Phytobacterium flavum GH8, bacterial agent and bacterial fertilizer based on same, and preparation method and application of bacterial agent and bacterial fertilizer

Abstract

The invention relates to a phytobacterium flavum GH8 in the technical field of microorganisms, and the preparation method of a microbial agent containing the phytobacterium flavum GH8 comprises the following steps: inoculating the phytobacterium flavum GH8 into an LB liquid culture medium, and culturing for 40-50 hours on a shaking table at the temperature of 25-35 DEG C and the speed of 100-200 rpm; and centrifuging for 4-6 minutes at the speed of 7500-8500 rpm, collecting thalli, removing supernate, washing the thalli by using sterile water, and suspending the thalli by using the sterile water to prepare bacterial suspension with OD600 of 0.5-0.6, namely the microbial agent. The vinasse bacterial fertilizer prepared by mixed fermentation of the microbial agent and vinasse can significantly promote growth of sorghum.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Flavobacterium GH8, and its-based inoculants, inoculants, preparation methods, and applications. Background Technology

[0002] As an important part of my country's traditional industry, the production of baijiu (Chinese liquor) has led to a significant increase in the amount of solid waste generated as a byproduct—distillery lees. Distillery lees mainly consist of the solid matter remaining after grain raw materials undergo fermentation, distillation, and other processes. Studies have shown that approximately 3 to 4 tons of fresh distillery lees are produced for every ton of baijiu produced. Distillery lees are rich in organic matter, such as protein, starch, cellulose, and fat, and have high potential for resource utilization. Using distillery lees to prepare fertilizer as a substitute for chemical fertilizers is a feasible resource utilization approach.

[0003] Distillers' grains microbial fertilizer is a microbial fertilizer prepared by mixing distillers' grains with appropriate microbial strains through relevant processes. It not only effectively utilizes the organic matter in the distillers' grains, reducing the input of other chemical fertilizers, but also improves soil structure and promotes crop growth through the action of microorganisms, resulting in significant economic and environmental benefits. Plant endophytic bacteria are microbial groups that live in plant tissues, interstitial spaces, and organs at certain or all stages of their life cycle. Some endophytic bacterial communities play crucial roles in plant development, nutrient acquisition, and oxidative stress tolerance.

[0004] Sorghum is the world's fifth largest cereal crop. It can be grown in plains, hills, waterlogged areas, and saline-alkali land. It has multiple uses, including food, brewing, feed, energy, and silage, and has great development potential.

[0005] 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

[0006] The present invention aims to provide a strain of Xanthomonas GH8 that can be compounded with distiller's grains to produce microbial fertilizer and can promote the growth of sorghum.

[0007] A type of Flavobacterium GH8, classified and named Plantibacter flavus GH8, the Flavophyte GH8, was deposited at the China Center for Type Culture Collection on September 25, 2023, with accession number CCTCC NO: M 20231773, and the deposit address is Wuhan University, Wuhan, China.

[0008] Preferably, as an improvement, the Flavobacterium GH8 is isolated from sorghum roots.

[0009] A microbial inoculant comprising the Flavobacterium GH8.

[0010] A microbial inoculant composed of the aforementioned Flavophyte GH8.

[0011] A method for preparing a microbial inoculant includes the following steps: Step 1: Inoculate Flavobacterium GH8 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 (Flavobacterium GH8 inoculant).

[0012] The application of the microbial inoculant in the preparation of microbial fertilizer.

[0013] Preferably, as an improvement, the microbial fertilizer is a distiller's grains microbial fertilizer.

[0014] A fermented fertilizer made from distiller's grains is obtained by fermenting the aforementioned microbial agent and distiller's grains together.

[0015] A method for producing a distiller's grains microbial fertilizer includes the following steps: (1) Preparation of microbial inoculum: Flavobacterium GH8 was inoculated into LB liquid medium and cultured on a shaker at 100-200 rpm at 25-35℃ for 40-50 h; the cells were collected by centrifugation at 7500-8500 rpm for 4-6 min, the supernatant was removed, the cells were washed with sterile water, and the 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.

[0016] 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

[0017] Figure 1 Comparison chart showing the growth of sorghum seedlings sampled for the first time.

[0018] Figure 2 Comparison chart of nitrogen and phosphorus content analysis from the first sampling.

[0019] Figure 3 Comparison chart showing the growth of sorghum seedlings from the second sampling.

[0020] Figure 4 Comparison chart of nitrogen and phosphorus content analysis from the second sampling.

[0021] Figures 1-4 In the diagram, CK represents the blank group, JZ represents the distiller's grains fertilizer group, and GH8 represents the GH8 distiller's grains microbial fertilizer group. Detailed Implementation

[0022] The following detailed description illustrates the specific implementation method: Example 1: Preparation of microbial inoculants Single colonies of *Flavobacterium GH8* were picked from LB agar plates and inoculated into LB liquid medium. The culture was 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. *Flavobacterium flavonoides* GH8 was deposited on September 25, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231773, located at Wuhan University, Wuhan, China. Classification and nomenclature... Plantibacter flavus GH8.

[0023] 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.

[0024] 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 GH8 distiller's grains microbial fertilizer in the following text.

[0025] I. Sorghum Pot Experiment (1) Substrate preparation Mix perlite and vermiculite in a 1:4 ratio and add them to the flowerpots. Add 200 g of substrate to each flowerpot and 50 g of GH8 distiller's grains microbial fertilizer to the flowerpots as the GH8 group. Add an equal amount of distiller's grains microbial fertilizer to the distiller's grains group. Do not add any distiller's grains microbial fertilizer or GH8 distiller's grains microbial fertilizer to the blank group.

[0026] (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.

[0027] (3) Sorghum planting Sow 30-35 newly sprouted sorghum seeds into flowerpots. During the seedling growth period, water the flowerpots with sterile water as needed.

[0028] (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.

[0029] 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, showing no significant difference compared to the control group. The average height of seedlings in the GH8 group was 13.113 cm, which was significantly higher than that of the control group by 21.08% and significantly higher than that of the distiller's grains group by 27.894%. Compared with the control group, the addition of distiller's grains did not affect the height of sorghum seedlings, while GH8 distiller's grains microbial fertilizer promoted the height of sorghum seedlings.

[0030] 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 GH8 group was 8.463 cm, showing no significant difference from the control group, but significantly higher than the distiller's grains group by 37.097%. Compared with the control group, the addition of distiller's grains fertilizer inhibited the root length of sorghum seedlings, while the addition of GH8 distiller's grains microbial fertilizer had no significant effect on root length.

[0031] 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 GH8 group was 0.515 g, which was significantly increased by 64.013% compared to the blank group and by 59.443% compared to the distiller's grains group. Adding distiller's grains fertilizer had no significant effect on the fresh weight of sorghum seedlings, while GH8 distiller's grains microbial fertilizer promoted the fresh weight of sorghum seedlings.

[0032] like Figure 1As shown in Figure D, the average dry weight of sorghum seedlings in the blank 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 blank group. The average dry weight of the GH8 group was 0.091 g, which was significantly increased by 49.180% compared to the blank group and by 44.444% compared to the distiller's grains group. Adding distiller's grains fertilizer had no significant effect on the dry weight of sorghum seedlings, but GH8 distiller's grains microbial fertilizer promoted the dry weight of sorghum seedlings.

[0033] (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 GH8 group was 39.407 g / kg, significantly higher than the control group by 109.790% and significantly higher than the distiller's grains group by 44.269%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer promoted the total nitrogen content of sorghum seedlings.

[0034] 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 GH8 group was 6.785 g / kg, significantly higher than the control group by 149.265% and significantly higher than the distiller's grains group by 48.892%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer has a promoting effect on the total phosphorus content of sorghum seedlings.

[0035] 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 GH8 group was 180.486 mg / kg, significantly higher than the control group by 241.635% and significantly higher than the distiller's grains group by 17.189%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer promoted the available nitrogen content in the rhizosphere substrate of sorghum seedlings.

[0036] like Figure 2 As shown in D, the average available phosphorus content in the rhizosphere substrate of sorghum seedlings in the blank group was 95.83 mg / kg, while the average available phosphorus content in the distiller's grains group was 312.802 mg / kg, which was significantly higher than that in the blank group by 226.413%. The average total phosphorus content in the GH8 group was 293.652 mg / kg, which was significantly higher than that in the blank group by 206.43% and significantly lower than that in the distiller's grains group by 6.122%.

[0037] (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 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 =0.7277X1+0.2723X2, Y The overall score is shown in Table 2. The overall score of the blank group was -1.598, the overall score of the distiller's grains group was -0.919, and the overall score of the GH8 distiller's grains microbial fertilizer group was 2.517. The results show that the GH8 distiller's grains microbial fertilizer group had the best effect on promoting the growth of sorghum seedlings.

[0038] Table 1. Principal component analysis results from the first sampling.

[0039] Table 2. Principal component factor scores and composite scores for different processing methods in the first sampling.

[0040] 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 blank group was 13.77 cm, and the average height of seedlings in the distiller's grains group was 12.783 cm, with no significant difference compared to the blank group; the average height of seedlings in the GH8 group was 14.567 cm, with no significant difference compared to the blank group, but significantly increased by 13.956% compared to the distiller's grains group.

[0041] like Figure 3 As shown in B, the average root length of sorghum seedlings in the blank group was 9.93 cm, and the average root length of the distiller's grains group was 9.237 cm, with no significant difference compared to the blank group; the average root length of the GH8 group was 9.427 cm, with no significant difference compared to the blank group and the distiller's grains group.

[0042] like Figure 3 As shown in Figure C, the average fresh weight of sorghum seedlings in the blank group was 0.877 g, and the average fresh weight in the distiller's grains group was 0.923 g, with no significant difference compared to the blank group. The average fresh weight of the GH8 group was 1.191 g, which was significantly higher than the blank group by 35.804% and significantly higher than the distiller's grains group by 29.036%. Adding distiller's grains fertilizer did not affect the fresh weight of sorghum seedlings; therefore, GH8 distiller's grains microbial fertilizer promoted the fresh weight of sorghum seedlings.

[0043] 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 GH8 group was 0.236 g, which was significantly higher than the control group by 30.387% and significantly higher than the distiller's grains group by 24.211%. Compared with the control group, distiller's grains did not affect the dry weight of sorghum seedlings, and GH8 distiller's grains microbial fertilizer had a promoting effect on the dry weight of sorghum seedlings.

[0044] (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 GH8 group was 45.389 g / kg, significantly higher than the control group by 133.446% and significantly higher than the distiller's grains group by 13.444%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer promoted the total nitrogen content of sorghum seedlings.

[0045] 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 GH8 group was 4.131 g / kg, significantly higher than the control group by 36.247% and significantly higher than the distiller's grains group by 32.404%. Compared to distiller's grains, GH8 distiller's grains microbial fertilizer promoted the total phosphorus content of sorghum seedlings.

[0046] 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 distiller's grains group was 131.783 mg / kg, significantly higher than the control group by 1613.693%. The average available nitrogen content in the GH8 group was 143.061 mg / kg, significantly higher than the control group by 1760.351% and significantly higher than the distiller's grains group by 8.558%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer promoted the available nitrogen content in the rhizosphere substrate of sorghum seedlings.

[0047] 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 distiller's grains group was 452.602 mg / kg, significantly higher than the control group by 11905.36%. The average available phosphorus content in the GH8 group was 587.718 mg / kg, significantly higher than the control group by 15489.337% and significantly higher than the distiller's grains group by 29.853%. Compared with distiller's grains, GH8 distiller's grains microbial fertilizer promoted the available phosphorus content in the rhizosphere substrate of sorghum seedlings.

[0048] (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 =0.7312 X1 + 0.2688X2, Y The overall score is shown in Table 4. The overall score of the blank group was -1.886, the overall score of the distiller's grains group was -0.559, and the overall score of the GH8 distiller's grains microbial fertilizer group was 2.445. According to the overall score, the GH8 distiller's grains microbial fertilizer group > the distiller's grains group > the blank group. The results show that the GH8 distiller's grains microbial fertilizer group has the best effect on promoting the growth of sorghum seedlings.

[0049] Table 3. Principal component analysis results from the second sampling.

[0050] Table 4. Principal component factor scores and composite scores for different processing methods in the second sampling.

[0051] 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. A Flavobacterium GH8, characterized in that: The *Flavobacterium GH8* strain was deposited on September 25, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231773, located at Wuhan University, Wuhan, China. It is classified and named accordingly. Plantibacter flavus GH8.

2. The Flavobacterium GH8 according to claim 1, characterized in that: The Flavophyta GH8 was isolated from sorghum roots.

3. A microbial inoculant, characterized in that: Includes Flavobacterium GH8 as described in claim 1 or 2.

4. The microbial inoculant according to claim 3, characterized in that: It is composed of the aforementioned Flavophyte GH8.

5. The method for preparing the microbial inoculant according to claim 4, characterized in that: Includes the following steps: Step 1: Inoculate Flavobacterium GH8 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: Flavobacterium GH8 was inoculated into LB liquid medium and cultured on a shaker at 100-200 rpm at 25-35℃ for 40-50 h; the cells were collected by centrifugation at 7500-8500 rpm for 4-6 min, the supernatant was removed, the cells were washed with sterile water, and the 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.

Images