Agrobacterium tumefaciens GH6, fungicide and bacterial fertilizer based on agrobacterium tumefaciens GH6, and preparation method and application of agrobacterium tumefaciens GH6
By combining Agrobacterium tumefaciens GH6 with distiller's grains to prepare distiller's grains microbial fertilizer, the problem of limited microbial strains in distiller's grains in existing technologies has been solved, which has significantly promoted the growth of sorghum and increased nitrogen and phosphorus content and rhizosphere matrix activity.
Patent Information
- Application Number
- CN202511183468.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-10-28
AI Technical Summary
In existing technologies, when distillers' grains are used as fertilizer for sorghum, the limited number of microorganisms makes it difficult to effectively promote sorghum growth, and the effect of distillers' grains alone on increasing the number of soil microorganisms and enzyme activity is limited.
Agrobacterium tumefaciens GH6 was compounded with distiller's grains to prepare distiller's grains microbial fertilizer. Through fermentation, the fertility of the distiller's grains was improved and the growth of sorghum was promoted.
It significantly improved the growth of sorghum, including indicators such as plant height, root length, fresh weight, dry weight, and nitrogen and phosphorus content. In particular, the available nitrogen and phosphorus content in the rhizosphere substrate was superior to that of distillers' grains alone.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to Agrobacterium tumefaciens GH6, and its inoculants, inoculants, and their preparation methods and applications. Background Technology
[0002] Baijiu (Chinese liquor) lees are a byproduct of the brewing industry. Studies have shown that producing 1 ton of baijiu generates approximately 3 to 4 tons of fresh lees. Although lees have a high organic matter content, their nutrient content is low. When producing lees-based microbial fertilizer, it is generally done by adding highly efficient phosphorus-solubilizing bacteria, nitrogen-fixing bacteria, cellulose-degrading bacteria, or by using compound microbial agents such as Bacillus, molds, and brewer's yeast to promote the composting process and improve the quality of the lees-based microbial fertilizer.
[0003] Sorghum is the world's fifth largest cereal crop, and it can be grown in plains, hills, waterlogged areas, and saline-alkali lands. It has multiple uses, including food, brewing, feed, energy, and silage, and possesses great development potential. When using distiller's grains microbial fertilizer to enhance sorghum fertility, the number of soil microorganisms and enzyme activity significantly increase with the amount of fertilizer applied, leading to a corresponding increase in sorghum yield. However, currently, the number of microbial strains that can be compounded with distiller's grains to produce microbial fertilizer and promote sorghum growth is very limited.
[0004] Therefore, developing microbial strains that can be compounded with distiller's grains to produce microbial fertilizer and promote sorghum growth is of great significance for promoting sorghum growth and the green and healthy development of the liquor industry. Summary of the Invention
[0005] The present invention aims to provide a strain of Agrobacterium tumefaciens GH6 that can be compounded with distiller's grains to produce microbial fertilizer and can promote the growth of sorghum.
[0006] A type of Agrobacterium tumefaciens GH6, classified and named Agrobacterium tumefaciens GH6, the Agrobacterium tumefaciens GH6, was deposited at the China Center for Type Culture Collection on October 28, 2024, with accession number CCTCC NO: M20242355, and deposit address: Wuhan University, Wuhan, China.
[0007] Preferably, as an improvement, the Agrobacterium tumefaciens GH6 is isolated from sorghum roots.
[0008] A microbial inoculant comprising the Agrobacterium tumefaciens GH6.
[0009] A microbial inoculant composed of Agrobacterium tumefaciens GH6.
[0010] A method for preparing a microbial inoculant includes the following steps: Step 1: Inoculate Agrobacterium tumefaciens GH6 into LB liquid medium; Step 2: Incubate at 25-35℃ on a shaker at 100-200 rpm for 40-50 hours; Step 3: Centrifuge at 7500-8500 rpm for 4-6 minutes to collect bacterial cells, remove the supernatant, wash the bacterial cells with sterile water, and then suspend the bacterial cells in sterile water to prepare OD. 600 =0.5~0.6 bacterial suspension, which is the microbial inoculant (Agrobacterium tumefaciens GH6 inoculant).
[0011] A fermented fertilizer made from distiller's grains is obtained by fermenting the aforementioned microbial agent and distiller's grains together.
[0012] A method for producing a distiller's grains microbial fertilizer includes the following steps: (1) Preparation of microbial inoculum: Agrobacterium tumefaciens GH6 was inoculated into LB liquid medium and cultured on a shaker at 100-200 rpm at 25-35℃ for 40-50 h; the bacterial cells were collected by centrifugation at 7500-8500 rpm for 4-6 min, the supernatant was removed, the bacterial cells were washed with sterile water, and the bacterial cells were then suspended in sterile water to prepare OD. 600 =0.5~0.6 bacterial suspension, which is a microbial inoculant; (2) Mixed fermentation: Mix the lees and microbial inoculants at a ratio of 10g:0.5-1.5mL, pile them up and ferment for 5-10 days to obtain lees microbial fertilizer.
[0013] Studies have found that the effect of distillers' grains microbial fertilizer in promoting sorghum growth is significantly better than that of distillers' grains alone. Therefore, this invention also seeks protection for the application of distillers' grains microbial fertilizer in promoting sorghum growth. Attached Figure Description
[0014] Figure 1 Comparison of sorghum seedling growth from the first sampling.
[0015] Figure 2 Comparison chart of nitrogen and phosphorus content analysis from the first sampling.
[0016] Figure 3 Comparison chart showing the growth of sorghum seedlings from the second sampling.
[0017] Figure 4 Comparison chart of nitrogen and phosphorus content analysis from the second sampling.
[0018] Figures 1-4 In the diagram, CK represents the blank group, JZ represents the distiller's grains fertilizer group, and GH6 represents the GH6 distiller's grains microbial fertilizer group. Detailed Implementation
[0019] The following detailed description illustrates the specific implementation method: Example 1: Preparation of microbial inoculants Agrobacterium tumefaciens GH6 was picked from LB plates. Agrobacterium tumefaciens Single colonies of GH6 were inoculated into LB liquid medium, placed on a shaker, and incubated at 150 rpm and 30 °C for 48 h. The cells were collected by centrifugation at 8000 rpm for 10 min, washed three times with sterile water, and then resuspended in sterile water to prepare a bacterial suspension. The concentration of the bacterial suspension was adjusted to OD0.05. 600 =0.5~0.6, yielding the microbial inoculum. Agrobacterium tumefaciens GH6 was deposited on October 28, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242355, located at Wuhan University, Wuhan, China. Classification and nomenclature... Agrobacterium tumefaciens GH6.
[0020] Example 2: Fertilizer Preparation Preparation of distiller's grains fertilizer: Weigh 1500g of distiller's grains and add them to a self-sealing bag. Add 150 mL of sterile water, leave the bag open, and pile it up for fermentation for 7 days to obtain distiller's grains fertilizer.
[0021] Preparation of distiller's grains microbial fertilizer: Weigh 1500g of distiller's grains and add them to a self-sealing bag. Add 150mL of the microbial agent prepared in Example 1, leave it open, and pile it up for fermentation for 7 days to obtain distiller's grains microbial fertilizer. To distinguish it more clearly from distiller's grains fertilizer, the distiller's grains microbial fertilizer will be referred to as GH6 distiller's grains microbial fertilizer in the following text.
[0022] I. Sorghum Pot Experiment (1) Substrate preparation Mix perlite and vermiculite in a 1:4 ratio and add them to the flowerpots, with 200 g of substrate added to each pot. Add 50 g of GH6 distiller's grains microbial fertilizer to each pot (GH6 group). The distiller's grains group receives an equal amount of distiller's grains fertilizer (GH6), while the control group receives neither distiller's grains fertilizer nor GH6.
[0023] (2) Sorghum seed treatment Select plump, disease-free red sorghum seeds and add enough sterile water to cover them. Soak at room temperature for 48 hours, until most of the seeds show signs of sprouting.
[0024] (3) Sorghum planting Sow the sorghum seeds that have just sprouted into white plumes into flowerpots, 30-35 seeds per pot. During the growth period of the sorghum seedlings, water the flowerpots with sterile water as needed.
[0025] (4) Sampling Sampling was conducted at 20 and 40 days after sowing. During sampling, the plants were carefully removed from the pots using tweezers, rinsed under running water to remove the root substrate, and then the surface moisture was absorbed using absorbent paper. The plant height, root length, fresh weight, dry weight, total nitrogen content, and total phosphorus content of the sorghum seedlings were measured. The available nitrogen and available phosphorus content in the root substrate of the sorghum seedlings were also determined.
[0026] II. Results Analysis 1. Analysis of the first sampling results (1) Analysis of sorghum seedling growth like Figure 1 As shown in Figure A, the average height of sorghum seedlings in the control group was 10.83 cm, and the average height of seedlings in the distiller's grains group was 10.253 cm, with no significant difference compared to the control group. The average height of seedlings in the GH6 group was 12.307 cm, which was significantly higher than that of the control group by 13.638% and significantly higher than that of the distiller's grains group by 20.033%. Compared with the control group, the addition of distiller's grains did not affect the height of sorghum seedlings, while GH6 distiller's grains microbial fertilizer had a promoting effect on the height of sorghum seedlings.
[0027] like Figure 1 As shown in Figure B, the average root length of sorghum seedlings in the control group was 8.99 cm, while the average root length in the distiller's grains group was 6.173 cm, significantly lower than the control group by 31.335%. The average root length in the GH6 group was 7.837 cm, showing no significant difference compared to the control group, but significantly higher than the distiller's grains group by 26.956%. The addition of distiller's grains fertilizer inhibited the root length of sorghum seedlings, while the addition of GH6 distiller's grains microbial fertilizer had no significant effect on root length.
[0028] like Figure 1 As shown in Figure C, the average fresh weight of sorghum seedlings in the blank group was 0.314 g, and the average fresh weight of the distiller's grains group was 0.323 g, with no significant difference compared to the blank group. The average fresh weight of the GH6 group was 0.513 g, which was significantly increased by 63.376% compared to the blank group and by 58.824% compared to the distiller's grains group. Adding distiller's grains fertilizer had no significant effect on the fresh weight of sorghum seedlings, but adding GH6 distiller's grains microbial fertilizer promoted the fresh weight of sorghum seedlings.
[0029] like Figure 1 As shown in Figure D, the average dry weight of sorghum seedlings in the control group was 0.061 g, and the average dry weight of the distiller's grains group was 0.063 g, with no significant difference compared to the control group. The average dry weight of the GH6 group was 0.094 g, which was significantly higher than the control group by 54.098% and significantly higher than the distiller's grains group by 49.206%. Adding distiller's grains fertilizer had no significant effect on the dry weight of sorghum seedlings, but adding GH6 distiller's grains microbial fertilizer promoted the dry weight of sorghum seedlings.
[0030] (2) Nitrogen and phosphorus content analysis like Figure 2 As shown in Figure A, the average total nitrogen content of sorghum seedlings in the control group was 18.784 g / kg, while the average total nitrogen content in the distiller's grains group was 27.315 g / kg, significantly higher than the control group by 45.416%. The average total nitrogen content in the GH6 group was 33.052 g / kg, significantly higher than the control group by 75.958% and significantly higher than the distiller's grains group by 21.003%. Compared with distiller's grains, GH6 distiller's grains microbial fertilizer had a promoting effect on the total nitrogen content of sorghum seedlings.
[0031] like Figure 2 As shown in Figure B, the average total phosphorus content of sorghum seedlings in the control group was 2.722 g / kg, while the average total phosphorus content in the distiller's grains group was 4.557 g / kg, significantly higher than the control group by 67.414%. The average total phosphorus content in the GH6 group was 6.05 g / kg, significantly higher than the control group by 122.263% and significantly higher than the distiller's grains group by 32.763%. Compared with distiller's grains, both GH6 distiller's grains microbial fertilizers promoted the total phosphorus content of sorghum seedlings.
[0032] like Figure 2 As shown in Figure C, the average available nitrogen content in the rhizosphere substrate of sorghum seedlings in the control group was 52.83 mg / kg, while the average available nitrogen content in the distiller's grains group was 154.013 mg / kg, significantly higher than the control group by 191.526%. The average available nitrogen content in the GH6 group was 188.919 mg / kg, significantly higher than the control group by 257.598% and significantly higher than the distiller's grains group by 22.664%. Compared with distiller's grains, the addition of GH6 distiller's grains microbial fertilizer promoted the available nitrogen content in the rhizosphere substrate of sorghum seedlings.
[0033] like Figure 2 As shown in Figure D, the average available phosphorus content in the rhizosphere substrate of sorghum seedlings in the control group was 95.83 mg / kg, while the average available phosphorus content in the distiller's grains group was 312.802 mg / kg, significantly higher than the control group by 226.413%. The average total phosphorus content in the GH6 group was 359.683 mg / kg, significantly higher than the control group by 275.334% and significantly higher than the distiller's grains group by 14.987%. Compared with distiller's grains, the addition of GH6 distiller's grains microbial fertilizer promoted the available phosphorus content in the rhizosphere substrate of sorghum seedlings.
[0034] (3) Comprehensive evaluation of the effects of different treatments on sorghum growth Principal component analysis was performed on the plant height, root length, fresh weight, dry weight, total nitrogen content, total phosphorus content, and available nitrogen and available phosphorus content in the rhizosphere matrix of sorghum seedlings under three treatments. Table 1 shows that the cumulative contribution rate of the first two principal components was 100%. Based on the weighted proportions of the variance contribution rates of the first two principal components to the cumulative contribution rate, a comprehensive evaluation model was constructed. Y =0.75352X1+0.24648X2, Y The overall score is shown in Table 2. The overall score for the control group was -1.94, for the distiller's grains group it was -0.608, and for the GH6 distiller's grains microbial fertilizer group it was 2.548. Based on the overall score, the GH6 distiller's grains microbial fertilizer group > the distiller's grains group > the control group. This indicates that the GH6 distiller's grains microbial fertilizer group had the best effect on promoting the growth of sorghum seedlings.
[0035] Table 1. Principal component analysis results from the first sampling.
[0036] Table 2. Principal component factor scores and composite scores for different processing methods in the first sampling.
[0037] 2. Analysis of the Second Sampling Results (1) Analysis of sorghum seedling growth like Figure 3 As shown in Figure A, the average height of sorghum seedlings in the control group was 13.77 cm, and the average height in the distillers' grains group was 12.783 cm, showing no significant difference compared to the control group. The average height of the GH6 group was 15.693 cm, significantly higher than the control group by 13.965% and significantly higher than the distillers' grains group by 22.765%. Compared to the control group, the addition of distillers' grains did not affect the height of sorghum seedlings, while the addition of GH6 distillers' grains microbial fertilizer promoted the height of sorghum seedlings.
[0038] like Figure 3 As shown in Figure B, the average root length of sorghum seedlings in the control group was 9.93 cm, and the average root length in the distiller's grains group was 9.237 cm, showing no significant difference compared to the control group. The average root length in the GH6 group was 9.197 cm, showing no significant difference compared to both the control group and the distiller's grains group. The addition of distiller's grains or GH6 distiller's grains microbial fertilizer had no significant effect on the root length of sorghum seedlings.
[0039] like Figure 3 As shown in Figure C, the average fresh weight of sorghum seedlings in the control group was 0.877 g, and the average fresh weight of the distiller's grains group was 0.923 g, with no significant difference compared to the control group. The average fresh weight of the GH6 group was 0.89 g, with no significant difference compared to both the control group and the distiller's grains group. Adding distiller's grains or GH6 distiller's grains microbial fertilizer had no significant effect on the fresh weight of sorghum seedlings.
[0040] like Figure 3 As shown in Figure D, the average dry weight of sorghum seedlings in the control group was 0.181 g, and the average dry weight of the distiller's grains group was 0.19 g, with no significant difference compared to the control group. The average dry weight of the GH6 group was 0.233 g, which was significantly higher than the control group by 28.729% and significantly higher than the distiller's grains group by 22.632%. Adding distiller's grains fertilizer had no significant effect on the dry weight of sorghum seedlings; however, adding GH6 distiller's grains microbial fertilizer promoted the dry weight of sorghum seedlings.
[0041] (2) Nitrogen and phosphorus content analysis like Figure 4 As shown in Figure A, the average total nitrogen content of sorghum seedlings in the control group was 19.443 g / kg, while the average total nitrogen content in the distiller's grains group was 40.01 g / kg, significantly higher than the control group by 105.781%. The average total nitrogen content in the GH6 group was 44.206 g / kg, significantly higher than the control group by 127.362% and significantly higher than the distiller's grains group by 10.487%. Compared with distiller's grains, GH6 distiller's grains microbial fertilizer has a promoting effect on the total nitrogen content of sorghum seedlings.
[0042] like Figure 4 As shown in Figure B, the average total phosphorus content of sorghum seedlings in the control group was 3.032 g / kg, and the average total phosphorus content in the distiller's grains group was 3.12 g / kg, showing no significant difference compared to the control group. The average total phosphorus content in the GH6 group was 3.891 g / kg, significantly higher than the control group by 28.331% and significantly higher than the distiller's grains group by 24.712%. Compared to distiller's grains, the addition of GH6 distiller's grains microbial fertilizer promoted the total phosphorus content of sorghum seedlings.
[0043] like Figure 4 As shown in Figure C, the average available nitrogen content in the rhizosphere substrate of sorghum seedlings in the control group was 7.69 mg / kg, while the average available nitrogen content in the distillers' grains group was 131.783 mg / kg, significantly higher than the control group by 1613.693%. The average available nitrogen content in the GH6 group was 139.76 mg / kg, significantly higher than the control group by 1717.425% and significantly higher than the distillers' grains group by 6.053%. Compared with distillers' grains, the addition of GH6 distillers' grains microbial fertilizer promoted the available nitrogen content in the rhizosphere substrate of sorghum seedlings.
[0044] like Figure 4As shown in Figure D, the average available phosphorus content in the rhizosphere substrate of sorghum seedlings in the control group was 3.77 mg / kg, while the average available phosphorus content in the distillers' grains group was 452.602 mg / kg, significantly higher than the control group by 11905.36%. The average total phosphorus content in the GH6 group was 532.929 mg / kg, significantly higher than the control group by 14036.048% and significantly higher than the distillers' grains group by 17.748%. Compared with distillers' grains, the addition of GH6 distillers' grains microbial fertilizer promoted the available phosphorus content in the rhizosphere substrate of sorghum seedlings.
[0045] (3) Comprehensive evaluation of the effects of different treatments on sorghum growth Principal component analysis was performed on the plant height, root length, fresh weight, dry weight, total nitrogen content, total phosphorus content, and available nitrogen and available phosphorus content in the rhizosphere matrix of sorghum treated with the three methods. Table 3 shows that the cumulative contribution rate of the first two principal components was 100%. Based on the weighted proportions of the variance contribution rates of the first two principal components to the cumulative contribution rate, a comprehensive evaluation model was constructed. Y =0.70182 X1 + 0.29818X2, Y The overall score is shown in Table 4. The overall score of the blank group was -1.986, the overall score of the distiller's grains group was -0.235, and the overall score of the GH6 distiller's grains microbial fertilizer group was 2.221. Based on the overall score ranking, the GH6 distiller's grains microbial fertilizer group > the distiller's grains group > the blank group. The results indicate that the GH6 distiller's grains microbial fertilizer group had the best effect on promoting the growth of sorghum seedlings.
[0046] Table 3. Principal component analysis results from the second sampling.
[0047] Table 4. Principal component factor scores and composite scores for different processing methods in the second sampling.
[0048] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. An Agrobacterium tumefaciens GH6, characterized in that: The Agrobacterium tumefaciens GH6 strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 28, 2024, with accession number CCTCC NO: M 20242355, located at Wuhan University, Wuhan, China. It is classified and named accordingly. Agrobacterium tumefaciens GH6.
2. The Agrobacterium tumefaciens GH6 according to claim 1, characterized in that: The Agrobacterium tumefaciens GH6 was isolated from sorghum roots.
3. A microbial inoculant, characterized in that: Includes Agrobacterium tumefaciens GH6 as described in claim 1 or 2.
4. The microbial inoculant according to claim 3, characterized in that: It is composed of Agrobacterium tumefaciens GH6.
5. The method for preparing the microbial inoculant according to claim 4, characterized in that: Includes the following steps: Step 1: Inoculate Agrobacterium tumefaciens GH6 into LB liquid medium; Step 2: Incubate at 25-35℃ on a shaker at 100-200 rpm for 40-50 hours; Step 3: Centrifuge at 7500-8500 rpm for 4-6 minutes to collect bacterial cells, remove the supernatant, wash the bacterial cells with sterile water, and then suspend the bacterial cells in sterile water to prepare OD. 600 =0.5~0.6 bacterial suspension, which is a microbial inoculant.
6. A fermented food fertilizer, characterized in that: It is obtained by fermentation of the microbial agent described in claim 3 and the distiller's grains.
7. The method for preparing the distiller's grains microbial fertilizer according to claim 6, characterized in that: Includes the following steps: (1) Preparation of microbial inoculum: Agrobacterium tumefaciens GH6 was inoculated into LB liquid medium and cultured on a shaker at 100-200 rpm at 25-35℃ for 40-50 h; the bacterial cells were collected by centrifugation at 7500-8500 rpm for 4-6 min, the supernatant was removed, the bacterial cells were washed with sterile water, and the bacterial cells were then suspended in sterile water to prepare OD. 600 =0.5~0.6 bacterial suspension, which is a microbial inoculant; (2) Mixed fermentation: Mix the lees and microbial inoculants at a ratio of 10g:0.5-1.5mL, pile them up and ferment for 5-10 days to obtain lees microbial fertilizer.
8. The application of the distiller's grains microbial fertilizer according to claim 6 in promoting sorghum growth.