mycelium facing down on the PDA plate coated with the bacterial solution and incubate it for 3 - 10 d, and set a control group (a plate without inoculating the strain). Use the cross - intersection method to measure the antibacterial effect ( Figure 9 ).

[0040] Antibacterial rate = (growth amount of the control group - growth amount of the treatment group) / growth amount of the control group × 100%

[0041] Results (Table 2) Pseudomonas paraversuta The antibacterial range of MPP2401 against the three pathogenic bacteria was 41.0 - 77.6%. It shows that this bacterium has the potential to control Fusarium graminearum, Verticillium tricorpus, and Magnaporthe oryzae.

[0042] Table 2 Pseudomonas paraversuta Antibacterial rate of MPP2401 against three pathogenic bacteria

[0043] Pathogen type Fusarium graminearum Fusarium verticillioides Magnaporthe oryzae Inhibitory rate 56.0±1.4 41.0±2.6* 77.6±3.7*

[0044] As can be seen from the above examples, the present invention screens out Pseudomonas paraversutaMPP2401 has strong environmental adaptability, can maintain its activity, grow stably and maintain high phosphate-solubilizing activity within a wide range of temperature, pH value and NaCl concentration. In addition, this strain also exhibits certain antibacterial functions and can effectively inhibit the growth of some pathogenic bacteria. In summary, this strain can be widely applied in the fields of biochar bacterial fertilizer development, agricultural sustainable development and biological control, etc.

Claims

1. Screening of a phosphate-solubilizing bacterium, characterized in that the strain from the corn straw biochar separated from the continuously planted rice for 3 years.

2. A method for screening phosphate-solubilizing bacteria with adaptability to the biochar microenvironment, wide temperature and salinity adaptability according to claim 1. It is characterized in that: (1)The basic culture medium formula is as follows: 10 g of glucose, 0.3 g of NaCl, 0.3 g of KCl, 0.3 g of MgSO4·7H2O, 0.5 g of (NH4)2SO4, 0.03 g of MnSO4·4H2O, 0.03 g of FeSO4·7H2O, 5 g of Ca3(PO4)2, 1 L of distilled water, and the pH is 7.0 - 7.

5. (2)The fermentation broth described in (1) is inoculated into the basic culture medium with different saline-alkali gradients, and the pH is set at gradients of 7, 8, 9, and 10, and the NaCl concentration is set at 0, 0.2, 0.4, 0.6, 0.8 mol·L -1 , to form inorganic phosphorus culture media with different saline-alkali gradients. (3)The inorganic phosphorus culture media with different saline-alkali gradients described in (2) are cultured at 30 °C, 15 °C, and 5 °C.

3. A phosphate-solubilizing bacterium screened according to claims 1 and 2 Pseudomonas paraversuta MPP2401, depositary institution: China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuchang District, Wuhan City, Hubei Province, deposit date: November 6, 2024, deposit number: CCTCC NO: M20242439.

4. A fermentation method of phosphate-solubilizing bacterium MPP2401 according to claim 3, characterized in that, After activating the phosphate-solubilizing bacterium MPP2401, inoculate it into LB culture medium and ferment and culture at 5 - 30 °C and 150 - 220 r·min -1 for 24 - 36 h to obtain a fermentation broth.

5. The application of the fermentation broth according to claim 4 for phosphorus solubilization under wide temperature and saline-alkali conditions; characterized in that, Convert insoluble phosphorus into soluble phosphorus in the range of 5 °C - 30 °C and pH 7 - 10.

6. Application of the fermentation broth according to claim 4 in preventing and controlling corn and rice diseases; the diseases include Fusarium graminearum, Fusarium verticillioides, and Magnaporthe oryzae.

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