Bacillus dextranolyticum and application of complex microbial inoculant thereof in preparation of bio-organic fertilizer

Abstract

The invention relates to the technical field of microorganisms, in particular to application of bacillus glucans and a complex microbial inoculant thereof in preparation of bio-organic fertilizers, the strain Mh85 is separated from the root system of apple rootstock Malus hupehensis treated by a research group through trichoderma TB2, has good IAA production, nitrogen fixation, inorganic phosphorus decomposition and siderophore production capacities, and can be used for preparing bio-organic fertilizers. The strain Mh85 not only can directly promote plant growth, but also has a good growth-promoting effect on trichoderma TB2, and when the strain is compounded with the trichoderma TB2 to form a complex microbial inoculant, tomato seedling pot experiments are carried out. The complex microbial inoculant shows a synergistic effect, effectively overcomes the problems of unstable growth promoting effect, insufficient synergistic effect among strains and the like of an existing single microbial preparation, provides an efficient and stable technical scheme for improving plant growth, improving root development and increasing crop yield, and is a good raw material for preparing a bio-organic fertilizer.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of a glucan-degrading bacillus strain and a composite bacterial agent thereof in the preparation of biological organic fertilizer. Background Art

[0002] In recent years, with the promotion of green agriculture and sustainable development concepts, the use of beneficial microorganisms to regulate plant growth, improve root development and improve plant health has become an important means to replace traditional fertilizers. At present, common biological agents mainly include single strains, such as Trichoderma ( Trichoderma spp. ) and various plant growth promoting bacteria (PGPR). However, these existing technologies still have the following shortcomings: (1) Limitations of single strain application: Although Trichoderma has certain advantages in regulating plant root growth and improving soil environment, when it is used alone, it is limited by environmental conditions and plant varieties and other factors, and it is often difficult to achieve a stable promotion effect under various conditions; (2) Insufficient optimization of compound schemes: Most of the existing microbial compound preparations adopt a simple superposition method, which fails to give full play to the complementary synergistic effect between different strains, resulting in an overall synergistic effect that is not ideal; (3) Single strain source: Most of the applied strains are derived from conventional soil or traditional microbial libraries, and there are few studies on endophytes screened for specific plant environments. Endophytes often have unique advantages in promoting plant growth due to long-term symbiosis with the host, but the relevant technology has not been fully developed; (4) Strain specificity. In the process of strain screening, we found that strains of the same species have great specificity between different strains. Strains of the same species and different strains often have different application ranges due to differences in physiological characteristics. Therefore, technicians in this field need to continuously screen strains and conduct functional verification to improve the germplasm resource library.

[0003] The technical solution of the present invention is based on the above-mentioned deficiencies. The research team of this application separated microorganisms that may have a growth-promoting effect on Trichoderma TB2 from the roots of the apple rootstock "Pingyi Sweet Tea" treated with Trichoderma TB2, and compounded them with Trichoderma TB2 to form a composite bacterial agent, in order to find strains that can promote the growth of Trichoderma TB2 and prepare them into a composite bacterial agent. The composite bacterial agent can synergistically enhance the effect, providing new technical ideas and application prospects for the development of efficient and stable biological preparations. Summary of the invention

[0004] In view of the above, it is necessary to isolate microorganisms that may have a growth-promoting effect on Trichoderma TB2 and compound them with Trichoderma TB2 to form a composite bacterial agent, in order to find strains that can promote the growth of Trichoderma TB2 and prepare them into a composite bacterial agent. The composite bacterial agent can synergistically enhance the effect and provide new technical ideas and application prospects for the development of efficient and stable biological agents.

[0005] To achieve the above object, a new strain, Bacillus glucanolyticus, was screened out in the present invention. Paenibacillus glycanilyticus Strain Mh85, and its taxonomic name is: Paenibacillus glycanilyticus Mh85, and its Chinese taxonomic name is: Bacillus glucanolyticus Mh85, and the deposit number is CCTCC NO: M2025410; this strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is March 10, 2025.

[0006] The present invention also includes a composite microbial agent containing the above-mentioned Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85.

[0007] Furthermore, the composite microbial agent is composed of Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 and Trichoderma brevicompactum Trichoderma brevicompactum Strain TB2; the Trichoderma brevicompactum Trichoderma brevicompactum Strain TB2, and its taxonomic name is Trichoderma brevicompactum TB2, and its deposit number is CCTCC NO: M20231913; this strain is deposited in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit date is October 16, 2023, and this strain has been disclosed in the patent application: 202311753128.0.

[0008] The present invention also includes the application of the above-mentioned Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 or the composite microbial agent in the preparation of bio-organic fertilizer.

[0009] The present invention also includes the application of the above-mentioned Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 or the composite microbial agent in promoting plant growth.

[0010] Furthermore, the plant is tomato.

[0011] The present invention also includes the application of the above-mentioned Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 or the composite microbial agent in the production of IAA, nitrogen fixation, solubilization of inorganic phosphorus and / or production of siderophores.

[0012] The present invention also includes a method for promoting plant growth by using the above-mentioned Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 or the composite microbial agent, and the method is: pouring the bacterial suspension of Bacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85 or the composite microbial agent onto the rhizosphere of plant seedlings.

[0013] Furthermore, the composite microbial agent is composed of Bacillus glucanolyticusPaenibacillus glycanilyticus Mh85 bacterial liquid and Trichoderma brevicompactum Trichoderma brevicompactum The TB2 bacterial liquid is obtained by mixing in a ratio of 1:1.

[0014] Furthermore, the Bacillus glucanolyticus Paenibacillus glycanilyticus The effective viable count of Mh85 is 10 7 CFU / g; the effective viable count of the Trichoderma brevicompactum Trichoderma brevicompactum The effective viable count of TB2 is 10 6 CFU / g.

[0015] The present invention has the following beneficial effects: The strain Mh85 of the present invention is isolated from the roots of the apple rootstock "Malus hupehensis Rehd." treated with Trichoderma sp. TB2 by the research group, and has good abilities of producing IAA, nitrogen fixation, dissolving inorganic phosphorus and producing siderophores. The strain Mh85 can not only directly promote plant growth. Co-culturing this strain with strains of the genus Trichoderma found that: the strain Mh85 has a good growth-promoting effect on Trichoderma sp. TB2 and has no obvious growth-promoting effect on Trichoderma 4742. After preparing the strain and Trichoderma sp. TB2 into a compound microbial agent, it is found that: this compound microbial agent exhibits a synergistic effect of 1+1>2 and has a significant growth-promoting effect on tomato seedlings. The present invention effectively overcomes the problems of unstable growth-promoting effects and insufficient synergistic effects between strains in existing single microbial agents. There is a good synergistic effect between the strains of the compound microbial agent, thereby providing an efficient and stable technical solution for improving plant growth, improving root development and increasing crop yield, and is a good raw material for preparing biological organic fertilizers. Description of the Drawings

[0016] Figure 1 It is a colony morphology diagram of the strain Mh85.

[0017] Figure 2 It is a phylogenetic tree diagram of the strain Mh85.

[0018] Figure 3 It is a hyphal growth diagram of the growth-promoting effect of the strain Mh85 on different Trichoderma strains (TB2, 4742).

[0019] Figure 4 It is a biomass result diagram of the growth-promoting effect of the strain Mh85 on the Trichoderma strain TB2.

[0020] Figure 5 It is a growth diagram of the strain Mh85 and the TB2+Mh85 compound microbial agent on tomato seedlings.

[0021] Figure 6 It is a result diagram of the above-ground dry weight of tomato seedlings by the strain Mh85.

[0022] Figure 7It is a result graph of the root dry weight of tomato seedlings by strain Mh85.

[0023] Figure 8 It is a result graph of the total dry weight of tomato seedlings by strain Mh85.

[0024] Figure 9 It is a result graph of the shoot dry weight of tomato seedlings by strain Mh85 and the composite bacterium agent TB2 + Mh85.

[0025] Figure 10 It is a result graph of the root dry weight of tomato seedlings by strain Mh85 and the composite bacterium agent TB2 + Mh85.

[0026] Figure 11 It is a result graph of the total dry weight of tomato seedlings by strain Mh85 and the composite bacterium agent TB2 + Mh85. Biological material preservation information

[0027] The preservation information of strain Mh85 in this application is: Paenibacillus glucanolyticus Paenibacillus glycanilyticus Strain Mh85, its taxonomic name is: Paenibacillus glycanilyticus Mh85, its Chinese taxonomic name is: Paenibacillus glucanolyticus Mh85, preservation number is CCTCC NO: M2025410; this strain is preserved in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is March 10, 2025.

[0028] The preservation information of strain TB2 in this application is: Trichoderma brevicompactum Trichoderma brevicompactum Strain TB2, its taxonomic name is Trichoderma brevicompactum TB2, its preservation number is CCTCC NO: M20231913; this strain is preserved in the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is October 16, 2023. This strain has been publicly disclosed in the patent application: 202311753128.0. Detailed implementation methods

[0029] All features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0030] Any feature disclosed in this specification (including any additional claims, abstract), unless specifically stated, each feature is only an example of a series of equivalent or similar features. Example 1

[0031] This example is about the isolation and identification of Paenibacillus glucanolyticus Mh85.

[0032] 1. Isolation and purification of strain Mh85: Approximately 1 g of root tissue was collected from the roots of the apple rootstock "Malus hupehensis Rehd." treated with Trichoderma harzianum TB2. After collection, the roots were first surface-sterilized to remove exogenous microorganisms attached to the surface and ensure the purity of endophytes. The sterilized root tissue was placed in a 50 mL sterile Erlenmeyer flask containing glass beads and 9 mL of SM buffer, and shaken at 30 °C and 170 rpm for 30 minutes to fully break the root tissue and release endophytes into the buffer, forming a bacterial suspension. The obtained suspension was serially diluted with sterile water (diluted to 10 -5 ~10 -7 concentration), and then spread on TSA medium and cultured at 30 °C for 48 hours. According to colony morphology, representative single colonies were picked by the streak plate method for repeated purification, and finally a pure culture strain was obtained.

[0033] 2. Identification of strain Mh85: ① Morphological identification: The specific operation was as follows. As Figure 1 shown, strain Mh85 was inoculated into TSB medium and cultured in a constant temperature incubator for 24 hours. After that, circular and smooth colonies were observed on the solid medium, and their morphological characteristics were consistent with the common characteristics of Paenibacillus polysaccharolyticus. ② 16S rRNA gene amplification and sequencing were performed on the purified strain. The sequence alignment results were as Figure 2 shown. This strain had a high similarity with Paenibacillus glycanilyticus , so it was named Paenibacillus glycanilyticus Mh85. Example 2

[0034] This example was for the determination of the growth-promoting function of strain Mh85.

[0035] 1. Qualitative determination of the IAA-producing function of the strain: Strain Mh85 was added to TSB medium containing L-tryptophan at an inoculation amount of 1%, and shaken at 30 °C and 170 r min -1 for 48 h. After that, the supernatant was obtained by centrifugation at 10000 rpm for 5 min. 100 μl of the supernatant was mixed with an equal volume of Salkowski colorimetric solution. A 100 μl of uninoculated medium mixed with an equal volume of Salkowski colorimetric solution was used as a blank control. The reaction was carried out for 30 min under light-shielded conditions. The solution turning red indicated that the strain had the ability to produce IAA.

[0036] 2. Determination of potassium-solubilizing ability of the strain: Add 10 μL of the activated Mh85 bacterial suspension to Ashby nitrogen-fixing medium, Meng Jinna organic phosphorus medium, and PKO inorganic phosphorus medium respectively, with three replicates for each. Incubate in an incubator at 28 °C for 5 days, and observe whether there is a halo or transparent circle on the medium. Judge the nitrogen-fixing, organic phosphorus-solubilizing, and inorganic phosphorus-solubilizing abilities of the strain according to the ratio of the halo or transparent circle (D) to the colony diameter (d).

[0037] 3. Determination of siderophore production ability: Referring to Schwyn and Neilands, centrifuge the bacterial suspension cultured in LNM medium for 48 h under iron-limited conditions through a well plate filter membrane at 3500 rpm for 10 min. Mix the obtained supernatant with the CAS detection solution at a ratio of 1:1, let it stand and react for 2 h, and measure the absorbance value (A) at 630 nm; the control is sterile LNM medium, which is also mixed with the CAS detection solution in an equal proportion to measure the absorbance value (Ar). Calculate the relative content of siderophores according to the formula [(Ar - As) / Ar]×100%.

[0038] The results of the above growth-promoting property determinations are shown in Table 1 below.

[0039]

[0040] As can be seen from Table 1, the strain Mh85 has the abilities of producing IAA, nitrogen fixation, inorganic phosphorus solubilization, and siderophore production. Example 3

[0041] This example is a study on the growth promotion effect of the strain Mh85 on Trichoderma brevicompactum.

[0042] (1) Experimental design: To evaluate Paenibacillus glycanilyticus the growth promotion effect of strain Mh85 (hereinafter referred to as: Mh85) on Trichoderma, the Trichoderma strains selected are: Trichoderma brevicompactum Trichoderma brevicompactum strain TB2 (hereinafter referred to as: TB2) and Trichoderma harzianum Trichoderma Harzianum NJAU 4742 with the preservation number CGMCC No. 12166 (hereinafter referred to as: 4742), and the following 4 treatment groups are set respectively.

[0043] Control group 1: Culture TB2 alone.

[0044] Treatment group 1: Co-culture TB2 and Mh85.

[0045] Control group 1: Culture 4742 alone.

[0046] Treatment group 1: Co-culture 4742 and Mh85.

[0047] (2)Cultivation conditions: To ensure the normal growth of bacteria, PDB liquid medium was selected, and the pH of the medium was adjusted to 7.0. The corresponding bacterial solutions were inoculated into each group and cultured under shaking conditions at 30 °C and 170 rpm for 48 hours.

[0048] (3)Determination of mycelial biomass: After the cultivation, Trichoderma mycelia were collected by filter paper, and then the mycelia were dried and weighed to determine their biomass. By comparing the mycelial biomass of the treatment group and the control group of Trichoderma, the promoting effect of strain Mh85 on the growth of Trichoderma strains was evaluated.

[0049] Test results.

[0050] 1. As Figure 3 shown: Strain Mh85 can significantly promote the growth of Trichoderma TB2 mycelia. Compared with the control, the volume of TB2 mycelia inoculated with Mh85 increased by 266%; while strain Mh85 had no obvious promoting effect on the mycelia of Trichoderma 4742.

[0051] 2. As Figure 4 shown, strain Mh85 can significantly promote the growth of Trichoderma TB2 mycelia and increase the biomass. After drying to a constant weight, compared with the control, the mycelial biomass of TB2 inoculated with Mh85 increased by 44%.

[0052] The above experiments showed that for Trichoderma strains of the same genus, strain Mh85 can significantly promote the growth of Trichoderma TB2, and has no obvious promoting effect on Trichoderma 4742. Example 4

[0053] This example is a study on the growth-promoting effect of strain Mh85 on tomato seedlings.

[0054] (1)Tomato seedling raising.

[0055] ① Seed treatment: Take healthy tomato seeds, first soak them in sterile water for 24 hours; then soak them in 75% alcohol for 30 seconds, and rinse them with sterile water 3 - 5 times; then, soak them in 2% sodium hypochlorite solution for 20 minutes, and finally rinse them thoroughly with a large amount of sterile water.

[0056] ② Germination acceleration and sowing: Spread the disinfected seeds on the medium padded with filter paper moistened with sterile water, and place them in a constant temperature incubator at 28 °C for 3 - 5 days for germination acceleration. Select the seeds with good germination and sow them in the sterilized medium or sterile substrate for seedling raising.

[0057] ③ Transplanting: After the grown tomato seedlings have 2 - 3 true leaves and grow evenly, select healthy and consistent seedlings and transplant them into sterilized substrate flowerpots for pot cultivation.

[0058] (2)Inoculation treatment.

[0059] ① Preparation of bacterial suspension: Obtained according to the aforementioned method Strain Mh85, and prepare it into a bacterial suspension, adjusting its final concentration to 10 7 CFU / g soil. The method for preparing the bacterial suspension of Trichoderma strain TB2 is a concentration of 10 6 CFU / g soil.

[0060] ② The bacterial suspensions of the four treatment groups are as follows.

[0061] CK group (control group): Do not inoculate any bacterial suspension and cultivate tomato seedlings alone.

[0062] TB2 group: Only inoculate the bacterial suspension of Trichoderma TB2, with a concentration of 10 6 CFU / g soil.

[0063] Mh85 group: Only inoculate the bacterial suspension of Mh85, with a concentration of 10 7 CFU / g soil.

[0064] TB2 + Mh85 group: Inoculate the bacterial suspension of Mh85 and the bacterial suspension of Trichoderma TB2 in a 1:1 ratio and inoculate it into the rhizosphere of tomato seedlings.

[0065] ③ Inoculation operation: Uniformly inoculate the prepared bacterial suspension into the rhizosphere of tomato seedlings and operate according to the above four treatments.

[0066] ④ Experimental design: Set 5 replicates for each treatment group and culture them under the same environmental conditions.

[0067] ⑤ Result collection: After 30 days of culture, measure and record the growth phenotypes of tomato seedlings in each group, including indicators such as plant height, root development, and biomass, to evaluate the growth promotion effect of the combination of strain Mh85 and Trichoderma TB2 on tomato seedlings.

[0068] (3) The test results are as follows.

[0069] ① As Figure 5 shown, inoculating strain Mh85 alone can significantly promote the growth of tomato seedlings. Compared with the control group (CK), as Figures 6 - 8 shown: The shoot dry weight, root dry weight, and total dry weight of tomato seedlings in the treatment group inoculated with strain Mh85 were significantly increased by 259.5%, 192.9, and 249.0% respectively.

[0070] ② Inoculation with the TB2 + Mh85 compound bactericide can significantly promote the growth of tomato seedlings. As Figures 9 - 11As shown, compared with the control group CK, the root dry weight, shoot dry weight, and total dry weight of tomato seedlings treated with the TB2 + Mh85 complex inoculant increased significantly by 497.6%, 591.4%, and 576.8%, respectively. Compared with the Mh85 strain, the root dry weight, shoot dry weight, and total dry weight of tomato seedlings treated with the TB2 + Mh85 complex inoculant increased significantly by 104.2%, 92.3%, and 93.9%, respectively. Compared with the TB2 strain, the root dry weight, shoot dry weight, and total dry weight of tomato seedlings treated with the TB2 + Mh85 complex inoculant increased significantly by 35.2%, 72.7%, and 66.3%, respectively.

[0071] In summary, the Bacillus amyloliquefaciens Mh85 screened by the applicant of the present invention has good abilities of producing IAA, nitrogen fixation, dissolving inorganic phosphorus, and producing siderophores. Moreover, this strain has a growth-promoting effect on Trichoderma brevicompactum TB2. Preparing the Bacillus amyloliquefaciens Mh85 and Trichoderma brevicompactum TB2 into a complex inoculant can more effectively promote the growth of plants, especially tomatoes. This shows that the strains and complex inoculants of this application can be promoted and utilized as biological organic fertilizer materials.

[0072] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Glucanolytic Bacillus Paenibacillus glycanilyticus The strain Mh85 has a deposit number of CCTCC NO: M2025410.

2. The method comprising the glucan-degrading bacillus according to claim 1 Paenibacillus glycanilyticus Composite bacterial agent of strain Mh85.

3. The composite bacterial agent according to claim 2, characterized in that: The composite bacterial agent is composed of glucan-degrading bacillus Paenibacillus glycanilyticus Trichoderma brevis Trichoderma brevicompactum The strain TB2 is composed of: the short dense Trichoderma Trichoderma brevicompactum The deposit number of strain TB2 is CCTCCNO: M20231913.

4. The dextranizing bacillus as claimed in claim 1 Paenibacillus glycanilyticus Use of strain Mh85 or the composite bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer.

5. The dextranizing bacillus as claimed in claim 1 Paenibacillus glycanilyticus Use of strain Mh85 or the composite bacterial agent as claimed in claim 2 in promoting plant growth.

6. The use according to claim 5, characterized in that: The plant is a tomato.

7. The dextranizing bacillus according to claim 1 Paenibacillus glycanilyticus Use of strain Mh85 or the composite bacterial agent as described in claim 2 in producing IAA, fixing nitrogen, decomposing inorganic phosphorus and / or producing iron carriers.

8. Use of the glucan-degrading bacillus as claimed in claim 1 Paenibacillus glycanilyticus The method for promoting plant growth by the strain Mh85 or the composite bacterial agent as claimed in claim 2, characterized in that: The method comprises: Paenibacillus glycanilyticus The bacterial suspension of strain Mh85 or the compound bacterial agent is poured into the root zone of plant seedlings.

9. The method according to claim 8, characterized in that The composite bacterial agent is composed of glucan-degrading bacillus Paenibacillus glycanilyticus Mh85 bacterial suspension and Trichoderma brevis Trichoderma brevicompactum The TB2 bacterial solution was mixed in a 1:1 ratio.

10. The method according to claim 9, characterized in that Paenibacillus glucanolyticus Paenibacillus glycanilyticus The effective viable count of Mh85 is 10 7 CFU / g; the short dense Trichoderma Trichoderma brevicompactum The effective viable count of TB2 is 10 6 CFU / g.

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