Pseudomonas chlororaphis subsp. Aurantiaca strain and application thereof

Abstract

The invention relates to the field of microbiology and fermentation engineering, in particular to a pseudomonas chlororaphis subsp. Aurantiacum strain and application thereof. The strain is named as PC-1, and is preserved in China Center for Type Culture Collection in Wuhan University in Wuhan City in China on March 24, 2025, and the preservation number is CCTCC NO: M 2025568. The strain has tolerance to 11 chemical pesticides such as dimethomorph, carbendazim, bromothalon.prochloraz and the like, and can be alternately or mixed with the pesticides for use. In addition, the strain has remarkable inhibitory activity on various plant pathogenic microorganisms such as phytophthora infestans, fusarium solani, fusarium oxysporum and fusarium tripteroides, and has broad-spectrum antibacterial performance. Application tests show that fermentation liquor or fermentation supernate of the strain can effectively prevent and treat potato late blight, potato dry rot, physalis alkekengi black spot, tobacco root rot and other crop diseases, and shows good prevention effect and environmental adaptability in field tests. The strain has good pesticide compatibility and growth promoting potential, and is suitable for biological prevention and control of plant diseases in green agriculture.

Description

Technical Field

[0001] The present invention relates to the fields of microorganisms and fermentation engineering, in particular to the technical field of agricultural microbial prevention and control, and specifically to a Pseudomonas chlororaphis subspecies orange strain and an application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Potatoes are nutritious, highly productive, and inexpensive, making them a staple food in much of the world. Potatoes are widely cultivated worldwide, with my country boasting the world's largest potato planting area and production. Along with rice, wheat, and corn, they are listed as one of my country's four major staple foods. Potato late blight, caused by Phytophthora infestans, is the most widespread and devastating potato disease worldwide. It is reported that Phytophthora infestans is one of the most important diseases in potato cultivation and production. Annual losses caused by Phytophthora infestans can account for 30% of my country's total potato planting, sometimes reaching 50%. In severe cases, it can even lead to crop failure in some areas, resulting in hundreds of millions of yuan in economic losses to potato cultivation.

[0004] Potato late blight is caused by the pathogen Phytophthora infestans, which infects the stems, leaves, and tubers of potatoes. When the leaves become infected, green-brown lesions typically appear at the tips or edges of the leaves. As the disease progresses, the lesions grow larger and darker, eventually covering the entire leaf, turning it brown, dry, and brittle. In humid conditions, a layer of white mold will grow on the leaf lesions. When Phytophthora infestans infects the stems and petioles of potatoes, brown stripes of lesions appear. In the later stages of the disease, the tissue at the affected area necrotizes and rots, causing the leaves on the upper stem to wilt and curl, and in severe cases, the entire plant to die. When potato tubers become infected, irregular brown lesions appear. As the disease progresses, the lesions continue to expand and begin to sink in from the center, followed by tuber rot. If these rotten tubers are not properly treated, they may serve as a source of infection and further infect surrounding plants.

[0005] Traditional control measures for potato late blight primarily rely on large-scale spraying of chemical fungicides. Commonly used chemical fungicides generally fall into the phenylamide and benzamide classes, with metalaxyl being a prominent example. These fungicides are relatively low in toxicity and highly effective against Phytophthora pathogens. However, their extensive and unscientific use has led to resistance in many pathogens. For example, metalaxyl-resistant strains emerged in Israel just one year after its use. In my country, where metalaxyl was widely used to control Phytophthora diseases in the 1980s, resistant strains were also discovered two years later. Globally, resistance to metalaxyl has been observed in various Phytophthora species, including Phytophthora infestans, Phytophthora capsici, Phytophthora cucumeris, and Phytophthora vinifera. According to a 2018 report by Lu Fen et al., resistance to metalaxyl has become widespread in potato late blight pathogens in Hebei, Inner Mongolia, and Jilin, reaching a 100% resistance frequency, an average resistance multiple of 34,934, and a resistance index of 0.97. In 2011-2012, moderately and highly resistant strains dominated the late blight population, while from 2013 to 2016, highly resistant strains dominated. The average resistance multiple of strains in Jilin and Inner Mongolia was slightly higher than that in Hebei. In 2011-2012, the resistance index of strains in Jilin and Inner Mongolia was slightly higher than that in Hebei, and from 2013 to 2016, the resistance index in all three regions reached a maximum of 1.00. In the field efficacy test, the control effects of 25% metalaxyl SC, 68% metalaxyl·mancozeb WG and 64% mancozeb·mancozeb WP were 37.0%, 67.5% and 50.9%-65.8% respectively after 4 sprays at the recommended concentration, which were significantly lower than the control effect of 80% mancozeb WP (72.8%). The control effects of 10% fluazifop OD, 50% fluopicolide WG, 50% dimethomorph WP and 687.5 g·L -1 Fluopyram + propamocarb SC, 16% fluthiazolin + azoxystrobin SC, and 26% fluthiazolin + mandiprop-amid SC demonstrate excellent control efficacy (83.9%-90.3%) against potato late blight and can be used as alternatives to metalaxyl. However, the overuse of chemical fungicides can lead to environmental pollution and residues, adversely affecting beneficial organisms such as nitrogen-fixing bacteria and mycorrhizal fungi, and causing irreversible damage to the ecological environment and human health. This is contrary to today's pursuit of environmental sustainability.

[0006] Against this backdrop, finding effective methods to control potato disease-causing Phytophthora has become a top priority. Currently, the main focus of potato disease research and control is a comprehensive control approach based on rational prediction, forecasting, and prevention, along with environmentally friendly biological control technologies. Biological control offers numerous advantages, including high efficiency, low toxicity, safety, and environmental friendliness, and has gradually become an effective approach to plant disease prevention and control. Summary of the Invention

[0007] The present invention aims to provide a strain of Pseudomonas chlororaphis subsp. aurantiacus that is highly effective in inhibiting Phytophthora infestans, a fermentation method for this strain, and its application in plant disease control. This approach aims to address the resistance and environmental pollution issues associated with traditional chemical pesticides, providing an environmentally friendly biocontrol method. Specifically, the present invention aims to address at least one of the following technical problems:

[0008] The widespread problem of resistance to chemical pesticides in the prevention and control of potato late blight, especially the severe situation where the resistance frequency of phenylamide fungicides such as metalaxyl is as high as 100%; the long-term and large-scale use of chemical pesticides has caused environmental pollution, drug residues and adverse effects on beneficial microorganisms; there is currently a lack of efficient biological control strain resources for potato late blight, and there is an urgent need to develop more microbial control products with practical application value.

[0009] Through screening and identification, the present invention obtained a strain of Pseudomonas chlororaphis subspecies orange PC-1 with significant inhibitory activity against Phytophthora infestans. The strain has the following technical effects:

[0010] The inhibition rate against Phytophthora infestans is as high as 90.19%, significantly higher than that of many existing biocontrol strains;

[0011] The control effect of the plant on potato late blight under field conditions reached 83.04%, which was much higher than the control effect of the conventional chemical pesticide dimethomorph (20.49%).

[0012] The strain can produce antimicrobial active substances such as phenazine-1-carboxylic acid, which has broad-spectrum inhibitory activity against a variety of plant pathogens;

[0013] It is tolerant to 11 commonly used pesticides, including dimethomorph and carbendazim, and can be used in conjunction with chemical pesticides;

[0014] After the fermentation conditions are optimized, the viable bacterial count can reach 12 billion CFU / mL, which has good potential for industrial production.

[0015] Specifically, the present invention provides the following technical features, and the combination of one or more of the following technical features constitutes the technical solution of the present invention.

[0016] In a first aspect of the present invention, a Pseudomonas chlororaphis subsp. aurantiaca is provided, which is named PC-1 and has been deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, China on March 24, 2025, with a deposit number of CCTCC NO: M 2025568.

[0017] The 16S rRNA gene sequence of the strain PC-1 is shown in SEQ ID NO: 1. It can grow in a temperature range of 4-42°C, can tolerate an environment with a pH value of 5-9, can tolerate a NaCl concentration of 5%, and can tolerate cadmium ions (Cd) of 5 mg / L. 2+ The strain PC-1 can produce phenazine-1-carboxylic acid, and its yield can reach 1.45 g / L after 28 hours of cultivation under optimized fermentation conditions.

[0018] In a second aspect of the present invention, a plant disease biological control agent composition is provided, wherein the composition comprises the above-mentioned Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 or a fermentation product thereof.

[0019] In some embodiments, the composition further comprises agriculturally acceptable carriers and / or adjuvants. For example, the carrier-like substances include solid carriers and liquid carriers, solid carriers such as kaolin, bentonite, diatomaceous earth, vermiculite, perlite, zeolite and other mineral carriers; organic carriers such as peat, compost, humus, biochar, rice husks, wheat bran and the like; liquid carriers such as water, glycerol, vegetable oil, mineral oil and the like. For example, the adjuvants can be selected from surfactants, adhesives, dispersants, stabilizers, protective agents and the like as needed. These carriers and adjuvants can be reasonably selected or combined according to the characteristics of the strain PC-1, the form of the formulation (such as suspension, wettable powder, water-dispersible granules, emulsion, etc.) and the application requirements to improve the stability, activity and use effect of the product. In certain application scenarios, the strain PC-1 or its fermentation product without carriers and adjuvants can also be used directly.

[0020] In some embodiments, the viable bacterial count concentration of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 in the composition is 1×10 7 CFU / mL to 1 × 10 12 CFU / mL, more preferably 1×10 8 CFU / mL to 1 × 10 11 CFU / mL, the most preferred is 1×10 9 CFU / mL to 1 × 10 10 CFU / mL.

[0021] In some embodiments, the composition may further comprise at least one chemical pesticide selected from the group consisting of dimethomorph, carbendazim, bromothiocarb, mancozeb, zhongshengmycin, thiophanate-methyl, oligosaccharide-chain protein, tetramycin-tetracycline, benzyl-prochloraz, oxadiazine and flutolane-tebuconazole.

[0022] In some embodiments, the plant disease is at least one selected from the following: potato late blight caused by Phytophthora infestans, potato dry rot caused by Fusarium solani, cotton verticillium wilt caused by Rhizoctonia solani, corn stalk rot caused by Fusarium graminearum, tobacco root rot caused by Fusarium commune, potato early blight caused by Alternaria solani, tomato root rot caused by Fusarium oxysporum, cucumber black spot caused by Alternaria cucumerina, tea leaf spot caused by Alternaria longipes, etc.

[0023] In a third aspect of the present invention, there is provided use of the Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 or a fermentation product thereof or the composition thereof in preparing a biological control agent for controlling plant diseases.

[0024] In some embodiments, the plant disease is at least one selected from the following: late blight of potato caused by Phytophthora infestans, dry rot of potato caused by Fusarium solani, verticillium wilt of cotton caused by Rhizoctonia solani, stalk rot of corn caused by Fusarium graminearum, root rot of tobacco caused by Fusarium commensal, early blight of potato caused by Alternaria solani, root rot of tomato caused by Fusarium oxysporum, black spot of cucumber caused by Alternaria cucurbitae, leaf spot of tea caused by Alternaria longibotia, etc.

[0025] In a fourth aspect of the present invention, there is provided use of the Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 or its fermentation product or the above composition in the preparation of an antibacterial agent.

[0026] In some embodiments, the antibacterial agent has an antibacterial effect on at least one selected from the group consisting of Phytophthora infestans, Fusarium solani, Fusarium oxysporum, Fusarium tricinctum, Fusarium chlamydosporum, Fusarium incarnatum, Alternaria alternata, and Rhizoctonia solani. The Alternaria is at least selected from the group consisting of Alternaria solani, Alternaria cucumerina, and Alternaria longipes.

[0027] In a fifth aspect of the present invention, a method for preventing and controlling plant diseases is provided, comprising applying the above-mentioned Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 or its fermentation product or the above-mentioned composition to plants (such as the whole plant, such as stems, leaves, roots, tubers, flowers and fruits, etc.), plant seeds, plant cultivation substrates or plant growth environments.

[0028] In some embodiments, the plant disease is at least one selected from the following: late blight of potato caused by Phytophthora infestans, dry rot of potato caused by Fusarium solani, verticillium wilt of cotton caused by Rhizoctonia solani, stalk rot of corn caused by Fusarium graminearum, root rot of tobacco caused by Fusarium graminearum, early blight of potato caused by Alternaria solani, root rot of tomato caused by Fusarium oxysporum, black spot of cucumber caused by Alternaria cucumerina, leaf spot of tea caused by Alternaria longipes, etc.

[0029] In some embodiments, the fermentation product of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 is a fermentation broth, a fermentation supernatant, or a fermentation filtrate of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1.

[0030] In this application, the fermentation broth refers to the entire culture medium obtained by fermenting strain PC-1 in an optimized medium, containing intact bacterial cells and various metabolites (such as phenazine-1-carboxylic acid). This form contains the combined effects of live bacteria and metabolites and is suitable for scenarios requiring sustained antibacterial activity and inducing plant resistance.

[0031] In this application, the fermentation supernatant refers to the supernatant after centrifugation or sedimentation of the fermentation broth. It primarily contains metabolites secreted by strain PC-1, such as phenazine-1-carboxylic acid, but contains no or minimal bacterial cells. This is suitable for control scenarios where only metabolites are needed, without the presence of live bacteria.

[0032] In the present invention, the fermentation filtrate refers to the filtrate obtained after the fermentation broth has been filtered (e.g., through a 0.22 μm membrane), which completely removes the bacterial cells and contains only soluble metabolites. It is particularly suitable for situations where sterilization is required or only the activity of metabolites is utilized.

[0033] In some embodiments, the amount of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 or its fermentation product is 10-500 times diluted fermentation liquid of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 and applied to plants.

[0034] In some embodiments, the application is foliar spraying, soil drench, root drench, seed treatment, tuber treatment, or a combination of any two or more of the foregoing.

[0035] In a sixth aspect of the present invention, a fermentation method of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 is provided, the method comprising:

[0036] 1) Cultivating Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 in a nutrient medium to prepare a seed solution;

[0037] 2) inoculating the obtained seed liquid into a fermentation medium containing a nutrient source;

[0038] 3) Fermenting under suitable conditions to obtain a fermentation broth of strain PC-1.

[0039] In some embodiments, the nutrient medium is a nutrient broth medium, and the fermentation medium is a glycerol peptone medium.

[0040] In some embodiments, the culture condition in step 1) is culturing at 28-30° C. for 12-24 hours.

[0041] In some embodiments, the inoculation amount in step 2) is 3-10%, more preferably 5%.

[0042] In some embodiments, the fermentation conditions in step 3) are a temperature of 25-30° C., a pH of 6.5-7.0, a stirring speed of 150-200 rpm, and a fermentation time of 20-30 hours.

[0043] In some embodiments, the viable cell count of strain PC-1 in the fermentation broth is 1×10 9 CFU / mL to 1 × 10 12CFU / mL, and the content of phenazine-1-carboxylic acid in the fermentation broth was 0.5-2.0 g / L.

[0044] In one embodiment, the fermentation method comprises: inoculating strain PC-1 into NB medium and culturing overnight in a shaking incubator at 30°C and 180 rpm to prepare a seed solution; inoculating the seed solution into modified GSP medium (pH 6.8) at a 5% inoculum; and fermenting at 30°C for 28 hours, wherein 0-4 hours is the lag phase, 4-12 hours is the logarithmic growth phase, and 12-28 hours is the stationary phase. At the end of fermentation, the viable count of strain PC-1 in the fermentation broth can reach 12 billion CFU / mL, and the phenazine-1-carboxylic acid content can reach 1.45 g / L. This method is simple, efficient, and suitable for large-scale industrial production.

[0045] Compared with the prior art, the advantages of the present invention include:

[0046] Highly effective in inhibiting Phytophthora infestans: The Pseudomonas chlororaphis subsp. aurantifolia strain PC-1, obtained through screening and screening, demonstrated an inhibition rate of 90.19% against Phytophthora infestans in in vitro antibacterial tests, significantly higher than other currently used biocontrol strains. In field trials, it demonstrated an 83.04% efficacy against potato late blight, significantly exceeding the 20.49% efficacy of the conventional chemical pesticide dimethomorph, demonstrating the strain's outstanding biocontrol potential.

[0047] Broad-spectrum antibacterial activity: Strain PC-1 not only has a significant inhibitory effect on pathogenic Phytophthora infestans, but also shows good inhibitory activity against a variety of plant pathogens such as Fusarium solani (inhibition rate 90.54%), Rhizoctonia solani (59.13%), Fusarium graminearum (78.38%), Fusarium graminearum (78.08%), Alternaria solani (87.12%), Fusarium oxysporum (85.91%), Alternaria cucurbitae (78.14%), and Alternaria longibotia (83.83%), showing a broad-spectrum control effect.

[0048] Efficient production of biologically active substances: Strain PC-1 can efficiently produce the antibacterial active substance phenazine-1-carboxylic acid, with a fermentation yield of up to 1.45 g / L in 28 hours. Its antibacterial activity increases with the extension of fermentation time, and the antibacterial rate can reach 72.27%, providing a material basis for biological control.

[0049] Compatibility with chemical pesticides: Strain PC-1 has good tolerance to 11 commonly used pesticides, including dimethomorph, carbendazim, bromocriptine and prochloraz, and can be used alternately or in combination with these agents, providing the possibility of building an integrated prevention and control system combining chemical pesticides and biological control agents.

[0050] Strong adaptability: Strain PC-1 can grow in a wide temperature range of 4-42°C, can tolerate an environment with a pH value of 5-9, and can tolerate a NaCl concentration of 5% and a cadmium ion of 5 mg / L, indicating that the strain has strong environmental adaptability and is conducive to its application in complex agricultural production environments.

[0051] Safe and environmentally friendly: As a biocontrol agent, strain PC-1 is non-toxic and residue-free, with no negative impacts on the environment or beneficial organisms. Wheat seed germination tests showed that a 500-fold diluted bacterial solution not only did not inhibit plant growth but actually significantly increased wheat seedling emergence and plant height, demonstrating its safety and growth-promoting effects on crops.

[0052] Industrial Potential: Optimization of fermentation conditions for strain PC-1 has been completed. After 28 hours of fermentation in a 20-L fermenter, the viable bacterial count can reach 12 billion CFU / mL, demonstrating excellent industrial production potential and meeting the needs of large-scale production.

[0053] Easy to use: This strain can be applied in a variety of ways, such as foliar spraying, soil irrigation, root irrigation, seed treatment, etc. It is suitable for disease prevention and control of different crops and different growth stages, and is flexible and convenient to use.

[0054] In summary, the Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 and its applications provided by the present invention not only have a highly effective control effect on potato late blight, but also exhibit good inhibitory activity against a variety of plant diseases. It also has the advantages of being safe, environmentally friendly, and having great potential for industrial production, and is expected to become an important biological control agent in the integrated plant disease prevention and control system. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute undue limitations thereon. The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0056] Figure 1 The results show the inhibitory effects of seven biocontrol bacteria on Phytophthora infestans.

[0057] Figure 2 Shown are microscopic images (scale bar: 10 μm) and colony pictures of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1.

[0058] Figure 3 Shown is a neighbor-joining phylogenetic tree of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 based on 16S rRNA gene sequences.

[0059] Figure 4The results of the sensitivity test of Pseudomonas chlororaphis subspecies orange strain PC-1 to chemical agents are shown; among them, 1. dimethomorph; 2. carbendazim; 3. bromothiocarb·prochloraz; 4. mancozeb; 5. cypermethrin; 6. chlorothalonil; 7. oligosaccharide·trencin; 8. tetramycin·methylenetetramycin; 9. benzyl·prochloraz; 10. oxadiazon; 11. flutolan·tebuconazole; 12. sterile water.

[0060] Figure 5 Shown are the effects of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 on wheat seed germination and growth.

[0061] Figure 6 The results of antimicrobial susceptibility tests of Pseudomonas chlororaphis subsp. aurantifolia strain PC-1 are shown; among them, penicillin (PEN), enrofloxacin (ENR), amoxicillin (AMX), cotrimoxazole (SXT), neomycin (N), erythromycin (E), streptomycin (S), cefepime (FEP), doxycycline (DO), gentamicin (GM), tetracycline (TET) and lincomycin (MY).

[0062] Figure 7 The graph shows the comparison of the protective effects of the fermentation broth of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 and dimethomorph against Phytophthora infestans.

[0063] Figure 8 The graph shows the comparative inhibition of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 fermentation supernatant and dimethomorph against Phytophthora infestans.

[0064] Figure 9 Shown are the results of a temperature range tolerance test of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1.

[0065] Figure 10 The results of the UV tolerance test of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 are shown.

[0066] Figure 11 The results of a pH tolerance test of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 are shown.

[0067] Figure 12 The effect of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 on Cd 2+ The tolerance test results.

[0068] Figure 13 The results of a tolerance test of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 to NaCl concentrations are shown.

[0069] Figure 14 The graph shows a comparison of the in vitro protective effects of Pseudomonas chlororaphis subsp. aurantiaca strain PC-1 and dimethomorph against Phytophthora infestans.

[0070] Figure 15 The results show the disease incidence and tuber formation of potato in experimental fields with different treatments.

[0071] Figure 16 Shown is a microscopic image of a seed solution of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1.

[0072] Figure 17 Shown are microscopic images every 2 hours from the 2nd to the 28th hour during the fermentation of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1.

[0073] Figure 18 The viable count and OD of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 at different times during the fermentation process are shown. 600 Record.

[0074] Figure 19 The figure shows the inhibition of Pseudomonas chlororaphis subsp. aurantiaca strain PC-1 fermentation supernatant at different times against Phytophthora infestans.

[0075] Figure 20 The graph shows the antibacterial effect of Pseudomonas chlororaphis subsp. aurantiaca strain PC-1 on Fusarium solani.

[0076] Figure 21 Shown are the in vitro pathogenesis of tubers.

[0077] Figure 22 The graph shows the antibacterial effect of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 on different pathogens.

[0078] Figure 23 The graph shows the antibacterial test of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 against various pathogens. DETAILED DESCRIPTION

[0079] The present invention will be further described with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope thereof. Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0080] Unless otherwise defined, all technical and scientific terms used herein shall have the meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the present invention can be obtained through conventional routes and used in accordance with conventional methods in the art or product specifications. In addition, any content similar or equivalent to the methods or materials described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0081] Material:

[0082] Potato dextrose agar (PDA): 6 g potato powder, 20 g agar, 20 g glucose, 1 L distilled water, pH 5.6 ± 0.2, sterilize at 121 °C for 20 min.

[0083] Soybean juice culture medium: 60g soybeans, soak overnight, use a wall breaker to break them up, then filter with double-layer gauze, 20g glucose, distilled water to 1L, natural pH, sterilize at 121℃ for 20min.

[0084] Nutrient broth (NB) medium: peptone 10 g, beef extract 3 g, sodium chloride 5 g, distilled water 1 L, pH natural, sterilized at 121 °C for 20 min.

[0085] NA medium: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar 20 g, distilled water 1 L, pH natural, sterilize at 121 °C for 20 min.

[0086] Glycerol peptone (GSP) medium: 49.5 g glycerol, 37.3 g peptone, 1.5 g potassium chloride, 0.75 g magnesium sulfate, 0.2 g potassium hydrogen phosphate, 1 L distilled water, pH 6.8, sterilized at 121°C for 20 min.

[0087] Physiological saline: 0.9 g NaCl, 100 mL distilled water, sterilized at 121°C for 20 min.

[0088] Instruments and equipment:

[0089] Clean bench, constant temperature shaker, oscillator, electric constant temperature incubator, high pressure sterilizer, electron microscope.

[0090] Test strains:

[0091] Escherichia coli ATCC25922 and 20 biocontrol strains were provided by the Culture Collection Center of Shandong Bilan Biotechnology Co., Ltd.; various pathogens such as Phytophthora infestans, Rhizoctonia solani, Fusarium graminearum, Fusarium graminearum, Alternaria solani, Alternaria alternata, Fusarium oxysporum, and Alternaria longibotia were all from the Culture Collection Center of Shandong Bilan Biotechnology Co., Ltd., and are all plant pathogens with clear identification and verified pathogenicity.

[0092] Pesticides: Dimethomorph, carbendazim, bromothiocarb·prochloraz, mancozeb, chlorothalonil, oligosaccharide·streptavidin, tetramycin·benzyl, benzyl·prochloraz, oxadiazine, fluazifop·tetracycline are all commercially available products.

[0093] Antimicrobial drug disks: penicillin (PEN), enrofloxacin (ENR), amoxicillin (AMX), cotrimoxazole (SXT), neomycin (N), erythromycin (E), streptomycin (S), cefepime (FEP), doxycycline (DO), gentamicin (GM), tetracycline (TET) and lincomycin (MY) were purchased from Changde Beekman Biotechnology Co., Ltd.

[0094] Example 1 Screening of biocontrol bacteria

[0095] 1 Test method

[0096] Twenty biocontrol strains were selected from the collection of Shandong Bilan Biotechnology Co., Ltd.'s strain collection center and inoculated into NB medium. Fermentation broths were obtained for 24 hours at 30°C and 180 rpm. A 5 mm diameter cake of Phytophthora infestans was placed in the center of each PDA plate. The fermentation broths were inoculated with toothpicks at 20 mm intervals around the cake. A control plate containing only Phytophthora infestans was set up. Three replicates were prepared for each treatment and control group. The plates were incubated at 28°C in the dark for 7 days. When the P. infestans in the control group was about to fill the entire plate, the inhibition rate was measured: inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0097] 2 Test results

[0098] The results are as follows Figure 1 As shown in Table 1, a total of 7 biocontrol bacteria (named PC-1, AC-2, AC-5, BC-3, BC-10, BC-22, and BC-42) with an inhibition rate of more than 75% against P. infestans were obtained, among which strain PC-1 had the best inhibition effect ( Figure 1 ), the inhibition rate was 90.19%, so the strain PC-1 was selected as the antagonistic bacterium against potato late blight.

[0099] Table 1 Inhibition rate of 7 biocontrol bacteria against Phytophthora infestans

[0100]

[0101] Example 2 Identification of strain PC-1

[0102] 1 Colony morphological characteristics

[0103] Streak a single colony of strain PC-1 onto a NA medium plate, place the plate upside down in a constant temperature incubator at 28°C, and culture for 24 hours. The colony will be round, with neat edges, opaque, orange-yellow in color, and easy to pick up (see Figure 2 right).

[0104] 2. Morphological characteristics of bacteria

[0105] The PC-1 strain was stained with crystal violet staining solution and the bacteria were observed to be small, rod-shaped and non-spore-forming under an optical microscope (see Figure 2 left).

[0106] 3 Molecular identification

[0107] Strain PC-1 was sequenced and identified based on the 16S rRNA gene sequence. The PC-1 genome was extracted using universal primers for 16S rRNA gene sequences (27F: 5'-AGAGTTTGATCCTGGCTCAG / 1492R: 5'-GGTTACCTTGTTACGACTT) and sent to Sangon Biotech Co., Ltd. for sequencing. Ezbiocloud analysis showed that the PC-1 strain belonged to the genus Pseudomonas. Strain PC-1 shared the highest 16S rRNA gene sequence similarity with Pseudomonas chlororaphis subsp. aurantiaca (99.65%), Pseudomonas chlororaphis subsp. aureofaciens (99.51%), and Pseudomonas chlororaphis subsp. piscium (99.37%). In the neighbor-joining phylogenetic tree based on 16S rRNA gene sequences, strain PC-1 formed a separate clade with Pseudomonas chlororaphis subsp. aurantiaca (see Figure 3 ).

[0108] The 16S rRNA gene sequence of strain PC-1 is shown in SEQ ID NO: 1 and is also as follows:

[0109]

[0110] Strain PC-1 was identified as Pseudomonas chlororaphis subsp. aurantiaca PC-1. This strain was deposited with the China Center for Type Culture Collection at Wuhan University, Wuhan, China, on March 24, 2025, with the accession number CCTCC NO: M 2025568. Pseudomonas chlororaphis subsp. aurantiaca PC-1 will continue to be referred to as strain PC-1 in the following examples.

[0111] Example 3 Sensitivity test of strain PC-1 to chemical pesticides

[0112] 1 Test method

[0113] The fermentation broth of strain PC-1 was prepared according to the method of Example 1. That is, strain PC-1 was inoculated into NB medium and cultured in a shaking incubator at 30°C and 180 rpm for 24 h. The viable bacterial count of the obtained fermentation broth was 1×10 9 CFU / mL, 100 μL of strain PC-1 fermentation liquid was spread on NA medium, and double-layer sterile filter paper was evenly distributed on the coated medium. Use sterile water to prepare chemical agent solutions according to the following recommended concentrations: dimethomorph (80% active ingredient content, diluted 2000 times), carbendazim (50% active ingredient content, diluted 1000 times), bromocriptine·prochloraz (30% active ingredient content, diluted 1000 times), mancozeb (65% active ingredient content, diluted 200 times), zhongshengmycin (3% active ingredient content, diluted 600 times), chlorothalonil (75% active ingredient content, diluted 500 times), oligosaccharide·chain protein (6% active ingredient content, diluted 800 times), tetramycin (2% active ingredient content, diluted 600 times), benzyl prochloraz (35% active ingredient content, diluted 1000 times), oxadiazine (98% active ingredient content, diluted 2000 times), flutolanil tebuconazole (35% active ingredient content, diluted 2500 times), 8 μL of the above chemicals and sterile water were respectively absorbed onto filter paper, and the samples were cultured at 28°C for 24 hours to observe the sensitivity of strain PC-1 to the chemicals.

[0114] 2 Test results

[0115] The results showed that strain PC-1 was resistant to all 11 chemical agents used (1. dimethomorph; 2. carbendazim; 3. bromothiocarb·prochloraz; 4. mancozeb; 5. zhongshengmycin; 6. chlorothalonil; 7. oligosaccharide·streptavidin; 8. cinnamomum camphor·tetramycin; 9. benzyl·prochloraz; 10. oxadipate; 11. flutolane·tebuconazole) (see Figure 4 , Table 2), and can be used alternately with these 11 pesticides later.

[0116] Table 2 Tolerance test of strain PC-1 to 11 drugs

[0117]

[0118]

[0119] Note: √ indicates that the strain is tolerant; × indicates that the strain is intolerant.

[0120] Example 4 Effects of strain PC-1 on wheat seed germination and growth

[0121] 1 Test method

[0122] The treatment group was treated with PC-1 fermentation broth (viable cell count was 1×10 9 CFU / mL, the fermentation broth was prepared in the same manner as in Example 3), and the fermentation broth diluted 10 times, 100 times, and 500 times were respectively poured into potted wheat seeds that had just been sown. The control group was watered with clean water. Ten wheat seeds were planted in each pot. Each group was repeated for 3 pots, and watering was performed once every 5 days. The growth of wheat in each group was observed after 15 days of treatment.

[0123] 2 Test results

[0124] In the wheat pot test, the fermentation liquid of strain PC-1 had a significant inhibitory effect on the growth of wheat seeds. The fermentation liquid of strain PC-1 diluted 10, 100, and 500 times had no inhibitory effect on the germination and growth of wheat. The fermentation liquid of strain PC-1 diluted 500 times could significantly promote the growth of wheat (Table 3, Figure 5 ).

[0125] Table 3 Effects of strain PC-1 on wheat growth

[0126]

[0127] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0128] Example 5 Antimicrobial susceptibility testing

[0129] 1 Test method

[0130] The PC-1 fermentation broth (1×10 9 CFU / mL, the fermentation broth was prepared as in Example 3) and the fermentation broth of the quality control strain Escherichia coli ATCC25922 (1×10 9 CFU / mL (CFU / mL) was determined by inoculating Escherichia coli ATCC25922 into NB medium and incubating the resulting fermentation broth at 37°C and 180 rpm for 24 hours. The resulting fermentation broth was evenly spread on the surface of a NA medium plate, covered, and allowed to dry for 5 minutes. Aseptically, antibiotic discs (penicillin (PEN), enrofloxacin (ENR), amoxicillin (AMX), cotrimoxazole (SXT), neomycin (N), erythromycin (E), streptomycin (S), cefepime (FEP), doxycycline (DO), gentamicin (GM), tetracycline (TET), and lincomycin (MY)) were placed on the surface of the plate, with six discs placed per plate. The plates were then incubated at 30°C for 24 hours and observed for the presence of inhibition zones around the antibiotic discs.

[0131] 2 Test results

[0132] The results showed that the quality control strain Escherichia coli ATCC25922 was sensitive to most drug-resistant bacteria, and the strain PC-1 was sensitive to enrofloxacin (ENR) and gentamicin (GM), indicating that the strain PC-1 can be controlled by at least two drugs ( Figure 6 ).

[0133] Example 6 Comparison of the inhibitory effects of strain PC-1 and dimethomorph on Phytophthora infestans

[0134] Comparison of the antibacterial effects of PC-1 fermentation broth and dimethomorph against Phytophthora infestans

[0135] 1.1 Test method

[0136] The fermentation broth of strain PC-1 (1×10 9 CFU / mL, the fermentation broth was prepared as in Example 3) and 1 mL of its 10-fold dilution and 100-fold dilution were added to 50 mL of PDA medium, respectively, so that the final dilution factors relative to the original fermentation broth were 50-fold, 500-fold, and 5000-fold, respectively. After thorough mixing, the mixture was poured onto plates.

[0137] The dimethomorph treatment was performed by diluting a commercially available 80% active ingredient stock solution of dimethomorph 40-fold with sterile water. 1 mL was added to 50 mL of PDA medium, mixed thoroughly, and plated. This treatment corresponded to a final dimethomorph dilution of 2000-fold (equivalent to the recommended field dose of 17-24 g / mu).

[0138] At the same time, a control group (CK group) was set up (no fermentation broth or drugs were added).

[0139] A 5 mm diameter cake of Phytophthora infestans was placed in the center of each PDA medium plate. Three replicates were set up for each group and incubated in the dark at 28°C for 7 days. When the P. infestans colonies in the control group were about to fill the plate, the diameter of each treated group's colonies was measured, and the inhibition rate was calculated: Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0140] 1.2 Test results

[0141] The results are shown in Table 4 and Figure 7 As shown, the fermentation broth of strain PC-1 exhibited significant inhibitory effects against Phytophthora infestans at various dilution ratios (50x, 500x, and 5000x), with inhibition rates exceeding 93%. In comparison, the inhibition rate of the dimethomorph-treated group was 21.75%. This result demonstrates that the fermentation broth of strain PC-1 significantly outperformed the chemical fungicide dimethomorph (80% active ingredient content, diluted 2000x, equivalent to the recommended field dose of 17-24g / mu) in inhibiting Phytophthora infestans under these experimental conditions.

[0142] Table 4 Comparison of the inhibitory effects of PC-1 fermentation broth and dimethomorph on Phytophthora infestans

[0143]

[0144] Strain PC-1 has excellent dynamic proliferation capacity, reaching the logarithmic growth phase within 4-12 hours of culture. Therefore, despite significantly different initial viable bacterial concentrations introduced at 50-fold, 500-fold, and even 5,000-fold dilutions, the bacteria continued to proliferate in PDA medium due to the presence of viable bacteria in the fermentation broth and the 7-day culture duration. Over time, strain PC-1 in all treatment groups likely reached similar population densities, resulting in similar antibacterial effects. This demonstrates that even under conditions of limited application dose or high dilution, strain PC-1 maintains excellent proliferation and disease suppression capabilities, demonstrating its practicality and application flexibility.

[0145] Comparison of the antibacterial effects of the fermentation supernatants of two strains PC-1 and dimethomorph against Phytophthora infestans

[0146] 2.1 Test method

[0147] Take the fermentation liquid of strain PC-1 (1×10 9CFU / mL. Fermentation broth was prepared as in Example 3. Bacteria were removed by centrifugation (8000 rpm, 10 min), and the fermentation supernatant was collected as the treated stock solution. The fermentation supernatant was diluted 10-fold, 40-fold, and 80-fold, respectively. 1 mL of each stock solution and dilution was added to 50 mL of PDA medium, resulting in final dilutions of 50-fold, 500-fold, 2000-fold, and 4000-fold relative to the original fermentation supernatant, respectively. The mixture was thoroughly mixed and plated.

[0148] The dimethomorph group was set up as follows: commercial dimethomorph (active ingredient content 80%) was diluted 40 times with sterile water, 1 mL was added to 50 mL of PDA culture medium, mixed thoroughly and poured into plates to make the final concentration equivalent to a 2000-fold dilution (equivalent to the recommended field application dose of 17-24 g / mu).

[0149] At the same time, a control group was set up (only inoculated with Phytophthora infestans, without adding treatment solution).

[0150] Three replicates were set up for each group. A 5 mm diameter P. infestans plug was inoculated in the center of each plate. All plates were incubated in the dark at 28°C for 7 days. When P. infestans colonies in the control group filled the plate, the diameter of each colony was measured and the inhibition rate was calculated: Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0151] 2.2 Test results

[0152] The results are shown in Table 5 and Figure 8 As shown, the fermentation supernatant of strain PC-1 exhibited moderate antibacterial activity at various dilution ratios. The inhibition rate of the 50-fold dilution group was 40.53%, higher than the 35.89% inhibition rate of the 2000-fold dimethomorph dilution group. The inhibition rate of the 500-fold dilution group was 34.61%, similar to that of the dimethomorph dilution group. These results demonstrate that the fermentation supernatant of strain PC-1 exhibits strong antibacterial activity against P. infestans over a wide dilution range (50-500 times), suggesting its potential as a biocontrol agent.

[0153] Table 5 Comparison of the inhibitory effects of PC-1 fermentation supernatant and dimethomorph on Phytophthora infestans

[0154]

[0155] Example 7 Tolerance test of strain PC-1

[0156] 1 Test method

[0157] The strain PC-1 was inoculated into NB medium and cultured in a shaking incubator at 28°C and 180 rpm for 24 h. The viable bacterial count of the resulting fermentation liquid was 1×10 9In each of the following tests, 50 μL of fermentation liquid was spread on NA plates for inoculation to observe its tolerance.

[0158] Salt content test: Evenly spread the fermentation broth of strain PC-1 onto plates containing 1%, 5%, 10%, 15%, and 20% NaCl, with three replicates per treatment. Incubate the plates at 28°C for 24 hours and then observe the growth of strain PC-1.

[0159] Different cadmium ions Cd 2+ Content test: The fermentation liquid of strain PC-1 was evenly spread on the Cd 2+ The plates were incubated at 28°C for 24 hours and the growth of strain PC-1 was observed.

[0160] Different pH tests: Evenly spread the fermentation broth of strain PC-1 onto NA medium plates at pH values of 5, 7, 9, and 12, with three replicates for each treatment. Incubate the plates at 28°C for 24 hours and then observe the growth of strain PC-1.

[0161] Different temperature tests: The fermentation broth of strain PC-1 was evenly spread on NA medium plates, and the plates were placed in incubators at 4, 25, 37, 42, and 52°C for 24 hours. The growth of strain PC-1 was observed, and 3 replicates were performed for each treatment.

[0162] Experiment with varying UV irradiation durations: The fermentation broth of strain PC-1 was evenly spread onto NA medium plates. The plates were then opened and exposed to a 254nm UV lamp (at a distance of 20cm) for 0, 1, 5, 15, 30, and 60 minutes. Three replicates were used for each treatment. After irradiation, the plates were incubated at 28°C for 24 hours, and the growth of strain PC-1 was observed.

[0163] 2. Test results

[0164] like Figure 9-13 As shown, strain PC-1 has strong environmental adaptability: it can grow in the range of 4-42℃ and in the presence of 5% NaCl and 5% Cd 2 Colonies can still form at concentrations up to 5 mg / L. Growth is good within the pH range of 5-9, demonstrating strong acid-base adaptability. Colony growth is inhibited after UV irradiation for more than 15 minutes, demonstrating a certain degree of UV sensitivity.

[0165] Example 8 Experiment on biocontrol of potato late blight by detached leaves and tubers

[0166] 1 Test method

[0167] Use a sterile cotton swab dipped in 75% alcohol to wipe the surface of potato leaves and tubers, then soak them in sterile water for 30 seconds to remove residual alcohol. After disinfection, place the leaves and tubers on sterile filter paper to absorb surface moisture. Use a sterile cotton swab to inoculate the PC-1 fermentation liquid (1×10 9 CFU / mL, the fermentation broth was prepared in the same manner as in Example 3) and diluted 10-fold, 50-fold and 250-fold solutions, as well as a dimethomorph dilution (active ingredient content 80%, diluted 2000 times, corresponding to a field dose of 17-24 g / mu). Subsequently, a spore suspension of Phytophthora infestans (concentration 10 5 spores / mL). A blank control group (CK) was set up for treatment with clear water, and a diseased control group (BCK) was set up for inoculation with only spores of Phytophthora infestans. All treated samples were incubated in 1.5% water agar medium in the dark for 5 days to observe lesion formation.

[0168] 2 Test results

[0169] Figure 14 The study demonstrated the control efficacy of PC-1 fermentation broth on detached potato leaves and tubers. Different concentrations of PC-1 fermentation broth were applied to leaves and cut tubers and then inoculated with Phytophthora infestans. The results showed that compared with the control group, the PC-1 treatment group significantly inhibited pathogen infection, reduced lesion area, and alleviated the degree of rot, demonstrating excellent biocontrol efficacy. PC-1 fermentation broth diluted 10-fold, 50-fold, and 250-fold showed comparable or even superior efficacy to the chemical agent dimethomorph. These results demonstrate that PC-1 has a strong inhibitory effect on Phytophthora infestans and has the potential to be used for the control of potato late blight.

[0170] Example 9 Field plot biocontrol experiment on potato late blight

[0171] 1 Test method

[0172] Before the potato bud stage, the experimental field was divided into three plots and treated as follows: PC-1 group: the fermentation liquid of strain PC-1 (1×10 9 CFU / mL, prepared as in Example 3) was diluted 40-fold and then applied to the roots of potato plants (100 mL / plant) and sprayed on both sides of the leaves until they were wet but not dripping. A dimethomorph group used a 2000-fold dilution of commercially available dimethomorph (80% active ingredient content) (corresponding to a field dose of 17-24 g / mu) and applied to the roots in the same manner as above. A diseased control group (BCK) received only root irrigation and water spraying without any other treatment. All three treatments were repeated every five days for a total of three treatments.

[0173] After the 3 treatments were completed, spores of Phytophthora infestans were sprayed (concentration 5×10 5 spores / mL) on the leaves (front and back) of the plants and water the roots in small amounts to simulate pathogen infection. After spraying the pathogen, maintain normal field management. After 15 days, investigate the disease situation and record the disease index, control effect, and tuber weight per plant.

[0174] Grading: Level 0, no lesions; Level 1, leaf lesion coverage rate <5%; Level 3, lesion coverage rate 6-10%; Level 5, lesion coverage rate 11-20%; Level 7, lesion coverage rate 21-50%; Level 9, lesion coverage rate >50%.

[0175] Disease index = [∑(number of diseased leaves at each level × relative level) / (total number of leaves surveyed × 9)] × 100%.

[0176] Control effect = [(BCK-PT) / BCK] × 100%;

[0177] BCK: disease index of the control group; PT: disease index of different treatments.

[0178] Figure 15 Table 6 shows the disease incidence and tuber set of different treatments in the potato experimental field. Table 6 shows the disease index and control efficacy of different treatments in the potato experimental field. The results show that the control efficacy of the dimethomorph and PC-1 treatments was 20.49% and 83.04%, respectively. The PC-1 treatment significantly reduced disease incidence, exhibited good plant growth, and produced higher tuber numbers and weights than the inoculated control and dimethomorph treatments. These results demonstrate that PC-1 strain effectively controls potato late blight under field conditions, significantly reducing disease incidence and increasing yield. Its biocontrol efficacy is superior to that of conventional chemical agents and suggests promising application prospects.

[0179] Table 6 Disease index and control effect of different treatments in potato experimental fields

[0180]

[0181] Example 10 Fermentation of strain PC-1

[0182] 1 Test method

[0183] Preparation of seed solution: strain PC-1 was inoculated into 600 mL of NB medium and cultured overnight at 30°C and 180 rpm.

[0184] Tank: Prepare 12L GSP liquid medium (pH 6.8), sterilize the tank, inoculate 5% of the PC-1 seed solution (see Figure 16 for microscopic examination of the seed solution), use acid solution to stabilize the pH at 6.8, take samples every 2 hours, perform microscopic examination, and calculate the OD 600Value measurement and viable bacteria count.

[0185] 2 Test results

[0186] During the fermentation process, samples were taken every 2 hours to record the number of viable bacteria and OD 600 Values are calculated and growth curves are drawn. Figure 17 The microscopic images of the fermentation process of Pseudomonas chlororaphis subsp. aurantiacus strain PC-1 every 2 hours from 2 hours to 28 hours are shown. Table 7 shows the number of viable cells and OD of strain PC-1 at different times. 600 Records show that the viable bacterial count reached 12.1 billion CFU / mL at 28 hours. Figure 18 The viable bacterial count and OD at different time points during the culture of strain PC-1 in a 20L small fermenter are shown. 600 The results showed that when the strain PC-1 was fermented in a 20L fermenter, the lag phase was 0-4h, the logarithmic growth phase was 4-12h, and the stationary phase was 12-28h, indicating that the strain had good adaptability and stability in liquid culture.

[0187] Table 7 Number of viable cells and OD of PC-1 at different times 600 Record

[0188]

[0189] Example 11 Comparison of antibacterial activity of supernatants from fermentation of strain PC-1 at different times

[0190] 1 Test method

[0191] To investigate the antibacterial activity of metabolites during fermentation of strain PC-1, 1 mL of fermentation supernatant obtained at different times (2, 6, 10, 14, 18, 20, 24, and 28 h) during the fermentation in Example 10 was added to 50 mL of PDA medium. After thorough mixing, the plates were poured onto the plates. A 5 mm cake of P. infestans was then placed in the center of each plate. A control group (CK) was not treated with fermentation supernatant; instead, a normal PDA plate was plated with P. infestans cake. All treatments were replicated in triplicate and incubated at 28°C for 7 days. When P. infestans colonies in the control group filled the plate, the diameter of the pathogen colonies was recorded, and the inhibition rate was calculated: Inhibition rate (%) = (CK colony diameter – treatment colony diameter) / CK colony diameter × 100%.

[0192] At the same time, the fermentation supernatant at each time point was used to determine the content of phenazine-1-carboxylic acid by high performance liquid chromatography. The determination method was based on the literature reference Wang Meng et al., High Performance Liquid Chromatographic Analysis of 3% Phenazine-1-carboxylic Acid Aqueous Suspension, Analytical Laboratory, 2015, 34(01): 95-98.

[0193] 2 Test results

[0194] As shown in Table 8, the fermentation supernatant of strain PC-1 began to exhibit significant inhibitory activity against Phytophthora infestans after 14 hours of fermentation, with the antibacterial activity continuously increasing with fermentation time. By 28 hours of fermentation, the diameter of P. infestans colonies had significantly decreased to 22.18 mm, with an inhibition rate of 72.27%. Furthermore, the content of the active metabolite, phenazine-1-carboxylic acid, also increased over time, reaching a peak of 1.45 g / L at 28 hours. These results demonstrate that strain PC-1 possesses excellent metabolic regulation and is suitable for collection of fermentation broth after 24-28 hours of fermentation for biocontrol agent preparation.

[0195] Table 8 Inhibition rate of PC-1 fermentation supernatant against Phytophthora infestans at different times

[0196]

[0197] Example 12 Biocontrol effects of strain PC-1 on different crop diseases

[0198] 1. Potato dry rot

[0199] 1.1 Isolation and identification of the pathogen: Two fungal strains were isolated from potato dry rot samples collected in Heilongjiang Province and labeled as MLS-1 and MLS-2. After ITS sequence analysis, MLS-1 and MLS-2 were determined to be Fusarium solani (see Table 9), which is the pathogen causing potato dry rot.

[0200] Table 9 Sequencing identification results

[0201]

[0202] 1.2 Plate antibacterial test: The PC-1 fermentation broth (1×10 9 CFU / mL, the fermentation broth preparation method is the same as that in Example 3) 1mL is added to 50mL PDA culture medium plate, and the final dilution ratio is 50 times. After thorough mixing, pour the plate. At the same time, a control group without bacterial solution is set up. A 5mm diameter Fusarium solani MLS-1 bacterial cake is placed in the middle of the above-mentioned culture medium plate. Each group has 3 replicates and is cultured at 28°C for 7 days. After the control group Fusarium solani MLS-1 has grown all over the plate, the inhibition rate is calculated: inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%. The results show that the inhibition rate of strain PC-1 against pathogen MLS-1 is as high as 90.54±0.56%. Figure 20 .

[0203] 1.3 In vitro tuber disease prevention test: The in vitro tuber experiment was conducted according to the method of Example 8. The specific operation was as follows: the surface of the tuber was wiped and disinfected with a sterile cotton swab soaked in 75% alcohol, and then the tuber was soaked in sterile water for 30 seconds to remove the residual alcohol, and the surface moisture was blotted dry. The PC-1 fermentation broth (1×10 9 CFU / mL) were applied on the surface of the tuber, and then the spore suspension of Phytophthora infestans (10 5 spores / mL). A blank control group (CK) was set up for treatment with clear water, and a control group (BCK) was set up for inoculation with only spores of Fusarium solani MLS-1. All treated samples were cultured in a 1.5% water agar medium in the dark and kept moist for 5 days.

[0204] like Figure 21 As shown, the tubers in the inoculated control group (BCK) showed obvious white mycelium and rot symptoms on their surfaces. However, tubers treated with different dilutions of PC-1 effectively inhibited the growth and infection of the pathogen, even at a dilution of up to 500 times. There were no obvious rot symptoms on the tubers, only color changes. This indicates that strain PC-1 has significant control effects against potato dry rot caused by Fusarium solani and maintains its biocontrol activity over a wide dilution range (50-500 times), suggesting promising application prospects.

[0205] 2. Inhibitory effect on Heilongjiang girl fruit diseases

[0206] 2.1 Isolation and Identification of Pathogens: Six fungal strains were isolated from diseased guiandra fruit in Heilongjiang Province, including two strains of Alternaria and four strains of Fusarium. Their species were identified by ITS sequencing as shown in Table 10. All of these fungi can cause plant disease. Alternaria can cause black spot on guiandra fruit; Fusarium species such as Fusarium trilineatum, Fusarium chlamydophyllum, and Fusarium seminatum can cause guiandra fruit rot; and Fusarium oxysporum can cause wilt.

[0207] Table 10 Sequencing identification results

[0208]

[0209] 2.2 Antibacterial Activity Test: To evaluate the antibacterial activity of strain PC-1, representative pathogens GN-1, GN-2, GN-3, GN-5, and GN-6 were selected and subjected to antibacterial tests according to the plate confrontation method of Example 1. The specific method was as follows: a 5 mm diameter bacterial cake of the above pathogens was placed in the center of a PDA plate, and the fermentation broth of strain PC-1 (1×10 9CFU / mL). Plates inoculated with pathogens alone served as controls, with three replicates for each treatment. Plates were incubated in the dark at 28°C for 7 days. When the pathogens in the control group had completely grown on the plate, the colony diameters of each pathogen were measured and the inhibition rate was calculated: Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0210] The results showed that PC-1 fermentation broth (1×10 9 CFU / mL) against the above five pathogenic fungi, the inhibition rate was over 80%, showing a broad-spectrum and stable antibacterial ability, and is suitable for the prevention and control of various fungal diseases of girl fruit. Figure 22 .

[0211] 3. Inhibitory effects on other crop diseases

[0212] 3.1 Antibacterial activity test: A 5 mm diameter plant pathogen cake (see Table 11 for pathogen species) was placed in the center of each PDA plate, and the fermentation liquid of strain PC-1 (1×10 9 CFU / mL), with the plate inoculated with plant pathogenic bacteria cake alone as the control group, and three replicates were performed. The plates were cultured in the dark at 28°C for 7 days. When the plant pathogenic bacteria cake in the control group was about to cover the entire culture dish, the inhibition rate was measured: inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0213] The test results are shown in Table 11 and Figure 23 As shown in the results, strain PC-1 exhibited significant inhibitory effects against seven representative plant pathogens in a plate standoff test, with inhibition rates ranging from 59.13% to 87.12%. The inhibition rates against Alternaria solani and Fusarium oxysporum reached 87.12% and 85.91%, respectively. This indicates that strain PC-1 not only possesses broad-spectrum antimicrobial activity but also exhibits significant biocontrol effects against a variety of common pathogens that cause root rot, leaf spot, and stem rot, providing experimental support for its use as a multi-target plant disease biocontrol agent.

[0214] Table 11 Antibacterial test of strain PC-1 against various plant pathogens

[0215]

[0216] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art, after reading this description, may make various modifications to the technical solution or replace some of the technical features with equivalents. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present invention shall be deemed to fall within the scope of protection of the present invention.

Claims

1. A Pseudomonas chlororaphis subsp. aurantiaca strain, named PC-1, which was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China on March 24, 2025, with the deposit number CCTCC NO: M 2025568.

2. A plant disease biological control agent composition, characterized in that: The composition comprises the Pseudomonas chlororaphis subspecies aurantiacus strain according to claim 1 or a fermentation product thereof.

3. The composition according to claim 2, characterized in that The composition further comprises an agriculturally acceptable carrier and / or adjuvant; Preferably, the viable bacterial count concentration of the Pseudomonas chlororaphis subspecies orange strain in the composition is 1×10 7 CFU / mL to 1 × 10 12 CFU / mL.

4. The composition according to claim 2, characterized in that The composition further comprises at least one chemical pesticide selected from the group consisting of dimethomorph, carbendazim, bromothiocarb·prochloraz, mancozeb, zhongshengmycin, chlorothalonil, oligosaccharide·chain protein, cinnamyl·tetramycin, benzyl·prochloraz, oxadiazine and flutolane·tebuconazole; Preferably, the plant disease is at least one selected from the following: late blight of potato caused by Phytophthora infestans, dry rot of potato caused by Fusarium solani, verticillium wilt of cotton caused by Rhizoctonia solani, stalk rot of corn caused by Fusarium graminearum, root rot of tobacco caused by Fusarium commensal, early blight of potato caused by Alternaria solani, root rot of tomato caused by Fusarium oxysporum, leaf spot of tea caused by Alternaria longipetalum, and black spot of cucumber caused by Alternaria cucurbitae.

5. Use of the Pseudomonas chlororaphis subsp. aurantiacus strain according to claim 1 or the composition according to claim 2 or 3 in the preparation of a biological control agent for controlling plant diseases.

6. The use according to claim 5, characterized in that The plant diseases are at least one selected from the following: late blight of potato caused by Phytophthora infestans, dry rot of potato caused by Fusarium solani, verticillium wilt of cotton caused by Rhizoctonia solani, stalk rot of corn caused by Fusarium graminearum, root rot of tobacco caused by Fusarium graminearum, early blight of potato caused by Alternaria solani, root rot of tomato caused by Fusarium oxysporum, leaf spot of tea caused by Alternaria longipetalum, and black spot of cucumber caused by Alternaria cucurbitae.

7. Use of the Pseudomonas chlororaphis subsp. aurantiacus strain according to claim 1 or the composition according to any one of claims 2 to 4 in the preparation of an antibacterial agent.

8. The use according to claim 7, characterized in that The antibacterial agent has an antibacterial effect on at least one selected from the group consisting of: Phytophthora infestans, Fusarium solani, Fusarium oxysporum, Fusarium tricinctum, Fusarium chlamydosporum, Fusarium incarnatum, Alternaria alternata, and Rhizoctonia solani; Preferably, the Alternaria species is at least selected from the group consisting of Alternaria solani, Alternaria cucumerina and Alternaria longipes.

9. A method for preventing and controlling plant diseases, characterized in that: The method comprises applying the Pseudomonas chlororaphis subsp. aurantiacus strain or a fermentation product thereof according to claim 1 or the composition according to any one of claims 2 to 4 to plants, plant seeds, plant cultivation media or plant growth environments.

10. The method according to claim 9, characterized in that The plant disease is at least one selected from the following: potato late blight caused by Phytophthora infestans, potato dry rot caused by Fusarium solani, cotton verticillium wilt caused by Rhizoctonia solani, corn stalk rot caused by Fusarium graminearum, tobacco root rot caused by Fusarium commensalis, potato early blight caused by Alternaria solani, tomato root rot caused by Fusarium oxysporum, tea leaf spot caused by Alternaria longipetrum, and cucumber black spot caused by Alternaria cucurbitae; Preferably, the fermentation product of the Pseudomonas chlororaphis subsp. aurantiacus strain is a fermentation broth, a fermentation supernatant or a fermentation filtrate of the Pseudomonas chlororaphis subsp. aurantiacus strain; Preferably, the amount of the Pseudomonas chlororaphis subsp. aurantiacus strain or its fermentation product is to dilute the fermentation liquid of the Pseudomonas chlororaphis subsp. aurantiacus strain 10-500 times and apply it to the plant; Preferably, the application is foliar spraying, soil drench, root drench, seed treatment, tuber treatment or a combination of any two or more of the foregoing.

Images