Microbacterium sp. and application thereof in plant disease prevention and treatment

Abstract

This invention belongs to the field of agricultural microbiology technology, specifically relating to a microbacterium strain and its application in plant disease control. This invention discloses a microbacterium strain screened from wolfberry roots, named... Microbacterium sp. R5-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34797, has the 16S rDNA sequence shown in SEQ ID NO:1. It secretes siderophores, cellulase, and protease. Experimental results demonstrate that this strain exhibits inhibitory activity against the growth of *Fusarium oxysporum*, *Rhizoctonia solani*, and *Verticillium dahliae*, and shows significant control efficacy against soil-borne fungal diseases of plants caused by these fungi. This invention provides an important microbial resource for the green control of soil-borne fungal diseases of plants.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a strain of microbacterium and its application in the prevention and control of plant diseases. Background Technology

[0002] In recent years, with the continuous expansion of goji berry planting area, soil-borne diseases, especially goji berry root rot, have become a core bottleneck restricting the high-quality development of the goji berry industry. The pathogens of this disease mainly include Fusarium oxysporum (…). Fusarium oxysporum Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani Filamentous fungi such as *Verticillium dahliae*, and others. Verticillium dahliae Although it does not directly cause root rot in wolfberries, infection with this pathogen causes the leaves of infected wolfberry plants to yellow and wilt from the bottom up, eventually leading to overall necrosis and rotting. These pathogens can survive in the soil for a long time, infecting wolfberries and causing symptoms such as root rot, yellowing and wilting of leaves, and slow growth; in severe cases, the entire plant may die.

[0003] Currently, the control of diseases affecting goji berries still relies primarily on chemical pesticides. However, long-term excessive use has led to increased pesticide resistance in pathogens, excessive pesticide residues in goji berry products, and damage to the soil ecosystem, deviating from the direction of green agriculture. Therefore, the development of efficient and safe control technologies is particularly urgent. Biological control, due to its unique advantages such as good environmental compatibility, low resistance rate, and safety for humans and animals, is considered one of the most promising directions for controlling soil-borne diseases. However, existing biocontrol agents have highly specific biocontrol strains, making it difficult to cope with the complex infection of multiple pathogens causing fungal diseases in goji berries. Therefore, screening for a high-quality biocontrol strain that can simultaneously control multiple diseases is key to overcoming the current bottleneck in the biological control of soil-borne fungal diseases in goji berries. Summary of the Invention

[0004] The purpose of this invention is to provide a novel biocontrol strain with significant antagonistic activity against various soil-borne fungal pathogens of wolfberry, thereby solving the problems of residue and pollution caused by existing chemical control methods and overcoming the technical bottleneck of the limited specificity of existing biocontrol agents. To address the above technical problems, this invention isolated a microbacterium from the root system of healthy wolfberry plants in a typical Northwest wolfberry producing area. Microbacterium sp. R5-1. This strain exhibits significant inhibitory activity against the main pathogens of wolfberry, including Fusarium oxysporum, Verticillium dahliae, and Rhizoctonia solani, providing a key strain resource for the development of broad-spectrum antifungal agents for the control of soil-borne fungal diseases.

[0005] On the one hand, the present invention provides a microbacterium, the microbacterium being named Microbacterium sp. R5-1. Specifically, this microbacterium... Microbacterium sp. R5-1 was obtained by screening and isolation from wolfberry roots, and its preservation information is as follows:

[0006] Strain name: R5-1;

[0007] Classification name: Microbes Microbacterium sp. ;

[0008] Date received by the collection center: July 4, 2025;

[0009] Date of issuance of preservation certificate: July 15, 2025;

[0010] Preservation institution: China General Microbiological Culture Collection Center (CGMCC);

[0011] Accession number: CGMCC No.34797.

[0012] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0013] Furthermore, the 16S rDNA sequence of the microbacterium is shown in SEQ ID NO:1.

[0014] Furthermore, the microbacteria can produce siderophores, cellulases, and proteases.

[0015] Secondly, a microbial agent containing the microbacteria described in this invention is also provided, said microbial agent containing microbacteria. Microbacterium sp. R5-1 bacteria and microbes Microbacterium sp. Fermentation broth and microbes of R5-1 Microbacterium sp. One or more of the fermentation broth supernatant of R5-1.

[0016] Thirdly, the invention also provides the application of the microbacterium or fungal agent described in this invention in the prevention and control of soil-borne fungal diseases of plants, wherein the soil-borne fungal diseases are caused by Fusarium oxysporum. Fusarium oxysporum Rhizoctonia solani Rhizoctonia solani and Verticillium dahliae Verticillium dahliae One or more of the following can trigger this.

[0017] Furthermore, in the application, the plant is either goji berry or tomato.

[0018] Fourthly, a method for controlling soil-borne fungal diseases of plants is also provided, the method comprising applying the fungal agent described in this invention to the roots of plants, wherein the soil-borne fungal disease is caused by Fusarium oxysporum. Fusarium oxysporum Rhizoctonia solani Rhizoctonia solani and Verticillium dahliae Verticillium dahliae One or more of the following can trigger this.

[0019] Furthermore, in the method, the application method is root irrigation, root dipping, or soil watering.

[0020] Furthermore, in the method, the plant is either wolfberry or tomato.

[0021] Furthermore, in the method, the plant disease is wolfberry root rot.

[0022] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0023] (1) This invention isolates a new strain of microbacterium from the roots of wolfberry. Microbacterium sp. R5-1, a microbacterium, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34797. Its 16S rDNA sequence is shown in SEQ ID NO:1. This microbacterium exhibits significant antagonistic activity against the main pathogens of wolfberry root rot (Fusarium oxysporum and Rhizoctonia solani) and Verticillium dahliae, breaking through the technical bottleneck of high specificity in existing biocontrol strains. This new strain—*R5-1*—is a microbacterium. Microbacterium sp. R5-1 has a broad antibacterial spectrum and multiple effects against one bacterium.

[0024] (2) Microbes provided by the present invention Microbacterium sp. R5-1 can simultaneously produce siderophores, cellulase, and protease, and synergistically inhibit pathogens through multiple mechanisms such as nutrient competition and cell wall lysis. Indoor pot experiments have confirmed its significant efficacy in preventing diseases in wolfberry and tomato.

[0025] In summary, this invention provides a green biocontrol technology solution for the prevention and control of soil-borne fungal diseases induced by Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae in plants such as wolfberry and tomato. It can replace or reduce the use of chemical pesticides, avoid pesticide residues and environmental pollution. In addition, the microbial agent is simple to prepare and easy to apply, and has good prospects for development and application. Attached Figure Description

[0026] Figure 1 Microbes Microbacterium sp. The morphology of R5-1 inside the dish.

[0027] Figure 2 Microbes Microbacterium sp. The image shows the test results of antagonistic activity between Fusarium oxysporum and Verticillium dahliae in R5-1 dish.

[0028] Figure 3 Microbes Microbacterium sp. Image showing the results of Gram staining with R5-1.

[0029] Figure 4 Microbes Microbacterium sp. The phylogenetic tree of R5-1.

[0030] Figure 5 Microbes Microbacterium sp.The result of the test on the inhibition rate of R5-1 fermentation broth supernatant against Fusarium oxysporum.

[0031] Figure 6 Microbes Microbacterium sp. The results of the detection of R5-1's ability to produce siderophores, cellulase and protease are shown in the figure.

[0032] Figure 7 Microbes Microbacterium sp. Figure 1 shows the test results of R5-1's efficacy against different pathogens in indoor potted plants. Detailed Implementation

[0033] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0034] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0035] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0036] Example 1

[0037] This example describes the isolation and identification of strain R5-1.

[0038] 1. Isolation and purification of strain R5-1

[0039] Rootlets from healthy wolfberry plants in Dulan County, Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province were collected and sterilized with 6‰ sodium hypochlorite solution for 5 minutes in the dark. The sterilized roots were then ground under aseptic conditions to obtain a tissue homogenate. The homogenate was spread onto LB agar plates using the dilution-spread method and incubated upside down at 28℃ for 1-3 days. After clear, morphologically distinct single colonies of varying sizes appeared on the plates, they were initially screened based on colony morphology, color, and edge characteristics. Single colonies with different morphologies were selected and purified onto new LB agar plates. This process was repeated 2-3 times until the desired homogenate was obtained. Figure 1 The pure cultures shown are defined as the primary screening biocontrol strains. Subsequently, the antagonistic activity of these strains against *Fusarium oxysporum* and *Verticillium dahliae* was verified using a plate confrontation screening method, as detailed below:

[0040] A well approximately 5 mm in diameter was prepared in the center of a PDA medium using a sterile punch, and 20 μL of the fermentation broth of the initially screened biocontrol strain was inoculated into it. Mycelial discs, approximately 5 mm in diameter, were prepared at the edges of *Fusarium oxysporum* and *Verticillium dahliae* colonies using the punch, and inverted on both sides of the well. A plate containing only an equal volume of sterile LB medium served as a negative control. The plates were incubated at 25°C until the pathogen colonies on both sides of the control plate were nearly identical. Photos were taken and the results observed. The results are as follows: Figure 2 As shown, a strain was found that produced clear inhibition bands against two pathogenic fungi of plant root rot, Fusarium oxysporum and Verticillium dahliae. This strain was labeled R5-1. This strain showed significant antagonistic effects against Fusarium oxysporum and Verticillium dahliae, the pathogenic fungi of plant root rot. The R5-1 strain was stored in a -80℃ low temperature freezer.

[0041] 2. Gram staining of strain R5-1

[0042] The preserved R5-1 strain was activated on LB agar plates, and single colonies were selected for Gram staining after 2 days. The Gram staining results of strain R5-1 are shown below. Figure 3 As shown, it appears purple and is a Gram-positive bacillus.

[0043] 3. Construct a phylogenetic tree for strain R5-1.

[0044] Genomic DNA was extracted from strain R5-1 as a template, and PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rDNA gene. The PCR product was purified and sent to a sequencing company for sequencing. The sequences of primers 27F and 1492R are as follows:

[0045] 27F (SEQ ID NO:2): 5'-AGAGTTTGATCCTGGCTCAG-3';

[0046] 1492R (SEQ ID NO:3): 5'-GGTTACCTTGTTACGACTT-3'.

[0047] The 16S rDNA sequence of strain R5-1, as shown in SEQ ID NO:1, was subjected to BLAST homology comparison in the National Center for Biotechnology Information (NCBI) database. Sequences with high homology were selected, and a phylogenetic tree was constructed using MEGA 11 software via neighbor-joining. The constructed phylogenetic tree is shown below. Figure 4 As shown, strain R5-1 and microbacteria ( Microbacterium They clustered together, showing the highest sequence similarity. Combined with their morphological characteristics and Gram staining, they were identified as microbes (Microbacteria). Microbacterium ), named Microbacterium Microbacterium sp. R5-1, abbreviated as (R5-1). This microbacteriumMicrobacterium sp. R5-1 was deposited on July 4, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34797.

[0048] Example 2

[0049] This example is for testing microbes. Microbacterium sp. Inhibition rate of R5-1 fermentation broth supernatant against Fusarium oxysporum.

[0050] To verify microbes Microbacterium sp. The ability of R5-1 to inhibit pathogens through secreted metabolites was evaluated using the plate confrontation method to determine microbes. Microbacterium sp. The activity of the supernatant from R5-1 fermentation broth. Microbes... Microbacterium sp. Strain R5-1 was cultured at 28℃ and 200 rpm for 48 h. The resulting fermentation broth was filtered three times through a 0.22 μm microporous membrane to remove bacterial cells, yielding microbes. Microbacterium sp. R5-1 fermentation broth supernatant. Microbes were added at volume fractions of 2.5%, 5%, 7.5%, 10%, and 12.5%. Microbacterium sp. The supernatant of R5-1 fermentation broth was mixed with PDA medium and poured into plates, with PDA plates containing an equal volume of sterile LB medium used as a control. Fusarium oxysporum mycelium was inoculated into the center of each plate and incubated at 25°C for 5 days. Colony diameter was measured and inhibition rate calculated. Results are as follows: Figure 5 As shown, microbes Microbacterium sp. The inhibitory effect of the supernatant of R5-1 fermentation broth on Fusarium oxysporum increased with increasing concentration, indicating that this strain can secrete secondary metabolites with antibacterial activity. The formula for calculating the inhibition rate is as follows:

[0051] Inhibition rate = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%

[0052] Example 3

[0053] This example is for evaluating microbes. Microbacterium sp. R5-1's biocontrol potential.

[0054] To assess microbes Microbacterium sp. The biocontrol potential of R5-1 was assessed using three selective culture media, the formulations of which are shown in Table 1. Microbes were determined by specific colorimetric reactions or hydrolysis zone methods. Microbacterium sp. R5-1's ability to produce siderophores, cellulase, and protease. The specific procedure is as follows: Punch a well in the center of a suitable culture medium plate and inoculate with 20 μL of microbes. Microbacterium sp. R5-1 fermentation broth (OD) 600=0.8), and the results were observed after incubation at 28℃ for 72 h. Siderophore production capacity was determined by the appearance of an orange-yellow halo on a CAS plate; cellulase production capacity was determined by the appearance of a clear hydrolysis zone after Congo red staining for 15 min, destaining with 1M NaCl for 15 min, and fixing with 5% acetic acid solution for 10 min; protease production capacity was determined by the appearance of a clear protein hydrolysis zone on a skim milk plate. Each experiment was performed in triplicate, and the test results are as follows: Figure 6 As shown, microbes Microbacterium sp. The R5-1 strain has the ability to produce siderophores, cellulases, and proteases.

[0055] Table 1. Culture medium formulation used in Example 3

[0056]

[0057] Example 4

[0058] This example uses microbes. Microbacterium sp. R5-1 test on the efficacy of indoor potted plants against different pathogens.

[0059] 1. Microbes Microbacterium sp. Efficacy test of R5-1 against Fusarium oxysporum on wolfberry

[0060] First, healthy goji berry seedlings about 10cm tall were transplanted into sterilized nutrient soil and allowed to recover for one week. In the experimental group, 10mL of microbial agents were applied to the root zone of each goji berry plant. Microbacterium sp. R5-1 fermentation broth (OD) 600 =0.8), the experimental group was treated with an equal volume of sterile water, and the control group was treated with an equal volume of sterile water. Treatment was performed every 2 days for three consecutive days. Two days after the last treatment, both the experimental and control groups were irrigated with 10 mL of Fusarium oxysporum spore suspension (10... 8 (cfu / mL), applied as a drench every 2 days for two consecutive applications. Observe plant disease incidence 42 days after inoculation with *Fusarium oxysporum* spore suspension. Results are as follows: Figure 7 As shown, after treatment with Fusarium oxysporum, wolfberry plants exhibited symptoms such as leaf drying, wilting, and stunted growth, displaying typical root rot symptoms. In contrast, the treatment with the biocontrol bacterium *Microbacterium*... Microbacterium sp. Lycium barbarum plants treated with R5-1 fermentation broth showed normal growth and significantly reduced disease incidence, indicating that Microbes... Microbacterium sp. R5-1 has a significant control effect against Fusarium oxysporum on wolfberry.

[0061] 2. Microbes Microbacterium sp. Efficacy test of R5-1 against Verticillium dahliae and Rhizoctonia solani on tomatoes

[0062] Verticillium dahliae is the main pathogen of Verticillium wilt in tomatoes, while Rhizoctonia solani is one of the diseases causing root rot in wolfberries and is also a major pathogen inducing damping-off in tomatoes and other plants. Further verification of microbial... Microbacterium sp. The broad-spectrum biocontrol effect of R5-1 was tested on tomato seedlings using microbes. Microbacterium sp. R5-1 against Verticillium dahliae ( Verticillium dahliae ) and Rhizoctonia solani ( Rhizoctonia solani The control effect of the pathogen was observed. The experimental method was the same as above, but due to the short growth cycle of tomatoes, observations were conducted 28 days after pathogen inoculation. The results are as follows: Figure 7 As shown, microbes Microbacterium sp. R5-1 pretreatment effectively alleviated tomato wilting and root and stem disease symptoms caused by Verticillium dahliae and Rhizoctonia solani, indicating that microbes... Microbacterium sp. R5-1 has a certain broad spectrum of host activity, and its application is not limited to wolfberry crops.

[0063] The above embodiments fully demonstrate that the microbacteria provided by the present invention Microbacterium sp. R5-1 is an excellent biocontrol strain with multiple biocontrol mechanisms, significant efficacy against root rot of wolfberry, and broad applicability.

[0064] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A strain of microbacterium, characterized in that, The microbacterium was named Microbacterium sp. R5-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34797.

2. The microbacteria according to claim 1, characterized in that, The 16S rDNA sequence of the microbacterium is shown in SEQ ID NO:

1.

3. The microbacteria according to claim 1, characterized in that, The microbacteria can produce siderophores, cellulase, and protease.

4. A biological control agent, characterized in that, The biocontrol agent contains one or more of the following: the microbacterium cells, its fermentation broth, and its fermentation broth supernatant as described in claim 1.

5. The application of the microbacterium as described in claim 1 or the biocontrol agent as described in claim 4 in the control of soil-borne fungal diseases of plants, characterized in that, The soil-borne fungal disease is caused by Fusarium oxysporum. Fusarium oxysporum Rhizoctonia solani Rhizoctonia solani and Verticillium dahliae Verticillium dahliae One or more of the following can trigger this.

6. The application according to claim 5, characterized in that, The plant in question is either wolfberry or tomato.

7. A method for controlling soil-borne fungal diseases of plants, characterized in that, This includes applying the biological control agent of claim 4 to the plant roots, wherein the soil-borne fungal disease is caused by Fusarium oxysporum. Fusarium oxysporum Rhizoctonia solani Rhizoctonia solani and Verticillium dahliae Verticillium dahliae One or more of the following can trigger this.

8. The method according to claim 7, characterized in that, The application method is root irrigation, root dipping, or soil watering.

9. The method according to claim 7, characterized in that, The plant in question is either wolfberry or tomato.

10. The method according to claim 7, characterized in that, The soil-borne fungal disease of the plant is wolfberry root rot.

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