A drug-resistant and stress-resistant Sinorhizobium freundii, a bacterial agent and its application

Abstract

The invention discloses a drug-resistant and stress-resistant Sinorhizobium freundii, a bacterial agent and an application thereof, belonging to the technical field of microbial application. The invention screens out a strain of Sinorhizobium freundii ( Sinorhizobium fredii ) NBL‑LR‑1, its biological deposit number is CCTCC NO: M 20241790; strain NBL‑LR‑1 has a wide range of drug resistance and can tolerate a variety of common soybean seed dressing agents, so the strain can be used in combination with a variety of commonly used seed dressing agents; strain NBL‑LR‑1 also has excellent salt resistance, alkali resistance and drought resistance, and can infect multiple soybean varieties in different regions such as Northeast China, North China, the Yangtze River Basin, Xing'an League of Inner Mongolia, and has a wide range of applications.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a drug-resistant and stress-resistant Sinorhizobium freundii, a bacterial agent and applications thereof. Background Art

[0002] As an important food and feed raw material, the demand for soybeans is increasing year by year. Therefore, it is an urgent practical problem to improve the yield and quality of soybeans without increasing the cultivated area.

[0003] Inducing nodules to form in the roots of leguminous crops and establishing a symbiotic nitrogen fixation system are the main ways for rhizobia to improve the yield and quality of leguminous crops. As a type of Gram-negative bacteria widely distributed in the soil, rhizobia play an important role in the nitrogen cycle of agricultural ecosystems. Symbiotic nodulation nitrogen fixation and non-leguminous combined nitrogen fixation are the two biological nitrogen fixation methods that can be applied to agricultural production so far. Compared with the latter, rhizobia have a stronger dependence on leguminous crops. By forming bacteroids in the invaded cells of soybean roots, rhizobia can provide soybeans with 1 / 2~1 / 3 of the nitrogen needed for growth. However, non-leguminous combined nitrogen fixation is easily affected by environmental stress (acid-base, high salt), resulting in low nitrogen fixation efficiency. Its nitrogen fixation in nature is generally only 1~50 kg pure nitrogen / hectare / year.

[0004] Seed dressing with microbial agents is the main way to inoculate rhizobia in the field. However, in order to prevent and control common soil-borne diseases such as soybean root rot, damping-off, and damping-off, as well as other underground pests, farmers usually use chemical agents to coat soybean seeds before sowing. Studies have shown that common ingredients in seed dressing agents such as metalaxyl, fludioxonil, carboxin, azoxystrobin, and thiram have strong bactericidal effects on Gram-negative bacteria. Rhizobia are Gram-negative bacteria, which limits their use in combination with seed dressing agents. Therefore, screening rhizobia with strong drug resistance and good nodulation and nitrogen fixation effects has important practical significance for their promotion and application. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a drug-resistant and stress-resistant Sinorhizobium freundii, a bacterial agent and its application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a drug-resistant and stress-resistant Sinorhizobium freundii ( Sinorhizobium fredii )NBL-LR-1; the strain was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on August 14, 2024, and its biological deposit number is CCTCCNO: M 20241790.

[0008] The Sinorhizobium favreii ( Sinorhizobium fredii )NBL-LR-1, has the following characteristics:

[0009] The bacteria are short rod-shaped, 0.5~0.9μm×1.2~3.0μm, non-spore-forming, Gram-negative, with red-stained diaphragms inside; the colonies are round or nearly round, sticky, smooth, slightly convex, milky white, moist on the surface, and 2~4 mm in diameter. The colonies are pink on YEMA Congo red medium.

[0010] The second aspect of the present invention provides a bacterial agent comprising the above-mentioned Sinorhizobium freundii.

[0011] Preferably, the bacterial agent may be in the form of a liquid, emulsion, suspension, powder, granule, wettable powder or water dispersant.

[0012] Preferably, in the bacterial agent, Sinorhizobium freundii exists in the form of cultured live bacteria, fermentation liquid or bacterial suspension.

[0013] Preferably, the fermentation broth of Sinorhizobium freundii is prepared by the following method:

[0014] The Chinese rhizobium frugiperda was inoculated into the YMB medium containing calcium carbonate, and the culture was carried out in a shaking incubator at 30°C and 180 rpm for 40-48 hours;

[0015] The YMB medium containing calcium carbonate is composed of: 10.0 g of mannitol, 3 g of calcium carbonate, 1.0 g of yeast extract powder, 0.2 g of magnesium sulfate heptahydrate, 0.5 g of dipotassium hydrogen phosphate, 0.1 g of sodium chloride, and 1 L of distilled water.

[0016] The third aspect of the present invention provides the use of the above-mentioned Sinorhizobium freundii or a bacterial agent containing Sinorhizobium freundii in combination with a seed dressing containing an antibacterial component in soybean seed treatment.

[0017] Preferably, the specific method of the above application is:

[0018] Mixing soybean seeds, a seed dressing agent containing antibacterial components and a bacterial agent containing Sinorhizobium freundii for seed dressing;

[0019] Alternatively, the soybean seeds are firstly dressed with a seed dressing agent containing antibacterial components, and then the bacterial agent containing Sinorhizobium freundii is sprayed on the soybean seeds.

[0020] Preferably, the antibacterial component is selected from tebuconazole, metalaxyl-M, thiamethoxam, imidacloprid, fludioxonil, azoxystrobin, prochloraz, thiram or carboxin.

[0021] In a fourth aspect of the present invention, the use of the above-mentioned Sinorhizobium freundii NBL-LR-1 in at least one of the following (1) to (5) is provided:

[0022] (1) Promote crop growth;

[0023] (2) Improve crop drought tolerance;

[0024] (3) Salt tolerance;

[0025] (4) Alkali resistance;

[0026] (5) Promote the increase of nodule number and fresh weight of crops.

[0027] Preferably, the crop is soybean.

[0028] Beneficial effects of the present invention:

[0029] 1. The strain Sinorhizobium freundii of the present invention ( Sinorhizobium fredii ) NBL-LR-1 has a wide range of drug resistance and can tolerate a variety of common seed dressing agents on the market that contain antibacterial components (tebuconazole, metalaxyl-M, thiamethoxam, imidacloprid, fludioxonil, myclobutanil, prochloraz, thiram, carboxin, etc.). Therefore, this strain can be used in combination with a variety of commonly used seed dressing agents, which have insecticidal and fungicidal effects, thereby protecting soybean seeds during sowing and reducing the risk of diseases and pests.

[0030] 2. The strain NBL-LR-1 described in the present invention also has good growth ability, especially it can still grow normally in an environment of salinity 3% and pH 11.0, and has excellent drug resistance, salt resistance and alkali resistance; in addition, the Sinorhizobium fredii NBL-LR-1 described in the present invention has a growth-promoting effect, promotes soybean nodulation, and can significantly promote soybean growth under drought conditions, and has excellent growth-promoting and drought-resistance capabilities.

[0031] 3. The "host specificity" of the currently reported rhizobia is relatively strong. Each species and strain of rhizobia can only infect certain legume species. The strain NBL-LR-1 described in the present invention can infect multiple soybean varieties in different regions such as the Northeast, North China, the Yangtze River Basin, and Xing'an League, Inner Mongolia, and has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the source of samples for screening strain NBL-LR-1 in Example 1;

[0033] Figure 2 is a colony morphology diagram of strain NBL-LR-1 in Example 1;

[0034] Figure 3is a bacterial morphology diagram of strain NBL-LR-1 in Example 1;

[0035] Figure 4 1 is a growth curve diagram of Sinorhizobium freundii NBL-LR-1 and a diagram showing changes in pH value of the fermentation broth in Example 2; wherein (a) is a growth curve diagram, and (b) is a diagram showing changes in pH value of the fermentation broth;

[0036] Figure 5 The results of the test of the resistance of Sinorhizobium freundii NBL-LR-1 to different seed dressing agents in Example 3;

[0037] Figure 6 This is a test diagram of promoting soybean drought resistance by Sinorhizobium freundii NBL-LR-1 in Example 4; wherein (a) is a test diagram of soybean drought resistance in the control group, and (b) is a test diagram of soybean drought resistance in the test group;

[0038] Figure 7 These are the test results of the germination of different varieties of soybeans by Sinorhizobium freundii NBL-LR-1 in Example 6. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0040] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0041] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0042] The culture medium used was as follows:

[0043] LB medium: peptone 10.0 g, yeast extract 5.0 g, sodium chloride 10.0 g, distilled water 1 L, pH 7.

[0044] YEMA Congo red medium: mannitol 10 g, yeast powder 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl 0.1 g, 0.4% Congo red 10 mL, agar powder 20 g, distilled water 1000 mL, sterilize at 121℃ for 20 min.

[0045] YMA medium: mannitol 10g, yeast powder 1g, K2HPO4 0.5g, MgSO4·7H2O 0.2g, NaCl 0.1g, agar powder 20g, distilled water 1000mL, sterilized at 121℃ for 20min. YMB medium is based on YMA medium without agar powder.

[0046] YMA medium containing calcium chloride: 10.0 g glucose, 1.0 g yeast powder, 0.2 g magnesium sulfate heptahydrate, 0.5 g potassium hydrogen phosphate, 0.1 g sodium chloride, 0.05 g calcium chloride, 20 g agar powder, 1 L distilled water, pH 7. YMB medium containing calcium chloride is YMA medium containing calcium chloride with agar powder omitted.

[0047] YMA medium containing calcium carbonate: 10.0 g mannitol, 3 g calcium carbonate, 1.0 g yeast extract powder, 0.2 g magnesium sulfate heptahydrate, 0.5 g potassium hydrogen phosphate, 0.1 g sodium chloride, 20 g agar powder, 1 L distilled water, pH 7. YMB medium containing calcium carbonate is based on YMA medium containing calcium carbonate, but agar powder is omitted.

[0048] SM medium: glucose 10.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.2 g, sodium chloride 0.1 g, calcium chloride 0.05 g, potassium nitrate 0.5 g, agar powder 20 g, distilled water 1 L, pH 7.

[0049] Glucose yeast medium: glucose 10 g, yeast extract 1 g, sodium chloride 10 g, agar powder 20 g, distilled water 1 L, pH 7;

[0050] Bean sprout extract medium (BSE medium): glucose 10 g, sodium chloride 10 g, bean sprout extract 1 L, pH 7.0;

[0051] Wherein the bean sprout extract is prepared by the following method:

[0052] Weigh 500g of fresh soybean sprouts, add 1000mL of water, boil on an uncovered 1200W induction cooker, set timer for 30min, filter out the liquid, repeat this 2-3 times, and adjust the volume of the collected liquid to 500mL.

[0053] Bean sprout juice medium: glucose 10 g, sodium chloride 10 g, soybean sprout juice 1 L, pH 7.0;

[0054] The soybean sprout extract is prepared by the following method: 500 g of fresh soybean sprouts are weighed, the juice is squeezed, diluted with water to 2000 mL, and then directly sterilized, and the supernatant is taken.

[0055] Hoagland nutrient solution: potassium sulfate 607 mg, diammonium phosphate 115 mg, magnesium sulfate 493 mg, sodium iron EDTA 20 mg, ferrous sulfate 15 mg, boric acid 2.86 mg, borax 4.5 mg, manganese sulfate 2.13 mg, copper sulfate 0.05 mg, zinc sulfate 0.22 mg, ammonium sulfate 0.02 mg, calcium nitrate 0.945 g, distilled water 1 L, sterilize at 115°C for 20 min.

[0056] Example 1: Isolation and identification of strains

[0057] 1. Isolation and screening of strains

[0058] Isolation of strains: Dig out the whole soybean plants with good growth ( Figure 1 ), put them into a plastic bag together with the soil around the roots. After returning to the laboratory, gently shake off the soil attached to the roots to expose the nodules, collect the light pink or pink plump nodules, and store them at 4℃ for later use.

[0059] Under sterile conditions, the collected nodules were rinsed with sterile water, soaked in 75% ethanol solution for 30 seconds, rinsed with sterile water 5 times, soaked in 5% sodium hypochlorite solution for 5 minutes, and rinsed with sterile water 8 times. Then, they were placed in a sterile mortar and ground with a sterile mortar stick to obtain the rhizobium stock solution. The dilution method (1 mL of the rhizobium stock solution was taken with a 1 mL sterile pipette and added to a 9 mL sterile water tube and shaken thoroughly) was used to obtain 10 -1 dilution, and so on) the rhizobium solution is mixed with the original solution, 10 -1 , 10 -2 The dilution multiples were spread on YEMA Congo red medium, cultured in a constant temperature box at 28℃, and the growth was observed at any time.

[0060] Purify on YMA medium using the dilution plate streak method, repeat 2-3 times until a single colony appears. Pick a single colony, inoculate it into YMA medium, and store it in a 4°C refrigerator for later use.

[0061] Screening of strains: Based on the characteristics of rhizobium colonies, which are round, opaque, milky white, convex, and moist and shiny surfaces, a suspected rhizobium strain was initially obtained for identification and named NBL-LR-1.

[0062] 2. Strain Identification

[0063] 2.1 Morphological observation

[0064] A single colony of strain NBL-LR-1 was streaked onto YMA solid plate medium, and the plate was inverted in a constant temperature incubator and cultured at 28°C for 3 days.

[0065] like Figure 2As shown, the colonies of NBL-LR-1 were round or nearly round, sticky, smooth, slightly convex, milky white, with a moist surface, and a diameter of 2 to 4 mm.

[0066] like Figure 3 As shown, the colonies on the YEMA Congo red medium are pink. Microscopic observation of strain NBL-LR-1 is short rod-shaped, 0.5~0.9 μm×1.2~3.0 μm, non-spore-forming, Gram-negative, and has a red-stained diaphragm inside the bacteria.

[0067] 2.2 Physiological and biochemical identification

[0068] The physiological and biochemical characteristics and sole carbon source utilization of strain NBL-LR-1 were determined using Haibo Biotechnology microbial biochemical identification strips. The detection method was referred to the Manual of Identification of Common Bacterial Systems, see Table 1.

[0069] Table 1 Physiological and biochemical characteristics and sole carbon source utilization of strain NBL-LR-1

[0070]

[0071] Note: "+" represents positive, "-" represents negative;

[0072] As shown in Table 1 , strain NBL-LR-1 can grow with mannitol, various sugars (glucose, lactose, fructose, xylose, cellobiose, D-mannose, D-galactose, D-arabinose, D-ribose, and D-turanose) and small organic molecules (L-glutamine and succinate) as the sole carbon sources and can reduce nitrate, indicating that it has a strong ability to adapt to the environment.

[0073] 2.3 Molecular Biological Identification

[0074] The 16S rDNA gene sequence analysis method was used to identify the genus and species of the isolated strain NBL-LR-1. The 16S rDNA gene sequence is shown in SEQ ID NO.1. The identification results showed that the strain NBL-LR-1 belonged to Sinorhizobium freundii ( Sinorhizobium fredii ).

[0075] Based on the results of morphological, physiological and biochemical, and molecular biological identification, strain NBL-LR-1 was identified as Sinorhizobium freundii and named as Sinorhizobium freundii ( Sinorhizobium fredii )NBL-LR-1. The strain was biologically deposited, and the deposit information is as follows:

[0076] Strain name: Sinorhizobium freundii NBL-LR-1

[0077] Latin name: Sinorhizobium fredii

[0078] Collection institution: China Center for Type Culture Collection

[0079] Abbreviation of depository institution: CCTCC

[0080] Address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0081] Storage date: August 14, 2024

[0082] Deposit number: CCTCC NO: M 20241790.

[0083] Example 2: Preparation of Sinorhizobium freundii NBL-LR-1 fermentation broth

[0084] 1. Culture medium optimization

[0085] Take the bacterial solution of Sinorhizobium freundii NBL-LR-1 in the glycerol tube and streak it into three zones in YMA medium, culture it at 30°C for 3 days, take a single colony from the three zones and inoculate it into fresh YMB medium, and culture it in a shaker at 30°C and 180 r / min for 24 hours to obtain the seed solution of Sinorhizobium freundii NBL-LR-1.

[0086] The seed liquid was inoculated into LB medium, YMB medium containing calcium chloride, YMB medium containing calcium carbonate, YMB medium, SM medium, glucose yeast medium, bean sprout extract medium (BSE medium) and bean sprout juice medium at an inoculum size of 2% (volume ratio), respectively, and cultured at 30°C and 180 r / min in a shaking incubator for 39 h. The culture was then spread and counted in YMA medium to screen the best fermentation medium, as shown in Table 2.

[0087] Table 2 The number of viable bacteria of Sinorhizobium freundii NBL-LR-1 fermented in different culture media

[0088]

[0089] As shown in Table 2, the optimal fermentation medium for Sinorhizobium freundii NBL-LR-1 was YMB medium containing calcium carbonate, with a viable cell count of 2.0 × 10 9 CFU / mL, and this culture medium will be used for subsequent fermentation.

[0090] 2. Preparation of Fermentation Broth

[0091] The seed liquid of Sinorhizobium freundii NBL-LR-1 was inoculated into YMB medium containing calcium carbonate at a 2% inoculation rate, and cultured in a constant temperature shaker at 30°C and 180 r / min for 48 h to obtain the fermentation liquid of Sinorhizobium freundii NBL-LR-1.

[0092] During the fermentation process, samples were taken every 2 h, and 3 mL was sampled each time for OD 600 and pH were measured, and the growth curve of Sinorhizobium freundii NBL-LR-1 was drawn.

[0093] like Figure 4 (a) and Figure 4 As shown in (b), the fermentation cycle of Sinorhizobium freundii NBL-LR-1 is 48 h, with a delay period of 0-14 h, a logarithmic growth period of 14-26 h, and a stable period of 26-48 h. The pH value in the culture medium gradually decreases, indicating that Sinorhizobium freundii NBL-LR-1 has the ability to produce acid.

[0094] Example 3: Drug resistance test of Sinorhizobium freundii NBL-LR-1

[0095] 1. Test materials

[0096] Seed dressing agents: Boshidun (Miaoboshi), Aobary (Bayer), Miaodeyi (Mingdelida), Liangdun (Syngenta), Yingdun (Yinghui), Jingqiao (Xianglong) are all commonly used commercially available soybean seed dressing agents.

[0097] 2. Test methods

[0098] 200 μL of the fermentation broth of Sinorhizobium fredii NBL-LR-1 prepared in Example 2 (2×10 9 CFU / mL), and spread evenly with a sterile coating rod. Immerse φ=8mm sterile filter paper in the above commonly used commercial soybean seed dressing agent, stick it on the YMA medium coated with the fermentation liquid of Sinorhizobium fredii NBL-LR-1, and culture at 25℃ for 7 days to observe the growth of the strain.

[0099] 3. Test results

[0100] Table 3 Commonly used soybean seed dressing agents on the market, their active ingredient concentrations, and the drug resistance of strain NBL-LR-1

[0101]

[0102] Note: “-” indicates drug resistance, with no transparent circle;

[0103] As shown in Table 3 and Figure 5 As shown, no transparent circle appeared around the filter paper soaked with the above seed dressing agents, indicating that Sinorhizobium fredii NBL-LR-1 can tolerate the above 6 seed dressing agents and has a wide adaptability.

[0104] Example 4: Effect of Sinorhizobium freundii NBL-LR-1 on soybean stress resistance

[0105] 1. Salt tolerance test of Sinorhizobium freundii NBL-LR-1

[0106] Sinorhizobium fredii NBL-LR-1 was streaked on YMA medium containing 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% NaCl, respectively. The medium without NaCl was used as a negative control. The culture was carried out at a constant temperature of 28°C for 5 days, and the growth of the colonies was observed.

[0107] 2. Alkali resistance test of Sinorhizobium freundii NBL-LR-1

[0108] Sinorhizobium fredii NBL-LR-1 was streaked on YMA medium with pH values ​​of 5.0, 6.0, 8.0, 9.0, 10.0, 11.0 and 12.0, respectively, with the natural pH value (7.0) as the negative control. The culture was carried out at 28℃ for 5-7 days, and the colony growth was observed.

[0109] 3. Effect of Sinorhizobium freundii NBL-LR-1 on drought tolerance of soybean

[0110] Plastic Holland pots (50cm×20cm×15cm) were used for soil culture. The pots were soaked in 1g / L potassium permanganate solution overnight, rinsed with sterile water, and dried for later use. Soybean seeds with basically the same size and intact seed coat were selected. The soybean variety was Hedou 33. They were treated with 95% ethanol for 1 min, washed with sterile water 2-3 times, treated with 1% sodium hypochlorite solution for 5 min, washed with sterile water 5-7 times, and placed in a sterile culture dish covered with filter paper at 28°C for germination. When the radicle grew to about 1 cm, the radicle was transplanted downward into a plastic Holland pot (50cm×20cm×15cm) containing about 7.5 kg of soil, with 8 seeds per pot.

[0111] In the treatment group, 1 mL of the fermentation liquid of Sinorhizobium fredii NBL-LR-1 prepared in Example 2 (2×10 9 CFU / mL), 1 mL of sterile water was added around each seed in the control group, and 1000 mL of Hoagland nutrient solution was poured into each pot of soybean every 10 days. Three pots were set up for the treatment group and the control group, and the soybean was watered once every 5 days. The amount of watering was determined according to the results of soil moisture measurement to keep the soil in moderate drought conditions (absolute moisture content 40%~45%). Plant height, stem diameter and leaf SPAD value were measured after soybeans grew to 60 days. The determination methods of various indicators are as follows:

[0112] (1) Plant height: Use a ruler to measure the height from the soil surface to the top growth point of the plant.

[0113] (2) Stem diameter: measured with a vernier caliper, 5 cm from the soil at the base of the stem.

[0114] (3) SPAD value: The SPAD value of soybean leaves was measured on a sunny morning using a SPAD 502 chlorophyll content meter.

[0115] 4. Test results

[0116] 4.1 Results of salt tolerance test of Sinorhizobium freundii NBL-LR-1

[0117] Table 4 Salt tolerance of Sinorhizobium freundii NBL-LR-1

[0118]

[0119] Note: “+” indicates growth, and “-” indicates no growth.

[0120] As shown in Table 4, Sinorhizobium freundii NBL-LR-1 can tolerate at least 3% salinity, indicating that it can tolerate high-salinity environments and has a wide range of adaptability.

[0121] 4.2 Alkali resistance test results of Sinorhizobium freundii NBL-LR-1

[0122] Table 5 Identification of alkaline stress resistance of Sinorhizobium freundii NBL-LR-1

[0123]

[0124] Note: "+" indicates growth, "-" indicates no growth, and "±" indicates reduced growth ability.

[0125] As shown in Table 5, Sinorhizobium freundii NBL-LR-1 can tolerate at least pH 11.0, indicating that Sinorhizobium freundii NBL-LR-1 can grow and reproduce in an alkaline environment and has a wide adaptability.

[0126] 4.3 Drought resistance of S. fredii NBL-LR-1

[0127] Table 6 Growth indexes of soybeans under different treatments

[0128]

[0129] Note: Compared with the control group, * indicates significant difference (P < 0.05).

[0130] As shown in Table 6, compared with the control group, the plant height, stem diameter and SPAD value of soybeans in the treatment group increased by 22.90%, 15.65% and 5.92%, respectively, indicating that Sinorhizobium fredii NBL-LR-1 has the ability to promote soybean growth under drought conditions and improve soybean drought tolerance and environmental adaptability. In addition, the SPAD value of plant leaves is positively correlated with the nitrogen level in the body, indicating that inoculation of rhizobia effectively increases the nitrogen supply level.

[0131] from Figure 6 It can be seen that under drought stress, the number of main stem nodes and branches of soybeans in the control group was significantly reduced, and serious dehydration phenomena such as necrosis of growing points and curling and yellowing of leaves occurred. The soybean leaves in the treatment group did not show the above symptoms, and the photosynthetic leaf area was significantly larger than that in the control group, which was conducive to the accumulation of photosynthetic products and soybean pod formation.

[0132] Example 5: Effect of Sinorhizobium freundii NBL-LR-1 combined with seed dressing on soybean nodulation

[0133] 1. Test methods

[0134] Plastic flower pots were used for soil culture test. The flower pots were soaked in 1g / L potassium permanganate solution overnight, rinsed with sterile water, and dried for use. Soybean seeds with basically the same size and intact seed coat were selected. The soybean variety was Heihe 43. They were treated with 95% ethanol for 1 min, washed with sterile water 2-3 times, treated with 1% sodium hypochlorite solution for 5 min, washed with sterile water 5-7 times, and dried for use.

[0135] The experiment set up four treatments:

[0136] Treatment 1: The commercially available seed dressing agent Liangdun (Syngenta) was used for coating at a dosage of 4 mL / kg of bean seeds; and the fermentation liquid of Sinorhizobium fredii NBL-LR-1 (2×10 9 CFU / mL) for seed dressing, the dosage is 3mL / kg bean seeds;

[0137] Treatment 2: The commercially available seed dressing agent Liangdun (Syngenta) was used for coating at a dosage of 4 mL / kg of bean seeds; and the fermentation broth of Sinorhizobium fredii NBL-B0180 (2×10 9 CFU / mL) for seed dressing, the dosage is 3mL / kg bean seeds;

[0138] Treatment 3: No coating, using the fermentation broth of Sinorhizobium freundii NBL-LR-1 prepared in Example 2 (2×10 9 CFU / mL) for seed dressing, the dosage is 3mL / kg bean seeds;

[0139] Treatment 4: No coating, using the fermentation broth of Sinorhizobium freundii NBL-B0180 (2×109 CFU / mL) for seed mixing, the dosage is 3mL / kg bean seeds.

[0140] The coating and seed dressing steps were omitted in the blank control group.

[0141] Immediately after seed dressing, soybean seeds were sown into plastic pots containing 3 kg of soil. All the above treatments were replicated in 6 pots, and Hoagland nutrient solution was watered once every 10 days, 500 mL each time, and other agronomic management measures remained the same. After 30 days of transplanting, the measured indicators included plant height, stem diameter, nodule number, and nodule fresh weight. The measurement methods of each indicator are as follows:

[0142] (1) Plant height: Use a ruler to measure the height from the soil surface to the top growth point of the plant.

[0143] (2) Stem diameter: measured with a vernier caliper, 5 cm from the soil at the base of the stem.

[0144] (3) Number of nodules: Count the nodules after cutting them off with a scalpel. The unit is pieces per plant.

[0145] (4) Fresh weight of nodules: The excised nodules are weighed using a 1 / 1000 balance, and the unit is g / plant.

[0146] 2. Test results

[0147] Table 7 Growth indexes of soybeans under different treatments

[0148]

[0149] As shown in Table 7, when no seed dressing agent was used to coat the soybean seeds, the fermentation liquid of Sinorhizobium freundii NBL-LR-1 and NBL-B0180 showed good growth-promoting effects. Compared with the blank control, when no coating was performed and only Sinorhizobium freundii was used for seed dressing, treatment 3 used Sinorhizobium freundii NBL-LR-1 for seed dressing, which increased the number of soybean nodules and the fresh weight of nodules by 9.23 times and 10.75 times, respectively; treatment 4 used Sinorhizobium freundii NBL-B0180 for seed dressing, which increased the number of soybean nodules and the fresh weight of nodules by 6.07 times and 6.83 times, respectively. In terms of nodulation and nitrogen fixation, the performance of Sinorhizobium freundii NBL-LR-1 was better than that of NBL-B0180.

[0150] Compared with the blank control, when the soybean seeds were coated with seed dressing agents, treatment 1 used Sinorhizobium freundii NBL-LR-1 for seed dressing, and the nodulation and growth promotion effects were not significantly affected. The number of soybean nodules and the fresh weight of nodules increased by 8.27 times and 10.25 times, respectively, which was not significantly different from the indicators measured in treatment 3. Treatment 2 used Sinorhizobium freundii NBL-B0180 for seed dressing, and the measured indicators were not significantly different from the blank control, which may be related to the fact that the strain could not tolerate the fungicidal components (fludioxonil and metalaxyl-M) in the seed dressing agent.

[0151] This example illustrates that Sinorhizobium freundii NBL-LR-1 and its preparation can be mixed with commercially available seed dressing agents without affecting the activity and function of the strain, which has important practical significance in promotion and application.

[0152] Example 6: Effects of Sinorhizobium freundii NBL-LR-1 on growth promotion and nodulation of potted soybean

[0153] 1. Test methods

[0154] 1.1 Effect of Sinorhizobium freundii NBL-LR-1 on soybean growth promotion

[0155] The experiment selected soybean varieties from multiple regions (Heihe 43, Suinong 43, Heike 86, Zhonghuang 37, Gaofeng 1, Xingnong 3, Zhongdou 43, Changjiangchun 2, Gandou 15, Qinong 7, Dengke 13, Suinong 30) for germination test. Soybean seeds with basically the same size and complete structure were treated with 5% sodium hypochlorite solution for 3 min, washed with sterile water 2-3 times, and the treated group was diluted 200 times with the fermentation liquid of Sinorhizobium fredii NBL-LR-1 prepared in Example 2 (2×10 9 CFU / mL) for 1 min, and the control group was soaked in sterile water for 1 min, and then placed on 0.7% agar medium (agar 7g, distilled water 1000mL, sterilized at 121℃ for 30min) and germinated at 28℃ for 72h, and the sprout length of each soybean seed was counted.

[0156] 1.2 Effect of Sinorhizobium freundii NBL-LR-1 on soybean nodulation

[0157] The experiment selected different varieties of soybeans from multiple regions, and used plastic flower pots for soil culture experiments. The flower pots were soaked in 1g / L potassium permanganate solution overnight in advance, rinsed with sterile water, and dried for use. Soybean seeds of basically the same size and intact seed coat were selected, treated with 95% ethanol for 1 min, washed with sterile water 2-3 times, treated with 1% sodium hypochlorite solution for 5 min, washed with sterile water 5-7 times, and placed in a sterile culture dish covered with filter paper at 28°C for germination. When the radicle grew to about 1 cm, the radicle was transplanted downward into a plastic flower pot filled with 3 kg of soil, with one seed per pot.

[0158] In the treatment group, 1 mL of the fermentation liquid of Sinorhizobium fredii NBL-LR-1 prepared in Example 2 (2×10 9 CFU / mL), 1 mL of sterile water was added around the seeds of the control group, and then sterile water was poured irregularly to keep the soil moist. In addition, 100 mL of Hoagland nutrient solution was poured into each pot of soybean every 10 days. Six pots were set up for both the treatment group and the control group. The plant height and root length of soybeans were measured when they grew to the early stage of pod formation, and the complete root system was taken out to count the number of nodules and fresh nodule weight.

[0159] 2. Test results

[0160] 2.1 Effect of Sinorhizobium fredii NBL-LR-1 on soybean growth promotion

[0161] like Figure 7 As shown, the soybean sprout length of Heihe 43, Gaofeng 1, Dengke 13 and Suinong 30 in the treatment group was significantly higher than that in the blank CK group, which had a growth-promoting effect.

[0162] 2.2 Effect of Sinorhizobium freundii NBL-LR-1 on soybean nodulation

[0163] Table 8 Growth indices of 12 soybean varieties treated with strain NBL-LR-1

[0164]

[0165] As shown in Table 8, Sinorhizobium freundii NBL-LR-1 can significantly increase the plant height of Heihe 43, Zhonghuang 37, Gaofeng 1 and Suinong 30 soybeans, has a growth-promoting effect, and can infect soybean varieties from different regions to promote soybean nodulation, which has a wide range of application value.

[0166] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A drug-resistant and stress-resistant Sinorhizobium freundii, characterized in that: This strain is Sinorhizobium freundii ( Sinorhizobium fredii )NBL-LR-1, its biological deposit number is CCTCC NO: M 20241790.

2. A bacterial agent, characterized in that The bacterial agent comprises the Sinorhizobium freundii described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The formulation of the bacterial agent is liquid, emulsion, suspension, powder, granule, wettable powder or water dispersant.

4. The bacterial agent according to claim 2, characterized in that In the bacterial agent, Sinorhizobium freundii exists in the form of cultured live bacteria, fermentation liquid or bacterial suspension.

5. The bacterial agent according to claim 2, characterized in that The fermentation broth of the Chinese rhizobium fredii is prepared by the following method: The Chinese rhizobium frugiperda was inoculated into the YMB medium containing calcium carbonate, and the culture was carried out in a shaking incubator at 30°C and 180 rpm for 40-48 hours; The YMB medium containing calcium carbonate is composed of: 10.0 g of mannitol, 3 g of calcium carbonate, 1.0 g of yeast extract powder, 0.2 g of magnesium sulfate heptahydrate, 0.5 g of dipotassium hydrogen phosphate, 0.1 g of sodium chloride, and 1 L of distilled water.

6. Use of the Sinorhizobium freundii described in claim 1 or the bacterial agent described in any one of claims 2 to 5 in combination with a seed dressing containing antibacterial components in soybean seed treatment.

7. The use according to claim 6, characterized in that: The specific methods of application are: Mixing soybean seeds, a seed dressing agent containing antibacterial components and a bacterial agent containing the Sinorhizobium freundii for seed dressing; Alternatively, the soybean seeds are firstly dressed with a seed dressing agent containing antibacterial components, and then the bacterial agent containing the Sinorhizobium freundii is sprayed on the soybean seeds.

8. The use according to claim 7, characterized in that: The antibacterial component is selected from tebuconazole, metalaxyl-M, thiamethoxam, imidacloprid, fludioxonil, azoxystrobin, prochloraz, thiram or carboxin.

9. Use of the Sinorhizobium freundii described in claim 1 or the bacterial agent described in any one of claims 2 to 5 in at least one of the following (1) to (5): (1) Promote soybean growth; (2) Improve soybean drought tolerance; (3) Salt tolerance; (4) Alkali resistance; (5) Promote the increase of soybean nodule number and nodule fresh weight.

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