Rhizobium jangularis LN-ZDJ-1, complex microbial inoculant containing rhizobium jangularis LN-ZDJ-2 and application of rhizobium jangularis LN-ZDJ-1 and complex microbial inoculant

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to rhizobium jangularis LN-ZDJ-1, a complex microbial inoculant containing the rhizobium jangularis LN-ZDJ-2 and application of the rhizobium jangularis LN-ZDJ-1 and the complex microbial inoculant. The rhizobium jangularis LN-ZDJ-1 is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number of the rhizobium jangularis LN-ZDJ-1 is CCTCC (China Center for Type Culture Collection) NO: M20241652; the bacillus amyloliquefaciens LN-ZDJ-2 is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number of the bacillus amyloliquefaciens LN-ZDJ-2 is CCTCC (China Center for Type Culture Collection): M 20241651; the compound microbial agent provided by the invention not only can remarkably improve the growth and yield of the Achyranthes bidentata, but also can improve the nutrient absorption capacity of plants, further promotes the healthy growth and resistance of the plants by secreting active substances such as auxin and siderophores, and is beneficial to improving the quality of pasture.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a composite bacterial agent containing Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and applications thereof. Background Art

[0002] Microbial agents, also known as microbial fertilizers, play an important role in promoting plant growth. These fertilizers contain beneficial microorganisms, such as nitrogen-fixing bacteria, phosphate-dissolving bacteria, and growth hormone-producing bacteria, which can coexist or synergize with plant roots to provide nutrients to plants and inhibit the growth of pathogenic microorganisms, thereby promoting plant growth and development. By enhancing the nutrient absorption efficiency and stress resistance of plants, microbial fertilizers can significantly improve the yield and quality of crops. Despite this, chemical fertilizers are still the dominant growth-promoting fertilizer products on the current market, occupying an absolute dominant market share. In contrast, the variety and quantity of biological growth-promoting agents are relatively small, and the market share is not high, which is significantly different from the needs of green prevention and control and green food production. Therefore, it is of great significance to actively screen and develop new varieties of efficient and low-toxic biological growth-promoting agents.

[0003] Lespedezapotaninii Vass., as an excellent legume forage, plays an important role in forage production and grassland improvement. The new variety "Tengger" cattle and branches (No.: GS-CWV-2020-007) domesticated and bred by Lanzhou University in 2022 has the characteristics of drought resistance, cold resistance and poverty tolerance, and is considered to be one of the key varieties for improving desert grasslands. In recent years, scholars have made significant progress in the fields of genetic breeding, physiological ecology, tillage management and planting technology of Lespedezapotaninii Vass. However, there are few studies on the interaction between Lespedezapotaninii and microorganisms to promote the growth of Lespedezapotaninii, especially the screening and application of beneficial strains.

[0004] During the research process, the inventors accidentally screened out Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, which can not only significantly improve the growth and yield of cattle branches, but also improve the plant's ability to absorb nutrients, and further promote the healthy growth and resistance of the plant by secreting active substances such as auxins and siderophores, which helps to improve the quality of forage. Summary of the invention

[0005] The primary purpose of the present invention is to provide a strain of Rhizobium giardinii LN-ZDJ-1, which was deposited in the China Center for Type Culture Collection on July 22, 2024, with a deposit number of CCTCCNO: M 20241652.

[0006] The second object of the present invention is to provide a bacterial agent, which contains the Rhizobium japonicum LN-ZDJ-1 described in claim 1.

[0007] The third object of the present invention is to provide a microbial composite agent, which includes the Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and the Bacillus amyloliquefaciens LN-ZDJ-2 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M20241651.

[0008] The fourth object of the present invention is to provide a plant fertilizer containing the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent.

[0009] The fifth object of the present invention is to provide a soil ecological environment regulator, containing the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent.

[0010] The sixth object of the present invention is to provide the use of the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent in activating the insoluble phosphorus in the soil.

[0011] The seventh object of the present invention is to provide the use of the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent as or in the preparation of a plant growth promoter or in promoting plant growth.

[0012] The eighth object of the present invention is to provide the use of the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent in improving the utilization rate of phosphate fertilizer.

[0013] The ninth objective of the present invention is to provide the use of the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent in preventing soil compaction, acidification, and accumulation of harmful substances caused by the application of phosphate fertilizers.

[0014] The tenth object of the present invention is to provide the use of the Rhizobium japonicum LN-ZDJ-1 or the bacterial agent or the composite bacterial agent in the preparation of plant fertilizers and / or soil ecological environment regulators.

[0015] Beneficial effects of the present invention:

[0016] (1) The present invention provides a strain of Rhizobium giardinii LN-ZDJ-1, which was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M20241652.

[0017] (2) The present invention provides a microbial composite agent, which includes the Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and the Bacillus amyloliquefaciens LN-ZDJ-2 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M20241651.

[0018] (3) The aboveground and underground biomass of the schizonepeta treated with the composite microbial agent increased: After the composite microbial agent was used, the aboveground biomass of the schizonepeta increased by 49.1%, and the underground biomass increased by 56.1%. This indicates that the composite microbial agent can significantly promote the increase of the overall biomass of the schizonepeta, including the growth of the aboveground part and the root system.

[0019] (4) The compound microbial agent can promote the absorption of nitrogen and phosphorus in the sedge: The compound microbial agent can increase the absorption capacity of sedge for nitrogen and phosphorus, and the absorption amount has increased by 31.82%. This is of great significance to the nutrient utilization efficiency and growth and development of plants. Increase the nitrogen content of leaves: The compound microbial agent can also increase the nitrogen content of sedge leaves, which further promotes the growth and metabolic activity of plants.

[0020] (5) The promotion effect of indoleacetic acid and siderophore secretion in the compound microbial agent on the growth of C. truncatula: The microorganisms in the compound microbial agent secrete indoleacetic acid and siderophore, which significantly promote the growth of C. truncatula. Indoleacetic acid is an important plant growth hormone that can promote the development of stems, buds and roots. Siderophores have a high affinity and can bind to free Fe in the rhizosphere. 3+ The combination can effectively reduce the iron deficiency around the root zone of plants, thereby inhibiting the reproduction of pathogens in the root zone and reducing damage to plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Colony images of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

[0022] Figure 2 Phylogenetic tree of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

[0023] Figure 3 Growth curves of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

[0024] Figure 4 Effects of Carbon Source and pH on the Activity of Rhizobium japonicum LN-ZDJ-1

[0025] Figure 5 Effects of different culture media on the activity of Bacillus amyloliquefaciens LN-ZDJ-2 strain

[0026] Figure 6 Effects of Temperature on the Activity of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

[0027] Figure 7 Effect of rotation speed on the activity of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 strains

[0028] Figure 8 Effects of each treatment on promoting the growth of Rhizoma Cistanches after adding fungi DETAILED DESCRIPTION

[0029] The technical solution claimed for protection of the present invention is described below in conjunction with specific implementation methods. It should be noted that the protection scope of the present invention is not limited by the following embodiments.

[0030] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0031] In the following examples, Excel 2010 was used to organize the data, SPSS 19.0 was used to perform one-way analysis of variance (P < 0.05) and significant difference test, and the Duncan method was used for multiple comparisons.

[0032] In the following examples, the modified PVK medium used: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g sodium chloride, 0.2 g potassium chloride, 0.03 g iron sulfate, 0.03 g manganese sulfate, 5 g tricalcium phosphate, 1000 mL distilled water, pH 6.5-7.0; sterilized at 115°C for 30 min.

[0033] In the following examples, the beef extract peptone medium formula used is: beef extract 3g, peptone 10g / L, NaCl5g, distilled water 1000mL, pH7.4-7.6; NA formula base formula peptone 10g, beef extract powder 3g, NaCl 5gL, 7.3±0.2.

[0034] In the following examples, the plant culture medium used was: 20 g of dry plant sample was placed in a beaker and boiled for 30 min. Stirring was performed with a glass rod to prevent the bottom from becoming sticky. The medium was then filtered through a double-layer gauze. The filtrate was taken and made up to 1000 mL, with a natural pH.

[0035] In the following examples, the formula of the YMA medium used is: mannitol 20 g, yeast powder 0.5 g, NaH2PO4 0.3 g, MgSO4·7H2O 0.5 g, NaCl 0.1 g, agar 15-20 g, distilled water 1000 mL;

[0036] In the following examples, the TSB medium formula used was: 15.0 g tryptone, 5.0 g soy peptone, 5.0 g NaCl, and 1000 mL distilled water.

[0037] In the following examples, the CAS medium formula is: chrome azurol S 0.605 g, hexadecyltrimethylammonium bromide (HOTMA) 0.729 g, FeCl3·6H2O 0.026 g, NaH2PO4·12H2O 0.295 g, Na2HPO4·12H2O 1.213 g, ammonium chloride 0.125 g, KH2PO40 . 375 g, NaCl 0.625 g, agar 9 g.

[0038] In the following examples, nitrogen-fixing solid culture medium: mannitol 20 g, KH2PO4 0.2 g, K2HPO4 0.8 g, MgSO4·7H2O 0.2 g, CaSO4·2H2O 0.1 g, yeast extract 0.5 g, trace amount of FeCl3, trace amount of Na2MoO4·2H2O, fixed to volume with ddH2O, and adjusted to pH 7.2.

[0039] Example 1: Bacteria screening

[0040] 1. Isolation, identification and screening of strains

[0041] (1) Strain culture

[0042] Transfer the root sample of C. truncatula to a sterile 50ml centrifuge tube containing 20ml sterile 10mM PBS solution, place it in a full-temperature shaker at 120r / min, and shake it at room temperature for 20min. Pick out the roots in the 50ml centrifuge tube, and centrifuge the remaining suspension at high speed (6000rpm, 4℃) for 20min to collect the rhizosphere soil. Remove the supernatant and the sediment left at the bottom of the test tube is the rhizosphere soil. Weigh 1g of rhizosphere soil and put it into a sterile centrifuge tube containing 9mL of 0.85% saline. Shake it for 10min to evenly disperse the soil sample in the diluent to form a soil suspension. Pipette 1ml of the soil suspension and transfer it to 9ml of sterile water. Dilute it in sequence by the ten-fold method, usually to 10 -7 : Use a sterile pipette tip to transfer 200ul of soil suspension to modified Pikovskaya (PVK) medium and YMA medium, use a spreading rod to evenly distribute the suspension on the medium, invert the medium and transfer it to a constant temperature incubator and culture it at 28℃ for 3-7 days, pick out different single colonies in the medium, streak them on new medium, and invert them in the incubator for culture, and repeat the previous operation 2-3 times to purify the colonies.

[0043] (2) Strain purification

[0044] In PVK and YMA medium, select strains with transparent circles, purify them on LB medium with an inoculation loop according to three-zone lines, and purify them at least 5 times. The purified strains are mixed with 40% glycerol and fresh bacterial solution in a ratio of 1:1 and stored in a -80℃ refrigerator. The colony after purification is shown in the figure below: Figure 1 As shown, two strains were obtained and named LN-ZDJ-1 and LN-ZDJ-2 respectively.

[0045] (3) Strain identification

[0046] Use a sterilized white pipette tip to gently pick up a single colony on the plate and place the white pipette tip in a 2mL centrifuge tube filled with 1mL sterile water. Mix thoroughly until the bacteria in the white pipette tip are completely dissolved in the sterile water to obtain a DNA template for colony PCR amplification. The total volume of the phosphate-solubilizing bacteria PCR reaction system is 25uL, the phosphate-solubilizing bacteria DNA template is 1uL, and the DNA template concentration is 0.1-10ng / uL.

[0047] Upper primer: 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′

[0048] Lower primer: 1492R: 5′-GGYTACCTTGTTACGACTT-3′

[0049] 1uL each, Mix (DNA enzyme and four nucleotides) 12.5uL.

[0050] The PCR reaction conditions were 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s, and an extension temperature of 72°C for 10 min.

[0051] After the PCR reaction is completed, gel electrophoresis is performed to observe the quality of the PCR product. The PCR product with a single and bright band in the PCR product electrophoresis result is selected and sent to Shanghai Biotechnology for sequencing. The sequencing results are compared for similarity sequences in NCBI (National Center for Biotechnology Information), and the strains with higher similarity are selected and their related sequences are downloaded. Multiple sequence alignment is performed in Mega software, and attention is paid to the consistency of sequence direction. The phylogenetic tree is constructed according to the NJ method, such as Figure 2 shown.

[0052] According to the Manual of Identification of Common Bacteria Systems and Bergey's Manual of Identification of Bacteria, the physiological and biochemical characteristics of LN-ZDJ-1 and LN-ZDJ-2 strains were tested. The physiological and biochemical tests included Gram staining, starch hydrolysis test, methyl red test, indole test, Volta-Pu test, and gelatin liquefaction test. The results are shown in Table 1.

[0053] Table 1 Physiological and biochemical characteristics of strains

[0054]

[0055] “+” indicates positive, and “-” indicates negative.

[0056] The 16S rRNA gene sequences of LN-ZDJ-1 and LN-ZDJ-2 were determined, and similar sequences were retrieved from the NCBI database for comparison. Combined with the physiological and biochemical test structure of 00491, it was determined that the strain LN-ZDJ-1 belonged to Rhizobium giardinii, with the Latin name Rhizobium giardinii. It was named Rhizobium giardiniiL LN-ZDJ-1, which was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M 20241652. The deposit address is Wuhan University, Wuhan, China, and the telephone number is 027-68754052;

[0057] The strain LN-ZDJ-2 belongs to Bacillus amyloliquefaciens, with a Latin name (Bacillus amyloliquefaciens). It is named Bacillus amyloliquefaciens LN-ZDJ-2. ​​It was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M 20241651. The deposit address is Wuhan University, Wuhan, China, and the telephone number is 027-68754052.

[0058] In the following examples, Rhizobium giardinii LN-ZDJ-1 is abbreviated as Rhizobium giardinii LN-ZDJ-1, and Bacillus amyloliquefaciens LN-ZDJ-2 is abbreviated as Bacillus amyloliquefaciens LN-ZDJ-2, which have the same meanings.

[0059] Example 2: Screening of composite bacterial agents in potted plant experiments

[0060] 1. Plant Materials

[0061] The seeds of Tengri Niuzhizi come from the Zhang Jiyu team of Lanzhou University and are drought-resistant and cold-resistant. The seeds were sterilized with 70% ethanol for 5 minutes, soaked in 2% HClO4 for 5 minutes, washed with sterile water 3 times, and placed in a culture dish for germination.

[0062] 2. Experimental treatment

[0063] The potted plant growth promotion experiment was conducted at the Grassland Microbiology Center of Lanzhou University. There were three treatments: treatments with Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and a control group with an equal amount of sterile water. Five pots were set up in each group, and each pot contained 200g of soil. When the seedlings of the Chinese taro had two leaves and one heart, 0.5ml (OD 600 =1.00) solution, and then watered once every week, for a total of four times. After the plants grew to two months, the seedlings were harvested, and the root fresh weight, root dry weight, aboveground fresh weight, total dry weight and plant height of the Niuzhizi in the treatment group and the control group were measured. The results are shown in Table 2.

[0064] Table 2 Effects of strain addition on plant height and fresh and dry weight of Rhizoma Coptidis

[0065]

[0066] Note: * indicates significant difference among treatments (p<0.05), ** indicates significant difference among treatments (p<0.01), *** indicates significant difference among treatments (p<0.001).

[0067] As shown in Table 2, the treatment with the inoculant has a promoting effect on the growth of C. chinensis. The treatment with the inoculant significantly increased the aboveground fresh weight, dry weight and plant height of the plants, among which the aboveground fresh weight, dry weight and plant height of Rhizobium japonicum LN-ZDJ-1 increased by 50.22%, 41.63% and 54.05% respectively compared with the control without the inoculant. The aboveground fresh weight, dry weight and plant height of Bacillus amyloliquefaciens LN-ZDJ-2 increased by 39.36%, 10.82% and 37.45% respectively compared with the control without the inoculant.

[0068] Example 3: Determination of the growth-promoting properties of two bacteria constituting the composite bacterial agent

[0069] 1. Determination of IAA production capacity:

[0070] IAA content detection: Reactivate the bacteria stored on the slant to LB medium to restore its activity, pick a loop of single colonies with an inoculation loop and inoculate it into LB medium containing L-tryptophan. 28℃, 180r·min -1 The culture was shaken for 4 days. The bacterial solution was taken and stirred at 160 r·min -1 Centrifuge for 10 min under the same conditions, take 100 ul of supernatant and add it to the ELISA plate, add an equal volume of Salkowski colorimetric reagent, mix well, let stand in the dark for 15 min, and measure the absorbance at a wavelength of 530 nm using an ELISA reader. Standard curve drawing: prepare 100 μg·mL-IAA standard solution, and dilute it to 0, 10, 20, 30, 50, 75 μg·mL -1 For the IAA standard solution with different concentrations, 0.1 mL of colorimetric solution and 0.1 mL of standard solution were respectively aspirated onto the ELISA plate, and after standing in the dark for 15 min, the absorbance was measured at a wavelength of 530 nm using an ELISA reader to draw a standard curve. The IAA content per unit volume of fermentation broth was calculated based on the standard curve.

[0071] 2. Determination of iron carrier production:

[0072] The test strain was inoculated into CAS detection medium by plate streak method. After culturing in a constant temperature incubator at 28℃ for 5 days, if an orange-yellow transparent halo appeared around the colony, it indicated that the strain could produce iron carriers.

[0073] 3. Determination of phosphorus dissolving capacity:

[0074] The activated single colony was transferred to the modified PVK medium by spot inoculation, with 4 replicates on each plate. The culture was carried out at 28°C for 5 days. If an orange-yellow transparent halo appeared around the colony, it indicated that the strain had the ability to solubilize phosphate.

[0075] 4. Determination of nitrogen fixation ability:

[0076] The activated single colony was transferred to the nitrogen-fixing solid culture medium with the addition of dye by dot inoculation, with 4 replicates in each culture dish. The culture was cultured at 28°C for 5 days. If an orange-yellow transparent halo appeared around the colony, it indicated that the strain had the ability to fix nitrogen.

[0077] The growth-promoting properties of the strains are shown in Table 3

[0078] Table 3 IAA and siderophore production capacity of strains

[0079]

[0080] “+” indicates positive, and “-” indicates negative.

[0081] It can be seen from Table 3 that the strains have strong IAA production and siderophore capacity, as well as phosphorus solubilization and nitrogen fixation capabilities, indicating that the two strains may directly promote plant growth and crop yields through nitrogen fixation, phosphorus solubilization, plant hormone production, and resistance to pathogens.

[0082] Example 4: Optimization of strain fermentation medium

[0083] 1. Optimization of fermentation medium of Rhizobium japonicum LN-ZDJ-1

[0084] 1) Culture of bacterial strains: Use an inoculation loop to pick a single colony and inoculate it into 50 mL / 250 mL of liquid seed solution. Cultivate in a constant temperature incubator at 28°C and 180 rpm until the bacterial solution concentrations of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 are OD600=0.6 and OD 600 =1.0, and the seed liquid is obtained.

[0085] 2) Determination of growth curve: Transfer the above seed liquid to LB medium at a 1% inoculation rate, shake and mix, and place in a constant temperature oscillator at 28°C and 180r / min for 36h; take out Rhizobium japonicum LN-ZDJ-1 every 6h and Bacillus amyloliquefaciens LN-ZDJ-2 every 2h, use sterile deionized water as a blank control, and determine the OD600um value of the bacterial suspension. Draw a growth curve with the culture time as the horizontal axis and the OD600um value as the vertical axis. Results are shown in Figure 3 .

[0086] 3) After consulting relevant literature and combining with actual production needs, YMA medium was used as the basic medium for Rhizobium japonicum LN-ZDJ-1, and the carbon source and pH in the fermentation medium of the strain were optimized. Among them, the carbon sources mainly selected glucose, sucrose, mannitol, soluble protein, sucrose and peptone; pH = 6.5, pH = 7, pH = 7.5, pH = 8 and pH = 8.5 were selected, and a single variable was changed respectively, and the above seed liquid was inoculated at a rate of 1% and inoculated into a 50mL / 250mL triangular flask, and the fermentation temperature was 28°C and the speed was 180r / min under shaking culture conditions for 24-36h. Determine the OD600nm value and dry weight of bacteria under different conditions (centrifuge the culture medium at 4000r / min for 10min, remove the supernatant, put the bacteria and the pre-weighed sterile 50ml centrifuge tube in a 105℃ oven until constant weight, weigh the total weight, the dry weight of bacteria is equal to the total weight minus the weight of the centrifuge tube), repeat the experiment six times and take the average value.

[0087] The results are as follows Figure 4 As shown in the figure, under the condition of mannitol as the carbon source of YMA medium, the activity of Rhizobium japonicum LN-ZDJ-1 is the highest, so mannitol is determined to be the best carbon source component of Rhizobium japonicum. Secondly, under the condition of pH = 6.5, the OD600um and dry weight of Rhizobium japonicum are the highest, which are 0.7796 and 1.08g / L respectively. Therefore, pH = 6.5 is determined to be the best culture pH of Rhizobium japonicum.

[0088] 2. Optimization of fermentation medium for Bacillus amyloliquefaciens LN-ZDJ-2

[0089] Relevant literature was consulted and combined with the laboratory's culture medium resources to explore the effects of different culture media (NA, TSB, LB, beef extract peptone and plant culture medium) on the growth of Bacillus amyloliquefaciens LN-ZDJ-2, and the fermentation medium was optimized with the OD600un value of the bacterial liquid and the dry weight of the bacteria as optimization indicators.

[0090] The results are as follows Figure 5 As shown in the results, under the conditions of TSB culture medium, the OD600um value and dry weight of the bacterial suspension of Bacillus amyloliquefaciens LN-ZDJ-2 were significantly higher than those of other culture media (P<0.05). The dry weight of the bacteria could reach 1.64 g / L, the fermentation efficiency was high, and TSB was economical. After comprehensive comparison, TSB was selected as the optimal culture medium for Bacillus amyloliquefaciens LN-ZDJ-2.

[0091] 3. Effects of different temperatures and shaking on the bacterial strains of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

[0092] Different temperatures (26°C, 28°C, 30°C, 33°C and 37°C) and shaking speeds (160r / min, 180r / min, 200r / min, 220r / min and 240r / min) were set, and the OD600um value and cell dry weight of the fermentation broth of the strain were measured using the optimized culture medium in Examples 3 and 4 according to the method in Example 3. The test was repeated six times and the average value was taken.

[0093] The results are as follows Figure 6 As shown in the figure, at 37℃ and 34℃, the OD600um value and dry weight of bacterial suspension of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 were higher than those at other temperatures. The OD600um of bacterial suspension were 1.1055 and 0.9412, and the dry weight of bacteria were 9.80g / L and 1.69g / L, respectively. Therefore, 37℃ and 34℃ were the optimal culture temperatures for Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2.

[0094] The results are as follows Figure 7 As shown in the figure, under the condition of 240r / min, the OD600um value and dry weight of bacterial suspension of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 were higher than those at other speeds. The OD600um of bacterial suspension were 0.8976 and 0.8553, and the dry weight of bacteria were 1.3275g / L and 2.9192g / L, respectively. Therefore, 240r / min was the optimal fermentation speed for Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2.

[0095] Example 5. Preparation of liquid bacterial agent and potted plant experiment on Coix lacryma-jobi

[0096] Preparation of liquid inoculant: Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 were inoculated into YMA and TSB liquid culture media at 28°C and 180 r·min. -1 Shake and culture for 24-36 hours until the liquid inoculum OD 600 When the value reaches 0.8, transfer the culture medium to a sterile 50 ml centrifuge tube and centrifuge at 4000 r / min -1 Centrifuge for 10 min, remove the supernatant, and resuspend the lower layer of cells with sterile water to OD 600 Adjust to 1.00 and set the above OD 600 =1 bacterial suspension was mixed in a volume ratio of 1:1.

[0097] Potted test of liquid bacterial agent: The potted test for promoting growth was conducted at the Grassland Microbiology Center of Lanzhou University. There were four treatments: a treatment group with the addition of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 alone, a treatment group with a mixture of Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and a control group with the addition of an equal amount of sterile water. Each treatment had 5 pots, and each pot contained 600g of soil. When the seedlings had two leaves and one heart, the roots of each pot were watered for the first time with 0.5ml (OD 600 =1.00) of bacterial solution, and then watered once every other week, for a total of four times of bacterial solution watering. Two months later, the seedlings were harvested, and the root fresh weight, root dry weight, stem fresh weight, stem dry weight, plant height, leaf nitrogen and phosphorus content, and soil nitrogen and phosphorus content of the Niuzhizi in each treatment group and the control group were measured. The results are shown in Tables 4 and 5.

[0098] Table 4 Effects of different treatments with fungi on root development and plant height of Rhizoma Coptidis

[0099]

[0100] Note: * indicates significant difference among treatments (p<0.05), ** indicates significant difference among treatments (p<0.01), *** indicates significant difference among treatments (p<0.001).

[0101] From Table 4 and Figure 8 It can be seen that the treatment with inoculants has different promoting effects on the growth of C. truncatum. The root fresh weight, root dry weight, aboveground dry weight, aboveground fresh weight and plant height of the experimental group with inoculants were increased compared with the control group without inoculants. Among them, the plant height of the treatment with Rhizobium japonicum LN-ZDJ-1 inoculant was significantly higher than that of the control group, increasing by 48.4%. At the same time, the application of liquid inoculants can promote the development of the root system of C. truncatum. Compared with the control, the root fresh weight and root dry weight increased to varying degrees. The aboveground dry weight of the treatment with Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 inoculants was higher than that of the control group. It was the treatment with the largest aboveground dry weight among all treatments and had the best growth promotion effect. The aboveground dry weight of the treatment with Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 increased by 10.6% and 17.6% compared with the treatment with Bacillus amyloliquefaciens LN-ZDJ-2 and Rhizobium japonicum LN-ZDJ-1, indicating that the mixed culture of the strains will produce a synergistic effect.

[0102] Table 5 Effects of bacterial agents on aboveground nitrogen content, phosphorus content, plant nitrogen accumulation and plant phosphorus accumulation in Cnidium monnieri

[0103]

[0104] Note: * indicates significant difference among treatments (p<0.05), ** indicates extremely significant difference among treatments (p<0.01).

[0105] As shown in Table 5, compared with the sterile control, the total nitrogen and nitrogen accumulation of the experimental group treated with Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 increased by 31.82% and 106.57% respectively compared with the control without the application of the bacterial agent, among which the increase of total nitrogen in the plant reached a significant level, and the increase of nitrogen accumulation in the plant reached an extremely significant level. Except for the treatment of Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2, the phosphorus content of the plants treated with the bacterial agents increased by 0.26% and 14.95% compared with the control. The phosphorus content of the Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 treatment decreased by 1.04% compared with the control. The significant decrease in the phosphorus content of the Rhizobium japonicum plant treated with the rhizobium may be due to the excessive consumption of phosphorus by the nodulation process of the rhizobium and the massive reproduction of microorganisms. The application of Bacillus amyloliquefaciens LN-ZDJ-2 inoculant can increase the phosphorus accumulation of Boletus chinensis by 65.63% compared with the control. In short, compared with the control, both single bacteria treatment and mixed bacteria addition can increase the total nitrogen and phosphorus content of Boletus chinensis, as well as the absorption of nitrogen and phosphorus. In short, inoculation of Rhizobium japonicum LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 can increase the absorption of N and P by Boletus chinensis and increase the amount of nodules.

[0106] In summary, the high-efficiency composite microbial agent produced by the present invention has a significant promoting effect on the growth of schizonepeta. Compared with the control group, schizonepeta treated with the composite microbial agent showed the following significant advantages: increase in aboveground and underground biomass: after using the composite microbial agent, the aboveground biomass of schizonepeta increased by 49.1%, and the underground biomass increased by 56.1%. This shows that the composite microbial agent can significantly promote the increase of the overall biomass of schizonepeta, including the growth of the aboveground part and the root system. Promote the absorption of nitrogen and phosphorus elements: the use of the composite microbial agent can increase the absorption capacity of schizonepeta for nitrogen and phosphorus, and the absorption amount is increased by 31.82%. This is of great significance to the nutrient utilization efficiency and growth and development of plants. Increase the nitrogen content of leaves: the composite microbial agent can also increase the nitrogen content of schizonepeta leaves, which further promotes the growth and metabolic activity of plants. The promoting effect of secreting indoleacetic acid and siderophores: the microorganisms in the composite microbial agent secrete indoleacetic acid and siderophores, which have a significant promoting effect on the growth of schizonepeta. Indoleacetic acid is an important plant growth hormone that can promote the development of stems, buds and roots. Siderophores have a high affinity for free Fe in the rhizosphere. 3+The combination effectively reduces the iron deficiency around the rhizosphere of plants, thereby inhibiting the reproduction of pathogens in the rhizosphere and reducing damage to plants. The composite microbial agent provided by the present invention can not only significantly improve the growth and yield of cattle branches, but also improve the plant's ability to absorb nutrients, and further promote the healthy growth and resistance of plants by secreting active substances such as auxins and iron carriers, which helps to improve the quality of forage.

[0107] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A strain of Rhizobium giardinii LN-ZDJ-1, characterized in that: The J. gibberellosis LN-ZDJ-1 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M20241652.

2. A bacterial agent, characterized in that The bacterial agent contains the Rhizobium japonicum LN-ZDJ-1 described in claim 1.

3. A microbial composite agent, characterized in that: The composite bacterial agent includes the Rhizobium japonicum LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 described in claim 1, and the Bacillus amyloliquefaciens LN-ZDJ-2 was deposited in the China Center for Type Culture Collection on July 22, 2024, with the deposit number: CCTCCNO: M20241651.

4. A plant fertilizer, characterized in that: Contains the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3.

5. A soil ecological environment regulator, characterized in that: Contains the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3.

6. Use of the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3 in activating sparingly soluble phosphorus in soil.

7. Use of the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3 as or in the preparation of a plant growth promoter or in promoting plant growth.

8. Use of the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3 in improving the utilization rate of phosphate fertilizer.

9. Use of the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3 in preventing soil compaction, acidification, and accumulation of harmful substances caused by the application of phosphate fertilizers.

10. Use of the Rhizobium japonicum LN-ZDJ-1 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 3 in the preparation of plant fertilizers and / or soil ecological environment regulators.

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