Marsinia strain and application thereof

Abstract

The invention provides a strain of Marsinia sp.LXY-5 and application thereof, the classification name of the Marsinia sp.LXY-5 is Massinia sp.LXY-5, the preservation unit is China General Microbiological Culture Collection Center, and the preservation number of the Marsinia sp.LXY-5 is CGMCC (China General Microbiological Culture Collection Center). 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 (China General Microbiological Culture Collection Center) No. 1.62472. The invention also provides an application of the marssonia in the aspect of microbial phosphate solubilization and an application of the marssonia in arid regions.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and relates to plant growth promotion technology and the application of microbial fertilizers in production, and particularly relates to a strain of Massilia bacteria and its application. Background Art

[0002] Phosphorus plays a crucial role in the growth and development of plants, and to a certain extent affects the growth potential, metabolic activities and stress adaptability of plants. As the core component of adenosine triphosphate (ATP), phosphorus ensures the effective storage and transfer of energy within plant cells, providing the necessary power source for anabolic metabolism and growth processes. Phosphorus is also involved in the photosynthesis mechanism, helping plants efficiently convert light energy into chemical energy, accelerating the growth and development rhythm of plants, and at the same time having a positive effect on the flowering and fruiting processes of plants, improving the quality of flowers and the yield of fruits. Phosphorus also promotes the vigorous growth and increased branching of roots, strengthens the ability of plants to absorb water and nutrients, and through the synergistic effect with other nutrient elements such as nitrogen and potassium, improves the overall nutrient utilization efficiency of plants. Finally, phosphorus can assist in enhancing the resistance of plants to adverse environmental conditions such as drought, pests and diseases, and improving the survival competitiveness of plants.

[0003] At the ecological environment level, microorganisms, as key regulators of the ecosystem, play an indispensable role. Among them, phosphate-solubilizing bacteria, with their unique phosphate-solubilizing ability, have become an important microbial group for improving the phosphorus status in soil. They convert insoluble phosphorus compounds (such as calcium phosphate in phosphate rock) that are originally difficult for plants to directly utilize in the soil into forms that plants can absorb by releasing metabolic products such as organic acids and enzymes, effectively increasing the available phosphorus content in the soil. This process not only improves the biological availability of phosphorus, but also promotes the optimization of soil structure and enhances the microbial activity in the soil, thus comprehensively improving the fertility level of the soil. The symbiotic relationship between phosphate-solubilizing bacteria and plant roots further promotes the absorption of other nutrient elements by plants and optimizes the nutrient utilization structure.

[0004] In the application of microbial fertilizers, phosphate-solubilizing bacteria, as an important microbial resource, have the ability to convert insoluble phosphorus in the soil into available phosphorus that plants can utilize, and play an important role in improving soil fertility and promoting crop growth. However, there are also some problems in the application of existing phosphate-solubilizing bacteria in microbial fertilizers, including difficult screening of phosphate-solubilizing bacteria, limited identification technology, poor environmental adaptability and unstable application effects.

[0005] Therefore, it has become an urgent problem to develop a phosphate-solubilizing bacterium that is environmentally friendly and significantly reduces the dependence on chemical fertilizers. Summary of the Invention

[0006] In order to solve the above problems, the object of the present invention is to provide a strain of Massilia bacteria, which has the effect of phosphate solubilization.

[0007] Another object of the present invention is to provide the application of Massilia bacteria.

[0008] To achieve the above object, the present invention provides a strain of Massilia bacteria, whose taxonomic name is: Massilia sp. LXY-5, and the preservation unit is: General Microbiology Center of China Committee for Culture Collection of Microorganisms; 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 the application of the above Massilia bacteria in microbial phosphorus solubilization.

[0010] The present invention also provides the application of the above Massilia bacteria in arid regions.

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

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

[0013] The present invention provides a strain of Massilia bacteria and its application. The Massilia bacteria have the function of dissolving phosphorus, which can not only increase the content of soluble phosphorus in the soil, but also promote the growth and development of crops, thereby increasing the yield and quality of agricultural crops. At the same time, it also has good drought tolerance and can play an environmental restoration function in arid environments. Description of the Drawings

[0014] Figure 1 It is a growth status diagram of Massilia bacteria LXY-5 provided by the present invention on the culture medium.

[0015] Figure 2 It is a phylogenetic tree of Massilia bacteria LXY-5 provided by the present invention based on 16S rDNA.

[0016] Figure 3 It is the OD of Massilia bacteria LXY-5 provided by the present invention in LB medium containing different concentrations of PEG-6000 600 Change diagram.

[0017] Figure 4 It is a change diagram of the phosphorus solubilization amount of Massilia bacteria LXY-5 provided by the present invention in Mengjinna inorganic phosphorus medium containing different concentrations of PEG-6000. Detailed Embodiments

[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 definite definition of the protection scope of the present invention.

[0019] Materials

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

[0021] Preparation of culture medium:

[0022] The formula of Mengjinna inorganic phosphorus liquid medium (1L) is: glucose 10.0g, ammonium sulfate 0.5g, yeast extract powder 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, magnesium sulfate 0.3g, ferrous sulfate 0.03g, manganese sulfate 0.03g, tricalcium phosphate 5.0g. After shaking well, adjust the pH to 7 with NaOH or HCl, sterilize at 121°C under high temperature and high pressure for 20 min, and then cool to obtain the Mengjinna inorganic phosphorus liquid medium for standby.

[0023] The Mengjinna inorganic phosphorus medium plate is prepared by adding 15g of agar powder to the Mengjinna inorganic phosphorus liquid medium, adjusting the pH to 7, sterilizing at 121°C under high temperature and high pressure for 20 min, and then pouring it into a sterile petri dish while it is hot, and cooling to obtain the Mengjinna inorganic phosphorus medium plate.

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

[0025] LB liquid medium (1L): yeast powder 5g, sodium chloride 10g, tryptone 10g. After shaking well, adjust the pH to 7 with NaOH or HCl, sterilize at 121°C under high temperature and high pressure for 20 min, and then cool for standby.

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

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

[0028] Example 1 Isolation and Identification of Strains

[0029] Collect soil samples from a lead-zinc smelter in Laibin, Guangxi. Use a sterilized shovel or sampler to collect soil samples with a depth of 40-100 cm. Put the samples into sterile plastic bags or containers and mark the sample information. Send the collected samples to the laboratory at low temperature for microbial isolation and screening operations.

[0030] Take 5 g of soil sample and add 45 mL of sterile normal saline. Shake well 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 for screening. Take 100 μL of the bacterial suspension and add it to 900 μL of sterile normal saline, and mix well to form a 10 -1 dilution. Take 100 μL of the 10 -1 dilution and add it to 900 μL of sterile normal saline to form a 10 -2 dilution, and so on until 10 -6 dilution. Take 200 μL of the 10 -4 、10 -5 and 10 -6 gradient dilutions and add them to the solid Mengjinna inorganic phosphorus medium. Use a sterile spreader to evenly spread the dilutions. Invert and place it in an incubator at 30 °C for 48 hours to observe the growth of the colonies.

[0031] After obvious single colonies grow on the solid Mengjinna inorganic phosphorus medium, select representative and well-growing colonies and pick them up with a sterile inoculation loop. Conduct streak culture on a new solid medium to ensure the isolation of single colonies. Invert and place it in an incubator at 30 °C for 48 hours. Observe the growth of the colonies and repeat the above streak process until a purified strain is obtained. As shown in Figure 1 is a photo of the growth status on the LB medium.

[0032] Enlarge and culture the strain LXY-5 with liquid LB medium. Use a DNA extraction kit to extract genomic DNA from it and perform PCR amplification. Electrophorese the PCR product on an agarose gel to confirm the amplification effect. After PCR amplification and sequencing of the 16S rRNA of this bacterium, the sequence shown in Seq ID No.1 is obtained. Through comparison with the Ezbiocloud database (https: / / www.ezbiocloud.net / ), the maximum similarity of the full-length 16S rRNA gene sequence of this strain to all standard strains in the genus Massilia is 98.35%, which is lower than the new species classification threshold of 98.5%. As shown in Figure 2 Combined with the physiological and biochemical characteristics, this bacterium is identified as a new species of the genus Massilia, named Massilia sp. LXY-5, abbreviated as Massilia LXY-5, and submitted for preservation. The preservation number is CGMCC No. 1.62472; the preservation date is October 15, 2024, and the preservation unit is the China General Microbiological Culture Collection Center. The preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] Example 2 Detection of the phosphate-solubilizing ability of the strain

[0034] The strain of *Marseillaea* LXY-5 obtained in Example 1 was inoculated into a liquid LB medium and cultured at 30 °C and 180 r / min for 24 h. The OD of the bacterial solution was adjusted 600 = 1 to prepare a seed solution. 10 μL of the seed solution was aspirated and inoculated onto a solid Mengjinna inorganic phosphorus medium plate, which was then placed in an incubator at 30 °C and incubated upside down for 3 d. The colony diameter and the diameter of the phosphorus-dissolving ring were measured.

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

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

[0037] Table 1 Determination of the phosphorus-dissolving ability of *Marseillaea* LXY-5 strain

[0038]

[0039] As can be seen from Table 1, after 3 days of culture, an obvious phosphorus-dissolving ring appeared around the colony, indicating that *Marseillaea* LXY-5 has the ability to dissolve insoluble tricalcium phosphate into soluble phosphorus elements, indicating that this bacterium can provide the phosphorus elements required for plant growth that can be absorbed and utilized. At the same time, the phosphorus element content in the supernatant of the liquid medium reached 164.1 mg / L, and the pH value of the culture solution decreased from the original pH 7.0 to pH 4.0, indicating that *Marseillaea* LXY-5 can secrete organic acids and dissolve phosphorus elements through the secreted organic acids.

[0040] Example 3 Adaptability of the strain to drought conditions

[0041] PEG-6000 is a hydrophilic polymer that can form a certain osmotic pressure in an aqueous solution. When PEG-6000 is added to the medium, it will increase the osmotic pressure of the system, and at the same time can attract water and retain it in its structure, thereby reducing the available water. Its characteristics reduce the water availability in the medium. It can be used to simulate the environment of water shortage under drought conditions.

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

[0043] Prepare Mengjinna liquid medium, dispense it into 50 mL conical flasks, and add different concentrations of polyethylene glycol 6000 (PEG-6000) to simulate drought conditions. Specifically, set up a total of 5 gradient experiments including an experimental group with a concentration of 10%, an experimental group with a concentration of 20%, an experimental group with a concentration of 30%, an experimental group with a concentration of 40%, and a control group without PEG-6000 (concentration of 0). They respectively correspond to osmotic potential levels of 0, -0.2, -0.6, -1.2, and -1.9 MPa (megapascals). Transfer the seed liquid of Mycobacterium sp. LXY-5 with OD 600 =1 to the above-mentioned LB medium according to an inoculation amount of 1%, and after culturing with shaking at 30 °C and 160 r / min for 24 h, measure the OD of the bacterial suspension of Mycobacterium sp. LXY-5. 600 value, and the results are as Figure 3 shown.

[0044] From Figure 3 it can be seen that the OD of the control group 600 =3.61. The OD of the experimental group with a concentration of 10% 600 =3.57 did not show an obvious decrease, indicating that Mycobacterium sp. LXY-5 can completely tolerate PEG-6000 with a concentration of 10%. The OD of the experimental group with a concentration of 20% 600 =2.58, and the OD value decreased slightly, indicating that Mycobacterium sp. LXY-5 has the ability to tolerate PEG-6000 with a concentration of 20%. The OD of the experimental group with a concentration of 30% 600 =1.34, and the growth of the strain was inhibited, but the bacterium could still grow, indicating that at a concentration of 30%, Mycobacterium sp. LXY-5 still has a certain tolerance. However, the growth of Mycobacterium sp. LXY-5 was significantly inhibited under the condition of 40% (simulating an extreme environment). This shows that in the natural environment, Mycobacterium sp. LXY-5 is tolerant in normal soil and moderately drought-stricken soil, and also has a certain tolerance in a severely drought-stricken environment, but it is difficult to survive only in an extreme environment. It shows that Mycobacterium sp. LXY-5 can tolerate a relatively extreme drought environment.

[0045] Use Mengjinna liquid medium, culture it under the same culture conditions, and then use the molybdenum antimony anti-colorimetric method to measure the water-soluble phosphorus content in the culture solution, asFigure 4 As shown in Figure 4 It can be seen that in the control group without PEG-6000, the soluble phosphorus content was 162.1 mg / L, the phosphorus solubilization amount in the experimental group with a concentration of 10% was 157.4 mg / L, and the phosphorus solubilization amount in the experimental group with a concentration of 20% was 168.2 mg / L. From this, it can be seen that although the addition of PEG-6000 will inhibit the growth of microorganisms, there is no obvious inhibition on the exertion of its phosphorus solubilization function, indicating that microorganisms can still normally exert their phosphorus solubilization function under the condition of 20%. At a concentration of 30%, the growth of the strain was more inhibited, and the function was also correspondingly inhibited, and the phosphorus solubilization amount decreased to 66.1 mg / L, indicating that at a concentration of 30%, the strain still has a certain phosphorus solubilization ability. However, the growth of the strain was significantly inhibited under the condition of 40%, and the function was basically lost. This shows that under normal to moderately arid environments, Mycobacterium massiliense LXY-5 can normally exert its phosphorus solubilization function, and it is only under extremely arid conditions that the function is basically lost due to obvious growth inhibition.

[0046] From Figure 3 and Figure 4 it can be seen that this bacterium also has a certain growth ability under drought conditions, has good adaptability to water absorption and high osmotic pressure stress, indicating that Mycobacterium massiliense LXY-5 can be applied to sites in arid and semi-arid regions.

[0047] Example 4 Promotion ability of the strain on plant growth under drought environment

[0048] Explore the influence of Mycobacterium massiliense LXY-5 on rape seedlings.

[0049] Culture Mycobacterium massiliense LXY-5 in LB liquid medium at 30 °C and 150 r / min until the logarithmic growth phase. Transfer the bacterial liquid to a sterile centrifuge tube in a laminar flow hood, centrifuge at 8000 r / min for 5 min, discard the supernatant, wash with sterile water, and adjust the bacterial liquid concentration to 10 8 CFU / ml. Take 100 g of air-dried soil, inoculate the bacterial suspension into the soil, and the inoculation amount is 10 6 CFU / g dry soil; set the non-inoculated treatment as the control group (CK), and add sterile water equal to the bacterial suspension as a substitute. Among them, each group has 5 parallels.

[0050] Select rape seeds with consistent germination and inoculate them into planting pots. The temperature is 25 °C, the light time is 16 h / d, and the dark time is 8 h / d. Cultivate for 30 d without watering in the middle. Conduct destructive sampling on the 30th day of cultivation, and measure the fresh weight, plant height and root length of the plants. The results are shown in Table 2.

[0051] Table 2 Determination of the plant growth promotion ability of the strain

[0052]

[0053] As can be seen from Table 2, the germination rate of rapeseed seeds in the control group was 79.6%, and the germination rate after treatment with strain LXY-5 was 90.3%, which was 13.4% higher than that of the CK group. In terms of the impact on growth and development, the rapeseed seedlings in the LXY-5 group increased by 33.3%, 73.9% and 58.4% respectively in fresh weight, plant height and root length compared with the CK group. From the above results, it can be seen that strain LXY-5 has a significant effect on improving the germination rate of plants, increasing fresh weight, plant height and root length, and has a good effect on promoting plant growth.

[0054] As can be seen from the above examples, the Mycobacterium massiliense LXY-5 provided by the present invention can grow effectively in the environment of the Pikovskaya's inorganic phosphorus medium and exert its unique phosphorus solubilizing effect. Through reasonable cultivation and regulation, the controllable large-scale production of this bacterium can be realized, and then it can be applied to the preparation of microbial bacterial fertilizers.

[0055] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A strain of Marseilles, characterized in that: The classification name of the Massilia sp. is Massilia sp.LXY-5, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration; the address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is: October 15, 2024, and the preservation number is: CGMCCNo.1.62472.

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

3. Use of the Marseille bacteria as claimed in claim 1 in microbial phosphate solubilization.

4. Use of the Marseille bacteria as claimed in claim 1 in arid areas.

5. Use of the Massillaria spp. as claimed in claim 1 to promote the growth of crops and improve the yield and quality of crops in arid areas.

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