Pseudomonas aeruginosa CWD-D and application thereof in preventing and treating plant diseases

Abstract

The application discloses a Pseudomonas aeruginosa CWD-D and application thereof in preventing and treating plant diseases. The Pseudomonas aeruginosa CWD-D strain is preserved in the China Center for Type Culture Collection on July 31, 2023, and the address of the preservation center is Wuhan, Wuhan University, China, and the preservation number is CCTCC No: M 20231386. The strain has a high efficient inhibiting effect on phytophthora colocasiae, phytophthora citri and phytophthora capsici, and also has an excellent effect in preventing and treating plant diseases caused by the pathogenic bacteria, and is significantly stronger than a Pseudomonas aeruginosa model strain PAO1. In addition, the bacterial suspension and the fermentation liquor prepared from the strain also have the same pathogenic bacteria inhibiting effect, the culture condition requirement is low, and the strain has a good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology. More specifically, it relates to a strain of Pseudomonas aeruginosa CWD-D and its application in the control of plant diseases. Background Technology

[0002] Tropical plants comprise about two-thirds of the world's higher plant species, including numerous economic crops. They possess extremely rich genetic diversity, providing humans with food, beverages, energy, medicine, timber, fuel, and various industrial raw materials. They also provide other plants and animals with essential oxygen, water, and a suitable habitat, playing an irreplaceable role in the health of ecosystems. However, the hot and humid conditions also create a breeding ground for many plant pathogens, which can cause severe biological disasters and lead to reduced crop yields.

[0003] taro( Colocasia esculenta Taro (Potentilla chinensis) is a perennial monocotyledonous herbaceous wetland plant originating in Southeast Asia. It is a dual-purpose crop (food and vegetable) with high economic value and promising prospects for industrial development. The fungus *Phytophthora tectorum* (Phytophthora tectorum) is active in hot and humid environments. Phytophthora colocasiae Taro blight, caused by [unspecified disease], is a significant disease in taro cultivation, widely distributed and highly prevalent in taro-producing areas. The disease can reduce taro yield by up to 30%, and in severe cases, even lead to total crop failure.

[0004] Phytophthora indicum ( Peronophythora litchi Downy mildew, caused by pyrethrum, seriously threatens the healthy development of my country's pyrethrum industry. It is the most serious and widespread disease affecting pyrethrum production and post-harvest. This disease can infect young leaves, young branches, flower spikes, and fruits, causing extensive rot of flower spikes and fruits. In normal years, it can cause a yield loss of 10% to 30%, while in epidemic years, it can cause a yield loss of 80%.

[0005] Phytophthora blight in peppers is caused by Phytophthora capsici ( Phytophthora capsici Phytophthora blight is a soil-borne disease caused by the fungus *Phytophthora capsici*, which severely impacts chili pepper yields. It occurs on a large scale in various regions, especially in hot and humid environments where outbreaks are more likely and the damage is more severe. *Phytophthora capsici* has a wide range of hosts, can be transmitted through soil, and can infect various dicotyledonous crops, including chili peppers, tomatoes, melons, and fruit trees, causing enormous losses to the global horticulture industry every year.

[0006] Currently, the control of plant diseases and fungi mainly relies on chemical control. However, the problems and risks associated with using fungicides alone are increasing daily. In contrast, modern biological control technologies, such as using antagonistic bacteria or utilizing the active substances produced by antagonistic bacteria to control crop diseases, have advantages such as safety, high efficiency, low toxicity, no pollution, short cycle, ease of research, ease of production, and no residue.

[0007] In response to the wide variety of plant diseases, it is necessary to develop a broad-spectrum antagonistic fungicide that can simultaneously control multiple plant diseases and reduce the economic losses caused by plant diseases. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and to provide a strain of Pseudomonas aeruginosa CWD-D and its application in the prevention and control of plant diseases.

[0009] The first objective of this invention is to provide a strain of Pseudomonas aeruginosa CWD-D.

[0010] A second objective of this invention is to provide a biocontrol agent.

[0011] A third objective of this invention is to provide the application of the biocontrol agent in inhibiting pathogens or in the preparation of pathogen-inhibiting products.

[0012] A fourth objective of this invention is to provide the application of the biocontrol agent in the prevention and control of plant diseases caused by pathogens.

[0013] A fifth object of the present invention is to provide the application of the biocontrol agent in the preparation of products for the prevention and control of plant diseases caused by pathogens.

[0014] The sixth objective of this invention is to provide a method for preventing and controlling taro blight.

[0015] The above-mentioned objective of this invention is achieved through the following technical solution:

[0016] This invention provides a strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa The CWD-D strain described in this invention was deposited on July 31, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: M 20231386.

[0017] The Pseudomonas aeruginosa described in this invention ( Pseudomonas aeruginosa The CWD-D strain originated from soil in a greenhouse at the College of Agriculture, South China Agricultural University. Soil samples were diluted and streaked onto LB agar plates to obtain multiple pure colonies. All colonies were then selected, and a strain exhibiting inhibitory activity against *Pseudomonas aeruginosa* was identified through plate contrast screening. This strain was subsequently identified as *Pseudomonas aeruginosa*. Therefore, this invention applies for protection of the aforementioned *Pseudomonas aeruginosa* strain. Pseudomonas aeruginosa )CWD-D strain.

[0018] This invention involves culturing *Pseudomonas aeruginosa* CWD-D around *Phytophthora tarois*, the pathogen of taro blight, and finding that it has a strong antibacterial effect and causes abnormal mycelial growth of *Phytophthora tarois*. This invention also involves uniformly spraying a suspension or fermentation broth of *Pseudomonas aeruginosa* CWD-D onto taro leaves, and finding that it has a significant effect in controlling taro blight.

[0019] Furthermore, this invention has discovered that, in addition to being effective against Phytophthora taroum (… Phytophthora colocasiae The *Pseudomonas aeruginosa* CWD-D strain of the present invention is effective against *Phytophthora lichee* (…). Peronophythora litchi ) and Phytophthora capsici ( Phytophthora capsici It also has excellent inhibitory effects, and the inhibitory effect is significantly better than that of the Pseudomonas aeruginosa model strain PAO1.

[0020] Therefore, this invention applies to protect the following applications of the Pseudomonas aeruginosa CWD-D strain:

[0021] This invention applies to protect a biocontrol agent containing the CWD-D strain or its bacterial suspension or fermentation broth.

[0022] This invention application protects the use of the biocontrol agent in inhibiting pathogens or in the preparation of pathogen-inhibiting products.

[0023] This invention application protects the use of the biocontrol agent in the prevention and control of plant diseases caused by pathogens.

[0024] This invention application protects the use of the biocontrol agent in the preparation of products for preventing and controlling plant diseases caused by pathogens.

[0025] Specifically, the pathogen is any one or more of Phytophthora taroii, Phytophthora litchii, and Phytophthora capsicum.

[0026] Specifically, the plant diseases mentioned include taro blight, litchi downy mildew, pepper blight, and tomato downy mildew, which are caused by Phytophthora taroii, Phytophthora litchiii, and Phytophthora capsici, respectively.

[0027] As one possible implementation, the preparation method of the bacterial suspension of the CWD-D strain is as follows: the CWD-D strain is inoculated into LB liquid medium and cultured with shaking.

[0028] Preferably, the pH value of the LB liquid culture medium is 6.8 to 7.2.

[0029] Preferably, the shaking culture conditions are as follows: place in a shaker at 28~30℃ and culture at 180~200 rpm for 16~24 h.

[0030] As one possible implementation, the fermentation broth of the CWD-D strain is prepared by further inoculating the bacterial suspension of the CWD-D strain into LB liquid medium, shaking and culturing, and then centrifuging to collect the supernatant.

[0031] Preferably, the density of the bacterial suspension of the CWD-D strain is: OD600 = 0.7~0.8; the shaking culture conditions are: 28~30℃, 180~200 rpm for 36~48 h; the centrifugation conditions are: 10000~12000 rpm for 5~10 min.

[0032] As one possible implementation, the supernatant is filtered through a 0.22 μm filter to remove bacterial cells.

[0033] The present invention also provides a method for preventing and controlling plant diseases, which involves uniformly spraying the above-mentioned biocontrol agent onto the plant surface to achieve the prevention and control of taro blight; the plant diseases include taro blight, lychee downy mildew, pepper blight, tomato downy mildew, etc.

[0034] As one possible implementation, when the biocontrol agent is a bacterial suspension, the OD600 of the bacterial suspension is 0.7~0.8; when the biocontrol agent is a fermentation broth, the fermentation broth is the secretion obtained after culturing 1% (v / v) bacterial suspension for 36~48 h.

[0035] Preferably, when the biocontrol agent is a bacterial suspension, the OD600 of the bacterial suspension is 0.8; when the biocontrol agent is a fermentation broth, the fermentation broth is the secretion obtained after culturing a 1% (v / v) bacterial suspension for 48 h.

[0036] The present invention has the following beneficial effects:

[0037] 1. The *Pseudomonas aeruginosa* CWD-D obtained in this invention was isolated from healthy taro soil, eliminating the risk of pathogenicity to the plant itself. Due to its long-term symbiotic relationship with the plant, applying the metabolites of *Pseudomonas aeruginosa* CWD-D to the plant surface will not have a negative impact on plant growth, nor will it cause any residual harm after application.

[0038] 2. The Pseudomonas aeruginosa CWD-D biocontrol agent (including bacterial suspension and fermentation broth) of the present invention is effective against Phytophthora tectorum (…). Phytophthora colocasiae ), Phytophthora lichee ( Peronophythora litchi ), Phytophthora capsici ( Phytophthora capsici All of them have strong inhibitory effects, and the inhibitory effect is significantly better than that of the Pseudomonas aeruginosa model strain PAO1. They also have excellent control effects on plant diseases caused by Phytophthora taroii, Phytophthora litchiensis, and Phytophthora capsici.

[0039] 3. The fermentation broth of Pseudomonas aeruginosa CWD-D obtained by this invention has low requirements for culture conditions and can be obtained by collecting the supernatant through centrifugation, which has great development and application value. Attached Figure Description

[0040] Figure 1 This image shows a single colony of Pseudomonas aeruginosa CWD-D on LB agar. A: Colony morphology of CWD-D strain on LB agar; B: Stereoscopic image of a CWD-D strain colony.

[0041] Figure 2 This image shows the inhibitory effect of a suspension of *Pseudomonas aeruginosa* CWD-D on *Phytophthora tectorum* on agar plates. A: *Phytophthora tectorum* colonies with normal growth; B: *Phytophthora tectorum* colonies whose growth is inhibited by *Pseudomonas aeruginosa* CWD-D.

[0042] Figure 3 The images show the abnormal hyphal growth of *Pseudomonas aeruginosa* CWD-D suspension induced by this bacteria. A: Normal hyphal morphology of *P. aeruginosa*; B: Abnormal hyphal growth of *P. aeruginosa* caused by *P. aeruginosa* inhibition.

[0043] Figure 4 The image shows the plate inhibition effect of Pseudomonas aeruginosa CWD-D fermentation broth on Phytophthora tectorum. A: Colony growth in LB and various proportions of CWD-D fermentation broth mixed with carrot medium inverted plates; B: Inhibition rate of colony growth in various proportions of CWD-D fermentation broth mixed with carrot medium at 3 and 6 days.

[0044] Figure 5 This image shows the preventive effect of Pseudomonas aeruginosa CWD-D on detached taro leaves infected with Phytophthora taro.

[0045] Figure 6 This image shows the therapeutic effect of Pseudomonas aeruginosa CWD-D on detached taro leaves infected with Phytophthora tarois.

[0046] Figure 7 The diagrams show the preventive effect of Pseudomonas aeruginosa CWD-D on Phytophthora taro leaves infected with Phytophthora taro. A: Sterile water sprayed on the leaves, without Phytophthora taro inoculation; B: LB liquid medium sprayed on the leaves, with Phytophthora taro inoculation; C: CWD-D bacterial suspension sprayed on the leaves, with Phytophthora taro inoculation; D: CWD-D fermentation broth sprayed on the leaves, with Phytophthora taro inoculation.

[0047] Figure 8This is a comparison of the pathogenic inhibition effects of *Pseudomonas aeruginosa* CWD-D and *Pseudomonas aeruginosa* PAO1. A: Plate contrast diagram of *Pseudomonas aeruginosa* CWD-D and PAO1 against *Phytophthora tuna*, *Phytophthora litchifolia*, and *Phytophthora capsici* on carrot agar, with yellow impurities representing carrot residue. B: Colony inhibition rates of *Pseudomonas aeruginosa* CWD-D and PAO1 against *Phytophthora tuna*, *Phytophthora litchifolia*, and *Phytophthora capsici*. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0049] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0050] The sources of the microbial materials used in the following examples are:

[0051] Phytophthora indicum: Phytophthora indicum ( Phytophthora colocasiae ) Lyd2019 strain, disclosed in "GenomeSequence Resource of Phytophthora colocasiae from China Using NanoporeSequencing Technology, Plant Dis. In references such as , 2021, 105 (12):4141-4145.

[0052] Phytophthora lichee: Phytophthora lichee Peronophythora litchi The SHS3 strain was disclosed in literature such as "Cloning and Functional Analysis of PLBZP32, a Pathogenic Gene of Phytophthora lichee, Chen Yubin, South China Agricultural University" and "Functional Study of Phytophthora lichee Effector PLAvh133 and Screening of Target Proteins, Shao Yi, South China Agricultural University".

[0053] Phytophthora capsici: Phytophthora capsici ( Phytophthora capsici The LT263 strain was disclosed in literature such as "Identification and Functional Analysis of PiAvr3alike Genes in Phytophthora capsici, Xu Jing, Nanjing Agricultural University".

[0054] The Pseudomonas aeruginosa PAO1 strain has the accession number CCTCC M 2014630 and is disclosed in patent CN104673704B.

[0055] The following LB solid medium is prepared as follows: Weigh 10 g of tryptone (Oxoid LTD LP0042, England), 5 g of yeast extract (Oxoid LTD LP0021, England), and 10 g of sodium chloride (NaCl, Sinopharm Chemical Reagent Co., Ltd., 10019318). Add water to 1000 mL and stir well. Then add 15 g of agar, heat to dissolve completely, dispense into containers, sterilize at 121℃ for 20 min, and store for later use.

[0056] The LB liquid culture medium described below is as follows: Weigh 10 g of tryptone (Oxoid LTD LP0042, England), 5 g of yeast extract (Oxoid LTD LP0021, England), and 10 g of sodium chloride (NaCl, Sinopharm Chemical Reagent Co., Ltd., 10019318), add water to 1000 mL, stir well, heat thoroughly to dissolve, dispense into containers, sterilize at 121℃ for 20 min, and store for later use.

[0057] The carrot culture medium described below is as follows: Weigh 300 g of carrots, juice them thoroughly using a juicer, filter them through 16 layers of gauze, add water to the filtrate to make up to 1000 mL, add 15 g of agar, heat thoroughly to dissolve, and dispense into Erlenmeyer flasks (100 mL of culture medium per flask), sterilize at 121℃ for 20 min, cool and store for later use.

[0058] Example 1: Isolation, purification, identification, and preservation of Pseudomonas aeruginosa

[0059] (1) Isolation and purification of bacteria to be screened:

[0060] Soil samples used for separation were collected from the greenhouse of the College of Agriculture, South China Agricultural University.

[0061] Weigh 1 g of soil sample and add it to 100 mL of PBS buffer. Mix well and incubate at 200 rpm for 30 min on a shaker at 30℃. Take 1 mL of supernatant and add it to 9 mL of PBS buffer for a 10-fold dilution. Prepare 9 PBS buffer serial dilutions. Spread 50 μL of each dilution onto LB agar plates and incubate at 37℃ in the dark for 48 h, with 3 replicates. Randomly select 32 single colonies from each plate (by selecting colonies from plates with high dilutions to avoid fungal contamination) and streak them on the LB agar plate to obtain pure bacteria. Add 100 μL of LB medium to each well of a 96-well plate. Use a toothpick to pick out all colonies and place them in the wells of the 96-well plate. Shake at 200-300 rpm for 18-24 h on a shaker at 30℃. Add 100 μL of 50% (v / v) glycerol to each well and store at -80℃.

[0062] (2) Screening and identification of strains that have inhibitory effects on Phytophthora tauraceae.

[0063] Strains exhibiting inhibitory effects against *Phytophthora tectorum* were screened using plate confrontation, and 16S rDNA sequencing of these strains was performed. The sequencing results are shown below (SEQ ID No. 1):

[0064]

[0065] Blast sequence alignment using data from the National Center for Biotechnology Information (NCBI) revealed that this strain is similar to... Pseudomonas aeruginosa strain Gxun-7 The sequence similarity reached 98%, which preliminarily confirms that the strain with inhibitory activity against Phytophthora tectorum screened in this invention belongs to Pseudomonas aeruginosa (Pseudomonas aeruginosa). Pseudomonas aeruginosa The strain was named Pseudomonas aeruginosa CWD-D.

[0066] The CWD-D strain was cultured on LB solid medium at 30°C for 24 h, and the results were as follows: Figure 1 As shown, the colony surface is transparent and smooth, yellowish-green in color, with a distinct colony halo spreading outwards.

[0067] (3) Preservation of Pseudomonas aeruginosa CWD-D:

[0068] In summary, the CWD-D strain was identified as Pseudomonas aeruginosa. Pseudomonas aeruginosa The strain was deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, China, on July 31, 2023, with accession number CCTCC No: M20231386.

[0069] Example 2: Inhibitory effect of Pseudomonas aeruginosa CWD-D bacterial suspension and fermentation broth on Phytophthora tectorum.

[0070] (1) Preparation of Pseudomonas aeruginosa CWD-D suspension: Pseudomonas aeruginosa CWD-D was streaked onto LB agar plates and activated for 24 h. Single colonies were then picked and streaked for preservation. The single colony was inoculated into LB liquid medium and incubated at 30℃ and 200 rpm for 24 h to obtain Pseudomonas aeruginosa CWD-D suspension.

[0071] (2) Preparation of Pseudomonas aeruginosa CWD-D fermentation broth: Adjust the bacterial suspension obtained in step (1) to OD600 = 0.8, take 2 mL of bacterial suspension and add it to a conical flask containing 200 mL of LB liquid medium, incubate at 200 rpm for 48 h, centrifuge at 12000 rpm for 10 min after the incubation, take the supernatant, and filter out the bacterial cells with a 0.22 μm filter to obtain Pseudomonas aeruginosa CWD-D fermentation broth.

[0072] (3) Activity assay:

[0073] ① Determination of the inhibitory effect of Pseudomonas aeruginosa CWD-D suspension on the growth of Phytophthora taurensis: Six-day-old colony plates were selected. A 3 mm diameter Phytophthora taurensis mycelium patch was inoculated into the center of a carrot agar plate. Two μL of Pseudomonas aeruginosa CWD-D suspension was inoculated at three points 2.5 cm away from the mycelium patch. LB liquid medium was used as the control group. Each group had three replicates. The plates were incubated at 28℃. On day 3, the width of the inhibition zone was measured, and the edge of the inhibition zone and the hyphal morphology of Phytophthora taurensis were observed. The results are as follows: Figure 2 and Figure 3 As shown.

[0074] ② Determination of the inhibitory effect of Pseudomonas aeruginosa CWD-D fermentation broth on the growth of Phytophthora tectorum: Pseudomonas aeruginosa CWD-D fermentation broth was added to carrot culture medium at volume ratios of 20%, 30%, 40%, and 50% to obtain mixed carrot culture medium. LB liquid medium was added to carrot culture medium in the same proportion as a control. Six-day-old colony plates were selected, and 3 mm diameter Phytophthora tectorum cakes at the growth edge were inoculated into the center of the mixed carrot culture medium plate. Colony size and morphology were measured on days 3 and 6, and the results are shown below. Figure 4 As shown.

[0075] Inhibition rate (%) = (Mycelial growth radius of control group - Mycelial growth radius of treatment group) / Mycelial growth radius of control group × 100%

[0076] (4) Results analysis:

[0077] Depend on Figure 2 , Figure 3 It can be seen that the suspension of Pseudomonas aeruginosa CWD-D has a significant inhibitory effect on the growth of Phytophthora tauraceae. The inhibition rate was 53.3% on the 3rd day of plate confrontation and reached 61.2% on the 6th day, and it also caused malformation of Phytophthora tauraceae hyphae. Figure 4 It can be confirmed that the fermentation broth of the CWD-D strain of the present invention has a significant inhibitory effect on the growth of Phytophthora taurensis. In particular, the mixed culture medium containing 50% Pseudomonas aeruginosa CWD-D fermentation broth has an inhibition rate of up to 100% on the mycelial growth of Phytophthora taurensis, achieving complete inhibition of Phytophthora taurensis.

[0078] Example 3: Control effect of Pseudomonas aeruginosa CWD-D on detached taro leaves infected with Phytophthora laurentii.

[0079] (1) Prevention of detached taro leaves infected by *Pseudomonas aeruginosa* CWD-D by *Pseudomonas aeruginosa*: Select healthy taro leaves with uniform growth status, cut them into 6×6 cm pieces, rinse them clean in running water, and air dry them. Then, place them in 10 mL LB liquid medium and the *Pseudomonas aeruginosa* CWD-D suspension (OD2) prepared in Example 2.600 =0.8) and the fermentation broth prepared in Example 2 (with OD 600 After soaking in a bacterial suspension (obtained from 0.8 OD600) for 10 min, remove and place in a petri dish when no more droplets drip. Select 6-day-old *Pseudomonas aeruginosa* colony plates and inoculate the center of the leaf block with a 3 mm diameter *Pseudomonas aeruginosa* cake at the growth edge. Incubate at 28℃ after inoculation and observe the leaf block condition regularly. Meanwhile, healthy leaf blocks not inoculated with *Pseudomonas aeruginosa* were set up as a blank control and treated in the same way in 10 mL LB liquid medium, *Pseudomonas aeruginosa* CWD-D bacterial suspension (OD600=0.8) prepared in Example 2, and fermentation broth prepared in Example 2 (obtained from bacterial suspension with OD600=0.8).

[0080] (2) Treatment of detached taro leaves infected with Phytophthora taro by Pseudomonas aeruginosa CWD-D: Healthy taro leaves with consistent growth were selected and cut into 6×6 cm pieces. After rinsing them in running water and drying them, 6-day-old Phytophthora taro colony plates were selected. A 3 mm diameter Phytophthora taro cake was inoculated from the edge of the growth plate into the center of the leaf piece. After inoculation, the plates were placed in a 28℃ environment for 3 days to allow Phytophthora taro to fully colonize. The size of the lesions was recorded. The plates were then soaked in 10 mL LB liquid medium, Pseudomonas aeruginosa CWD-D suspension, and fermentation broth for 10 min respectively. After soaking, the plates were placed in petri dishes when the liquid droplets stopped dripping. Normal diseased leaves without treatment were set up as a blank group. After 3 days of treatment (i.e., 6 days after the onset of the disease), the change in the size of the leaf lesions was recorded, the growth of the colony radius was measured, and the treatment results were statistically analyzed.

[0081] Treatment rate (%) = (3-day colony radius growth in the control group - 3-day colony radius growth in the treatment group) / 3-day hyphae growth in the control group × 100%

[0082] (3) Experimental results show that:

[0083] Three days after inoculation, compared with uninoculated leaves, leaves treated with LB liquid medium showed significant disease in the inoculated leaves, while leaves treated with Pseudomonas aeruginosa CWD-D suspension and fermentation broth showed no disease yet. Figure 5 .from Figure 5 The control group showed that healthy taro leaves did not develop the disease after treatment with Pseudomonas aeruginosa CWD-D suspension and fermentation broth, indicating that the Pseudomonas aeruginosa CWD-D strain of the present invention is not a pathogenic fungus for taro leaves. At the same time, the experimental results also showed that healthy taro leaves did not develop the disease after being treated with Pseudomonas aeruginosa CWD-D suspension and fermentation broth and then inoculated with the pathogen Phytophthora taroii. Therefore, the Pseudomonas aeruginosa CWD-D strain of the present invention can be used as a strain for the prevention of taro blight.

[0084] Figure 6 The results showed that, when treating diseased leaves already colonized with *Pseudomonas aeruginosa*, both the bacterial suspension and fermentation broth of *Pseudomonas aeruginosa* CWD-D significantly reduced the production of white mold on the leaf surface compared to the blank control and LB liquid medium treatment groups. Furthermore, the treatment rate of the *P. aeruginosa* CWD-D bacterial suspension averaged around 40%, significantly higher than that of the LB liquid medium, thus inhibiting the spread of the disease caused by *P. aeruginosa* to a certain extent. This experiment demonstrates that the bacterial suspension and fermentation broth of *P. aeruginosa* CWD-D have a considerable ability to block the rapid spread of *P. aeruginosa* during an outbreak of taro blight.

[0085] Example 4: The preventive effect of Pseudomonas aeruginosa CWD-D on Phytophthora taro infection in live taro leaves.

[0086] Taro potted plants with similar growth periods were selected. 50 mL of *Pseudomonas aeruginosa* CWD-D suspension and fermentation broth were evenly sprayed onto the taro leaves. Potted plants sprayed with LB liquid medium served as a negative control, while those sprayed with water but not inoculated with *Phytophthora taroica* served as a blank control. Two potted taro plants were used in each group, with three healthy leaves selected from each pot. After the leaves dried, 6-day-old actively growing *Phytophthora taroica* cakes with a diameter of 3 mm were inoculated onto the leaves. Regular moisturizing treatments were performed, and the leaf condition was observed. The taro potted plants were cultured in a natural environment at 26–36℃.

[0087] Six days after inoculation with *Phytophthora laurentii*, taro leaves in the LB liquid medium treatment group showed significant disease, while taro leaves treated with *Pseudomonas aeruginosa* CWD-D bacterial suspension and fermentation broth did not show disease. Figure 7 The infection of Phytophthora taroii on taro leaves treated with Pseudomonas aeruginosa CWD-D bacterial suspension and fermentation broth was completely inhibited, with an inhibition rate of 100%. This indicates that both Pseudomonas aeruginosa CWD-D bacterial suspension and fermentation broth have significant preventive effects against taro blight.

[0088] Example 5: Inhibitory effect of Pseudomonas aeruginosa CWD-D on Phytophthora lichei and Phytophthora capsici.

[0089] The bacterial concentration of Pseudomonas aeruginosa CWD-D was adjusted to OD. 600 =0.8.

[0090] Six-day-old colony plates were selected. Three mycelial discs of *Phytophthora tarota*, *Phytophthora litchifolia*, and *Phytophthora capsici* with a diameter of 3 mm at the growth edge were inoculated into the center of carrot agar plates. Two μL of *Pseudomonas aeruginosa* CWD-D suspension was inoculated at three points 2.5 cm away from each mycelial disc. LB broth was used as the control group, and each group had three replicates. The plates were incubated at 28℃. On day 3, the width of the inhibition zone was measured, and the edge of the inhibition zone and the hyphal morphology of *Phytophthora tarota* were observed. The results are as follows: Figure 8 As shown.

[0091] The results showed that, in addition to being effective against *Phytophthora tarota*, *Pseudomonas aeruginosa* CWD-D was also effective against *Phytophthora litchifolia*. Peronophythora litchi ) and Phytophthora capsici ( Phytophthora capsici It has a strong inhibitory effect and has the potential to be used as a broad-spectrum antibacterial agent.

[0092] Comparative Example 1: Comparison of the antibacterial effects of Pseudomonas aeruginosa PAO1 suspension

[0093] The bacterial concentration of Pseudomonas aeruginosa PAO1 was adjusted to OD. 600 =0.8.

[0094] Six-day-old colony plates were selected. Mycelial cakes of *Phytophthora tarota*, *Phytophthora litchifolia*, and *Phytophthora capsici* with a diameter of 3 mm at the growth edge were inoculated into the center of carrot agar plates. Two μL of *Pseudomonas aeruginosa* PAO1 suspension was inoculated at three points 2.5 cm away from each mycelial cake. LB broth served as the control group, and each group had three replicates. The plates were incubated at 28℃. On day 3, the width of the inhibition zone was measured, and the edge of the inhibition zone and the hyphal morphology of *Phytophthora tarota* were observed. Results are shown below. Figure 8 As shown.

[0095] The results showed that Pseudomonas aeruginosa CWD-D had a significantly better inhibitory effect on Phytophthora taro, Phytophthora litchii, and Phytophthora capsici than the Pseudomonas aeruginosa model strain PAO1, indicating that it is more effective in controlling plant diseases caused by these pathogens.

[0096] In summary, the *Pseudomonas aeruginosa* CWD-D and its bacterial suspension and fermentation broth of the present invention have inhibitory effects on *Phytophthora tarota*, *Phytophthora litchii*, and *Phytophthora capsici*, and have excellent control effects on plant diseases caused by these pathogens, with a very broad application prospect.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa CWD-D strain, characterized in that, The CWD-D strain was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on July 31, 2023, with accession number CCTCC No: M 20231386.

2. A biocontrol agent, characterized in that, Contains the CWD-D strain of claim 1, or its bacterial suspension or fermentation broth.

3. The biocontrol agent according to claim 2, characterized in that, The density of the bacterial suspension is: OD600 = 0.7~0.

8.

4. The biocontrol agent according to claim 2, characterized in that, The fermentation broth is the secretion obtained after culturing a 1% (v / v) bacterial suspension for 36-48 h.

5. The use of the CWD-D strain of claim 1 or any of the biocontrol agents of claims 2-4 in inhibiting pathogens or preparing pathogen-inhibiting products, characterized in that, The pathogens are any one or more of Phytophthora taroides, Phytophthora litchiides, and Phytophthora capsicum.

6. The application of the CWD-D strain of claim 1 or the biocontrol agent of any one of claims 2-4 in the control of plant diseases caused by pathogens, characterized in that, The pathogens are any one or more of Phytophthora taroides, Phytophthora litchiides, and Phytophthora capsicum.

7. The use of the CWD-D strain of claim 1 or the biocontrol agent of any one of claims 2-4 in the preparation of products for controlling plant diseases caused by pathogens, characterized in that, The pathogens are any one or more of Phytophthora taroides, Phytophthora litchiides, and Phytophthora capsicum.

8. The application according to claim 6 or 7, characterized in that, The plant diseases mentioned include taro blight, lychee downy mildew, pepper blight, and tomato downy mildew.

9. A method for preventing and controlling plant diseases, characterized in that, The biocontrol agent according to any one of claims 2-4 is evenly sprayed onto the plant surface; the plant diseases include taro blight, lychee downy mildew, pepper blight, and tomato downy mildew.

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