Complex microbial inoculant for efficiently degrading tobacco straws as well as preparation method and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for efficiently degrading tobacco straws as well as a preparation method and application of the complex microbial inoculant, and relates to the technical field of microorganisms. The compound bacterial agent is named as a compound bacterial agent J-2024-2, and the compound bacterial agent comprises bacillus zanthoxylum ZY2 (Bacillus zanthoxylum ZY2), bacillus subtilis WP-2022 (Bacillus subtilis WP-2022) and bacillus YP-2023-01 (Bacillus YP-2023-01), and the compound bacterial agent comprises bacillus subtilis WP-2022 (Bacillus subtilis WP-2022), bacillus subtilis WP-2022 (Bacillus subtilis WP-2022) and bacillus subtilis YP-2023-01 (Bacillus subtilis YP-2023-01). The multifunctional efficient degradation complex microbial inoculant J-2024-2 provided by the invention can be directly used on tobacco straws in the field to accelerate degradation of lignin and cellulose in the tobacco straws, has very strong decomposition capability on the tobacco straws, and also has no significant influence on tobacco growth. The innovation, the ecological adaptability and the crop friendliness of the complex microbial inoculant in the field of straw degradation are fully explained, and the market potential and the practical application value of the complex microbial inoculant are fully shown.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite bacterial agent for efficiently degrading tobacco straws, and a preparation method and application thereof. Background Art

[0002] At present, the total output of straw resources produced by crops in my country is as high as 865 million tons, which has great potential utilization value. Therefore, it is particularly important to effectively treat and utilize straw. Among them, straw, especially tobacco straw, is difficult to degrade due to its high cellulose and lignin content. Secondly, adverse climatic conditions in the natural environment, such as low temperature and drought, will further inhibit the activity of microorganisms. For example, when the temperature exceeds 50°C, many microorganisms will be subjected to heat stress, reducing the degradation efficiency; when it is below 10°C, the microbial metabolism slows down, the degradation process slows down, and even enters a dormant state. In addition, in addition to adverse climatic factors, complex interactions between microorganisms, such as nutrient competition and cross-feeding, can also significantly affect their ability to degrade cellulose and lignin. For example, bacteria may secrete secondary metabolites such as antibiotics and enzymes to inhibit the growth of certain fungi, or inhibit the reproduction of other bacteria by releasing antibiotics or volatile organic compounds. Similarly, fungi may also affect the survival of bacteria by secreting chemicals. This inhibitory effect between microorganisms leads to their insignificant efficiency in straw degradation. Therefore, it is urgent to find a microbial agent that can efficiently degrade straw in a natural environment.

[0003] However, on the one hand, it is difficult for a single microorganism to secrete a complete enzyme system. On the other hand, the natural environment in the field has a significant impact on straw degradation, and the existing composite bacterial agents are not effective in this environment. Due to factors such as soil conditions, climate change, and the diversity of microbial communities, the activity and degradation ability of composite bacterial agents are restricted and cannot fully exert their due effects. This causes the degradation process of straw to become slow, affecting the effective use of resources. Therefore, it is particularly important to invent a composite bacterial agent to study its adaptability to the natural environment in order to enhance its degradation effect under field conditions.

[0004] Therefore, inventing a composite bacterial agent that can adapt to the natural environment to enhance its degradation effect under field conditions and has no negative impact on crop growth is an urgent problem to be solved by the present invention. Summary of the invention

[0005] The present invention provides a composite bacterial agent for efficiently degrading tobacco straw, and a preparation method and application thereof, aiming to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses a composite bacterial agent for efficiently degrading tobacco straw. The composite bacterial agent is named composite bacterial agent J-2024-2, which includes Bacillus zanthoxyli ZY2, Bacillus subtilis WP-2022 and Bacillus YP-2023-01.

[0008] In a preferred embodiment of the present invention, the preservation number of the Bacillus zanthoxylum ZY2 is CCTCC NO: M2021897, the preservation number of the Bacillus subtilis WP-202 is CCTCC NO: M 20241187, and the preservation number of the Bacillus YP-2023-01 is CCTCC NO: M 20241188.

[0009] In a preferred embodiment of the present invention, the composite bacterial agent is fermented by adding seed liquid of Bacillus zanthoxylum ZY2, Bacillus subtilis WP-202 and Bacillus YP-2023-01 at a live bacteria count ratio of 1:1:1.

[0010] In a preferred embodiment of the present invention, the effective viable bacteria count in the composite bacterial agent is not less than 1×1010 CFU.

[0011] In a second aspect, the present invention discloses a method for preparing a composite bacterial agent for efficiently degrading tobacco straw as described in the first aspect, comprising the following steps:

[0012] 1) After enrichment and culture of tobacco-growing soil samples, the supernatant was collected and shaken, and the diluted bacterial solution was spread on a sterile culture medium for culture, and representative colonies were selected for streaking and purification until a single colony was obtained;

[0013] 2) The isolated and purified strains were inoculated on skim milk powder medium, with 3 replicates for each strain, cultured at 37°C, and the size of the hydrolysis transparent zone was observed to screen out the strain with the strongest protein degradation ability;

[0014] 3) Inoculate a single strain on CMC-Na medium, with 3 replicates per strain, culture at 30°C, and then detect using Congo red clearing zone staining to select the cellulose-degrading strain with the largest clearing zone;

[0015] 4) The strains were inoculated on PDA-aniline blue and PDA-guaiacol culture media, with 3 replicates for each strain, and cultured at 30°C for 24 hours to observe whether fading or coloring circles were produced, and to select the strain with the strongest lignin degradation ability;

[0016] 5) An antagonistic experiment is performed on the three strains of bacteria screened. If there is no antagonistic effect, the seed liquid of the three strains of bacteria is added at a ratio of 1:1:1 of live bacteria count for fermentation, that is, compounded into the composite bacterial agent J-2024-2.

[0017] In a third aspect, the present invention discloses an application of the composite bacterial agent for efficiently degrading tobacco straw as described in the first aspect in degrading tobacco straw.

[0018] In a preferred embodiment of the present invention, the composite bacterial agent is sprayed on tobacco straw.

[0019] Preferably, the composite bacterial agent is sprayed on tobacco straw at an inoculation rate of 6%.

[0020] In a preferred embodiment of the present invention, the composite bacterial agent degrades cellulose and lignin in the straw.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The multifunctional and efficient degradation composite bacterial agent J-2024-2 provided by the present invention can be directly used on tobacco straws in the field to accelerate the degradation of lignin and cellulose in tobacco straws, has a strong decomposition ability on tobacco straws, and is found to have no significant effect on tobacco growth. This fully demonstrates the innovation, ecological adaptability and crop friendliness of the composite bacterial agent in the field of straw degradation, and fully demonstrates its market potential and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a comparison chart of the degradation rate of the composite bacterial agent J-2024-2 and single bacteria;

[0025] Figure 2 This is the decomposition rate of tobacco stalks degraded by the composite bacterial agent J-2024-2;

[0026] Figure 3 This is a graph showing the antagonism results between strains of the composite bacterial agent J-2024-2. DETAILED DESCRIPTION

[0027] The following examples are provided to further illustrate the present invention, but not to limit the present invention. Specific experimental conditions and methods are not specified in the following examples, and the technical means adopted are conventional means well known to those skilled in the art.

[0028] Example 1 Preparation of composite bacterial agent

[0029] 1. Isolation and purification of degradation strains

[0030] Tobacco soil samples were collected from Anshun City, Guizhou Province. 10.00 g of fresh soil samples were weighed and cultured in enrichment medium for 1 hour. After standing for 5 minutes, 2 mL of supernatant was placed in a new sterilized and cooled enrichment medium and cultured at 28°C and 180 r / min for 3 days. According to the requirements of aseptic operation, the bacterial solution was diluted to 10 -3 , 10 -4 , 10 -5 0.1 mL was spread on sterile culture medium and inverted at 28°C for 2-3 days. Three parallel experiments were set for each gradient. Representative colonies were selected according to colony morphology and color and purified by streaking until a single colony was obtained.

[0031] 2. Strain Function Screening Method

[0032] 2.1 Screening of protein degradation ability

[0033] The isolated and purified strains were inoculated on skimmed milk powder culture medium, with 3 replicates for each strain, and cultured at 37°C for 24 hours. The size of the hydrolysis transparent zone was observed to screen the strain with the strongest protein degradation ability.

[0034] 2.2 Screening of cellulose degradation ability

[0035] The strains were inoculated on CMC-Na medium with 3 replicates for each strain. After culturing at 30°C for 24 hours, the strains were detected by Congo red transparent zone staining to screen the cellulose-degrading strains with the largest transparent zone.

[0036] 2.3 Lignin degradation ability screening

[0037] The strains were inoculated on PDA-aniline blue and PDA-guaiacol culture media, with 3 replicates per strain, and cultured at 30°C for 24 hours to observe whether fading circles (ability to secrete MnP and LiP) or color circles (ability to secrete Lac) were produced, so as to screen the strain with the strongest lignin degradation ability.

[0038] 3. Molecular Biological Identification of the Strains

[0039] The 16S rDNA was amplified by PCR, and the different functional strains screened were identified by molecular biology. After the strain DNA was extracted, PCR amplification and agarose gel electrophoresis were performed using 16S rDNA universal primers, and the PCR products were sequenced and compared with the sequences in the GeneBank database for homology to determine the species of the strain.

[0040] The 16S rRNA identification results showed that they were Bacillus zanthoxyli ZY2, a highly efficient protein-degrading bacterium; Bacillus subtilis WP-2022, a highly efficient cellulose-degrading bacterium; and Bacillus YP-2023-01, a highly efficient lignin-degrading bacterium. Antagonistic experiments were performed on the three strains, and the results showed that there was no antagonistic effect between the three strains (such as Figure 3 ). The seed liquid of the above three strains of bacteria was added in a ratio of 1:1:1 in terms of live bacteria count for fermentation to prepare the composite bacterial agent J-2024-2.

[0041] Test Example 1 Bacterial Antagonism Experiment

[0042] The different functional single strains isolated and purified were subjected to a cross-streak inoculation experiment on sterile LB solid culture medium: first, the strain to be tested was inoculated along a straight line at one end of the plate; then, the strain to be tested was inoculated in the same way at the other end, so that the streaked areas of the two bacteria were close to each other but not overlapping. The inoculated plates were cultured at 28°C until the strains formed obvious colonies or bacterial ribbons, and then the growth inhibition zone at the junction of the two bacteria was observed. If there was an inhibition zone, it indicated the presence of antagonism. Each group of experiments was repeated 3 times to ensure reliable results.

[0043] This application screened out three strains of Bacillus with different functions from tobacco-growing soil in Yangwu Township, Anshun City, Guizhou Province. The three strains were identified by 16SrRNA as follows: Bacillus zanthoxyli ZY2, an efficient protein-degrading bacterium; Bacillus subtilis WP-2022, an efficient cellulose-degrading bacterium; and Bacillus YP-2023-01, an efficient lignin-degrading bacterium. Antagonistic experiments were conducted on the three strains, and the results showed that there was no antagonistic effect between the three strains (such as Figure 3 ), so it can be compounded into the composite bacterial agent J-2024-2.

[0044] Test Example 2 Comparative experiment on the ability of composite bacteria and single bacteria

[0045] Select the strains without antagonistic effects to form a composite bacteria, and mix them with the single bacteria at a 6% inoculation rate (strain content ≥ 1×10 10 CFU) was sprayed on 500g tobacco stems, mixed with an appropriate amount of soil and poured into pots. The straw was taken out at 0d, 7d, 15d, 21d and 35d, washed with distilled water, and then a dilute acid solution was used to remove the attached bacteria and CaCO 3 The residue was precipitated and washed with distilled water for 3 times and dried at 80℃ to constant weight. The straw degradation rate was calculated by weight loss method: Straw degradation rate % = (W 0 -Wt) / W 0 ×100%

[0046] Among them, W 0 is the initial dry weight of the straw, and Wt is the dry weight of the straw after cultivation for t time.

[0047] The degradation rates of composite bacterial agent J-2024-2 and single bacterial strains are as follows Figure 1 As shown. The ZY2 strain is mainly a protein-degrading bacterium, so it is not effective in straw degradation. In contrast, the degradation rates of the lignin-degrading bacteria YP-2023-01 and the cellulose-degrading bacteria WP-2022 were significantly higher than those of the control group (CK), among which the degradation rate of the cellulose-degrading bacteria WP-2022 was significant, showing its good degradation potential. Overall, the composite bacterial agent J-2024-2 always performed better than a single strain throughout the degradation process, indicating that it has a more stable and more effective degradation ability. This advantage may be due to the synergistic effect of multiple functional strains in the composite bacterial agent, which enables it to more comprehensively decompose various organic components in the straw.

[0048] Experimental Example 3 Study on the Effect of Field Application of Composite Microbial Agents

[0049] In the field experiment, tobacco straw was selected as the material, and a composite bacterial agent J-2024-2 treatment group and a sterile water treatment group (CK) were set up, with 3 replicates in each group. 5% bacterial solution or sterile water was added to each replicate, and the effective viable bacterial count of the bacterial solution was ≥1×10 -8 The degradation effect was evaluated by calculating the straw decomposition rate. To further verify the effect of the composite microbial agent on tobacco growth, the treated straw was returned to the field, tobacco was planted, and the growth of tobacco was recorded according to the "Survey and Measurement Methods of Tobacco Agronomic Traits" (YC / T 142-2010).

[0050] The results are as follows Figure 2 As shown: After 105 days of treatment, the composite agent J-2024-2 showed excellent straw degradation efficiency. The degradation rate of the composite agent was always significantly higher than that of the control group (CK), indicating that the composite agent maintained high degradation ability throughout the treatment period. On the 105th day, the degradation rate of the composite agent reached a peak, which was 64.20% for the composite agent treatment group and 26.75% for the control group. During the same period, the degradation rate of the J-2024-2 agent was significantly better than the degradation rates of the composite strains F and G reported in the literature, which were 32.96% and 36.24%, respectively (Wang Ziyuan, 2024). These data show that the composite agent J-2024-2 has a sustained and efficient degradation effect, has a broad application prospect, and can effectively solve the degradation problem in the process of returning straw to the field.

[0051] Experimental Example 4 Effect of Composite Bacterial Agent J-2024-2 on Tobacco Growth

[0052] High-quality composite microbial agents should not only have efficient straw degradation capabilities, but also have no adverse effects on crop growth. To this end, the present invention returns the straw treated with the composite microbial agent and the straw of the control group to the field, and plants tobacco. By investigating the agronomic traits of tobacco during the cluster period, including indicators such as leaf number, maximum leaf length, maximum leaf width, plant height, and stem girth, the results are shown in Table 1. There is no significant difference between the composite microbial agent treatment group and the control group in the above growth indicators. This shows that the composite microbial agent has no adverse effect on tobacco growth, further proving the friendliness and feasibility of the composite microbial agent in agriculture.

[0053] Table 1 Effects of compound fungicide J-2024-2 on agronomic traits of tobacco during the bush stage

[0054]

[0055] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composite bacterial agent for efficiently degrading tobacco straw, characterized in that: The composite bacterial agent is named as composite bacterial agent J-2024-2, which includes Bacillus zanthoxyli ZY2, Bacillus subtilis WP-2022 and Bacillus YP-2023-01.

2. The composite bacterial agent for efficiently degrading tobacco straw according to claim 1, characterized in that: The preservation number of the Zanthoxylum bungeanum ZY2 is CCTCC NO: M 2021897, the preservation number of the Bacillus subtilis WP-202 is CCTCC NO: M20241187, and the preservation number of the Bacillus YP-2023-01 is CCTCC NO: M 20241188.

3. The composite bacterial agent for efficiently degrading tobacco straw according to claim 1, characterized in that: The composite bacterial agent is fermented by adding seed liquid of Bacillus zanthoxylum ZY2, Bacillus subtilis WP-202 and Bacillus YP-2023-01 at a live bacteria count ratio of 1:1:

1.

4. The composite bacterial agent for efficiently degrading tobacco straw according to claim 1, characterized in that: The effective live bacteria count in the composite bacterial agent is not less than 1×1010 CFU.

5. A method for preparing a composite bacterial agent for efficiently degrading tobacco straw according to any one of claims 1 to 4, characterized in that: The steps include: 1) After enrichment and culture of tobacco-growing soil samples, the supernatant was collected and shaken, and the diluted bacterial solution was spread on a sterile culture medium for culture, and representative colonies were selected for streaking and purification until a single colony was obtained; 2) The isolated and purified strains were inoculated on skim milk powder medium, with 3 replicates for each strain, cultured at 37°C, and the size of the hydrolysis transparent zone was observed to screen out the strain with the strongest protein degradation ability; 3) Inoculate a single strain on CMC-Na medium, with 3 replicates per strain, culture at 30°C, and then detect using Congo red clearing zone staining to select the cellulose-degrading strain with the largest clearing zone; 4) The strains were inoculated on PDA-aniline blue and PDA-guaiacol culture media, with 3 replicates for each strain, and cultured at 30°C to observe whether fading or coloring circles were produced, and to select the strain with the strongest lignin degradation ability; 5) An antagonistic experiment is performed on the three strains of bacteria screened. If there is no antagonistic effect, the seed liquid of the three strains of bacteria is added at a ratio of 1:1:1 of live bacteria count for fermentation, that is, compounded into the composite bacterial agent J-2024-2.

6. Use of the composite bacterial agent for efficiently degrading tobacco straw as described in any one of claims 1 to 4 in degrading tobacco straw.

7. The use according to claim 6, characterized in that: The composite bacterial agent is sprayed on tobacco stalks.

8. The use according to claim 7, characterized in that: The composite bacterial agent is sprayed on tobacco straw at an inoculation rate of 6%.

9. The use according to claim 6, characterized in that: The composite bacterial agent degrades cellulose and lignin in tobacco straw.

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