Brachybacterium horti and application thereof

Abstract

The invention provides a Brachium horti strain and application thereof, and relates to the technical field of microorganisms. The invention provides a strain of Brachium hortiNH1 / 13, the strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the strain is CGMCC No.29851; the strain has good starch degradation capacity, cellulose degradation capacity and plant disease prevention and control capacity, can be further used for straw degradation, and has great potential in agricultural application such as straw returning, plant disease prevention and control and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Brachybacterium horti strain and application thereof. Background Art

[0002] The annual production of straw is enormous, yet a significant amount of crop straw has yet to be rationally utilized and developed. Direct straw return to the field or return after composting is the most effective way to balance cost and harmlessness. Microorganisms play a crucial role in the degradation of straw during return. Tobacco, a major cash crop, also produces a significant amount of straw waste annually, which is not effectively utilized and causes environmental pollution. Furthermore, tobacco straw contains nicotine, which has a certain inhibitory effect on microbial growth and metabolism. Therefore, we isolated and identified microorganisms using tobacco straw as raw material in order to obtain microbial resources that can effectively degrade straw, particularly tobacco straw.

[0003] However, it should be noted that returning straw to the field directly or after composting can easily lead to soil-borne pests and diseases, which greatly limits its application in production. Therefore, obtaining microbial strains that can both degrade straw and control plant diseases and pests is of great significance for the practical application of straw returning to the field. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a Brachybacterium horti and its application. The Brachybacterium horti has good cellulose and starch degradation properties, and also has the functions of straw degradation and plant disease prevention. It has great potential in agricultural applications related to straw return to fields and plant disease prevention.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a Brachybacterium horti strain, which is named as NH1 / 13 and has a preservation number of CGMCC No. 29851.

[0007] The present invention also provides a fermentation broth comprising the NH1 / 13.

[0008] The present invention also provides a method for preparing the fermentation broth, comprising inoculating the NH1 / 13 into a culture medium and culturing the culture medium to obtain the fermentation broth.

[0009] The present invention also provides a degradation liquid comprising the NH1 / 13 or the fermentation liquid.

[0010] The present invention also provides use of the Brachybacterium horti, the fermentation liquid or the degradation liquid in degrading cellulose and / or starch.

[0011] The present invention also provides use of the Brachybacterium horti, the fermentation liquid or the degradation liquid in degrading straw.

[0012] The present invention also provides use of the Brachybacterium horti or the fermentation liquid in preparing a product for preventing and treating pathogens.

[0013] Preferably, the pathogenic bacteria include Ralstonia solanacearum.

[0014] The present invention also provides use of the Brachybacterium horti or the fermentation liquid in preventing and controlling plant diseases.

[0015] Preferably, the plant disease includes bacterial wilt.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention isolated a strain of Brachybacterium hortiNH1 / 13 from discarded tobacco stems. This strain has been deposited with the China General Microbiological Culture Collection Center under the accession number CGMCC No. 29851. This strain has excellent starch-melting capabilities, cellulose degradation, and the ability to control plant diseases. It can be further used for straw degradation and has great potential in agricultural applications such as straw return to fields and plant disease control.

[0018] Biodeposit Information:

[0019] The Brachybacterium horti of the present invention has a strain name of NH1 / 13, is classified as Brachybacterium horti, is deposited in the China General Microorganism Culture Collection Center, abbreviated as CGMCC, has a strain accession number of CGMCC No. 29851, is deposited on February 2, 2024, and is deposited at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 4 is the colony morphology of strain NH1 / 13, where the left is the early stage of culture and the right is the late stage of culture.

[0021] Figure 2 This is the phylogenetic tree of strain NH1 / 13.

[0022] Figure 3This is the filter paper disintegration test result of strain NH1 / 13.

[0023] Figure 4 This is the result of Galanz iodine solution staining of strain NH1 / 13 on starch culture medium.

[0024] Figure 5 This is the antagonistic effect of strain NH1 / 13 against the pathogen Ralstonia solanacearum. DETAILED DESCRIPTION

[0025] The invention provides a Brachybacterium horti strain, which is named as NH1 / 13 and has a deposit number of CGMCC No. 29851.

[0026] In the present invention, NH1 / 13 was isolated from discarded cigarette stalks and deposited with the China General Microbiological Culture Collection on February 2, 2024. NH1 / 13 exhibits smooth, raised, pale yellow, circular colonies on NA solid culture medium. After prolonged cultivation, the colonies transform to bright yellow. Amplification of this strain using universal 16S rRNA primers yielded a 1401-bp sequence fragment, as shown in SEQ ID NO. 3.

[0027] In the present invention, the sequences of the 16S rRNA universal primers include: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0028] The present invention also provides a fermentation liquid comprising the NH1 / 13. In the present invention, the fermentation liquid has the ability to degrade cellulose and starch, and also has the function of degrading straw and / or preventing and controlling plant diseases.

[0029] The present invention also provides a method for preparing the fermentation broth, comprising inoculating the NH1 / 13 in a culture medium to obtain a fermentation broth. In the present invention, the culture medium is a NA liquid culture medium or a NB liquid culture medium. The culture temperature is 25 to 30°C, more preferably 28°C. As an embodiment, the fermentation broth includes OD 600 Bacterial suspension with a value of 0.4-0.6.

[0030] The present invention also provides a degradation liquid comprising the Brachybacterium horti or the fermentation liquid. In the present invention, the degradation liquid can be used to degrade cellulose and / or starch. In one embodiment, the degradation liquid can be used to degrade cellulose- and / or starch-containing crops, such as straw, including one or more of tobacco straw, wheat straw, and rice straw.

[0031] The present invention also provides the use of the Brachybacterium horti, the fermentation liquid or the degradation liquid in degrading cellulose and / or starch. As an embodiment, the product can be used to degrade waste with a high starch content, such as kitchen waste.

[0032] The present invention also provides the use of the Brachybacterium horti, the fermentation liquid or the degradation liquid in degrading straw. In the present invention, the straw includes one or more of tobacco straw, wheat straw and rice straw.

[0033] The present invention also provides the use of the Brachybacterium horti or the fermentation broth in preparing a pathogen control product. Preferably, the pathogen includes bacterial wilt, more preferably Ralstonia solanacearum.

[0034] The present invention also provides the use of the Brachybacterium horti or the fermentation liquid in preventing and treating plant diseases. In the present invention, the plant disease includes bacterial wilt, and the pathogenic bacteria of the bacterial wilt include Ralstonia solanacearum.

[0035] The present invention does not specifically limit the product type. In the product, the Brachybacterium hortiNH1 / 13, the fermentation broth, or the degradation broth can be the sole active ingredient, or other active ingredients can be included. The product of the present invention can be used alone or in combination with other excipients and / or products. The present invention does not specifically limit the concentration of the bacterial broth in a specific application and can be conventionally selected according to actual needs.

[0036] In the present invention, bacterial wilt is a devastating soil-borne disease caused by infection with Ralstonia solancearum. R. solanacearum can infect more than 200 plant species from more than 50 families, such as tobacco, tomato, pepper, eggplant, ginger, potato, peanut, strawberry and banana.

[0037] In the present invention, the discarded tobacco stems were harvested in September 2023 from the Licha tobacco field in Jiaozhou, Qingdao. After being dried on the field for two months, they were brought back to the laboratory.

[0038] In the present invention, the Ralstonia solanacearum described in the following examples is Ralstonia solanacearum RS10, and RS10 is a known strain, see Chu D., Ilyas N., Peng L., Wang X., Wang D., Xu Z., Gao Q., Tan X., Zhang C., Li Y. and Yuan Y. Genomic insights on fighting bacterial wilt by a novel Bacillus amyloliquefaciens strain in Cas02. Microb. biotechnology., 2021, 15: 1152-1167.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] Example 1

[0041] 1. Isolation of Brachybacterium horti

[0042] 10 g of discarded tobacco stems were added to 90 mL of sterile ultrapure water, shaken at 180 rpm at 28°C for 30 min, mixed thoroughly and then allowed to stand. 1 mL of the suspension was taken for gradient dilution. 100 μL of the diluted suspension was spread on NA solid medium (beef extract powder, peptone, sodium chloride, agar), and cultured in an incubator at 28°C for 5 days. The colonies on the NA solid medium were streaked and purified repeatedly 5 to 8 times. The purified single strain was cultured on NA solid medium. The colony morphology on the culture medium was a smooth and raised light yellow circular colony. After long-term culture, the color of the colony turned bright yellow. The strain was numbered NH1 / 13.

[0043] 2. Identification of Brachybacterium horti

[0044] Genomic DNA from NH1 / 13 was extracted and amplified using Novagen 2× Taq Plus Master Mix II DNA polymerase using the extracted genomic DNA as a template and 16S rDNA universal primers. The sequences of the 16S rRNA universal primers are as follows: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2). The PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2. PCR amplification was performed on each of the NH1 / 13 bacterial strains. PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0045] Table 1 PCR amplification system

[0046] Reagent name Dosage / μL DNA template (genomic DNA of NH1 / 13) 1 Upstream primer 1 Downstream primer 1 2×TaqPlusMasterMixⅡ 12.5 <![CDATA[ddH2O]]> 9.5

[0047] Table 2 PCR amplification program

[0048]

[0049] Gene sequence alignment and analysis were performed using EzBioCloud (https: / / www.ezbiocloud.net / ) and NCBI Blast, and sequences of bacteria with high similarity to the strain were downloaded. Phylogenetic trees were constructed using MEGA 11 software.

[0050] like Figure 2 As shown, strain NH1 / 13 was most closely related to Brachybacterium horti|THG S15-4|KM073966 at 95%. The results confirmed that strain NH1 / 13 belonged to Brachybacterium horti.

[0051] The amplified 16s rDNA sequence of NH1 / 13 is as follows (1401 bp):

[0052]

[0053] The NH1 / 13 was deposited in the China General Microorganism Culture Collection Center, abbreviated as CGMCC, on February 2, 2024. The culture collection number is CGMCC No. 29851, and the collection address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0054] Example 2

[0055] 1. Brachybacterium horti degradation function test

[0056] (1) Brachybacterium horti has the function of degrading cellulose

[0057] In order to test the cellulose degradation ability of the strain NH1 / 13 screened in Example 1, a filter paper disintegration experiment was set up, with no bacteria added as a blank control.

[0058] The specific test method is as follows: 0.05g filter paper strips were used as substrates, 50mL NB liquid medium was used as basal medium to perform filter paper disintegration test on strain NH1 / 13, and two filter paper strips were placed in each treatment. 600 A suspension of NH1 / 13 with a pH of 0.4-0.6 was inoculated into the culture medium at a concentration of 1% and cultured at 180 rpm and 28°C for 10 days. The degree of filter paper disintegration was observed to evaluate the cellulose degradation ability of strain NH1 / 13.

[0059] The results are as follows Figure 3 As shown, strain NH1 / 13 has a certain ability to degrade filter paper.

[0060] (2) Brachybacterium horti has the function of degrading starch

[0061] The specific experimental method is as follows: 2g / L soluble starch was used as substrate and NA medium was used as basal medium to test the starch degradation of strain NH1 / 13 obtained in Example 1. 600 Inoculate 2.5 μL of a suspension of NH1 / 13 bacteria with a pH value of 0.4-0.6 in the center of an indicator plate and incubate in the dark at 28°C for 48 hours. After removing the NH1 / 13 colonies, stain the plate with iodine-potassium iodide solution (1 g / L). Observe the color change and assess the strain's ability to degrade starch.

[0062] Results: The strain NH1 / 13 was stained with Galanz iodine solution on starch medium. Figure 4 As shown, a color change zone appeared in the center of the culture medium after the NH1 / 13 colony was removed, indicating that the strain has the ability to degrade starch.

[0063] (3) Brachybacterium horti has the function of degrading straw

[0064] In order to verify the degradation effect of the strain NH1 / 13 obtained in Example 1 on real straw materials, the following experiment was set up: tobacco straw / wheat straw / rice straw powder was added to distilled water to a final concentration of 20 g / L, and then 15.15 g / LNa2HPO4·12H2O, 3 g / LKH2PO4, 0.5 g / LNaCl, and 1 g / LNH4Cl were added and sterilized for use.

[0065] OD 600 An NH1 / 13 bacterial suspension with a pH value of 0.4-0.6 was inoculated into the above straw culture medium, and a fermentation medium without bacterial liquid was used as a control. After culturing at 30°C and 180 rpm for 10 days, the supernatant was discarded by centrifugation, and the straw was repeatedly rinsed with distilled water. After centrifugation, the relative degradation rate of the straw was determined by the weight loss method according to the following formula.

[0066] Y=(m1-m2) / m1*100%

[0067] Where: Y is the degradation rate; m1 is the dry mass of straw before fermentation; m2 is the dry mass of straw after fermentation.

[0068] The results are shown in Table 3.

[0069] Table 3 Relative degradation rates of different straws by NH1 / 13 bacterial suspension

[0070] Group tobacco straw wheat straw rice straw CK 16.54±0.13％b 19.00±0.00％b 17.33±0.67％b NH1 / 13 bacterial suspension 21.66±0.24％a 24.00±0.58％a 23.33±0.33％a

[0071] (4) Brachybacterium horti has the function of antagonizing Ralstonia solanacearum

[0072] The specific experimental method is as follows: the strain was activated by NB medium and the OD 600 2.5 μL of the bacterial suspension of the NH1 / 13 strain obtained by screening in Example 1 with a value of 0.4-0.6 was inoculated in the center of the NA culture medium and cultured at 28°C for 24 hours. The pathogen Ralstonia solanacearum suspension was evenly sprayed into the culture medium and cultured at 28°C for another 24 hours to observe whether there was an antagonistic effect. The antagonistic activity of the strain was judged by the size of the inhibition zone.

[0073] Antagonistic effects such as Figure 5 As shown, the radius of the inhibition zone of strain NH1 / 13 is about 0.7 cm, indicating that strain NH1 / 13 not only has the ability to degrade straw, but also has the effect of antagonizing bacterial wilt.

[0074] The results of the above examples show that strain NH1 / 13 has the functions of both straw degradation and plant disease prevention and control, and has great potential in the application of straw returning to the field.

[0075] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Brachybacterium horti, characterized in that The Brachybacterium horti is named NH1 / 13 and has a deposit number of CGMCC No. 29851.

2. A fermentation broth, characterized in that: Containing the NH1 / 13 described in claim 1.

3. The method for preparing the fermentation broth according to claim 2, characterized in that: The method comprises inoculating the NH1 / 13 into a culture medium and culturing the culture medium to obtain a fermentation liquid.

4. A degradation liquid, characterized in that Comprising the NH1 / 13 according to claim 1 or the fermentation broth according to claim 2.

5. Use of the Brachybacterium horti according to claim 1, the fermentation liquid according to claim 2 or the degradation liquid according to claim 4 in degrading cellulose and / or starch.

6. Use of the Brachybacterium horti of claim 1, the fermentation liquid of claim 2 or the degradation liquid of claim 4 in degrading straw.

7. Use of the Brachybacterium horti according to claim 1 or the fermentation liquid according to claim 2 in preparing a product for preventing and treating pathogens.

8. The use according to claim 7, characterized in that The pathogenic bacteria include Ralstonia solanacearum.

9. Use of the Brachybacterium horti according to claim 1 or the fermentation liquid according to claim 2 in preventing and controlling plant diseases.

10. The use according to claim 7, characterized in that The plant diseases include bacterial wilt.

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