Rhizobium jiaxingense ln-zdj-1, complex microbial inoculant containing same and bacillus amyloliquefaciens ln-zdj-2 and application

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a rhizobium jiaxingense LN-ZDJ-1, a complex microbial inoculant containing the rhizobium jiaxingense LN-ZDJ-1 and a bacillus amyloliquefaciens LN-ZDJ-2, and application thereof. The rhizobium jiaxingense LN-ZDJ-1 is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number is CCTCC 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 is CCTCC NO: M20241651. The complex microbial inoculant provided by the present application can not only significantly improve the growth and yield of cowhage, but also improve the nutrient absorption capacity of the plant, and further promote the healthy growth and resistance of the plant by secreting active substances such as auxin and iron carrier, which is helpful to improve the quality of the pasture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to Rhizobium giardinii LN-ZDJ-1, a complex microbial inoculant containing the same and Bacillus amyloliquefaciens LN-ZDJ-2, and application thereof. BACKGROUND

[0002] Microbial inoculants, also known as microbial fertilizers, play an important role in promoting plant growth. These fertilizers contain beneficial microorganisms, such as nitrogen-fixing bacteria, phosphorus-dissolving bacteria, and bacteria that produce growth hormones, which can symbiotically or synergistically interact with plant roots to provide nutrients and inhibit the growth of pathogenic microorganisms, thereby promoting the growth and development of plants. By enhancing the nutrient absorption efficiency and stress resistance of plants, microbial fertilizers can significantly improve crop yield and quality. However, chemical fertilizers still dominate the current market, occupying a dominant market share. In contrast, the variety and quantity of biological growth-promoting inoculants are relatively small, and their market share is not high, which is significantly different from the demand for green prevention and control and green food production. Therefore, it is of great significance to actively screen and develop new varieties of biological growth-promoting inoculants with high efficiency and low toxicity.

[0003] Lespedeza potaninii Vass. as a kind of excellent leguminous forage grass, plays an important role in forage production and grassland improvement. The new variety "Tenggeli" Lespedeza potaninii (number: GS-CWV-2020-007) domesticated and bred by Lanzhou University in 2022 has the characteristics of drought resistance, cold resistance and poor tolerance, and is considered as one of the key varieties for improving desert grassland. In recent years, scholars have made significant progress in the fields of genetics and breeding, physiological ecology, cultivation management and planting technology of Lespedeza potaninii. However, there are few studies on the interaction between Lespedeza potaninii and microorganisms to promote the growth of Lespedeza potaninii, especially the screening and application of beneficial strains.

[0004] The inventors accidentally screened Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 during the research process, which not only can significantly improve the growth and yield of Lespedeza potaninii, but also can improve the nutrient absorption capacity of the plant, and further promote the healthy growth and resistance of the plant by secreting active substances such as auxin and iron carrier, which is helpful to improve the quality of forage grass. SUMMARY

[0005] The first object of the present application is to provide a strain of Rhizobium giardinii LN-ZDJ-1, which was deposited with the China Center for Type Culture Collection on July 22, 2024, and has a deposit number of CCTCC NO: M 20241652.

[0006] The second object of the present application is to provide a microbial agent containing the Rhizobium giardinii LN-ZDJ-1 according to claim 1.

[0007] The third object of the present application is to provide a microbial compound agent, which comprises the Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, which was deposited with the China Center for Type Culture Collection on July 22, 2024, and has a deposit number of CCTCC NO: M 20241651.

[0008] The fourth object of the present application is to provide a plant fertilizer containing the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent.

[0009] The fifth object of the present application is to provide a soil ecological environment regulator containing the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent.

[0010] The sixth object of the present application is to provide the use of the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent in activating soil insoluble phosphorus.

[0011] The seventh object of the present application is to provide the use of the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent as or in the preparation of a plant growth promoter or in promoting plant growth.

[0012] The eighth object of the present application is to provide the use of the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent in improving the utilization rate of phosphorus fertilizer.

[0013] The ninth object of the present application is to provide the use of the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent in preventing soil compaction, acidification and harmful substance accumulation caused by the application of phosphorus fertilizer.

[0014] The tenth object of the present application is to provide the use of the Rhizobium giardinii LN-ZDJ-1 or the microbial agent or the microbial compound agent in the preparation of a plant fertilizer and / or a soil ecological environment regulator.

[0015] The beneficial effects of the present application are:

[0016] (1) The present application provides a strain of Rhizobium giardinii LN-ZDJ-1, which was deposited with the China Center for Type Culture Collection on July 22, 2024, and has the accession number CCTCC NO: M20241652.

[0017] (2) The present application provides a microbial complex microbial agent, which comprises the Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, which was deposited with the China Center for Type Culture Collection on July 22, 2024, and has the accession number CCTCC NO: M20241651.

[0018] (3) The aboveground and underground biomass of the treated Millettia pachynema increases: after using the complex microbial agent, the aboveground biomass of Millettia pachynema increases by 49.1%, and the underground biomass increases by 56.1%. This shows that the complex microbial agent can significantly promote the increase of the overall biomass of Millettia pachynema, including the growth of the aboveground part and the root system.

[0019] (4) The use of the complex microbial agent treated Millettia pachynema promotes the absorption of nitrogen and phosphorus elements: the use of the complex microbial agent can increase the absorption capacity of Millettia pachynema to nitrogen and phosphorus, and the absorption amount increases by 31.82%. This is of great significance to the nutrient use efficiency and growth and development of plants. Increase the nitrogen content of leaves: the complex microbial agent can also increase the nitrogen content of the leaves of Millettia pachynema, which further promotes the growth and metabolic activity of plants.

[0020] (5) The use of the complex microbial agent treated Millettia pachynema promotes the secretion of indole acetic acid and siderophore: the secretion of indole acetic acid and siderophore by the microorganisms in the complex microbial agent has a significant promoting effect on the growth of Millettia pachynema. Indole acetic acid is an important plant growth regulator that can promote the development of stems, buds and roots. Siderophore has high affinity and can bind to free Fe 3+ around the rhizosphere, effectively reducing the lack of iron elements around the rhizosphere of plants, thereby inhibiting the reproduction of pathogenic bacteria in the rhizosphere and reducing the damage to the plants. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Colony figure of Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

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

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

[0024] Figure 4 Effects of carbon source and pH on the activity of Rhizobium jinzhongensis LN-ZDJ-1 strain

[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 jinzhongensis LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 strains

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

[0028] Figure 8 Potting effects of each treatment for promoting the growth of Millettia pachynema after adding microbial agents DETAILED DESCRIPTION

[0029] The technical solutions claimed in the present application will be described below in conjunction with specific embodiments, and it should be noted that the protection scope of the present application is not limited by the following embodiments.

[0030] In the following examples, the instruments, reagents, materials, etc. involved, if not specifically stated, are conventional instruments, reagents, materials, etc. already existing in the prior art, which can be obtained through regular commercial channels. In the following examples, the experimental methods, detection methods, etc. involved, if not specifically stated, are conventional experimental methods, detection methods, etc. already existing in the prior art.

[0031] In the following examples, Excel 2010 was used to organize data, SPSS19.0 was used for single-factor variance analysis (P<0.05) and significant difference test, and Duncan method was used for multiple comparisons.

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

[0033] The following examples, the use of beef extract protein peptone medium formula: beef extract 3g, protein peptone 10g / L, NaCl 5g, distilled water 1000ml, pH 7.4-7.6; NA formula base formula protein peptone 10g, beef extract powder 3g, NaCl 5gL, 7.3±0.2.

[0034] The following examples, the use of plant culture medium: 20g plant dry sample into a beaker boiled for 30min, pay attention to stirring with glass rod to prevent paste bottom, then with double layer gauze filter, take its filtrate, make up to 1000ml, natural pH.

[0035] The following examples, the use of YMA medium formula: mannitol 20g, yeast powder 0.5g, NaH2PO40.3g, MgSO4·7H2O 0.5g, NaCl 0.1g, agar 15-20g, distilled water 1000ml;

[0036] The following examples, the use of TSB medium formula: tryptone 15.0g, soybean protein peptone 5.0g, NaCl 5.0g, distilled water 1000ml.

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

[0038] The following examples, nitrogen fixation solid medium: mannitol 20g, KH2PO40.2g, K2HPO40.8g, MgSO4·7H2O 0.2g, CaSO4·2H2O 0.1g, yeast extract 0.5g, FeCl3trace, Na2MoO4·2H2O trace, with ddH2O constant volume, adjust pH to 7.2.

[0039] Example 1, strain screening

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

[0041] (1) strain culture

[0042] The root system sample of the Radix Broussonetiae was transferred into a sterile 50 ml centrifuge tube containing 20 ml of sterile 10 mM PBS solution, and placed in a constant temperature shaker at 120 r / min and shaken at room temperature for 20 min. The root system in the 50 ml centrifuge tube was picked out, and the remaining suspension was centrifuged (6000 rpm, 4 DEG C) for 20 min to collect the rhizosphere soil. The supernatant was removed, and the sediment at the bottom of the test tube was the rhizosphere soil. 1 g of the rhizosphere soil was weighed and placed in a sterile centrifuge tube containing 9 mL of 0.85% physiological saline, shaken for 10 min to uniformly disperse the soil sample in the diluent to form a soil suspension. 1 ml of the soil suspension was transferred into 9 ml of sterile water, sequentially diluted according to the ten-fold method, and usually diluted to 10 -7 : 200 ul of the soil suspension was taken with a sterile gun head and transferred into a modified Pikovskaya inorganic phosphorus culture medium (Pikovskaya, PVK) and a YMA culture medium. The suspension was uniformly distributed on the culture medium with a coating rod. The culture medium was inverted and transferred into a constant temperature incubator for culture at 28 DEG C for 3-7 days. Different single colonies in the culture medium were picked out, streaked on new culture medium, and inverted in the incubator for culture. The above steps were repeated for 2-3 times to purify the colonies.

[0043] (2) Strain purification

[0044] In the PVK and YMA culture media, the strains with transparent circles were selected, and the inoculation ring was used for three-zone streaking purification on the LB culture medium. The streaking purification was performed at least 5 times. The purified strains were mixed with fresh bacterial liquid at 1:1 with 40% glycerol and stored in a -80 DEG C refrigerator. The colony after purification is shown in Figure 1 , and 2 strains were obtained, which were named as LN-ZDJ-1 and LN-ZDJ-2, respectively.

[0045] (3) Strain identification

[0046] A sterile white gun head was used to gently pick a single colony on the plate, and the white gun head was placed in a 2 ml centrifuge tube containing 1 ml of sterile water. The mixture was fully mixed until the bacteria in the white gun head were completely dissolved into the sterile water to obtain a DNA template for colony PCR amplification. The total volume of the PCR reaction system of the phosphate-solubilizing bacteria was 25 ul, and the DNA template of the phosphate-solubilizing bacteria was 1 ul. The concentration of the DNA template was 0.1-10 ng / uL.

[0047] The upper primer was 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'

[0048] The lower primer was 1492R: 5'-GGYTACCTTGTTACGACTT-3'

[0049] 1 ul each, and 12.5 ul of Mix (DNAse and four nucleotides).

[0050] PCR reaction condition 94℃ 4min, then 94℃ 30s, 56℃ 30s, 72℃ 60s, 30 cycles, extension temperature 72℃ 10min.

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

[0052] According to the "Common Bacteria System Identification Manual" and "Berger's Bacteria Identification Manual", the physiological and biochemical characteristics of LN-ZDJ-1 and LN-ZDJ-2 strains were detected. The physiological and biochemical tests included gram staining, starch hydrolysis test, methyl red test, indole test, V-P test, and gelatin liquefaction test, and the results are shown in Table 1.

[0053] Table 1 Physiological and biochemical characteristics of strains

[0054]

[0055] Among them, "+" represents positive; "-" represents 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, aligned, and combined with the physiological and biochemical test structure to determine that the strain LN-ZDJ-1 belongs to Rhizobium giardinii, and the Latin name is Rhizobium giardinii. It is named Rhizobium giardinii (Rhizobium giardinii L) LN-ZDJ-1, which was preserved in China Center for Type Culture Collection on July 22, 2024, with the preservation number CCTCC NO: M 20241652, and the preservation address is Wuhan University, Wuhan, China, with the telephone number 027-68754052;

[0057] ​The strain LN-ZDJ-2 belongs to Bacillus amyloliquefaciens, and its Latin name is Bacillus amyloliquefaciens. It is named Bacillus amyloliquefaciens LN-ZDJ-2, and was preserved in the China Center for Type Culture Collection on July 22, 2024, with the preservation number CCTCC NO: M 20241651, and the preservation address is Wuhan University, Wuhan, China, telephone 027-68754052.

[0058] In the following examples, Rhizobium giardinii L 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 meaning.

[0059] Example II, screening of potting test of composite microbial agent

[0060] 1. Plant material

[0061] “Tenggeli” Caragana korshinskii was derived from Zhang Jiyu team of Lanzhou University, and has the characteristics of drought tolerance and cold resistance. The seeds were treated with 70% ethanol for 5 min, then soaked with 2% HCIO4 for 5 min, and then washed with sterile water for 3 times. Then they were placed in a culture dish for germination.

[0062] 2. Test treatment

[0063] The growth-promoting potting test was carried out in the grassland microbial center of Lanzhou University. Three treatments were set up: adding Rhizobium giardinii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and adding the same amount of sterile water as the control group. Each group set 5 pots, each containing 200g soil. When the seedlings of Caragana korshinskii had two leaves and one heart, 0.5ml (OD 600 =1.00) of bacterial solution was first poured, and then every week, a total of four times of bacterial solution. After the plants grew for two months, the seedlings were treated, and the root fresh weight, root dry weight, aboveground fresh weight, total dry weight and plant height of Caragana korshinskii in the treatment group and the control group were measured, and the results are shown in Table 2.

[0064] Table 2 Effect of strain addition on plant height and fresh and dry weight of Caragana korshinskii

[0065]

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

[0067] From Table 2, it can be seen that the bacterial agent treatment has a promoting effect on the growth of Millettia pachynema. The bacterial agent treatment significantly increases the plant aboveground fresh weight, dry weight and plant height, wherein the Rhizobium jolliffi LN-ZDJ-1 is increased by 50.22%, 41.63% and 54.05% respectively compared with the control without bacterial agent. The Bacillus amyloliquefaciens LN-ZDJ-2 is increased by 39.36%, 10.82% and 37.45% respectively compared with the control without bacterial agent.

[0068] Example 3, determination of the growth-promoting properties of two bacteria constituting the complex bacterial agent

[0069] 1. Determination of IAA production capacity:

[0070] IAA content detection: the bacteria preserved in the slope were reactivated in the LB medium to restore their activity, and a single colony was picked up with an inoculation ring and inoculated into the LB medium containing L-tryptophan. The culture was incubated at 28°C, 180r·min -1 for 4d. The bacterial liquid was centrifuged at 160r·min -1 for 10min, 100ul of the supernatant was added to the enzyme-labeled plate, and an equal volume of Salkowski color reagent was added, mixed well, and placed in the dark for 15min. The absorbance was measured at a wavelength of 530nm using an enzyme-labeled instrument. Standard curve preparation: prepare 100μg·mL-IAA standard solution, dilute to 0, 10, 20, 30, 50, 75μg·mL -1 concentration of IAA standard solution, respectively, 0.1mL of colorimetric solution and 0.1mL of standard solution were taken in the enzyme-labeled plate, and the absorbance was measured at a wavelength of 530nm using an enzyme-labeled instrument after being placed in the dark for 15min. The standard curve was drawn. The content of IAA in unit volume of fermentation liquid was calculated according to the standard curve.

[0071] 2. Determination of siderophore production:

[0072] The test strains were inoculated on the CAS detection medium by the plate streaking method, and cultured in a 28°C constant temperature incubator for 5d. If orange transparent halos appear around the colonies, it indicates that the strain can produce siderophores.

[0073] 3. Determination of phosphorus solubilizing capacity:

[0074] The activated single colony was transferred to the modified PVK medium by point inoculation, 4 repeats were inoculated on each plate, and cultured at 28°C for 5d. If orange transparent halos appear around the colonies, it indicates that the strain has phosphorus solubilizing capacity.

[0075] 4. Determination of nitrogen fixation capacity:

[0076] The activated single colony was inoculated on the nitrogen-fixing solid medium with dye added in a point inoculation manner, 4 repeats were inoculated in each dish, and the culture was incubated at 28℃ for 5d. If orange transparent halos appeared around the colonies, the strain had nitrogen-fixing ability.

[0077] Strain growth-promoting characteristics are shown in Table 3

[0078] Table 3: IAA and siderophore production abilities of the strains

[0079]

[0080] where "+" represents positive; "-" represents negative.

[0081] As shown in Table 3, the strains have strong IAA and siderophore production abilities, and also have phosphorus solubilization and nitrogen fixation abilities. It is indicated that the two strains can promote plant growth and crop yield through the ways of nitrogen fixation, phosphorus solubilization, plant hormone production, and pathogenic bacteria resistance.

[0082] Example 4: Optimization of fermentation medium of the strains

[0083] 1. Optimization of fermentation medium of Mesorhizobium huakuii LN-ZDJ-1

[0084] 1) Strain culture: single colonies were picked up with an inoculation loop and inoculated in 50mL / 250mL liquid seed solution, and the culture was incubated at 28℃ in a constant temperature incubator at 180r / min until the concentration of Mesorhizobium huakuii LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 reached OD600=0.6 and OD600=1.0, respectively, i.e. the seed solution was prepared. 600

[0085] 2) Determination of growth curve: the above seed solution was inoculated in LB medium at an inoculation amount of 1%, and then mixed and placed in a constant temperature shaker at 28℃ and 180r / min for 36h. Mesorhizobium huakuii LN-ZDJ-1 was taken out every 6h, and Bacillus amyloliquefaciens LN-ZDJ-2 was taken out every 2h, with sterile deionized water as blank control. The OD600um value of the bacterial suspension was determined. The culture time was taken as abscissa, and the OD600um value was taken as ordinate to draw the growth curve. The results are shown in Figure 3 .

[0086] ​3) According to the relevant literature and the actual production needs, YMA medium was used as the basic medium for Rhizobium jilinense LN-ZDJ-1, and the carbon source and pH in the fermentation medium of the strain were optimized. The carbon source mainly selected glucose, sucrose, mannitol, soluble protein, sucrose and proteose peptone; pH = 6.5, pH = 7, pH = 7.5, pH = 8 and pH = 8.5 were selected to change the single variable, respectively, and the above seed liquid was inoculated into the medium with a volume of 50 mL / 250 mL in a triangular flask with an inoculation amount of 1%, and the fermentation temperature was 28℃, the rotation speed was 180r / min, and the shaking culture was carried out for 24-36h. The OD600nm value and the dry weight of the bacterial body under different conditions were determined (the culture solution was centrifuged in a centrifuge with a rotation speed of 4000r / min for 10min, the supernatant was removed, the bacterial body and the pre-weighed sterile 50ml centrifuge tube were placed in a 105℃ oven until the constant weight, the total weight was weighed, and the dry weight of the bacterial body was equal to the total weight minus the weight of the centrifuge tube), and the test was repeated six times to take the average value.

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

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

[0089] According to the relevant literature and the culture medium resources in the laboratory, the influence of different culture media (NA, TSB, LB, beef extract proteose peptone and plant culture medium) on the growth of Bacillus amyloliquefaciens LN-ZDJ-2 was explored, and the OD600un value and the dry weight of the bacterial body were used as the optimization indexes for the optimization of the fermentation medium.

[0090] The results are shown in Figure 5 As shown in the table, under the condition of TSB medium, the OD600um value and the dry weight of the bacterial body of Bacillus amyloliquefaciens LN-ZDJ-2 were significantly higher than those of other culture media (P<0.05), among which the dry weight of the bacterial body reached 1.64g / L, the fermentation efficiency was high, and the price of TSB was economical. After comprehensive comparison, TSB was selected as the best culture medium for Bacillus amyloliquefaciens LN-ZDJ-2.

[0091] 3. Influence of different temperatures and shaking bed strains on the bacterial body of Rhizobium jilinense LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2

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

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

[0094] The results are shown in Table 3. Figure 7 As shown in Table 3, under the condition of 240r / min, the OD600um value and dry weight of the bacterial suspension of Rhizobium jilinense LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 were higher than those at other speeds, the OD600um value of the bacterial suspension was 0.8976 and 0.8553, and the dry weight of the bacterial body was 1.3275g / L and 2.9192g / L, respectively. Therefore, 240r / min was the optimal fermentation speed for Rhizobium jilinense LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2.

[0095] Example Five, Preparation of Liquid Fungicide and Pot Experiment for Broussonetia papyrifera

[0096] Preparation of liquid fungicide: Rhizobium jilinense LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2 strains were inoculated into YMA and TSB liquid medium, 28℃, 180r·min -1 shaking culture for 24-36h, until the OD 600 value of the liquid fungicide reached 0.8, the culture medium was transferred to a sterile 50ml centrifuge tube at 4000r·min -1 centrifugation for 10min, the supernatant was removed, and the lower bacterial body was resuspended with sterile water to an OD 600 of 1.00, and the bacterial suspension with an OD 600 of 1 was compounded at a volume ratio of 1:1.

[0097] Pot experiment of liquid inoculants: The pot experiment was carried out in the Center of Grassland Microorganism of Lanzhou University. Four treatments were designed: single application of Rhizobium jinzhongensis LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, mixed application of Rhizobium jinzhongensis LN-ZDJ-1 and Bacillus amyloliquefaciens LN-ZDJ-2, and control with equal amount of sterile water. Five pots were used for each treatment, and each pot contained 600 g soil. When the seedlings had two leaves and one heart, 0.5 ml (OD 600 =1.00) of the bacterial solution was applied to the roots of each plant in the first treatment, followed by application every other week for a total of four times. After two months, the seedlings were harvested, and the root fresh weight, root dry weight, stem fresh weight, stem dry weight, plant height, leaf nitrogen content, phosphorus content, and soil nitrogen content and phosphorus content were measured for each treatment and the control. The results are shown in Tables 4 and 5.

[0098] Table 4 Effect of different treatments of bacterial inoculants on root development and plant height of B. peregrina

[0099]

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

[0101] As shown in Tables 4 and Figure 8 It can be seen that the bacterial inoculant treatments had different effects on the growth of B. peregrina. The root fresh weight, root dry weight, aboveground dry weight, aboveground fresh weight, and plant height of the experimental groups with bacterial inoculants were all increased compared to the control without bacterial inoculants. The plant height of the treatment with Rhizobium jinzhongensis LN-ZDJ-1 was extremely significantly higher than that of the control, with an increase of 48.4%. The application of liquid bacterial inoculants could also promote the development of the root system of B. peregrina, with different degrees of increase in root fresh weight and root dry weight compared to the control. The aboveground dry weight of the treatment with Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 was the largest among all treatments, with the best growth-promoting effect. The aboveground dry weight of the treatment with Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 was increased by 10.6% and 17.6% compared to the treatments with Bacillus amyloliquefaciens LN-ZDJ-2 and Rhizobium jinzhongensis LN-ZDJ-1, respectively, indicating that mixed culture of the two strains could produce a synergistic effect.

[0102] Table 5 Effect of bacterial inoculants on aboveground nitrogen content, aboveground phosphorus content, plant nitrogen accumulation, and plant phosphorus accumulation of B. peregrina

[0103]

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

[0105] As shown in Table 5, compared with the sterile control, the total nitrogen and nitrogen accumulation of the plants in the experimental group applying Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 inoculant increased by 31.82% and 106.57% respectively compared with the control without inoculant, among which the increase of total nitrogen reached a significant level, and the increase of nitrogen accumulation reached a very significant level. Except for Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 treatment, the phosphorus content of plants treated with each inoculant increased by 0.26% and 14.95% compared with the control. The phosphorus content of Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 treatment decreased by 1.04% compared with the control, and the significant decrease of phosphorus content in plants inoculated with rhizobium was probably caused by the consumption of excessive phosphorus during the process of nodule formation and the large-scale reproduction of microorganisms. Applying Bacillus amyloliquefaciens LN-ZDJ-2 inoculant could increase the phosphorus accumulation of Millettia pachynema, which increased by 65.63% compared with the control. In summary, compared with the control, both single bacteria treatment and mixed bacteria addition could increase the total nitrogen and phosphorus content of Millettia pachynema, as well as the absorption of nitrogen and phosphorus. In summary, inoculation of Rhizobium jinzhongensis LN-ZDJ-1 + Bacillus amyloliquefaciens LN-ZDJ-2 could improve the absorption of N and P in Millettia pachynema and increase the amount of nodulation.

[0106] In summary, the high-efficiency complex microbial inoculant produced by the present application has a significant promoting effect on the growth of Millettia pachynema. Compared with the control group, Millettia pachynema treated with the complex microbial inoculant shows the following significant advantages: increase in aboveground and underground biomass: after using the complex microbial inoculant, the aboveground biomass of Millettia pachynema increased by 49.1%, and the underground biomass increased by 56.1%. This indicates that the complex microbial inoculant can significantly promote the increase of the overall biomass of Millettia pachynema, including the growth of aboveground parts and roots. Promote the absorption of nitrogen and phosphorus elements: the use of complex microbial inoculant can increase the absorption capacity of Millettia pachynema for nitrogen and phosphorus, and the absorption amount increases 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 complex microbial inoculant can also increase the nitrogen content of the leaves of Millettia pachynema, which further promotes the growth and metabolic activity of the plant. The promoting effect of secreting indole acetic acid and iron carrier: the microorganisms in the complex microbial inoculant secrete indole acetic acid and iron carrier, which have a significant promoting effect on the growth of Millettia pachynema. Indole acetic acid is an important plant growth hormone that can promote the development of stems, buds and roots. Iron carrier has high affinity and can bind to free Fe 3+The combination effectively reduces iron element deficiency around the rhizosphere of the plants, thereby inhibiting the reproduction of the pathogenic bacteria in the rhizosphere and reducing the damage to the plants. The composite microbial agent provided by the application can not only significantly improve the growth and yield of the cow-tail trees, but also improve the nutrient absorption capacity of the plants, and further promote the healthy growth and resistance of the plants by secreting active substances such as auxin and iron carrier, which is helpful to improve the quality of the pasture.

[0107] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features can be replaced by equivalents for ordinary skilled persons in the art; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A strain of Rhizobium giardinii LN-ZDJ-1, characterized in that, The Rhizobium jilinense LN-ZDJ-1 is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number is CCTCC NO: M20241652.

2. An inoculant characterized in that, The bacterial agent contains the Rhizobium jilinense LN-ZDJ-1 in claim 1.

3. A microbial complex inoculant, characterized in that, The composite bacterial agent contains the Rhizobium jilinense LN-ZDJ-1 in claim 1 and Bacillus amyloliquefaciens LN-ZDJ-2, which is preserved in the China Center for Type Culture Collection on July 22, 2024, and the preservation number is CCTCC NO: M 20241651.

4. A plant fertilizer, characterized by, The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3.

5. A soil eco-environmental conditioner, characterized in that, The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3.

6. The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3 for activating soil insoluble phosphorus.

7. The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3 for use as or preparation of a plant growth promoter or for promoting plant growth.

8. The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3 for use in improving phosphorus fertilizer utilization rate.

9. The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3 for use in preventing soil hardening, acidification, and harmful substance accumulation caused by the application of phosphorus fertilizer.

10. The Rhizobium jilinense LN-ZDJ-1 in claim 1 or the bacterial agent in claim 2 or the composite bacterial agent in claim 3 for use in preparing a plant fertilizer and / or a soil ecological environment regulator.

Images