A Pectin-Degrading Strain of Sinomicrobium 7-7 and Its Application

Abstract

The present invention discloses a pectin-degrading Chinese microbe 7-7 and its application. The pectin-degrading Chinese microbe (Sinomicrobium pectinilyticum) 7-7 provided by the present invention has a preservation number of CGMCC No.31313. The bacterium can tolerate 120gL ‑1 The present invention has the potential growth-promoting ability of producing IAA, iron carriers and forming biofilms, etc.; the bacteria can improve the germination rate of rice / tomato seeds under NaCl stress; the strain is inoculated into the rhizosphere of potted corn, and can promote the growth and development of corn under 700mM NaCl stress. The present invention is of great significance for developing functional microbial fertilizers, tapping the potential of saline-alkali land, achieving high and stable yields of saline-alkali crops, and ensuring my country's food security.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural biological product production, and in particular to the preparation and application of a rhizosphere growth-promoting bacterium capable of enhancing the salt tolerance of crops and a bacterial agent thereof. Background Art

[0002] Soil salinization is a major soil degradation process that threatens ecosystems and is considered one of the major issues threatening agricultural production, food security and sustainability in arid and semi-arid regions worldwide. The total area of ​​saline soil in China is about 36 million hm2. 2 , accounting for 4.88% of the country's available land area. The salinized cultivated land area reached 9.2 million hm 2 , accounting for 6.62% of the country's cultivated land area. The main factors causing soil salinization are various soluble salts, including sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3). The accumulation of these salts makes the soil salinized and alkaline. The formation of saline-alkali land is affected by natural factors such as drought, large evaporation, upward movement of groundwater salt and seawater intrusion, as well as human factors such as unreasonable irrigation, poor drainage and excessive land use. The soil structure of saline-alkali land deteriorates, crops have difficulty absorbing water, ion poisoning, nutritional imbalance, loss of soil biodiversity, plant growth inhibition, and reduced crop yield and quality. Therefore, how to grow crops normally in saline-alkali soil and ensure high and stable yields, and achieve the expansion, quality improvement and efficiency of cultivated land resources is of great significance to the present and future when urbanization is accelerating and land resources are becoming increasingly constrained. The management of saline-alkali land is a complex process that consumes huge manpower and material resources. It is mainly improved through water conservancy, physical, chemical and biological measures. Among them, biological improvement has promotion advantages due to its low cost, high efficiency, energy and water saving and lasting effect.

[0003] Under salt stress, plant probiotics can help plants cope with salt stress through a variety of mechanisms, including secreting plant hormones (such as IAA, ABA, ACC deaminase), organic acids, antioxidants and other functions to improve plant nutrient absorption. In addition, growth-promoting bacteria can also improve plant salt tolerance by regulating the ion balance in plants, enhancing the activity of antioxidant enzymes and stimulating the plant's own defense mechanisms. In recent years, more and more research has focused on screening and optimizing salt-tolerant microbial strains, developing microbial fertilizers and biological agents, improving saline-alkali soil health, and increasing crop salt tolerance and yield. The research and application of salt-tolerant microbial strains provide new solutions for the effective management and improvement of agricultural saline-alkali land, especially in saline-alkali land management and low-input ecological agricultural production.

[0004] Although the ability of microorganisms to directly or indirectly reduce plant salt stress damage has been verified in the breeding of a variety of economic crops, the development of salt-tolerant microbial resources is still restricted by the difficulty of cultivation, pathogenicity, and lack of efficient strains. In order to solve the problem of the lack of commercial excellent strains, the development of microbial resources in diverse special habitats is particularly necessary. Therefore, developing microbial resources that can promote the salt-tolerant growth of crops from the rhizosphere of desert halophytes, using them to create microbial fertilizers, and then applying them to the improvement of agricultural saline-alkali land is a forward-looking biotechnology strategy, which is of great significance to the food security and green and sustainable development of agriculture in my country and even the world. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain capable of enhancing the salt stress tolerance of crops, thereby increasing the biomass of crops under salt stress environment and promoting the effective development and utilization of saline-alkali land.

[0006] The technical solution of the present invention is: a strain of Sinomicrobium pectinilyticum 7-7, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postal Code: 100101) on July 16, 2024, and the deposit number is CGMCC No.31313.

[0007] A bacterial agent containing the above-mentioned Sinomicrobium pectinilyticum 7-7.

[0008] The use of the above-mentioned Sinomicrobium pectinilyticum 7-7 or bacterial agent in improving the salt tolerance of plants.

[0009] The application of the above-mentioned Sinomicrobium pectinilyticum 7-7 or bacterial agent in increasing plant biomass under salt stress environment.

[0010] Use of the above-mentioned Sinomicrobium pectinilyticum 7-7 or the bacterial agent according to claim 2 in improving the germination rate of plant seeds under salt stress environment.

[0011] Furthermore, the plant is rice, tomato or corn.

[0012] The present invention provides a strain capable of improving the salt stress tolerance of plants (rice, tomato and corn), and the strain can tolerate 120 g L-1 The strain can survive in a strong saline-alkali environment of 100% NaCl and pH=11, and has the potential plant growth-promoting abilities of producing IAA, siderophores and biofilm, and is a rhizosphere growth-promoting bacterium. The strain can improve the germination rate and average root length of rice and tomato seeds under salt stress; it can significantly reduce the malondialdehyde (MDA) content in rice and tomato young roots, and significantly increase the catalase (CAT) activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity in rice and tomato young roots. When the bacteria was inoculated into the roots of potted corn, the growth indicators of corn (fresh weight, dry weight and plant height, etc.) could be significantly improved under high salt stress; the net photosynthesis rate, transpiration rate and stomatal conductance of corn plant leaves could be significantly increased, and the intercellular CO2 concentration of corn could be significantly reduced; the proline (Pro) content in the aboveground part of corn and the total soluble sugar in corn plants could be significantly increased; the malondialdehyde (MDA) content in the aboveground part of corn could be significantly reduced, the catalase (CAT) activity and peroxidase (POD) activity in the aboveground part of corn could be significantly increased, and the superoxide dismutase (SOD) activity could be significantly reduced; the malondialdehyde (MDA) content and catalase (CAT) activity in the underground part of corn could be significantly increased, and the superoxide dismutase (SOD) activity could be significantly reduced.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The effective strains in the microbial agent provided by the present invention are derived from saline-alkali soil, have strong adaptability to saline-alkali environment, and have potential plant growth-promoting functions such as producing IAA, producing iron carriers and forming biofilms. The hemolytic reaction is negative and is harmless to the human body, which meets the requirements of functional strains of microbial fertilizers.

[0015] 2. The microbial agent provided by the present invention can increase the biomass of corn at a concentration of 700 mM NaCl.

[0016] 3. The microbial agent of the present invention can significantly improve the germination rate of plant seeds under salt stress environment.

[0017] Collection information:

[0018] Sinomicrobium pectinilyticum 7-7 was deposited on July 16, 2024 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit number is CGMCC No.31313. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The following are photos of the bacterial colony cell phenotypes.

[0020] Figure 2Growth curve of strains was determined.

[0021] Figure 3 To test the salt and alkali tolerance of the strain.

[0022] Figure 4 To test the biofilm forming ability of strains.

[0023] Figure 5 It is the rice seed growth index under different treatments in the strain-promoted salt-tolerance germination test in Example 3.

[0024] Figure 6 These are the MDA content and antioxidant enzyme activity-related indicators of rice seeds under different treatments in the strain salt-tolerance germination test in Example 3.

[0025] Figure 7 It is the tomato seed growth index under different treatments in the strain-promoted salt-tolerance germination test in Example 3.

[0026] Figure 8 These are the MDA content and antioxidant enzyme activity related indicators of tomato seeds under different treatments in the strain salt-tolerance germination test in Example 3.

[0027] Fig. 9 This is the hemolytic ability test of the strain in Example 4.

[0028] Fig.10 These are the phenotypic photos and growth indicators of corn under different treatments in the salt tolerance pot experiment in Example 5.

[0029] Fig.11 These are the photosynthesis-related indicators of corn under different treatments in the salt-tolerance pot experiment in Example 5.

[0030] Fig.12 It is the osmotic substance content of corn under different treatments in the salt tolerance pot experiment in Example 5.

[0031] Fig.13 These are the MDA content and antioxidant enzyme activity related indicators of corn under different treatments in the salt tolerance pot experiment in Example 5. DETAILED DESCRIPTION

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0033] The quantitative tests in the following examples were repeated more than three times, and the results were averaged.

[0034] Rice variety Nanjing 46: Available from various fruiting stores and online stores.

[0035] Tomato variety Cooperation 903: Available from various fruit stores and online stores.

[0036] Corn variety Mingyu 1203: Available from various physical stores and online stores.

[0037] Example 1. Strain origin, morphology, growth ability test and preservation

[0038] 1. Sample collection and strain isolation

[0039] Rhizosphere soil of halophytes in the Qinghai desert was collected, and the soil suspension was gradient diluted. A suitable gradient was selected and spread on TSB medium, and cultured at 30°C overnight. The strains on the plate were separated and purified, and strain 7-7 was screened.

[0040] 2. Molecular identification of strain 7-7

[0041] The 16S rDNA sequence of strain 7-7 was amplified and sequenced, and the sequencing result is shown in SEQ ID No.1. After identification, it can be determined that strain 7-7 belongs to the genus Sinomicrobium. This genus has been rarely studied, and only 5 species have been uploaded to the NCBI (National Center for Biotechnology Information) database. Strain 7-7 is closest to Sinomicrobium pectinilyticum strain 5DNS001, with a similarity of 97.82%. This species is Sinomicrobium pectinilyticum, and there is only one genome data in the database. Therefore, the bacterium is named Sinomicrobium pectinilyticum 7-7, referred to as Sinomicrobium 7-7.

[0042] 3. Morphological observation of Sinomicrobacterium 7-7

[0043] 1. Colony phenotype observation

[0044] Use an inoculation loop to dip the strain storage solution of Sinomicrobacterium 7-7 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 in A, the colony of S. sinensis 7-7 is milky white, has a wrinkled surface, is dull, slightly transparent, has a sticky texture, has complete edges, and has a cap structure in the center of the colony.

[0045] 2. Cell phenotype observation

[0046] 2.1. Strain preparation: After activating S. sinensis 7-7, take 1% of the inoculum into TSB liquid culture medium and culture at 30°C and 200 rpm for 1 day.

[0047] 2.2. Sample preparation:

[0048] (1) Pre-fixation: fix in 2.5% glutaraldehyde solution at 4°C overnight;

[0049] (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;

[0050] (3) Refixation: Fix the sample with 1% osmium acid solution for 1-2 hours;

[0051] (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;

[0052] (5) Dehydration: Dehydrate the samples with ethanol solutions of gradient concentrations (including 50%, 70%, 80%, 90% and 95%), with each concentration treated for 15 min; then treat with 100% ethanol twice, each time for 20 min;

[0053] (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;

[0054] (7) Critical point drying;

[0055] (8) Coating and observation.

[0056] 2.3. Electron microscopy: Use Hitachi 8100 electron microscope to observe and take photos of the strain morphology.

[0057] The electron microscopic results of Sinomicrobium 7-7 are as follows Figure 1 As shown in B, the cell diameter is about 2.0 μm, and it is short rod-shaped with a rough surface and uniform cell morphology.

[0058] IV. Growth ability test of Sinomicrobacterium 7-7

[0059] 1. Use an inoculation loop to dip the preservation solution of S. sinensis 7-7 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.

[0060] 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.

[0061] 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 60 minutes. 600 The value was measured, shaken for 30 seconds before and after the measurement, and cultured for 25 hours to complete the growth test of the bacteria throughout the entire cycle. Three wells were set up for replication, and a blank TSB medium without strain seed solution was used as a control. After the experiment, the data was exported and Excel and Origin software were used for data analysis and chart drawing.

[0062] Specific results such as Figure 2 The growth curve of strain 7-7 is S-shaped, with 0-3h as the lag phase, 3-20h as the logarithmic growth phase, and reaching the plateau phase after 20h. The 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.

[0063] V. Storage of Sinomicrobacterium 7-7

[0064] The Sinomicrobium pectinilyticum 7-7 provided by the present invention has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC; address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postal Code: 100101) on July 16, 2024, with the deposit number CGMCC No. 31313.

[0065] Example 2: Test of plant growth-promoting related indicators of Sinomicrobacterium 7-7

[0066] 1. Test of salt and alkali resistance

[0067] a. Salt tolerance test

[0068] The seed solution of S. sinensis 7-7 was prepared by the method in Example 1, and inoculated with 1% inoculum into the NaCl contents of 5, 20, 40, 60, 80, 100, and 120 g L -1 The culture medium was freshly prepared and dispensed into a 96-well plate at 200 μL per well. The plate was placed in a 30°C, 200 rpm shaking incubator. After 48 h, the OD value of the bacterial solution was measured using an ELISA reader. 600 Data analysis and graph drawing were performed using Excel and Prism software.

[0069] b. Alkali resistance test

[0070] The seed solution of S. sinensis 7-7 was prepared by the method in Example 1, and inoculated into fresh TSB medium with pH values ​​of 6, 7, 8, 9, 10, and 11 at a 1% inoculum, and dispensed into 96-well plates at 200 μL per well. The well plates were placed at 30°C and 200 rpm for shaking culture. After 48 h, the OD value of the bacterial solution was measured using an ELISA reader. 600 Data analysis and graph drawing were performed using Excel and Prism software.

[0071] Specific results such as Figure 3 The results showed that Sinomicrobacterium 7-7 had the ability to tolerate 120 g L -1 The ability to adapt to the strong alkaline environment of NaCl and pH=11 can ensure the normal survival of Chinese microbacterium 7-7 in saline-alkali environment and the stable performance of its plant growth-promoting function.

[0072] 2. IAA production capacity determination

[0073] S. sinensis 7-7 was inoculated with 1 g L -1 The culture medium was incubated with L-tryptophan in Landy medium at 30°C and 200 rpm for 72 hours, and 1 mL of the culture solution was centrifuged at 12,000 rpm for 5 minutes to obtain the supernatant, which was assayed using an Elisa kit (enzyme immunosorbent assay). The IAA production (nmol / L) of Sinomicrobacterium 7-7 was calculated.

[0074] Plant hormones (IAA) have multiple biological functions such as promoting plant cell division, elongation, differentiation and root development. Therefore, the synthesis and secretion of IAA is considered to be one of the key mechanisms for promoting plant growth. Probiotics can significantly improve plant growth conditions and enhance plant adaptability to adversity by secreting IAA, especially in soil environments with saline-alkali stress. The results showed that Sinomicrobium 7-7 has a certain IAA production capacity, and the yield can reach 16.77±1.2nmol / L, indicating that it has a certain potential for promoting plant growth.

[0075] 3. Determination of iron carrier production capacity

[0076] The seed solution of Chinese microorganism 7-7 was prepared by the method in Example 1, and inoculated into TSB liquid medium at a 1% inoculum, cultured at 30°C, 200 rpm for 24 h, centrifuged at 12000 rpm for 15 min, and the supernatant was removed by membrane sterilization, mixed with an equal volume of CAS detection solution, reacted fully, and the OD was measured. 630 , and the absorbance value As was obtained, and double distilled water was used as a control to adjust the value to zero; uninoculated culture medium and CAS detection solution were mixed in equal volumes, and OD was measured. 630, and the absorbance value Ar was obtained. The As / Ar value represents the relative content of siderophore in the sample. The siderophore content determination was repeated 3 times. As / Ar ranged from 1.0 to 0 with an interval of 0.2. Every time As / Ar decreased by 0.2, a "+" was added. There were five levels from "+" to "+++++". The higher the level, the stronger the siderophore production ability of the strain.

[0077] CAS blue detection solution formula: Solution A: dissolve 60.5 mg CAS in 50 mL deionized water, add 10 mL FeCl3 solution (containing 1 mM FeCl3·6H2O, 10 mM HCl); Solution B: dissolve 72.9 mg HDTMA (hexadecyltrimethylammonium bromide) in 40 mL deionized water; slowly inject solution A into solution B along the wall of the beaker to obtain CAS blue detection solution.

[0078] Siderophores are substances that chelate iron and are secreted by microorganisms under low intracellular iron concentrations. Most beneficial bacteria can secrete low-molecular-weight, strong-chelating siderophores in low-iron environments, which improve the absorption and transportation of iron ions by microorganisms, increase the mobility of iron ions in the environment, and promote plant growth in iron-deficient environments. In addition, the affinity of bacterial siderophores is generally higher than that of fungal siderophores, and bacteria can use the siderophores they produce to deprive pathogenic fungi of the iron ions chelated by siderophores. Therefore, the ability to produce siderophores is an important evaluation indicator of the growth-promoting / disease-resistant ability of probiotic microorganisms. The siderophore production capacity of Sinomicrobacterium 7-7 is at the fourth level "++++", indicating that it has a strong ability to produce siderophores, that is, it has potential plant growth-promoting functions.

[0079] 4. Biofilm formation ability test

[0080] The seed solution of S. sinensis 7-7 was prepared by the method in Example 1, and inoculated into a 48-well plate containing 1 mL of fresh TSB medium at a 1% inoculum. The plate was sealed with plastic wrap and cultured statically in a 30°C biochemical incubator. The biofilm formation was observed at different time points and photographed. The biofilm-forming ability of the strain is the key to the strain colonizing on the root surface of the plant and exerting its growth-promoting ability. The stronger its biofilm-forming ability is, the stronger its ability to colonize the root surface is. The results are as follows: Figure 4 S. sinensis 7-7 has the ability to form biofilms, but its biofilm-forming ability is weak, indicating that its plant growth-promoting effect may not occur through direct colonization on the plant surface, but may be caused by the release of growth-promoting substances or colonization in the plant roots.

[0081] Example 3: Test on promoting salt-tolerant germination of seeds by using Sinomicrobacterium 7-7

[0082] a. Preparation of bacterial suspension

[0083] After activation, the Chinese microbacterium 7-7 strain was inoculated in TSB liquid medium at an inoculum volume of 1% and cultured at 30°C and 200rpm. The supernatant was removed by centrifugation to collect the bacteria, and the bacteria were washed 3 times with a sterile NaCl solution (250mM for rice seeds and 100mM for tomato seeds) to a final concentration of OD = 1. The bacterial suspension at OD = 1 was diluted with a sterile NaCl solution (250mM for rice seeds and 100mM for tomato seeds) to obtain the test bacterial suspensions: the original concentration bacterial suspension (7-7_1), the bacterial suspension diluted 10 times (7-7_2), and the bacterial suspension diluted 100 times (7-7_3).

[0084] b. Seed treatment

[0085] Select an appropriate amount of rice / tomato seeds with full grains and comparable grain size, clean the surface dust with distilled water, soak them in 1% NaClO solution for 2 minutes, soak them in 75% alcohol for 5 minutes, rinse them with sterile deionized water for 5 to 7 times, and soak the sterilized rice / tomato seeds in sterile deionized water at room temperature for 24 hours for later use.

[0086] c. Germination test

[0087] Take 20 rice / tomato seeds and place them evenly in a 9 cm petri dish covered with two layers of filter paper. Five treatments were set up in the experiment: ddH2O treatment (10 mL sterile ddH2O), NaCl treatment [10 mL sterile NaCl solution (250 mM for rice seeds and 100 mM for tomato seeds)], 7-7_1 treatment (10 mL 7-7_1 bacterial suspension), 7-7_2 treatment (10 mL 7-7_2 bacterial suspension), 7-7_3 treatment (10 mL 7-7_3 bacterial suspension). Use a pipette to slowly inject the solution in the treatment into the edge of the petri dish (note that the liquid should not directly impact the seeds), and then shake the petri dish slightly to spread the solution evenly on the petri dish. Three replicates of each treatment were set up in a light incubator (25°C, 80% RH) and cultured until the germination rate of seeds in the ddH2O group reached more than 90%. The experiment was then terminated and a series of data such as germination rate, seed root length (seeds germinate first and then root, so seed root length was calculated), root MDA content, and antioxidant enzyme activity were recorded and measured.

[0088] In addition to the ion toxicity of the high salt environment, it also forms hyperosmotic stress, which jointly inhibits seed germination. The Chinese microbacterium 7-7 of the present invention can significantly alleviate the inhibition of rice seed germination by the high salt environment, and improve the germination rate and root length of rice seeds in the high salt environment ( Figure 5). When plants are subjected to salt stress, reactive oxygen species (ROS) will accumulate in the body, causing serious damage to cells and tissues. Malondialdehyde (MDA) is one of the products of plasma membrane peroxidation. Its content can indicate the degree of membrane peroxidation, that is, the degree of damage to the cell membrane, and can reflect the degree of damage to cells or tissues under oxidative stress conditions. Sinomicrobium 7-7 can significantly reduce the content of malondialdehyde (MDA) in rice seedlings, indicating that it can reduce the degree of damage to rice caused by salt stress. Catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), as important components of the antioxidant enzyme system in plants, can work together to remove ROS produced by plants under salt stress and maintain the redox balance in cells. Sinomicrobacterium 7-7 can significantly increase the activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in rice seedlings, proving that Sinomicrobacterium 7-7 can alleviate the oxidative damage caused by salt stress in rice seedlings by activating the antioxidant enzyme system, thereby promoting the germination of rice seeds ( Figure 6 ).

[0089] Sinomicrobacterium 7-7 can significantly alleviate the inhibition of high-salt environment on tomato seed germination and promote the germination rate and root length of tomato seeds under high-salt stress ( Figure 7 ). Inoculation with S. chinensis 7-7 can significantly reduce the malondialdehyde (MDA) content in tomato seedlings, indicating that it can alleviate the damage of salt stress to tomatoes. At the same time, the bacteria can also significantly increase the activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in tomato seedlings, proving that S. chinensis 7-7 can reduce the oxidative damage caused by salt stress in tomatoes by activating the antioxidant enzyme system, thereby promoting tomato seed germination ( Figure 8 ).

[0090] Example 4, hemolytic reaction of S. sinensis 7-7

[0091] S. sinensis 7-7 was inoculated on a sheep blood plate (purchased from Hangzhou Shanghe Biotechnology Co., Ltd.), incubated at 30°C for 24 hours, and no hemolysis zone was observed ( Fig. 9 ), that is, the hemolytic reaction is negative and can be used for the development and utilization of fertilizers.

[0092] Example 5: Potted plant experiment on bacterial strains promoting salt tolerance growth

[0093] 1. After surface disinfection of Mingyu 1203 corn seeds, sow them in moist vermiculite and place them in a greenhouse for cultivation. At the cotyledon stage, select seedlings with uniform growth and transplant them into pots (1 L in volume, with a tray containing 800 mL of substrate) filled with seedling cultivation peat.

[0094] 2. 5 days after corn seedlings are transplanted, the potted plants are treated as follows:

[0095] 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.

[0096] NaCl group: A total of 32.18 g NaCl was added (making the final salt concentration in the substrate about 700 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 550 mM NaCl solution was added every 3 days, for a total of 10 times.

[0097] NaCl+7-7 group: In addition to the addition of NaCl to the NaCl treatment group, Chinese Microbacterium 7-7 was inoculated when the NaCl solution was first added, so that the final concentration of Chinese Microbacterium 7-7 was 1×10 7 CFU g -1 earth.

[0098] 3. The potted plants were cultivated in the greenhouse for another 6 weeks, and the growth indexes of corn plants (fresh weight, dry weight, plant height and root length), photosynthesis-related indexes (net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration), plant cell osmosis-related indexes (proline Pro content and total soluble sugar), plant oxidative damage and antioxidant enzyme system (MDA content, CAT activity, POD activity and SOD activity in the aboveground part, MDA content, CAT activity, POD activity and SOD activity in the underground part) were observed, recorded and measured. The photosynthetic efficiency of corn leaves was measured using the advanced photosynthetic fluorescence meter LI-6800 (the specific parameters were set as Flow: Flow 500.0 μmol s -1 ΔP: 0.1kPa; H2O: RH_air: 60%; CO2: 400μmol mol -1 ;Fan:Speed ​​10,000rpm;Temperature:off;Light:1500μmol m -2 s -1 ). The MDA content, CAT activity, POD activity, SOD activity, Pro content and soluble total sugar in the aboveground and underground parts were determined using commercial kits (all purchased from Solebow Biotechnology Co., Ltd.). The determination method is detailed in the kit instructions. P is the P value, and FC (Fold Change) is the difference fold.

[0099] The results showed that Sinomicrobacterium 7-7 could improve the growth indexes of corn (plant height, fresh weight and dry weight, etc.) under salt stress and ensure the normal growth of corn ( Fig.10). It shows that Sinomicrobacterium 7-7 has a superior growth-promoting effect on corn under extremely severe salt stress (700mM NaCl), and the microbial agent developed using it has broad prospects in saline-alkali land improvement and crop growth guarantee.

[0100] Net photosynthetic rate, transpiration rate, stomatal conductance and intercellular carbon dioxide concentration are key physiological indicators for measuring plant photosynthesis and water use. There is a close relationship between them and they are of great significance to plant growth and environmental adaptability. Net photosynthetic rate directly affects the carbon fixation capacity and growth rate of plants. A high photosynthetic rate means that plants can more effectively convert carbon dioxide into organic matter, promoting their growth and development. Transpiration plays an important role in regulating water balance in plants and promoting nutrient absorption. Moderate transpiration helps to obtain carbon dioxide and cool leaves, maintaining a high photosynthetic efficiency. Stomatal conductance reflects the absorption efficiency of carbon dioxide and the transpiration of water during photosynthesis. High stomatal conductance usually means that plants can better absorb carbon dioxide for photosynthesis, thereby increasing the photosynthetic rate. Intercellular CO2 concentration reflects the utilization efficiency of carbon dioxide in the photosynthetic process of plants. Low intercellular CO2 concentration usually indicates that carbon dioxide is being actively used, while high intercellular CO2 concentration may indicate that stomata are closed or photosynthesis is inhibited. Sinomicrobacterium 7-7 can significantly increase the net photosynthesis rate, transpiration rate and stomatal conductance of corn, while significantly reducing the intercellular CO2 concentration of corn, indicating that the strain effectively promotes the photosynthesis efficiency of corn. The increase in stomatal conductance promotes the entry of carbon dioxide, enhances the carbon dioxide fixation capacity during photosynthesis, and allows more CO2 to be quickly utilized, thereby reducing the intercellular CO2 concentration. At the same time, the increase in transpiration rate shows that the opening of stomata not only promotes the absorption of carbon dioxide, but also enhances the loss of water, which helps to regulate leaf temperature, prevent overheating, and improve the water transport efficiency of plants. This shows that Sinomicrobacterium 7-7 significantly promotes photosynthesis by increasing stomatal conductance, enhancing carbon absorption and regulating transpiration. This effect helps plants maintain efficient photosynthetic metabolism under environmental stress, promotes healthy growth and increases yield of plants ( Fig.11 ).

[0101] Under salt stress conditions, plants usually experience osmotic stress and ion stress, which lead to intracellular water loss, ion toxicity and metabolic disorders. In response to these stresses, plants produce and accumulate some osmotic regulating substances to maintain intracellular water balance, protect cell structure and reduce salt damage. Sinomicrobium 7-7 can significantly increase the proline (Pro) content in the aboveground part of corn and the total soluble sugar in corn plants, ensure the stability of the internal water content of corn under hyperosmotic stress, and prevent it from losing water and dying under salt stress ( Fig.12 ).

[0102] Sinomicrobium 7-7 can reduce the MDA content in the aboveground and underground parts of corn, indicating that it can reduce the damage of corn to salt stress. Sinomicrobium 7-7 can significantly increase the activity of catalase (CAT) and peroxidase (POD) in the aboveground part of corn, and significantly reduce the activity of superoxide dismutase (SOD) in the aboveground part of corn; significantly reduce the activity of catalase (CAT) in the underground part of corn, and significantly increase the activity of superoxide dismutase (SOD) in the underground part of corn. This shows that Sinomicrobium 7-7 can alleviate the stress effect of corn plants under salt adversity by regulating the above three antioxidant enzyme systems ( Fig.13 ).

Claims

1. A strain of Chinese microorganisms of pectin degrading bacteria ( Sinomycrobium pectinolyticum )7-7, and its deposit number is CGMCCNo.31313.

2. A composition containing the pectin-degrading Chinese microorganisms according to claim 1 ( Sinomycrobium pectinolyticum )7-7 of the fungal agent.

3. The pectin-degrading Chinese microorganism of claim 1 ( Sinomycrobium pectinolyticum ) 7-7 or the use of the bacterial agent described in claim 2 in increasing plant biomass under salt stress environment, wherein the plant is corn.

4. The pectin-degrading Chinese microorganism of claim 1 ( Sinomycrobium pectinolyticum ) 7-7 or use of the bacterial agent described in claim 2 in improving the germination rate of plant seeds under salt stress environment, wherein the plant is rice or tomato.

Images