Application of Pseudomonas chlororaphis strain Rhs-P27 in prevention and treatment of pine wood nematode disease

Abstract

The application provides a Pseudomonas chlororaphis strain Rhs-P27 in the application of preventing and treating pine wood nematode disease, relates to the field of agricultural environmental microorganism technology. Specifically relates to a Pseudomonas chlororaphis strain Rhs-P27 isolated from the rhizosphere soil of Pinus tabulaeformis and the application of the strain in the prevention and treatment of pine wood nematode disease. The strain is preserved in China Center for Type Culture Collection of Wuhan University on December 18, 2024, and the preservation number is CCTCCM20242846. The in vitro toxic killing activity of the strain Rhs-P27 to pine wood nematode can reach 100% in 24 hours. The fermentation filtrate and fermentation bacteria suspension of the strain Rhs-P27 have significant inhibitory effect on the reproduction of pine wood nematode. The prevention effect of the fermentation bacteria suspension of the strain Rhs-P27 on the pine wood nematode disease of two-year-old Pinus tabulaeformis pot seedlings can reach 45%, and the density of pine wood nematode in the pine tree can be significantly reduced. The Pseudomonas chlororaphis strain Rhs-P27 of the application has a good application prospect in the field of nuisance-free prevention and treatment of pine wood nematode disease.

Description

Technical Field

[0001] This invention relates to the field of agricultural environmental microbiology, specifically to the application of a strain of Pseudomonas aeruginosa, Rhs-P27, in the prevention and control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a devastating forest disease caused by the pine nematode (Bursaphelenchus xylophilus). It can affect a variety of pine species, including 58 species of Japanese black pine and Masson pine, as well as 13 non-pine coniferous species. In 1982, the disease was first discovered in my country on Japanese black pine at the Sun Yat-sen Mausoleum in Nanjing.

[0003] Due to the complexity of its pathogenic mechanism and the serious damage it causes to pine trees, scientists have been exploring effective control measures. Traditional control methods include physical and chemical methods. Physical methods mainly involve the removal of infected trees, which, while simple and effective, consumes a large amount of resources and is costly. Chemical methods involve trunk injection and aerial spraying, which can effectively reduce the incidence of disease but may bring about the "3R" problem (reduction, redundancy, and loss of resources). Biological control, as an environmentally friendly and efficient method, has attracted much attention. The use of biocontrol bacteria to inhibit pine wood nematode invasion has been proven to be an effective means. These bacteria with nematode-resistant activity originate from various bacterial genera, providing a scientific basis for the development of new biocontrol agents. Biological control is one of the key pathways for the pollution-free control of plant diseases.

[0004] *Pseudomonas aeruginosa* produces various antimicrobial compounds during its metabolism, such as antimicrobial substances synthesized by ribosomes, enzymes, and antibiotics. These substances have bactericidal activity against other microorganisms, helping to control plant diseases. Secondly, this bacterium has the ability to form biofilms, promoting plant growth and controlling pests and diseases. This characteristic gives *P. aeruginosa* great potential and advantages in agricultural applications. Furthermore, because this bacterium originates from naturally occurring bacterial communities in micro-ecosystems and has no toxic side effects, it is environmentally friendly and pollution-free, posing no harm to the environment or human health in practical applications. Therefore, using *P. aeruginosa* for plant disease control is an efficient, safe, environmentally friendly, and sustainable method. With further in-depth and extensive research and application of this technology, it will bring broad development prospects to the agricultural and forestry industries in the future and provide important support for achieving sustainable agriculture and forestry. Based on this, this innovative technological solution is proposed. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides the application of a strain of Pseudomonas aeruginosa Rhs-P27 in the prevention and control of pine wilt disease, solving the problem of a lack of green and pollution-free prevention and control technologies for pine wilt disease.

[0007] (II) Technical Solution

[0008] Another object of the present invention is to provide results on the toxic activity of Pseudomonas aeruginosa strain Rhs-P27 against pine wood nematode.

[0009] Another object of the present invention is to provide results on the inhibitory effect of Pseudomonas aeruginosa strain Rhs-P27 on the reproduction of pine wood nematode.

[0010] Another objective of this invention is to provide the application of *Pseudomonas aeruginosa* strain Rhs-P27 in the control of pine wilt disease in potted *Pinus tabuliformis* seedlings.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A strain of Pseudomonas aeruginosa, Rhs-P27, was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCC NO: M20242846.

[0013] Furthermore, based on the application of the aforementioned strain Rhs-P27 in the prevention and control of pine wilt disease.

[0014] Furthermore, *Pseudomonas aeruginosa* strain Rhs-P27 was cultured in LB liquid medium for liquid fermentation. Under stable culture conditions (25℃, 150 rpm, dark culture for 4 days), a 1× fermentation suspension and a 1× fermentation filtrate (containing no bacterial cells) of Rhs-P27 were obtained. These suspensions were then diluted with sterile water to prepare 5-fold (0.2×) and 10-fold (0.1×) dilutions of the fermentation suspension and filtrate. Next, the fermentation suspensions and filtrates at various dilutions were co-incubated with pine wood nematodes for 24 hours. Based on the count of surviving and dead nematodes, the in vitro toxic activity of Rhs-P27 against pine wood nematodes was evaluated by analyzing the corrected mortality rate of the pine wood nematodes.

[0015] Furthermore, *Pseudomonas aeruginosa* strain Rhs-P27 was cultured in LB liquid medium for liquid fermentation. Under the established stable culture conditions (25℃, 150 rpm, dark culture for 4 days), a fermentation suspension and fermentation filtrate (without bacterial cells) of this strain were successfully obtained. Next, *Botrytis cinerea* was inoculated onto PDA medium plates, and after 7 days of culture, 50 µL of a suspension containing 600 pine wood nematodes was added to the center of the *Botrytis cinerea*. Simultaneously, 100 µL of Rhs-P27 fermentation filtrate or fermentation suspension was added to the inoculation site, with an equal volume of LB liquid medium as a control. Notably, the concentration of the fermentation suspension was 9.35 × 10⁸ CFU / mL. Subsequently, the samples were incubated in a constant temperature incubator for 7 days, and the number of surviving nematodes and eggs was counted. The effect of Rhs-P27 on the reproduction of pine wood nematodes was evaluated by analyzing the reproductive coefficient and egg production of pine wood nematodes.

[0016] Furthermore, based on an artificial inoculation system for pine wilt disease, the trunk-drilling injection method was used to evaluate the preventive and therapeutic effects of *Pseudomonas aeruginosa* Rhs-P27 bacterial suspension on pine wilt disease. In the prevention group, Rhs-P27 bacterial suspension was injected before inoculation with pine wilt nematodes; in the treatment group, pine wilt nematodes were inoculated before injection of Rhs-P27 bacterial suspension. The concentration of the fermentation suspension was 9.35 × 10⁸ CFU / mL. The incidence and severity of disease in potted *Pinus tabuliformis* seedlings were statistically analyzed, and the disease index and biological control efficacy for each treatment were calculated. Simultaneously, the density of pine wilt nematodes in pine trees under different treatments was measured to observe the effect of Rhs-P27 on their reproduction, thereby comprehensively evaluating the efficacy of Rhs-P27 bacterial suspension in the prevention and treatment of pine wilt disease.

[0017] (III) Beneficial Effects

[0018] This invention provides the application of a *Pseudomonas aeruginosa* strain Rhs-P27 in the control of pine wilt disease. It has the following beneficial effects:

[0019] 1. This invention successfully screened a biocontrol strain, Pseudomonas aeruginosa strain Rhs-P27, which has significant toxic activity against pine wood nematodes.

[0020] 2. By measuring the effects of the fermentation suspension and fermentation filtrate of Pseudomonas aeruginosa strain Rhs-P27 on the reproduction of pine wood nematodes, and using the pine wood nematode reproduction coefficient and oviposition as detection indicators, the results showed that the strain had a significant inhibitory effect.

[0021] 3. The preventive and therapeutic effects of *Pseudomonas aeruginosa* strain Rhs-P27 on pine wilt disease in potted Pinus tabuliformis seedlings were evaluated. The study found a more significant preventive effect, with a control efficacy of up to 45% against pine wilt disease, and a significant reduction in nematode density within the pine trees. This indicates that this strain has broad application potential in biological control and provides a crucial foundation for its subsequent product development, forest application, and commercialization, offering a new biological resource for controlling pine wilt disease using biological control techniques.

[0022] 4. This invention lays the foundation for the development and commercialization of microbial agents using this strain. Replacing chemical pesticides with microbial products can effectively avoid the problems of chemical pesticide residues, resistance, and potential resurgence of pests. This will help reduce or replace the use of other chemical pesticides in the control of pine wilt disease, promoting a more environmentally friendly approach to this disease. Attached Figure Description

[0023] Figure 1 The morphology of pine wood nematode in its living (A) and dead (B) state.

[0024] Figure 2 Analysis of the toxic activity of Pseudomonas aeruginosa strain Rhs-P27 against pine wood nematode.

[0025] Figure 3 Colony morphology (A) and spore staining (B) of Pseudomonas aeruginosa strain Rhs-P27.

[0026] Figure 4 Analysis of the inhibitory effect of Pseudomonas aeruginosa strain Rhs-P27 on the reproduction of pine wood nematode.

[0027] Figure 5 The growth status of Pinus tabuliformis seedlings under different treatment groups of Pseudomonas aeruginosa strain Rhs-P27.

[0028] Figure 6 This study analyzed the potted plant control efficacy of *Pseudomonas aeruginosa* strain Rhs-P27 against pine wilt disease in *Pinus tabuliformis* seedlings. The results included: disease incidence rate of *Pinus tabuliformis* seedlings under different treatments (A); disease index of *Pinus tabuliformis* seedlings under different treatments (B); control effect of strain Rhs-P27 against pine wilt disease (C); and density of pine wilt nematodes in *Pinus tabuliformis* seedlings under different treatments (D). Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Isolation of biocontrol strains and determination of their toxic activity against pine wood nematode

[0031] A biocontrol strain of *Pseudomonas chlororaphis*, Rhs-P27, for pine wilt disease, was deposited at the China Center for Type Culture Collection (CCTCC) on December 18, 2024, with accession number CCTCC NO: M20242846. The biocontrol strain Rhs-P27 was isolated from rhizosphere soil microorganisms of *Pinus tabuliformis*. A co-incubation method revealed that strain Rhs-P27 exhibited significant toxic activity against pine wilt nematodes. Its effect on pine wilt nematode reproduction was subsequently determined. The methods for isolating the biocontrol strain Rhs-P27 and determining its toxic activity against pine wilt nematodes are as follows:

[0032] (1) Isolation of biocontrol strains

[0033] ① Sampling location and sampling method

[0034] Location: Rhizosphere soil samples from Pinus tabuliformis were collected from Huanshan Park, Chengguan Town, Ningshan County (N33°18′25″, E108°18′39″).

[0035] Sampling method: Select four healthy 20-year-old Pinus tabuliformis trees and set up rhizosphere soil sampling points 15cm from the base of the trunk. Dig up soil 10-20cm from the topsoil, collect soil containing Pinus tabuliformis fine roots (diameter <2mm), and put it into a sealed plastic bag.

[0036] ② Soil sample processing and preparation of soil suspension

[0037] Take 2.5g of each of four rhizosphere soil samples, mix them thoroughly, and transfer them to sterile glass Erlenmeyer flasks containing 90mL of sterile water. Shake well for 15min, let stand for 5min, and take the supernatant as a 10⁻¹ g / mL soil suspension. Prepare soil dilutions of different concentrations (10⁻² g / mL, 10⁻³ g / mL, and 10⁻⁴ g / mL) based on the 10⁻¹ g / mL suspension.

[0038] ③ Soil microbial isolation and culture

[0039] Take 30 µL of soil suspensions of each of the above concentrations and add them dropwise onto PDA plates, spreading them evenly. Prepare three plates for each concentration. Seal the petri dishes and incubate them at 25°C for 30 days. Observe the single colonies growing on the culture medium daily, and inoculate and purify single colonies with different morphologies using sterile toothpicks.

[0040] ④ Strain preservation

[0041] The obtained strain was placed in 30% glycerol and stored at a low temperature (-80℃).

[0042] The method for preparing the PDA culture medium is as follows: Take 200g of peeled potato chunks, add 700-900mL of deionized water, boil for 20min, filter with gauze, collect the filtrate, then add 20g of glucose to the filtrate, and after it is completely dissolved, make up to 1000mL with deionized water, add 15-20g of agar powder, set the pH to natural, and autoclave before use.

[0043] The above steps illustrate the entire process from sampling to colony isolation and purification, and finally to strain preservation, and provide a method for preparing the PDA medium used. These procedures provide detailed guidance for the isolation and subsequent experiments of the biocontrol strain Rhs-P27.

[0044] (2) Determination of the toxic activity of rhizosphere soil microorganisms of Pinus tabuliformis against pine wood nematode

[0045] ① Fermentation culture of soil microorganisms in the rhizosphere of Pinus tabuliformis

[0046] The isolated microbial strains from the rhizosphere soil of Pinus tabuliformis were cultured in LB liquid medium for 4 days at 25℃, 150 rpm and in the dark. The resulting fermentation suspension (1×) and fermentation filtrate (1×) were obtained. The strains were then diluted with sterile water to prepare 5-fold (0.2×) and 10-fold (0.1×) fermentation suspensions and filtrates, respectively.

[0047] ② Preparation of pine wood nematode suspension

[0048] Botrytis cinerea was inoculated onto PDA medium and cultured for 7 days until the entire plate was covered with colonies. Pine wood nematodes were then inoculated onto Botrytis cinerea on a clean bench and cultured for another 7 days at 25°C without darkness until the surface of the medium showed a glossy appearance. Subsequently, the pine wood nematodes were rinsed with sterile water and collected from the culture dish to form a pine wood nematode suspension with a concentration of 100 nematodes / 10 μL.

[0049] ③ Co-incubation of pine rhizosphere soil microbial fermentation suspension / filtrate with pine wood nematode

[0050] Add 480 μL of each fermentation bacterial suspension (1×, 0.2×, and 0.1×) and fermentation filtrate (1×, 0.2×, and 0.1×) to sterile centrifuge tubes, and then add 20 μL of pine wood nematode suspension (a mixture of larvae and adults, totaling 200 nematodes) to each centrifuge tube. Place the tubes in a constant temperature incubator and incubate at 25°C in the dark for 24 hours. Then count the surviving and dead pine wood nematodes.

[0051] ④ Calculation of corrected mortality rate of pine wood nematodes under each treatment

[0052] The number of surviving and dead nematodes in each treatment group was detected by combining safranin staining and morphological observation. Surviving nematodes were not stained and remained active. Figure 1 A); Dead nematodes are stained red with safranin and are shaped like a "C" or "J", and do not respond to physical stimuli. Figure 1 B). The toxic activity of various isolated strains of microorganisms from the root soil of *Pinus tabuliformis* against pine wood nematodes was systematically tested and converted into corrected mortality rates. It was found that the fermentation suspension and fermentation filtrate (excluding cell bodies) of a bacterium Rhs-P27 had a significant lethal effect on pine wood nematodes. When treated with the original fermentation broth of Rhs-P27 (1×), the corrected mortality rate of pine wood nematodes reached 100%. After a 5-fold dilution (0.2×), the corrected mortality rates of pine wood nematodes treated with the Rhs-P27 fermentation suspension and fermentation filtrate remained at 37.77% (fermentation suspension) and 28.88% (fermentation filtrate), respectively.

[0053] The mortality rate and corrected mortality rate of the isolated microbial strains from the rhizosphere soil of Pinus tabuliformis against pine wilt nematodes were calculated using the following formula.

[0054] Mortality rate (%) =

[0055] Corrected mortality rate (%) =

[0056] In the formula, Nd is the number of nematode deaths in the treatment group; Nt is the total number of nematodes; d is the nematode mortality rate (%) in the treatment group; and c is the nematode mortality rate (%) in the control group.

[0057] Example 2: Identification of biocontrol strain Rhs-P27

[0058] DNA was extracted from strain Rhs-P27 using the CATB method. The 16S rDNA sequence was amplified and sequenced using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction volume was 25 μL, containing: 12.5 μL of 2×ESTaqMasterMix, 1 μL of template DNA, 1 μL of 27F primers, 1 μL of 1492R primers, and 9.5 μL of sterile distilled water. PCR was performed in a PCR thermal cycler, with 30 cycles of pre-denaturation at 94℃ for 3 minutes, followed by 30 cycles of denaturation at 94℃ for 30 seconds, annealing at 52℃ for 30 seconds, extension at 72℃ for 45 seconds, and a final extension at 72℃ for 10 minutes. After PCR, the length of the amplified fragments was detected by agarose gel electrophoresis, and the products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. According to the NCBI database comparison results, strain Rhs-P27 was identified as *Peribacilllus frigoritolerans*. This strain was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCCM20242847.

[0059] Sequencing results of the 16S rDNA amplified fragment of strain Rhs-P27:

[0060]

[0061] Example 3: Detection of the effect of biocontrol strain Rhs-P27 on the reproduction of pine wood nematode

[0062] Rhs-P27 was cultured in LB liquid medium under the following conditions: 25℃, 150 rpm, and dark culture for 4 days. The resulting fermentation suspension and fermentation filtrate (without cells) of strain Rhs-P27 were obtained.

[0063] The *Pseudomonas aeruginosa* strain Rhs-P27 was cultured in LB liquid medium at 25°C, 150 rpm, and in the dark for 4 days, yielding a fermentation suspension and fermentation filtrate (without bacterial cells). *Botrytis cinerea* was inoculated onto PDA agar plates. After 7 days of culture, 50 µL of a suspension containing 600 pine wood nematodes was added dropwise to the center of each *Botrytis cinerea* colony. Simultaneously, 100 µL of either the Rhs-P27 fermentation filtrate (T1 group) or the fermentation suspension (T2 group) was added to the inoculation site. LB liquid medium served as the control group (CK group), and the concentration of the fermentation suspension was 9.35 × 10⁸ CFU / mL. The plates were then incubated at 25°C for 7 days. Pine wood nematodes were then aseptically collected from each petri dish. Using safranin staining and morphological observation, the number of surviving pine wood nematodes and eggs in each treatment group were counted under a microscope and compared with the CK group to analyze the effect of Rhs-P27 on the reproduction of pine wood nematodes.

[0064] The experimental results showed that both the fermentation filtrate and the fermentation suspension of strain Rhs-P27 significantly inhibited the reproduction of pine wood nematodes. The reproduction coefficient and egg production of the CK group were 59.56 and 4165, respectively, while the reproduction coefficients of the T1 and T2 groups were only 0.79 and 1.39, respectively. Furthermore, no new eggs were produced in the T1 group, and the egg production of the T2 group was only 59. Figure 4 This indicates that both the fermentation filtrate and the fermentation suspension of Rhs-P27 can effectively inhibit the reproduction of pine wood nematodes.

[0065] The reproduction coefficient of pine wood nematodes on each PDA plate is calculated using the following formula:

[0066] Reproduction coefficient =

[0067] Where Pf is the number of surviving nematodes in each plate after 7 days of culture, and Pi is the initial inoculation number of nematodes in each plate.

[0068] Example 4: Application of strain Rhs-P27 in pine wilt disease

[0069] The research subjects were five-year-old healthy Pinus tabuliformis seedlings grown in greenhouses at Northwest A&F University. Five different treatment groups were designed, as follows:

[0070] Control group (CK group): treated with LB liquid medium only;

[0071] Pine wood nematode treatment group (PWN group): Inoculated with pine wood nematodes only;

[0072] Rhs-P27 treatment group (Rhs-P27 group): treated with only Rhs-P27 fermentation suspension;

[0073] Prevention group (Rhs-P27→PWN group): First, inject the Rhs-P27 strain fermentation suspension, then inoculate with pine wood nematodes.

[0074] Treatment group (PWN→Rhs-P27 group): Pine wood nematode inoculation was performed first, followed by injection of Rhs-P27 fermentation bacteria suspension.

[0075] All Pinus tabuliformis seedlings will be cultured and observed in the greenhouse of Northwest A&F University. Each treatment will be replicated in triplicate, with each replicate containing 3 Pinus tabuliformis seedlings. The Rhs-P27 bacterial suspension used will be prepared by fermentation in LB liquid medium, with a concentration of 9.35 × 10⁸ CFU / mL.

[0076] The specific procedure is as follows: Before treatment, first clean the surface of the branches and trunks of healthy pine seedlings with damp, sterile cotton. Then, use a handheld electric drill to drill two small holes in the stem of each pine seedling, each hole being 4.5 mm in diameter and 5 mm deep, with a distance of 10 cm between the two holes. The specific procedures for different treatment groups are as follows:

[0077] CK group: Each well was covered with sterile cotton and injected with 5 ml of LB medium. The well openings were then sealed with plastic film to prevent rapid evaporation of moisture. The cotton and plastic film were removed after 7 days of treatment.

[0078] PWN group: Each well was inoculated with 50 µL of a suspension containing 3000 PWN larvae (a mixture of larvae and adults), and then the well opening was sealed with plastic film. The plastic film was removed after 7 days of treatment.

[0079] Rhs-P27 group: Same as CK group, but 5mRhs-P27 strain fermentation suspension was used instead of LB medium.

[0080] Rhs-P27→PWN group: Initial treatment was the same as the Rhs-P27 group. Seven days after Rhs-P27 injection, the plastic film and cotton were removed, and 50 µL of pine wood nematode suspension (containing 3000 PWNs) was inoculated into each well. The well openings were sealed with plastic film, and the plastic film was removed after 7 days of treatment.

[0081] PWN→Rhs-P27 group: The initial treatment was the same as that of the PWN group. Seven days after inoculation with PWN, 5 ml of Rhs-P27 strain fermentation suspension was injected into each well using the same method as the Rhs-P27 group. The well openings were sealed with plastic film, and the plastic film was removed after 7 days of treatment.

[0082] Disease incidence was observed and recorded in each treatment group of *Pinus tabuliformis* seedlings at 7, 14, 21, 28, and 35 days after inoculation with PWN, and the incidence rate was calculated. Simultaneously, the seedlings were divided into five equal segments, and the severity of disease in each segment was statistically analyzed. Five severity levels (S1–S5) were established, with representative values ​​of 0, 1, 2, 3, and 4, respectively. The specific representations for each level are as follows:

[0083] Grade S1: All needles are in a healthy, green state;

[0084] S2 level: Yellowing needles appear, but the number does not exceed one-quarter;

[0085] S3 level: Dead needles are present, and yellowing or dead needles account for between one-quarter and one-half of the total number of needles.

[0086] Grade S4: Yellowing or dead needles are present in one-half to two-thirds of the needles, and the pine logs show slight wilting.

[0087] Grade S5: More than two-thirds of the needles are yellowed or dead, and the pine seedlings show obvious signs of water loss and wilting.

[0088] Then, the disease index of pine wilt disease under each treatment was calculated, and the efficacy of Rhs-P27 preventive treatment (Rhs-P27→PWN group) and treatment (PWN→Rhs-P27 group) in controlling pine wilt disease was further evaluated. Through this method and data analysis, a more comprehensive understanding of the control effects of different Rhs-P27 treatments on pine wilt disease can be obtained.

[0089] Thirty-five days after inoculation with pine wood nematodes, all Pinus tabuliformis seedlings in the PWN group were observed to be wilted and dead. Subsequently, each seedling from the PWN, Rhs-P27→PWN, and PWN→Rhs-P27 groups was collected, and its roots, stems, and leaves were cut into small pieces, mixed thoroughly, and weighed. Then, the Bellman funnel method was used to isolate pine wood nematodes from all tissues of each seedling, and the nematode count was performed under a microscope (Number of nematodes, NN). The density of pine wood nematodes in Pinus tabuliformis seedlings under different treatments is expressed as the number of nematodes per gram of fresh weight (FW) of Pinus tabuliformis (NN·g⁻¹FW). This method allows for a comprehensive understanding of the effects of different treatments on the number of pine wood nematodes within Pinus tabuliformis seedlings, and further evaluates the effectiveness of the Rhs-P27 strain in reducing or controlling the number of pine wood nematodes in Pinus tabuliformis.

[0090] Incidence rate (%) = ×100

[0091] Disease index = ×100

[0092] Prevention and control effect (%) = ×100

[0093] Pine wood nematode density (NN·g) -1 FW) =

[0094] Based on the results of the pot experiment, strain Rhs-P27 was found to be non-toxic to pine seedlings. The seedlings treated with Rhs-P27 showed essentially the same growth pattern as the control group, remaining healthy. Thirty-five days after inoculation with pine wilt nematodes, all needles in the PWN group of pine seedlings became chlorotic and withered, exhibiting significant wilting. The disease incidence in the prevention group (Rhs-P27 → PWN group) was significantly lower than in the PWN group, with less than two-thirds of the needles on each seedling showing chlorosis or withering. The treatment group (PWN → Rhs-P27 group) also showed some mitigation of pine wilt disease, but the number of dead needles was significantly higher than in the prevention group. These results indicate that both preventative and therapeutic treatments are effective in inhibiting or reducing pine wilt disease, with preventative treatment showing better results. Figure 5 ).

[0095] Analysis of the incidence and disease index of pine wilt disease in different treatment groups at 7, 14, 21, 28, and 35 days after inoculation with P. wilt nematode revealed that the dominant stage of pine wilt disease in five-year-old Pinus tabuliformis seedlings appeared on day 14 after PWN inoculation. As the disease progressed, symptoms gradually worsened, and by day 35, all seedlings showed obvious disease manifestations, with a disease index as high as 100 (…). Figure 6 AB). Both the prevention group (Rhs-P27→PWN) and the treatment group (PWN→Rhs-P27) of strain Rhs-P27 also showed mild symptoms on day 14, but the severity of the disease was milder, especially in the prevention group. During the course of pine wilt disease, the incidence gradually increased, reaching 83.33% in the Rhs-P27 prevention group at day 35, while the incidence in the treatment group was 100%. Figure 6 A). Although there was no significant difference in incidence between the two groups, the disease index in the prevention group (62.5) was significantly lower than that in the treatment group (79.2) and the PWN group (100). Figure 6 B). Calculations show that the control efficacy of the prevention group against pine wilt disease ranged from 34.37% to 45.00%, while the efficacy of the treatment group was lower than that of the prevention group, ranging from 18.75% to 30.83%. Figure 6C). Therefore, in the control of pine wilt disease, strain Rhs-P27 has a better preventive and protective effect. These results provide an important reference for further optimizing control strategies.

[0096] At 35 days post-inoculation with PWN, strain Rhs-P27 significantly reduced the PWN density in pine seedlings. Compared to the PWN group, the Rhs-P27→PWN group showed a significant 59.13% reduction in pine wilt nematode density, while the PWN→Rhs-P27 group also showed a significant 34.61% reduction. This indicates that strain Rhs-P27 effectively inhibits the reproduction of pine wilt nematodes in Pinus tabuliformis seedlings, and the inhibitory effect of the prevention group (Rhs-P27→PWN group) was significantly stronger than that of the treatment group (PWN→Rhs-P27 group).

[0097] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A strain of *Pseudomonas chlororaphis*, Rhs-P27, characterized by: The *Pseudomonas aeruginosa* strain Rhs-P27 was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCCM20242846.

2. The application of strain Rhs-P27 according to claim 1 in the prevention and control of pine wilt disease.

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