Separation and identification of Burkholderia sp. TC129QW202409 and application of Burkholderia sp. TC129QW202409

Abstract

The invention discloses separation and identification of a Burkholderia sp. TC129QW202409 strain and an application of the Burkholderia sp. TC129QW202409 strain. The invention provides Burkholderia sp. TC129QW202409, the preservation number of the Burkholderia sp. TC129QW202409 is CGMCC NO.31999, and the application of a culture solution of the Burkholderia sp. Or a bacterial solution of the Burkholderia sp. Or a fermentation solution of the Burkholderia sp. In preparation of products with the following functions: 1) inhibiting fusarium; 2) treating plant diseases caused by fusarium; and 3) preventing plant diseases caused by fusarium. The invention discloses a Burkholderia sp. TC129QW202409, and the Burkholderia sp. TC129QW202409 strain can be used as a biocontrol bacterium to antagonize a plurality of F. fusarium zeae (F. fusarium zeae, F. graminearum), and can be used for treating plant diseases caused by compound infection of the plant fusarium zeae.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-fusarium fungus and relates to the separation, identification and application of a Burkholderia TC129_QW202409. Background Art

[0002] Fusarium spp. can cause wilt, root rot / stem rot, etc. in many crops (including corn), seriously affecting growth and yield, and is an important disease threatening crop production safety. Researchers from Yunnan Agricultural University found that Fusarium can cause corn diseases through single or combined infection (Yuan Ding et al., 2017, Journal of Southwest Agriculture). It is worth noting that F. oxysporum, as a wilt pathogen of many crops (celery, asparagus, banana, oil palm and strawberry), has been included in the list of quarantine pests for imported plants of the People's Republic of China to strengthen the supervision of its spread and diffusion.

[0003] Fusarium is mainly spread through soil and seeds. In severe cases, it can cause the death of the entire crop plant, causing huge losses to agricultural production. At present, there is no effective treatment technology for wilt and root rot / stem rot caused by Fusarium. Therefore, the technology for this disease is mostly focused on tillage management and prevention strategies for breeding disease-resistant varieties. However, the problems with these methods cannot be ignored. For example, the effects of tillage management and disease-resistant varieties are limited by many factors. Therefore, it is urgent to develop technologies to resist Fusarium spp. to ensure crop production safety and reduce economic losses. The development of new biological control strategies, especially the exploration and utilization of antagonistic strains, is of great practical significance for improving maize disease resistance. Summary of the invention

[0004] The technical problem solved by the invention is to provide a strain for inhibiting plant diseases caused by Fusarium.

[0005] In order to solve the above technical problems, in a first aspect, the present invention provides Burkholderia sp. TC129_QW202409, whose deposit number is CGMCC NO.31999.

[0006] In a second aspect, the present invention provides the use of the bacteria or its culture solution or its bacterial solution or its fermentation solution according to the first aspect in preparing a product having any of the following functions:

[0007] 1) Inhibit Fusarium;

[0008] 2) Treat plant diseases caused by Fusarium;

[0009] 3) Prevent plant diseases caused by Fusarium.

[0010] In the application described above, the Fusarium is Fusarium graminearum, Fusarium oxysporum and / or Fusarium verticillium.

[0011] In the application described above, the product is a bacterial agent.

[0012] Furthermore, the product is a biocontrol agent.

[0013] In a third aspect, the present invention provides a culture solution of the bacteria described in the first aspect, which is a culture solution obtained by culturing the bacteria described in the first aspect.

[0014] In a fourth aspect, the present invention provides a product, the active ingredient of which is the bacteria or its culture solution or its bacterial solution or its fermentation solution as described in the first aspect.

[0015] In the applications described above, the product has any of the following functions:

[0016] 1) Inhibit Fusarium;

[0017] 2) Treat plant diseases caused by Fusarium;

[0018] 3) Prevent plant diseases caused by Fusarium.

[0019] In the application described above, the product is a biocontrol agent.

[0020] In the above, the plant may be a dicotyledonous plant or a monocotyledonous plant.

[0021] Furthermore, the monocotyledonous plant may be a Poaceae plant.

[0022] Furthermore, the grass plant may be corn.

[0023] The present invention obtains a Burkholderia sp. TC129_QW202409, which is isolated from the rhizosphere of corn disease plants in black soil, and is specifically enriched by pathogens, and is verified for the first time to be effective against plant Fusarium complex infection. The strain has the ability to antagonize corn Fusarium, and at the same time enhances the ability of corn to prevent Fusarium complex infection diseases, and can be used as a biocontrol bacterium to antagonize multiple corn Fusarium (F.oxysporum, F.verticillioides, F.graminearum) and treat plant diseases caused by Fusarium complex infection of plants.

[0024] Collection Instructions

[0025] Classification name: Burkholderia sp.

[0026] Strain ID: TC129_QW202409

[0027] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0028] Abbreviation of depository unit: CGMCC

[0029] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0030] Deposit date: September 19, 2024

[0031] CGMCC Registration Number: CGMCC No.31999 BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 To carry out sterilization and scale-up verification for corn rhizosphere soil infected with Fusarium disease on black soil.

[0033] Figure 2 Morphological identification of Burkholderia TC129_QW202409.

[0034] Figure 3 Confrontation experiment of Burkholderia TC129_QW202409 (OD600=1.0) against F. graminearum (A), F. oxysporum (B), and F. verticillioides (C) on PDA plates; the fungi were inoculated with a bacterial block with a diameter of 7.5 mm.

[0035] Figure 4 The ability of antagonistic bacteria to resist the mixed fungal disease of three strains of Fusarium was monitored by stem injection; A is bacteria (Burkholderia TC129_QW202409, OD600=1.0), B is MgCl2, and C is a mixed fungal strain of three strains (F.oxysporum, spore amount: about 3×10 7 / mL; F. verticillioides, about 3×10 7 / mL; F.graminearum, mycelium broken), D is a mixture of three bacterial and fungal strains, and E is a scoring statistical chart. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0039] Some of the reagents in the following examples are as follows:

[0040] LB solid medium: 10 g tryptone, 5 g yeast powder, 10 g NaCl, 14 g agar, add distilled water to 1 L, sterilize at 116°C for 20 min.

[0041] LB liquid culture medium: 10 g tryptone, 5 g yeast powder, 10 g NaCl, add distilled water to 1 L, sterilize at 116°C for 20 min.

[0042] PDA solid culture medium: 3 g potato powder, 20 g glucose, 14 g agar, add distilled water to make up to 1 L, sterilize at 116℃ for 20 min.

[0043] Example 1: Isolation and screening of antagonistic bacteria to Fusarium

[0044] 1. Isolation of Antagonistic Bacteria TC129_QW202409 to Fusarium

[0045] Fusarium oxysporum, Fusarium verticillioides, and Fusarium graminearum isolated from the diseased parts of corn leaves and stems in the black soil were used to enrich the potential functional strains in the rhizosphere soil of corn. Figure 1 As shown, potential antagonistic bacteria were enriched from soil samples of susceptible corn using three strains of Fusarium (F. oxysporum, F. verticillioides, and F. graminearum), respectively, and primary screening was performed using Fusarium plates, and then the inhibitory effect of the antagonistic bacteria on Fusarium was rescreened using bacterial plates.

[0046] The strain TC129_QW202409 with the strongest inhibitory effect was found.

[0047] 2. Identification of the Antagonistic Bacteria TC129_QW202409 to Fusarium

[0048] 1. Morphological identification

[0049] The strain TC129_QW202409 was cultured in LB solid medium for 36 h. Figure 2 As shown in A, it can be seen that after short-term cultivation (36h), the colony morphology is light yellow with a depression in the middle.

[0050] The strain TC129_QW202409 was cultured in LB solid medium for 96 h. Figure 2 As shown in B, it can be seen that after long-term cultivation, the surface secretes extracellular polysaccharides, becomes smooth and plump, and the depression in the middle disappears.

[0051] 2. Molecular identification

[0052] The genomic DNA of strain TC129_QW202409 was extracted, and the full-length 16S rDNA fragment was amplified using universal primers 27F and 1492R. 16S sequencing was performed to determine the type of bacteria isolated from the soil.

[0053] Bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3').

[0054] Results The nucleotide sequence of 16S of strain TC129 was sequence 1.

[0055] The sequence was compared and it was found that the strain matched 99.72% (100% Query Cover) with the Burkholderia sp. strain TC129 reported by NCBI. Therefore, the strain belonged to Burkholderia sp.

[0056] Burkholderia TC129_QW202409 was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on September 19, 2024, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.31999, and the classification name is Burkholderia sp.

[0057] Example 2: Application of Burkholderia TC129_QW202409 in inhibiting Fusarium

[0058] 1. Preparation of Burkholderia TC129_QW202409 bacterial solution and pathogenic fungi

[0059] Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides stored at -80°C were streaked on PDA plates for activation and cultured in a 28°C incubator for 5 days.

[0060] Fusarium graminearum, Fusarium oxysporum, and Fusarium verticillioides were isolated from leaves and stems of corn infected with Fusarium wilt, and the target strains were identified by ITS gene amplification and sequencing. The simplified process is as follows: the diseased leaves and stems of corn were crushed with sterile PBS buffer, and then three different concentration gradients (10 -3 , 10 -4 , 10 -5 ), diluted and spread on PDA medium, single colonies were selected for preservation and sent for sequencing for identification.

[0061] The Burkholderia strain TC129_QW202409 stored at -80°C was streaked on an LB plate for activation. After culturing in a 28°C incubator for 24 hours, a single colony was picked and inoculated into LB liquid culture medium, and cultured at 28°C and 220 rpm for 16 hours to obtain the Burkholderia TC129_QW202409 bacterial solution.

[0062] 2. Antibacterial activity detection method

[0063] Pour 30 mL of melted PDA solid culture medium into a 9 cm × 9 cm round culture dish, blow dry, and then make holes with a blue gun tip with an inner diameter of 7.5 mm at the socket, and remove the culture medium in the hole with a sterilized toothpick.

[0064] like Figure 3 As shown, a blue pipette tip with an inner diameter of 7.5 mm at the socket was used to take bacterial cakes at the new mycelium at the edge of Fusarium graminearum, Fusarium oxysporum, and Fusarium verticillioides and transferred them to the center of the PDA plate respectively, and then the OD600 of the Burkholderia TC129_QW202409 bacterial solution obtained above was adjusted to 1.0, and 20 μL of Burkholderia TC129_QW202409 bacterial solution (bacterial solution) and LB culture medium (CK) were injected into different wells around, blown dry, sealed, and placed in an incubator at 28°C for 48 hours to observe whether there was an obvious antibacterial zone.

[0065] The results are as follows Figure 3 As shown, it can be seen that Burkholderia TC129_QW202409 can inhibit the growth of Fusarium graminearum, Fusarium oxysporum and Fusarium verticillioides.

[0066] 3. Application of Burkholderia TC129_QW202409 as a biocontrol agent in the treatment of plant diseases

[0067] 1. Experimental preparation:

[0068] 10mM MgCl2, sterile toothpick, PDA plate, LB solid, LB liquid

[0069] 2. Experimental steps:

[0070] 1) Preparation of fungal culture liquid

[0071] Take the Fusarium oxysporum, Fusarium verticillioides, and Fusarium graminearum stored at -80°C and streak them on PDA plates for activation, and then culture them in a 28°C incubator for 4 days; Fusarium oxysporum and Fusarium verticillioides together with the culture medium are cut into small pieces and placed in a 50 ml centrifuge tube, and 10 ml of 10 mM MgCl2 aqueous solution is added, shaken and filtered through a cell filter to obtain the spore solution of Fusarium oxysporum (spore concentration is about 3×10 7 / mL) and Fusarium verticillaster spore solution (spore concentration of about 3×10 7 / mL).

[0072] Use a sterile toothpick to scrape off the hyphae of Fusarium graminearum (90mm 2 All the hyphae on the cake were put into a 2 ml centrifuge tube, and after the hyphae were aseptically ground and broken, 1 ml of 10 mM MgCl2 aqueous solution was added and shaken to obtain the broken hyphae liquid of Fusarium graminearum.

[0073] 2) Preparation of biocontrol bacteria solution

[0074] The Burkholderia TC129_QW202409 strain stored at -80°C was streaked on LB solid culture medium for activation. After culturing in a 28°C incubator for 24 hours, a single colony was picked and inoculated into LB liquid culture medium. The culture was carried out at 28°C and 220 rpm for 16 hours to obtain the Burkholderia TC129_QW202409 bacterial solution (bacterial concentration was OD600 = 1.0).

[0075] 3) Inoculation

[0076] The experimental treatments included control, pathogens, biocontrol bacteria, and a mixture of pathogens and biocontrol bacteria.

[0077] Control group (denoted as MgCl2 in the figure): 10 mM MgCl2 aqueous solution.

[0078] Fungi: The spore liquid of Fusarium oxysporum, the spore liquid of Fusarium verticillium and the broken mycelium liquid of Fusarium graminearum were mixed in the ratio of 2:2:1 per unit volume of the liquid.

[0079] Bacteria: Burkholderia TC129_QW202409 bacterial solution (bacterial concentration is OD600=1.0).

[0080] Bacteria + fungi: Burkholderia TC129_QW202409 bacterial solution and fungi mixed with equal volumes of bacterial solution (Fusarium oxysporum spore solution, Fusarium verticillatus spore solution and Fusarium graminearum hyphae broken bacterial solution are mixed in a ratio of 2:2:1 per bacterial solution unit volume.)

[0081] Each group of the above experimental treatments was injected into the stem of the second leaf of corn (Zhengdan 958) seedlings (three-leaf stage or four-leaf stage), 200 μl was injected, and the new leaves were observed and photographed two weeks later. Three pots of corn were repeated for each treatment, with 3 corns in each pot.

[0082] Leaf Wilting Score: 0-5 points.

[0083] 0 means no disease, no yellowing at the pinholes, and no symptoms;

[0084] 1 is a mild disease with slight yellowing symptoms at the pinholes;

[0085] 2 is a mild disease with a large area of ​​yellowing around the pinholes;

[0086] 3 is moderate disease, with severe yellowing of leaves;

[0087] 4 is a more serious disease, with severe yellowing of leaves and deformation of leaves;

[0088] 5 is a severe disease. The leaves are severely rotten and curled.

[0089] The results are as follows Figure 4 As shown, it can be seen that in the leaf experiment of plant disease test, the control leaves (MgCl2) are very healthy, and there is no yellowing or other symptoms at the pinholes ( Figure 4 B); Leaves inoculated with antagonistic bacteria (bacteria only) are healthier and have no obvious signs of disease ( Figure 4 A); Only inoculated with fungi (true only mixed three) "leaves show severe lesions, leaves rot, wither and deform ( Figure 4 C); the leaf disease of the leaves inoculated with antagonistic bacteria and pathogens (bacteria + true mixed three) was significantly reduced ( Figure 4 D), Figure 4 The scoring results shown in E are consistent with the phenotype diagram on the left ( Figure 4 A-4D) are consistent.

[0090] It shows that the Burkholderia TC129_QW202409 of the present invention can inhibit Fusarium oxysporum, Fusarium verticillioides and Fusarium graminearum, thereby treating plant diseases caused by these fungal infections.

[0091] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Burkholderia sp. TC129_QW202409, whose accession number is CGMCCNO.31999.

2. Use of the bacteria or its culture solution or its bacterial solution or its fermentation solution according to claim 1 in preparing a product having any of the following functions: 1) Inhibit Fusarium; 2) Treat plant diseases caused by Fusarium; 3) Prevent plant diseases caused by Fusarium.

3. The use according to claim 2, characterized in that: The Fusarium is Fusarium graminearum, Fusarium oxysporum and / or Fusarium verticillium.

4. The culture solution of the bacterium according to claim 1, which is a culture solution obtained by culturing the bacterium according to claim 1.

5. A product, the active ingredient of which is the bacterium according to claim 1 or its culture solution or bacterial solution or fermentation solution.

6. The use according to claim 5, characterized in that: The product has any of the following functions: 1) Inhibit Fusarium; 2) Treat plant diseases caused by Fusarium; 3) Prevent plant diseases caused by Fusarium.

7. The product according to claim 5 or 6, characterized in that: The product is a biocontrol agent.

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