Defense pseudomonas JR-4 and application thereof in preventing and treating rice blast

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to defensive pseudomonas JR-4 and application thereof in preventing and treating rice blast. The pseudomonas defensive JR-4 provided by the invention has the preservation number of CCTCC NO: M 20252405, is separated from rhizosphere soil of dry land high-quality rice-Yunnan rice you 615 in a healthy land parcel adjacent to a rice blast occurrence land parcel, has the capabilities of fixing nitrogen, dissolving cellulase, dissolving phosphorus and producing siderophores, can effectively inhibit rice blast bacteria, and has a good application prospect. Especially, hypha growth of pyricularia oryzae with strong pathogenicity can be effectively inhibited, and the bacteriostasis rate reaches 46.71%. Moreover, results of in-vitro leaf inoculation and root irrigation tests show that the fermentation broth for preventing the pseudomonas JR-4 provided by the invention can effectively inhibit the occurrence of the rice blast, and has good prevention and treatment effects on the rice blast of the rice, especially the rice variety of Yunnan rice Yunyou 615.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a strain of Pseudomonas protegens JR-4 and application thereof in preventing and treating rice blast. BACKGROUND

[0002] High-quality rice in dry land is a kind of high-quality rice variety planted in dry land, and the yield and quality of which have no significant difference with that of rice; it has the characteristics of strong topsoil capacity, developed root system, vigorous tillering, good drought resistance, high yield, and rice quality reaching the national or Ministry of Agriculture and Rural Affairs high-quality rice standard. The dry land growth environment makes the main and secondary diseases of high-quality rice in dry land change significantly compared with the growth environment in paddy field. However, rice blast ( Magnaporthe oryzae ) is still the main disease to be prevented and treated for high-quality rice in dry land, and reasonable prevention and treatment of rice blast is of great significance to the production of high-quality rice in dry land.

[0003] At present, the prevention and treatment of rice blast mainly adopts chemical prevention and treatment. The advantages of chemical prevention and treatment are quick effect, low cost, convenience, and multi-disease treatment. The disadvantages are that long-term and unreasonable use of chemicals will lead to reduced prevention and treatment effect, and the high toxicity and high residual characteristics of chemicals will not only harm human and livestock health, but also cause serious pollution to soil, water, and atmosphere, and destroy the ecological balance. At the same time, due to the rapid variation of rice blast pathogen and the diversification of physiological races, the use amount and frequency of pesticides are increasing, and the drug resistance of the pathogen is increasing. Chemical prevention and treatment has good effect at the beginning, but with the passage of time, the physiological races of the pathogen are continuously differentiated, and the pathogen will have different degrees of drug resistance.

[0004] With the improvement of plant disease prevention and treatment technology and the high attention of human beings to the environment, the use of beneficial microorganisms to prevent and treat diseases is friendly to human beings and the environment and has good prevention and treatment effect, and has good application prospect in the prevention and treatment of rice blast of high-quality rice in dry land. At present, the research on beneficial microorganism groups for preventing and treating rice blast mainly focuses on Bacillus, and there is no research report on Pseudomonas protegens. For example, the existing research (Chen C, Cui T B, Yu P Q. Identification and antibacterial research of a strain of antifungal Bacillus amyloliquefaciens [J]. Modern Food Science and Technology, 2011, 27 (01): 36-39. DOI:10.13982 / j.mfst.1673-9078.2011.01.018.) isolates a strain of Bacillus amyloliquefaciens HN06 producing antifungal substances from soil in Guangzhou, which has good inhibitory effect on Magnaporthe oryzae ( Magnaporthe oryzae ). SUMMARY

[0005] The present application aims to provide a strain of Pseudomonas protegens JR-4 and its application in preventing and treating rice blast, effectively inhibiting Magnaporthe oryzae, especially effectively inhibiting Magnaporthe oryzae with strong pathogenicity, inhibiting the occurrence of rice blast, and preventing and treating rice blast.

[0006] The present application provides a strain of Pseudomonas protegens JR-4, with a preservation number of CCTCC NO: M 20252405.

[0007] The present application provides a bacterial agent containing the Pseudomonas protegens JR-4 according to the above technical solution.

[0008] Preferably, the bacterial agent is a fermentation broth of the Pseudomonas protegens JR-4.

[0009] Preferably, the effective viable cell count of the Pseudomonas protegens JR-4 in the fermentation broth is 1×10 7 ~1×10 8 CFU / mL.

[0010] The present application provides a preparation method of the bacterial agent according to the above technical solution, inoculating the Pseudomonas protegens JR-4 into a fermentation medium for fermentation culture, collecting the fermentation broth, and obtaining the bacterial agent.

[0011] Preferably, the fermentation medium comprises an LB liquid medium, and the fermentation culture is performed at a temperature of 28-32℃ for 24-72h.

[0012] The present application provides an application of the Pseudomonas protegens JR-4 according to the above technical solution or the bacterial agent according to the above technical solution or the bacterial agent obtained by the preparation method according to the above technical solution in inhibiting Magnaporthe oryzae and / or preventing and treating rice blast.

[0013] Preferably, the Magnaporthe oryzae comprises Magnaporthe oryzae YL22H7, and the rice blast comprises leaf blast.

[0014] Preferably, the rice varieties corresponding to the Magnaporthe oryzae and the rice blast are both Dianhewu 615.

[0015] The present application provides a method for preventing and treating rice blast, comprising the following steps: applying the Pseudomonas protegens JR-4 according to the above technical solution or the bacterial agent according to the above technical solution or the bacterial agent obtained by the preparation method according to the above technical solution to a rice crop.

[0016] Beneficial effects: The *Pseudomonas* JR-4 strain provided by this invention, with preservation number CCTCC NO: M 20252405, was isolated from the rhizosphere soil of high-quality dryland rice variety Dianheyou 615 in a healthy plot adjacent to a rice blast outbreak area. It possesses nitrogen-fixing, cellulase-degrading, phosphorus-solubilizing, and iron-carrier-producing abilities, effectively inhibiting rice blast pathogens, especially the mycelial growth of highly pathogenic rice blast pathogens, with an inhibition rate of 46.71%. Furthermore, in vitro leaf inoculation and root drenching experiments showed that the fermentation broth of the *Pseudomonas* JR-4 strain provided by this invention effectively inhibits the occurrence of rice blast, exhibiting good preventive and curative effects against rice blast, especially in the Dianheyou 615 rice variety.

[0017] Biological Preservation Information The bacterium *Pseudomonas* JR-4 is classified and named *Pseudomonas* JR-4. Pseudomonas protegens JR-4 was deposited on November 3, 2025, at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCC NO: M 20252405. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 Colony morphology of Pseudomonas JR-4 on a plate; Figure 2 Phylogenetic tree for defense against Pseudomonas JR-4; Figure 3 The results of biological function verification for defense against Pseudomonas JR-4; from left to right, the results are the functional verification results for nitrogen fixation, phosphorus solubilization, protease solubilization, amylase solubilization, silicate solubilization, laccase solubilization, siderophoresis production, and cellulase solubilization. Figure 4 The results of the flat plate confrontation experiment in Example 3; Figure 5 The results of colony diameter analysis for rice blast fungus in the control and treatment groups of Example 3 are as follows; Figure 6 The lesion condition of detached leaves in the control and treatment groups of Example 4; Figure 7 The above are the statistical results of the lesion area of ​​the detached leaves of the control group and the treatment group in Example 4; Figure 8 The lesion condition of leaves in the control and treatment groups of Example 5 root drenching experiment; Figure 9 The results show the statistical findings of lesion area on leaves in the control and treatment groups during the root drenching experiment in Example 5. Figure 10 Leaf spot conditions of the root irrigation test leaves of the control group and the treatment group of Example 6 were observed; Figure 11 Leaf spot area statistics of the root irrigation test leaves of the control group and the treatment group of Example 6 were observed; wherein, indicates P <0.05, indicates P <0.001. DETAILED DESCRIPTION

[0020] The application provides a defense pseudomonas JR-4, and the preservation number is CCTCC NO: M 20252405.

[0021] The defense pseudomonas JR-4 is separated from rhizosphere soil of high-quality dryland rice Diankeyou 615 in a healthy plot adjacent to a rice blast plot, and the colony morphology characteristics on a plate are as follows: the colony is light yellow, the surface is smooth and lustrous, the texture is viscous, the shape is round and raised, and the edge is neat. The defense pseudomonas JR-4 has the abilities of nitrogen fixation, cellulase, phosphorus solubilization and iron carrier production, can effectively inhibit the growth of rice blast fungus, especially the mycelium growth of rice blast fungus with strong pathogenicity, and the inhibition rate reaches 46.71%. In addition, the results of in vitro leaf inoculation and root irrigation experiments show that the fermentation liquor of the defense pseudomonas JR-4 can effectively inhibit the occurrence of rice blast, and has good prevention and treatment effects on rice blast of rice, especially Diankeyou 615 rice.

[0022] The application provides a bacterial agent, which contains the defense pseudomonas JR-4.

[0023] As an implementation form, the bacterial agent is a fermentation liquor of the defense pseudomonas JR-4. As an implementation form, the effective viable cell number of the defense pseudomonas JR-4 in the fermentation liquor is 1×10 7 ~1×10 8 CFU / mL; as another implementation form, the effective viable cell number of the defense pseudomonas JR-4 in the fermentation liquor is 1×10 8 CFU / mL.

[0024] The application provides a preparation method of the bacterial agent.

[0025] In one embodiment, the fermentation medium of the present invention includes LB liquid medium. In one embodiment, the fermentation temperature of the present invention is 28-32°C; in another embodiment, the fermentation temperature of the present invention is 28°C. In one embodiment, the fermentation time of the present invention is 24-72 hours; in another embodiment, the fermentation time of the present invention is 36-48 hours.

[0026] This invention provides the application of the above-described protective agent against Pseudomonas jR-4, or the inoculant described in the above-described technical solution, or the inoculant prepared by the above-described technical solution, in inhibiting rice blast fungus and / or controlling rice blast.

[0027] In one embodiment, the rice blast fungus of the present invention includes rice blast fungus YL22H7. In one embodiment, the rice blast disease of the present invention includes leaf blast. In one embodiment, the rice blast fungus and the rice variety corresponding to the rice blast disease of the present invention are both Dianheyou 615.

[0028] This invention provides a method for preventing and controlling rice blast, comprising the following steps: applying the anti-pseudomonas JR-4 as described in the above technical solution, or the inoculant as described in the above technical solution, or the inoculant obtained by the preparation method described in the above technical solution, to rice crops.

[0029] In one embodiment, the application method of the anti-Pseudomonas JR-4 agent or the inoculant described in this invention includes root irrigation. In one embodiment, the application amount of the inoculant is 40-50 mL / 9 plants; in another embodiment, the application amount of the inoculant is 50 mL / 9 plants. In one embodiment, the rice crop described in this invention is a rice plant at the 3-leaf-1-heart stage. In one embodiment, the rice variety described in this invention is Dianheyou 615.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Pseudomonas japonicus JR-4 provided by the present invention and its application in the control of rice blast, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 Isolation and identification of strains Bacteria were isolated from the rhizosphere soil of high-quality dryland rice (Dianheyou 615) in healthy plots adjacent to rice blast-affected plots using the dilution plating method. The specific procedure was as follows: Soil samples were weighed and placed in sterile water at a ratio of 1 g:10 mL, and incubated with shaking at 28°C for 30 min to prepare 10... -1 Soil suspension; diluted sequentially in 10-fold gradients to 10... -6Finally, a diluted soil suspension was obtained. 100 μL of the suspension was pipetted onto LB medium and incubated upside down in a 28°C incubator for 24 h. Purification was performed using the streak plate method to obtain pure culture strain JR-4. Colonies were pale yellow, smooth and glossy, viscous in texture, rounded and raised in shape, and had neat edges. Figure 1 Finally, the purified strain was identified using 16S rRNA. The results showed that the full-length 16S rRNA sequence of strain JR-4 was 1373 bp, similar to that of *Pseudomonas aeruginosa*. Pseudomonas protegens L21 and defense against Pseudomonas aeruginosa Pseudomonas protegens The sequence similarity of BSS21 was 100%, and it was also similar to that of Pseudomonas aeruginosa. Pseudomonas protegens The strain is in a phylogenetic branch ( Figure 2 Therefore, strain JR-4 was identified as a defensive pseudomonad. Pseudomonas protegens And to preserve them biologically.

[0032] Example 2 Validation of biological function of defense against Pseudomonas JR-4 Activated Pseudomonas aeruginosa JR-4 for 24 hours was inoculated onto functional media containing nitrogen fixation, phosphorus solubilization, protease production, amylase production, silicate, laccase, siderophoresis, and cellulase, respectively. After incubation at 28°C upside down for 72 hours, the presence of clear zones around the colonies was observed. The formation of clear zones indicated the presence of the corresponding functions. The results showed that Pseudomonas aeruginosa JR-4 produced clear zones on nitrogen fixation, phosphorus solubilization, siderophoresis, and cellulase functional media, indicating that the Pseudomonas aeruginosa JR-4 obtained in Example 1 possesses nitrogen fixation, phosphorus solubilization, siderophoresis production, and cellulase functions, but lacks silicate, protease, laccase, and amylase functions. Figure 3 ).

[0033] Example 3 Inhibition rate of Pseudomonas JR-4 against rice blast fungus 1. Preparation of fermentation broth: The anti-Pseudomonas JR-4 strain obtained in Example 1 was picked and inoculated into LB liquid medium and cultured at 28°C and 180 r / min for 72 h to obtain the anti-Pseudomonas JR-4 fermentation broth.

[0034] 2. Rice blast pathogen: Rice blast fungus YL22H7, isolated from susceptible plants of Dianheyou 615, exhibits the strongest pathogenicity and is published in Pathogenicity and Genetic Variations in... Magnaporthe oryzaeIsolates from One Rice Variety Planting in Paddy and Upland Fields (https: / / doi.org / 10.3390 / agronomy13051246).

[0035] 3. Use a sterile puncher with a diameter of 5 mm to punch a Pyricularia oryzae cake and inoculate it in the center of a PDA medium plate. The Pseudomonas protegens JR-4 fermentation liquid is inoculated around the pathogenic fungus at a distance of 2 cm. An equal amount of liquid LB medium is inoculated as a control group (CK). Cultivate at 28°C. When the pathogenic fungus of the control group covers the culture plate, measure the colony diameter of the pathogenic fungus of the control group and the treatment group (JR-4) by the cross method. Calculate the inhibition rate of the strain by the formula. The results are shown in Table 1 and Figure 4~5 .

[0036] Table 1 Detection results of the antibacterial rate of Pseudomonas protegens JR-4 on Pyricularia oryzae

[0037] According to Table 1 and Figure 4~5 , it can be seen that the Pseudomonas protegens JR-4 provided by the present application can effectively inhibit the growth of the strong pathogenicity pathogenic fungus (Pyricularia oryzae YL22H7) of high-quality upland rice Diankeyou 615, and the antibacterial rate reaches 46.71%.

[0038] Example 4 Ex vivo leaf detection The treatment group (JR-4) selects healthy upland high-quality rice Diankeyou 615 plant leaves, cuts 5 cm long leaf segments, immerses them in 1×10 8 CFU / mL of Pseudomonas protegens JR-4 fermentation liquid, and shakes and cultures at 150 r / min for 1 h. Sterile water is used as a negative control (CK). Place the treated leaves in a sterile culture plate, lightly prick the surface of the leaves with a sterilized toothpick, prick one point on each leaf, and inoculate 10 μL of spore suspension (1×10 5 spores / mL) of Pyricularia oryzae YL22H7 on the pricked points. Place the inoculated leaves in an artificial climate box and cultivate at 28°C for 48 h (light for 12 h / day, darkness for 12 h / day, light intensity of 4000 Lux). Investigate the relative lesion area of the inoculation point of rice blast. The results show that the Pseudomonas protegens JR-4 provided by the present application can effectively inhibit the occurrence of plant rice blast ( Figure 6~7 ).

[0039] Example 5 Preventive effect of Pseudomonas protegens JR-4 on rice blast A pot experiment was conducted with a planting density of 9 plants per pot. Once the dry-grown rice plants (Dianheyou 615) reached the 3-leaf, 1-heart stage, the irrigation concentration per pot was 1×10⁻⁶. 8 50 mL of fermentation broth containing CFU / mL of the protective *Pseudomonas aeruginosa* JR-4 was added, with three replicates per treatment. Sterile water was used as a negative control. After 24 h of treatment, the spores of *Bacillus oryzae* YL22H7 (1×10⁻⁶) were inoculated. 5 Inoculation was performed by spraying at a uniform angle (spores / mL), followed by constant temperature incubation at 22℃, with the first 24 hours in darkness and humidity. Approximately 7 days after inoculation, once the disease had fully developed, the relative lesion area was calculated on a leaf-by-leaf basis. The results showed that, in disease prevention treatment, compared to the negative control (CK), the treatment group (JR-4) showed a significant reduction in the relative lesion area of ​​rice leaves after treatment with this strain, with a decrease of up to 26%. Figure 8~9 ).

[0040] Example 6 The therapeutic effect of Pseudomonas JR-4 on rice blast A pot experiment was conducted with a planting density of 9 plants per pot. Once the dry-grown rice plants (Dianheyou 615) reached the 3-leaf-1-heart stage, each pot was first inoculated with a spore suspension (1×10⁻⁶) of rice blast fungus YL22H7. 5 (spores / mL), inoculated by spraying at a uniform angle, followed by 24 hours of dark, humidified culture. The treatment group (JR-4) was then watered with a concentration of 1×10⁻⁶. 8 50 mL of fermentation broth of *Pseudomonas aeruginosa* JR-4 (CFU / mL) was added to each treatment in triplicate. The negative control (CK) was treated with sterile water in triplicate, and all treatments were incubated at 22°C. After full disease development, the relative lesion area was calculated on a leaf-by-leaf basis. The results showed that, compared to the negative control (CK), the relative lesion area of ​​rice leaves treated with the strain (JR-4) was significantly reduced by 58% in the treatment group. Figure 10~11 ).

[0041] As can be seen from the above, the anti-pseudomonas JR-4 provided by the present invention has the ability to fix nitrogen, dehydrogenate cellulase, dehydrogenate phosphorus and produce iron carriers, and can effectively inhibit the growth of mycelium of highly pathogenic fungus of high-quality dryland rice - Dianheyou 615, and inhibit the occurrence of rice blast disease in the plant.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of defensive pseudomonads ( Pseudomonas protegens JR-4, characterized in that, The accession number is CCTCCNO: M 20252405.

2. A microbial agent, characterized in that, It includes the defense against Pseudomonas JR-4 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is the fermentation broth for defending against Pseudomonas JR-4.

4. The microbial agent according to claim 3, characterized in that, The effective viable count of Pseudomonas japonicus JR-4 in the fermentation broth was 1 × 10⁻⁶. 7 ~1×10 8 CFU / mL.

5. The method for preparing the microbial agent according to any one of claims 2 to 4, characterized in that, The anti-Pseudomonas aeruginosa JR-4 was inoculated into a fermentation medium for fermentation culture, and the fermentation broth was collected to obtain the bacterial agent.

6. The preparation method according to claim 5, characterized in that, The fermentation medium includes LB liquid medium; the fermentation temperature is 28~32℃ and the time is 24~72h.

7. The agent for defending against Pseudomonas JR-4 as described in claim 1, or the agent for defending against Pseudomonas japonicus as described in any one of claims 2 to 4, or the agent for defending against Pseudomonas japonicus as described in claim 5 or 6, in inhibiting rice blast fungus ( Magnaporthe oryzae Applications in the prevention and control of rice blast.

8. The application according to claim 7, characterized in that, The rice blast fungus includes rice blast fungus YL22H7; the rice blast disease includes leaf blast.

9. The application according to claim 7 or 8, characterized in that, The rice blast fungus and the rice variety corresponding to the rice blast disease are both Dianheyou 615.

10. A method for preventing and controlling rice blast, characterized in that, The process includes the following steps: applying the anti-pseudomonas JR-4 agent as described in claim 1, or the agent as described in any one of claims 2 to 4, or the agent obtained by the preparation method described in claim 5 or 6 to rice crops.

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