A strain of Marseilles and its application

Abstract

The present invention provides a strain of Massilia sp. LXY-5 and its use. The strain is classified as Massilia sp. LXY-5 and deposited at the General Microbiology Center of the China Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is: October 15, 2024, and the deposit number is: CGMCC No. 1.62472. The present invention also provides the use of the strain in microbial phosphate solubilization and its use in arid areas.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology, relates to plant growth promotion technology and application of microbial fertilizer in production, and specifically relates to a Marseille mushroom and application thereof. Background Art

[0002] Phosphorus plays a vital role in plant growth and development, influencing a plant's growth potential, metabolic activity, and resilience to adverse conditions. As a core component of adenosine triphosphate (ATP), phosphorus ensures the efficient storage and transfer of energy within plant cells, providing the necessary power source for anabolism and growth. Phosphorus also participates in photosynthesis, helping plants efficiently convert light energy into chemical energy, accelerating the pace of plant growth and development. It also positively impacts flowering and fruiting, improving flower quality and fruit yield. Phosphorus also promotes robust root growth and branching, enhancing a plant's ability to absorb water and nutrients. By synergizing with nutrients like nitrogen and potassium, it improves a plant's overall nutrient utilization efficiency. Finally, phosphorus helps strengthen a plant's resilience to adverse environmental conditions such as drought, pests and diseases, and enhances its competitiveness.

[0003] At the ecological and environmental level, microorganisms play an indispensable role as key regulators of the ecosystem. Among them, phosphate-solubilizing bacteria, with their unique ability to dissolve phosphorus, have become an important microbial group for improving soil phosphorus conditions. They convert insoluble phosphorus compounds in the soil (such as calcium phosphate in phosphate rock) that were originally difficult for plants to directly utilize into a form that can be absorbed by plants by releasing metabolites such as organic acids and enzymes, effectively increasing the effective phosphorus content of the soil. This process not only improves the biological effectiveness of phosphorus, but also promotes the optimization of soil structure and enhances soil microbial activity, thereby comprehensively improving the fertility level of the soil. The symbiotic relationship between phosphate-solubilizing bacteria and plant roots further promotes the absorption of other nutrients by plants and optimizes the nutrient utilization structure.

[0004] Phosphate-solubilizing bacteria (PSBs), a key microbial resource in the application of microbial fertilizers, have the ability to convert insoluble phosphorus in the soil into available phosphorus for plant use, playing a vital role in improving soil fertility and promoting crop growth. However, existing PSBs in microbial fertilizers also face challenges, including difficulty in screening, limited identification techniques, poor environmental adaptability, and unstable application results.

[0005] Therefore, developing an environmentally friendly phosphate-solubilizing bacterium that significantly reduces dependence on chemical fertilizers has become an urgent problem to be solved. Summary of the Invention

[0006] In order to solve the above problems, the object of the present invention is to provide a strain of Massillaria thunbergii having phosphate-solubilizing effect.

[0007] Another object of the present invention is to provide applications of Massillaria thunbergii.

[0008] In order to achieve the above-mentioned object, the present invention provides a strain of Massiliasp., which is classified and named as Massiliasp.LXY-5, and the preservation unit is: General Microbiology Center of China Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is: October 15, 2024, and the preservation number is: CGMCC No.1.62472.

[0009] The present invention also provides application of the above-mentioned Massillaria thaliana in microbial phosphate solubilization.

[0010] The present invention also provides application of the above-mentioned Massillaria thunbergii in arid areas.

[0011] The Marseille bacteria provided by the present invention can convert insoluble phosphates into dissolved phosphate ions that can be efficiently absorbed and utilized by plants under drought conditions, providing a large amount of nutrients for crop production, and has great potential in producing high-efficiency bio-organic fertilizers.

[0012] The beneficial effects of the present invention are:

[0013] The present invention provides a strain of Massillaria and its application. The Massillaria has a phosphorus-dissolving effect, which can not only increase the content of soluble phosphorus in the soil, but also promote the growth and development of crops, thereby improving the yield and quality of crops. At the same time, it also has good drought resistance and can play an environmental repair function in arid environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram showing the growth status of the Marseilles strain LXY-5 on a culture medium provided by the present invention.

[0015] Figure 2 The present invention provides a phylogenetic tree of the species LXY-5 based on 16S rDNA.

[0016] Figure 3 The OD value of the strain LXY-5 provided by the present invention in LB medium containing different concentrations of PEG-6000 is 600 Change graph.

[0017] Figure 4 This is a graph showing changes in the amount of solubilized phosphorus of the Marseille strain LXY-5 provided by the present invention in a Montkina inorganic phosphorus culture medium containing different concentrations of PEG-6000. DETAILED DESCRIPTION

[0018] The embodiments of the present invention will be described in detail and comprehensively below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0019] Material

[0020] 1. DNA extraction kit was purchased from Beijing Jinsha Biotechnology Co., Ltd., product number: DE703-50.

[0021] Prepare culture medium:

[0022] The formula for 1 L of Montkina Inorganic Phosphate Liquid Medium is as follows: 10.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast extract powder, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, and 5.0 g tricalcium phosphate. After thorough shaking, adjust the pH to 7 with NaOH or HCl. Sterilize by autoclaving at 121°C for 20 min, and cool to obtain the Montkina Inorganic Phosphate Liquid Medium.

[0023] The Montkina inorganic phosphate culture medium plate is prepared by adding 15 g of agar powder to the Montkina inorganic phosphate liquid culture medium, adjusting the pH to 7, sterilizing the medium at 121° C. and high-pressure sterilization for 20 minutes, then pouring the medium into a sterile culture dish while hot, and cooling the medium to obtain the Montkina inorganic phosphate culture medium plate.

[0024] The Montkina inorganic phosphate liquid medium and the Montkina inorganic phosphate medium plate are used to separate and detect the ability of the screened phosphate-solubilizing bacteria to dissolve tricalcium phosphate.

[0025] LB liquid medium (1 L): 5 g yeast powder, 10 g sodium chloride, and 10 g tryptone. Shake thoroughly, adjust the pH to 7 with NaOH or HCl, and sterilize at 121°C under high temperature and high pressure for 20 min. Cool and set aside.

[0026] LB solid culture medium plates were prepared by adding 15 g of agar powder to LB liquid culture medium, adjusting the pH to 7, sterilizing the medium at 121° C. and high pressure for 20 min, then pouring the medium into a sterile culture dish while hot, and cooling the medium to obtain LB solid culture medium plates.

[0027] LB medium is used for the expansion culture of phosphate-solubilizing bacteria.

[0028] Example 1 Isolation and identification of strains

[0029] Soil samples were collected from a lead-zinc smelter in Laibin City, Guangxi Zhuang Autonomous Region. Using a sterilized shovel or sampler, soil samples were collected at a depth of 40-100 cm. The samples were placed in sterile plastic bags or containers and labeled. The collected samples were then shipped to a laboratory at low temperatures for microbial isolation and screening.

[0030] Take 5g of soil sample and add 45mL of sterile saline. Shake thoroughly for 10 minutes to fully suspend the soil particles. Let the soil mixture stand for 10 minutes to precipitate the solid particles. The supernatant is the bacterial suspension used for screening. Take 100μL of bacterial suspension and add it to 900μL of sterile saline, mix thoroughly, and form 10 -1 diluent. Take 100μL 10 -1 The dilution was added to 900 μL of sterile saline to form 10 -2 dilution, and so on, up to 10 -6 Dilution. Take 200 μL of 10 -4 , 10 -5 and 10 -6 Add the gradient dilutions to the solid Montana inorganic phosphate medium and spread the dilutions evenly with a sterile applicator. Place the plate upside down in a 30°C incubator for 48 hours and observe the growth of the colonies.

[0031] After a single colony has grown on the solid Montana inorganic phosphate medium, select a representative and well-growing colony and pick it up with a sterile inoculation loop. Perform streak culture on a new solid medium to ensure that a single colony is isolated. Place the culture upside down in a 30°C incubator and culture for 48 hours. Observe the growth of the colony and repeat the streak process until a purified strain is obtained. Figure 1 Shown are photos of the growth conditions on LB medium.

[0032] The strain LXY-5 was expanded in liquid LB medium, and genomic DNA was extracted from it using a DNA extraction kit and PCR amplification was performed. The PCR product was subjected to agarose gel electrophoresis to confirm the amplification effect. The 16S rRNA of the strain was PCR amplified and sequenced to obtain the sequence shown in Seq ID No. 1. According to the Ezbiocloud database (https: / / www.ezbiocloud.net / ), the maximum similarity of the full-length 16S rRNA gene sequence of this strain with all standard strains of the Massilia genus was 98.35%, which is lower than the 98.5% threshold for new species classification. Figure 2 As shown, combined with physiological and biochemical characteristics, the bacterium was identified as a new species of the genus Massilia, named Massilia sp. LXY-5, or Massilia LXY-5 for short. It was deposited with the CGMCC No. 1.62472 on October 15, 2024, and is deposited with the General Microbiology Center of the China Culture Collection of Microorganisms. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Example 2 Phosphate-dissolving ability test of bacterial strains

[0034] The strain of Massillaria LXY-5 obtained in Example 1 was inoculated into liquid LB medium and cultured at 30°C and 180 rpm for 24 h. 600 = 1, and prepare the seed solution. Pipette 10 μL of the seed solution onto a solid Montana inorganic phosphate medium plate, incubate it upside down in a 30°C incubator for 3 days, and measure the colony diameter and the diameter of the phosphate ring.

[0035] The seed liquid was inoculated at a 1% inoculation rate into 100 mL of liquid Montana inorganic phosphate liquid culture medium (3 parallels), and cultured in a shaker at 30°C and 180 r / min for 3 days. The water-soluble phosphorus content in the culture medium was determined by the molybdenum antimony colorimetric method.

[0036] The colony diameter, phosphate-solubilizing ring diameter, and water-soluble phosphorus content are shown in Table 1.

[0037] Table 1 Determination of phosphate solubilization ability of Massillaria LXY-5 strain

[0038]

[0039] As shown in Table 1, after three days of culture, a clear phosphorus-dissolving ring appeared around the colony. This indicates that LXY-5 can dissolve insoluble tricalcium phosphate into soluble phosphorus, demonstrating that it can provide the necessary phosphorus for plant growth. Furthermore, the phosphorus content in the supernatant of the liquid culture reached 164.1 mg / L, and the pH of the culture fluid decreased from its original pH 7.0 to pH 4.0, indicating that LXY-5 can secrete organic acids, which dissolve phosphorus.

[0040] Example 3 Adaptability of strains to drought conditions

[0041] PEG-6000 is a hydrophilic polymer that creates a certain osmotic pressure in aqueous solutions. When added to culture media, PEG-6000 increases the system's osmotic pressure while simultaneously attracting and retaining water within its structure, thereby reducing available water. These properties reduce the availability of water in the culture medium, making it useful for simulating water-deficient environments such as drought conditions.

[0042] The water potential (osmotic potential) calculation formula may have certain calculation differences depending on different conditions. By comprehensively considering the various variable conditions in the calculation formula, it can be roughly concluded that the range of drought degrees simulated by PEG-6000 is as follows: Generally, the mass percentage concentration of PEG-6000 is 5-10%, corresponding to the osmotic potential of the aqueous solution is -0.06 to -0.2 MPa, which can simulate mild drought; 15% to 20% corresponds to the osmotic potential of the aqueous solution is -0.2 to -0.6 MPa, which can simulate moderate drought; 25% to 30% corresponds to the osmotic potential of the aqueous solution is -0.6 to -1.2 MPa, which can simulate severe drought.

[0043] Prepare Montana liquid culture medium, dispense into 50 mL conical flasks, add different concentrations of polyethylene glycol 6000 (PEG-6000) to simulate drought conditions, specifically set up 10% concentration experimental group, 20% concentration experimental group, 30% concentration experimental group and 40% concentration experimental group and no PEG-6000 (control group, concentration is 0) a total of 5 gradient experiments. Corresponding to the osmotic potential levels of 0, -0.2, -0.6, -1.2 and -1.9 MPa (mega Pascal). The OD 600 = 1, the seed liquid of Marseilles LXY-5 was inoculated into the above LB medium at a 1% inoculum amount, and the suspension of Marseilles LXY-5 was cultured at 30°C and 160 r / min for 24 h, and the OD of the suspension was measured. 600 Value, the result is Figure 3 shown.

[0044] from Figure 3 It can be seen that the OD of the control group 600 =3.61. OD of 10% concentration experimental group 600 =3.57 did not show a significant decrease, indicating that the Marseille strain LXY-5 can completely tolerate 10% concentration of PEG-6000. The OD of the 20% concentration experimental group was 600 =2.58, and the OD value decreased slightly, indicating that the LXY-5 strain has the ability to tolerate 20% concentration of PEG-6000. 600 =1.34 strain growth was inhibited, but the bacterium was still able to grow, indicating that LXY-5 had some tolerance at a 30% concentration. However, LXY-5's growth was significantly inhibited at 40% (simulating an extreme environment). This indicates that, in natural environments, LXY-5 is tolerant to both normal and moderately arid soils, and has some tolerance even in severe drought conditions, but struggles to survive only in extreme conditions. This suggests that LXY-5 can tolerate relatively extreme drought conditions.

[0045] The culture was carried out using the same culture conditions using the Montana liquid medium, and then the water-soluble phosphorus content in the culture medium was determined using the molybdenum antimony colorimetric method. Figure 4 As shown. Figure 4 As can be seen, in the control group without PEG-6000, the soluble phosphorus content was 162.1 mg / L, while the 10% concentration experimental group solubilized 157.4 mg / L. The 20% concentration experimental group solubilized 168.2 mg / L. This suggests that while the addition of PEG-6000 inhibited microbial growth, it did not significantly inhibit its phosphate solubilization function, indicating that the microorganism could still function normally at 20% concentration. At a 30% concentration, the strain's growth was further inhibited, and its function was also correspondingly suppressed, with the phosphorus capacity dropping to 66.1 mg / L, indicating that at 30% concentration, the strain still had some phosphate solubilization capacity. However, at 40% concentration, the strain's growth was significantly inhibited, and its function was largely lost. This indicates that Marseilles LXY-5 can function normally in normal to moderate drought environments, and only in extreme drought conditions does its growth become significantly inhibited, essentially losing its function.

[0046] from Figure 3 and Figure 4 It can be seen that the fungus also has a certain growth ability under drought conditions, and has a good adaptability to water absorption and high osmotic pressure stress, indicating that Marseille bacteria LXY-5 can be used in sites in arid and semi-arid areas.

[0047] Example 4 Ability of strains to promote plant growth in arid environments

[0048] To investigate the effects of Marsellus LXY-5 on rapeseed seedlings.

[0049] The Marseille strain LXY-5 was cultured in LB liquid medium at 30°C and 150 rpm until the logarithmic growth phase. The bacterial solution was transferred to a sterile centrifuge tube in a clean bench and centrifuged at 8000 rpm for 5 min. The supernatant was discarded and the tube was washed with sterile water. The concentration of the bacterial solution was adjusted to 10 8 CFU / ml. Take 100g of air-dried soil and inoculate the bacterial suspension into the soil. The inoculation amount is 10 6 CFU / g dry soil; a control group (CK) was set up without inoculation, and sterile water equal to the amount of bacterial suspension was added as a substitute. Each group had 5 replicates.

[0050] Rapeseed seeds with consistent germination were inoculated into pots at 25°C, with a light intensity of 16 hours per day and a dark intensity of 8 hours per day. The plants were incubated for 30 days without watering. Destructive sampling was performed on the 30th day of incubation, and the fresh weight, plant height, and root length of the plants were measured. The results are shown in Table 2.

[0051] Table 2 Determination of plant growth promoting ability of strains

[0052]

[0053] As shown in Table 2, the germination rate of rapeseed seeds in the control group was 79.6%, while that in the LXY-5-treated group was 90.3%, a 13.4% increase compared to the CK group. Regarding growth and development, the fresh weight, plant height, and root length of rapeseed seedlings in the LXY-5-treated group increased by 33.3%, 73.9%, and 58.4%, respectively, compared to the CK group. These results demonstrate that the LXY-5 strain significantly increases plant germination rate, fresh weight, plant height, and root length, demonstrating its beneficial effect in promoting plant growth.

[0054] As can be seen from the above examples, the bacterium LXY-5 provided by the present invention can effectively grow in the inorganic phosphorus medium environment of Montina and exert its unique phosphate solubilization effect. Through reasonable cultivation and regulation, it can achieve controllable large-scale production of this bacterium and then apply it to the preparation of microbial fertilizer.

[0055] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A strain of Marseilles, characterized in that: The taxonomic name of this Marseille fungus is: Massiliasp. The depository is: General Microbiology Center of China Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is: October 15, 2024, and the deposit number is: CGMCC No. 1.62472.

2. A microbial fertilizer, characterized in that: The method comprises the Massillaria thunbergii as claimed in claim 1.

3. Use of the Massillaria thaliana as claimed in claim 1 in microbial phosphate dissolution.

4. The use of the Massillaria thunbergii for promoting crop growth, improving crop yield and quality in arid areas according to claim 1, characterized in that: The crop is rapeseed.

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