Application of pantoea agglomerans RMM02 and its products in prevention and treatment of rice false smut

Abstract

The application discloses application of Pantoea agglomerans RMM02 and a product thereof in prevention and treatment of rice smut. Pantoea agglomerans The classification name of the Pantoea agglomerans RMM02 is , the preservation number is GDMCC No. 65516, the preservation unit is Guangdong Microbial Culture Collection Center, and the preservation time is November 29, 2024. The Pantoea agglomerans RMM02 is separated from a soil of a rice smut occurrence plot, and is screened through indoor confrontation test and has significant inhibitory activity on Ustilaginoidea virens. Through indoor bacteriostatic test, it is shown that the active bacteria have significant inhibitory activity on Ustilaginoidea virens. Through indoor and field tests, it is shown that the bacteria have significant inhibitory effect on growth of Ustilaginoidea virens, and can control development of a disease during a field smut occurrence period. A biocontrol fungicide prepared after fermentation of the bacteria can effectively prevent and treat smut hazards, and guarantees high yield and high quality of rice.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Pantotheca acuminata RMM02 and its products in the prevention and control of rice false smut. Background Technology

[0002] Rice false smut is a rice disease caused by the fungus Ustilaginoidea virens, which is widely distributed in major rice-producing areas around the world.

[0003] Rice false smut fungus primarily invades rice during the flowering stage, manifests symptoms during the grain-filling stage, and becomes a major disease outbreak at maturity. Currently, rice false smut control still mainly relies on chemical methods. However, due to the short control window (5-7 days before heading) and susceptibility to weather conditions such as rain, widespread and severe outbreaks of rice false smut are still unavoidable in actual production. To improve the effective control of rice false smut, farmers use chemical pesticides repeatedly, which increases production costs and also raises the risk of pesticide residue contamination in rice and the environment.

[0004] Active microbial biocontrol agents are an ideal alternative to chemical pesticides due to their advantages such as environmental safety, non-polluting nature, and lack of residue.

[0005] Given the widespread and recurring occurrence of Aspergillus oryzae, developing more, safer, and more effective biocontrol agents is of great application significance. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Pantotheca acuminata RMM02 and its products in the prevention and control of rice false smut.

[0007] To address the problems existing in the prior art, the technical solution adopted in this invention is:

[0008] In the first aspect, the present invention provides a strain of Pantoea agglomerans RMM02, which is classified as Pantoea agglomerans, with accession number GDMCC No. 65516; depositary institution: Guangdong Provincial Center for Microbial Culture Collection; deposit date: November 29, 2024.

[0009] In a second aspect, the present invention provides a microbial agent comprising the fermentation broth of the Pantotheca RMM02 described in the first aspect above.

[0010] Thirdly, the present invention provides the application of the above-described Pantotheca agglomerata RMM02 or the above-described microbial agent in the preparation of products for the prevention and control of rice false smut.

[0011] Furthermore, the product is a biocontrol agent.

[0012] Fourthly, the present invention provides a biocontrol agent comprising the clustering pantothecin RMM02 described in the first aspect and / or the microbial agent described in the second aspect, wherein the number of viable bacteria in the biocontrol agent is not less than 1 × 10⁻⁶. 5 cfu / mL.

[0013] Preferably, the number of viable bacteria in the biocontrol agent is 1×10⁻⁶. 5 cfu / mL -1×10 10 cfu / mL.

[0014] More preferably, the number of viable bacteria in the biocontrol agent is 1×10⁻⁶. 9 cfu / mL -1×10 10 cfu / mL, such as 1×10 9 cfu / mL, 2×10 9 cfu / mL, 3×10 9 cfu / mL, 4×10 9 cfu / mL, 5×10 9 cfu / mL

[0015] 6×10 9 cfu / mL, 7×10 9 cfu / mL, 8×10 9 cfu / mL, 9×10 9 cfu / mL, 1×10 10 cfu / mL, etc., and other point values ​​within this range can be selected.

[0016] Furthermore, the biocontrol agent also includes a protectant and sterile water, wherein the protectant is one or more of carboxymethyl cellulose, methyl cellulose, starch, cyclodextrin, chitosan, and lignin; and the concentration of the protectant in the biocontrol agent is 1-10% (m / m).

[0017] Fifthly, the present invention also provides a method for preparing the biocontrol agent described in the fourth aspect above, comprising the following steps:

[0018] ① Inoculate the clump-forming pantothecin RMM02 into LB liquid medium and place it in a constant temperature shaker at 26-33℃ for 24-60 hours to obtain the fermentation broth;

[0019] ② Add a protective agent solution and adjust the concentration of the cultured fermentation broth to a viable cell count of 1×10⁻⁶. 5 cfu / mL -1×10 10 cfu / mL.

[0020] Furthermore, the fermentation broth prepared in step ① was measured to have an OD value ≥ 0.8 at 600 nm.

[0021] In a sixth aspect, the present invention provides a method for preventing and controlling rice false smut, comprising the following steps:

[0022] The biocontrol agent described in the fourth aspect above, or the biocontrol agent prepared according to the method described in the fifth aspect above, is sprayed 15 days before the rice heads break and 5 days after the heading stage. The spraying concentration of the biocontrol agent is 1×10⁻⁶. 5 cfu / mL -1×10 10 cfu / mL. If it rains within 4 hours after application, apply again at the same concentration after the rain stops.

[0023] Furthermore, the period from 15 days before rice heading to 5 days after heading refers to 10-15 days before heading, 0-5 days before heading, or 0-5 days after heading.

[0024] Furthermore, the biocontrol agent according to any one of claims 5-6 or the biocontrol agent prepared according to the method of claim 7 is sprayed 10-15 days before the rice heading stage, at a concentration of 1×10⁻⁶. 5 cfu / mL -1×10 7 cfu / mL.

[0025] When spraying the biocontrol agent according to any one of claims 5-6 or the biocontrol agent prepared according to the method of claim 7 0-5 days before the rice heads break or 0-5 days after heading, a supplementary spray is required 3 days after the first spray, with a spray concentration of 1×10 8 cfu / mL -1×10 10 cfu / mL.

[0026] Preservation instructions:

[0027] Strain name: Pantoea agglomerans RMM02;

[0028] Accession number: GDMCC No. 65516;

[0029] Taxonomic name: Pantoea agglomerans;

[0030] Deposit date: November 19, 2024;

[0031] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0032] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] The advantages and beneficial effects of this invention are:

[0034] The RMM02 microbial agent of this invention, produced through indoor antibacterial tests, demonstrates significant inhibitory activity against rice false smut. Both indoor and field trials show that this agent significantly inhibits the growth of rice false smut pathogens and can control disease development during the rice false smut outbreak. The biocontrol agent prepared from the fermentation of this agent can effectively prevent and control rice false smut damage, ensuring high and high yields of rice. Attached Figure Description

[0035] Figure 1 To illustrate the antagonistic effect of Pantotheca acuminata RMM02 on Aspergillus oryzae of the present invention, wherein: A represents the growth status of Aspergillus oryzae control on PDA medium; B represents the growth status of Aspergillus oryzae on PDA medium containing Pantotheca acuminata RMM02.

[0036] Figure 2 The colony morphology of the pantothecin RMM02 of this invention is shown.

[0037] Figure 3 To illustrate the field control effect of the biocontrol agent with RMM02 strain of this invention as the main component on rice false smut, Figure A shows rice panicles before control and Figure B shows rice panicles after control. Detailed Implementation

[0038] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, distilled water to make up to 1000ml, pH adjusted to 7.0-7.2, sterilized at 121℃.

[0041] LB solid plate medium: Based on LB liquid medium, add agar powder at a concentration of 15 g / L.

[0042] PDA solid plate culture medium: 200g potato, 20g glucose, distilled water to make up to 1000ml, add agar powder at a concentration of 15g / L, sterilize at 121℃.

[0043] Example 1: Isolation, purification and identification of Pantotheca acuminata RMM02

[0044] 1. Bacterial isolation and initial screening

[0045] Soil samples were collected from rice (Yongyou 31) fields in Yuecheng District, Shaoxing City, Zhejiang Province, where rice false smut was present. The samples were sealed in sterile self-sealing bags and brought back to the laboratory, where they were stored at 4°C for the purpose of isolating single colonies.

[0046] The target pathogen used in the experiment was *Ustilaginoidea virens*, which was isolated by our team from rice plants in a diseased area of ​​rice variety Yongyou 31 in Yuecheng District, Shaoxing City, Zhejiang Province. The bacterial culture medium was LB liquid medium, and the fungal culture medium was PDA solid agar plate medium.

[0047] Bacteria were isolated using the soil dilution method and continuously streaked until purified. The antagonistic effect of the bacteria on *Aspergillus oryzae* was determined using growth rate as an indicator. *Aspergillus oryzae* mycelial cakes were prepared using a 5mm diameter punch and inoculated onto PDA plates (control group) and PDA plates containing pre-mixed antagonistic bacteria (antagonistic group), respectively, and incubated in the dark at 28℃ for 8 days. The growth diameter of *Aspergillus oryzae* in the antagonistic group was measured, and strains showing significant antagonistic effects compared to the control group were preserved. The experiment showed that in the antagonistic group induced by *RMM02*, *Aspergillus oryzae* growth was inhibited, and the effect was more significant compared to other antagonistic bacteria. The results of the plate antagonistic effect of strain RMM02 of this invention on *Aspergillus oryzae* are as follows: Figure 1 As shown, Figure 1 Figure A shows the growth status of Aspergillus oryzae in the control group on PDA medium; Figure 1 Figure B shows the growth status of Aspergillus oryzae on PDA medium containing RMM02.

[0048] 2. Strain identification

[0049] 2.1 Morphological identification:

[0050] like Figure 2 As shown, the colony morphology of the antagonistic bacterium RMM02 is as follows: the colonies are round, yellow, with neat, low-convex and smooth edges.

[0051] 2.2 The antagonistic bacteria RMM02 screened out were identified using molecular biological methods.

[0052] PCR amplification was performed using universal primers (27F and 1492R) for bacterial identification of 16S rDNA.

[0053] PCR reaction system (50 μL system): 2 μL each primer, 25 μL 2×Mix, and ddH2O added to bring the volume to 50 μL. The 16S rDNA primer sequences were 27F (5'-AGAGTTT GATCCTGGCTCAG-3') (SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO.2). Reaction program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 55 s, 50℃ annealing for 50 s, 72℃ extension for 1 min, 72℃ extension for 10 min, 35 cycles, and then stored at 16℃. After detection by 1.5% agarose gel electrophoresis, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. The 16S rDNA gene sequence of the antagonistic bacterium RMM02 is shown in SEQ ID NO.3. The sequence was BLAST-aligned using the Eziocloud database. BLAST analysis confirmed that the antagonistic strain RMM02 is *Pantoea agglomerans*. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 65516 and deposit date of November 19, 2024.

[0054] SEQ ID NO.3

[0055]

[0056] Example 2

[0057] This embodiment provides a biocontrol agent, the preparation method of which includes the following steps:

[0058] ① The clump of pantothecin RMM02 was inoculated into LB liquid medium and cultured in a constant temperature shaker at 30℃ and 200r / min for 24h to obtain the fermentation broth. The OD value of the fermentation broth was measured to be 0.8 under 600nm conditions.

[0059] ② Add a preservative and sterile water to the fermentation broth, and adjust the concentration of the cultured fermentation broth to a viable cell count of 1×10⁻⁶. 10 The biocontrol agent was obtained by reacting cfu / mL. The protectant was carboxymethyl cellulose, and the concentration of the protectant in the biocontrol agent was 6% (m / m).

[0060] Example 3

[0061] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the protective agent used is starch and carboxymethyl cellulose, and the mass ratio of starch to carboxymethyl cellulose is 1:1. All other aspects are the same as in Embodiment 2.

[0062] Example 4

[0063] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the protective agent used is lignin, and all other aspects are the same as in Embodiment 2.

[0064] Example 5

[0065] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the protective agent used is starch, and all other aspects are the same as in Embodiment 2.

[0066] Example 6

[0067] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the protective agent used is chitosan, and all other aspects are the same as in Embodiment 2.

[0068] Example 7

[0069] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the concentration of the protectant in the biocontrol agent is 1% (m / m), and all other aspects are the same as in Embodiment 2.

[0070] Example 8

[0071] This embodiment provides a biocontrol agent. The only difference between this embodiment and Embodiment 2 is that the concentration of the protectant in the biocontrol agent is 10% (m / m), and all other aspects are the same as in Embodiment 2.

[0072] Comparative Example 1

[0073] This comparative example provides a biocontrol agent. The only difference between this comparative example and Example 2 is that it does not contain a protectant. In step ② of this comparative example, only sterile water is added to the fermentation broth, and the rest is the same as in Example 2.

[0074] Example 9

[0075] This embodiment provides a method for preventing and controlling rice false smut, including the following steps:

[0076] 10-15 days before the rice heading stage, spray the biocontrol agent described in Example 2 at a concentration of 1×10⁻⁶. 5 cfu / mL~1×10 7 CFU / mL. If it rains within 4 hours after application, apply again with the same concentration after the rain stops. Application time: evening, when the temperature does not exceed 30℃.

[0077] Example 10

[0078] This embodiment provides a method for preventing and controlling rice false smut, including the following steps:

[0079] Spray the biocontrol agent described in Example 2 0-5 days before the rice heading stage, at a concentration of 1×10⁻⁶. 8 cfu / mL~1×10 10 CFU / mL. If it rains within 4 hours after application, apply a second application at the same concentration after the rain stops. Application time: evening, when the temperature does not exceed 30℃. Apply a second application at the same concentration 3 days after the first application.

[0080] Example 11

[0081] This embodiment provides a method for preventing and controlling rice false smut, including the following steps:

[0082] Five days after rice heading, the biocontrol agent described in Example 2 was sprayed at a concentration of 1×10⁻⁶. 8 cfu / mL~1×10 10 CFU / mL. If it rains within 4 hours after application, apply a second application at the same concentration after the rain stops. Application time: evening, when the temperature does not exceed 30℃. Apply a second application at the same concentration 3 days after the first application.

[0083] Experimental Example 1: Field Trial of the Biocontrol Agent of the Present Invention against Rice False smut

[0084] 1.1 Overview of the experimental site: From August to September 2024, a control experiment was conducted in rice fields where rice false smut was present. The experimental site was located in Shanghuizao Village, Lanting Street, Keqiao District, Shaoxing City, Zhejiang Province. The experimental rice fields had good fertility management and covered a total area of ​​10 mu.

[0085] 1.2 Experimental Grouping: The experimental field was divided into four large areas: a control area and three experimental areas. A 2-meter rice paddy spacing was maintained between each experimental area. The control area was 10 m². 2 Each experimental area included eight treatments: the biocontrol agent group prepared in Examples 2-8 and the control agent group prepared in Comparative Example 1. Each treatment was repeated three times, resulting in 24 plots in each experimental area, with each plot measuring 10 m². 2 All experimental areas followed the same planting pattern, and fertilizer and water management were carried out normally in each area during the experiment. Cultivation, water and fertilizer management, and weed control were consistent with local rice production. The rice variety used in the experiment was Yongyou 31.

[0086] Test method:

[0087] Water control area: No pesticides were applied throughout the growing season; only water was sprayed during the experiment.

[0088] Experimental Area 1: Control measures were applied 10-15 days before rice heading. The specific application time for this experiment was the evening of August 20, 2024, when the temperature did not exceed 30℃. The spraying concentration was 1×10⁻⁶. 7 cfu / mL.

[0089] Experimental Area 2: Control measures were applied 0-5 days before rice heading, with two applications. Specifically, the applications were made on September 2nd and 5th, 2024, in the evening when the temperature did not exceed 30℃. The spraying concentration was 1×10⁻⁶. 9 cfu / mL.

[0090] Experimental Area 3: Control measures were applied 0-5 days after rice heading, with two applications. Specifically, the applications were made on September 8th and 11th, 2024, in the evening when the temperature did not exceed 30℃. The spray concentration was 1×10⁻⁶. 9 cfu / mL.

[0091] 3. Survey on rice false smut control: A survey was conducted on September 20th in each plot. A random 5-point sampling method was used in each plot, with 10 rice panicles sampled at each point. The number of diseased grains was recorded, and the control rate was calculated. The statistical results are shown in Tables 1-4.

[0092] Particle control efficacy (%) = (Number of diseased particles in the control area - Number of diseased particles in the treatment area) / (Number of diseased particles in the control area) × 100%.

[0093] Table 1. Control efficacy (%) of one seed in the experimental area:

[0094]

[0095] Table 2. Control efficacy (%) of 2 seeds in the experimental area

[0096]

[0097]

[0098] Table 3. Control efficacy (%) of 3 seeds in the experimental area

[0099]

[0100] Table 4. Grain control efficacy (%) in the water control group

[0101]

[0102] As shown in Tables 1-4, spraying the biological agent of this invention 10-15 days before heading, 0-5 days before heading, or 0-5 days after heading has a good control effect on rice false smut. Figure 3 The control effect of the biological agent prepared in Example 2 was observed in experimental area 3. Figure 3 As shown, even before prevention and control, rice panicles already showed relatively severe rice false smut, and the rice false smut balls had already developed ( Figure 3 A) Seven days after the second application of the biological agent prepared according to this invention, the further development of rice blast fungus was successfully inhibited, and the rice blast balls shrank and fell off. Figure 3 B).

[0103] The comparison between Examples 2-8 and Comparative Example 1 shows that the addition of a protectant to Pantotheca cum Clindamycin RMM02 significantly improves the control effect. In particular, the biocontrol agent prepared in Example 2 using carboxymethyl cellulose as a protectant has the best effect. Using carboxymethyl cellulose and other protectants can provide more suitable growth conditions for Pantotheca cum Clindamycin RMM02, prevent the live bacteria from being inactivated during storage or transportation, effectively maintain the survival rate of Pantotheca cum Clindamycin RMM02, and effectively protect the strain from colonization and growth on rice, thereby exerting the control effect.

[0104] The biological agent containing Pantothecin RMM02 of this invention not only has a very good control effect on rice false smut, but also extends the control time of rice false smut from the traditional 5-7 days before rice heading to 15 days before rice heading to 5 days after heading, which greatly reduces the difficulty and intensity of control work.

[0105] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A biocontrol agent, characterized in that: The biocontrol agent contains either Pantotheca acuminata RMM02 or Pantotheca acuminata RMM02, wherein the viable count of Pantotheca acuminata RMM02 in the biocontrol agent is 1×10⁻⁶. 5 cfu / mL -1×10 10 cfu / mL; the classification name of the clump-forming pantothecin RMM02 is: Pantoea agglomerans Accession number: GDMCC No: 65516; Depository institution: Guangdong Provincial Center for Microbial Culture Collection; Deposit date: November 29, 2024; It also includes a preservative and sterile water, wherein the preservative is one or more of carboxymethyl cellulose, starch, chitosan, and lignin; The concentration of the protective agent in the biocontrol agent is 1-10% m / m.

2. The application of the biocontrol agent according to claim 1 in the preparation of products for controlling rice false smut.

3. The method for preparing the biocontrol agent according to claim 1, characterized in that, Includes the following steps: ① Inoculate the clump-forming pantothecin RMM02 into LB liquid medium, place it on a constant temperature shaker at 26-33 ℃ and shake for 24-60 h to obtain the fermentation broth; ② Add a preservative and sterile water, and adjust the concentration of the cultured fermentation broth to a viable cell count of 1×10⁻⁶. 5 cfu / mL -1×10 10 cfu / mL.

4. A method for preventing and controlling rice false smut, characterized in that, Includes the following steps: Spray the biocontrol agent according to claim 1 or the biocontrol agent prepared according to the method of claim 3 10-15 days before the rice heading stage, at a concentration of 1×10⁻⁶. 5 cfu / mL -1×10 7 If it rains within 4 hours after application, apply the same concentration again after the rain stops.

5. A method for preventing and controlling rice false smut, characterized in that, Includes the following steps: Spray the biocontrol agent according to claim 1 or the biocontrol agent prepared according to the method of claim 3 0-5 days before heading or 0-5 days after heading, with a spraying concentration of 1×10⁻⁶. 8 cfu / mL -1×10 10 CFU / mL. If it rains within 4 hours after application, apply again at the same concentration after the rain stops. Apply again at the same concentration 3 days after the first application.

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