Pseudomonas chlororaphis PCJ81 for preventing and treating tobacco mosaic virus as well as extract and application of pseudomonas chlororaphis PCJ81

Abstract

The invention provides pseudomonas chlororaphis PCJ81 for preventing and treating tobacco mosaic virus as well as an extract and application thereof, and belongs to the technical field of biological prevention and treatment. The preservation number of the pseudomonas chlororaphis PCJ81 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.29687, the strain can be used for remarkably inhibiting infection of common tobacco mosaic viruses, and a half-leaf method biocontrol experiment result shows that the protection effect is greater than 90%. Meanwhile, the invention also provides an extract of the pseudomonas chlororaphis PCJ81, and experiments prove that the extract has an excellent effect in the aspect of resisting TMV (tobacco mosaic virus), and the effect of preventing and treating the TMV is superior to that of ningnanmycin.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to a Pseudomonas aeruginosa PCJ81 strain for controlling tobacco mosaic virus disease, its extract, and its applications. Background Technology

[0002] Tobacco mosaic virus (TMV) is a widely distributed plant virus, affecting 268 species of plants across 38 families, particularly Solanaceae crops such as tobacco, tomato, and pepper, where infection rates are high. TMV primarily infects plants during the seedling stage. Due to the relatively weak resistance of seedlings and suitable temperature and humidity conditions, the seedling stage becomes a peak period for TMV infection. If infection occurs before or after transplanting, abnormal plant growth occurs, leading to symptoms such as mosaic patterns, blisters, deformities, and necrosis in the leaves, severely impacting tobacco quality and yield, with losses reaching approximately 50%. Comparatively, infection during the vigorous growth stage has a smaller impact on yield, while infection during the budding stage has little effect on yield and quality. Statistics show that annual tobacco losses due to TMV infection reach as high as $100 million, making this a significant challenge for the international tobacco industry in virus control.

[0003] Currently, the main control methods for tobacco mosaic virus include purchasing virus-free seedlings, promptly removing diseased plants, and using chemical pesticides to kill virus-transmitting aphids. While these methods are effective to some extent in controlling tobacco mosaic virus, they have some drawbacks. Purchasing virus-free seedlings is costly and cannot guarantee absolute virus-free status; removing diseased plants requires significant manual labor and cannot completely eliminate the virus. Although chemical pesticides can assist in virus control, their irrational use can threaten human and animal safety and the ecological environment, which is inconsistent with the requirements of sustainable development in green ecological agriculture.

[0004] In the pursuit of green and ecological agriculture, low-toxicity and residue-free biological agents have gradually gained attention and occupy a core position in the integrated management of crop diseases and pests. Therefore, there is an urgent need to develop a biological agent targeting tobacco mosaic virus to replace or supplement existing control methods. Domestic research on the biological control of tobacco mosaic virus has yielded some results, but most studies are limited to screening rhizosphere biocontrol strains in soil and pot experiments. Research on plant endophytic biocontrol strains and their antagonistic small-molecule metabolites is relatively limited. Summary of the Invention

[0005] The purpose of this invention is to provide a Pseudomonas aeruginosa PCJ81 strain and its extract for the prevention and control of tobacco mosaic virus disease, and its application. The Pseudomonas aeruginosa PCJ81 strain and its extract of this invention can significantly inhibit the infection of tobacco common mosaic virus.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a Pseudomonas chlororaphis PCJ81 strain, with the strain preservation number CGMCC No. 29687.

[0008] This invention provides an extract of *Pseudomonas aeruginosa* PCJ81, and the preparation method of the extract includes the following steps:

[0009] The bacterial culture of Pseudomonas aeruginosa PCJ81 was mixed with macroporous adsorption resin to obtain macroporous adsorption resin for adsorbing fermentation metabolites.

[0010] The macroporous adsorption resin that adsorbs fermentation metabolites is first eluted with methanol to obtain a methanol eluent, and then eluted with a mixture of methanol and dichloromethane to obtain a methanol and dichloromethane eluent.

[0011] Combine the above eluents, filter, and dry to obtain the extract.

[0012] This invention provides a biological agent, the main components of which include the aforementioned Pseudomonas aeruginosa PCJ81 and / or the aforementioned extract.

[0013] This invention provides the application of the aforementioned microbial agent in inhibiting tobacco mosaic virus.

[0014] This invention provides the application of the aforementioned microbial agent in the prevention and control of plant diseases caused by tobacco mosaic virus.

[0015] The present invention discloses the following technical effects

[0016] The plant endophytic fungus *Pseudomonas chlororaphis* strain obtained in this study exhibits significant resistance to tobacco mosaic virus (TMV). Half-leaf experiments showed that the fermentation broth of strain PCJ81 showed an inhibition rate of up to 95.9% against TMV, clearly demonstrating the strain's strong antiviral ability. The strain provides effective protection against TMV infection for at least 7 days. Furthermore, the crude extract of metabolites from the strain's fermentation broth also showed significant efficacy, with a 500-fold dilution achieving 80% protection against TMV. This indicates that the strain's metabolites also have a significant effect in controlling TMV and possess practical value in production.

[0017] This invention aims to explore the characteristics and advantages of endophytic biocontrol bacteria in the biological control of tobacco mosaic virus (TMV). By screening plant endophytic antagonistic strains with significant control efficacy and extracting and separating the antagonistic active substances produced during their fermentation, this invention seeks to apply these substances to control plant diseases caused by TMV. Furthermore, the yield loss in tobacco production due to TMV depends specifically on the growth stage of the infected tobacco, the type of virus, and its severity. Therefore, the successful application of this invention will directly alleviate the economic burden on the tobacco industry, improve yield and quality, and is expected to significantly reduce annual losses caused by TMV infection. This will provide new ideas and approaches for solving the problem of tobacco mosaic virus control and contribute to the sustainable development of the tobacco industry. Attached Figure Description

[0018] Figure 1 The inactivation effect of Pseudomonas aeruginosa PCJ81 on TMV is shown in the figure.

[0019] Figure 2 The diagram shows the effect of Pseudomonas aeruginosa PCJ81 on inducing resistance in tobacco.

[0020] Figure 3 The protective effect of Pseudomonas aeruginosa PCJ81 on tobacco is shown in the figure.

[0021] Figure 4 Colony morphology and Gram staining of Pseudomonas aeruginosa PCJ81;

[0022] Figure 5 Figure 1 shows the physiological and biochemical characteristics of Pseudomonas aeruginosa PCJ81 (Note: A: Starch hydrolysis reaction; B: Citrate utilization; C: Sucrose utilization; D: Phenol red staining; E: Methyl red staining; F: Bromocresol purple staining).

[0023] Figure 6 Phylogenetic tree diagram constructed based on the 16sRNA gene of Pseudomonas aeruginosa PCJ81;

[0024] Figure 7 The image shows the antibacterial spectrum test results for Pseudomonas aeruginosa PCJ81.

[0025] Biological Preservation Information

[0026] *Pseudomonas chlororaphis*, deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 29687 and strain number PCJ81. Detailed Implementation

[0027] This invention provides a Pseudomonas chlororaphis PCJ81 strain, with the strain preservation number CGMCC No. 29687.

[0028] This invention tested the inactivation effect of different endophytic strains on TMV using the half-leaf method. A mixture of 10-fold diluted crude TMV-GFP extract and endophytic bacterial suspension was inoculated onto tobacco leaves. Results showed that the PCJ81 bacterial suspension inhibited TMV infection with an inhibition rate of approximately 80%, indicating that PCJ81 has a strong inactivation effect on TMV. This invention further determined the preventive effect of PCJ81 bacterial suspension on TMV infection. PCJ81 bacterial suspension was applied to tobacco leaves, and TMV was inoculated 24 hours later. Results showed that the average inhibition rate of PCJ81 bacterial suspension against TMV infection reached 95.9%, effectively preventing TMV infection. Furthermore, this invention, by applying PCJ81 bacterial suspension to tobacco leaves and inoculating with TMV 7 days later, showed that the inhibition rate against TMV infection remained at approximately 80% after 7 days of treatment with PCJ81 bacterial suspension, indicating that the PCJ81 strain provides sustained protection against TMV infection for at least 7 days. This invention further involved treating tobacco roots with PCJ81 bacterial solution, followed by leaf inoculation with TMV 24 hours later. The results showed that PCJ81 strain could induce systemic resistance to TMV infection in tobacco and inhibit the spread and translocation of TMV in tobacco plant tissues. Therefore, *Pseudomonas aeruginosa* PCJ81 exhibits a strong inhibitory effect on TMV and can induce systemic resistance to TMV infection in tobacco, inhibiting the spread and translocation of TMV in tobacco plant tissues.

[0029] This invention provides an extract of *Pseudomonas aeruginosa* PCJ81, and the preparation method of the extract includes the following steps:

[0030] The bacterial culture of Pseudomonas aeruginosa PCJ81 was mixed with macroporous adsorption resin to obtain macroporous adsorption resin for adsorbing fermentation metabolites.

[0031] The macroporous adsorption resin that adsorbs fermentation metabolites is first eluted with methanol to obtain a methanol eluent, and then eluted with a mixture of methanol and dichloromethane to obtain a methanol and dichloromethane eluent.

[0032] Combine the above eluents, filter, and dry to obtain the extract.

[0033] This invention involved spraying tobacco leaves with a 500-fold dilution of *Pseudomonas aeruginosa* PCJ81 extract, fermentation supernatant, and a 500-fold dilution of Nanningmycin, respectively. After 24 hours, the leaves were inoculated with TMV. The results showed that the 500-fold dilution of the *Pseudomonas aeruginosa* PCJ81 extract provided 80% protection against TMV, and its effect on controlling tobacco mosaic virus was superior to that of Nanningmycin. Therefore, the BHG91 bacterial extract exhibits superior efficacy against TMV.

[0034] In this embodiment of the invention, the tobacco variety is Bunsen tobacco.

[0035] In this embodiment of the invention, the calculation formulas for TMV inhibition rate, disease index, and prevention and treatment effect are shown in Formulas I to III.

[0036] TMV inhibition rate (%) = (Number of control fluorescent spots - Number of treated fluorescent spots) / Number of control fluorescent spots × 100% Formula I Disease index = [Σ(number of diseased leaves at each level × corresponding disease grade) / total number of leaves surveyed × 9] × 100 Formula II Prevention and control efficacy (%) = (Disease index of control - Disease index of treatment) / Disease index of control × 100% Formula III

[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] Isolation and identification of endophytic bacteria in apples and preparation of fermentation broth

[0040] 1. Apple sample collection

[0041] Three healthy 10-year-old Fuji apple trees were selected at the Hebei Agricultural University Orchard Experimental Base. Tissue samples were collected from different parts of the trees to isolate different types of endophytic bacteria. Fruits, apple branches with a diameter of approximately 1–1.5 cm, and lateral root samples were collected from different trees at a depth of 20–30 cm and 1 m from the trunk. Each sample was labeled and recorded.

[0042] 2. Isolation and purification of apple endophytic bacteria

[0043] The collected plant stems, roots, and fruit tissues were surface-sterilized. After rinsing with tap water and then sterile water, the samples were blotted dry with filter paper. Each sample group was then placed in a 100mL beaker and soaked in 1% sodium hypochlorite for 3 minutes, followed by rinsing with sterile water. Finally, the samples were soaked in 75% ethanol for 3 minutes, followed by three rinses with sterile water, each lasting 1 minute. Excess moisture was then blotted dry with filter paper. The surface-sterilized samples were cut into 3-5mm pieces with a sterile scalpel and manually ground using a mortar and pestle, adding a suitable amount of sterile water during the process to fully release endophytic bacteria.

[0044] Then, the ground samples were serially diluted with sterile water at 10-fold, 100-fold, and 1000-fold ratios. 100 μL of each diluted sample was pipetted onto solid LB agar for isolation, and the plates were incubated at 28°C. After 1–2 days, single colonies with distinct morphological differences were streaked for isolation, and purification was performed at least three times to obtain a single endophytic strain. Ultimately, a total of 50 strains were isolated and purified from roots, stems, and fruits collected from the apple experimental orchard of Hebei Agricultural University, including 15 strains from roots, 13 strains from stems, and 22 strains from fruits.

[0045] 3. Preparation of apple endophytic bacteria fermentation broth

[0046] To obtain the apple endophytic bacteria fermentation broth, a single colony of the purified strain was first inoculated into 3 mL of LB liquid medium and cultured for 24 hours at 28°C and 180 rpm with shaking. Subsequently, seed culture medium was inoculated into 250 mL of LB liquid medium at a ratio of 1:100 and fermented under the same conditions for 48 hours. Finally, the fermentation broth was diluted with sterile water to a concentration of 1×10⁻⁶. 8 The concentration of cfu / mL was determined and stored at 4°C for later use.

[0047] Example 2

[0048] Determination of the inhibitory effect of antagonistic bacteria on TMV infection using the half-leaf method

[0049] In our previous work, we constructed an infectious TMV-GFP clone labeled with green fluorescent protein using the method described by Shailaja Shivprasad (Virology, 1999, 255:312-323). The infectious RNA transcript transcribed from this clone was used to infect tobacco leaves. One to two days after inoculation, the green fluorescence in the inoculated leaves and the entire plant could be observed using a handheld UV lamp (wavelength 490-500 nm) to determine whether tobacco mosaic virus infection was successful. The severity of tobacco mosaic virus disease could be assessed by comparing the number and size of fluorescent spots in the treatment and control groups. This method, by counting the number of visually visible necrotic spots, is more accurate than traditional methods for evaluating tobacco mosaic virus symptoms. This is because the symptoms caused by tobacco mosaic virus infection are diverse, and necrotic spots are only one of the symptoms in the later stages of infection; furthermore, the formation of necrotic spots cannot rule out cell damage caused by mechanical friction during inoculation. By observing the number and size of fluorescent spots after TMV-GFP infection, we can not only accurately determine the success rate of TMV infection and the severity of TMV infection during follow-up, but also observe the transfer path and disease severity of the virus from infected leaves to other parts after infection.

[0050] 1. Preparation of crude extract of TMV virus inoculation:

[0051] Five fresh tobacco leaves (approximately 2g) infected with the TMV-GFP infectious clone were thoroughly ground in a sterile mortar with an appropriate amount of silicon carbide and phosphate-PBS buffer (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4 and 8mM K2HPO4, pH 7.2). The mixture was then filtered through sterile double-layered gauze, and finally, phosphate-PBS buffer was added to bring the volume to 100mL to obtain the crude virus extract.

[0052] 2. Half-leaf friction inoculation:

[0053] Healthy tobacco plants were selected. First, the leaf surface was rinsed with sterile water to remove impurities. Then, silicon carbide was evenly sprinkled onto the leaves. A sterile cotton swab was used to apply crude virus extract or endophytic bacteria suspension to the leaves for TMV inoculation. Ten minutes after inoculation, the leaves were rinsed with sterile water to prevent other bacteria from entering the tobacco plant through the micro-wounds created during the inoculation process. On days 3, 5, 7, and 9 post-inoculation, the number of fluorescent infection points was observed under ultraviolet light, and TMV inhibition rate and disease index were statistically analyzed.

[0054] Table 1 Grading standards for tobacco mosaic disease (GB / T23222-2008)

[0055]

[0056] 3. Test on the inactivation effect of apple endophytic bacteria on TMV:

[0057] The inactivation effect of the endophytic strains isolated from apple tissue on TMV was tested using the half-leaf method. First, a 10-fold diluted crude TMV-GFP extract and endophytic bacterial suspension were mixed in equal volumes and, after 30 minutes, evenly spread on the left half of the leaf as the experimental group. Then, a 10-fold diluted crude TMV-GFP extract and liquid LB medium were mixed in equal volumes and, after 30 minutes, evenly spread on the right half of the leaf as the control group. Three leaves were inoculated each time. Three days after inoculation, the number of fluorescent spots in the experimental group (left side) and control group (right side) was counted, and the inhibitory effect of the antagonistic bacteria on TMV was calculated. The results are shown in Table 2. Figure 1 As shown, 12 strains had high inhibition rates, exceeding 50%. Among them, the fermentation broth of strain PCJ81 showed the best inhibitory effect on TMV, with an inhibition rate of about 80%, indicating that this strain may have produced resistant substances that inhibit TMV activity during its growth and metabolism.

[0058] Table 2. Effect of apple endophytic fungi on the number of green fluorescent spots produced by tobacco mosaic virus infection in Nicotiana Bunsenata (passivation effect)

[0059]

[0060]

[0061] 4. Tests on the prevention of TMV infection by apple endophytic fungi

[0062] The inhibitory effects of endophytic strains PCJ81 and PGG2 isolated from apple tissue on TMV infection were tested using the half-leaf method. First, the bacterial culture of the endophytic strains was evenly spread on the left half of the leaf as the experimental group, and liquid LB medium was evenly spread on the right half of the leaf as the control group. After 24 hours, an appropriate amount of silicon carbide powder was evenly sprinkled on the treated leaves, and 10-fold crude TMV-GFP extract was evenly spread on the entire leaf. Three days after inoculation, the number of fluorescent spots in the experimental group on the left and the control group on the right were counted, and the inhibitory effect of the antagonistic bacteria on TMV was calculated. The results are shown in Table 3. The average inhibition rate of TMV infection by the fermentation broth of strain PCJ81 reached 95.9%, while strain PGG2 showed virtually no resistance to TMV infection, indicating that strain PCJ81 can effectively prevent TMV infection.

[0063] Table 3. Effects of apple endophytic strains on the number of infection sites produced by tobacco mosaic virus infection in Nicotiana Bunsenata (preventive effect).

[0064]

[0065] 5. Test on the protective period of apple endophytic bacteria against tobacco's resistance to TMV

[0066] Similar to the method used to test the inhibitory effect of apple endophytic bacteria on TMV, the inhibitory effects of the endophytic strains PCJ81 and PGG2 isolated from apple tissue on TMV were tested using the half-leaf method. The difference was that the TMV inoculation was delayed by 7 days after spraying the endophytic bacteria. The bacterial solution of the test endophytic bacteria was evenly spread on the left half of the leaf as the experimental group, and the right half of the leaf was evenly spread with liquid LB medium as the control group. After 7 days, an appropriate amount of silicon carbide powder was evenly sprinkled on the treated leaves, and 10-fold crude TMV-GFP extract was evenly spread on the entire leaf. Three days after inoculation, the number of fluorescent spots in the experimental group on the left side of the leaf and the control group on the right side of the leaf were counted, and the inhibitory effect of the antagonistic bacteria on TMV was calculated. The results are shown in Table 4. After 7 days of treatment with the fermentation solution of strain PCJ81, the inhibition rate of TMV infection was still maintained at about 80%, indicating that the effective protection period of strain PCJ81 against TMV infection was no less than 7 days.

[0067] Table 4. Results of the survey on the number of TMV infection sites 7 days after apple endophytic fungus treatment (protection period evaluation)

[0068]

[0069] 6. Test on the induction of TMV resistance in tobacco by apple endophytic bacteria:

[0070] To test whether antagonistic strains can induce systemic resistance to TMV infection in tobacco, the fermentation broth of strain PCJ81 (1×10⁻⁶) was first prepared. 8 Nicotiana Bunsenata was pretreated with root irrigation (CFU / mL), while the control group received LB liquid medium. TMV crude solution was inoculated by friction 24 hours later. From day 3 after inoculation, the infection and spread of TMV-GFP were observed under UV light for 9 consecutive days. Results are shown in Table 5. Figure 2 As shown, three days after inoculation, sporadic fluorescent spots were observed on tobacco leaves treated with PCJ81 strain through root drenching. Subsequent investigations revealed that the area of ​​fluorescent lesions only expanded on the inoculated tobacco leaves, with very little TMV migration and diffusion to other leaves. In contrast, the number of fluorescent spots on the tobacco leaves of the control group was significantly higher, and there were signs of migration and diffusion. Statistical analysis based on the disease severity criteria in Table 1 showed a significant difference in disease index between the PCJ81 strain root drenching treatment and the control group. The control efficacy at 5, 7, and 9 days after inoculation reached 76.95%, 84.44%, and 81.58%, respectively. This indicates that PCJ81 strain can induce systemic resistance to TMV infection in tobacco and inhibit the spread and transfer of TMV in tobacco plant tissues.

[0071] Table 5. Induced protective effect of apple endophytic fungus PCJ81 against tobacco mosaic virus infection in *Nicotiana benthamiana*.

[0072]

[0073] Note: DPI represents the number of days after inoculation with TMV crude extract.

[0074] Example 3

[0075] Identification and Antibacterial Spectrum Determination of Apple Endophytic Bacterium PCJ81

[0076] 1. Colony morphology identification

[0077] Inoculated onto LB solid medium and incubated at 28°C for 3 days, followed by morphological observation. Results are as follows: Figure 4 As shown, PCJ81 colonies are regular in shape, round, with raised bumps, and opaque yellow; the surface is smooth and moist, and the edges are smooth without wrinkles.

[0078] 2. Physiological and biochemical identification

[0079] The physiological and biochemical parameters of the screened strains were determined according to the "Handbook of Bacterial Identification". The results are shown in Table 7 and 8. Figure 5 Consulting the "Handbook of Bacterial Identification" reveals that these physiological and biochemical characteristics are similar to those of Pseudomonas.

[0080] Table 7. Physiological and biochemical characteristics of apple endophytic bacteria strain PCJ81

[0081]

[0082] Note: "+" indicates a positive reaction or that the product can be used; "-" indicates a negative reaction or that the product cannot be used.

[0083] 3. Molecular biological identification

[0084] After 16S rDNA molecular identification (16S rDNA sequence shown in SEQ ID NO:1), strain PCJ81 showed the highest similarity to P. chlororaphis. A phylogenetic tree was constructed using the NJ method. Figure 6 Based on morphological and physiological and biochemical test results, it was preliminarily identified as Pseudomonas chlororaphis.

[0085] 4. Antibacterial spectrum of antagonistic strains

[0086] From Table 8 and Figure 7 It is known that strain PCJ81 has a good inhibitory effect on seven important fungal pathogens of other plants, including apple rot fungus, corn sheath blight fungus, apple ring rot fungus, apple anthracnose leaf blight fungus, potato early blight fungus, and apple leaf spot fungus. The mycelial growth inhibition rate is 36.00% to 65.77%, especially showing a strong inhibitory effect on fruit diseases such as apple rot fungus and ring rot fungus.

[0087] Table 8. Antibacterial spectrum of Pseudomonas aeruginosa strain PCJ81

[0088]

[0089]

[0090] Example 4

[0091] Extraction and anti-TMV effects of metabolites from apple endophytic bacteria PCJ81

[0092] 1. Macroporous resin addition and fermentation:

[0093] Add 30g of XAD16 macroporous resin to 500mL of liquid fermentation medium to adsorb small molecule metabolites produced by Pseudomonas aeruginosa PCJ81 strain during fermentation. Inoculate with 5mL of BHG91 seed culture. After 48 hours of fermentation, filter with a Buchner funnel to obtain macroporous adsorption resin.

[0094] 2. Extraction of biocontrol bacteria metabolic active products:

[0095] All the macroporous adsorption resin was transferred to an Erlenmeyer flask containing 200 mL of methanol, and the active ingredient was extracted for half an hour with the aid of ultrasound. The extraction was repeated three times with methanol. Then, the extraction was repeated three times with a 1:1 mixture of 200 mL of methanol and dichloromethane. The extract was filtered through an organic filter membrane with a pore size of Ψ = 0.22 micrometers.

[0096] Finally, the mixture was concentrated under reduced pressure using a rotary evaporator at a heating bath temperature of 60°C, a vacuum degree of 95–98 kPa (absolute pressure of 2–5 kPa), and a cooling medium temperature of 20°C, followed by drying. These steps yielded approximately 3 g of crude extract of the metabolically active product, which was used as an extract of *Pseudomonas aeruginosa* PCJ81 for testing.

[0097] 3. To conduct an in-depth comparison of the efficacy of Pseudomonas aeruginosa PCJ81 extract, fermentation supernatant, and other antiviral agents against tobacco mosaic virus disease, we adopted the following experimental procedures:

[0098] First, the fermentation supernatant of the PCJ81 strain was prepared by high-speed centrifugation: the 48-hour fermentation broth was centrifuged at 12,000 rpm for 10 minutes at a low temperature of 4°C to remove bacterial cells, obtaining a fermentation supernatant containing PCJ81 extracellular metabolites. Simultaneously, using Nanningmycin and LB liquid medium as references, we compared the resistance effects of Pseudomonas aeruginosa PCJ81 extract, fermentation supernatant, and other antiviral agents against TMV using the half-leaf method described in Example 2. Four- to five-leaf stage tobacco seedlings were selected as experimental materials. Each treatment was sprayed with a 500-fold dilution of Pseudomonas aeruginosa PCJ81 extract, fermentation supernatant, a 500-fold dilution of Nanningmycin, and LB liquid medium, with three replicates per treatment and four tobacco plants per replicate. After 24 hours, the topmost true leaf of each tobacco plant was inoculated with TMV-GFP via friction. Starting from day 3 after inoculation, the disease incidence in tobacco plants was observed and recorded at one-day intervals. Disease index and control efficacy were calculated. The results showed that tobacco plants sprayed with LB liquid medium exhibited more severe disease and relatively lower TMV control efficacy. In contrast, tobacco plants sprayed with extracts of *Pseudomonas aeruginosa* PCJ81 and ningnanmycin showed darker green leaves, milder disease, fewer necrotic spots, and better TMV control efficacy. The control efficacy of *P. aeruginosa* PCJ81 extract and ningnanmycin treatments reached 80.06% and 77.41% respectively by day 7 after inoculation. This experiment demonstrates that the extract of *P. aeruginosa* PCJ81 has superior efficacy against TMV and possesses potential value for biological control applications.

[0099] Table 9. Protective effects of different component treatments on TMV in *Nicotiana tabacum* strain PCJ81.

[0100]

[0101] Note: DPI represents the number of days after inoculation with TMV crude extract.

[0102] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A species of Pseudomonas aeruginosa ( Pseudomonas chlororaphis PCJ81, strain preservation number CGMCCNo.29687.

2. An extract of *Pseudomonas aeruginosa* PCJ81 as described in claim 1, characterized in that, The method for preparing the extract includes the following steps: The bacterial culture of Pseudomonas aeruginosa PCJ81 as described in claim 1 is mixed with macroporous adsorption resin to obtain macroporous adsorption resin for adsorbing fermentation metabolites. The macroporous adsorption resin that adsorbs fermentation metabolites is first eluted with methanol to obtain a methanol eluent, and then eluted with a mixture of methanol and dichloromethane to obtain a methanol and dichloromethane eluent. Combine the above eluents, filter, and dry to obtain the extract.

3. The extract according to claim 2, characterized in that, The macroporous resin includes styrene-based resins.

4. The extract according to claim 2, characterized in that, The volume ratio of methanol to dichloromethane is 1:

1.

5. The extract according to any one of claims 2 to 4, characterized in that, The filtration is performed using a filter membrane with a pore size of 0.22 μm.

6. A biological agent, characterized in that, The main components include Pseudomonas aeruginosa PCJ81 as described in claim 1 and / or the extract as described in any one of claims 2 to 5.

7. The use of the microbial agent according to claim 6 in inhibiting tobacco mosaic virus.

8. The application of the microbial agent according to claim 6 in the prevention and control of plant diseases caused by tobacco mosaic virus.

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