Strain R3 of spathiphyllum conifer and complex microbial inoculant thereof, and application of strain R3 and complex microbial inoculant in prevention and control of late blight

Abstract

The invention provides a spathiphyllum conifer strain R3, a complex microbial inoculant thereof and application of the spathiphyllum conifer strain R3 in prevention and control of late blight, and belongs to the technical field of biocontrol bacteria. The invention relates to a spathiphylli strain R3, and the preservation number of the spathiphylli strain R3 is CGMCC (China General Microbiological Culture Collection Center) No.36025. The spathiphylli strain R3 has the advantages that the preservation number is CGMCC No.36025; the bacterial strain R3 has good bacteriostatic activity on phytophthora infestans, and meanwhile, the biocontrol agent formed by compounding the bacterial strain R3 with at least one of the bacillus safensis bacterial strain B1 and the leisei bacterial strains L1 and L3 or the combination of the bacterial strain R3 and the L6 has good prevention and control effects in pot experiments and field experiments.

Description

Technical Field

[0001] This application belongs to the field of biocontrol bacteria technology, specifically involving a strain of *Spathobacterium tumefaciens* (…). Rhodanobacter spathiphylli Strain R3 and its compound inoculant and its application in the prevention and control of late blight. Background Technology

[0002] Late blight is caused by the pathogen Phytophthora (Phytophthora infestans). Phytophthora infestans Late blight (Phytophthora indicum) is a disease with a wide range of infectivity, affecting multiple organs of the plant, including leaves, stems, and fruits. It can occur from the seedling stage to the mature plant stage. Late blight develops rapidly; under suitable conditions (high humidity, low temperature), the disease can spread to the entire plant within days, even causing large-scale outbreaks in the field. After infection, photosynthesis is hindered, leading to fruit rot. Infected fruits develop brown hard spots or rot, losing their commercial value; even mild infections reduce the fruit's storability. Furthermore, the pathogen can spread through air currents, rainwater, irrigation water, and agricultural operations, easily leading to secondary infections in the field, making control difficult. The primary hosts of Phytophthora indicum are Solanaceae crops, with potatoes and tomatoes being the most affected. Some wild Solanaceae weeds (such as groundcherry and black nightshade) can also serve as intermediate hosts, providing sources of infection for disease transmission.

[0003] While there are reports on various biocontrol bacteria for late blight, there are currently no reports on the biocontrol efficacy of Spatholobus suberectus, nor on its role in controlling late blight. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Spathobacterium tumefaciens R3 that has the activity of inhibiting pathogenic Phytophthora, and exerts a biocontrol effect on the control of late blight in the field.

[0005] This invention provides a strain of *Spatholobus suberectus* (Spatholobus suberectus). Rhodanobacter spathiphylli The strain R3, with accession number CGMCC No.36025, is used.

[0006] This invention provides a biocontrol composition comprising Bacillus sabolicii (Bacillus safranin) Bacillus safensis ) strain B1 or Reissella genus ( Leifsonia sp. strain L1 and strain R3 of *Spatholobus suberectus*; The preservation number of the *Bacillus sarcodactylis* strain B1 is CGMCC No. 36024; The preservation number of the Reiberella strain L1 is CGMCC No. 36021.

[0007] Preferably, when strain B1 is included, it also includes one of the following: Reissella strain L1, Reissella strain L3 and Reissella strain L6. The preservation number of the Reissella strain L1 is CGMCC No. 36021; The preservation number of the Reissella strain L3 is CGMCC No. 36022; The preservation number of the Reiberella strain L6 is CGMCC No. 36023.

[0008] Preferably, when strain B1 is included, it also includes at least one of Reissella strain L1 and Reissella strain L6 and Reissella strain L3. The preservation number of the Reissella strain L1 is CGMCC No. 36021; The preservation number of the Reissella strain L3 is CGMCC No. 36022; The preservation number of the Reiberella strain L6 is CGMCC No. 36023.

[0009] The present invention provides a biocontrol agent comprising the *Spatholobus suberectus* strain R3 or the biocontrol composition and excipients.

[0010] Preferably, the biocontrol agent includes an aqueous solution and / or a dry powder.

[0011] Preferably, the number of viable bacteria in the biocontrol agent is not less than 10. 8 CFU / g or 10 8 CFU / mL.

[0012] This invention provides the use of the *Phytophthora indicum* strain R3 or the biocontrol composition in the preparation of pathogenic *Phytophthora* (…). Phytophthora infestans Application in antibacterial agents.

[0013] This invention provides the application of the *Spathobacterium tumefaciens* strain R3, the biocontrol composition, or the control agent in the prevention and control of late blight.

[0014] Preferably, the late blight includes at least one of the following: potato late blight, tomato late blight, eggplant late blight, and pepper late blight.

[0015] This invention provides a strain of *Spatholobus suberectus* (Spatholobus suberectus). Rhodanobacter spathiphylli Strain R3, with accession number CGMCC No. 36025, was identified by morphological observation and molecular identification as belonging to *Spatholobus suberectus* (Spatholobus suberectus). Rhodanobacter spathiphylli Plate inhibition experiments showed that strain R3 was effective against pathogenic *Phytophthora* (…). Phytophthora infestans It exhibits good antibacterial activity. Furthermore, pot experiments and field experiments show that strain R3 has a good control effect against late blight. The strain R3 provided by this invention offers a new means for the biological control of late blight.

[0016] This invention provides a biocontrol composition, consisting of Bacillus salsa (… Bacillus safensis The combination formed by strain B1 alone or in combination with Reissler L1 or Reissler L3, or further in combination with Reissler L1 and Reissler L3, and Reissler L6, and strain R3, is more effective against pathogenic *Phytophthora* than strain R3. Phytophthora infestans It exhibits stronger antibacterial activity; furthermore, pot experiments and field experiments show that the biocontrol composition has better control effect against late blight. The biocontrol composition provided by this invention offers a new approach for the biological control of late blight. Attached Figure Description

[0017] Figure 1 The images show the colony morphology of the strains screened in this invention during plate culture. Figure 2 Phylogenetic trees constructed for the strains screened in this invention; Figure 3 These are the results of a plate antibacterial experiment. Figure 4 Diagram of bacterial microbial community composition; Figure 5 Diagram of fungal microbial community composition; Figure 6 The control (0 d) amplifies the electrophoresis results of microorganisms on the surface of potato leaves; Figure 7 The control (7 days) amplified the electrophoresis results of microorganisms on the surface of potato leaves; Figure 8 The results show the colonization of five strains on leaves 7 days after the application of synthetic microbial communities.

[0018] Biological material preservation survival certificate Spatholobus suberectus ( Rhodanobacter spathiphylli Strain R3 was deposited on September 22, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC No. 36025.

[0019] Bacillus sarfusae ( Bacillus safensis Strain B1 was deposited on September 22, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC No. 36024.

[0020] Lai's genus ( Leifsonia sp.Strain L1 was deposited on September 22, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the bio-accession number CGMCC No. 36021.

[0021] Lai's genus ( Leifsonia sp. Strain L3 was deposited on September 22, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the biological collection number CGMCC No. 36022.

[0022] Lai's genus ( Leifsonia sp. Strain L6 was deposited on September 22, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the accession number CGMCC No. 36023. Detailed Implementation

[0023] This invention provides a strain of *Spatholobus suberectus* (Spatholobus suberectus). Rhodanobacter spathiphylli The strain R3, with accession number CGMCC No.36025, is used.

[0024] In this invention, strain R3 was isolated and screened from soil, and morphological observation and molecular identification determined it to belong to *Spatholobus suberectus* (Spatholobus suberectus). Rhodanobacter spathiphylli The strain R3 is effective against pathogenic Phytophthora ( ). Phytophthora infestans It exhibits good antibacterial activity. Pot experiments and field experiments show that strain R3 has good control effect against late blight.

[0025] In this invention, the cultivation method of strain R3 preferably includes the following steps: The strain R3 was inoculated into a culture medium for seed culture to obtain a seed solution; The seed culture was inoculated into a culture medium for expansion culture to obtain a culture solution.

[0026] In this invention, the culture medium is preferably NB liquid culture medium. The temperature for seed culture or scale-up culture is preferably 28-32°C, and can be 30°C. The seed culture or scale-up culture is preferably accompanied by shaking. The shaking speed is preferably 180-220 rpm, and can be 200 rpm. The seed culture time is preferably 15-17 h, and can be 16 h. The scale-up culture time is preferably 40-54 h, and can be 48 h.

[0027] This invention provides a biocontrol composition comprising Bacillus sabolicii (Bacillus safranin)Bacillus safensis ) strain B1 or Reissella genus ( Leifsonia sp. The strains are L1 and R3 of the *Spatholobus suberectus* strain; the *Bacillus sabovellatus* strain B1 has the accession number CGMCC No. 36024; and the *Reisi* strain L1 has the accession number CGMCC No. 36021.

[0028] In this invention, when strain B1 is included, the biocontrol composition preferably further includes one of the following: Reissella strain L1, Reissella strain L3, and Reissella strain L6; the preservation number of Reissella strain L1 is CGMCC No. 36021; the preservation number of Reissella strain L3 is CGMCC No. 36022; and the preservation number of Reissella strain L6 is CGMCC No. 36023.

[0029] In this invention, when strain B1 is included, the biocontrol composition preferably also includes at least one of Reidella strain L1 and Reidella strain L6 and Reidella strain L3.

[0030] This invention does not impose any particular limitation on the viable cell ratio of the various strains in the biocontrol composition; any ratio known in the art can be used, such as mixing them according to an equal volume ratio of the cultured bacterial solutions. The culture methods for strains B1, L1, L3, and L6 are the same as those for strain R3, and will not be described in detail here.

[0031] In this invention, the biocontrol composition has a significant advantage over the single strain R3 in inhibiting the activity of pathogenic Phytophthora. In addition, the compound microbial agent composed of two or more strains that can control crop diseases has the advantages of more stable disease control effect, stronger environmental adaptability, more diverse disease control mechanism, simultaneous control of multiple pathogens, and stronger competitive ability against rhizosphere and pathogen infection sites. Therefore, the biocontrol composition is more suitable for field control in complex environments.

[0032] In one embodiment of the present invention, in order to obtain a biocontrol composition with high antibacterial activity against pathogenic Phytophthora, any one, two, three, or four of strains B1, L1, L3, and L6 are combined with strain R3. The compositions formed by different combinations exhibit varying degrees of antibacterial activity. For example, L3R3, B1R3, L1B1R3, L3B1R3, L6B1R3, L1L6B1R3, L3L6B1R3, L1L3B1R3, and L1L3L6B1R3 have a significant antibacterial advantage over strain R3 alone in inhibiting pathogenic Phytophthora; while L1R3, L6R3, L1L3R3, L3L6R3, L1L6R3, and L1L3L6R3 have significantly reduced antibacterial activity against pathogenic Phytophthora compared to strain R3 alone.

[0033] In another embodiment of the present invention, to further investigate the interactions among the strains in the biocontrol composition, different strains from different sources were cultured. The results showed that the combination of strain R3 with the metabolites of any other strain promoted the growth of those other strains, while the absence of R3's metabolites inhibited the growth of other strains. This indicates that the secretion of metabolites by strain R3 is beneficial in promoting the growth of other strains in the biocontrol composition.

[0034] The present invention provides a biocontrol agent comprising the *Spatholobus suberectus* strain R3 or the biocontrol composition and excipients.

[0035] In this invention, the biocontrol agent preferably comprises an aqueous solution and / or a dry powder. The aqueous solution is preferably prepared by mixing the culture medium of *Spathobacterium spathulatum* strain R3 with a cryoprotectant. The cryoprotectant preferably comprises glycerol. The volume ratio of the culture medium of *Spathobacterium spathulatum* strain R3 to the cryoprotectant is preferably 0.8~1.2:0.8~1.2, and can be 1:1. When the biocontrol composition is the active ingredient, the corresponding culture medium of each biocontrol composition is compounded with the culture medium of strain R3 according to the composition of each strain, and then mixed with the cryoprotectant to obtain an aqueous biocontrol composition.

[0036] In this invention, the dry powder preferably comprises solid-liquid separation of the culture medium of *Spatholobus suberectus* strain R3, collection of bacterial cells, mixing with a freeze-drying protectant, and freeze-drying to obtain the dry powder. The solid-liquid separation method is preferably centrifugation. The centrifugation speed is preferably 10000~14000 rpm, and can be 12000 rpm. The centrifugation time is preferably 50~70 seconds, and can be 60 minutes. The freeze-drying protectant preferably includes at least one of the following: sugars, polyols, polymers, amino acids, proteins or peptides, antioxidants, inorganic salts, etc. The sugars, polyols, or polymers mainly play an anti-damage role. The sugars preferably include at least one of sucrose, trehalose, glucose, lactose, etc., which stabilize cell membranes and protein structures through hydrogen bonds. The polyols preferably include at least one of glycerol, mannitol, sorbitol, etc., which can regulate osmotic pressure and reduce mechanical damage to cells from ice crystals. The polymers preferably include polyvinylpyrrolidone (PVP) and hydroxyethyl starch (HES), which can form a protective film and reduce damage caused by rapid water loss. Amino acids, proteins or peptides, and antioxidants enhance stability or adapt to cellular needs. The amino acids preferably include glutamic acid, glycine, and proline, which protect the spatial structure of proteins and alleviate freeze-drying stress. The proteins or peptides preferably include bovine serum albumin (BSA) and gelatin, which act as fillers and stabilizers, reducing intercellular adhesion. The antioxidants preferably include vitamin C and glutathione, which scavenge free radicals generated during freeze-drying and reduce oxidative damage. Inorganic salts act as osmotic pressure regulators (maintaining intracellular and extracellular balance). The inorganic salts preferably include sodium chloride, potassium chloride, and potassium dihydrogen phosphate, which regulate the osmotic pressure of the system and prevent cell rupture due to drastic changes in osmotic pressure.

[0037] In this invention, the number of viable bacteria in the biocontrol agent is preferably not less than 10. 8 CFU / mL, which can be 5 × 10 8 CFU / mL ~100×10 8 CFU / mL. This invention does not impose any particular limitation on the preparation method of the biocontrol agent; any preparation method well-known in the art can be used. The preferred dosage of the biocontrol agent is 3 L~10 L / acre, and can be 5 L / acre.

[0038] This invention provides the application of the *Spathobacterium tumefaciens* strain R3 or the biocontrol composition in the preparation of a bacteriostatic agent against pathogenic *Phytophthora*.

[0039] This invention provides the application of the *Spathobacterium tumefaciens* strain R3, the biocontrol composition, or the control agent in the prevention and control of late blight.

[0040] In this invention, the late blight preferably includes at least one of the following: potato late blight, tomato late blight, eggplant late blight, and pepper late blight. This embodiment of the invention uses potato late blight as an example to illustrate the control effect of strain R3 or the biocontrol composition on late blight. The control of potato late blight includes reducing the disease index, improving control efficiency, and increasing potato yield.

[0041] In one embodiment of the present invention, the strain R3 or the biocontrol composition can colonize the plant leaves after application, which is beneficial to maintaining its long-term efficacy in controlling late blight.

[0042] In another embodiment of the present invention, after application of strain R3 or the biocontrol composition, the composition of the phyllosphere microbial community (fungi and bacteria) was measured. Treatment with the biocontrol agent formed by the combination of five strains altered both the bacterial and fungal communities in the potato phyllosphere, changing the relative abundance of dominant species at the bacterial and fungal genus levels, and regulating the microbial community structure. Leifsonia and bacillus They all occupy a dominant position, thus having a significant competitive inhibitory effect on pathogens.

[0043] The following detailed description, in conjunction with embodiments, illustrates a strain R3 of *Spathobacterium tumefaciens* and its compound bacterial agent, as well as their application in controlling late blight. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0044] Example 1 A strain of Spatholobus suberectus ( Rhodanobacter spathiphylli Methods for the isolation and identification of strain R3 First, healthy potato rhizosphere soil samples collected in Guizhou in 2018 were passed through a 20-mesh sieve. 0.2 g of the sieved soil was placed in a 50 mL centrifuge tube, and 25 mL of sterile water was added. The mixture was then vortexed to mix thoroughly. The suspension was then serially diluted with 1000 mL of 1 / 10 TSA or NA liquid medium.

[0045] Table 1. Dilution of suspension

[0046] Note: The amount added refers to the amount of soil suspension added to 1 L of liquid culture medium.

[0047] Vortex the diluent to mix thoroughly, pour into 13 cm × 13 cm square dishes, and use a multi-pipette to add 160 μL to each well of a 96-well plate. Perform 2-3 replicates for each concentration, with 3 blank control wells per plate. Seal the 96-well plates with sealing film and incubate at 30°C. After 7-14 days, select wells that have become turbid and streak them onto TSA solid medium plates, incubating at 30°C. Purify the grown single colonies (strains L1, L3, L6, B1, and R3) by streaking twice. Select single colonies and incubate them in liquid TSA medium at 28°C and 200 rpm for 24 h. Mix with 30% glycerol at a 1:1 volume ratio and store at -80°C.

[0048] Figure 1 The plate morphology of strains numbered L1, L3, L6, B1, and R3 is shown.

[0049] 2. Molecular identification of strains 2.1 Extraction of bacterial genomic DNA (1) Use sterile toothpicks to pick up the strains stored at -80℃, streak them on NA medium plates to activate them, and incubate them in a 30℃ constant temperature incubator for 12 h for later use; (2) Use a sterile 200 μL pipette tip to pick up a single colony with clear morphology of the test strain and inoculate it into a glass test tube containing 5 mL NB liquid medium. Incubate at 30℃ and 200 rpm for 12 h. (3) Take 1.5 mL of bacterial culture and add it to a 2 mL centrifuge tube. Centrifuge at 12000 rpm for 1 min, discard the supernatant, and collect the bacterial cells. (4) Add 1 mL ddH2O to the centrifuge tube, use a vortex mixer for 30 s to mix the bacterial suspension until there are no obvious bacterial clumps suspended, centrifuge again, and discard the supernatant. (5) Add 60 μL of lysozyme with a concentration of 20 mg / mL to the tube, invert it to mix thoroughly, and incubate it in a water bath at 37°C for 40 min. Invert it again every 10-20 min during this period. (6) Add 650 μL of nucleic acid lysis buffer to the tube and invert it to mix thoroughly; (7) Add 250 μL of protein precipitation solution to the tube and vortex it for about 1 min. (8) Centrifuge at 12000 rpm for 5 min to obtain 900 μL of supernatant, and transfer it to a new centrifuge tube using a pipette; (9) Take the isopropanol that has been pre-cooled at -20°C and add it to the tube. Slowly invert the tube 10 times to mix it thoroughly. (10) Centrifuge at 12000 rpm for 5 min and discard the supernatant; (11) Add 500 μL of 75% ethanol solution to the tube, invert it several times to mix it thoroughly, centrifuge at 14000 rpm for 2 min, and discard the supernatant. (12) Repeat step (11), and then place the centrifuge tube in the clean bench to dry the ethanol in the tube; (13) Add 60 μL of ddH2O to the tube to resuspend the DNA, and store it in a -20℃ refrigerator for later use.

[0050] 2.2 PCR amplification (1) PCR reaction system: The DNA template of the strain was amplified using 16S rDNA gene fragments as primers (27F: AGAGTTTGATCMTGGCTCAG, SEQ ID NO:1; 1492R: GGTTACCTTGTTACGACTT, SEQ ID NO:2). The reaction system is shown in Table 2 below, and the reaction procedure is shown in Table 3.

[0051] Table 2 PCR reaction system

[0052] Table 3 PCR amplification conditions

[0053] The amplified PCR products were sent to Beijing Nuosai Genome Research Center Co., Ltd. for sequencing.

[0054] After molecular identification, the strains numbered L1, L3, L6, B1, and R3 were sequenced and aligned in NCBI. A phylogenetic tree was then constructed using the Mega7 software method.

[0055] See phylogenetic tree Figure 2 Based on the sequence alignment results and the clustering of the phylogenetic tree, the identification results shown in Table 4 are obtained.

[0056] Table 4 Molecular identification results

[0057] Example 2 Plate antibacterial test 1. Preparation method of bacterial culture medium: (1) Take the test strain stored in the -80℃ ultra-low temperature freezer, use a sterile toothpick to take a small amount of the strain solution in the ultra-clean workbench and activate it on the NA plate. Seal the plate with sealing film and invert it in the 30℃ constant temperature incubator for 24 h for later use. (2) Pick a single colony of the test strain with a sterile pipette tip and inoculate it into 5 mL of NB liquid medium. Shake and incubate at 30℃ and 200 rpm for 16 h to obtain the seed culture. (3) Inoculate the seed culture into 200 mL of NB liquid medium at a volume ratio of 1:50, and incubate at 30℃ and 200 rpm for 48 h to prepare the culture medium. Adjust the viable cell concentration in the culture medium to 1.0 × 10⁻⁶. 8 CFU / mL available for use.

[0058] 2. Plate confrontation test between single or combined strains and the pathogen causing late blight. (1) Prepare the culture medium of the strain according to step 1 above; determine the antibacterial activity of strain R3 and the compound bacterial solution formed by combining strain R3 with one, two, three or four of strains L1, L3, L6 and B1 respectively; wherein the compound bacterial solution is prepared by mixing the culture medium of a single strain in equal volume ratio.

[0059] (2) Activate and culture pathogenic Phytophthora in rye agar plates with a diameter of 90 mm. Phytophthora infestans (See the following technical article: Li Lei, Lu Jie, Bao Yazhou, et al. Effects of four compounds on potato resistance to late blight and analysis of related defense gene expression [J]. Plant Protection News, 2020, 47(06):1277-1286). Inoculate the mycelium cake onto the center of a new rye plate. Inoculate single-strain culture or compound culture into Oxford cup wells equidistantly around the late blight fungus cake. Leave one Oxford cup well untreated as a control on each plate. Incubate in the dark at 18℃. Repeat each treatment three times. After four days of incubation, observe and record the antagonistic effect against late blight.

[0060] See results Figure 3 Compared to the control group, strain R3 alone showed a significant inhibitory advantage in suppressing pathogenic *Phytophthora*. However, the combination strains composed of strain R3 exhibited inconsistent antimicrobial activity. For example, L3R3, B1R3, L1B1R3, L3B1R3, L6B1R3, L1L6B1R3, L3L6B1R3, L1L3B1R3, and L1L3L6B1R3 all showed a significant inhibitory advantage over strain R3 alone in suppressing pathogenic *Phytophthora*. Conversely, L1R3, L6R3, L1L3R3, L3L6R3, L1L6R3, and L1L3L6R3 showed significantly reduced antimicrobial activity against pathogenic *Phytophthora* compared to strain R3 alone, indicating that inappropriate strains antagonized the antimicrobial activity of R3.

[0061] Example 3 1. Test on the efficacy of potted plants against late blight Select healthy potato potted plants with similar growth patterns and place them in an artificial climate chamber (22℃, 95% humidity). First, spray with biocontrol agents. The biocontrol agents include culture solutions of individual strains L1, L3, L6, B1, and R3, as well as compound bacterial solutions B1R3, L1B1R3, L3R3B1, and L1L3L6B1R3. After centrifuging the culture solutions of individual strains, collect the bacterial cells, resuspend them in PBS, and adjust the viable cell concentration of strain R3 to 10. 8 CFU / mL; Compound bacterial solution B1R3 is a mixture of equal volumes of culture media from strains B1 and R3, adjusted to a concentration of 10. 8 CFU / mL; the compound bacterial culture L1B1R3 is a mixture of equal volumes of culture media of strain L1, strain B1, and strain R3, adjusted to a concentration of 10. 8 CFU / mL; Compound bacterial culture L3R3B1 is a mixture of equal volumes of culture media of strain L3, strain B1, and strain R3, adjusted to a concentration of 10. 8 CFU / mL; the compound bacterial culture L1L3L6B1R3 is a mixture of equal volume cultures of strain L1, strain L3, strain L6, strain B1, and strain R3, adjusted to a concentration of 10. 8 CFU / mL; the amount of bacteria applied for single strains and compound bacterial solutions was the same; a control group (CK) was also set up, which was sprayed with the same volume of sterile water. Two days later, the pathogen was sprayed (a suspension of pathogenic *Phytophthora* spores was prepared using sterile water pre-cooled to 4°C, to a final concentration of 10). 5 CFU / mL), sprayed to the maximum liquid capacity of the leaves, and cultured for about 4 days to observe the disease incidence (see Table 5). The incidence rate was counted and the control effect was calculated according to Formula I and Formula II.

[0062] Disease index = Σ (number of plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100% (Formula I) Relative prevention and control effect (%) = (Control disease index - Treatment control disease index) / Control disease index × 100% Formula II.

[0063] Table 5 Grading Standards for Potato Late Blight

[0064] The results are shown in Table 6. Compared with the control group, the pot experiment confirmed that strain B1 and other single strains could significantly inhibit the occurrence of potato late blight, and the control efficacy of each compound bacterial solution was significantly higher than that of any single strain.

[0065] Table 6. Pot experiment verification of the efficacy of compound bacterial solution.

[0066] Note: The letters abcd are used to indicate significant differences between different groups.

[0067] 2. The method of controlling late blight using compound bacterial solution L1L3L6B1R3 through both foliar spraying and root irrigation.

[0068] Centrifuge the compound bacterial culture L1L3L6B1R3 at 12000 rpm for 1 min to separate the bacterial cells and fermentation broth for later use.

[0069] Healthy potato potted plants with similar growth were selected and divided into five groups: the first group was the control (CK); the second group was treated with foliar spray of compound bacterial solution L1L3L6B1R3 (referred to as the mycorrhizal group); the third group was treated with root irrigation of compound bacterial solution L1L3L6B1R3 (referred to as the mycorrhizal group); the fourth group was treated with foliar spray of fermented compound bacterial solution L1L3L6B1R3 (referred to as the fermented liquid leaf group); and the fifth group was treated with root irrigation of fermented compound bacterial solution L1L3L6B1R3 (referred to as the fermented liquid root group). Five groups of potted plants were cultured in an artificial climate chamber (22℃, 95% humidity). For the first group, 3 mL of the compound bacterial solution L1L3L6B1R3 was sprayed onto the leaves, and 3 mL of the compound bacterial solution L1L3L6B1R3 was applied to the roots. Additionally, 3 mL of the fermented broth of the compound bacterial solution L1L3L6B1R3 was sprayed onto the leaves, and 3 mL of the culture medium of the compound bacterial solution L1L3L6B1R3 was applied to the roots. The control group received sterile water.

[0070] Two days later, spray with the pathogen (prepared using a suspension of pathogenic *Phytophthora* spores in pre-cooled sterile water at 4 degrees Celsius, to a final concentration of 10). 5 (CFU / mL), spray to the maximum liquid load on the leaves, incubate for about 4 days to observe the disease incidence, count the incidence rate and calculate the control effect according to the above method.

[0071] Table 7. Pot experiment verification of the efficacy of different application methods of compound bacterial solution.

[0072] Note: The letters abcd are used to indicate significant differences between different groups.

[0073] The results are shown in Table 7. The compound bacterial solution L1L3L6B1R3 can effectively control the occurrence of late blight by both foliar spraying and root application. The metabolic products of the bacterial community can also effectively control the occurrence of late blight, but the bacterial cells are more advantageous in controlling late blight than the metabolic products.

[0074] Example 4 Experiment on the interaction relationships among strains in compound bacterial culture L1L3L6B1R3 1. Test method: (1) Take the test strains (L1, L3, L6, B1 and R3) stored in the -80℃ ultra-low temperature freezer, use a sterile toothpick to take a small amount of bacterial solution of the strains in the ultra-clean workbench and activate it on the NA plate. Seal the plate with sealing film and invert it in the 30℃ constant temperature incubator for 24 h for later use. (2) Pick a single colony of the test strain with a sterile pipette tip and inoculate it into 5 mL of NB liquid medium. Shake and incubate at 30℃ and 200 rpm for 16 h to obtain the seed culture. (3) Inoculate the seed culture into 200 mL of NB liquid culture medium at a volume ratio of 1:50, shake culture at 30℃ and 200 rpm for 48 h, centrifuge at 8000 rpm for 10 min, and then filter with a disposable sterile filter to obtain sterile metabolic liquid.

[0075] (4) Using 100 mL of NB liquid medium as the control, and NB liquid medium + strain metabolic solution as the experimental group (20 mL of metabolic solution for each strain, total volume 100 mL), the strain metabolic solution can be one or two kinds, and then 100 μL of culture medium of any strain is added. The culture is carried out at 30℃ and 200 rpm for 48 h, and the OD is measured. 600 value.

[0076] 2. Experimental Results: The results are shown in Tables 8 to 19.

[0077] Table 8. Metabolites of R3 and OD values ​​of other strains 600 Value (48h)

[0078] Table 9. OD of a bacterial strain cultured from the metabolites of R3 and B1. 600 Value (48h)

[0079] Table 10 OD of a bacterial culture containing metabolites of R3 and L3 600 Value (48h)

[0080] Table 11 OD of a bacterial strain containing metabolites of R3 and L1 600 Value (48h)

[0081] Table 12 OD of a bacterial culture containing metabolites of R3 and L6 600 value

[0082] Table 13 OD of L1 and L6 metabolites cultured from a single strain 600 value

[0083] Table 14 OD of L1 and L3 metabolites cultured from a single bacterial strain 600 value

[0084] Table 15 OD of L3 and L6 metabolites cultured from a single strain 600 value

[0085] Table 16 OD of L1 and B1 metabolites cultured from a single strain 600 value

[0086] Table 17 OD of L3 and B1 metabolites cultured from a single strain 600 value

[0087] Table 18 OD of L1 and B1 metabolites cultured from a single strain 600 value

[0088] After co-culturing the strains and removing one strain, co-culturing for 48 hours, and then measuring the OD... 600 After discovering the absence of R3, the OD value of the co-cultured strains was... 600 The value is the lowest (Table 19).

[0089] Table 19 OD of co-cultured strains 600 value

[0090] Note: The letters abcd are used to indicate significant differences between different groups.

[0091] The results of the metabolic symbiotic relationship of the compound bacterial solution showed that R3 was crucial to the growth of the other four strains in the compound bacterial solution. The combination of R3 with the metabolites of any other strain could promote the growth of the other strains, while the combination of the metabolites of strains without R3 would inhibit the growth of the other strains.

[0092] Example 5 Field test for late blight control efficacy Test location: Bimoga, Yi, Hui and Miao Autonomous Prefecture, Xueshan Town, Weining County, Bijie City, Guizhou Province; Experimental variety: Weiyu No. 5; Planting method: Each row is 40 cm wide and 120 m long, with a 20 cm spacing between each potato tuber and a planting depth of 10 cm. Two rows are designed as one treatment group, and two protective rows are designed between different treatments, with water as a blank control.

[0093] Application method of biocontrol bacteria: Dilute 1L of culture medium of strain R3, compound bacterial solution B1R3, compound bacterial solution L1B1R3, compound bacterial solution L1L3R3, compound bacterial solution L3R3B1, and compound bacterial solution L1L3L6B1R3 to make 10L of liquid bacterial agent. Spray at the seedling stage of potato plants, with a spraying rate of 5L / acre each time, spraying once every 7 days, for a total of 6 sprays. Six days after the last spray, conduct a survey. The survey method is a five-point sampling method, taking 10 samples at each point, for a total of 50 plants per treatment. Investigate the disease incidence and calculate the control effect according to the method in Example 3.

[0094] Table 20 Field efficacy verification of biocontrol bacteria

[0095] Note: The letters abcd are used to indicate significant differences between different groups.

[0096] As shown in Table 20, compared with the control group, the R3 strain alone can effectively control the disease index of potato late blight and has a certain field control effect. At the same time, the compound bacterial solution formed by combining the R3 strain with B1, L1 and L3 strains can further reduce the disease index and improve the field control effect. Moreover, the compound bacterial solution L1L3L6B1R3 has the best control effect and there is no significant difference in control effect compared with mancozeb.

[0097] Example 6 Determination of leaf microbial community of potatoes treated with compound bacterial solution L1L3L6B1R3 1. Collection of potato leaves Using sterile scissors, cut leaves from potato plants treated with the compound bacterial solution L1L3L6B1R3. Place the leaves in numbered sterile containers, weigh 10g of each leaf, and add them to 40mL of sterile PBS buffer. Shake at 200 rpm for 30 minutes, then sonicate twice for 30 seconds each time, and let stand for 5-10 minutes. Pass the buffer containing microbial cells through a 0.22 μm filter membrane, and store the resulting membrane in a sterile petri dish at -80°C.

[0098] Microbial DNA was extracted according to the instructions of the Fast DNAS SPIN Kit for soil extraction kit, and the extracted microbial DNA was sent to Meiji Biotechnology Co., Ltd. for high-throughput sequencing.

[0099] See results Figure 4 and Figure 5 After applying the compound bacterial solution L1L3L6B1R3, Pseudomonas, Chryseobacterium, Leifsonia, bacillus As the number of bacteria increases, unclassified f Enterobacteriaceae, unclassified o Enterobacterales, microbacterium The number of bacteria and fungi has decreased. Fungi_gen_ Incertae_se, Meyerozyma, Naganishia, Cystobasidium, Pseudallescheria When the fungal genus increases ; Alternaria, Cladosporium, unclassified_k_Fungi, Penicillium, Fusarium, Filobasidium The number of fungal genera decreased. Analysis of the phyllosphere bacterial and fungal community composition of potato plants revealed changes in both the bacterial and fungal communities treated with the compound bacterial solution L1L3L6B1R3. These changes altered the relative abundance of dominant species at the bacterial and fungal genera level, thus regulating the microbial community structure. Leifsonia and bacillus They all occupy a dominant position, thus having a significant competitive inhibitory effect on pathogens.

[0100] Example 6 Colonization capacity test of compound bacterial culture L1L3L6B1R3 The genomes of five bacterial strains in the compound bacterial culture L1L3L6B1R3 were analyzed to identify genes unique to each strain. Primers were designed and amplified to identify unique primers for each strain. Microorganisms were also extracted from potato leaves to verify whether the unique primers could amplify microorganisms on potato leaves.

[0101] Before spraying the compound bacterial solution L1L3L6B1R3 for 7 days, potato leaves were collected and leaf microorganisms were amplified using specific primers (Table 21). The amplification system is shown in Table 22 and the amplification program is shown in Table 23. The amplification products were detected by agarose gel electrophoresis to observe whether there were bands. A control was set up at 7 days that was not sprayed with the compound bacterial solution L1L3L6B1R3.

[0102] Seven days after spraying with the compound bacterial solution L1L3L6B1R3, microorganisms were extracted from potato leaves. Using sterile scissors, potato plant leaves were cut and placed in numbered sterile containers. 10g of each leaf was weighed and placed in 40mL of sterile PBS buffer. The mixture was shaken at 200 rpm for 30 minutes, then sonicated twice for 30 seconds each time, followed by standing for 5-10 minutes. The buffer containing microbial cells was filtered through a 0.22 μm filter and extracted using the Fast DNAS SPIN Kit for soil extraction. Amplification was then performed according to the above protocol.

[0103] PCR amplification was performed using primers containing genes specific to five strains of the synthetic microbial community to amplify the microorganisms on potato leaves, and the presence of bands was observed.

[0104] Table 21 Selection of strain-specific primers

[0105] Table 22 PCR Reaction System

[0106] Table 23 PCR Amplification Conditions

[0107] See results Figures 6 - 8 After spraying with the compound bacterial solution L1L3L6B1R37d7d, the plant leaves were able to amplify bands, while the plants that were not sprayed with the synthetic bacterial community could not amplify bands. This indicates that all five strains in the compound bacterial solution L1L3L6B1R37d7d can colonize the plant leaves and have long-term control stability.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Heliotrophic bacillus* from *Spatholobus suberectus* ( Rhodanobacter spathiphylli strain R3, characterized in that, The accession number is CGMCC No.36025.

2. A biocontrol composition, characterized in that, Including Bacillus safortiformis ( Bacillus safensis ) strain B1 or Reissella genus ( Leifsonia sp. Strain L1 and the Spathobacterium tumefaciens strain R3 as described in claim 1; The preservation number of the *Bacillus sarcodactylis* strain B1 is CGMCC No. 36024; The preservation number of the Reiberella strain L1 is CGMCC No. 36021.

3. The biocontrol composition according to claim 2, characterized in that, When strain B1 is included, it also includes one of the following: Reissler strain L1, Reissler strain L3 and Reissler strain L6; The preservation number of the Reiberella strain L1 is CGMCCGMCC No.36021; The preservation number of the Reissella strain L3 is CGMCC No. 36022; The preservation number of the Reiberella strain L6 is CGMCC No. 36023.

4. The biocontrol composition according to claim 2, characterized in that, When strain B1 is included, it also includes at least one of Reissler strains L1 and L6 and Reissler strain L3. The preservation number of the Reissella strain L1 is CGMCC No. 36021; The preservation number of the Reissella strain L3 is CGMCC No. 36022; The preservation number of the Reiberella strain L6 is CGMCC No. 36023.

5. A biocontrol agent, characterized in that, It includes the *Spathobacterium tumefaciens* strain R3 as described in claim 1 or the biocontrol composition and excipients as described in any one of claims 2 to 4.

6. The biocontrol agent according to claim 5, characterized in that, The biocontrol agent includes aqueous and / or dry powder formulations.

7. The biocontrol agent according to claim 5 or 6, characterized in that, The biocontrol agent contains no less than 10 live bacteria. 8 CFU / g or 10 8 CFU / mL.

8. The *Spatholobus suberectus* strain R3 of claim 1 or the biocontrol composition of any one of claims 2-4 in the preparation of pathogenic *Phytophthora* ( Phytophthora infestans Application in antibacterial agents.

9. The application of the Spathobacterium tumefaciens strain R3 of claim 1, the biocontrol composition of any one of claims 2 to 4, or the biocontrol agent of any one of claims 5 to 7 in the prevention and control of late blight.

10. The application according to claim 9, characterized in that, The late blight includes at least one of the following: potato late blight, tomato late blight, eggplant late blight, and pepper late blight.

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