A psyllium plant and its use in the prevention and control of rice diseases

Abstract

This invention discloses a plantago rhizosphere strain and its application in the control of rice diseases. The strain is *Pleurotus erythroptera* RSP5, and its classification name is *Pleurotus erythroptera* (…). Talaromyces purpureogenus The strain described in this application, with accession number CGMCC No. 41720, is a purple basket-forming bacterium RSP5 isolated from the rhizosphere soil of Plantago asiatica. This strain not only promotes the growth of rice plants but also exhibits good growth inhibition against rice sheath blight, rice blast, bacterial leaf streak, and bacterial blight. It can be used alone or in combination with pesticides for the control of rice-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a plantago rhizosphere strain and its application in the control of rice diseases. Background Technology

[0002] Rice sheath blight, rice blast, bacterial leaf streak, and bacterial leaf blight are important diseases of rice, affecting photosynthesis and nutrient transport, leading to plant wilting and reduced yield. Currently, the control of these rice diseases mainly relies on chemical fungicides. However, long-term use of chemical fungicides causes environmental pollution, disrupts the ecological balance, and results in pesticide residues. From an environmental protection perspective, biological control offers high specificity, is environmentally safe, and can better maintain the balance of the ecosystem. It represents a new approach to controlling rice diseases, reducing the use of pesticides and fertilizers while promoting increased grain production. Currently, there are few commercially available strains for the biological control of rice diseases.

[0003] Basket-shaped bacteria ( Talaromyces *Penicillium* sp. is an important type of fungus belonging to the kingdom Fungi, phylum Ascomycota, class Ascomycetes, order Ascomycetes, family Ascomycetes. Originally the sexual form of *Penicillium*, it possesses the potential to be a highly effective biocontrol microorganism. Some *Penicillium* sp. species exhibit enhanced disease resistance, stress tolerance, and growth promotion effects. However, there are currently no reports on the application of *Penicillium* sp. in the control of rice sheath blight, rice blast, bacterial leaf streak, and bacterial blight. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a Plantago asiatica rhizosphere strain and its application in rice disease control. This strain not only promotes rice plant growth but also exhibits good growth inhibition against *Rhizoctonia solani*, *Bacillus oryzae*, bacterial leaf streak, and *Bacillus thuringiensis*. It can be used alone or in combination with pesticides for the control of rice-related diseases. Specifically, the Plantago asiatica rhizosphere strain RSP5 primarily demonstrates competitive inhibition of the growth of *Rhizoctonia solani* and *Bacillus oryzae*.

[0005] The technical solution of the present invention is as follows: A plantago asiatica rhizosphere strain, RSP5, specifically *RSP5*, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41720. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is December 16, 2024. The suggested classification name is *RSP5*. Talaromyces purpureogenus This strain was independently isolated from the rhizosphere soil of plantain plants at the Bencao Shiguang Rice-Plantain Rotation Experimental Base in Yongxiu County, Jiujiang City, Jiangxi Province in May 2023.

[0006] Preferably, the 16S rRNA gene sequence of the purple basket-producing bacterium RSP5 is shown in SEQ ID NO: 1.

[0007] Preferably, single colonies of the purple basket-forming bacterium RSP5 are obtained by dilution and plating, and single colonies are picked and inoculated onto PDA medium and preserved in a refrigerator at 4 ℃.

[0008] Preferably, the culture conditions for the purple basket-producing bacterium RSP5 are: cultured in PDA medium at a temperature of 27°C.

[0009] Preferably, the main biological characteristics of the purple basket-producing bacterium RSP5 are: the aerial hyphae are white and filamentous; after being cultured on the culture medium for a period of time, the hyphae of the purple basket-producing bacterium RSP5 can break off on their own to form hyphal fragments.

[0010] The present invention also provides a spore liquid of a Plantago rhizosphere strain.

[0011] Preferably, the spore solution is obtained by culturing the purple basket-producing bacterium RSP5 on PDA medium. The culture temperature is 25-30 °C, and the culture time is 7-15 days.

[0012] Preferably, the concentration of the spore solution is 10. 5 ~10 7 CFU / mL.

[0013] This invention also provides a fermentation broth of a Plantago rhizosphere strain, comprising a bacterial fermentation broth and a sterile fermentation broth, the preparation method of which includes: inoculating Plantago rhizosphere strains into PDB medium for culture as a seed culture medium; inoculating the seed culture medium into PDB medium for culture to obtain a fermentation broth; and diluting the fermentation broth to a concentration of 10. 5 ~10 7 The bacterial fermentation broth containing CFU / mL was centrifuged to obtain the supernatant, which was then filtered to obtain the sterile fermentation broth.

[0014] Preferably, the culture conditions for the Plantago rhizosphere strains or seed culture medium include: culture in a constant temperature shaker at 25~30 ℃ and 100~150 rpm / min, with the culture time for the Plantago rhizosphere strains being 20~30 h and the culture time for the seed culture medium being 40~50 h.

[0015] Preferably, the centrifugation conditions for the bacterial fermentation broth include: centrifugation at 5000~10000 rpm / min for 8~15 min, and the filter membrane used for filtering the supernatant is a 0.2~0.5 μm microporous membrane.

[0016] The present invention also provides a microbial preparation comprising the aforementioned Plantago rhizosphere strain, spore liquid, or fermentation broth.

[0017] The present invention also provides a rice growth promoter, comprising the aforementioned Plantago rhizosphere strain, spore liquid, or fermentation broth.

[0018] The present invention also provides a rice disease control agent, comprising the aforementioned Plantago rhizosphere strain, spore liquid, or fermentation broth.

[0019] The present invention also provides a rice disease control agent, comprising a mixture of the aforementioned plantain rhizosphere strain, spore liquid or fermentation broth and pesticide.

[0020] The present invention also provides the application of the aforementioned Plantago rhizosphere strain, spore liquid or fermentation broth in promoting rice growth or controlling rice diseases.

[0021] The present invention also provides an application of the aforementioned Plantago rhizosphere strain, spore liquid or fermentation broth and pesticide mixture in the control of rice diseases.

[0022] Preferably, the rice diseases include any one or both of fungal and bacterial diseases.

[0023] Preferably, the fungal disease includes any one or both of rice sheath blight and rice blast.

[0024] Preferably, the bacterial disease includes any one or both of bacterial leaf streak and bacterial blight.

[0025] Preferably, the pesticide includes any one or more of jinggangmycin, tricyclazole, and thiamethoxam.

[0026] Preferably, in a mixture of plantain rhizosphere strains, spore liquid, or fermentation broth and pesticide, every 10 4 ~10 6 Add 4-1000 μg of pesticide to CFU Plantago asiatica rhizosphere strains and mix.

[0027] Preferably, the rice variety includes any one of Nipponbare, Xiangzhan, and Shen 9. More preferably, the rice variety is Nipponbare or Shen 9.

[0028] Preferably, the rice disease control includes inhibiting any one or more of the following: rice sheath blight fungus, rice blast fungus, bacterial leaf streak fungus, and bacterial blight fungus.

[0029] More preferably, the rice disease control includes competitively inhibiting the growth of rice sheath blight fungus or rice blast fungus.

[0030] Preferably, the application method in promoting rice growth or controlling rice diseases is any one or more of soil treatment, leaf treatment, seed treatment, and root treatment.

[0031] More preferably, the application method for promoting rice growth is soil treatment.

[0032] More preferably, the application method in rice disease control is leaf treatment.

[0033] The beneficial effects of this invention are: 1. The *Plantago asiatica* rhizosphere strain (purple-bearing bacterium) RSP5 isolated in this invention not only promotes the growth of rice plants but also exhibits good growth inhibition effects against *Rhizoctonia solani*, *Bacillus oryzae*, bacterial leaf streak, and *Bacillus thuringiensis*, making it applicable for the control of rice-related diseases. Specifically, *Plantago asiatica* rhizosphere strain RSP5 primarily demonstrates competitive inhibition of the growth of *Rhizoctonia solani* and *Bacillus oryzae*.

[0034] 2. Experiments have confirmed that the Plantago rhizosphere strain RSP5 of this invention promotes rice growth by 25.4%; inhibits the growth of sclerotia of rice sheath blight fungus by 40.8%, reducing the incidence of sheath blight by 33.4%; the colony diameter of the inhibition zone against rice blast fungus reaches 70.8 mm; the growth inhibition rate against rice bacterial leaf streak fungus reaches 87.7%; and the growth inhibition rate against rice bacterial blight fungus reaches 85.6%.

[0035] 3. The *Plantago asiatica* rhizosphere strain RSP5 of this invention can also be used in combination with pesticides for the control of rice-related diseases. Experiments have confirmed that the combination of *Plantago asiatica* rhizosphere strain RSP5 and pesticides can significantly inhibit the occurrence of fungal diseases (sheath blight, rice blast) and bacterial diseases (bacterial leaf streak, bacterial leaf blight) in rice, and reduce the development of lesions. Under certain disease control effects, the *Plantago asiatica* rhizosphere strain RSP5 can reduce the use of pesticides.

[0036] 4. Experiments show that the rhizosphere strain RSP5 of Plantago asiatica of the present invention has different disease control effects on different rice varieties. Its control effect on rice sheath blight infection of different varieties is as follows: Nipponbare > Shen 9 > Xiangzhan. Attached Figure Description

[0037] Figure 1 The mycelial morphology of Plantago asiatica rhizosphere strain RSP5; Figure 2 The spore morphology of Plantago asiatica rhizosphere strain RSP5; Figure 3 This illustrates the effect of Plantago asiatica rhizosphere strain RSP5 in Example 1 on inhibiting the mycelial growth of rice sheath blight fungus; Figure 4 This is an electrophoresis image of the PCR product of the Plantago rhizosphere strain RSP5 in Example 1 (B is a magnified view of the first column in A). Figure 5This is a phylogenetic tree diagram of the Plantago rhizosphere strain RSP5 constructed in Example 1 (RSP-5). Figure 6 This illustrates the effect of Plantago asiatica rhizosphere strain RSP5 on inhibiting the mycelial growth of rice blast fungus in Example 3. Figure 7 This illustrates the effect of Plantago asiatica rhizosphere strain RSP5 in Example 4 on inhibiting the mycelial growth of rice bacterial leaf streak fungus; Figure 8 This illustrates the effect of Plantago asiatica rhizosphere strain RSP5 in Example 5 on inhibiting the mycelial growth of rice bacterial blight fungus; Figure 9 The effect of Plantago asiatica rhizosphere strain RSP5 alone or in combination with pesticides on rice leaf infection with rice sheath blight in Example 6; Figure 10 The effect of Plantago asiatica rhizosphere strain RSP5 alone or in combination with pesticides on rice leaf infection with rice blast in Example 6; Figure 11 The effect of Plantago asiatica rhizosphere strain RSP5, alone or in combination with pesticides, on rice leaf infection with bacterial leaf streak in Example 6; Figure 12 The effect of Plantago asiatica rhizosphere strain RSP5 alone or in combination with pesticides on rice leaf infection with rice bacterial blight in Example 6; Figure 13 The effect of Plantago rhizosphere strain RSP5 on detached leaves of rice (Nipponbare) infected with sheath blight in Example 8; Figure 14 The effect of Plantago rhizosphere strain RSP5 on detached leaves of rice (Xiangzhan) infected with sheath blight in Example 8; Figure 15 The effect of Plantago rhizosphere strain RSP5 on detached leaves of rice (Shen 9) infected with sheath blight in Example 8. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0040] The rice rhizoctonia solani of this invention (Rhizoctonia solani) Thanatephorus Cucumeris The sample was obtained from the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO.3.724.

[0041] The rice blast fungus of the present invention ( Magnaporthe oryzae The strain is Guy11, strain number FXPS001; bacterial streak pathogen ( Xanthomonas oryzae pv. oryzicola , Xoc The strain is RS105, strain number FXPB002; bacterial blight pathogen ( Xanthomonas oryzae pv. oryzae , Xoo The strain number was FXPB004; all were purchased from Hangzhou Fengxun Biotechnology Co., Ltd.

[0042] The potato dextrose agar (PDA) culture medium of this invention is formulated as follows: 1 L of 20% potato juice, 20.0 g of glucose, 3 g of KH2PO4, 1.5 g of MgSO4•7H2O, 8.0 mg of vitamin B1, 20.0 g of agar, and natural pH. Preparation method of 20% potato juice: Take 200.0 g of peeled potatoes, cut them into small pieces, add 1 L of water and boil for 20 minutes. Filter out the potato pieces, and add the filtrate to make up to 1 L.

[0043] The potato dextrose broth (PDB) culture medium of the present invention is formulated as follows: 1 L of 30% potato juice and 20.0 g of glucose. Preparation method of 30% potato juice: Take 300.0 g of peeled potatoes, cut them into small pieces, add 1 L of water and boil for 20 minutes. Filter out the potato pieces, and add the filtrate to make up to 1 L.

[0044] The nutrient agar culture medium formula of the present invention is as follows: 10 g peptone, 3 g beef extract powder, 5 g sodium chloride, 15 g agar, add 1 L water, stir and heat to boiling until completely dissolved, add the filtrate to 1 L, dispense into test tubes or Erlenmeyer flasks, and autoclave at 121 °C for 15 min.

[0045] The formula for the glucose-yeast-beef extract culture medium of the present invention is as follows: 10 g glucose, 10 g yeast extract, 4 g beef extract, 4 g peptone, 2.5 g sodium chloride, 30 g agar, 1 L water, stirring and heating to boiling until completely dissolved, adding the filtrate to 1 L, dispensing into test tubes or Erlenmeyer flasks, and autoclaving at 121 °C for 15 min.

[0046] Example 1: Isolation and Identification of Plantago rhizosphere Strains RSP5 1. Isolation of Plantago rhizosphere strain RSP5 In May 2023, soil samples were collected from the rhizosphere of *Plantago asiatica* plants at the Bencao Shiguang Rice-Plantago asiatica Rotation Experimental Base in Yongxiu County, Jiujiang City, Jiangxi Province, using a 5-point sampling method. The collected soil samples were quickly placed into numbered plastic bags and brought back to the laboratory for separation. After rinsing the soil samples three times with sterile water, 1.0 g of soil sample was weighed and mixed with 9 mL of sterile water in a laminar flow hood, sonicated for 3 min, and then ground. The supernatant of the homogenate was then subjected to 10... -1 10 -2 10 -3 10 -4 Serial dilutions were performed, with 100 μL of each diluted soil sample solution spread onto solid PDA agar plates. Each dilution was repeated once, and the plates were incubated upside down at 27 ℃. Based on colony morphology (color, size, elevation, edge characteristics, etc.), single colonies of different morphologies were selected and purified using PDA medium through streak plating. The purified Plantago rhizosphere bacteria were then screened for strains with good resistance to rice sheath blight using a plate confrontation experiment.

[0047] The plate confrontation experiment was conducted as follows: Purified Plantago rhizosphere fungi were evenly ablated into circular mycelial discs with a diameter of 6 mm from the outer edge of the colony using a sterilized punch. These mycelial discs were then inoculated into the center of a 90 mm diameter solid PDA medium plate. Rice sheath blight pathogens were then streaked around the plate 35 mm from the center. Solid PDA medium plates without Plantago rhizosphere fungal discs served as a control group. Each treatment was repeated three times. The plates were incubated upside down in a 28℃ incubator for 2–7 days, and the diameter of colonies with a clear inhibition zone was observed and measured. Strains exhibiting good inhibitory effects on the growth of Rice sheath blight pathogens were screened.

[0048] As a result, a plantago rhizosphere strain, RSP5, was selected. Figure 1 The aerial hyphae are white and filamentous. After being cultured on a medium for a period of time, the hyphae of *RSP5* spontaneously break apart, forming hyphal fragments. Its spore morphology is as follows... Figure 2 The spores are round.

[0049] The results of the rhizosphere strain RSP5 of Plantago asiatica inhibiting rice sheath blight are shown in Figure 3 The colony diameter of its inhibition zone was 75.5 mm, indicating that it had a good inhibitory effect on the growth of rice sheath blight pathogen. Under sufficient nutrient conditions, the growth of rice sheath blight pathogen was inhibited, and its antibacterial mechanism was mainly manifested in the competitive inhibition of rice sheath blight pathogen growth by the rhizosphere strain RSP5 of Plantago asiatica.

[0050] 2. Identification of Plantago rhizosphere strain RSP5 Genomic DNA was extracted from the rhizosphere strain RSP5 of Plantago asiatica using a bacterial genomic DNA extraction kit, following the instructions. The DNA was then amplified using a PCR instrument. Primer design, PCR reaction system, and amplification program are shown in Tables 1-3.

[0051] Table 1 Primer Design

[0052] Table 2 PCR amplification reaction system

[0053] Table 3 PCR amplification reaction procedure

[0054] After PCR amplification, 3 μL of the PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR amplification fragments. The electrophoresis image of the PCR product of Plantago rhizosphere strain RSP5 is shown below. Figure 4 Bright bands were detected.

[0055] The rhizosphere strain RSP5 of Plantago asiatica was collected and 16S rDNA sequencing was performed using an ABI3730-XL sequencer (commissioned by Shenzhen Microcom Technology Group Co., Ltd.). The sequence of Plantago asiatica rhizosphere strain RSP5 is shown in SEQ ID NO: 1.

[0056] The 16S rDNA sequence of the obtained Plantago rhizosphere strain RSP5 was compared with data in the NCBI nucleic acid database, and a phylogenetic tree was constructed using MEGA11 software (see...). Figure 5 The species information with the highest sequence similarity to the target species is obtained, which is the result of the strain identification. The final identification of the Plantago rhizosphere strain RSP5 as *Phyllostachys purpureus* (98% gene sequence similarity) was performed. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41720, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on December 16, 2024. The recommended classification name is *Phyllostachys purpureus*. Talaromyces purpureogenus ).

[0057] Example 2: Effects of Plantago rhizosphere strain RSP5 on sclerotia of rice sheath blight pathogen. Rice sheath blight pathogens were inoculated onto solid PDA medium plates and incubated at 28 ℃ for 10 days. Then, spores of the pathogens were scraped from the plates using a spreader to prepare a concentration of 1×10⁻⁶. 6 CFU / mL rice sheath blight spore suspension, stored at 4 ℃ for later use.

[0058] The rhizosphere strain RSP5 of Plantago asiatica was inoculated onto solid PDA medium plates and cultured at 28 ℃ for 10 days. Then, Plantago asiatica rhizosphere spores were scraped from the plates using a spreader to prepare a solution with a concentration of 1×10⁻⁶. 6 A CFU / mL suspension of Plantago asiatica rhizosphere strain RSP5 spores was stored at 4 ℃ for later use.

[0059] The experiment consisted of two culture groups: Culture group 1: Add 100 μL of rice sheath blight spore suspension and 100 μL of sterile water to 100 mL of PDB medium; Culture group 2: Add 100 μL of Plantago rhizosphere strain RSP5 spore suspension and 100 μL of rice sheath blight spore suspension to 100 mL of PDB medium.

[0060] Each culture group was set up in 3 replicates. After culturing in a constant temperature shaker at 28 ℃ and 150~180 rpm / min for 10 days, the number of sclerotia of rice sheath blight was counted, and the growth inhibition rate of the rhizosphere strain RSP5 of Plantago asiatica against the sclerotia of rice sheath blight was calculated. The results are shown in Table 4.

[0061] Inhibition rate (%) = 100% × (number of sclerotia in culture group 1 - number of sclerotia in culture group 2) / number of sclerotia in culture group 1.

[0062] Table 4. Effects of each group on the sclerotia of rice sheath blight pathogen ( (n=3)

[0063] As shown in Table 4, the rhizosphere strain RSP5 of Plantago asiatica inhibited the growth of sclerotia of rice sheath blight by 40.8%, which can effectively inhibit the growth of sclerotia of rice sheath blight.

[0064] Example 3: Plate confrontation experiment of Plantago asiatica rhizosphere strain RSP5 against rice blast fungus Purified Plantago rhizosphere bacteria were evenly ablated into 6 mm diameter circular mycelial cakes from the outer edge of the colony using a sterilized punch. These mycelial cakes were then inoculated into the center of 90 mm diameter solid PDA agar plates. Rice blast fungus was streaked around the perimeter of the plate 35 mm from the center. Solid PDA agar plates without Plantago rhizosphere mycelial cakes served as a control group. Each treatment was repeated three times. The plates were incubated upside down in a 28°C incubator for 2–7 days, and the colony diameter of the inhibition zone was observed and measured.

[0065] See results Figure 6The inhibition zone of the Plantago asiatica rhizosphere strain RSP5 had a colony diameter of 70.8 mm, indicating that it had a good inhibitory effect on the growth of rice blast fungus. Under sufficient nutrient conditions, the growth of rice blast fungus was inhibited, and its antibacterial mechanism was mainly manifested in the competitive inhibition of rice blast fungus growth by Plantago asiatica rhizosphere strain RSP5.

[0066] Example 4: Effect of Plantago rhizosphere strain RSP5 on the growth of bacterial leaf streak causal agent. A 6 mm diameter mycelium of the Plantago asiatica rhizosphere strain RSP5 was inoculated into PDB medium and cultured at 28 ℃ and 130 rpm / min for 24 h to obtain the seed culture. 100 μL of the seed culture was inoculated into an Erlenmeyer flask containing 150 mL of PDB medium and cultured at 28 ℃ and 130 rpm / min for 48 h to obtain the fermentation broth of Plantago asiatica rhizosphere strain RSP5. The fermentation broth of Plantago asiatica rhizosphere strain RSP5 was diluted to a concentration of 1×10⁻⁶. 6 Fermentation broth containing the plantago rhizosphere strain RSP5 (CFU / mL) was prepared for use.

[0067] Bacterial leaf spot pathogens were inoculated onto nutrient agar slant medium and incubated at 28 °C for 24 h. The slant culture was then washed with PBS and diluted to 1 × 10⁻⁶. 9 CFU / mL bacterial streak suspension is available for use.

[0068] 100 μL of bacterial leaf streak suspension was transferred to a 96-well cell culture plate, and 100 μL of fermentation broth containing Plantago rhizosphere strain RSP5 was added to each well. The mixture was thoroughly mixed to obtain a mixed bacterial suspension (RSP5 fermentation broth group). A control group containing 100 μL of sterile PDB medium was used. Both groups were co-cultured at 28 ℃ for 24 h. After 24 h, the mixed bacterial suspension was transferred to 90 mm inner diameter culture dishes and inoculated onto nutrient agar medium using the pour method. The mixture was cultured continuously for 48 h, with each treatment repeated three times. Results are shown below. Figure 7 Compared with the control group, the growth of bacterial streak pathogens in the RSP5 fermentation broth group was significantly inhibited.

[0069] The number of bacterial streak causal agents in each group was counted, and the inhibition rate (%) was calculated as follows: = 100% × (number of colonies in the control group - number of colonies in the RSP5 fermentation broth group) / number of colonies in the control group. The results showed that the inhibition rate of the RSP5 fermentation broth containing Plantago asiatica rhizosphere strain against bacterial streak causal agents was 87.7%.

[0070] Example 5: Experiment on the effect of Plantago asiatica rhizosphere strain RSP5 on the growth of bacterial blight pathogen. 1×10 was prepared according to the method in Example 4. 6 Fermentation broth containing the plantago rhizosphere strain RSP5 (CFU / mL) was prepared for use.

[0071] Bacterium oxysporum, the pathogen of bacterial blight, was inoculated into glucose yeast extract and beef extract agar slant culture medium and incubated at 28 ℃ for 24 h. The slant culture was then washed with PBS and diluted to 1×10⁻⁶. 9 CFU / mL suspension of bacterial blight pathogen is available for use.

[0072] Remove 1×10 9 100 μL of CFU / mL *Bacillus thuringiensis* suspension was added to a 96-well cell culture plate, followed by 100 μL of *Plantago asiatica* rhizosphere strain RSP5 fermentation broth in each well. The mixture was thoroughly mixed to obtain a mixed bacterial suspension (RSP5 fermentation broth group). 100 μL of sterile PDB medium was used as a control group. The mixtures were thoroughly mixed and co-cultured at 28 ℃ for 24 h. After 24 h, the mixed bacterial suspension was transferred to 90 mm inner diameter culture dishes and inoculated onto glucose yeast extract / beef extract medium using the pour method. The culture was continued for 48 h, with each treatment repeated three times. Results are shown below. Figure 8 Compared with the control group, the growth of bacterial blight pathogens in the RSP5 fermentation broth group was significantly inhibited.

[0073] The number of bacterial blight colonies in each group was counted, and the inhibition rate of bacterial blight was calculated according to the method in Example 4. The results showed that the fermentation broth containing the Plantago rhizosphere strain RSP5 had an inhibition rate of 85.6% against bacterial leaf streak.

[0074] Example 6: Effects of Plantago asiatica rhizosphere strains alone or in combination with pesticides on rice leaf infection with rice sheath blight, rice blast, bacterial leaf streak, and bacterial blight. 1. Basic Test Methods A 6 mm diameter mycelium of the Plantago asiatica rhizosphere strain RSP5 was inoculated into PDB medium and cultured at 28 ℃ and 130 rpm / min for 24 h as a seed culture. 100 μL of this seed culture was inoculated into an Erlenmeyer flask containing 150 mL of PDB medium and cultured at 28 ℃ and 130 rpm / min for 48 h to obtain the fermentation broth of Plantago asiatica rhizosphere strain RSP5. The fermentation broth of Plantago asiatica rhizosphere strain RSP5 was then diluted to a concentration of 1×10⁻⁶. 6 CFU / mL, 2×10 6 Fermentation broth containing *Plantaina rhizosphere strain RSP5* (CFU / mL) was prepared for use. 60 mL of the fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm microporous membrane for sterilization, yielding 1×10⁻⁶ CFU / mL of the culture. 6 CFU / mL, 2×10 6 Aseptic fermentation broth of Plantago rhizosphere strain RSP5 at CFU / mL.

[0075] The selected rice variety was Xiangzhan. Healthy rice leaves with equal tillering stage and uniform growth were taken, and both ends were cut off, leaving a 5cm leaf segment. The leaves were first disinfected with 2.5% sodium hypochlorite for 30 seconds, and then washed 3 times with sterile water. They were randomly divided into 9 treatment groups and 1 blank control group (inoculated with the corresponding culture medium). Rice leaves from nine different treatment groups were immersed for 20 min in 10 mL of RSP5 bacterial fermentation broth, RSP5 sterile fermentation broth, RSP5 bacterial fermentation broth + high-dose pesticide, RSP5 bacterial fermentation broth + low-dose pesticide, RSP5 sterile fermentation broth + high-dose pesticide, RSP5 sterile fermentation broth + low-dose pesticide, high-dose pesticide, low-dose pesticide, and sterile water (model group). Then, the rice leaves were removed and placed in petri dishes with sterile moistened filter paper, with three rice leaves in each dish. 6 mm diameter mycelium of rice sheath blight, rice blast, rice bacterial blight, and rice bacterial leaf streak were inoculated into the middle of the rice leaves of each treatment group. Each treatment was repeated three times. Separately, 6 mm diameter culture medium was inoculated into the middle of rice leaves as a blank control group. The leaves were sealed with sealing film and cultured for 7 days at 28 ℃ under 14 h light / 10 h dark conditions. The disease incidence on rice leaves was observed, and the relative lesion length of each group was calculated. Relative lesion length = 100% × (total lesion length / total leaf length).

[0076] in: Take 5 mL of RSP5 fermentation broth containing bacteria (2×10⁻⁶) 6 Mix 5 mL of high-dose pesticide with (CFU / mL) to obtain the RSP5 bacterial fermentation broth + high-dose pesticide group.

[0077] Take 5 mL of RSP5 fermentation broth containing bacteria (2×10⁻⁶) 6 Mix 5 mL of low-dose pesticide with (CFU / mL) to obtain the RSP5 bacterial fermentation broth + low-dose pesticide group.

[0078] Take 5 mL of RSP sterile fermentation broth (2×10⁻⁶) 6 Mix 5 mL of high-dose pesticide with (CFU / mL) to obtain the RSP sterile fermentation broth + high-dose pesticide group.

[0079] Take 5 mL of RSP sterile fermentation broth (2×10⁻⁶) 6 Mix 5 mL of low-dose pesticide with (CFU / mL) to obtain the RSP sterile fermentation broth + low-dose pesticide group.

[0080] Take 10 mL of RSP5 fermentation broth containing bacteria (1×10) 6 The CFU / mL count represents the RSP5 bacterial fermentation broth group.

[0081] Take 10 mL of RSP5 sterile fermentation broth (1×10⁻⁶) 6The CFU / mL ratio represents the RSP5 aseptic fermentation broth group.

[0082] 2. Rice leaves infected with rice sheath blight Jinggangmycin, a commonly used pesticide for controlling rice sheath blight (Shanghai Yuanye Biotechnology Co., Ltd., batch number: J24HS189094), was selected. A final concentration of jinggangmycin of 400 μg / mL was designated as the high-dose group, and a final concentration of 4 μg / mL was designated as the low-dose group. The blank control group used PDA medium. Results are shown below. Figure 9 See Table 5.

[0083] Table 5. Effects of Plantago asiatica rhizosphere strain RSP5 alone or in combination with jinggangmycin on the relative length of rice sheath blight lesions ( (n=9)

[0084] Table 5 shows that the rhizosphere strain RSP5 of Plantago asiatica, used alone or in combination with pesticides, can significantly inhibit the occurrence of rice sheath blight and reduce the development of lesions. In particular, the combination of RSP5 fermentation broth containing the bacteria and a high dose of jinggangmycin is superior, showing a significantly better control effect against sheath blight than other treatment groups. Among them, the fermentation broth containing the bacteria of RSP5 and high or low doses of jinggangmycin, as well as the sterile fermentation broth of RSP5 and low doses of jinggangmycin, showed significant synergistic effects.

[0085] In addition, the control effect of RSP5 sterile fermentation broth + high dose of jinggangmycin on sheath blight was comparable to that of the high dose of jinggangmycin group. Although the synergistic effect of RSP5 sterile fermentation broth and high dose of pesticide jinggangmycin was not obvious, under the same sheath blight control effect, the rhizosphere strain RSP5 of Plantago asiatica can still reduce the use of pesticide jinggangmycin.

[0086] 3. Rice leaves infected with rice blast disease Tricyclazole (75% wettable powder), a commonly used pesticide for controlling rice blast, was selected from Jiangsu Fengdeng Aseptic Protection Co., Ltd., batch number: 20240726A33080. A final tricyclazole concentration of 1000 μg / mL was designated as the high-dose group, and a final concentration of 10 μg / mL was designated as the low-dose group. The blank control group used PDA medium. Results are shown below. Figure 10 See Table 6.

[0087] Table 6. Effects of Plantago asiatica rhizosphere strain RSP5 alone or in combination with tricyclazole on the relative length of rice blast lesions ( (n=9)

[0088] Table 6 shows that the rhizosphere strain RSP5 of Plantago asiatica, used alone or in combination with pesticides, can significantly inhibit the occurrence of rice blast and reduce the development of lesions. Furthermore, the control effect of Plantago asiatica rhizosphere strain RSP5 combined with tricyclazole on rice blast is superior to the corresponding single treatment groups, demonstrating a significant synergistic effect.

[0089] Among them, the rice blast control effect of the RSP5 bacterial fermentation broth + tricyclazole low dose group was even better than that of the tricyclazole high dose group. Under a certain rice blast control effect, the Plantago rhizosphere strain RSP5 can significantly reduce the use of the pesticide tricyclazole.

[0090] 4. Rice leaves infected with bacterial leaf streak Thiazole zinc, a commonly used pesticide for controlling bacterial leaf streak in rice (Zhejiang Xinong Chemical Co., Ltd., batch number: 202509161613), was selected. A final concentration of 666.7 μg / mL was designated as the high-dose group, and a final concentration of 6.7 μg / mL was designated as the low-dose group. The blank control group used nutrient agar medium. Results are shown below. Figure 11 See Table 7.

[0091] Table 7. Effects of Plantago asiatica rhizosphere strain RSP5 alone or in combination with thiamethoxam zinc on the relative length of bacterial leaf streak in rice. (n=9)

[0092] Table 7 shows that the rhizosphere strain RSP5 of Plantago asiatica, used alone or in combination with pesticides, can significantly inhibit the occurrence of bacterial leaf streak in rice and reduce the development of lesions. In particular, the group containing RSP5 fermentation broth plus a high dose of thiamethoxam zinc showed better control of bacterial leaf streak than other treatment groups. Among them, the RSP5 fermentation broth and the high dose of thiamethoxam zinc showed a significant synergistic effect.

[0093] Furthermore, although the synergistic effect of RSP5 bacterial fermentation broth with low-dose thiamethoxam and the aseptic fermentation broth of RSP5 with high or low doses of thiamethoxam was not significant, the control effect of RSP5 bacterial fermentation broth + low-dose thiamethoxam on bacterial leaf streak was still higher than that of the low-dose thiamethoxam group. The control effect of RSP5 aseptic fermentation broth + high-dose thiamethoxam on bacterial leaf streak was comparable to that of the high-dose thiamethoxam group. The control effect of RSP5 aseptic fermentation broth + low-dose thiamethoxam on bacterial leaf streak was comparable to that of the low-dose thiamethoxam group. Under certain control effects on bacterial leaf streak, the rhizosphere strain RSP5 of Plantago asiatica can still reduce the use of the pesticide thiamethoxam.

[0094] 5. Rice leaves infected with rice bacterial blight Thiazole zinc, a commonly used pesticide for controlling bacterial blight in rice (Zhejiang Xinong Chemical Co., Ltd., batch number: 202509161613), was selected. A final concentration of 666.7 μg / mL was designated as the high-dose group, and a final concentration of 6.7 μg / mL was designated as the low-dose group. The blank control group used glucose yeast extract and beef extract agar. Results are shown below. Figure 12 See Table 8.

[0095] Table 8. Effects of Plantago asiatica rhizosphere strain RSP5 alone or in combination with thiamethoxam zinc on the relative lesion length of rice bacterial blight ( (n=9)

[0096] Table 8 shows that the rhizosphere strain RSP5 of Plantago asiatica, used alone or in combination with pesticides, can significantly inhibit the occurrence of bacterial blight in rice and reduce the development of lesions. Furthermore, the control effect of Plantago asiatica rhizosphere strain RSP5 combined with thiamethoxam zinc on bacterial blight is better than that of the corresponding single treatment groups, showing a significant synergistic effect.

[0097] Among them, the control effect of RSP5 bacterial fermentation broth + low dose of thiamethoxam zinc on bacterial blight was even better than that of the high dose of thiamethoxam zinc group. Under a certain control effect on bacterial blight, the rhizosphere strain RSP5 of Plantago asiatica can significantly reduce the use of pesticide thiamethoxam zinc.

[0098] 6. Conclusion The above results indicate that the rhizosphere strain RSP5 of Plantago asiatica, used alone or in combination with pesticides, can significantly inhibit the occurrence of fungal diseases (sheath blight, rice blast) and bacterial diseases (bacterial leaf streak, bacterial leaf blight) in rice, and reduce the development of lesions. Under certain conditions of effective control of rice diseases, the rhizosphere strain RSP5 of Plantago asiatica can reduce pesticide use to varying degrees.

[0099] Example 7: Effects of Plantago asiatica rhizosphere strain RSP5 on rice growth Prepare concentrations of 1×10 according to the method in Example 2. 6 CFU / mL rice sheath blight spore suspension and Plantago rhizosphere strain RSP5 spore suspension, stored at 4℃ for later use.

[0100] The selected rice variety was Nipponbare. Rice seeds were disinfected and pre-germinated. When the radicle reached 1 cm in length, they were transplanted into seedling pots. When the seedlings reached the two-leaf stage, rice seedlings with similar growth and height were selected for the experiment. Every 5 days, 5000 μL each of a suspension of rice sheath blight spores and a suspension of Plantago rhizosphere strain RSP5 spores were applied to the nutrient soil, for a total of 5 applications. Each treatment consisted of 6 seedlings, with 3 replicates. Sterile water was used as a control. The group setups are as follows: (1) Control group: Irrigated with 10,000 μL of sterile water; (2) Treatment group 1: Irrigation with 5000 μL sterile water + 5000 μL rice sheath blight spore suspension; (3) Treatment group 2: 5000 μL sterile water + 5000 μL Plantago rhizosphere strain RSP5 spore suspension for irrigation; (4) Treatment group 3: 5000 μL rice sheath blight spore suspension + 5000 μL plantain rhizosphere strain RSP5 spore suspension for irrigation.

[0101] Thirty days later, the fresh weight and disease incidence of rice seedlings in each group were observed and recorded. The growth promotion rate and the incidence of sheath blight were calculated. The effect of the RSP5 spore suspension of Plantago asiatica on rice growth was studied. The results are shown in Table 9.

[0102] The growth promotion rate and the incidence of sheath blight are calculated using the following formula: Growth promotion rate (%) = 100% × (fresh weight of rice seedlings in different treatment groups - fresh weight of rice seedlings in the control group) / fresh weight of rice seedlings in the control group.

[0103] Incidence rate of sheath blight (%) = 100% × (number of healthy seedlings in the control group - number of healthy seedlings in different treatment groups) / number of healthy seedlings in the control group.

[0104] Table 9. Effects of each group on rice growth and sheath blight incidence ( (n=18)

[0105] As shown in Table 9, the rhizosphere strain RSP5 of Plantago asiatica in treatment group 2 promoted rice growth by 25.4%. Compared with treatment group 1, the rhizosphere strain RSP5 of Plantago asiatica in treatment group 3 reduced the incidence of rice sheath blight by 33.4%. This indicates that the rhizosphere strain RSP5 of Plantago asiatica has the effect of promoting rice plant growth and inhibiting rice sheath blight.

[0106] Example 8: Effects of Plantago rhizosphere strain RSP5 on detached leaves of different rice varieties infected with sheath blight Prepare 1×10 according to the method of Example 6 6 Both the fermentation broth containing CFU / mL of Plantago asiatica rhizosphere strain RSP5 and the sterile fermentation broth of RSP5 are prepared for use.

[0107] Healthy rice leaves at the tillering stage with uniform growth were collected, and both ends were cut off, leaving a 5cm leaf segment. The leaves were first disinfected with 2.5% sodium hypochlorite for 30 seconds, then rinsed three times with sterile water. They were randomly divided into three treatment groups and one blank control group (PDA medium). The rice leaves from the three treatment groups were immersed in 10 mL of RSP5 bacterial fermentation broth, RSP5 sterile fermentation broth, and sterile water (model group), respectively, for 20 minutes. The rice leaves were then removed and placed in petri dishes lined with sterile, moistened filter paper, with three leaves placed in each dish. A 6mm diameter rice sheath blight fungal cake, cultured for 3 days, was inoculated into the center of each treatment group's rice leaves. A separate 6mm diameter PDA medium was inoculated into the center of the rice leaves as a blank control group. The dishes were sealed with sealing film and cultured at 28℃ under 14 h light / 10 h dark conditions for 7 days. The disease incidence on the rice leaves was observed, and the relative lesion length for each group was calculated, with three dishes per group. Relative lesion length = 100% × (total lesion length / total leaf length). Results are shown in Table 10 and... Figures 13-15 .

[0108] Table 10. Effects of each group on the relative lesion length (%) of the three rice varieties ( (n=9)

[0109] The results above show that both the fermentation broth containing the plantago rhizosphere strain RSP5 and the sterile fermentation broth containing RSP5 have a control effect on the leaves of rice varieties Nipponbare, Xiangzhan, and Shen 9 infected with sheath blight. The fermentation broth containing the plantago rhizosphere strain RSP5 is significantly better than the sterile fermentation broth containing RSP5. Furthermore, the control effect of plantago rhizosphere strain RSP5 on different rice varieties infected with sheath blight is in the following order: Nipponbare > Shen 9 > Xiangzhan.

[0110] Meanwhile, the results of the detached rice leaf experiment showed that the metabolites of the Plantago rhizosphere strain RSP5 have a certain allelopathic effect on rice sheath blight pathogen, and this strain may exert its effect on the sheath blight pathogen through its own metabolic secretions.

[0111] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A plantago rhizosphere strain, characterized in that: The strain was RSP5, and its classification name was *Purple Basket-producing* (RSP5). Talaromyces purpureogenus (The accession number is CGMCC No.41720).

2. A spore liquid or fermentation broth of the Plantago rhizosphere strain according to claim 1.

3. The spore liquid or fermentation broth according to claim 2, characterized in that: Its concentration is 10 5 ~10 7 CFU / mL.

4. The fermentation broth according to claim 2, characterized in that: The fermentation broth includes a bacterial fermentation broth and a sterile fermentation broth, and its preparation method includes: inoculating Plantago asiatica rhizosphere strains into PDB medium for culture as a seed culture medium; inoculating the seed culture medium into PDB medium for culture to obtain the fermentation broth; and diluting the fermentation broth to a concentration of 10. 5 ~10 7 The bacterial fermentation broth containing CFU / mL was centrifuged to obtain the supernatant, which was then filtered to obtain the sterile fermentation broth.

5. A microbial preparation, characterized in that: This includes the Plantago rhizosphere strain as described in claim 1, or the spore liquid or fermentation broth as described in claim 2.

6. The application of the Plantago rhizosphere strain of claim 1, or the spore liquid or fermentation broth of claim 2, in promoting rice growth or controlling rice diseases, characterized in that: The rice diseases mentioned include any one or more of the following: rice sheath blight, rice blast, bacterial leaf streak, and bacterial leaf blight.

7. The application of the Plantago rhizosphere strain of claim 1, or the mixture of spore liquid or fermentation broth of claim 2 and pesticide in the control of rice diseases, characterized in that: The pesticides include any one or more of jinggangmycin, tricyclazole, and thiamethoxam.

8. The application according to claim 6, characterized in that: The rice variety includes any one of Nipponbare, Kazan, and Shen 9.

9. The application according to claim 6, characterized in that: The rice disease control measures include competitively inhibiting the growth of rice sheath blight fungus or rice blast fungus.

10. The application according to claim 8, characterized in that: The rice varieties mentioned include Nipponbare or Shen 9.

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