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

Abstract

The invention provides application of a pseudomonas chlororaphis strain Rhs-P27 in prevention and treatment of pine wood nematode diseases, and relates to the technical field of agricultural environmental microorganisms. In particular to a Pseudomonas chlororaphis strain Rhs-P27 separated from rhizosphere soil of Chinese pines and application of the Pseudomonas chlororaphis strain Rhs-P27 in prevention and treatment of pine wood nematode diseases. The strain is preserved in the China Center for Type Culture Collection in Wuhan University on December 18, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) 20242846. The in-vitro poisoning activity of the strain Rhs-P27 on pine wood nematodes can reach 100% within 24 hours; a fermentation filtrate and a zymophyte suspension of the strain Rhs-P27 have a remarkable inhibition effect on the propagation of the pine wood nematodes; the prevention effect of a zymophyte suspension of the strain Rhs-P27 on the pine wood nematode disease of biennial pinus tabulaeformis potted seedlings can reach 45%, and the density of pine wood nematode in pine trees can be remarkably reduced; the pseudomonas chlororaphis strain Rhs-P27 disclosed by the invention has a good application prospect in the field of pollution-free prevention and treatment of pine wood nematode diseases.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural environmental microorganisms, and specifically to application of a Pseudomonas chlororaphis strain Rhs-P27 in preventing and controlling pine wood nematode disease. Background Art

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

[0003] Due to the complexity of its pathogenic mechanism and the serious harm to pine trees, scientists have been exploring effective prevention and control measures. Traditional prevention and control methods include physical and chemical control. The physical method is mainly to remove the diseased wood. Although the operation is simple and effective, it consumes a lot of resources and has high costs. Chemical methods involve trunk injection and aerial control, which can effectively reduce the incidence rate but may bring about the "3R" problem. Biological control has attracted much attention as an environmentally friendly and efficient means. The use of biocontrol bacteria to inhibit the invasion of pine wood nematodes has been proven to be an effective means. These bacteria with anti-nematode activity come from a variety of bacterial genera and provide a scientific basis for the development of new biocontrol agents. Biological control is one of the key paths for pollution-free prevention and control of plant diseases.

[0004] During the metabolism, Pseudomonas chlororaphis produces a variety of antimicrobial compounds, such as ribosomally synthesized antibacterial substances, enzymes and antibiotics, which have bactericidal activity against other microorganisms and help control the occurrence of plant diseases. Secondly, the bacteria have the ability to form biofilms, promote plant growth, and prevent and treat pests and diseases. This characteristic gives Pseudomonas chlororaphis great potential and advantages in agricultural applications. In addition, since the bacteria are derived from naturally existing bacterial communities in microecological systems, and have no toxic side effects, are environmentally friendly and pollution-free, they will not cause harm to the environment and human body during actual application. Therefore, the use of Pseudomonas chlororaphis for plant disease prevention and control is an efficient, safe, environmentally friendly and sustainable method. As people conduct more 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 the realization of sustainable agriculture and forestry. Based on this, this technical innovation plan is proposed. Summary of the invention

[0005] 1. Technical issues to be resolved In view of the deficiencies in the prior art, the present invention provides an application of a Pseudomonas chlororaphis strain Rhs-P27 in the prevention and treatment of pine wood nematode disease, which solves the problem of the lack of green and pollution-free prevention and control technology for pine wood nematode disease.

[0006] (II) Technical solution Another object of the present invention is to provide the results of the toxicity activity of Pseudomonas chlororaphis strain Rhs-P27 against pine wood nematodes.

[0007] Another object of the present invention is to provide the results of the inhibition of reproduction of pine wood nematodes by the Pseudomonas chlororaphis strain Rhs-P27.

[0008] Another object of the present invention is to provide an application of the Pseudomonas chlororaphis strain Rhs-P27 in the prevention and treatment of pine wood nematode disease in potted pine seedlings. To achieve the above objectives, the present invention is implemented through the following technical solutions: A strain of Pseudomonas chlororaphis Rhs-P27 was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO: M20242846.

[0009] Furthermore, the strain Rhs-P27 described above is used in the prevention and treatment of pine wood nematode disease.

[0010] Furthermore, the chlororaphis strain Rhs-P27 was fermented in LB liquid medium. Under stable culture conditions (25°C, 150rpm, dark culture for 4 days), the fermentation suspension (1×) and fermentation filtrate (without bacteria, 1×) of Rhs-P27 were obtained respectively. Afterwards, these suspensions were diluted with sterile water to prepare 5-fold dilution (0.2×) and 10-fold dilution (0.1×) of fermentation suspension and fermentation filtrate. Next, the fermentation suspension and fermentation filtrate at various dilutions were incubated with pine wood nematodes for 24 hours. On the basis of counting the number of surviving and dead nematodes, the in vitro toxicity of Rhs-P27 against pine wood nematodes was evaluated by analyzing the corrected mortality rate of pine wood nematodes.

[0011] Furthermore, LB liquid medium was used to carry out liquid fermentation culture of the Pseudomonas chlororaphis strain Rhs-P27. Under the set stable culture conditions (25℃, 150rpm, dark culture for 4 days), the fermentation suspension and fermentation filtrate (without bacteria) of the strain were successfully obtained. Next, Botrytis cinerea was inoculated on a PDA medium plate, and after 7 days of culture, 50µL of a suspension containing 600 pine wood nematodes was added to the center of the Botrytis cinerea. At the same time, 100µL of Rhs-P27 fermentation filtrate or fermentation suspension was dripped at the inoculation site, and an equal amount of LB liquid medium was used as a control. It is worth noting that the concentration of the fermentation suspension was 9.35×108CFU / mL. Subsequently, the samples were placed 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 reproduction coefficient and egg laying of pine wood nematodes.

[0012] Furthermore, based on the artificial inoculation system of pine wilt disease, the trunk punch injection method was used to evaluate the preventive and therapeutic effects of Pseudomonas chlororaphis Rhs-P27 suspension on pine wilt disease. Among them, the prevention group was first injected with Rhs-P27 suspension and then inoculated with pine wilt nematodes, and the treatment group was first inoculated with pine wilt nematodes and then injected with Rhs-P27 suspension. The concentration of the fermented bacterial suspension was 9.35×108CFU / mL. The incidence and severity of potted pine seedlings were counted, and the disease index and biological control effect of each treatment were calculated. At the same time, the density of pine wilt nematodes in pine trees under different treatments was measured to observe the effect of Rhs-P27 on its reproduction, so as to comprehensively evaluate the effect of Rhs-P27 suspension in the prevention and treatment of pine wilt disease.

[0013] (III) Beneficial effects The present invention provides an application of a Pseudomonas chlororaphis strain Rhs-P27 in preventing and treating pine wood nematode disease. The application has the following beneficial effects: 1. The present invention successfully screened a biocontrol strain, Pseudomonas chlororaphis strain Rhs-P27, which has significant toxic activity against pine wood nematodes.

[0014] 2. By measuring the effects of the fermentation suspension and fermentation filtrate of Pseudomonas chlororaphis strain Rhs-P27 on the reproduction of pine wood nematodes, and using the reproduction coefficient and egg-laying amount of pine wood nematodes as detection indicators, the results showed that the strain has a significant inhibitory effect.

[0015] 3. The preventive and therapeutic effects of the Pseudomonas chlororaphis strain Rhs-P27 on pine wilt disease in potted pine seedlings were evaluated. The study found that the preventive effect was more significant, with a control effect of up to 45% on pine wilt disease, and significantly reduced the nematode density in pine trees. This shows that the strain has a wide range of application potential in biological control, and provides a key basis for its subsequent product development, forest application and commercialization, providing a new biological resource for controlling pine wilt nematodes using biological control technology.

[0016] 4. This invention lays a foundation for the development of microbial agents using this strain and its commercialization. Using microbial products to replace chemical pesticides can effectively avoid the problems that may be caused by chemical pesticide residues, drug resistance problems, and the re-emergence of harmful organisms. This will help reduce or replace the use of other chemical pesticides in the prevention and control of pine wood nematode disease, and promote the disease to move towards a more environmentally friendly direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The morphology of living (A) and dead (B) pine wood nematodes.

[0018] Figure 2To analyze the toxicity of Pseudomonas chlororaphis strain Rhs-P27 against pine wood nematodes.

[0019] Figure 3 Colony morphology (A) and spore staining (B) of Pseudomonas chlororaphis strain Rhs-P27.

[0020] Figure 4 This study analyzes the inhibitory effect of Pseudomonas chlororaphis strain Rhs-P27 on the reproduction of pine wood nematodes.

[0021] Figure 5 This is the growth status of Pinus tabulaeformis seedlings in different treatment groups of Pseudomonas chlororaphis strain Rhs-P27.

[0022] Figure 6 This is an analysis of the potted control effect of Pseudomonas chlororaphis strain Rhs-P27 on pine wood nematode disease in Pinus tabulaeformis seedlings. The incidence of Pinus tabulaeformis seedlings under different treatments (A); the disease index of Pinus tabulaeformis seedlings under different treatments (B); the control effect of strain Rhs-P27 on pine wood nematode disease (C); the density of pine wood nematodes in Pinus tabulaeformis seedlings under different treatments (D). DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1: Isolation of biocontrol strains and determination of their toxicity against pine wood nematodes A Pseudomonas chlororaphis strain Rhs-P27 for pine wood nematode disease has been deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO:M20242846. The biocontrol strain Rhs-P27 was isolated from rhizosphere soil microorganisms of Pinus tabulaeformis. The co-incubation method was used to find that the strain Rhs-P27 showed significant toxicity to pine wood nematodes, and then the effect on the reproduction of pine wood nematodes was determined. The isolation of the biocontrol strain Rhs-P27 and the determination of its toxicity to pine wood nematodes are as follows: (1) Isolation of biocontrol strains ① Sampling location and sampling method Location: Rhizosphere soil of Pinus tabulaeformis was collected from Huanshan Park, Chengguan Town, Ningshan County (N33°18′25″, E108°18′39″).

[0025] Sampling method: Select four 20-year-old healthy Pinus tabulaeformis trees and set up rhizosphere soil sampling points 15 cm from the base of the trunk. Dig out the soil 10 to 20 cm from the topsoil, collect the soil containing Pinus tabulaeformis fine roots (diameter < 2 mm), and put it into a plastic bag.

[0026] ②Soil sample processing and preparation of soil suspension Take 2.5g of each of the four rhizosphere soil samples, mix them well and transfer them to a sterile glass flask containing 90mL of sterile water. After oscillating and mixing for 15 minutes, let it stand for 5 minutes and take the upper layer solution as a 10-1g / mL soil suspension. Prepare soil dilutions of different concentrations (10-2g / mL, 10-3g / mL and 10-4g / mL) based on the 10-1g / mL suspension.

[0027] ③Isolation and cultivation of soil microorganisms Take 30 µL of the soil suspension of each concentration and drop it onto the PDA plate, and spread it evenly. Prepare 3 plates for each concentration. Seal the culture plate and place it in a 25°C incubator for 30 days. Observe the single colonies grown on the culture medium every day, and use a sterile toothpick to inoculate and purify and preserve single colonies with different morphologies.

[0028] ④Strain preservation The obtained strains were placed in 30% glycerol and stored in a low temperature environment (-80°C).

[0029] The preparation method of the PDA culture medium comprises the following steps: taking 200 g of peeled potato pieces, adding 700-900 mL of deionized water, boiling for 20 minutes, filtering with gauze, collecting the filtrate, and then adding 20 g of glucose to the filtrate, and after the glucose is completely dissolved, making the volume to 1000 mL with deionized water, adding 15-20 g of agar powder, setting the pH to natural, and sterilizing with high pressure steam before use.

[0030] The above steps describe the whole process from sampling to isolation and purification of colonies to strain preservation, and provide the preparation method of the PDA medium used. These operations provide detailed guidance for the isolation of the biocontrol strain Rhs-P27 and subsequent experiments.

[0031] (2) Determination of the toxicity of rhizosphere soil microorganisms against pine wood nematodes ① Fermentation culture of rhizosphere soil microorganisms of Pinus tabulaeformis LB liquid culture medium was used to carry out liquid fermentation culture of the isolated strains of rhizosphere soil microorganisms of Pinus tabulaeformis. The culture was carried out at 25°C, 150 rpm and dark culture conditions for 4 days to obtain the fermentation suspension (1×) and fermentation filtrate (1×) of the strain, which were then diluted with sterile water to prepare 5-fold diluted (0.2×) and 10-fold diluted (0.1×) fermentation suspension and fermentation filtrate, respectively.

[0032] ② Preparation of pine wood nematode suspension Botrytis cinerea was inoculated onto PDA culture medium and cultured for 7 days until the entire plate was covered with colonies. Pine wood nematodes were inoculated onto the Botrytis cinerea colonies on an ultra-clean workbench and continued to be cultured at 25°C without darkness for 7 days until the surface of the culture medium became shiny. The pine wood nematodes were then rinsed with sterile water and collected from the culture dish to form a pine wood nematode suspension with a concentration of 100 heads / 10 μL.

[0033] ③ Co-incubation of Pinus tabulaeformis rhizosphere soil microbial fermentation suspension / filtrate with pine wood nematode Add 480 μL of each fermentation bacteria suspension (1×, 0.2× and 0.1×) and fermentation filtrate (1×, 0.2× and 0.1×) into a sterile centrifuge tube, then add 20 μL of pine wood nematode suspension (mixed larvae and adults, a total of 200) into each centrifuge tube, place them in a constant temperature incubator at 25°C, away from light and incubate for 24 h, and then count the living and dead pine wood nematodes.

[0034] ④ Calculation of corrected mortality of pine wood nematodes under each treatment The number of surviving and dead nematodes in each treatment group was detected by combining safranin staining and morphological observation. The surviving nematodes were not colored and remained active ( Figure 1 A); Dead nematodes are stained red by safranin and take the shape of a "C" or "J" and do not respond to physical stimulation ( Figure 1 B). Through systematic testing of the toxicity of various isolated strains of soil microorganisms from the root of Pinus tabulaeformis to pine wood nematodes and converting them into corrected mortality rates, it was found that the fermented bacterial suspension and fermented filtrate (without cell bodies) of a bacterial strain Rhs-P27 had a significant lethal effect on pine wood nematodes. When the fermented stock solution of Rhs-P27 was treated (1×), the corrected mortality rate of pine wood nematodes could reach 100%; after being diluted 5 times (0.2×), the corrected mortality rate of pine wood nematodes could be maintained at 37.77% (fermented bacterial suspension) and 28.88% (fermented filtrate) when treated with Rhs-P27 fermented bacterial suspension and fermented filtrate.

[0035] The mortality rate and corrected mortality rate of isolated strains of rhizosphere soil microorganisms against pine wood nematodes were calculated according to the following formula.

[0036] Mortality rate (%) = Corrected mortality rate (%) = Wherein, Nd is the number of nematodes killed in the treatment group; Nt is the total number of nematodes; d is the nematode mortality rate in the treatment group (%); c is the nematode mortality rate in the control group (%).

[0037] Example 2: Identification of biocontrol strain Rhs-P27

[0038] DNA of strain Rhs-P27 was extracted by CATB method, and the 16SrDNA sequence was amplified and sequenced by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') of 16SrDNA. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The PCR reaction system was 25 μL, including: 12.5 μL of 2×ESTaqMasterMix, 1 μL of template DNA, 1 μL of 27F primer, 1 μL of 1492R primer and 9.5 μL of sterile distilled water. PCR was performed in a PCR thermal cycler. After pre-denaturation at 94°C for 3 minutes, 30 cycles were performed: denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 45 seconds; and final extension at 72°C for 10 minutes. After PCR, the length of the amplified fragment was detected by agarose gel electrophoresis, and the sequencing product was commissioned to Sangon Biotech (Shanghai) Co., Ltd. According to the comparison results of the NCBI database, the strain Rhs-P27 was identified as Peribacilllus frigoritolerans. The strain was stored in the China Center for Type Culture Collection on December 18, 2024, with the accession number CCTCCM20242847.

[0039] Sequencing results of the 16SrDNA amplified fragment of strain Rhs-P27: Example 3: Detection of the effect of the biocontrol strain Rhs-P27 on the reproduction of pine wood nematodes Rhs-P27 was subjected to liquid fermentation culture using LB liquid medium. The culture conditions were 25°C, 150 rpm, and dark culture for 4 days, and the fermentation bacterial suspension and fermentation filtrate (without bacterial cells) of strain Rhs-P27 were obtained respectively.

[0040] The LB liquid medium was used to carry out liquid fermentation culture of the Pseudomonas chlororaphis strain Rhs-P27. The culture conditions were 25℃, 150rpm, and dark culture for 4 days to obtain the fermentation suspension and fermentation filtrate (without bacteria). Botrytis cinerea was inoculated on a PDA medium plate. After 7 days of culture, 50μL of suspension containing 600 pine wood nematodes was dripped into the center of the Botrytis cinerea colony. At the same time, 100μL of Rhs-P27 fermentation filtrate (T1 group) or fermentation suspension (T2 group) was dripped into the inoculation site. The LB liquid medium was used as the control group (CK group). The concentration of the fermentation suspension was 9.35×108CFU / mL. The plate was then placed in a constant temperature incubator and incubated at 25℃ for 7 days, and then the pine wood nematodes were collected from each culture dish by aseptic operation. Combining safranin staining and morphological observation methods, the number of surviving pine wood nematodes and eggs in each treatment group was counted under a microscope and compared with the CK group to analyze the effect of Rhs-P27 on the reproduction of pine wood nematodes.

[0041] The test results showed that the fermentation filtrate and 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. 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 bacterial suspension of Rhs-P27 can effectively inhibit the reproduction of pine wood nematodes.

[0042] The reproduction coefficient of pine wood nematodes on each PDA plate was calculated according to the following formula: Reproduction coefficient = Wherein, Pf is the number of surviving nematodes in each plate after 7 days of cultivation, and Pi is the initial inoculum number of nematodes in each plate.

[0043] Example 4: Application of strain Rhs-P27 in pine wood nematode disease The research subjects were five-year-old healthy Pinus tabulaeformis seedlings planted in greenhouse pots at Northwest Agriculture and Forestry University. Five different treatment groups were designed, as follows: Control group (CK group): treated with LB liquid medium only; Pine wood nematode treatment group (PWN group): only inoculated with pine wood nematodes; Rhs-P27 treatment group (Rhs-P27 group): treated with Rhs-P27 fermentation bacteria suspension only; Prevention group (Rhs-P27→PWN group): first injected with Rhs-P27 fermented bacterial suspension and then inoculated with pine wood nematodes Treatment group (PWN→Rhs-P27 group): first inoculated with pine wood nematodes and then injected with Rhs-P27 fermentation bacteria suspension All Pinus tabulaeformis seedlings will be cultured and observed in the greenhouse of Northwest Agriculture and Forestry University. Three replicates will be set for each treatment, and each replicate contains three Pinus tabulaeformis seedlings. The Rhs-P27 bacterial suspension used is prepared by fermentation in LB liquid medium, and the concentration of the bacterial suspension is 9.35×108CFU / mL.

[0044] The specific operation process is as follows: before treatment, first use wet sterile cotton to clean the surface of the branches and trunks of healthy Pinus tabulaeformis seedlings. Then, use a handheld electric drill to drill two small holes on the stem of each Pinus tabulaeformis seedling. Each hole has a diameter of 4.5 mm and a depth of 5 mm, and the distance between the two holes is 10 cm. The specific operations for different treatment groups are as follows: CK group: Each well was covered with sterile cotton and injected with 5 ml of LB medium, then the well mouth was sealed with plastic film to prevent rapid evaporation of water, and the cotton and plastic film were removed after 7 days of treatment.

[0045] PWN group: Each well was inoculated with 50 µL of a suspension containing 3,000 PWN (a mixture of larvae and adults), and then the wells were sealed with plastic film. The plastic film was removed 7 days after treatment.

[0046] Rhs-P27 group: Same as CK group, but 5 mL Rhs-P27 strain fermentation suspension was used instead of LB medium.

[0047] Rhs-P27→PWN group: The initial treatment was the same as that of the Rhs-P27 group. The plastic film and cotton were removed 7 days after the Rhs-P27 injection, and 50 µL of pine wood nematode suspension (containing 3,000 PWN) was inoculated into each well. The wells were sealed with plastic film, and the plastic film was removed 7 days after the treatment.

[0048] PWN→Rhs-P27 group: The initial treatment was the same as that of the PWN group. Seven days after inoculation of PWN, 5 mL of Rhs-P27 fermentation suspension was injected into each well using the same method as the Rhs-P27 group. The wells were sealed with plastic film, and the plastic film was removed after 7 days of treatment.

[0049] The disease conditions of the pine seedlings in each treatment group were observed and recorded at 7, 14, 21, 28 and 35 days after inoculation with PWN, and the incidence rate was calculated. At the same time, the pine seedlings were divided into 5 sections on average, and the severity of the disease in each section was counted. Five levels (S1-S5) were formulated according to the severity, with representative values ​​of 0, 1, 2, 3, and 4, respectively. The specific levels are represented as follows: S1: All needles are healthy and green; S2: Yellowing needles appear, but the number does not exceed one quarter; S3: Dead needles appear, and the number of yellow or dead needles is between one quarter and one half; S4: Yellowing or dead needles account for between one-half and two-thirds of the wood, and the pine wood segments show slight wilting; Grade S5: More than two-thirds of the needles are yellowing or dead, and the Pinus tabulaeformis seedlings are obviously dehydrated and wilting.

[0050] Then, the disease index of pine wilt disease under each treatment was calculated, and the prevention and control effects of Rhs-P27 prevention treatment (Rhs-P27→PWN group) and treatment treatment (PWN→Rhs-P27 group) on pine wilt disease were further evaluated. Through such methods and data analysis, we can have a more comprehensive understanding of the prevention and control effects of different treatments of Rhs-P27 on pine wilt disease.

[0051] 35 days after inoculation with pine wood nematodes, the whole pine seedlings in the PWN group were observed to be in a wilting and dead state. Subsequently, each pine seedling in the PWN group, Rhs-P27→PWN group, and PWN→Rhs-P27 group was collected, and its roots, stems, and leaves were cut into small pieces, mixed evenly, and weighed. Then, the pine wood nematodes in all tissues of each seedling were separated by the Baermann funnel method, and the number of nematodes (Number of nematodes, NN) was counted under a microscope. The density of pine wood nematodes in pine seedlings under different treatments will be expressed as the number of nematodes per gram of fresh weight (FW) of pine (NN g-1FW). This method can fully understand the effects of different treatments on the number of pine wood nematodes in pine seedlings, and further evaluate the effect of the Rhs-P27 strain on reducing or controlling the number of pine wood nematodes in pine.

[0052] Incidence (%) = ×100 Disease index = ×100 Control effect (%) = ×100 Density of pine wood nematodes (NN g -1 FW) = According to the results of the potted test, it was found that the strain Rhs-P27 had no toxicity to pine seedlings. The growth conditions of the pine seedlings treated with Rhs-P27 alone were basically the same as those of the control group, and both remained healthy. 35 days after inoculation with pine wood nematodes, all the needles of the Chinese pine seedlings in the PWN group lost their green color, dried up, and showed a significant wilting state. The disease in the prevention group (Rhs-P27→PWN group) was significantly lighter than that in the PWN group, and the number of green or dried needles of each pine seedling was less than two-thirds. The treatment group (PWN→Rhs-P27 group) also had a certain effect on alleviating the occurrence of pine wood nematode disease, but the number of dead needles was significantly more than that in the prevention group. These results show that both preventive and therapeutic treatments have a certain effect in inhibiting or alleviating pine wood nematode diseases, and the preventive treatment is more effective ( Figure 5 ).

[0053] According to the analysis of the incidence and disease index of pine wilt disease in each treatment group at 7, 14, 21, 28 and 35 days after inoculation with pine wood nematodes, it was found that in five-year-old Chinese pine seedlings, the dominant stage of pine wilt disease appeared on the 14th day after inoculation with PWN. As the disease progressed, the symptoms gradually worsened. At 35 days, all Chinese pine seedlings showed obvious disease manifestations, and the disease index was as high as 100 ( Figure 6 AB). The Chinese pine seedlings in the Rhs-P27 prevention group (Rhs-P27→PWN) and treatment group (PWN→Rhs-P27) also showed mild symptoms on the 14th day, but the severity of the disease was milder, especially in the prevention group. During the course of pine wilt disease, the incidence rate gradually increased, reaching 83.33% in the Rhs-P27 prevention group at 35 days, while the incidence rate in the treatment group was 100% ( Figure 6 A). Although there was no significant difference in the incidence between the two groups, the disease index of the prevention group (62.5) was significantly lower than that of the treatment group (79.2) and the PWN group (100) ( Figure 6 B). Calculations show that the prevention group's control effect on pine wood nematode disease ranged from 34.37% to 45.00%, while the treatment group's effect was lower than the prevention group, ranging from 18.75% to 30.83% ( Figure 6 C). Therefore, in the prevention and control of pine wood nematode disease, strain Rhs-P27 has a better preventive and protective effect. These results provide an important reference for further optimizing control strategies.

[0054] At 35 days after inoculation with PWN, strain Rhs-P27 was found to significantly reduce the density of PWN in pine seedlings. Compared with the PWN group, the density of pine wood nematodes in the Rhs-P27→PWN group was significantly reduced by 59.13%, while the density of pine wood nematodes in the PWN→Rhs-P27 group was also significantly reduced by 34.61%. This shows that strain Rhs-P27 can effectively inhibit the reproduction of pine wood nematodes in Chinese pine seedlings, and the inhibitory effect of the prevention group (Rhs-P27→PWN group) is significantly stronger than that of the treatment group (PWN→Rhs-P27 group).

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Pseudomonas chlororaphis strain Rhs-P27, characterized in that: The Pseudomonas chlororaphis strain Rhs-P27 was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCCM20242846.

2. Use of the strain Rhs-P27 according to claim 1 in preventing and controlling pine wood nematode disease.

3. The in vitro toxicity of a strain of Pseudomonas chlororaphis Rhs-P27 against pine wood nematodes according to claim 1, characterized in that: The components of the fermentation medium were 10 g of trypsin, 3 g of yeast extract, 10 g of sodium chloride, and 1 L of water. The fermentation conditions were 25°C, 150 rpm, and dark culture for 4 days. The collected fermentation bacterial suspension and fermentation filtrate (without bacteria) had significant in vitro toxic activity against pine wood nematodes. After 24 hours of co-incubation, the corrected mortality rate of pine wood nematodes reached 100%.

4. The method for determining the effect of a Pseudomonas chlororaphis strain Rhs-P27 on the reproduction of pine wood nematodes according to claim 1, characterized in that: The fermentation filtrate and fermentation bacterial suspension of Rhs-P27 strain had a significant inhibitory effect on the reproduction coefficient and egg-laying amount of pine wood nematode, and the concentration of the fermentation bacterial suspension was 9.35×108CFU / mL.

5. The effect of the fermented bacterial suspension of Pseudomonas chlororaphis strain Rhs-P27 on preventing pine wood nematode disease in potted pine seedlings according to claim 1, characterized in that: The control effect on pine wood nematode disease is 45%, and the density of pine wood nematodes in Chinese pine is significantly reduced.

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