Potato late blight antagonistic bacterium and application thereof

Abstract

The invention discloses a potato late blight antagonistic bacterium and application thereof, belongs to biological agents, and provides more strain resources for prevention and treatment of potato late blight. The potato late blight antagonistic bacterium is Pseudomonas chlororaphis blT-49 and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is December 27, 2021, and the preservation number is CGMCC No.24203. The invention further discloses a preparation method of the potato late blight antagonistic bacterium. The strain disclosed by the invention not only can inhibit the occurrence and growth of potato late blight, but also can regulate and control the ecological system of the micro-ecology of potatoes, reduce the infection ability of potato late blight and improve the disease resistance of potatoes. In the growth and development of the potatoes, the growth and development of the potatoes can be promoted, and the single-plant yield is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculants, specifically relating to a potato late blight antagonist and its application. Background Technology

[0002] Potato late blight is a disease caused by the pathogenic fungus *Phytophthora infestans (Mont.) de Bary*. Currently, a series of ecological control measures have been developed to address the outbreak of potato late blight, with varying degrees of effectiveness, but all have a positive impact on its control. Ecological control of potato diseases can be broadly categorized into three types: agricultural practices, improved seed breeding, and microbial control, with microbial control showing significant success. Microbial control is currently the main research direction for potato late blight control. In recent years, research on antagonistic bacterial strains for potato late blight has increased annually. Antagonistic strains are mainly classified as fungi, bacteria, and actinomycetes, with fungi and bacteria being the most common. While strains with antibacterial functions against potato late blight are diverse, few have been successfully applied industrially. Summary of the Invention

[0003] The present invention aims to provide a strain that effectively inhibits late blight, thereby providing more strain resources for the prevention and control of potato late blight.

[0004] A potato late blight antagonist bacterium of the present invention, *Pseudomonas chlororaphis* subsp. chlororaphis blT-49, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 27, 2021, with accession number CGMCC No. 24203.

[0005] The present invention relates to the application of a potato late blight antagonist bacterium, wherein the *Pseudomonas chlororaphis* blT-49 is used to control potato late blight and simultaneously promote the growth and development of potato plants.

[0006] Furthermore, the aforementioned control of potato late blight reduces the infectivity of potato late blight and enhances the disease resistance of potatoes.

[0007] Furthermore, the aforementioned Pseudomonas chlororaphis subsp. blT-49 can be prepared into bacterial liquid, bacterial suspension, bacterial cell lysate, or sterile fermentation broth for application.

[0008] Furthermore, the original suspension of *Pseudomonas chlororaphis* blT-49 was diluted to 10... -1 ~10 -8 use.

[0009] The application of a potato late blight antagonist strain of the present invention, wherein the *Pseudomonas chlororaphis* subsp. *blT-49* is used to inhibit the growth of *Fusarium oxysporum* cucumber-specific type, *Geotrichum candida*, *Fusarium solani*, and *Rhizoctonia solani*.

[0010] The present invention relates to the application of a potato late blight antagonist bacterium, wherein the described Pseudomonas chlororaphis subsp. blT-49 is used as a protective agent for detached potato leaves and tubers.

[0011] The present invention relates to a biochar inoculant for a potato late blight antagonistic bacterium.

[0012] Furthermore, the method for preparing the biochar agent is as follows:

[0013] Fermented Pseudomonas chlororaphis blT-49 bacterial suspension was added to LB liquid medium, and sterilized biochar was added. The medium was then incubated at 30-35℃ for 130-150 min, with 30-35 mg of biochar added per mL of bacterial suspension.

[0014] Furthermore, the fermentation conditions for the fermented Pseudomonas chlororaphis subsp. blT-49 were pH=7 and fermentation time was 45-50h.

[0015] The present invention has the following beneficial effects:

[0016] The blT-49 antagonistic bacterium of this invention exhibits an inhibition rate as high as 95%, with the sterile fermentation broth showing an inhibition rate of 92.25%, and the cell lysate also achieving an inhibition rate of 76.14%. The secondary metabolites of blT-49 may contain substances that inhibit potato late blight and possess heat-resistant properties. Significant antibacterial activity was demonstrated in both detached leaf and tuber tests. Furthermore, infection tests on detached potato leaves and tubers showed that the blT-49 antagonistic bacterium not only protects potato leaves and tubers but also has no pathogenicity to the potato plant itself. Layer-by-layer separation of the antibacterial substances from the strain provides a pathway and direction for regulating the antibacterial function of the antagonistic bacterium. The optimal effect of biochar encapsulation of the potato late blight antagonist, *Pseudomonas aeruginosa*, was achieved under the conditions of 34.4 mg / mL biochar application and encapsulation at 31°C for 145 min. In terms of its antibacterial ability in preventing potato late blight, the relative protection rate of the blT-49 antagonistic biochar agent is approximately 60%. The *Pseudomonas aeruginosa* blT-49 biochar agent of this invention not only inhibits the occurrence and growth of potato late blight, thus regulating the microecological ecosystem of potatoes and reducing the infectivity of potato late blight, but also enhances the disease resistance of potatoes. Furthermore, it can promote potato growth and development, increasing the yield per plant. Attached Figure Description

[0017] Figure 1 Figure a shows the antibacterial activity of antagonistic bacteria on blT-49 plates; Figure b shows the comparison of antibacterial activity rates of antagonistic bacteria. Each value represents the mean ± standard deviation (n = 3), and the letters indicate that there is a significant difference between groups at the p < 0.05 level.

[0018] Figure 2 The diagram shows the broad-spectrum antibacterial activity of the blT-49 antagonistic strain in a confrontational culture. Among them, A) is Fusarium oxysporum, a cucumber-specific strain; B) is Fusarium solani; C) Rhizoctonia solani; and D) Geotrichum candida.

[0019] Figure 3 Figure 1 shows the colony and microscopic morphological identification of the blT-49 antagonistic strain; Figure 2a shows the morphology of the blT-49 antagonistic bacteria on the plate, and Figure 3b shows the Gram staining.

[0020] Figure 4 A phylogenetic tree of the strains;

[0021] Figure 5 The diagram shows the confrontation between different components of blT-49 antagonistic bacteria; where A) is bacterial culture, B) is bacterial suspension, C) is sterile fermentation broth, and D) is bacterial cell lysate.

[0022] Figure 6The figure shows a comparison of the antibacterial functions of the components of blT-49 antagonistic bacteria; in the figure, a, b, c, and d represent significant differences at the p<0.05 level.

[0023] Figure 7 Photographs showing the antibacterial activity of blT-49 inoculant and Bacillus subtilis at different dilutions; A): Bacillus subtilis inoculant, B): blT-49 inoculant; a), b), c), d), e), f), g), h), i): from 10 0 -10 -8 Gradient dilution plot;

[0024] Figure 8 The graph shows the inhibition rate of antagonistic bacterial suspensions after gradient dilution; a) blT-49, b) Bacillus subtilis reference strain; each value represents the mean ± standard deviation (n=3); different lowercase letters indicate significant differences between groups at the p<0.05 level; * indicates significant correlation at the p<0.05 level; *** indicates significant correlation at the p<0.001 level;

[0025] Figure 9 This diagram illustrates the disease damage caused by the blT-49 antagonistic strain to detached potato leaves and tubers. In the diagram, A) and C) represent the application of the antagonistic bacteria followed by the pathogen, while B) and D) represent the application of only the antagonistic bacteria.

[0026] Figure 10 The graph shows the relative protection rate of antagonistic bacteria in detached leaves and tubers; in the graph, a) : disease index and relative protection rate of detached leaves, b) : disease index and relative protection rate of detached tubers; each value represents the mean ± standard deviation (n=3);

[0027] Figure 11 The graph shows the stability of blT-49 biochar inoculant storage; each value in the graph represents the mean ± standard deviation (n=3).

[0028] Figure 12 Photographs comparing the antibacterial activity of blT-49 antagonistic biochar inoculant with various fungicides; in the figure, A): recommended dose diluted 10 times for the same row, B): recommended dose for the same row; a): 95% cyazofamid, b): 99% fluazinam, c): 98% azoxystrobin, d): blT-49 biochar inoculant, e): Shengxinglong potato seed dressing agent, f): 58% metalaxyl-mancozeb, g): CK;

[0029] Figure 13This is a comparison chart of the antibacterial abilities of different bactericides; in the chart, each value represents the mean ± standard deviation (n=3); a, b, c, d, and e represent significant differences between groups at the p<0.05 level at the recommended dosage; A, B, C, D, and E represent significant differences between groups at the p<0.05 level after the recommended dosage is diluted 10 times. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0031] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0032] Example 1

[0033] 1. Experimental Materials

[0034] 1.1 Test Sample

[0035] Test samples: The tested potato variety was Eugene 885, provided by the Qiqihar Branch of the Heilongjiang Academy of Agricultural Sciences. The field test site was the experimental farm of Hulan Campus of Heilongjiang University (46°00′N, 126°38′E).

[0036] 1.2 Experimental Culture Medium

[0037] (1) LB culture medium (g / L): 10g tryptone, 5g yeast extract, 10g sodium chloride, 15-20g agar powder, dissolved in sterile water (1000mL), pH adjusted to 7.0 with 10% sodium hydroxide, and sterilized in an autoclave at 121℃ for 30min.

[0038] (2) Rye culture medium (g / L): 80g rye, 20g glucose, 15-20g agar powder, dissolved in sterile water (1000mL), sterilized in an autoclave at 121℃ for 30min.

[0039] (3) Basic inorganic salt culture medium: K2HPO4 1.79 g, KH2PO4 0.45 g, MgSO4·7H2O 0.2 g, NaCl 0.4 g, distilled water 1000 mL, pH 7.0.

[0040] 2. Experimental Methods

[0041] 2.1 Screening of antagonistic bacteria strains and determination of their antibacterial spectrum

[0042] 2.1.1 Isolation, purification, and preservation of bacterial strains

[0043] (1) Strain isolation: Weigh 2g of diseased potatoes infected with potato late blight from the junction of diseased and healthy tissue. Disinfect the surface of the sample with 75% alcohol. After frying, grind the treated sample thoroughly with 10mL of sterile water in a sterile mortar to form a paste. Take 1mL of the upper layer of the grinding liquid and add it to a centrifuge tube containing 9mL of sterile water. Serially dilute the grinding liquid in the tube with sterile water (10 ... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take 100 μL of 10 -4 10 -5 10 -6 The concentrated grinding solution was ball-coated onto LB solid medium and incubated upside down in a constant temperature incubator at 28°C for 24 hours.

[0044] (2) Strain purification: Based on phenotypic characteristics such as color and shape, colonies with significant phenotypic differences were selected from the culture medium and streaked onto LB solid medium for purification. The purified strains were then inoculated into test tubes containing 5 mL of LB liquid medium and cultured at 28°C and 180 rpm in a shaker. When the OD600 value reached 1.0, the culture was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the culture was mixed with 80% glycerol by pipetting and stored at -20°C.

[0045] 2.1.2 Antibacterial test of antagonistic strains

[0046] (1) Initial screening of antagonistic bacterial strains: The plate confrontation culture method was adopted. A piece of late blight pathogen cake (d=8.3mm) cultured for 7 days was taken out by punching a hole in an Oxford cup and placed in the center of the rye medium. The inoculum cultured for 24 hours was picked and inoculated at four positions (0°, 90°, 180°, 270°) 2cm away from the center. The culture was carried out at 28℃ for 7 days. The antagonistic bacterial confrontation test of each group needs to be repeated 3 times to select the strains that have significant antibacterial function against the pathogen and have a long-lasting antibacterial effect.

[0047] (2) Screening of antagonistic bacterial strains: The plate confrontation culture method was adopted. The late blight pathogen cake was placed in the center of the rye culture medium. The antagonistic strains that had been screened in the initial screening were inoculated at two positions (90° and 180°) 2 cm away from the center. The control group was inoculated with only pathogens. The antagonistic bacterial confrontation test of each group needed to be repeated 3 times. The culture was carried out at 21℃ for 7 days. The inhibition bandwidth was measured every 24 hours until the pathogens in the control group were about to fill the culture medium. The inhibition rate was then measured.

[0048] 2.1.3 Determination of the antibacterial spectrum of antagonistic strains

[0049] The plate confrontation method was used to detect the antibacterial effect of antagonistic bacteria on different pathogens. Specific procedures are detailed in section 2.1.2, "Antagonistic Bacterial Rescreening Procedure." The tested pathogens included *Fusarium oxysporum* cucumber-specific strain, *Geotrichum candida*, *Fusarium solani*, and *Rhizoctonia solani*. Each pathogen confrontation test was repeated three times. When the mycelium of the pathogen on the control plate was about to completely cover the plate, the mycelial diameter was measured, and the inhibition rate was calculated.

[0050] 2.2 Identification of antagonistic strains

[0051] 2.2.1 Morphological identification of antagonistic strains

[0052] Prepare LB solid medium and inoculate the antagonistic bacteria onto the medium using the three-zone streak inoculation method. Incubate at 30°C for 24 hours. Observe the shape, color, size, and edge regularity of single colonies of the antagonistic bacteria on the medium according to Bergey's Manual of Bacteriology, and take photos for recording. Pick a single colony for Gram staining and observe the morphology of the antagonistic bacteria under an optical microscope.

[0053] 2.2.2 Physiological and biochemical identification of antagonistic strains

[0054] The antagonistic bacteria were subjected to 15 physiological and biochemical tests, including starch hydrolysis, methyl red test, and VP test. Escherichia coli and Staphylococcus aureus were used as control groups. Each test was repeated three times. The highly effective antagonistic bacteria were then classified according to the "Handbook of Common Bacteria" and "Bergey's Manual of Bacteriology" to preliminarily infer the genus and species of the antagonistic bacteria.

[0055] 2.2.3 Identification of 16S rDNA sequences of antagonistic strains

[0056] (1) Bacterial genomic DNA extraction using thermal cell disruption method: Single colonies of antagonistic bacteria were inoculated into test tubes containing LB liquid medium. The incubator was set to 30℃ and cultured for 24 h. 1 mL of the cultured bacterial solution was transferred to a 1.5 mL centrifuge tube and centrifuged at 5000 r / min for 5 min. The supernatant was removed, and 200 μL of sterile water was added and mixed thoroughly. The mixture was then incubated in a boiling water bath at 100℃ for 8–10 min and centrifuged at 10000 r / min for 10 min. The supernatant was transferred to a 1.5 mL sterile centrifuge tube. 5 μL of the supernatant was taken from the centrifuge tube and spotted. The DNA was labeled with DL2000 and analyzed by 1% agarose gel electrophoresis. The DNA was then frozen at -20℃ for later use.

[0057] (2) 16S rDNA sequence analysis

[0058] The 16S rDNA of the antagonistic bacteria was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing. The antagonistic bacterial sequence was corrected using Chromas sequence assembly software. The corrected gene sequence was then compared with existing genes in the NCBI database (http: / / blast.ncbi.nlm.nih.gov / ) for homology analysis, and the antagonistic bacterial sequence was submitted to NCBI to obtain a accession number. A phylogenetic tree was constructed using MEGA 7.0 software with the Neighbor-Joining method to determine the species of the antagonistic bacterial strain.

[0059] 2.3 Antagonistic Bacterial Inhibition Test

[0060] 2.3.1 Antibacterial test of antagonistic bacterial suspension

[0061] Preparation of antagonistic bacterial suspension: A single colony of antagonistic bacteria in the logarithmic growth phase (24 h) was picked from LB solid medium using a disposable inoculation loop and inoculated into a test tube containing 6 mL of LB liquid medium. The culture was incubated for 24 h in a shaker at 30℃ and 180 r / min to obtain the antagonistic bacterial suspension. 100 μL of the antagonistic bacterial suspension with a LOD600 of 1.0 was taken. Using the Oxford cup method and the confrontation experiment, a late blight pathogen cake was placed in the center of a rye medium. 100 μL of bacterial suspension was injected into each of the Oxford cup wells (90° and 180°) at two locations 2 cm from the center. Physiological saline was injected as a control group. Each group of antagonistic bacterial suspensions was subjected to three replicates of the inhibition test. The culture was carried out at 21℃ for 7 days, and the inhibition band was measured every 24 h until the pathogen in the control group almost completely covered the medium. The inhibition rate was then measured.

[0062] 2.3.2 Antibacterial test of antagonistic bacterial suspension

[0063] Preparation of bacterial suspension: Pipette 1 mL of antagonistic bacterial suspension into a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 2 minutes, discard the supernatant, add 0.9% physiological saline, and vortex thoroughly using a Votex shaker. Measure the OD600 value of the bacteria using an ELISA reader, and then standardize the OD value to OD600 = 1.0 with physiological saline to obtain the bacterial suspension. Perform an antibacterial test with 100 μL of the bacterial suspension against late blight, following the same procedure as in 2.3.1. Each group of antagonistic bacterial suspensions should be tested three times in a single test.

[0064] 2.3.3 Detection of Antibacterial Rate by Gradual Dilution of Antagonistic Strains

[0065] The bacterial suspension with OD600 = 1 was diluted with sterile water to a concentration of 10. -1 10 -2 10 -3 ,10- 4 10 -5 10 -610 -7 10 -8 The original solution (100) and various gradient dilutions were used to conduct antibacterial tests on late blight. An equal amount of Bacillus subtilis reference bacterial solution and sterile water were used as control groups. The antibacterial test of each group of antagonistic strains was repeated 3 times.

[0066] 2.3.4 Antibacterial test of antagonistic bacteria sterile fermentation broth

[0067] Sterile fermentation broth: 5 mL of antagonistic bacterial suspension was inoculated into an Erlenmeyer flask containing 45 mL of LB liquid medium. The flask was incubated for 2 days at 30℃ and 180 rpm on a shaker. The fermented broth was then transferred to a 50 mL sterile centrifuge tube and centrifuged at 4500 rpm for 10 min. The supernatant was filtered through a 0.22 μm bacterial filter to remove bacterial cells, resulting in sterile fermentation broth. To prevent the unfiltered strains from exerting their effects, the sterile fermentation broth was placed in a 100℃ water bath for 10 min. 10 mL of the treated sterile fermentation broth was frozen at -20℃ and then freeze-dried for 24 h. 1 mL of sterile distilled water was added to fully dissolve the sterile fermentation broth powder, and the powder was shaken well. 100 μL of the 10-fold concentrated sterile fermentation broth was used to conduct an antibacterial experiment against late blight, following the same procedure as in 2.3.1. Each group of sterile fermentation broth was subjected to three replicates for the antibacterial experiment.

[0068] 2.3.5 Antibacterial test of antagonistic bacterial cell lysate

[0069] The method for preparing the bacterial cell lysate is as follows: Centrifuge the bacterial suspension grown for 48 hours at 4500 rpm for 10 minutes, remove the fermentation supernatant, and obtain 1 g of antagonistic bacterial cells. Wash the antagonistic bacterial cells twice with phosphate buffer solution, then resuspend the cells in 20 mL of phosphate buffer. Place the resuspended cells in ice water and disrupt them using an ultrasonic cell disruptor (ultrasonic disruption for 5 seconds, pause for 10 seconds, for a total of approximately 20 minutes) until the suspension becomes clear. Centrifuge the ultrasonically disrupted suspension at 4500 rpm for 10 minutes at 4°C; the resulting supernatant is the bacterial cell lysate. To prevent the undisturbed strain from exerting its effect, place the bacterial cell lysate in a 100°C water bath for 10 minutes. Perform an antibacterial test on the treated bacterial cell lysate against late blight, following the procedure described in 2.3.1. Each group of bacterial cell lysate was subjected to three replicates for the antibacterial test.

[0070] 2.3.6 Determination of the relative protection rate of antagonistic strains against isolated potato tissues

[0071] Healthy leaves (3rd and 4th leaves) were harvested from 30-day-old potato plants with uniform growth. 2cm × 2cm × 1.5cm slices were cut from healthy potato tubers. Both leaves and tubers were sterilized with 1% NaClO for 10 minutes, rinsed with sterile water, and placed in the center of agar plates containing agar. The treated detached leaves and tubers were divided into two groups. In one group, 20 μL of antagonistic bacterial suspension was evenly spread onto the surface of both the potato leaves and tuber slices. An equal amount of sterile water was used as a control group. The plants were incubated at 20℃ for 7 days to observe the effects of the antagonistic bacteria on the detached potato leaves and tubers. Late blight mycelium grown for 7 days was scraped off with a sterile spreading stick and placed in a centrifuge tube containing 10 mL of sterile water. The tubes were shaken for 3 minutes, and the resulting mixture was filtered through a 300-mesh sieve to remove mycelium, yielding a sporangium suspension. The sporangium suspension was adjusted to 1.0 × 10⁻⁶. 6 After applying cfu / mL, the sample was placed in a 4℃ refrigerator for 3 hours to release zoospores. In another group, after applying an equal amount of antagonistic bacterial suspension and culturing under the same conditions for 2 days, 20 μL of late blight sporangium suspension was evenly applied to the surface of the treated potato leaves. Potato late blight fungus cakes (8.3 mm) grown for 7 days were inoculated onto the surface of potato tuber slices and cultured for 7 days under light conditions of 20℃ and a light-dark ratio of 16:8. The relative protection rate of each group of antagonistic bacteria against potato ex vivo tissue was determined in 3 replicates. The disease index was statistically analyzed according to the disease classification criteria (Table 1) (Equation 1), and the relative protection rate was calculated (Equation 2).

[0072] Table 1. Grading Standards for General Severity of Potato Ex vivo Tissue

[0073]

[0074]

[0075] 2.4 Microbial biochar inoculants

[0076] 2.4.1 Determination of storage stability of antagonistic strain biochar inoculant

[0077] Add 2.5 mL of antagonistic bacterial suspension and 1.72 g of sterilized biochar to an Erlenmeyer flask containing 47.5 mL of LB liquid medium and shake at 160 rpm and 31 °C for 145 min. Add 2.5 mL of antagonistic bacterial suspension to another Erlenmeyer flask containing 47.5 mL of LB liquid medium and shake under the same shaking conditions for 135 min. Transfer the liquids from both treatments to sterile 50 mL centrifuge tubes and centrifuge at 4500 rpm and 4 °C for 10 min. Remove the supernatant and freeze the centrifuge tubes at -20 °C for 12 h. Freeze-dry the frozen centrifuge tubes in a vacuum freeze dryer for 12 h, then add 10 mL of sterile water. Measure the viable cell count by dilution and coating. Store the remaining freeze-dried bacterial agents at room temperature and measure the viable cell count of both agents at 0, 5, 30, and 60 days. Each treatment group was set up with 3 replicates to analyze the effect of different treatments on the number of viable bacteria.

[0078] 2.4.2 Determination of the inhibition rate of antagonistic biochar inoculant against potato late blight

[0079] Weigh out 95% cyazofamid, 99% fenpropathrin, 98% azoxystrobin, 58% metalaxyl-mancozeb, antagonistic biochar inoculant, and Shengxinglong potato seed dressing inoculant stored at room temperature. Dissolve each in a certain amount of organic solvent, then add sterile water to prepare a 1000 mg / L stock solution. Then, dilute sequentially to 10, 5, 6.4, 2, 10, and 10 mg / L according to the recommended dosage. Further serially dilute the pesticides 10-fold. Perform plate confrontation tests on potato late blight using both dilutions to compare the antibacterial activity of the antagonistic biochar inoculant. The plate confrontation test is the same as in 2.3.2, with three replicates for each treatment. Statistical analysis is performed.

[0080] 2.4.3 Bioassay of the antibacterial function of microbial agents

[0081] 2.4.3.1 Effects of inoculant seed treatment on the growth and development of potted potatoes

[0082] Healthy seed potatoes (Eugene 885) were cut into small pieces according to their eyes and divided into 5 groups, with 5 treatments according to Table 2, 9 pots per treatment. The treated tubers were planted in pots containing peat moss and vermiculite (mixture ratio 7:3) and placed in a greenhouse at 20℃, 70% relative humidity, and a 16h / 8h light / dark ratio. No watering was done for the first 2 days after planting to prevent the fungicide from being washed away. Watering was done on the 3rd day after planting, and then every 7 days thereafter. A 1.0×10⁻⁶ concentration was sprayed on the tubers 30 days after planting. 6A CFU / ml suspension of potato late blight zoospores was sprayed onto each plant at a rate of 20 mL. The greenhouse environment was set to 17℃ and 90% relative humidity. After 24 hours of incubation, the original greenhouse environment was restored, and the potato late blight infection status was observed. Each treatment group was set up with 3 replicates. The disease disease, disease index, and relative protection rate of potato plants in pots were observed and statistically analyzed according to Table 3. The yield of each group of plants was also calculated.

[0083] Table 2 Potting Treatment for Potato Late Blight

[0084]

[0085] Table 3. Potato Late Blight Disease Index Scoring Table

[0086]

[0087]

[0088] 2.4.4 Effects of Seed Treatment with Microbial Agents on Potato Growth and Development in the Field

[0089] The potato planting for the field experiment was on April 26th. Five treatment groups were designed (TA, TB, TC, TD, TE), with treatment methods as shown in Table 2-5. Each treatment group had 10 plants. The experimental field was divided into 5 rows, with 30 plants per row, spaced 20cm apart, and 60cm apart between rows. Healthy seed potatoes (Eugene 885) were cut into pieces, and the treated seed potatoes were planted in the experimental field. After 30 days, each group was sprayed with a concentration of 1.0 × 10⁻⁶. 6 A 200 mL suspension of CFU / mL potato late blight sporangia was used to observe potato late blight infection. Each treatment group was set up with 3 replicates, and the plant disease index (Equation 1) and the relative protection rate of the inoculant (Equation 2) were calculated.

[0090] 3 Results and Analysis

[0091] 3.1 Screening of antagonistic bacterial strains

[0092] 130 antagonistic bacteria against potato late blight were screened through plate confrontation tests, and highly resistant strains were selected based on the determination of their inhibition rate. Figure 1 It has an antibacterial rate of over 90%.

[0093] 3.2 Determination of the antibacterial spectrum of antagonistic strains

[0094] Antagonistic bacteria possess broad-spectrum antibacterial activity. They exhibit inhibitory effects against *Fusarium oxysporum* (cucumber type), *Fusarium solanum*, *Rhizoctonia solani*, and *Geotrichum candida*. Figure 2 (Table 4).

[0095] Table 4. Verification of the broad-spectrum antibacterial activity of antagonistic strains.

[0096]

[0097] 3.3 Identification of antagonistic strains

[0098] 3.3.1 Morphological identification of antagonistic strains

[0099] Morphological observation of the antagonistic bacteria on LB medium revealed that they were all rod-shaped. The bacterial cells were orange-yellow, opaque, and round, producing orange pigment; blT-49 stained red with Gram stain, indicating it was a Gram-negative bacterium. Figure 3 ).

[0100] 3.3.2 Physiological and biochemical identification of antagonistic strains

[0101] The strains were subjected to 12 physiological and biochemical tests, including glucose oxidation fermentation and indole assay. Each test was repeated three times. Based on Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria, the genus or species unit of the strains was preliminarily inferred. The results are shown in Table 5.

[0102] Table 5 Physiological and biochemical characteristics of antagonistic strains

[0103]

[0104] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0105] 3.3.3 Identification of 16S rDNA sequences of antagonistic bacterial strains

[0106] Submit the determined sequences and information to the Genbank database to obtain the accession number: ON063447.

[0107] The full-length 16S rDNA gene fragment of this strain is 1479 bp. This strain shows the closest genetic distance to *Pseudomonas chlororaphis*, both belonging to the same minimal branch with 100% homology. Based on the morphological and physiological-biochemical characteristics of strain blT-49, it is preliminarily identified as *Pseudomonas chlororaphis*. Because it only produces orange pigment, it is further identified as *Pseudomonas chlororaphis* subsp. *aurantiaca*. Figure 4 The strain was named blT-49.

[0108] 3.3.4 Antagonistic strains inhibit bacterial growth

[0109] All four components of strain blT-49 exhibit antibacterial activity. The antibacterial ability of the components, from highest to lowest, is as follows: bacterial culture = bacterial suspension > sterile fermentation broth > cell lysate (see [reference]). Figure 5 The bacterial culture and bacterial suspension exhibited extremely high antibacterial activity, with an inhibition rate of 100%. In the plate confrontation test, the mycelium of potato late blight on the bacterial cake could hardly survive. Although the inhibition rate of the sterile fermentation broth was significantly lower than that of the bacterial culture and bacterial suspension, its inhibition rate was still as high as 95% (see [link to relevant documentation]). Figure 6 This indicates that blT-49 can inhibit bacteria by producing antibacterial substances through fermentation, or by inhibiting bacteria through substances within the bacterial cell. The high antibacterial rate of the bacterial suspension reflects the synergistic effect of these two methods in exerting their antibacterial function.

[0110] Using an equal amount of Bacillus subtilis reference bacteria as a control, the antibacterial activity of blT-49 bacterial suspension was determined under serial dilution. The results showed that the antibacterial activity of blT-49 antagonistic bacteria was positively correlated with the bacterial concentration (see [link to study]). Figure 7 , Figure 8 ). blT-49 bacterial suspension in undiluted and diluted 10 -1 The antibacterial rate was consistent throughout, both exhibiting 100% antibacterial activity at a dilution of 10%. -2 At that time, the inhibition rate was 99.40%. The inhibition rates of the first five dilution gradients did not differ significantly, but were significantly higher than the last five dilution gradients. Among the nine dilution gradients, seven had inhibition rates above 90%, and nine had rates above 80.00%. Furthermore, the antagonistic bacterial suspensions of all nine dilution gradients were significantly higher than the blank control group (p<0.001). Among the Bacillus subtilis reference bacteria diluted from the bacterial suspension, the inhibition rates of the first four dilution gradients were significantly higher than those of the remaining five dilution gradients. Among the nine dilution gradients, two had inhibition rates above 90%, four above 80%, and three below 50%. The dilution gradient with the lowest inhibition rate was 10 times that of the Bacillus subtilis reference bacterial suspension. -8 Its antibacterial rate was only 7.39%. The Bacillus subtilis suspensions in the first 8 dilution gradients were significantly higher than those in the blank control group (p<0.001).

[0111] 3.3.5 Relative protection rate of antagonistic strains against isolated potato tissues

[0112] In potato tissue experiments, the late blight disease severity index of blT-49 was 11.11%, and the relative protection rate was 85.68% (see [reference needed]). Figure 10 Leaves treated with only blT-49 bacterial suspension showed only some antagonistic bacterial pigment residue, while leaves treated with both blT-49 bacterial suspension and late blight sporangium suspension showed small areas of water-soaked lesions (see...). Figure 9 ).

[0113] In potato tuber experiments, no obvious infection characteristics were observed in potato tubers treated with blT-49 bacterial suspension and late blight sporangium suspension (see [link]). Figure 9 Furthermore, the tubers treated with this method showed the lowest degree of late blight infection, with a disease index of 3.7% and a protection rate of 92.31% (see [link to relevant documentation]). Figure 10 ).

[0114] 3.4blT-49 Biochar Agent

[0115] 3.4.1 Storage stability of blT-49 biochar inoculant

[0116] The blT-49 biochar inoculant showed higher survival rate and stronger storage stability after vacuum freeze-drying. Figure 11 The viable count in the blT-49 bacterial suspension treatment group was 6.7 × 10⁻⁶. 7 CFU / 100μL, compared to the viable cell count in blT-49 biochar agent (6.4×10⁻⁶). 7 The cfu / 100μL concentration was slightly higher, but the inactivation rate of viable bacteria in the bacterial solution was high within 0–10 days. At 10 days, the number of viable bacteria in the inoculum was higher than that in the bacterial solution. At 60 days, the viable count of the bacterial strain in the blT-49 inoculum was 4.0 × 10⁻⁶. 7 The cfu / 100μL concentration resulted in a survival rate of 62.50%, while the bacterial cell count in the culture was 1.3 × 10⁻⁶. 7 With a cfu / 100 μL concentration, the survival rate was only 19.40% compared to 0 days.

[0117] 3.4.2 Determination of the inhibition rate of blT-49 biochar inoculant against potato late blight

[0118] according to Figure 12 and Figure 13 It can be seen that blT-49 inoculant had the highest inhibition rate, reaching 93.69%, significantly higher than the inhibition rates of several other commonly used pesticides on the market (p<0.01). Among them, there was no significant difference in the inhibition ability between flufenoxuron and potato seed dressing inoculant, and there was also no significant difference in the inhibition ability between flufenoxuron and metalaxyl-mancozeb. The lowest inhibition rate was achieved by azoxystrobin, with an inhibition rate of only 46.82%. When all fungicides were diluted by half based on the recommended agricultural dosage, the inhibition rate of blT-49 biochar inoculant decreased by only 1.47%, still significantly higher than the other five. Azoxystrobin's inhibition ability remained the weakest, at only 13.10%. As can be seen from the figure, fungicides are more effective at inhibiting bacteria than chemical fungicides. The five fungicides are ranked from most effective to least effective in inhibiting bacteria as follows: blT-49 biochar fungicide > potato seed dressing fungicide > 95% fumigant > 58% metalaxyl-mancozeb > 99% fluazinam > 98% azoxystrobin.

[0119] 3.5 Bioassay of Microbial Antibacterial Function

[0120] 3.5.1 Effects of inoculant seed treatment on the growth and development of potted potatoes

[0121] In pot experiments, the potato disease index of the group treated with blT-49 biochar inoculant was lower, and the growth and development were better. The blT-49 biochar inoculant group had a significantly lower disease index than the other groups, and the highest relative protection rate. The group treated with blT-49 liquid powder was the second highest. The relative protection rate of the group treated with 58% metalaxyl-manganese zinc was lower than the first two groups, and biochar also had a certain inhibitory effect on late blight. The antibacterial ability of the five groups, from highest to lowest, was: blT-49 biochar inoculant > blT-49 liquid powder > 58% metalaxyl-manganese zinc > biochar > CK (Table 6). Meanwhile, compared with the blank control group, the plants treated with blT-49 biochar inoculant showed a 21.00% increase in plant height, a 35.82% increase in main stem diameter, and a 17.92% increase in yield per plant (Table 7).

[0122] Table 6. Disease index and relative protection rate of 5 different treatments in the pot experiment.

[0123]

[0124] Note: Each value represents the mean ± standard deviation (n=3); different lowercase letters indicate significant differences between groups at the p<0.05 level.

[0125] Table 7. Effects of five different treatments on potato plant growth in the greenhouse experiment.

[0126]

[0127]

[0128] Note: Each value represents the mean ± standard deviation (n=3); different lowercase letters indicate significant differences between groups at the p<0.05 level.

[0129] 3.5.1 Effects of Seed Treatment with Microbial Agents on Potato Growth and Development in the Field

[0130] The results of field trials (Table 8) showed that among the five experimental groups, the group treated with blT-49 antagonistic biochar had the lowest disease index (32.96%) and the highest relative protection rate (59.22%), exhibiting the strongest antibacterial ability. The blT-49 bacterial liquid powder was the second strongest, with a disease index of 45.93% and a relative protection rate of 43.18%. The control group had a disease index of 80.74%. The antibacterial ability of the five groups, ranked from highest to lowest, was: blT-49 antagonistic biochar > blT-49 bacterial liquid > 58% metalaxyl-manganese zinc > biochar > CK.

[0131] Table 8 Disease index and relative protection rate of 5 different treatments in the field trial.

[0132]

[0133] Note: Each value represents the mean ± standard deviation (n=3); different lowercase letters indicate significant differences between groups at the p<0.05 level.

Claims

1. A potato late blight antagonist, characterized in that... It is Pseudomonas chlororaphis subsp. chlororaphis blT-49, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 27, 2021, with accession number CGMCC No. 24203.

2. The application of a potato late blight antagonist as described in claim 1, characterized in that... The aforementioned Pseudomonas chlororaphis blT-49 is used to control potato late blight and promote the growth and development of potato plants.

3. The application according to claim 2, characterized in that... The aforementioned control of potato late blight reduces the infectivity of potato late blight and enhances the disease resistance of potatoes.

4. The application according to claim 2 or 3, characterized in that... The aforementioned Pseudomonas chlororaphis subsp. blT-49 can be prepared into bacterial liquid, bacterial suspension, bacterial cell lysate, or sterile fermentation broth for application.

5. The application according to claim 4, characterized in that... The original suspension of *Pseudomonas chlororaphis* blT-49 was diluted to 10... -1 ~10 -8 use.

6. The application of a potato late blight antagonist as described in claim 1, characterized in that... The aforementioned *Pseudomonas chlororaphis* subsp. *chlororaphis* blT-49 is used to inhibit the growth of *Fusarium oxysporum* cucumber-specific type, *Geotrichum candida*, *Fusarium solani*, and *Rhizoctonia solani*.

7. The application of a potato late blight antagonist as described in claim 1, characterized in that... The aforementioned Pseudomonas chlororaphis subsp. blT-49 is used as a protective agent for detached potato leaves and tubers.

8. The inoculant for a potato late blight antagonist as described in claim 1, characterized in that... The bacterial agent mentioned is a biochar bacterial agent.

9. The inoculum agent of a potato late blight antagonist according to claim 8, characterized in that... The preparation method of the biochar agent is as follows: Fermented Pseudomonas chlororaphis blT-49 bacterial suspension was added to LB liquid medium, and sterilized biochar was added. The medium was then incubated at 30-35℃ for 130-150 min, with 30-35 mg of biochar added per mL of bacterial suspension.

10. The inoculum agent of a potato late blight antagonist according to claim 9, characterized in that... The fermentation conditions for the fermented Pseudomonas chlororaphis subsp. blT-49 were pH=7 and fermentation time was 45-50h.

Images