Application of pantoea agglomerans RMM02 and product of pantoea agglomerans RMM02 in preventing and treating rice false smut

Abstract

The invention discloses pantoea agglomerans RMM02 and application of a product of the pantoea agglomerans RMM02 in preventing and treating rice false smut. The Pantoea agglomerans RMM02 is classified and named as Pantoea agglomerans, and the preservation number of the Pantoea agglomerans RMM02 is GDMCC (China General Microbiological Culture Collection Center) No.65516; the preservation unit is Guangdong Microbial Culture Collection Center; the preservation time is November 29, 2024. The pantoea agglomerans RMM02 is separated from soil of a rice false smut occurrence plot, is screened through an indoor confrontation test, and has remarkable inhibition activity on the false smut. Indoor bacteriostasis tests show that the active bacterium has remarkable inhibition activity on Ustilaginoidea virens, and indoor and field tests show that the bacterium has a remarkable inhibition effect on the growth of Ustilaginoidea virens and can control the development of the disease condition during the onset period of field Ustilaginoidea virens. The biocontrol microbial inoculum prepared after fermentation of the strain can effectively prevent and treat rice false smut damage and guarantee high yield and high quality of rice.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to application of Pantoea agglomerans RMM02 and its products in preventing and controlling rice false smut. Background Art

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

[0003] Rice smut primarily invades rice during the flowering stage, becomes apparent during the grain filling phase, and becomes a major devastation during the ripening phase. Currently, rice smut relies primarily on chemical control. However, due to the short control window (5-7 days before rice grain burst) and susceptibility to weather conditions such as rain, widespread and recurring rice smut is still unavoidable in actual production. To effectively control rice smut, farmers frequently use chemical pesticides, which increases production costs and the risk of pesticide residue contamination in rice and the environment.

[0004] Active microbial biocontrol agents are an ideal alternative to chemical pesticides because of their advantages such as being environmentally safe, pollution-free and leaving no residue.

[0005] In view of the widespread and recurring occurrence of rice rust, the development of more safer and more effective biocontrol agents has important application significance. Summary of the Invention

[0006] The present invention aims to provide application of Pantoea agglomerans RMM02 and its products in preventing and controlling rice false smut.

[0007] In order to solve the problems existing in the prior art, the technical solution adopted by the present invention is:

[0008] In the first aspect, the present invention provides a strain of Pantoea agglomerans RMM02, the classification name of the Pantoea agglomerans RMM02 is: Pantoea agglomerans, the preservation number is: GDMCC No.65516; the preservation unit is: Guangdong Provincial Microbial Culture Collection Center; the preservation time is: November 29, 2024.

[0009] In a second aspect, the present invention provides a bacterial agent comprising the fermentation broth of Pantoea agglomerans RMM02 described in the first aspect.

[0010] In a third aspect, the present invention provides use of the Pantoea agglomerans RMM02 described in the first aspect or the bacterial agent described in the second aspect in the preparation of a product for preventing and controlling rice false smut.

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

[0012] In a fourth aspect, the present invention provides a biocontrol agent, which comprises the Pantoea agglomerans RMM02 described in the first aspect and / or the agent described in the second aspect, and the number of viable bacteria in the biocontrol agent is not less than 1×10 5 cfu / mL.

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

[0014] More preferably, the number of viable bacteria in the biocontrol agent is 1×10 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. Other point values within this numerical range can be selected.

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

[0017] In a fifth aspect, the present invention further provides a method for preparing the biocontrol agent described in the fourth aspect, comprising the following steps:

[0018] ① Inoculate Pantoea agglomerans RMM02 into LB liquid medium, place in a constant temperature shaker at 26-33°C, shake and ferment for 24-60 hours to obtain a fermentation broth;

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

[0020] Furthermore, the OD value of the fermentation liquid prepared in step ① is measured at 600 nm and is ≥0.8.

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

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

[0024] Furthermore, 10-15 days before the rice breaks, 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 at a concentration of 1×10 5 cfu / mL-1×10 7 cfu / mL.

[0025] Spray 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 rice rupture or 0-5 days after heading, and it is necessary to spray once more 3 days after the first spraying, and the spraying concentration is 1×10 8 cfu / mL-1×10 10 cfu / mL.

[0026] Preservation Instructions:

[0027] Strain name: Pantoea agglomerans RMM02;

[0028] Deposit number: GDMCC No.65516;

[0029] Taxonomic name: Pantoea agglomerans;

[0030] Deposit date: November 19, 2024;

[0031] Depository: Guangdong Provincial Microbial Culture Collection Center;

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

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

[0034] Indoor antibacterial tests have shown that the active strain Pantoea agglomerans RMM02 exhibits significant inhibitory activity against rice smut fungi. Indoor and field tests have also shown that the strain significantly inhibits the growth of the pathogen and can control the progression of rice smut in the field. The biocontrol agent prepared by fermentation of the strain effectively prevents and controls the damage caused by rice smut, ensuring high and quality rice yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The antagonistic effect of Pantoea agglomerans RMM02 on koji rice is shown in Figure 1, wherein: A is the growth state of koji rice control on PDA medium; B is the growth state of koji rice on PDA medium containing Pantoea agglomerans RMM02;

[0036] Figure 2 This is the colony morphology of Pantoea agglomerans RMM02 of the present invention;

[0037] Figure 3 The figure shows the field control effect of the biocontrol agent with the strain RMM02 of the present invention as the main component on rice false smut. Figure A shows the rice ear before control, and Figure B shows the rice ear after control. DETAILED DESCRIPTION

[0038] The following will be combined with the accompanying drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of this application.

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

[0040] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and distilled water to 1000 ml, adjust the pH to 7.0-7.2, and sterilize at 121°C.

[0041] LB solid plate medium: Add agar powder with a concentration of 15g / L to the LB liquid medium.

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

[0043] Example 1 Isolation, purification and identification of Pantoea agglomerans RMM02

[0044] 1. Bacterial isolation and initial screening

[0045] Soil was collected from a rice field (Yongyou 31) with false smut in Yuecheng District, Shaoxing City, Zhejiang Province, sealed in sterile ziplock bags, brought back to the laboratory, and stored at 4°C for isolation of single colonies.

[0046] The target pathogen was Ustilaginoidea virens, isolated by the present invention team from rice plants in the diseased area of rice (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 plate medium.

[0047] The soil dilution method was used to isolate the bacteria, and continuous streaking culture was performed until pure. The growth rate index was used to determine the antagonistic effect of bacteria on rice rust. A 5mm diameter puncher was used to prepare rice rust cakes, which were inoculated on PDA plates (control group) and PDA plates premixed with antagonistic bacteria (antagonistic group), and placed in a 28°C biochemical incubator for dark culture for 8 days. By measuring the growth diameter of the rice rust in the antagonistic group, the strains with obvious antagonistic effects compared with the control group were preserved. The experiment found that in the antagonistic bacteria RMM02 antagonistic group, the growth of rice rust has been inhibited, and the effect is more obvious compared with other antagonistic bacteria. The results of the plate antagonism of the strain RMM02 of the present invention on rice rust are shown as follows: Figure 1 As shown, Figure 1 A shows the growth status of the rice urticae in the control group on PDA medium; Figure 1 B shows the growth status of rice rust 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 bacteria RMM02 is: the colonies are round, yellow, with neat, low-convex and smooth edges.

[0051] 2.2 Molecular biological methods were used to identify the screened antagonistic bacteria RMM02.

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

[0053] PCR reaction system (50 μL system): 2 μL of each primer, 25 μL of 2× Mix, and ddH2O were added to make up 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). The reaction procedure was as follows: 94°C pre-denaturation for 3 min, 94°C denaturation for 55 s, 50°C annealing for 50 s, 72°C extension for 1 min, and 72°C extension for 10 min. After 35 cycles, the reaction was stored at 16°C. PCR products were detected on 1.5% agarose gel electrophoresis and 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 sequences were compared using the Eziocloud database using BLAST. Analysis using the BLAST program indicated that the antagonistic strain RMM02 was Pantoea agglomerans. The strain was deposited in the Guangdong Provincial Center for Microbiological Culture Collection, located at 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with the deposit number GDMCC No. 65516 and the date of deposit on November 19, 2024.

[0054] SEQ ID NO.3

[0055]

[0056] Example 2

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

[0058] ① Pantoea agglomerans RMM02 was inoculated into LB liquid culture medium and cultured in a constant temperature shaker at 30°C and 200 rpm for 24 h to obtain a fermentation broth. The OD value of the fermentation broth was measured at 600 nm to be 0.8.

[0059] ② Add protective agent and sterile water to the fermentation liquid and adjust the concentration of the fermentation liquid to a viable bacterial count of 1×10 10 cfu / mL, and a biocontrol agent is obtained. The protective agent is carboxymethyl cellulose, and the concentration of the protective agent in the biocontrol agent is 6% (m / m).

[0060] Example 3

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

[0062] Example 4

[0063] This embodiment provides a biocontrol agent. The only difference between this embodiment and Example 2 is that the protective agent used is lignin. The rest is the same as Example 2.

[0064] Example 5

[0065] This embodiment provides a biocontrol agent. The only difference between this embodiment and Example 2 is that the protective agent used is starch. The rest is the same as Example 2.

[0066] Example 6

[0067] This embodiment provides a biocontrol agent. The only difference between this embodiment and Example 2 is that the protective agent used is chitosan. The rest is the same as Example 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 protective agent in the biocontrol agent is 1% (m / m). The rest is the same as embodiment 2.

[0070] Example 8

[0071] This embodiment provides a biocontrol agent. The only difference between this embodiment and Example 2 is that the concentration of the protective agent in the biocontrol agent is 10% (m / m). The rest is the same as Example 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 protective agent. In step ② of this comparative example, only sterile water is added to the fermentation broth. The rest is the same as Example 2.

[0074] Example 9

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

[0076] 10-15 days before the rice breaks, the biocontrol agent described in Example 2 was sprayed at a concentration of 1×10 5 cfu / mL~1×10 7 cfu / mL. If it rains within 4 hours after application, apply the pesticide once more at the same spraying concentration after the rain stops. Application time: in the evening when the temperature does not exceed 30℃.

[0077] Example 10

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

[0079] 0-5 days before the rice breaks, the biocontrol agent described in Example 2 was sprayed at a concentration of 1×10 8 cfu / mL~1×10 10 cfu / mL. If it rains within 4 hours after spraying, apply the same spray concentration once after the rain stops. Application time: evening, when the temperature does not exceed 30°C. Apply the same spray concentration again 3 days after the first spraying.

[0080] Example 11

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

[0082] The biocontrol agent described in Example 2 was sprayed 5 days after rice heading, and the spraying concentration of the biocontrol agent was 1×10 8 cfu / mL~1×10 10 cfu / mL. If it rains within 4 hours after spraying, apply the same spray concentration once after the rain stops. Application time: evening, when the temperature does not exceed 30°C. Apply the same spray concentration again 3 days after the first spraying.

[0083] Experimental Example 1 Field test 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 prevention and control experiment was carried out in rice fields infected with rice false smut. The experimental site was Shanghuizao Village, Lanting Street, Keqiao District, Shaoxing City, Zhejiang Province. The fertility of the experimental rice fields was well managed, and the experimental field area was 10 mu.

[0085] 1.2 Experimental Grouping: The experimental field was divided into four large areas, namely the control area and three experimental areas. A 2-meter rice separation zone was reserved between each experimental area. The control area was 10m 2 Each experimental area was set up with 8 treatments, including 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, that is, each experimental area had 24 plots, each plot had an area of 10m 2 All experimental plots were planted in the same pattern. During the trial, fertilizer and fertilization management was maintained as normal in each plot. Cultivation, fertilizer and water management, and weed control practices were consistent with local rice production. The rice variety tested was Yongyou 31.

[0086] Test method:

[0087] Clean water control area: No pesticides were applied during the entire growth period, and only clean water was sprayed during the test.

[0088] Experimental area 1: The control was carried out 10-15 days before the rice burst. The specific application time of this experiment was: the evening of August 20, 2024, when the temperature did not exceed 30℃. The spraying concentration was 1×10 7 cfu / mL.

[0089] Experimental area 2: Control was carried out 0-5 days before the rice burst, and the pesticide was applied twice. The specific application time of this experiment was September 2 and September 5, 2024, in the evening when the temperature did not exceed 30℃. The spraying concentration was 1×10 9 cfu / mL.

[0090] Experimental area 3: The control was carried out 0-5 days after the rice heading, and the pesticide was applied twice. The specific application time of this experiment was: September 8 and September 11, 2024, in the evening, when the temperature did not exceed 30℃. The spraying concentration was 1×10 9 cfu / mL.

[0091] 3. Rice False Smut Control Survey: On September 20th, a survey was conducted in each plot. A random sampling method was used at five locations within each plot. Ten rice ears were sampled at each location, and the number of diseased grains was recorded to calculate the control rate. The statistical results are shown in Tables 1-4.

[0092] Grain prevention effect (%) = (number of diseased grains in the control area - number of diseased grains in the treated area) / (number of diseased grains in the control area) × 100%.

[0093] Table 1: Effect of one pill in the experimental area (%):

[0094]

[0095] Table 2: Effectiveness of 2 tablets in experimental area (%)

[0096]

[0097]

[0098] Table 3: Control effect of 3 pills in the experimental area (%)

[0099]

[0100] Table 4: Control group of water for preventing and controlling granules (%)

[0101]

[0102] As shown in Tables 1-4, spraying the biological agent of the present invention 10-15 days before rice rupture, 0-5 days before rupture, or 0-5 days after heading has a relatively good control effect on rice false smut. Figure 3 The control effect of the biological agent prepared in Example 2 in Experimental Area 3 is shown in Figure 2. Figure 3 As shown in the figure, even before the prevention and control, the rice ears had already suffered from severe rice false smut and the rice smut balls had developed into shape ( Figure 3 A), 7 days after the second spraying of the biological agent prepared by the present invention, the further development of rice koji fungi has been successfully inhibited, and the rice koji balls have shrunk and fallen off ( Figure 3 B).

[0103] By comparing Examples 2-8 with Comparative Example 1, it can be seen that after adding a protective agent to Pantoea agglomerans RMM02, the control effect is significantly improved, especially the biocontrol agent prepared in Example 2 with carboxymethyl cellulose as a protective agent has the best effect. Using carboxymethyl cellulose and the like as a protective agent can provide more suitable growth conditions for Pantoea agglomerans RMM02, prevent the inactivation of live bacteria during storage or transportation, effectively maintain the survival rate of Pantoea agglomerans RMM02, and effectively protect the strain from colonizing and growing on rice during application, thereby exerting a control effect.

[0104] The biological agent containing Pantoea agglomerans RMM02 of the present invention not only has a very good control effect on rice false smut, but also extends the control period of rice false smut from 5 to 7 days before rice smut to 15 days before rice smut and 5 days after heading, greatly reducing the difficulty and intensity of the control work.

[0105] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of this application, ordinary technicians in this field can also make many forms without departing from the purpose of this application and the scope of protection of the claims, all of which are within the protection of this application.

Claims

1. A strain of Pantoea agglomerans RMM02, characterized by: The classification of the Pantoea agglomerans RMM02 is named as: Pantoea agglomerans , deposit number: GDMCC No.65516; deposit unit: Guangdong Provincial Microbiological Culture Collection Center; deposit time: November 29, 2024.

2. A bacterial agent, characterized in that: Comprising the Pantoea agglomerans RMM02 and / or the fermentation broth of Pantoea agglomerans RMM02 according to claim 1.

3. Use of the Pantoea agglomerans RMM02 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for preventing and controlling rice false smut.

4. The use according to claim 3, characterized in that: The product is a biocontrol agent.

5. A biocontrol agent, characterized in that: The biocontrol agent comprises the Pantoea agglomerans RMM02 according to claim 1 and / or the microbial agent according to claim 2, wherein the number of viable bacteria in the biocontrol agent is 1×10 5 cfu / mL-1×10 10 cfu / mL.

6. The biocontrol agent according to claim 5, characterized in that: The biocontrol agent further comprises a protective agent and sterile water. The protective agent is one or more of carboxymethyl cellulose, methyl cellulose, starch, cyclodextrin, chitosan and lignin. The concentration of the protective agent in the biocontrol agent is 1-10% (m / m).

7. The method for preparing the biocontrol agent according to any one of claims 5 to 6, characterized in that: The following steps are involved: ① Inoculate Pantoea agglomerans RMM02 into LB liquid medium, place in a constant temperature shaker at 26-33°C, and shake and ferment for 24-60 hours to obtain a fermentation broth; ② Add protective agent and sterile water, and adjust the concentration of the fermentation liquid to a viable bacterial count of 1×10 5 cfu / mL-1×10 10 cfu / mL.

8. A method for preventing and controlling rice false smut, characterized in that: The following steps are involved: Spray the biocontrol agent according to any one of claims 5-6 or the biocontrol agent prepared according to the method of claim 7 15 days before rice rupture and 5 days after heading, wherein the spraying concentration of the biocontrol agent is 1×10 5 cfu / mL-1×10 10 cfu / mL. If it rains within 4 hours after application, apply the pesticide again at the same spraying concentration after the rain stops.

9. The method for preventing and controlling rice false smut according to claim 8, wherein: The 15 days before rice bursts to 5 days after heading refers to: 10-15 days before rice bursts, 0-5 days before rice bursts or 0-5 days after heading.

10. The method for preventing and controlling rice false smut according to claim 9, wherein: Spray the biocontrol agent according to any one of claims 5 to 6 or the biocontrol agent prepared according to the method of claim 7 10 to 15 days before the rice breaks, with a spraying concentration of 1×10 5 cfu / mL-1×10 7 cfu / mL; Spray 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 rice rupture or 0-5 days after heading, and spray once more 3 days after the first spraying, with a spraying concentration of 1×10 8 cfu / mL-1×10 10 cfu / mL.

Images