Pseudomonas protegens gdp-1 and application thereof

Abstract

The application discloses a Pseudomonas protegens strain GDP-1 and application thereof. The Pseudomonas protegens strain GDP-1 is isolated from soil, and has been preserved in the Guangdong Microbial Culture Collection Center on July 9, 2025, with a preservation number of GDMCC NO.66656. It is found through experiments that the strain can inhibit the growth of various different Pythiums, and has good tolerance to common pesticides and heavy metal ions, and has a good application prospect in the field of biological control.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a strain of defensive Pseudomonas GDP-1 and its applications. Background Technology

[0002] Phytophthora is a highly destructive plant pathogen, classified as an oomycete. Although long considered a fungus, it is more distantly related to modern fungi and closer to algae, making it less sensitive to many traditional fungicides and more difficult to control. Phytophthora can infect all parts of a plant, including roots, stems, leaves, and fruits, causing disease. Its most typical mode of damage is causing plant rot and damping-off. For example, the pathogen that causes late blight in potatoes can cause potato leaves and stems to turn black and wilt within a short time, as if scalded by boiling water, leading to the rotting of underground tubers. Similarly, blight in peppers and tomatoes causes brown sunken lesions (stem rot) on the stems, leading to rapid wilting and death of the plant; while in citrus, Phytophthora damages the root collar and root system, causing foot rot or root rot, ultimately leading to the decline and death of the entire tree. This direct tissue damage severely weakens plant growth and yield, and can even cause crop failure.

[0003] Phytophthora primarily reproduces and spreads through the production of zoospores. These zoospores are flagellated and can move in water droplets or moist soil. High humidity environments are particularly conducive to the production of large numbers of sporangia by Phytophthora, which then spread rapidly in the field through wind, rain, flowing water, and agricultural operations. Furthermore, Phytophthora is extremely resilient; it can form thick-walled oospores in infected plant debris, soil, or diseased seed potatoes. These dormant spores can withstand adverse environments and survive for several years, becoming the primary source of infection for the next growing season. This "environment-driven" epidemic pattern makes the disease particularly rampant in rainy, humid seasons or regions. Control relies heavily on weather forecasts and precise preventative pesticide application; once an outbreak occurs, it is often too late to take action. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain for defending against Pseudomonas GDP-1.

[0005] Another object of the present invention is to provide the above-mentioned application of defense against Pseudomonas GDP-1.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A strain of defensive Pseudomonas protegens GDP-1 was isolated from soil and named Pseudomonas protegensGDP-1.

[0008] The aforementioned defensive Pseudomonas GDP-1, with accession number GDMCC NO.66656, was deposited on July 9, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The nucleotide sequence of the 16S rDNA for defense against Pseudomonas GDP-1 is shown in SEQ ID NO.1.

[0010] The aforementioned Pseudomonas GDP-1 has the function of inhibiting the growth of Phytophthora.

[0011] The aforementioned Pseudomonas GDP-1 has the function of promoting plant growth.

[0012] The aforementioned *Phytophthora* includes at least one of *Phytophthora infestans*, *Phytophthora capsici*, *Phytophthora parasitica*, and *Pythium ultimum*.

[0013] The aforementioned protective Pseudomonas GDP-1 is resistant to pesticides and / or heavy metals.

[0014] The pesticides mentioned include at least one of chlorothalonil and / or pyraclostrobin.

[0015] The heavy metals mentioned include at least one of copper and / or manganese.

[0016] The application of the aforementioned defense against Pseudomonas GDP-1 in inhibiting the growth of Phytophthora.

[0017] The application of the aforementioned anti-pseudomonas GDP-1 in the preparation of antifungal biocontrol agents.

[0018] The aforementioned *Phytophthora* includes at least one of *Phytophthora infestans*, *Phytophthora capsici*, *Phytophthora parasitica*, and *Pythium ultimum*.

[0019] The aforementioned defense against Pseudomonas GDP-1 can promote plant growth.

[0020] The application of the aforementioned protective Pseudomonas GDP-1 in the preparation of plant growth-promoting agents.

[0021] An antifungal biological control agent, comprising the aforementioned defense against Pseudomonas GDP-1.

[0022] A plant growth promoter comprising the aforementioned anti-pseudomonas GDP-1.

[0023] The plant in question is Nicotiana benthamiana.

[0024] The present invention has the following advantages and effects compared with the prior art:

[0025] This invention isolated a strain of *Pseudomonas protegens* GDP-1 from soil, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 9, 2025, with accession number GDMCC NO. 66656. Experimental verification showed that this bacterium can inhibit the growth of various different *Phytophthora* species and exhibits good tolerance to common pesticides and heavy metal ions, demonstrating promising application prospects in the field of biological control. Attached Figure Description

[0026] Figure 1 It is the colony morphology of Pseudomonas GDP-1 on plate culture medium.

[0027] Figure 2 This is the experimental result of the plate antibacterial experiment in Example 2.

[0028] Figure 3 This is the experimental result of the *Tobacco Benedict* inoculation test in Example 2.

[0029] Figure 4 These are the experimental results of the potato inoculation experiment in Example 2.

[0030] Figure 5 This is the experimental result of the chlorothalonil resistance test in Example 3.

[0031] Figure 6 This is the experimental result of the azoxystrobin resistance test in Example 3.

[0032] Figure 7 This is the experimental result of the copper ion tolerance test in Example 3.

[0033] Figure 8 This is the experimental result of the manganese ion tolerance test in Example 3.

[0034] Figure 9 These are the experimental results of the plant growth-promoting effect experiment in Example 4. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0036] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0037] The tested pathogenic strains *Phytophthora infestans*, *Phytophthora capsici*, *Phytophthora parasitica*, and *Pythium ultimum* were preserved by the Potato Research Laboratory of the Crop Research Institute, Guangdong Academy of Agricultural Sciences. *Phytophthora infestans* was preserved on rye solid medium at 13℃, while *Phytophthora capsici*, *Phytophthora parasitica*, and *Pythium ultimum* were preserved on 10% V8 solid medium at 13℃.

[0038] Example 1 Sequencing and Identification

[0039] In preliminary experiments, three microorganisms with potential biocontrol activity were screened from soil samples. After DNA extraction and 16S rDNA sequence amplification, the sequences were sequenced and compared with existing strains in the database. Two *Pseudomonas* strains and one *Bacillus* strain were identified. Based on the species names of their closest existing strains, they were named *Pseudomonas protegens GDP-1* (PpGDP-1, its 16S rDNA is shown in SEQ ID NO.1), *Pseudomonas protegens 1-32* (Pp1-32, its 16S rDNA is shown in SEQ ID NO.2), and *Bacillus velezensis* 1-28* (Bv1-28, its 16S rDNA is shown in SEQ ID NO.3). Phylogenetic analysis revealed that these strains are closely related. The colony morphology of the biocontrol-promoting *Pseudomonas protegens GDP-1* on agar plates is shown in the figure below. Figure 1 As shown.

[0040] Pseudomonas protegens GDP-1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 9, 2025, with accession number GDMCC NO.66656. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0041] Example 2: Verification of the effectiveness of biological control

[0042] 2.1 Activation and culture of the strain

[0043] The frozen strain was inoculated into LB liquid medium and cultured with shaking at 30–37°C and 180 rpm for 12–16 hours until the logarithmic growth phase (OD200). 600 = 1.0), used for subsequent experiments.

[0044] 2.2 Plate Antibacterial Test

[0045] Inoculate the center of each plate containing the corresponding type of solid culture medium with the pathogens: *Phytophthora virulence*, *Phytophthora capsici*, *Phytophthora parasitica*, and *Pythium tertii*. Then, use sterile forceps to pick up filter paper discs and immerse them in the culture until the OD reaches the target concentration. 600 Prepare a culture medium of the test bacteria (PpGDP-1, Pp1-32, or Bv1-28) with a concentration of 1.0, ensuring complete saturation. Smoothly attach the soaked filter paper discs to the surface of the bacterial plate, gently pressing them with the tip of tweezers to ensure full contact between the filter paper discs and the culture medium surface, without air bubbles. A control group of filter paper discs soaked only in sterile culture medium is also included. The antibacterial effect is observed based on the growth time of different pathogens: 7 days (Phytophthora pathogenica), 2 days (Phytophthora capsici, Phytophthora parasitica), and 1 day (Pythium oxysporum). Photos are taken and the antibacterial effect is statistically analyzed.

[0046] Experimental results are as follows Figure 2 As shown, PpGDP-1 exhibits significantly stronger antibacterial activity against several Phytophthora species compared to Pp1-32 or Bv1-28, producing distinct inhibition zones with larger diameters, demonstrating that PpGDP-1 possesses broad-spectrum antibacterial activity against a variety of Phytophthora species.

[0047] 2.3 Inoculation test of Tobacco Benedict

[0048] 2.3.1 Preparation of the tested Nicotiana benthamiana seedlings

[0049] Nicotiana benthamiana was sown in plastic pots (d=10cm) filled with vermiculite (2-4mm) and placed in a greenhouse at 25℃ with 14 hours of light (strong light) / 10 hours of darkness. After 7 days of growth, when two true leaves had emerged, the plants were transplanted into a 5:1 mixture of vermiculite, black soil, and potting soil and grown for 30 days. Leaves from the same 3rd, 4th, and 5th leaf positions were used for inoculation experiments.

[0050] 2.3.2 Inoculation and Observation of Tobacco Benedict

[0051] Select tobacco leaves of suitable growth age, and use the detached leaf inoculation method, employing OD... 600The culture solution of the test bacteria with a concentration of 1.0 was sprayed onto the leaf surface. One hour later, Phytophthora virulence, Phytophthora capsici, Phytophthora parasitica, and Pythium oxysporum were inoculated separately. After inoculation, the induction effect was observed after standing for a period of time, specifically 5 days (Pytophthora virulence), 2 days (Pytophthora capsici and Phytophthora parasitica), and 1 day (Pythium oxysporum). The results were then photographed and recorded, and the diameter of the lesions was counted.

[0052] 2.3.3 Experimental Results

[0053] Experimental results are as follows Figure 3 As shown, the results in the experiment on tobacco leaves were consistent with those in the plate culture medium. Only PpGDP-1 showed significant antibacterial ability, while the other two control groups did not show any resistance to Phytophthora.

[0054] 2.4 Potato Inoculation Trial

[0055] 2.4.1 Preparation of potato seedlings for testing

[0056] Potato tubers were planted in 10cm diameter plastic pots and placed in a greenhouse with 14 hours of light (strong light) and 10 hours of darkness at a temperature of 20℃. After 1.5-2 months of growth, they could be used for inoculation experiments.

[0057] 2.4.2 Potato Inoculation Experiment

[0058] Select potato leaves of suitable growth age and use the detached leaf inoculation method, employing OD... 600 The culture solution of the test bacteria with a concentration of 1.0 was sprayed onto the leaf surface. One hour later, the pathogenic fungus Phytophthora and Pythium oxysporum were inoculated separately to observe their resistance effects. Specifically, the induction and resistance effects were observed for 5 days (Pytophthora) and 2 days (Pythium oxysporum). The results were then photographed and recorded, and the diameter of the lesions was counted.

[0059] 2.4.3 Experimental Results

[0060] Experimental results are as follows Figure 4 As shown, by calculating the lesion area, the conclusion is the same as that of the previous experiment: PpGDP-1 has a more obvious antibacterial effect and can achieve similar effects in different species, proving that this bacterium has a wide range of applications as a biocontrol agent.

[0061] 2.5 Discussion

[0062] In summary, the experimental results show that Pseudomonas protegens GDP-1 can achieve antibacterial effects against various Phytophthora species on both plates and experimental plants, while another strain in the same genus, Pseudomonas protegens 1-32, cannot achieve the same effect. Furthermore, this strain does not have any adverse effects on the growth of the plants themselves, proving that this strain can be widely used in the field of antibacterial treatment of related pathogens and has the potential to prepare biocontrol agents.

[0063] Example 3 Tolerance Test

[0064] 3.1 Chlorothalonil resistance test

[0065] When preparing LB solid medium, chlorothalonil was added to prepare medium containing eight gradient concentrations of 0, 200, 400, 600, 800, 1000, 1200 and 1600 μg / mL. The medium was poured into plates, sterilized, and cooled. The culture of the test bacteria (cultured according to the method in 2.1) was inoculated into the center of the medium. After culturing at 37°C for 24 h, the experimental results were observed and photographed.

[0066] Experimental results are as follows Figure 5 As shown, the growth rate of both decreased with increasing chlorothalonil concentration. In plate culture, Pseudomonas protegens 1-32 could not grow at concentrations above 600 μg / mL, while Pseudomonas protegens GDP-1 maintained good growth at a concentration of 1600 μg / mL of chlorothalonil, proving that the bacteria's tolerance to chlorothalonil was significantly better than that of Pseudomonas protegens 1-32.

[0067] 3.2 Azoxystrobin Resistance Test

[0068] Following the method described in 3.1, prepare LB solid plates containing azoxystrobin, inoculate with the bacterial suspension of the test bacteria, and record the experimental results after culturing under the same conditions for 24 hours.

[0069] Experimental results are as follows Figure 6 As shown, similar to the experimental results of chlorothalonil, Pseudomonas protegens 1-32 could not grow or form colonies when the concentration of azoxystrobin exceeded 600 μg / mL, while Pseudomonas protegens GDP-1 maintained normal growth up to a concentration of 1600 μg / mL, proving that this strain has a certain tolerance to common pesticides and is significantly better than other strains.

[0070] 3.3 Copper ion tolerance test

[0071] Following the method described in 3.1, copper sulfate was added to the culture medium to prepare Cu solutions with three gradient concentrations: 0, 200, and 400 μg / mL. 2+ The LB solid plate medium was used, and the test bacterial culture was inoculated. After culturing under the same conditions for 24 hours, the experimental results were recorded.

[0072] Experimental results are as follows Figure 7 As shown, in plate culture medium, the growth rate of both decreased with increasing copper ion concentration. Pseudomonas protegens 1-32 could not grow at concentrations above 200 μg / mL, while Pseudomonas protegens GDP-1 could not grow until 400 μg / mL, proving that Pseudomonas protegens GDP-1 was more tolerant to copper ions than Pseudomonas protegens 1-32.

[0073] 3.4 Manganese ion tolerance test

[0074] Following the method described in 3.1, manganese sulfate was added to the culture medium to prepare eight gradient concentrations of Mn: 0, 200, 400, 600, 800, 1000, 1200, and 1600 μg / mL. 2+ The LB solid plate medium was used, and the test bacterial culture was inoculated. After culturing under the same conditions for 24 hours, the experimental results were recorded.

[0075] Experimental results are as follows Figure 8 As shown, the trend of manganese ion tolerance is similar to that of copper ions. As the concentration of metal ions increases, the growth ability of the strain decreases. However, both strains have better tolerance to manganese ions. Pseudomonas protegens GDP-1 can still grow and form colonies in a medium with 1600 μg / mL of manganese ions, while Pseudomonas protegens 1-32 cannot form visible colonies at concentrations above 800 μg / mL.

[0076] Example 4: Experiment on plant growth-promoting effects

[0077] 4.1 Experimental Methods

[0078] Nicotiana benthamiana was sown in plastic pots (d=10cm) filled with vermiculite (2-4mm) and placed in a greenhouse at 25℃ with 14 hours of light (strong light) / 10 hours of darkness. After 7 days, when two true leaves had emerged, the seedlings were transplanted into a 5:1 mixture of vermiculite, black soil, and potting soil to continue growing. Three days after transplanting, the seedling height was measured, and the seedlings were cultivated to OD (difference of growth). 600 Centrifuge Pseudomonas protegens GDP-1 broth at 1.0 CFU, and resuspend the cells in deionized water to a final concentration of 1 × 10⁻⁶ CFU. 7 After obtaining 10 mL of deionized water, the roots of tobacco seedlings were irrigated with the same amount of deionized water. The control group was irrigated with the same amount of deionized water. Each group was repeated 5 times. After 30 days of continued cultivation, the plant height was measured and the growth promotion rate was calculated.

[0079] 4.2 Experimental Results

[0080] The experimental results showed that after root irrigation with Pseudomonas protegens GDP-1 culture solution, the seedlings of Tobacco Benzovia grew faster than the control group. The average height of the experimental group was 25 cm, while the height of the control group was only 19 cm, with a growth promotion rate of 31.58%, proving that this strain also has a certain function of promoting plant growth.

[0081] 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 that defends against Pseudomonas GDP-1, characterized by: Name is Pseudomonas protegens GDP-1, with accession number GDMCC NO.66656, was deposited on July 9, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The defense against Pseudomonas GDP-1 according to claim 1, characterized in that: The aforementioned Pseudomonas GDP-1 has the function of inhibiting the growth of pathogens; The pathogenic bacteria mentioned are selected from Phytophthora infestans (Phytophthora infestans) Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ), Phytophthora parasiticum ( Phytophthora parasitica ), ultimate phytosis ( Pythium ultimum At least one of the following.

3. The defense against Pseudomonas GDP-1 according to claim 1, characterized in that: The aforementioned Pseudomonas GDP-1 is resistant to pesticides and heavy metals.

4. The application of the anti-Pseudomonas GDP-1 as described in any one of claims 1 to 3 in inhibiting the growth of pathogenic bacteria; The pathogenic bacteria mentioned are selected from Phytophthora infestans (Phytophthora infestans) Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ), Phytophthora parasiticum ( Phytophthora parasitica ), ultimate phytosis ( Pythium ultimum At least one of the following.

5. The use of the anti-pseudomonas GDP-1 according to any one of claims 1 to 3 in the preparation of anti-pathogenic biological control agents; The pathogenic bacteria mentioned are selected from Phytophthora infestans (Phytophthora infestans) Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ), Phytophthora parasiticum ( Phytophthora parasitica ), ultimate phytosis ( Pythium ultimum At least one of the following.

6. The application of the defensive Pseudomonas GDP-1 according to any one of claims 1 to 3 in the preparation of plant growth-promoting agents, characterized in that: The plant in question is Nicotiana benthamiana.

7. The application of the defense against Pseudomonas GDP-1 as described in any one of claims 1 to 3 in promoting plant growth, characterized in that: The plant in question is Nicotiana benthamiana.

8. A disease-resistant biological control agent, characterized in that: Includes the defense against Pseudomonas GDP-1 as described in any one of claims 1 to 3; The aforementioned disease resistance refers to resistance to pathogens; The pathogenic bacteria mentioned are selected from Phytophthora infestans (Phytophthora infestans) Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ), Phytophthora parasiticum ( Phytophthora parasitica ), ultimate phytosis ( Pythium ultimum At least one of the following.

9. A plant growth promoter, characterized in that... Includes the defense against Pseudomonas GDP-1 as described in any one of claims 1 to 3; The plant in question is Nicotiana benthamiana.

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