Vicia sativa growth-promoting and adversity-resisting complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms and biological fertilizers, in particular to a vicia sativa growth-promoting and stress-resisting complex microbial inoculant and a preparation method thereof.The complex microbial inoculant is formed by mixing arthrobacter, bacillus pumilus, bacillus flexus and brevundimonas intermedia according to the proportion of the arthrobacter, the bacillus pumilus, the bacillus flexus and the brevundimonas intermedia, and the effective viable count in the complex microbial inoculant is 1 * 10 < 9 >-10 < 10 > CFU / g; the synthetic bacterial agent produced by the invention can be applied to the planting and production process of vicia sativa as bacterial fertilizer, and is convenient to apply and remarkable in growth promoting effect. The microbial agent not only can accelerate the accumulation of vicia sativa biomass and improve the production performance, but also can help vicia sativa to resist various environmental stresses, so that the viability of vicia sativa in a severe environment is guaranteed. The method has important significance for improving the production capacity of the common vetch and promoting the development of animal husbandry.

Description

Technical Field

[0001] The present invention relates to the technical fields of microorganisms and biological fertilizers, and specifically to a growth-promoting and stress-resistant composite microbial agent for Vicia sativa, its preparation method and application. Background Art

[0002] Vicia sativa L. has high nutritional, forage and ecological values. It is one of the most widely cultivated green manure and forage grass varieties in China, and is also a high-quality grass species that maintains the stability of grassland plant communities and soil functions. Vicia sativa also has broad application prospects in environmental protection and ecological restoration. Vicia sativa has strong environmental adaptability, being cold-resistant, salt-alkali-tolerant, drought-tolerant and barren-tolerant. It is usually the first batch of immigrant vegetation in barren or degraded soils and has great potential in improving land desertification and salinization.

[0003] The construction of synthetic microbial communities is a frontier research field in the field of microbial ecology. It involves screening and combining microorganisms with complementary functions to form stable and efficient microbial communities. Synthetic microbial communities can improve the adaptability and survival ability to environmental changes by simulating the diversity of microbial communities in natural ecosystems. In the agricultural field, the technology of constructing synthetic microbial communities has been gradually applied to crop cultivation. By constructing microbial communities suitable for specific crops, the yield and quality of crops can be improved. For Vicia sativa, by constructing the core intra-species microbial community, problems in its growth process, such as improving nutrient utilization efficiency and enhancing stress resistance, can be targeted to effectively assist the production of Vicia sativa. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a growth-promoting and stress-resistant composite microbial agent for Vicia sativa, its preparation method and application.

[0005] In order to achieve the above object, the following technical solutions are provided:

[0006] A growth-promoting and stress-resistant composite microbial agent for Vicia sativa, characterized in that: the composite microbial agent is composed of Arthrobacter pascens, Bacillus Pulimus, Bacillus flexus and Brevundimonas intermedia in equal proportions, and the effective viable count in the composite microbial agent is 1×10 9 -10 10 CFU / g;

[0007] The Arthrobacter pascens strain was deposited at the China Center for Type Culture Collection on January 6, 2025. The deposit address is: Wuhan University, Wuhan, China. The taxonomic name is Arthrobacter pascens G33, and the deposit number is CCTCC M 2025019;

[0008] The Bacillus Pulimus strain was deposited at the China Center for Type Culture Collection on January 6, 2025. The deposit address is: Wuhan University, Wuhan, China. The taxonomic name is Bacillus pulimus 66, and the deposit number is CCTCC M 2025017;

[0009] The Bacillus flexus strain was deposited at the China Center for Type Culture Collection on January 6, 2025. The deposit address is: Wuhan University, Wuhan, China. The taxonomic name is Bacillus flexus A18, and the deposit number is CCTCC M 2025016;

[0010] The Brevundimonas intermedia strain was deposited at the China Center for Type Culture Collection on January 6, 2025. The deposit address is: Wuhan University, Wuhan, China. The taxonomic name is Brevundimonas intermedia A74, and the deposit number is CCTCC M 2025018.

[0011] A preparation method of a composite bacterial agent for promoting the growth and resisting adversity of Vicia sativa, which is characterized by comprising the following steps:

[0012] Step 1: Respectively inoculate Arthrobacter pascens, Bacillus Pulimus, Bacillus flexus, and Brevundimonas intermedia into TSB solid medium, and culture at 28°C for about 12 h. After single colonies grow, pick the single colonies and inoculate them into TSB liquid medium for liquid fermentation. The fermentation production conditions are: pH 6.0 - 7.5, fermentation temperature is 26 - 29°C, stirring speed is 180 revolutions per minute, and fermentation time is 20 - 24 h, as the seed liquid;

[0013] Step 2: Respectively inoculate the 4 kinds of seed liquids obtained in Step 1 into a 7L fermenter for liquid fermentation, and the viable bacteria count reaches 1×10 10 CFU / ml or more to obtain the fermentation broth;

[0014] Step 3: Mix the 4 kinds of fermentation broths obtained in Step 2 with the carrier at a weight ratio of 1:1, and then place them in a low-temperature dryer at 40°C. After drying, mix them to obtain the finished compound microbial agent. It is measured that the concentration of the target strain in the microbial agent is not less than 1×10 9 cfu / g.

[0015] Furthermore, in Step 2, the fermentation parameters of Arthrobacter pascens are as follows: the culture medium is TSB liquid medium, the fermentation temperature is 30°C, the fermentation time with an inoculum size of 10% is 10 - 14 hours, and the fermentation pH value is 7.5 - 7.8; the fermentation parameters of Bacillus pulimus are as follows: the culture medium is B liquid medium, the fermentation temperature is 37°C, the fermentation time with an inoculum size of 10% is 10 - 14 hours, and the fermentation pH value is 7.2 - 7.6; the fermentation parameters of Bacillus flexus are as follows: the culture medium is TSB liquid medium, the fermentation temperature is 27°C, the fermentation time with an inoculum size of 10% is 14 - 18 hours, and the fermentation pH value is 6.5 - 7.0; the fermentation parameters of Brevundimonas intermedia are as follows: the culture medium is TSB liquid medium, the fermentation temperature is 29°C, the fermentation time with an inoculum size of 10% is 10 - 14 hours, and the fermentation pH value is 7.2 - 7.6.

[0016] Preferably, the composition of the TSB liquid medium is: 1L of deionized water, 8.5g of tryptone, 1.5g of peptone, 2.5g of NaCl, 1.25g of KH2PO4, 1.25g of C6H 12 O6, dissolved with deionized water, adjusted to a pH of 7.1 - 7.5 with NaOH, and autoclaved at 121°C for 20 min; TSB solid medium: add 20g of agar to the above TSB liquid medium.

[0017] Preferably, the composition of the NB liquid medium is: 1L of deionized water, 10.0g of peptone, 3.0g of beef extract powder, 5.0g of NaCl, stirred and dissolved, adjusted to a pH of 7.1 - 7.5 with NaOH, and autoclaved at 121°C for 20 min.

[0018] Furthermore, in Step 3, the carrier is uniformly mixed by rice husk powder and diatomite at a mass ratio of 1:1.

[0019] Application of the Vicia sativa growth-promoting and stress-resistant compound microbial agent of the present invention in promoting the growth of Vicia sativa.

[0020] The synthetic bacterial agent produced by the present invention can be used as a bacterial fertilizer in the planting and production process of Vicia sativa, which is convenient to apply and has a remarkable growth-promoting effect. This bacterial agent can not only accelerate the accumulation of the biomass of Vicia sativa and improve its production performance, but also help Vicia sativa resist various environmental stresses, ensuring its survival ability in harsh environments. The present invention is of great significance for improving the production capacity of Vicia sativa and promoting the development of animal husbandry.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. Rice bran powder is added to the carrier of the present invention, which not only reduces the production cost but also helps to increase the content of soil organic matter.

[0023] 2. The strains used in the present invention are derived from the isolation and screening of the core intra-species microorganisms of Vicia sativa. Under the field planting environment, the strains of this product can migrate to the plant body and finally transfer to the seeds to achieve the purpose of repairing or supplementing the endophytic bacteria of Vicia sativa seeds. Description of the Drawings

[0024] Figure 1 It is a diagram of the results of the plate confrontation experiment of 4 bacteria in Example 2 of the present invention;

[0025] Figure 2 It is a comparison diagram of the fresh weight and dry weight of Vicia sativa seeds inoculated with Arthrobacter pascens in Example 1 and the control group;

[0026] Figure 3 It is a comparison diagram of the fresh weight and dry weight of Vicia sativa seeds inoculated with Bacillus pulimus in Example 1 and the control group;

[0027] Figure 4 It is a comparison diagram of the fresh weight and dry weight of Vicia sativa seeds inoculated with Bacillus flexus in Example 1 and the control group;

[0028] Figure 5 It is a comparison diagram of the fresh weight and dry weight of Vicia sativa seeds inoculated with Brevundimonas intermedia in Example 1 and the control group;

[0029] Figure 6 It is a comparison diagram of the growth-promoting effect and the control group after applying the compound bacterial agent produced in Example 4 in the greenhouse experiment of Example 5 of the present invention;

[0030] Figure 7 It is a comparison diagram of the fresh weight and dry weight and the control group after applying the compound bacterial agent produced in Example 4 in the greenhouse experiment of Example 5 of the present invention; Detailed Embodiments

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0032] Example 1 Isolation and Screening of Functional Bacteria

[0033] 1. Sterile Seedling Cultivation

[0034] Take the seeds of Vicia sativa L. cv. LanJian No. 3 and place them in a 4°C refrigerator for vernalization for 3 days. Then disinfect the seed surface (disinfect with 1% sodium hypochlorite for 1 minute, 70% ethanol for 30 seconds, wash with sterile water 3 - 6 times, and blot the surface moisture with filter paper), soak and swell in sterile water for 24 hours, and then place them in a sterile glass culture tube (diameter: 48 mm; height: 200 mm) filled with sterilized 1 / 2 MS medium, and culture in an artificial climate incubator for 21 days (day: 16 h / 25°C; night: 8 h / 20°C), with 10 replicates for each generation.

[0035] 2. Strain Isolation

[0036] Harvest and grind the sterile seedlings, extract the total genomic DNA using the FastDNA Spin Kit for Soil, and sequence using the Illumina P250 platform with a paired-end read length of 250 bp to identify endophytic bacteria species.

[0037] Select Vicia sativa L. plants with good growth and no disease spots for the isolation experiment. Disinfect the surface of Vicia sativa L. seeds. First, wash them with running water, air dry, then transfer them to a laminar flow hood. Rinse with 75% ethanol for 30 seconds in the hood, wash with sterile water, then rinse with 1% sodium hypochlorite for 30 seconds, wash with sterile water 6 times, and blot the surface moisture with filter paper. Place the Vicia sativa L. tissue in a mortar and grind it completely, then let it stand for 3 hours. Absorb the supernatant and dilute it with ultrapure water, and set 4 concentration gradients from 10 -1 ~10 -4 . Make 4 replicates for each concentration gradient, coat them on TSB medium, and use the sterile water from the last wash as a control to coat the plate for culturing to check whether the disinfection is thorough. Culture in an incubator at 28°C for 1 - 2 days. When various morphological colonies grow on the plate, use an inoculation loop to streak and culture the strains with significantly different morphologies on TSB medium. Perform 16S rRNA sequencing on the isolated endophytic bacteria and compare the results with the sequencing results of the sterile seedlings. Those with consistent comparison results are the ones inside the Vicia sativa L. seeds.

[0038] 4. Identification of the Growth Promotion Effect of Single Bacteria

[0039] Pot experiments were conducted on core endophytes and key endophytes to screen out strains with growth-promoting effects. The vetch seeds used in the experiment were purchased from Jiangsu Chunzhiqiu Technology Ecology Co., Ltd., and the experimental site was the greenhouse of the Grassland Microorganism Center of Lanzhou University. 50 ml of TSB liquid medium was filled in a 200 ml conical flask, inoculated with a single colony of the key endophyte strain, and cultured in a shaker at 28 °C and 180 rpm for 24 h to obtain the bacterial liquid for the experiment. For the treatment group of potted plants, 10 ml of the bacterial liquid was mixed per kilogram of soil, and for the control group of potted plants, 10 ml of blank medium was mixed per kilogram of soil. Each group was set with 3 replicates and cultured in a suitable environment (air temperature 24 - 26 °C, humidity 45 - 55% RH, soil field water holding capacity 80% - 90%, light intensity 20000 Lux, light duration 12 h / day, light source was white light LED lamps) for 20 days. A total of 4 bacteria with significant growth-promoting effects were screened out, namely Arthrobacter pascens, Bacillus Pulimus, Bacillus flexus, and Brevundimonas intermedia. Figure 2 (a, b) show the comparison of dry weight and fresh weight between vetch seeds inoculated with Arthrobacter pascens and non-inoculated ones. It can be seen that the dry weight and fresh weight increased significantly after inoculation; Figure 3 (a, b) show the comparison of dry weight and fresh weight between vetch seeds inoculated with Bacillus Pulimus and non-inoculated ones. It can be seen that the dry weight and fresh weight increased significantly after inoculation; Figure 4 (a, b) show the comparison of dry weight and fresh weight between vetch seeds inoculated with Bacillus flexus and non-inoculated ones. It can be seen that the dry weight and fresh weight increased significantly after inoculation; Figure 5 (a, b) show the comparison of dry weight and fresh weight between vetch seeds inoculated with Brevundimonas intermedia and non-inoculated ones. It can be seen that the dry weight and fresh weight increased significantly after inoculation.

[0040] Example 2 Evaluation of the antagonistic relationship between strains

[0041] The absence of obvious antagonistic effects between selected strains is an important criterion for constructing synthetic microbial communities. Therefore, in order to verify whether the 4 core bacteria within vetch seeds in Example 1 can coexist amicably, a plate confrontation experiment based on the filter paper method was conducted in this example.

[0042] The bacterial strains were activated with TSB liquid medium until the logarithmic growth phase. 50 μL of 4 kinds of bacterial suspensions were respectively pipetted and evenly spread on TSB solid plates. Each kind of bacterium was spread on 3 plates repeatedly. Sterile filter paper was picked up with forceps and soaked in the other 3 kinds of bacterial suspensions except the already spread strain, and the filter paper was laid flat on the plate. It was cultured at 28 °C for 48 h, and whether a clear zone was formed around the filter paper was observed.

[0043] Figure 1 For the confrontation experiment results of these 4 kinds of bacteria on the plate, no obvious inhibition zone was observed, indicating that there was no obvious antagonistic effect between any two kinds of bacteria.

[0044] Example 3 Optimization of fermentation parameters

[0045] To determine the suitable conditions for the fermentation of the 4 kinds of bacteria described in Example 2, the fermentation parameters of the 4 kinds of bacteria were tested. The optional media in the test were TSB (tryptone soy broth) liquid medium, NB (nutrient broth) liquid medium, and LB (lysogeny broth) liquid medium. The set temperature gradients were 26 °C, 28 °C, 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C, 42 °C, and the set pH gradients were pH = 4, pH = 5, pH = 6, pH = 7, pH = 8, pH = 9. The test method was to perform permutations and combinations of the above media and culture conditions. Each combination was a treatment. Samples of the liquid medium of all treatments were taken every two hours to measure the absorbance and draw the fermentation curve. Table 1 shows the recommended fermentation parameters obtained from the analysis of the test results.

[0046] Table 1 Recommended fermentation parameters

[0047]

[0048] The composition of TSB liquid medium is: 1 L of deionized water, 8.5 g of tryptone, 1.5 g of plant peptone, 2.5 g of NaCl, 1.25 g of KH2PO4, 1.25 g of C6H 12 O6. Stir and dissolve, adjust the pH to 7.1 - 7.5 with NaOH, and sterilize at 121 °C for 20 min to obtain TSB liquid medium; TSB solid medium: add 20 g of agar to the above liquid medium to obtain TSB solid medium.

[0049] The composition of NB liquid medium is: 1 L of deionized water, 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of sodium chloride. Stir and dissolve, adjust the pH to 7.1 - 7.5 with NaOH, and sterilize at 121 °C for 20 min to obtain NB liquid medium.

[0050] Example 4 Production of compound bacterial agent

[0051] (1) The 4 strains of bacteria screened in Example 1 were respectively inoculated into TSB solid medium and cultured at 28°C for about 12 h. After single colonies grew, single colonies were picked and inoculated into TSB liquid medium for liquid fermentation. The fermentation production conditions were as follows: pH 6.0 - 7.5, fermentation temperature range 26 - 29°C, stirring speed 180 revolutions per minute, and fermentation time 20 - 24 h, serving as the seed liquid.

[0052] (2) The seed liquid produced in step (1) was inoculated into fermentation tanks containing 7 L of liquid medium respectively for fermentation culture. It was inoculated into the liquid medium according to the fermentation parameters provided in Example 2 and fermented, and the inoculation amount was 5%. After measurement, the viable bacteria count reached 1×10 10 CFU / ml or more.

[0053] (3) The bacterial liquid produced in step (2) was mixed with the carrier at a weight ratio of 1:1. After the bacterial liquid and the carrier were evenly mixed, they were placed in a constant temperature at 28°C for 72 h, and then dried at low temperature. The finished bacterial agent was obtained after mixing. After measurement, the concentration of the target strain in the bacterial agent was not less than 1×10 9 cfu / g.

[0054] The carrier was obtained by evenly mixing rice hull powder and diatomite at a mass ratio of 1:1.

[0055] Example 5 Greenhouse Test

[0056] The growth-promoting pot experiment was carried out in the greenhouse of Lanzhou University. The vetch seeds used in the experiment were purchased from Jiangsu Chunzhiqiu Technology Ecology Co., Ltd. A total of one control group and one treatment group were set up. The treatment group mixed the bacterial fertilizer produced in Example 3 in the pot. The control group and the treatment group were placed in three simulated stress environments and a suitable environment for cultivation, and 5 replicates were set for the control group and the treatment group in each environment. The four environments were as follows:

[0057] ① Normal suitable environment (air temperature 24 - 26°C, humidity 45 - 55%RH, soil water content 80% - 90%, light intensity 20000 Lux, light duration 12 h / day, light source white light LED lamp)

[0058] ② Simulated drought stress environment (controlling the soil water content at 40% - 50%, and the other conditions were the same as those in group ①)

[0059] ③ Simulated salt stress environment (controlling the soil salt content at 10 mmol / Kg, and the other conditions were the same as those in group ①)

[0060] ④ Simulated cold stress environment (air temperature 10°C, and the other conditions were the same as those in group ①)

[0061] After 20 days of cultivation, the plants and the potting soil in the experimental group and the control group were separated, and the relevant growth traits were measured.

[0062] 1) Growth of the above-ground part: As Figure 6 shown, the overall growth of the treatment group was better than that of the control group in the four simulated environments.

[0063] 2) Fresh weight: As Figure 7 shown in a, the fresh weight of the treatment group was significantly greater than that of the control group in the four simulated environments.

[0064] 3) Dry weight: As Figure 7 shown in b, the dry weight of the treatment group was significantly greater than that of the control group in the four simulated environments.

[0065] The overall test results show that the compound microbial agent developed in this patent can promote the growth of Vicia sativa plants.

[0066] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention. Any reference signs in the claims should not be construed as limiting the claimed invention.

Claims

1. A composite microbial inoculum for promoting the growth of Vicia sativa and resisting adversity, characterized in that: The composite bacterial agent is composed of Arthrobacter pascens, Bacillus Pulimus, Bacillusflexus, and Brevundimonas intermedia in equal proportions, and the effective viable count in the composite bacterial agent is 1×10 9 -10 10 CFU / g; the preservation number of the Arthrobacter pascens strain: CCTCC M 2025019; the preservation number of the Bacillus Pulimus strain: CCTCC M2025017; the preservation number of the Bacillusflexus strain: CCTCC M 2025016; the preservation number of the Brevundimonas intermedia strain: CCTCC M 2025018.

2. A preparation method of a composite microbial inoculum for promoting the growth of Vicia sativa and resisting adversity, characterized in that, It includes the following steps: Step 1: Inoculate Arthrobacter pascens, Bacillus Pulimus, Bacillus flexus, and Brevundimonas intermedia into TSB solid medium respectively, culture at 28°C for 10 - 14 hours. After single colonies grow, pick the single colonies and inoculate them into TSB liquid medium for liquid fermentation. The fermentation conditions are: pH 6.0 - 7.5, fermentation temperature 26 - 29°C, stirring speed 180 revolutions per minute, fermentation time 20 - 24 h, as the seed liquid; Step 2: Inoculate the 4 kinds of seed liquids obtained in Step 1 into a 7 L fermenter respectively for liquid fermentation until the viable cell count reaches 1×10 10 CFU / ml or more to obtain fermentation broth; Step 3: Mix the 4 kinds of fermentation broths obtained in Step 2 with the carrier at a weight ratio of 1:1, and then place them in a low-temperature dryer at 40°C. After drying, mix them to obtain the finished composite microbial agent. It is measured that the concentration of the target strain in the microbial agent is not lower than 1×10 9 cfu / g.

3. The preparation method of a composite microbial agent for promoting the growth of vetch and resisting adversity according to claim 1, characterized in that: In Step 2, the fermentation parameters of Arthrobacter pascens are: the medium is TSB liquid medium, the fermentation temperature is 30°C, the fermentation time with 10% inoculum is 10 - 14 hours, and the fermentation pH value is 7.5 - 7.8; the fermentation parameters of Bacillus Pulimus are: the medium is NB liquid medium, the fermentation temperature is 37°C, the fermentation time with 10% inoculum is 10 - 14 hours, and the fermentation pH value is 7.2 - 7.6; the fermentation parameters of Bacillus flexus are: the medium is TSB liquid medium, the fermentation temperature is 27°C, the fermentation time with 10% inoculum is 14 - 18 hours, and the fermentation pH value is 6.5 - 7.0; the fermentation parameters of Brevundimonas intermedia are: the medium is TSB liquid medium, the fermentation temperature is 29°C, the fermentation time with 10% inoculum is 10 - 14 hours, and the fermentation pH value is 7.2 - 7.

6.

4. The preparation method of a composite microbial agent for promoting the growth and resisting adversity of vetch according to claim 3, characterized in that: The composition of the TSB liquid medium is as follows: 1 L of water, 8.5 g of tryptone, 1.5 g of peptone, 2.5 g of NaCl, 1.25 g of KH2PO4, 1.25 g of C6H 12 O6, dissolved in deionized water, adjusted to pH 7.1 - 7.5 with NaOH, and autoclaved at 121 °C for 20 min; TSB solid medium: Add 20 g of agar to the above TSB liquid medium.

5. The preparation method of a composite microbial inoculum for promoting the growth of Vicia sativa and resisting adversity according to claim 3, characterized in that: The composition of the NB liquid medium is: 1 L of deionized water, 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of NaCl, stir to dissolve, adjust the pH to 7.1 - 7.5 with NaOH, and autoclave at 121°C for 20 min.

6. The preparation method of a composite microbial inoculum for promoting the growth and resisting adversity of Vicia sativa according to claim 2, characterized in that: In Step 3, the carrier is uniformly mixed by rice hull powder and diatomaceous earth in a mass ratio of 1:

1.

7. Application of the vetch growth - promoting and stress - resistant compound microbial agent according to claim 1 in promoting the growth of vetch.

Images