Leuconostoc mesenteroides a3 antagonizing xanthomonas oryzae pv. oryzae and application thereof

Abstract

This invention discloses a *Leuconostoc mesenteroides* a3 strain antagonistic to *Xanthomonas oryzae* and its applications, belonging to the field of microbial technology. This *Leuconostoc mesenteroides* a3 strain was deposited at the China Center for Type Culture Collection (CCTCC) on April 7, 2025, with the strain accession number CCTCC No: M 2025646. It exhibits high antagonistic activity against both *Xanthomonas oryzae* and *Xanthomonas oryzae*, the causal agent of rice bacterial blight. Biocontrol agents prepared using this strain show good biocontrol effects against both rice bacterial blight and rice bacterial leaf streak, reducing the use of chemical pesticides, improving food and ecological security, and bringing significant economic and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a type of Leuconostoc mesenteriae a3 that antagonizes Xanthomonas oryzae and its application. Background Technology

[0002] Xanthomonas oryzae, comprising two pathogenic species—Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola—causes rice bacterial blight (BB) and rice bacterial leaf streak (BLS), respectively, which are extremely detrimental to rice production. It affects a wide area year-round, severely impacting rice yield, typically causing 5%-10% loss, but in severe cases, exceeding 50% or even total crop failure. In recent years, influenced by factors such as the promotion of simplified rice planting models, the expansion of two-line hybrid indica rice cultivation, increasingly frequent germplasm resource exchanges, and the pathogenicity variation of pathogens, two bacterial diseases have not only continued to cause damage in the southern indica rice-growing areas but have also rapidly spread to the mixed indica-japonica rice-growing areas of the Yangtze River Basin and the northern japonica rice-growing areas, posing a severe challenge to rice safety production in all provinces of my country. Currently, chemical pesticides are the main means of controlling rice bacterial blight, such as thiamethoxam, thiamethoxam, thiamethoxam zinc, and trichloroisocyanuric acid. However, the long-term and large-scale use of chemical pesticides has brought many problems. On the one hand, it has led to the gradual increase of pathogen resistance, reduced control efficacy, and the need to blindly increase pesticide dosages; on the other hand, pesticide residues pose a serious threat to food safety and the ecological environment. Meanwhile, the development of new chemical fungicides is costly and carries high investment risks, resulting in a scarcity of new chemical fungicides registered for the control of crop bacterial diseases in recent years.

[0003] Biological control, as a green and sustainable method of pest control, boasts advantages such as safety, no residue, and environmental friendliness, making it a key focus of international green pesticide development. Microorganisms play a crucial role in biological control, with various microorganisms, including Bacillus and Pseudomonas, proven to have the potential to control plant diseases. However, the control efficacy varies significantly among different microbial strains, exhibiting strain specificity. Currently, there are few reports on the control efficacy of Leuconostoc mesenteroides against bacterial diseases of rice, making the screening and development of highly effective biocontrol strains of great significance. Summary of the Invention

[0004] This invention provides Leuconostocmesenteroides, with accession number CCTCC NO: M 2025646.

[0005] The present invention also provides a fermentation broth obtained from the fermentation of the above-mentioned Leuconostoc mesenteroides.

[0006] Preferably, the fermentation broth is obtained by fermentation using NB liquid culture medium.

[0007] More preferably, the fermentation temperature is 27-29℃.

[0008] More preferably, the fermentation temperature is 28°C.

[0009] The present invention also provides a microbial agent containing at least one of the following: Leuconostoc mesenteroides, a suspension of Leuconostoc mesenteroides, the fermentation broth, and a sterile filtrate of the fermentation broth.

[0010] The present invention also provides the application of the above-mentioned Leuconostoc mesenteroides, the above-mentioned Leuconostoc mesenteroides bacterial suspension, the above-mentioned fermentation broth and / or the above-mentioned sterile filtrate of the fermentation broth in the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

[0011] The present invention also provides the application of the above-mentioned microbial agents in the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

[0012] The present invention also provides the application of the above-mentioned Leuconostoc mesenteroides, the above-mentioned Leuconostoc mesenteroides bacterial suspension, the above-mentioned fermentation broth and / or the above-mentioned sterile filtrate of the fermentation broth in the preparation of products for the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

[0013] Preferably, the product is a liquid bacterial agent.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The *Leuconostoc mesenteriae* a3 strain provided by this invention is a newly discovered lactic acid bacteria biocontrol resource that exhibits high antagonistic activity against both rice bacterial blight and rice bacterial leaf streak. Biocontrol agents prepared using the strain of this invention show good biocontrol effects against both rice bacterial blight and rice bacterial leaf streak, which can reduce the use of chemical pesticides, ensure my country's food security, and have good economic and ecological benefits.

[0016] Biological preservation instructions for Leuconostoc mesenteroides a3:

[0017] Preservation institution: China Center for Type Culture Collection;

[0018] Accession number: CCTCC NO: M 2025646;

[0019] Deposit date: March 31, 2025;

[0020] Location of collection: Wuhan University, Wuhan, China;

[0021] Taxonomic name: Leuconostoc mesenteroides a3. Attached Figure Description

[0022] Figure 1 The antagonistic activity of a preliminarily identified biocontrol bacterium in Example 1 against rice bacterial blight pathogen (Xoo:PXO99), where Xoo is rice bacterial blight pathogen;

[0023] Figure 2 This is a statistical analysis of the inhibition zone diameter of a preliminarily identified biocontrol bacterium in Example 1 against bacterial leaf streak pathogen (Xoc:Rs105), where Xoc is the bacterial leaf streak pathogen of rice.

[0024] Figure 3 This is a statistical analysis of the inhibition zone diameters of a preliminarily identified biocontrol bacterium in Example 1 against rice bacterial blight and bacterial leaf streak strains.

[0025] Figure 4 The colony morphology and Gram staining of Leuconostoc mesenteriae a3 on MRS agar plates in Example 1;

[0026] Figure 5 Phylogenetic analysis of the 16S rDNA gene sequence of Leuconostoc mesenteroides a3 in Example 1;

[0027] Figure 6 The inhibitory effect of Leuconostoc mesenteriae a3 suspension on the expansion of bacterial blight lesions in rice in Example 4;

[0028] Figure 7 The control effect of Leuconostoc mesenteriae a3 suspension on rice bacterial blight in Example 4;

[0029] Figure 8 The inhibitory effect of Leuconostoc mesenteriae a3 suspension on the spread of bacterial leaf streak in rice in Example 5;

[0030] Figure 9 This demonstrates the control effect of Leuconostoc mesenteriae a3 suspension on bacterial leaf streak in rice in Example 5. Detailed Implementation

[0031] The NB medium components described below are: 5 g peptone, 10 g sucrose, 1 g yeast extract, 3 g beef extract, diluted with water to 1000 mL, pH 6.8~7.2, sterilized at 121 ℃ for 20 min; NA is a solid medium, with 15 g / L agar powder added to the NB medium.

[0032] The following MRS culture medium components are: 10 g peptone, 5 g beef extract, 4 g yeast extract, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g triammonium citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 1 g Tween 80. The mixture is brought to a final volume of 1000 mL with water, pH 6.2 ± 0.2, and sterilized at 121°C for 20 min. For solid culture media, 15 g / L of agar powder is added.

[0033] Example 1: Isolation and identification of Leuconostoc mesentery strain a3

[0034] (1) Isolation of microbial resources to be screened: 50 g of soil was randomly collected from the rhizosphere of pear trees, and a total of 10 soil samples were taken. The samples were mixed evenly, and 10 g of soil was placed in a 250 mL Erlenmeyer flask containing 20 fine glass beads and 100 mL of sterile deionized water. The flask was placed on a shaker at 220 r / min and 28 ℃ for 1 h. After standing for 15 min, 1 mL of the supernatant was taken and serially diluted 10 times with sterile deionized water. 100 μL of each concentration gradient was taken and spread on NA solid plates. The plates were incubated at 28 ℃ for 36 h. Single colonies were picked with an inoculation loop and streaked twice on NA solid medium for purification. The purified single colonies of different morphologies were transferred to NA solid plates and incubated upside down at 28 ℃ for 48 h. The plates were then stored at 4 ℃ for later use.

[0035] (2) Plate antagonistic screening of biocontrol strains: Each strain purified on NA solid plates was cultured in NB liquid medium at 220 r / min, 28 ℃, and 36 h to prepare seed culture. In addition, glycerol strains of rice bacterial blight pathogen PXO99 and rice bacterial leaf streak pathogen Rs105 were streaked onto NA solid plates and cultured at 28 ℃ for 48 h. Then, single colonies were picked and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB liquid medium and cultured at 220 r / min and 28 ℃ for 36 h. The culture solution (OD600) was then used to screen the antagonistic strains. nm≈1.0) was added to liquid low-temperature NA agar medium at a ratio of 1% (v / v), and quickly mixed to prepare NA solid nutrient plates. 2.5 µL of the seed culture of the test strain was spotted onto the prepared NA solid nutrient plates. Using plates without antagonistic bacteria as a control, the treated antagonistic plates were incubated at 28 ℃ for 48 h. The antagonistic activity of the test strains against *P. xerophyte* PXO99, the causal agent of rice bacterial leaf blight, and *R. xerophyte* Rs105, was assessed by the diameter of the antagonistic zone. Then, strains with an antagonistic zone diameter greater than 10 mm were selected. The results showed that among the 295 strains tested, strain a3 performed best, with an antagonistic zone diameter of 17.701 mm ~ 32.35 mm against *P. xerophyte* and 16.355 mm ~ 19.592 mm against *R. xerophyte*. Figure 1 , Figure 2 and Figure 3 As shown. Each strain was replicated in triplicate.

[0036] (3) Morphological observation of Leuconostoc mesenteroides strain a3: Bacterial cells were picked from a3 plates stored at 4 ℃ using a sterile inoculation loop and streaked onto NA solid plates. Colony characteristics after 36 h of incubation at 28 ℃: After 48 h of incubation on MRS solid medium plates at 28 ℃, the colony characteristics of Leuconostoc mesenteroides a3 were as follows: Single colonies were white or milky white, relatively flat, approximately circular in shape, and no more than 1 mm in diameter. The colony surface was smooth, uniform in texture, and the colony edges were neat, without obvious serrations or irregular protrusions. The cells were Gram-positive, spherical or oval in shape, and usually arranged in pairs or chains. Its cell wall structure contained a thick peptidoglycan layer, which caused it to stain purple during Gram staining. The colony morphology and Gram staining of a3 are as follows: Figure 4 As shown.

[0037] (4) Molecular identification of biocontrol strain a3: Using a single colony on a plate as a template, PCR amplification was performed using the universal primers 27F (SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3') for bacterial 16S rDNA. PCR reaction system (20 μL): 1 μL each of forward and reverse primers, 10 μL of 2×Rapid Tag Master Mix, 1 μL of template DNA, and 7 μL of ddH2O. Reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 1 min 30 s, 30 cycles; 72℃ extension for 10 min. The PCR amplification product was taken out, and an appropriate amount (generally 5-10 μL) was spotted into the prepared 1% gel wells for electrophoresis. Electrophoresis was performed at 125V, with the electrophoresis time varying depending on the size of the target fragment and the length of the electrophoresis tank, typically 30-60 min. The remaining PCR products were sequenced, and the sequencing results were analyzed using BLAST on the NCBI website. Based on the obtained 16S rDNA gene sequence and the GenBank database, phylogenetic analysis of strain a3 was performed using MEGA 11 software. This strain aggregated with *Leuconostoc mesenteroides*. *Leuconostoc mesenteroides*, such as... Figure 5 Show.

[0038] a3 16S rDNA gene sequence (SEQ ID NO.3):

[0039]

[0040] Example 2 Preparation of biocontrol agent for Leuconostoc mesentery strain a3

[0041] Glyceryl culture of *Leuconostoc mesenteriae* strain a3 of this invention was streaked onto NA agar plates and incubated at 28 °C for 36 h. A single colony of a3 was picked up with a sterile toothpick and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of NB liquid medium. The flask was incubated at 28 °C for approximately 16 h to obtain the seed culture. The seed culture was then inoculated at a ratio of 1% (v / v) into a 1000 mL Erlenmeyer flask containing 200 mL of NB medium and incubated at 28 °C for 60 h. Conventional adjuvants were added to the fermentation broth to adjust the pH to approximately 4–5, thus obtaining the a3 biocontrol agent. The mixture was then aseptically filled and stored at room temperature.

[0042] Example 3 Preparation of Leuconostoc mesentery strain a3 suspension

[0043] The a3 seed culture was prepared according to the method in Example 2 above. The a3 seed culture was inoculated at a ratio of 1% (v / v) into a 250 mL Erlenmeyer flask containing 50 mL of 50% MRS liquid medium, and cultured at 28 ℃ for 48 h. The prepared a3 fermentation broth was centrifuged at 5000 rpm / min for 10 min, the supernatant was removed, the bacterial cells were collected, and the cells were resuspended in sterile water. This washing process was repeated three times, and the cell density was adjusted to 1.0 × 10⁻⁶. 8 Add Tween 80 to a final concentration of 0.1% (v / v) at approximately cfu / mL to prepare a biocontrol bacterial suspension.

[0044] Example 4: Control test of Leuconostoc mesentery strain a3 suspension against rice bacterial leaf blight

[0045] The rice bacterial blight pathogen strain PXO99, preserved in glycerol, was taken from a -80 ℃ ultra-low temperature freezer and streaked onto NA solid plates. It was then incubated at 28 ℃ for 48 h. Single colonies of PXO99 were picked using sterile toothpicks and inoculated into 250 mL Erlenmeyer flasks containing 50 mL of NB liquid medium. The flasks were incubated at 220 r / min and 28 ℃ for 48 h. The bacterial concentration was then adjusted to 1.0 × 10⁻⁶ cells / mL using sterile water. 8 Add approximately cfu / mL of Tween 80 to a final concentration of 0.1% (v / v) as the pathogen seed culture.

[0046] The rice variety Nipponbare was selected, and three biocontrol treatments were set up: Treatment 1, first sprayed with 1.0×10 8A CFU / mL suspension of *Leuconostoc mesenteriae* a3 biocontrol agent was applied, slightly moistening the leaf surface. After 24 hours, leaves were cut approximately 3 cm from the leaf tip and inoculated with *PXO99* seed solution, designated as a3+PXO99. For treatment 2, leaves were first cut approximately 3 cm from the leaf tip and inoculated with *PXO99* seed solution. After 24 hours, a 1.0 × 10⁻⁶ CFU / mL solution was sprayed. 8 A CFU / mL suspension of the a3 biocontrol bacterium was used, just enough to slightly moisten the leaf surface, and labeled as PXO99+a3. Treatment 3 involved cutting leaves approximately 3 cm from the leaf tip and inoculating them with PXO99 seed solution, labeled as PXO99, as a control. Ten rice plants were inoculated for each treatment, with the top two leaves inoculated from each plant. Lesion length was assessed 14 days post-inoculation. Results showed that compared to the control treatment inoculated only with PXO99, the lesion length in both biocontrol treatments 1 and 2 was significantly reduced (P<0.05), and the difference between the two treatments was also significant (P<0.05). Figure 6 and Figure 7 As shown.

[0047] Example 5: Control test of Leuconostoc mesentery strain a3 suspension against bacterial leaf streak in rice.

[0048] The rice bacterial blight pathogen strain Rs105, preserved in glycerol from a -80 ℃ ultra-low temperature freezer, was streaked onto NA agar plates and incubated at 28 ℃ for 48 h. A single Rs105 colony was then picked with a sterile toothpick and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of NB liquid medium. The flask was incubated at 220 r / min and 28 ℃ for 48 h. The bacterial concentration was then adjusted to 1.0 × 10⁻⁶ cells / mL using sterile water. 8 Add approximately cfu / mL of Tween 80 to a final concentration of 0.1% (v / v) as the pathogen seed culture.

[0049] Using the a3 biocontrol bacteria suspension prepared in Example 3 above, three biocontrol experimental treatments were set up using the same rice variety, Nipponbare: Treatment 1, first sprayed with 1.0 × 10 8 Treatment 1: Inoculate the leaves with a CFU / mL *Leuconostoc mesenteriae* a3 biocontrol suspension, just enough to slightly moisten the leaves. After 24 hours, cut a leaf approximately 3 cm from the leaf tip and inoculate with rice bacterial leaf streak seed solution, labeling it a3+Rs105. Treatment 2: First, cut a leaf approximately 3 cm from the leaf tip and inoculate with rice bacterial leaf streak Rs105 seed solution. After 24 hours, spray with 1.0 × 10⁻⁶... 8A CFU / mL suspension of the a3 biocontrol bacterium was used, just enough to slightly moisten the leaf surface, and labeled as Rs105+a3. Treatment 3 involved cutting leaves approximately 3 cm from the leaf tip and inoculating with seed solution of the rice bacterial leaf streak pathogen Rs105, labeled as Rs105, as a control. Ten rice plants were inoculated for each treatment, with the top two leaves of each plant inoculated. Lesion length was assessed 14 days post-inoculation. Results showed that compared to the control treatment inoculated only with pathogen Rs105, the leaf lesion length in both biocontrol treatments 1 and 2 was significantly reduced (P<0.05), but there was no significant difference between the two biocontrol treatments (P>0.05). Figure 8 and Figure 9 As shown.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Leuconostoc mesenteroides (ATCC No: M 2025646 Leuconostoc mesenteroides ).

2. The fermentation broth obtained from the fermentation of Leuconostoc mesenteroides as described in claim 1.

3. The fermentation broth of claim 2, wherein, The fermentation broth was obtained by fermentation using NB liquid culture medium.

4. The fermentation broth of claim 3, wherein, The fermentation temperature is 27-29℃.

5. The fermentation broth according to claim 4, characterized in that, The fermentation temperature is 28°C.

6. A microbial inoculant, characterized in that, The microbial agent contains at least one of the following: Leuconostoc mesenteroides as described in claim 1, a suspension of Leuconostoc mesenteroides as described in claim 1, and a fermentation broth as described in claim 2.

7. The use of Leuconostoc mesenteroides as described in claim 1, the bacterial suspension of Leuconostoc mesenteroides as described in claim 1, and / or the fermentation broth as described in claim 2 in the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

8. The application of the microbial agent according to claim 6 in the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

9. The use of Leuconostoc mesenteroides as described in claim 1, the bacterial suspension of Leuconostoc mesenteroides as described in claim 1, and / or the fermentation broth as described in claim 2 in the preparation of products for the prevention and control of rice bacterial leaf blight and / or rice bacterial leaf streak.

10. Use according to claim 9, characterized in that, The product is a liquid bacterial agent.

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