Application of a Paenibacillus isoglycolyticus strain and its composite bacterial agent in the preparation of bio-organic fertilizer

Abstract

The present invention relates to the field of microbial technology, and in particular to an application of a heteroglycan-decomposing Bacillus and a composite microbial agent thereof in the preparation of biological organic fertilizer. The strain Mh85 of the present invention is isolated by a research team from the root system of the apple rootstock "Pingyi Sweet Tea" treated with Trichoderma TB2. The strain Mh85 has good IAA production, nitrogen fixation, inorganic phosphorus decomposition and siderogen production capabilities. The strain Mh85 can not only directly promote plant growth, but also has a good growth-promoting effect on Trichoderma TB2. When the strain is compounded with Trichoderma TB2 to form a composite microbial agent and then subjected to a tomato seedling pot experiment, it is found that the composite microbial agent exhibits a synergistic effect, effectively overcoming the problems of unstable growth-promoting effects and insufficient synergy between strains in existing single microbial preparations, thereby providing an efficient and stable technical solution for improving plant growth, improving root development and increasing crop yield. The strain Mh85 is a good raw material for preparing biological organic fertilizer.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to application of a Paenibacillus isoglycan decomposing 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 enhance plant health has become an important means to replace traditional chemical 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 used alone, it is limited by factors such as environmental conditions and plant varieties, and it is often difficult to achieve a stable promotion effect under various conditions; (2) Insufficient optimization of compound schemes: Most 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 applied strains are derived from conventional soil or traditional microbial libraries, while 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 yet been fully developed; (4) Strain specificity. In the process of strain screening, we found that different strains of the same species have great specificity. Different strains of the same species often have different application ranges due to differences in physiological characteristics. Therefore, those skilled in the art need to continuously screen strains and perform functional verification to improve the germplasm resource library.

[0003] The technical solution of the present invention is based on the above-mentioned shortcomings. The research team of this application isolated microorganisms that may have a growth-promoting effect with 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] In order to achieve the above purpose, the present invention screened out a new strain: Paenibacillus spp. Paenibacillus glycanilyticus Strain Mh85, its taxonomic designation is: Paenibacillus glycanilyticus Mh85, Chinese classification name: Bacillus subtilis Mh85, preservation number: CCTCC NO: M2025410; the strain is deposited 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.

[0006] The present invention also includes the bacillus sp. Paenibacillus glycanilyticus Composite bacterial agent of strain Mh85.

[0007] Furthermore, the composite bacterial agent is composed of Bacillus saccharidogenicus Paenibacillus glycanilyticus strain Mh85 and Trichoderma brevis Trichoderma brevicompactum strain TB2 composition;

[0008] Trichoderma brevis Trichoderma brevicompactum strain TB2, classified as Trichoderma brevicompactum TB2, its preservation number is CCTCC NO: M20231913; the strain is deposited 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. The strain has been disclosed in patent application: 202311753128.0.

[0009] The present invention also includes the Bacillus sp. Paenibacillus glycanilyticus Application of the strain Mh85 or the composite bacterial agent in the preparation of biological organic fertilizer.

[0010] The present invention also includes the Bacillus sp. Paenibacillus glycanilyticus Application of strain Mh85 or the composite bacterial agent in promoting plant growth.

[0011] Furthermore, the plant is tomato.

[0012] The present invention also includes the Bacillus sp. Paenibacillus glycanilyticus Application of strain Mh85 or the composite bacterial agent in producing IAA, fixing nitrogen, decomposing inorganic phosphorus and / or producing siderophores.

[0013] The present invention also includes the Bacillus sp. Paenibacillus glycanilyticus The method for promoting plant growth by using strain Mh85 or the composite bacterial agent is as follows: Paenibacillus glycanilyticus The strain Mh85 bacterial suspension or compound bacterial agent is poured into the rhizosphere of the plant seedlings.

[0014] Furthermore, the composite bacterial agent is composed of Bacillus saccharidogenicus Paenibacillus glycanilyticus Mh85 bacterial solution and Trichoderma brevis Trichoderma brevicompactum TB2 bacterial solution was mixed in a ratio of 1:1.

[0015] Furthermore, the Paenibacillus sp. 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.

[0016] The present invention has the following beneficial effects: the strain Mh85 of the present invention was isolated by the research team from the root system of the apple rootstock "Pingyi Sweet Tea" treated with Trichoderma TB2, and has good IAA production, nitrogen fixation, inorganic phosphorus decomposition and siderophore production capabilities. The strain Mh85 can not only directly promote plant growth, but also co-cultivate the strain with the Trichoderma strain and find that the strain Mh85 has a good growth-promoting effect on Trichoderma TB2, but has no obvious growth-promoting effect on Trichoderma 4742. After the strain is compounded with Trichoderma TB2 to prepare a composite microbial agent, it is found that the composite 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 species of existing single microbial preparations. The species of the composite microbial agent have good synergistic effects, thereby providing an efficient and stable technical solution for improving plant growth, improving root development and increasing crop yield. It is a good raw material for preparing biological organic fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the colony morphology of strain Mh85.

[0018] Figure 2 This is the phylogenetic tree of strain Mh85.

[0019] Figure 3 This is a graph of mycelial growth showing the growth-promoting effect of strain Mh85 on different Trichoderma strains (TB2, 4742).

[0020] Figure 4 This is a graph showing the biomass results of the growth-promoting effect of strain Mh85 on Trichoderma strain TB2.

[0021] Figure 5 This is a graph showing the growth effects of strain Mh85 and TB2+Mh85 composite agent on tomato seedlings.

[0022] Figure 6 This is the result of the effect of strain Mh85 on the aboveground dry weight of tomato seedlings.

[0023] Figure 7 This is the result of the effect of strain Mh85 on the dry weight of tomato seedling roots.

[0024] Figure 8 This is the result graph of the effect of strain Mh85 on the total dry weight of tomato seedlings.

[0025] Figure 9 This is the result of the effects of strain Mh85 and TB2+Mh85 composite agent on the aboveground dry weight of tomato seedlings.

[0026] Figure 10 This is the result of the effects of strain Mh85 and TB2+Mh85 composite agent on the dry weight of tomato seedling roots.

[0027] Figure 11 This is the result of the effects of strain Mh85 and TB2+Mh85 composite agent on the total dry weight of tomato seedlings.

[0028] Biomaterial deposit information

[0029] The deposit information of the strain Mh85 of this application is: Paenibacillus sp. Paenibacillus glycanilyticus Strain Mh85, its taxonomic designation is: Paenibacillus glycanilyticus Mh85, Chinese classification name: Bacillus subtilis Mh85, preservation number: CCTCC NO: M2025410; the strain is deposited 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.

[0030] The deposit information of the TB2 strain of this application is: Trichoderma brevis Trichoderma brevicompactum strain TB2, classified as Trichoderma brevicompactum TB2, its deposit number is CCTCC NO: M20231913; the 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. The strain has been disclosed in patent application: 202311753128.0. DETAILED DESCRIPTION

[0031] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0032] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features. Example 1

[0033] This example is the isolation and identification of Paenibacillus heterosaccharidogenicus Mh85.

[0034] 1. Isolation and purification of strain Mh85: About 1 gram of root tissue was collected from the root system of the apple rootstock "Pingyi Sweet Tea" that had been treated with Trichoderma TB2. After collection, the root system was first surface disinfected to remove exogenous microorganisms attached to the surface and ensure the purity of the endophytes; the disinfected root tissue was placed in a 50 mL sterile Erlenmeyer flask containing glass beads and 9 mL SM buffer, and shaken at 30°C and 170 rpm for 30 minutes to fully break the root tissue and release the endophytes into the buffer to form a bacterial suspension; the obtained suspension was gradiently diluted with sterile water (diluted to 10 -5 ~10 -7 The strain was then plated on TSA medium and incubated at 30°C for 48 hours. Based on colony morphology, representative single colonies were selected by streaking and repeatedly purified to obtain a pure culture strain.

[0035] 2. Identification of strain Mh85: ① Morphological identification: Figure 1 As shown in the figure, the Mh85 strain was inoculated into TSB medium and cultured in a constant temperature incubator for 24 hours. Round, smooth-surfaced colonies were observed on the solid medium. Their morphological characteristics were consistent with the common characteristics of polysaccharide-degrading Paenibacillus. ② The 16S rRNA gene of the purified strain was amplified and sequenced. The sequence alignment results are shown in the figure. Figure 2 As shown, this strain Paenibacillus glycanilyticus They are highly similar, hence the name Paenibacillus glycanilyticus Mh85. Example 2

[0036] This example is a growth-promoting function assay of strain Mh85.

[0037] 1. Qualitative determination of IAA production function of strains: 1% inoculum of Mh85 strain was added to TSB medium containing L-tryptophan, 30℃, 170 rpm -1 After shaking for 48 hours, centrifuge at 10,000 rpm for 5 minutes to obtain the supernatant. Take 100 μl of the supernatant and mix it with an equal volume of Salkowski colorimetric solution. Mix 100 μl of uninoculated culture medium with an equal volume of Salkowski colorimetric solution as a blank control. React in the dark for 30 minutes. If the solution turns red, it means that the strain has IAA ability.

[0038] 2. Determination of the strain's potassium-solubilizing ability: Add 10 μL of the activated MH85 bacterial suspension to each of the Axubei nitrogen-fixing medium, the Montgina organophosphate medium, and the PKO inorganic phosphorus medium, with three replicates. Culture in a 28°C incubator for 5 days. Observe the presence of halos or transparent circles on the culture medium. The ratio of the halo or transparent circle (D) to the colony diameter (d) is used to determine the strain's ability to fix nitrogen and solubilize organic and inorganic phosphorus.

[0039] 3. Determination of siderophore production capacity: Referring to Schwyn and Neilands, the bacterial suspension cultured in LNM medium for 48 h under iron-limited conditions was passed through a perforated filter and centrifuged at 3500 rpm for 10 min. The supernatant was mixed with CAS detection solution in a 1:1 ratio and allowed to react for 2 h. The OD value was measured. 630 The absorbance at 400 nm (A) was measured. A control was sterile LNM medium, also mixed with CAS assay solution in equal proportions, and the absorbance (Ar) was measured. The relative siderophore content was calculated using the formula [(Ar-As) / Ar] × 100%.

[0040] The results of the above growth-promoting properties are shown in Table 1.

[0041]

[0042] As shown in Table 1, strain Mh85 has the ability to produce IAA, fix nitrogen, decompose inorganic phosphorus and produce siderophores. Example 3

[0043] This example is a study on the growth-promoting effect of strain Mh85 on Trichoderma brevis.

[0044] (1) Experimental design: To evaluate Paenibacillus glycanilyticus The promoting effect of strain Mh85 (hereinafter referred to as Mh85) on the growth of Trichoderma, wherein the Trichoderma strain is selected: Trichoderma brevis Trichoderma brevicompactum strain TB2 (hereinafter referred to as: TB2) and Trichoderma harzianum Trichoderma Harzianum NJAU 4742, accession number CGMCC No. 12166 (hereinafter referred to as 4742), was set up with the following four treatment groups:

[0045] Control group 1: culture TB2 alone;

[0046] Treatment group 1: TB2 and Mh85 were co-cultured.

[0047] Control group 1: culture 4742 alone;

[0048] Treatment group 1: 4742 was co-cultured with Mh85.

[0049] (2) Culture conditions: To ensure normal bacterial growth, PDB liquid culture medium was selected and the pH of the culture medium was adjusted to 7.0. Each group was inoculated with the corresponding bacterial solution and cultured at 30°C and 170 rpm for 48 hours.

[0050] (3) Mycelial biomass determination: After the incubation period, the mycelia of Trichoderma were collected by filter paper, dried, and weighed to determine their biomass. The biomass of the mycelia of the treated group was compared with that of the control group to evaluate the effect of the Mh85 strain on the growth of Trichoderma strains.

[0051] Test results:

[0052] 1. If Figure 3 The results show that strain Mh85 significantly promoted the growth of Trichoderma TB2 mycelium. Compared with the control, the volume of mycelium in TB2 treated with Mh85 increased by 266%. However, strain Mh85 had no significant effect on the growth of Trichoderma 4742 mycelium.

[0053] 2. If Figure 4 As shown, strain Mh85 significantly promoted the growth of Trichoderma TB2 mycelium and increased its biomass. After drying to constant weight, the mycelial biomass of TB2 inoculated with Mh85 increased by 44% compared to the control.

[0054] The above experiments showed that for Trichoderma strains of the same genus, strain Mh85 could significantly promote the growth of Trichoderma TB2, but had no obvious promoting effect on Trichoderma 4742. Example 4

[0055] This example is a study on the growth-promoting effect of strain Mh85 on tomato seedlings.

[0056] (1) Tomato seedling cultivation:

[0057] 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 then 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 plenty of sterile water.

[0058] ② Germination and sowing: Spread the sterilized seeds on a culture medium lined with filter paper soaked in sterile water and place them in a 28°C constant temperature incubator for germination for 3-5 days. Select seeds with good germination and sow them on a sterilized culture medium or sterile substrate for seedling cultivation.

[0059] ③ Transplantation: After the tomato seedlings have grown to 2-3 true leaves and are growing evenly, select healthy and uniform seedlings and transplant them into sterilized substrate pots for potted cultivation.

[0060] (2) Inoculation treatment:

[0061] ① Preparation of bacterial suspension: Obtain according to the above method Mh85 strain was prepared into bacterial suspension and its final concentration was adjusted to 10 7 CFU / g soil. The preparation method of the bacterial suspension of Trichoderma TB2 strain is a concentration of 10 6 CFU / g soil.

[0062] ②The bacterial solutions of the four treatment groups are as follows:

[0063] CK group (control group): No bacterial solution was inoculated and tomato seedlings were cultivated alone.

[0064] TB2 group: Inoculated only with Trichoderma TB2 liquid at a concentration of 10 6 CFU / g soil.

[0065] Mh85 group: only inoculated with Mh85 bacterial solution, the concentration was 10 7 CFU / g soil.

[0066] TB2+Mh85 group: Mh85 bacterial solution and Trichoderma TB2 bacterial solution were mixed in a ratio of 1:1 and inoculated into the rhizosphere of tomato seedlings.

[0067] ③ Inoculation operation: Evenly inoculate the prepared bacterial suspension into the rhizosphere of tomato seedlings and perform the above four treatments.

[0068] ④ Experimental design: Each treatment group was set up with 5 replicates and cultured under the same environmental conditions.

[0069] ⑤Result collection: After 30 days of cultivation, the growth phenotypes of tomato seedlings in each group were measured and recorded, including plant height, root development, biomass and other indicators, to evaluate the growth-promoting effect of the combination of Mh85 strain and Trichoderma TB2 on tomato seedlings.

[0070] The test results are as follows:

[0071] ① Such as Figure 5 As shown in Figure 2, inoculation of Mh85 strain alone significantly promoted the growth of tomato seedlings. Figures 6 - 8 As shown in the results, the aboveground dry weight, root dry weight and total dry weight of tomato seedlings in the group inoculated with Mh85 strain increased significantly by 259.5%, 192.9% and 249.0%, respectively.

[0072] ②TB2+Mh85 compound fungicide inoculation can significantly promote the growth of tomato seedlings. Figures 9 - 11As shown, compared with the control (CK) group, tomato seedlings inoculated with the TB2+Mh85 complex significantly increased their root dry weight, aboveground dry weight, and total dry weight by 497.6%, 591.4%, and 576.8%, respectively. Compared with the Mh85 strain, tomato seedlings inoculated with the TB2+Mh85 complex significantly increased their root dry weight, aboveground dry weight, and total dry weight by 104.2%, 92.3%, and 93.9%, respectively. Compared with the TB2 strain, tomato seedlings inoculated with the TB2+Mh85 complex significantly increased their root dry weight, aboveground dry weight, and total dry weight by 35.2%, 72.7%, and 66.3%, respectively.

[0073] In summary, the Bacillus sp. Mh85 screened by the applicant of the present invention has good IAA production, nitrogen fixation, inorganic phosphorus decomposition and siderophore production capabilities, and the strain has a growth-promoting effect on Trichoderma brevis TB2. The preparation of a composite bacterial agent of Bacillus sp. Mh85 and Trichoderma brevis TB2 can more effectively promote the growth of plants, especially tomatoes. This shows that the strains and composite bacterial agents of the present application can be promoted and utilized as biological organic fertilizer materials.

[0074] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Paenibacillus saccharidogenicus Paenibacillus glycanilyticus strain Mh85, its deposit number is CCTCC NO: M2025410.

2. The method comprises the bacillus sp. of claim 1 Paenibacillus glycanilyticus The composite bacterial agent of strain Mh85 is characterized in that: The composite bacterial agent is made from Paenibacillus saccharidogenicus Paenibacillus glycanilyticus strain Mh85 and Trichoderma brevis Trichoderma brevicompactum strain TB2; the short dense Trichoderma Trichoderma brevicompactum The deposit number of strain TB2 is CCTCC NO: M20231913.

3. Paenibacillus sp. as claimed in claim 1 Paenibacillus glycanilyticus Use of the strain Mh85 or the composite bacterial agent as claimed in claim 2 in the preparation of biological organic fertilizer.

4. Paenibacillus sp. as claimed in claim 1 Paenibacillus glycanilyticus Use of the strain Mh85 or the composite bacterial agent as claimed in claim 2 in promoting plant growth, wherein the plant is tomato.

5. Paenibacillus sp. as claimed in claim 1 Paenibacillus glycanilyticus Application of strain Mh85 in producing IAA, fixing nitrogen, decomposing inorganic phosphorus and / or producing siderophores.

6. Use of the Paenibacillus sp. as claimed in claim 1 Paenibacillus glycanilyticus The method for promoting plant growth by the strain Mh85 or the composite bacterial agent according to claim 2, characterized in that: The method comprises: Paenibacillus glycanilyticus The bacterial suspension of strain Mh85 or the composite bacterial agent is watered into the rhizosphere of the plant seedlings, wherein the plant is tomato.

7. The method according to claim 6, characterized in that The composite bacterial agent is made from Paenibacillus saccharidogens Paenibacillus glycanilyticus Mh85 bacterial solution and Trichoderma brevis Trichoderma brevicompactum TB2 bacterial solution was mixed in a ratio of 1:

1.

8. The method according to claim 7, characterized in that Paenibacillus saccharidogenins 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.

Images