A strain of Planococcus lebituo 8-4 and its application

Abstract

The present invention discloses a Planococcus rifietoensis 8‑4 and its application, belonging to the field of microorganisms. The Planococcus rifietoensis 8‑4 provided by the present invention has a deposit number of CGMCC No.31311. The strain can tolerate 120 g / L of NaCl, has potential plant growth-promoting capabilities such as producing IAA and forming biofilms; the strain is inoculated into the rhizosphere of potted tomatoes, and can improve the growth index of tomatoes under 450 mM salt stress. The strain can be used to prepare functional microbial fertilizers, used to enhance crop resistance to salt stress, and improve saline-alkali land crop yields, which is of great significance to saline-alkali land improvement.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Planococcus lebitovici 8-4 and an application thereof. Background Art

[0002] Saline-alkali land is widely distributed throughout the world. Excessive salt will inhibit the growth of crops and reduce crop yields. It is an important issue facing global agricultural development. Strengthening the improvement and utilization of saline-alkali land is of great significance to ensuring world food security. The area of ​​saline-alkali land in my country is about 1.5 billion mu, of which about 500 million mu is usable. The effective and efficient use of saline-alkali land resources is an important way to increase land increment and realize the expansion, quality improvement and efficiency of cultivated land resources. Saline-alkali land is a type of land containing a relatively high concentration of soluble salt ions, of which the soluble salt ions are mainly sodium (Na + ), magnesium (Mg 2+ ), calcium (Ca 2+ ), potassium (K + ), chloride (Cl - ), sulfate (SO4 2- ), bicarbonate (HCO3 - ) and carbonates (CO3 2- ) etc., among which sodium ion (Na + ). Its causes are diverse, including climate, geography, soil texture and groundwater, river lateral infiltration, seawater irrigation and improper farming management. High concentrations of salt in saline-alkali land can cause osmotic stress, ion poisoning, and nutritional imbalance to plants. At present, the main solutions for improving saline-alkali land are physical improvement (leaching and salt removal, deep plowing and film covering), chemical improvement (gypsum, ferrous sulfate and aluminum sulfate), organic improvement (adding organic fertilizer and returning straw to the field), and biological improvement (planting salt-tolerant plants and green manure crops and applying microbial fertilizers). Physical improvement methods require huge investment and are difficult to implement in water-scarce areas; improper application of chemicals in chemical improvement methods can cause soil structure deterioration and environmental pollution; in organic improvement, the transportation cost of organic fertilizer is high, and the straw decomposes slowly and takes a long time; in biological improvement, the plant planting cycle is long and the results are not significant in the short term. However, microbial fertilizer improvement uses plant growth-promoting microorganisms to enhance the salt stress tolerance of saline-alkali land plants and ensure their normal growth, and does not have the defects of the above improvement methods.

[0003] Under salt stress, plant growth-promoting microorganisms can alleviate plant salt stress through various pathways, including: improving plant nutrient utilization (nitrogen fixation, phosphorus dissolution and potassium dissolution, etc.), producing plant hormones, producing antioxidants, producing ACC deaminase, increasing osmoprotectants, inhibiting harmful microorganisms, and inducing plant systemic resistance. Microbial agents are a type of microbial fertilizer, a class of products containing microorganisms with specific functions, used in agriculture, horticulture, and environmental management to improve soil health, promote plant growth, and increase crop yields. They have the advantages of being environmentally friendly and sustainable, and can provide important support for agricultural production. By developing functional microbial agents that can enhance crop tolerance to salt stress and increase crop yields in saline-alkali land, the soil conditions of saline-alkali land can be effectively improved. This type of microbial agent can not only promote the healthy growth of crops, but also improve the efficiency of land use, ultimately bringing significant economic and ecological benefits to agricultural production. Summary of the invention

[0004] The technical problem to be solved by the present invention is: in view of the practical problem that the crop yield in saline-alkali land is low and the land is difficult to use in agricultural production, a strain is provided that can improve the salt stress tolerance of saline-alkali land crops and increase the biomass of crops on saline-alkali land.

[0005] The technical solution of the present invention is: a strain of Planococcus rifietoensis 8-4, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 16, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit number is CGMCC No.31311.

[0006] A bacterial agent containing the above-mentioned Planococcus rifietoensis 8-4.

[0007] The use of the Planococcus rifietoensis 8-4 or bacterial agent described above in improving the salt tolerance of plants.

[0008] The use of the Planococcus rifietoensis 8-4 or bacterial agent described above in increasing the biomass of plants in saline-alkali land.

[0009] Furthermore, the plant is tomato.

[0010] The present invention provides a strain capable of improving the salt stress tolerance of tomatoes. The strain is inoculated into the rhizosphere of potted tomatoes, and the growth indexes of tomatoes (plant height, aboveground fresh weight, root weight, total fresh weight, aboveground dry weight, root dry weight, total dry weight, root length, etc.) can be improved under salt stress; the relative water content (DW) in tomato leaves is improved, the malondialdehyde (MDA) content in tomato leaves is reduced, the catalase (CAT) activity and peroxidase (POD) activity in tomato leaves are improved, and the superoxide dismutase (SOD) activity is reduced; the malondialdehyde (MDA) content in tomato roots is reduced, and the catalase (CAT) activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity in tomato roots are improved. In addition, the zoosporic coccus 8-4 also has potential plant growth-promoting abilities such as tolerance to 120 g / L NaCl, production of IAA and formation of biofilm, and is a rhizosphere growth-promoting zoosporic coccus.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. The effective strains in the microbial agent provided by the present invention are derived from saline-alkali soil, have very strong adaptability to saline-alkali environment, have the ability to produce IAA and form biofilm, and have negative hemolytic reaction, and are harmless to humans;

[0013] 2. The Motile Coccus 8-4 provided by the present invention has an excellent growth-promoting effect on tomatoes under extremely severe salt stress (450 mM NaCl), and the microbial agent developed using it has broad prospects in saline-alkali land improvement and crop growth guarantee. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a photo of the colony phenotype of strain 8-4.

[0015] Figure 2 This is an electron microscopic photograph of the cell phenotype of strain 8-4.

[0016] Figure 3 This is the growth curve determination of strain 8-4.

[0017] Figure 4 This is a test of the salt tolerance of strain 8-4.

[0018] Figure 5 The biofilm formation ability of strain 8-4 was tested.

[0019] Figure 6 This is a test of the hemolytic ability of strain 8-4.

[0020] Figure 7 These are the phenotypic photos and growth indicators of tomatoes under different treatments in the salt tolerance pot experiment in Example 4.

[0021] Figure 8 These are the physiological indicators of tomatoes under different treatments in the salt tolerance pot experiment in Example 4.

[0022] Fig. 9 These are the indicators related to the salt stress resistance of the aboveground parts of tomatoes under different treatments in the salt tolerance pot experiment in Example 4.

[0023] Fig.10 These are the indicators related to the resistance of the underground part of tomatoes to salt stress under different treatments in the salt tolerance pot experiment in Example 4. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0025] Tomato variety Cooperation 903: Available from various fruit stores and online stores.

[0026] TSB liquid culture medium: 17.0 g tryptone; 3.0 g soy peptone; 5.0 g sodium chloride; 2.5 g dipotassium hydrogen phosphate; 2.5 g glucose; pH value 7.3±0.2.

[0027] Example 1. Strain origin, morphology, and growth ability test

[0028] 1. Sample collection and strain isolation

[0029] Rhizosphere soil of Qinghai desert halophytes was collected, the soil suspension was gradient diluted, and a suitable gradient was selected to be spread on TSB medium, cultured at 37°C overnight, and the strains on the plate were separated and purified to screen strain 8-4.

[0030] 2. Molecular identification of strains

[0031] The 16S rDNA sequence of strain 8-4 was amplified and sequenced, and the sequencing result is shown in SEQ ID No. 1. After identification, it can be determined that strain 8-4 belongs to Planococcus rifietoensis. Planococcus rifietoensis 8-4 is referred to as Planococcus rifietoensis 8-4. Planococcus rifietoensis 8-4 has only one genome in the Genome database of NCBI (National Center for Biotechnology Information), and is a rare species that has been rarely studied.

[0032] 3. Morphological observation of strains

[0033] 1. Colony phenotype observation

[0034] Use an inoculation loop to dip the strain storage solution of Motile Coccus 8-4 in a glycerol tube, streak it on a TSB solid plate, place it in a 30℃ biochemical incubator for constant temperature culture and activation until a single colony grows, take a photo and record the colony phenotype. Figure 1 As shown, the colonies of strain 8-4 are round and raised, orange-yellow in color, smooth, shiny, translucent, sticky, and have complete edges.

[0035] 2. Cell phenotype observation

[0036] 2.1. Strain preparation: After activating Mosquito Coccus 8-4, take 1% of the inoculum into TSB liquid culture medium and culture at 30°C and 200 r·min-1 for 1 day.

[0037] 2.2. Sample preparation:

[0038] (1) Pre-fixation: fix in 2.5% glutaraldehyde solution at 4°C overnight;

[0039] (2) Rinsing: Pour off the fixative solution and rinse the sample three times with 0.1 M phosphate buffer (pH 7.0), 15 min each time;

[0040] (3) Refixation: Fix the sample with 1% osmium acid solution for 1-2 hours;

[0041] (4) Rinse: Pour off the fixative solution and rinse the sample three times with 0.1 M phosphate buffer (pH 7.0), 15 min each time;

[0042] (5) Dehydration: The samples were dehydrated with ethanol solutions of gradient concentrations (including 50%, 70%, 80%, 90% and 95%), with each concentration treated for 15 min; then treated twice with 100% ethanol, each time for 20 min;

[0043] (6) Overtreatment: Treat the sample with a mixture of ethanol and isoamyl acetate (V / V = 1 / 1) for 30 min, and then treat the sample with pure isoamyl acetate for 1-2 h;

[0044] (7) Critical point drying;

[0045] (8) Coating and observation.

[0046] 2.3. Electron microscopy: Use Hitachi 8100 electron microscope to observe and take photos of the strain morphology.

[0047] The electron microscopy results of strain 8-4 are as follows Figure 2 As shown, the cell diameter is about 0.8 μm, roughly spherical, with bumps on the surface, arranged in a unisphere, without flagella and spores.

[0048] 4. Growth ability test of strains

[0049] 1. Use an inoculation loop to dip the preservation solution of strain 8-4 in the glycerol tube, streak it on a TSB solid plate, and place it in a 30℃ biochemical incubator for constant temperature culture and activation until a single colony grows.

[0050] 2. Use a sterile pipette tip to pick up a single colony, inoculate it into a test tube containing 3 mL of TSB liquid culture medium, and culture it in a 30°C constant temperature shaker for 24 hours to prepare the seed solution.

[0051] 3. Inoculate the seed solution into fresh TSB liquid medium at a 1% inoculum volume, and dispense 200 μL per well into a sterile 100-well plate dedicated to the bacterial automatic growth curve analyzer. Place the well plate in the instrument, culture at 30°C, and measure the OD every 30 minutes. 600 The value was measured, shaken for 30 seconds before and after the measurement, and cultured for 50 hours to complete the growth test of the bacteria throughout the entire cycle. Five wells were set up for replication, and blank TSB medium without strain seed solution was used as a control. After the experiment, the data was exported and analyzed and graphed using Excel software.

[0052] Specific results such as Figure 3 As shown, the growth curve of strain 8-4 is S-shaped, with 0-5h as the lag phase, 5-20h as the logarithmic growth phase, reaching the plateau phase after 20h, and still in the plateau phase after 30h. This strain has a short lag phase, grows rapidly, can reach a high population density in the plateau phase, is easy to culture, and is convenient for liquid fermentation of microbial agents.

[0053] V. Deposit of strain 8-4

[0054] The Planococcus rifietoensis 8-4 provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration on July 16, 2024, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.31311.

[0055] Example 2: Test of plant growth-promoting related indicators of Mosquitococcus 8-4

[0056] 1. Salt tolerance test

[0057] The seed solution of Mosinococcus 8-4 was prepared by the method in Example 1, and inoculated into fresh TSB medium with NaCl content of 5, 20, 40, 60, 80, 100, and 120 g / L at a 1% inoculation amount, and dispensed into 96-well plates at 200 μL per well. The well plates were placed at 30°C and 170 rpm for shaking culture. After 48 h, the OD of the bacterial solution was measured using an ELISA instrument. 600Data analysis and chart drawing were performed using Excel software.

[0058] Specific results such as Figure 4 The results showed that Mosquitoes 8-4 could still reach a high population density at a salt concentration of 120 g / L and had a strong tolerance to salt stress, which met the prerequisite for the development of its function of promoting plant growth in saline-alkali land.

[0059] 2. IAA production capacity determination

[0060] The Motile Coccus 8-4 was inoculated into a medium containing 1 g L -1 The culture medium was incubated with L-tryptophan in Landy medium at 25°C and 140 rpm for 72 h. 1 mL of the culture medium was centrifuged at 12,000 × g for 5 min. The supernatant was added with an equal volume of Salkowski reagent. After color development at room temperature in the dark for 30 min, the OD was measured. 530 The blank medium was used as control, and the OD value corresponding to the standard IAA was 530 The values ​​were used as standard curve to calculate the IAA production (mg L -1 ).

[0061] IAA is a plant hormone that directly promotes plant growth and is involved in the entire growth and development cycle of plants. The results showed that Mosquitoes 8-4 has a certain ability to produce IAA, and the yield can reach 16.13±0.23nmol / L, indicating that it has a certain potential to promote plant growth.

[0062] 3. Biofilm formation ability test

[0063] The seed liquid of Motile Coccus 8-4 was prepared by the method in Example 1, and inoculated into a 48-well plate containing 1 mL of fresh TSB culture medium at a 1% inoculum. The plate was sealed with plastic wrap and statically cultured in a biochemical incubator at 30°C. The biofilm formation was observed at different time points and photographed for record. The biofilm-forming ability of the strain is the key to the strain's colonization on the plant root surface and its growth-promoting ability. The stronger its film-forming ability is, the stronger its root surface colonization ability is.

[0064] The results are as follows Figure 5 As shown in the results, Mosquitoes 8-4 has a strong ability to form biofilms. Biofilms were formed at 24 hours, and a solid and complex biofilm structure was formed at 40 hours. The biofilm structure became more and more complex at 72 hours. This shows that the strain has a strong potential to colonize plant root surfaces, and it may be through direct colonization on the surface of plant roots to establish a symbiotic relationship, thereby promoting plant growth and development.

[0065] Example 3: Hemolytic reaction of Motile Coccus 8-4

[0066] The motile cocci 8-4 were inoculated on sheep blood plates (purchased from Beekman Biotechnology Co., Ltd.) and incubated at 30°C for 12 h. No hemolysis zone was observed (e.g. Figure 6 ), that is, the hemolytic reaction is negative and can be used for the development and utilization of fertilizers.

[0067] Example 4: Potted plant experiment on bacterial strains promoting salt tolerance growth

[0068] 1. After surface disinfection, the Hezuo 903 tomato seeds were sown in moist vermiculite and placed in a light incubator for seedling cultivation (25°C, 70% RH, 2500LUX). Seedlings with uniform growth were selected at the cotyledon stage and transplanted into pots (1 L in volume, with a tray containing 800 mL of substrate) filled with seedling cultivation peat.

[0069] 2. 5 days after transplanting the tomato seedlings, treat the potted plants as follows:

[0070] H2O group: Only tap water was added during the planting period, and 100 mL of tap water was added every 3 days, for a total of 10 times.

[0071] NaCl group: A total of 21.21 g NaCl was added (making the final salt concentration in the substrate about 450 mM), and each addition was made by pouring the salt solution into the root system. The experiment adopted the interval multi-frequency salt addition method: 100 mL of 360 mM NaCl solution was added every 3 days, for a total of 10 times.

[0072] NaCl+8-4 group: In addition to the addition of NaCl as in the NaCl treatment group, the Motile Coccus 8-4 was inoculated when the NaCl solution was first added, so that the final concentration of Motile Coccus 8-4 was 1×10 7 CFU g -1 earth.

[0073] 3. The potted plants were cultured in a light incubator for 5 weeks, and the growth indexes (plant height, root length, fresh weight of aboveground part and fresh weight of underground part), physiological indexes (proline content, soluble sugar content, relative water content of leaves and photosynthesis efficiency) and salt stress tolerance related indexes (MDA content, CAT activity, POD activity and SOD activity of aboveground part, MDA content, CAT activity, POD activity and SOD activity of underground part) of tomato plants were observed, recorded and measured. The relative water content of leaves was calculated by the following formula: {(fresh weight of leaves - dry weight of leaves) / (water-saturated weight of leaves - dry weight of leaves)}×100%; the photosynthetic efficiency of tomato leaves was measured by advanced photosynthetic fluorescence meter LI-6800; the MDA content, CAT activity, POD activity, SOD activity, proline content and soluble sugar content of aboveground and underground parts were measured by commercial kits (all purchased from Solebow Biotechnology Co., Ltd.), and the measurement method is detailed in the kit instructions.

[0074] The results showed that the Mobilococcus 8-4 could improve the growth indexes of tomatoes (plant height, root length, fresh weight of aboveground parts, fresh weight of underground parts, etc.) under salt stress, ensuring the normal growth of tomatoes. Its biomass was even higher than that of tomatoes in the control group without salt addition (e.g. Figure 7 ). It shows that Mosococcus 8-4 has a superior growth-promoting effect on tomatoes under extremely severe salt stress (450mM NaCl), and the microbial agent developed using it has broad prospects in saline-alkali land improvement and crop growth guarantee.

[0075] Mobilococcus 8-4 increased the proline content, soluble sugar content, relative water content (DW) and photosynthesis efficiency (e.g. Figure 8 ). Proline and soluble sugars can increase the osmotic pressure in plant tissues, ensure the stability of the internal water content of plants under hyperosmotic stress, and even make their growth ability better (compared with the water treatment group).

[0076] Malondialdehyde (MDA) is a sign of plant tissue damage, and the higher its content, the more serious the damage to the plant tissue. Planococcus 8-4 can reduce the content of malondialdehyde (MDA) in tomato leaves and roots, reducing the degree of damage to plant tissues under salt stress. Catalase (CAT), peroxidase (POD) and reduction of superoxide dismutase (SOD) are key mechanisms for plants to resist damage caused by reactive oxygen species (ROS). The results showed that Planococcus 8-4 increased the activity of catalase (CAT) and peroxidase (POD) in tomato leaves, reduced the activity of superoxide dismutase (SOD), and increased the activity of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in tomato roots. It shows that Planococcus 8-4 can relieve the stress effect of tomatoes under salt stress by regulating the activities of the above three enzymes ( Fig. 9 , Fig.10 ).

Claims

1. A strain of Cytosporium lebitoxum ( Planococcus rifietoensis )8-4, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31311.

2. A method comprising the Lebituococcus according to claim 1 ( Planococcus rifietoensis )8-4 of the bacterial agent.

3. The Lebituococcus according to claim 1 ( Planococcus rifietoensis ) Use of the bacterial agent described in 8-4 or claim 2 to improve the salt tolerance of tomatoes.

4. The Lebito Cytosporium according to claim 1 ( Planococcus rifietoensis ) 8-4 or the use of the bacterial agent described in claim 2 to increase the biomass of tomatoes in saline-alkali soil.

Images