A myxobacterium resistant to plant pathogenic fungi and bacteria

Abstract

This invention discloses a myxobacterium resistant to plant pathogenic fungi and bacteria. The strain is named *Myxococcus sp.* R23, with accession number GDMCC No: 63212. The methanol extract of the fermentation supernatant of *Myxococcus sp.* R23 inhibits the growth of *Rhizoctonia solani* Kühn GDMCC 3.513, which causes rice sheath blight. Furthermore, this strain can prey on *Ralstonia solanacearum* GIM 1.70, which causes bacterial wilt in tomatoes, demonstrating its application value in the biological control of plant diseases.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a biocontrol myxococcus strain Myxococcus sp.R23 that inhibits the growth of plant pathogenic fungi and preys on plant pathogenic bacteria. Background Technology

[0002] Plant diseases are a major limiting factor restricting the high-quality and high-yield production of crops. It is estimated that disease losses in major crops worldwide account for approximately 20-40% of total yield, resulting in direct economic losses of hundreds of billions of US dollars annually. Currently, large-scale yield reductions caused by plant pathogenic fungi or bacteria infecting various major crops have brought increasing attention to food security, making it a critical issue for human survival and development. Various control methods exist in practice, with chemical control being the most widespread. However, while chemical control is fast-acting, the secondary pollution it causes can damage the environment unpredictably. Therefore, based on the concept of green development, the development and utilization of microorganisms to inhibit the growth of plant pathogens, thereby establishing environmentally friendly biological control technologies for plant diseases, has gradually become an important goal in plant disease control. However, the difficulty in colonizing biocontrol strains and the unstable effectiveness are significant factors limiting the large-scale application of biological control methods. Excellent biocontrol agents, in addition to possessing good biocontrol effects, should also be able to colonize in various soil environments and adapt to different environments to exert their effects. Myxobacteria exhibit complex multicellular social behavior and were the first reported bacteria with predatory capabilities. Their multicellular, group-based predation behavior is known as "wolf pack" predation. During predation, the colony of myxobacterial cells produces large amounts of bactericidal substances and lysins, creating high concentrations of "weapons" within a localized area that efficiently kill and decompose prey cells. Myxobacteria can also form highly resistant fruiting bodies and myxospores, giving them excellent environmental adaptability. Therefore, their predatory characteristics and good environmental adaptability make them highly promising for plant disease control, and they are considered novel biocontrol microorganisms. Thus, actively developing new myxobacterial resources and utilizing their characteristics for plant disease control is of great significance for my country's development of eco-friendly control strategies. Summary of the Invention

[0003] The purpose of this invention is to provide a potential new species of Myxococcus sp. R23, which possesses predatory and antagonistic properties against plant pathogenic fungi and bacteria. The Myxococcus sp. R23 provided by this invention was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China (Guangdong Academy of Sciences Institute of Microbiology), Guangdong Province, China, accession number: GDMCC No: 63212.

[0004] A second objective of the present invention is to provide a biocontrol agent comprising the aforementioned Myxococcus sp. R23.

[0005] The biocontrol agent uses Myxococcus sp. R23 culture or its fermentation broth as the active ingredient.

[0006] Preferably, the biocontrol agent further comprises excipients that can prolong the activity time of the strain, or other excipients acceptable to biocontrol agents.

[0007] A third objective of this invention is to provide the application of the aforementioned Myxococcus sp. R23 or biocontrol agent in the biological control of plant diseases.

[0008] Preferably, the pathogens causing the plant diseases include Rhizoctonia solani Kühn and Ralstonia solanacearum.

[0009] A fourth objective of this invention is to provide the application of the methanol extract of Myxococcus sp. R23 or its fermentation broth in the biological control of rice sheath blight.

[0010] The fifth objective of this invention is to provide the application of the aforementioned Myxococcus sp. R23 or its culture in the biological control of bacterial wilt of tomato.

[0011] A sixth object of the present invention is to provide a method for controlling plant diseases, comprising the step of contacting the aforementioned Myxococcus sp. R23 or a biocontrol agent with the plant. Preferably, the plant diseases include rice sheath blight and tomato bacterial wilt.

[0012] The present invention has the following beneficial effects:

[0013] The Myxococcus sp. R23 provided by this invention is a new species of the Myxococcus genus. The methanol extract of the fermentation broth of this strain can inhibit the growth of Rhizoctonia solani Kühn, and this strain can prey on Ralstonia solanacearum of the Solanaceae family. Therefore, it can be applied to the biological control of plant diseases such as rice sheath blight and tomato bacterial wilt.

[0014] Myxococcus sp. R23 was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. Accession number: GDMCC No. 63212. Attached Figure Description

[0015] Figure 1 The morphological characteristics of Myxococcus sp. R23 are as follows: a) Colony morphology of strain R23 on VY / 2 solid medium; b) Fruiting body morphology of strain R23 under a stereomicroscope; c) Vegetative cell morphology of strain R23 under a phase contrast microscope.

[0016] Figure 2 It is a phylogenetic tree constructed based on the tandem of core genes from Myxococcus sp. R23 and its closely related species.

[0017] Figure 3 The methanol extract of fermentation supernatant of Myxococcus sp. R23 at different concentrations inhibited the growth of Rhizoctonia solani Kühn, the pathogen of rice sheath blight.

[0018] Figure 4 These are photographs of Myxococcus sp. R23 preying on Ralstonia solanacearum, the solanaceous bacterium that causes bacterial wilt in tomatoes, during the early (left) and late (right) stages. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Example 1: Isolation, purification and identification of Myxococcus R23

[0021] Approximately 200g of soil from the 0-20cm soil layer of diseased tomato rhizosphere was collected from Zhanjiang City, Guangdong Province. The soil was sieved through a 2mm sieve and mixed thoroughly. About 100g of the soil sample was placed in a sterile self-sealing bag and stored at 4℃. The remaining 100g of soil sample was placed in a cool, dry place and allowed to air dry naturally.

[0022] Weigh 5 g of soil from freshly collected rhizosphere soil stored at 4 °C into a 150 mL Erlenmeyer flask containing 45 mL of sterilized normal saline. Place the Erlenmeyer flask on a shaker and shake it at 180 rpm for 2 h, then take it out and let it stand for 10 min. Take the supernatant and inoculate it into 500 mL of R2A liquid medium (tryptone 0.25 g / L, acid hydrolyzed casein 0.5 g / L, yeast extract powder 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate 0.1 g / L, sodium pyruvate 0.3 g / L, peptone 0.25 g / L, glucose 0.5 g / L, solvent: water; preparation: dissolve each component in water, stir and mix evenly, sterilize to obtain) at an inoculation amount of 1% by volume, and place it on a shaker at 28 °C and 180 rpm for shaking culture for 24 h. Centrifuge to collect the bacterial cells, and resuspend them with TPM buffer (potassium dihydrogen phosphate 1 mM, Tris-HCl 10 mM, magnesium sulfate 8 mM, pH 7.6, solvent: water) to an OD 600 = 10; Take 200 μL of the bacterial suspension and draw a "field" character on the surface of WCX medium (calcium chloride dihydrate 1 g / L, agar 15 g / L, pH 7.2, solvent: water; preparation: dissolve each component in water, adjust the pH, stir and mix evenly, sterilize to obtain), and place the plate in a laminar flow hood to dry.

[0023] Take 10 g of air-dried soil sample and place it in a 50 mL centrifuge tube, and add 15 mL of 100 μg / mL cycloheximide to soak overnight. After centrifugation, pour out the liquid. Use the WCX medium inoculated with rhizosphere bacterial cells to isolate bacteriolytic myxobacteria. Inoculate the soil treated with cycloheximide into the blank spaces of the "field" character on the WCX medium, and place it at 30 °C for cultivation. After 7 days of cultivation, start to observe under a stereomicroscope and pick the fruiting bodies to the purification medium VY / 2 (yeast 5 g / L, calcium chloride dihydrate 1 g / L, VB 12 0.5 mg / L, agar 15 g / L, pH 7.2, solvent: water; preparation: dissolve each component in water, adjust the pH, stir and mix evenly, sterilize to obtain). Purify the myxobacteria by repeatedly transferring the bacterial cells at the edge of the colony, and inoculate the visually observed purified myxobacteria into nutrient broth medium (peptone 10 g / L, beef extract powder 3 g / L, and sodium chloride 5 g / L, pH 7.2, solvent: water; preparation: dissolve each component in water, adjust the pH, stir and mix evenly, sterilize to obtain) for overnight shaking culture for purity verification. If no bacterial cells grow, it indicates that the myxobacteria strain has been successfully purified. Collect the purified myxobacteria cells for identification. Strain R23 forms yellow spreading colonies on VY / 2 medium and forms an obvious hydrolysis zone along the periphery of the colony. In the later stage of growth on VY / 2 medium, it can form round, stalked, yellow-brown fruiting bodies. Under a phase contrast microscope, long rod-shaped vegetative cells ( Figure 1 ) are observed, and the characteristics observed above are similar to those of other known species of Myxococcus.

[0024] Strain R23 was identified and classified using genomic DNA-based indicators. First, the 16S rRNA gene sequence of the strain (SEQ ID NO.1) was amplified and sequenced for preliminary identification. Then, classification and identification were performed based on the genomic sequence. Fresh bacterial cells were scraped from VY / 2 medium, and genomic DNA was extracted using the CTAB method. The DNA was then sent to Meiji Biotechnology Co., Ltd. for Illumina sequencing. After quality control, the data was assembled using SPAdes software. Different Kmer values ​​were selected for assembly, and the quality of the assembled contigs was evaluated using checkM and QUAST software to obtain the assembled genome draft sequence. The average nucleotide similarity (ANI) and hybridization value (dDDH) between the genomes of strain R23 and its closely related species were calculated using FastANI software and the web-based tool GGDC. Table 1 shows that the dDDH values ​​between the R23 genome and closely related species of the genus *Myxococcus* ranged from 23.1% to 35.7% (the species classification threshold of 70%), and the ANI values ​​ranged from 80.4% to 88.7% (the species classification threshold of 95% to 96%). Genome tandem phylogenetic analysis showed that strain R23 belongs to the genus *Myxococcus*, and is related to *Myxococcus dinghuensis* K15C18031901. T Closest kinship ( Figure 2 In summary, based on morphological and phylogenetic analysis, strain R23 was identified as a new species of the genus Myxococcus and named Myxococcus sp. R23. It was deposited on October 10, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070, China, Accession No.: GDMCC No.: 63212.

[0025] Table 1. Mean nucleotide similarity (ANI) and hybridization value (dDDH) between strain R23 and its closely related species.

[0026]

[0027] Myxococcus sp. R23 16S rRNA gene sequence (shown in SEQ ID NO.1):

[0028] CCTTAGTAGAGCGGCGCACGGGTGCGTAACACGTGGATAATCTGCCTGGATGCCTGG

[0029] ATAACCAGTCGAAAGATTGGCTAATACCGGATAAGCCCACGGTTTCTTCGGAGACTGA

[0030] GGGAAAAGGTGGCCTCTGTATACAAGCTATCACAACCAGATGAGTCCGCGGCCCATCA

[0031] GCTAGTTGGCGGGGTAATGGCCCACCAAGGCAACGACGGGTAGCTGGTCTGAGAGGA

[0032] CGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG

[0033] GGAATTTTGCGCAATGGGCGAAAGCCTGACGCAGCAACGCCGCGTGTGTGATGAAGG

[0034] TCTTCGGATTGTAAAGCACTTTCGACCGGGACGAAAACCCGTAGCCTAACACGCTACG

[0035] GCTTGACGGTACCGGGAGAAGAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAA

[0036] TACAGAGGGTGCAAGCGTTGTTCGGAATTATTGGGCGTAAAGCGCGTGTAGGCGGCGT

[0037] GACAAGTCGGGTGTGAAAGCCCTCAGCTCAACTGAGGAAGTGCGCCCGAAACTGTCG

[0038] TGCTTGAGTGCCGGAGAGGGTGGCGGAATTCCCCAAGTAGAGGTGAAATTCGTAGATA

[0039] TGGGGAGGAACACCGGTGGCGAAGGCGGCCACCTGGACGGTAACTGACGCTGAGAC

[0040] GCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGA

[0041] TGAGAACTAGGTGTCGTGGGAGTTGACCCCCGCGGTGCCGAAGCTAACGCATTAAGTT

[0042] CTCCGCCTGGGAAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCG

[0043] CACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGCAGAACCTTACCTGGTCT

[0044] TGACATCCTCGGAATGCCTCAGAGATGAGGCGGTGCCCGCAAGGGAACCGAGAGACA

[0045] GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG

[0046] AGCGCAACCCTCGCCTTTAGTTGTCGCGCAAGCGAATCTCTAGAGGGACTGCCGGTGT

[0047] TAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGACCAGGGCTA

[0048] CACACGTGCTACAATGGCCGGTACAGAGCGTTGCCAACCCGCGAGGGGGAGCTAATC

[0049] GCATAAAACCGGTCTCAGTTCAGATTGGAGTCTGCAACTCGACTCCATGAAGGCGGAA

[0050] TCGCTAGTAATCGCAGATCAGCACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCGATTGCTCCAGAAGTCACCTCACC.

[0051] Example 2: Inhibition of the growth of *Rhizoctonia solani* R23 fermentation broth methanol extract on rice sheath blight pathogen *Rhizoctonia solani*.

[0052] Strain R23 was inoculated into VY / 2 liquid medium, and sterilized macroporous adsorption resin XAD-16 was added at a volume ratio of 2%. The mixture was incubated at 180 rpm and 30°C for 7 days with shaking. The macroporous adsorption resin containing the fermentation supernatant was collected by filtration through gauze. The resin remaining on the gauze was washed with an equal volume of methanol, and then placed on a shaker for 1 hour to extract secondary metabolites of strain R23. After a short period of settling, the methanol was poured off, and fresh methanol was added to the macroporous adsorption resin for repeated extraction until the extract showed no color change. The methanol collected from several extractions was combined and evaporated to dryness using a rotary evaporator to obtain a crude extract (methanol extract of the fermentation supernatant). The crude extract was weighed and dissolved in 45% DMSO + 55% H2O to different concentrations, then filtered to remove bacteria before use.

[0053] Fresh *Rhizoctonia solani* Kühn GDMCC 3.513 stipes grown on PDA solid medium (potato 200 g / L, glucose 20 g / L, peptone 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, agar 20 g / L, solvent: water; preparation: dissolve all components in water, stir well, and sterilize) were inoculated into the center of a new PDA medium, and four holes were punched 2 cm from the center in four vertical directions. Methanol extracts of the fermentation supernatant of strain R23 were added to the wells at concentrations of 10 mg / mL, 25 mg / mL, 50 mg / mL, and 100 mg / mL, respectively. 100 mg / mL of G418 was used as a positive control, and 45% DMSO + 55% H2O was used as a negative control. The plates were incubated at 28°C, and the growth of the pathogenic fungus was observed. The results showed that crude extract concentrations greater than 25 mg / mL inhibited the growth of Rhizoctonia solani GDMCC 3.513. Figure 3 ).

[0054] Example 3: Predation of *Myxococcus* R23 on *Raylorhizium anisopliae*, the pathogen of bacterial wilt of tomato (Solanaceae family).

[0055] Inoculate 1.70 to 5 mL of nutrient broth with *Ralstonia solanacearum* GIM (Solanaceae family) and culture at 28°C with shaking at 180 rpm for 24 h to obtain a seed culture. Then, inoculate the seed culture into fresh nutrient broth at a volume fraction of 1% and culture at 28°C with shaking at 180 rpm for 24 h. After centrifugation, collect the bacterial cells, wash three times with TPM buffer, and resuspend the cells to a cell concentration of 1.0 × 10⁻⁶. 11Cells / mL, take 100 μL of bacterial suspension and inoculate it into the center of TPM solid medium (potassium dihydrogen phosphate 1 mM, Tris-HCl 10 mM, magnesium sulfate 8 mM, agar 15 g / L, pH 7.6, solvent: water; preparation: dissolve all components in water, adjust pH, stir well, and sterilize). Place it in a clean bench to dry for later use. Inoculate strain R23 into the center of GIM 1.70 plaques, incubate at 30℃, and observe the formation of predator-trapping zones. Figure 4 As shown, a clear predatory transparent zone was observed 24 hours after inoculation with strain R23, and the GIM 1.70 plaque was completely lysed about 4 days later, with fruiting bodies forming in the late predatory stage.

[0056] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Myxococci ( Myxococcus sp.)R23, with accession number: GDMCC No:63212.

2. A biocontrol agent, characterized in that, Contains the myxococcus as described in claim 1 ( Myxococcus sp.)R23.

3. The biocontrol agent according to claim 2, characterized in that, The biocontrol agent is based on Myxococcus ( Myxococcus sp.) R23 culture or its fermentation broth as the active ingredient.

4. The biocontrol agent according to claim 2 or 3, characterized in that, The biocontrol agent also includes excipients acceptable to the agent.

5. The Myxococcus as described in claim 1 ( Myxococcus The application of the biocontrol agent according to claim 2 or sp.) R23 in the biological control of plant diseases; the pathogen of the plant disease is Rhizoctonia solani (sp.) Rhizoctonia solani Kühn), Solanaceae Raulella ( Ralstonia solanacearum ).

6. The Myxococcus as described in claim 1 ( Myxococcus Application of the methanol extract of sp. R23 or its fermentation broth in the biological control of rice sheath blight.

7. The Myxococcus as described in claim 1 ( Myxococcus Application of sp.)R23 or its culture in the biological control of bacterial wilt of tomato.

8. A method for preventing and controlling plant diseases, characterized in that, Including the myxococcus as described in claim 1 ( Myxococcus The step of contacting the biocontrol agent with the plant as described in sp.) R23 or claim 2; the plant disease is rice sheath blight and tomato bacterial wilt.

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