Endophytic Pseudomonas gillieri and application thereof

Abstract

The invention provides an endophytic pseudomonas kirscheri and an application of the endophytic pseudomonas kirscheri. The pseudomonas is endophytic pseudomonas kilonensis Pk-5, and the preservation number of the pseudomonas kilonensis Pk-5 is CGMCC (China General Microbiological Culture Collection Center) No. 32570. The strain can improve the biomass of plants under normal and sick conditions by inhibiting the pathogenic effect of plasmodiophora brassicae; after plants are inoculated with plasmodiophora brassicae, the pseudomonas Pk-5 can obviously promote the growth of the plants.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application and isolation method of an endophytic *Pseudomonas kilum*, and more specifically to the isolation of a novel endophytic *Pseudomonas kilum* from plant rhizosphere. Pseudomonas kilonensis The Pk-5 strain can inhibit infection by *Plomboma brassicae* and increase plant biomass. Background Technology

[0002] Clubroot disease is caused by *Platycota brassicae*, a member of the Oomycetes. Plasmodiophora Brassicae Clubroot is a soil-borne fungal disease caused by *Cladosporium wilt* (woron), which severely damages the roots of cruciferous plants and is a devastating soil-borne plant disease worldwide. Due to its high infectivity, rapid spread, multiple transmission routes, and difficulty in control, clubroot has spread rapidly in my country, causing significant impact on cruciferous crop production in almost all provinces. (Note: The last sentence about bok choy appears to be unrelated and likely refers to a different topic.) Brassica campestris *Sp. chinensis* L. (Brassica oleracea genus of the Brassicaceae family), also known as Chinese cabbage, has had its production severely restricted in my country due to the spread of clubroot disease.

[0003] *Platycotyle brassicae* is a strictly obligate parasite. Its life cycle is complex and can be broadly divided into two stages: the first stage is root hair infection, where dormant spores release primary zoospores that penetrate cell walls to reach the surface of root hairs; the second stage involves the formation of several secondary zoospores within the sporangia, which penetrate the root cortex and develop within the cortical cells for several weeks, leading to abnormal root swelling. The formation of root nodules hinders the upward transport of water and nutrients, causing the above-ground parts to wither and develop stuntedly, resulting in a decrease in yield and quality.

[0004] Currently, the control of clubroot disease in cruciferous plants mainly relies on crop rotation and treatments such as lime application, soil sun exposure, and fungicide application to reduce the spread of pathogens and lower pathogen concentrations in the soil. However, none of these methods can completely eliminate the clubroot pathogen. Selecting resistant varieties is the most economical and effective method, but resistance may be affected by changes in the physiological races of the pathogen. Various chemical agents have varying effects on clubroot control, and their high residue levels and toxicity prevent their widespread use in the Shanghai area.

[0005] Because the pathogen causing clubroot in cruciferous vegetables can survive for a long time in the soil, it is desirable to find a microorganism that can antagonize the germination and infection of the pathogen. Controlling clubroot disease through beneficial endophytic or rhizosphere microorganisms is a reasonable, economical, and environmentally friendly method. Numerous studies have demonstrated the potential antagonistic ability of plant-associated microorganisms to pathogens; these microorganisms can promote plant growth and improve soil ecosystems. Summary of the Invention

[0006] This invention addresses the aforementioned shortcomings of existing clubroot pathogen control technologies by providing an endophytic Pseudomonas kilum and its applications.

[0007] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a Pseudomonas bacterium, wherein the Pseudomonas bacterium is *Pseudomonas endophyticus*. Pseudomonas kilonensis Pk-5, with accession number CGMCC No. 32570.

[0008] Secondly, the present invention provides the use of the above-mentioned Pseudomonas as a biocontrol agent for plants.

[0009] Preferably, the biological control agent is used to control clubroot disease in plants.

[0010] Furthermore, the plant clubroot disease is caused by the fungus *Cladosporium brassicum*. Plasmodiophora brassicae Woronin cause.

[0011] Preferably, the plant is a cruciferous vegetable.

[0012] Thirdly, the present invention provides the use of the above-mentioned Pseudomonas bacteria in promoting plant growth.

[0013] Preferably, the growth promotion is manifested in increasing plant biomass, especially the biomass of plants infected with clubroot fungus.

[0014] Preferably, the biomass includes at least one of root fresh weight, aboveground dry weight, aboveground fresh weight, number of leaves, and leaf area.

[0015] Fourthly, a method for promoting plant growth includes applying Pseudomonas bacteria as described above to the plants. Pseudomonas kilonensis Pk-5 Preferably, the application rate is 1.00 × 10⁻⁶. 5 CFU / mL ~1.00×10 7 CFU / mL.

[0016] Fifthly, a method for promoting plant growth includes applying the aforementioned Pseudomonas bacteria to the plants. Pseudomonas kilonensis Pk-5 And clubroot pathogen.

[0017] The dosage of the Pseudomonas was 1.00 × 10⁻⁶. 5 CFU / mL ~1.00×10 7 CFU / mL; The dosage of the clubroot pathogen used was 1.00 × 10⁻⁶. 6 •mL -1 ~1.00×10 8 •mL -1 .

[0018] The bacterial suspension of the clubroot pathogen is prepared through the following steps: (1) Select swollen root pieces from the infected Chinese cabbage roots as material for pathogen spore extraction; (2) Add the selected root tubers to sterile water, break the cell wall and homogenize, then filter and collect the filtrate; (3) Centrifuge the filtrate, collect the precipitate, dilute with deionized water, and obtain a suspension of dormant spores.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The Pseudomonas Pk-5 strain of the present invention exhibits a high inhibitory effect on the germination of dormant spores and can be selected as a potential biocontrol agent; 2. The Pseudomonas Pk-5 strain of the present invention can improve plant biomass under both normal and diseased conditions by inhibiting the pathogenicity of clubroot pathogens. 3. This invention is the first to discover that the substance produced by the antagonistic effect between Pseudomonas Pk-5 and Plasmodium falciparum significantly promotes plant growth. Attached Figure Description

[0020] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The morphological characteristics of the 15 bacterial strains in Example 1 of this invention; Figure 2 The inhibitory effect of 15 bacterial strains in Example 1 of this invention on dormant sporangia of clubroot disease in Brassica rapa; Figure 3 The inhibitory effect of Pseudomonas Pk-5 on clubroot pathogens; Figure 4 The effect of Pseudomonas Pk-5 treatment on the fresh weight of Chinese cabbage roots; Figure 5 The effect of Pseudomonas Pk-5 treatment on the morphology of Chinese cabbage; Figure 6 The effects of Pseudomonas Pk-5 treatment on plant leaf area, aboveground fresh weight, number of leaves, and aboveground dry weight. Detailed Implementation

[0021] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0022] In this invention: Preservation instructions for PK-5 strain: Classification and nomenclature: Pseudomonas kilgaris Strain name: PK-5 Latin name: Pseudomonas kilonensis Accession number: CGMCC No. 32570 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Date of preservation: November 11, 2024.

[0023] Example 1 1. Strains Isolation This invention isolated 34 bacterial strains from the rhizosphere soil of healthy Chinese cabbages in a green production base in Xinyecun Village, Zhuangxing Town, Fengxian District, Shanghai. 1g of the treated powder was added to 9ml of sterile distilled water, shaken thoroughly, and then diluted sequentially with sterile water to 10⁻¹⁰ and 10⁻¹⁰ concentrations. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Then, bacterial purification was performed. Each 60 μL dilution was placed on LB medium and incubated at 28 degrees Celsius for one week. Fifteen bacterial strains were selected, including Bm1, Bm2, Bv1, Bc1, Bc2, Bc3, Bs1, Bs2, Bs3, Bs4, Pk1, Pk2, Pk3, Pk4, and Pk5. Figure 1 Morphological characteristics of 15 bacterial strains.

[0024] The inhibitory effects of the above 15 strains on the germination rate and viability of dormant sporangia of *Cladosporium cladosporium* were tested respectively, and the test methods are as follows: Preparation of bacterial culture for each strain: Each strain was cultured separately in LB liquid medium at 28℃ and 180 rpm using a shaker. After 24 hours, bacterial samples were collected and diluted to 1×10⁻⁶. 7 cfu.mL -1 .

[0025] Preparation of clubroot spore suspension: Clubroot spores were extracted from root samples of infected bok choy (taken from the Xinyecun Green Production Base, Fengxian District, Shanghai). Significantly swollen root pieces were selected, added to sterile water, and homogenized using a high-speed blender. The mixture was then filtered through 25µm gauze. The filtrate was centrifuged at 5000 rpm for 10 minutes, the supernatant was removed, and the centrifugation was repeated four times to obtain a precipitate. The precipitate was diluted with deionized water to a concentration of 1×10⁻⁶. -7 •mL -1 To obtain a suspension of dormant spores at a concentration of [specific concentration], [the concentration was determined].

[0026] In sterile test tubes, 1 mL of the above 15 bacterial cultures were mixed with 1 mL of *Cladosporium brassicae* spore suspension and incubated at 25°C for 2 days to detect the viability of dormant spores. For each treatment, an equal volume of phosphate buffer (pH 7.4) was added and mixed with 2 mL of Evans blue solution. The mixture was then cooled for 30 minutes and rinsed four times with sterile water. The viability of dormant spores was detected using a BS203 microscope. Inactive spores appeared dark blue. Sterile LB medium was used as a control. Three replicates were performed.

[0027] The results are as follows Figure 2 Pk-5 showed the highest inhibitory effect on the germination of dormant sporangia of clubroot disease in Brassica rapa (e.g., Figure 2 ).

[0028] Figure 2 Different lowercase letters (a, b, bc, etc.) indicate significant differences at the p-value < 0.05 level according to Duncan's new multiple range test. Treatment groups labeled with the same letter showed no statistically significant difference in inhibition rate; different letters indicated significant differences.

[0029] For example: The inhibition rates of the Bv1 group (marked "a") and the Bc1 group (marked "a") were not significantly different, indicating that the two groups had similar inhibitory effects on clubroot pathogens. The letters of the Bs2 group (marked "f") are different from those of the Bv1 group (marked "a") and the Bc1 group (marked "a"), indicating that its antibacterial rate is significantly lower than these groups, and there is a significant difference in the inhibitory effect.

[0030] Therefore, Pk-5 can be selected as a potential biocontrol agent.

[0031] 2. Molecular biological identification The Pk-5 strain was identified by 16S rRNA sequencing (results shown in SEQ ID NO.1), and was identified as *Pseudomonas endophyticus*. It showed approximately 99% similarity to *Pseudomonas endophyticus* (accession number: MN826554.1).

[0032] The specific sequence of 16S rRNA is as follows: GGAAGTACGACTGACCTGAGGTGCGAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGGGGACGGTTACCACGGTGTGATTCATGACTGGGGTGAATCGAAAGGGGGGGAC。

[0033] Example 2 In this example, the inhibitory effect of the bacterial isolate Pk-5 (1×10 6 CFU / mL) against Plasmodiophora brassicae was detected under greenhouse conditions; the specific steps are as follows: Preparation method of dormant spore suspension: Clubroot pathogen spores were extracted from root samples of infected bok choy (taken from the Xinyecun Green Production Base, Fengxian District, Shanghai). Significantly swollen root pieces were selected, added to sterile water, and homogenized using a high-speed blender. The mixture was then filtered through 25µm gauze. The filtrate was centrifuged at 5000 rpm for 10 minutes, the supernatant was removed, and the centrifugation was repeated four times to obtain a precipitate. The precipitate was diluted with deionized water to a concentration of 1×10⁻⁶. -7 •mL -1 To obtain a dormant spore suspension at a concentration of [specific concentration], store at 4°C for later use.

[0034] Preparation of bacterial culture medium: Pk-5 strain was cultured in LB liquid medium at 28℃ and 180 rpm using a shaker. After 24 hours, bacterial samples were collected and diluted to 1×10⁻⁶. 7 cfu.mL -1 .

[0035] Choose healthy, uniformly sized bok choy. Brassica campestris sp. chinensis L Seeds were disinfected with 1% sodium hypochlorite for 5 minutes, then rinsed three times with sterile water. The bok choy seeds were sown in plastic trays containing 3 liters of pressurized sterilized perlite and 800 ml of MS medium solution. Seedlings were grown in a growth chamber at a daytime temperature of 28°C and a nighttime temperature of 15°C, a light / dark cycle of 16 hours / 8 hours, and a relative humidity of 60%. After 7 days, the seeds were sown into 7.5 cm × 7.5 cm pots on the plastic trays, with 30 pots per tray. Cultivation was carried out using a method where the seedlings received 600 ml of 1×10⁻⁶ MS medium solution. 7 •m L-1 The experimental group (PBPK) received a suspension of dormant spores and 300 mL of bacterial culture medium, while the control group (PB) received 600 mL of dormant spore suspension and 300 mL of sterile water. On days 7 and 14 after the initial infection, seedlings in the experimental group were again inoculated with 300 mL of bacterial suspension, while the control group received the same volume of deionized water. Three separate experiments were conducted. Ten pots were used for each treatment.

[0036] Compared to plants infected only with clubroot fungus (control group), bacterial isolate Pk-5 was an effective biocontrol agent against clubroot fungus and effectively suppressed clubroot disease (e.g., by reducing root nodule formation). Figure 3 ).

[0037] Example 3 1. Preparation of clubroot spore suspension: Take the swollen root pieces of infected Chinese cabbage from the green production base of Xinyecun Village, Fengxian District, Shanghai, add sterile water and homogenize with a high-speed blender. Filter through 25μm gauze, centrifuge the filtrate at 5000rpm for 10 minutes, discard the supernatant, repeat the centrifugation 4 times, collect the precipitate, add sterile water to the precipitate, and form a uniform bacterial suspension by mechanical dispersion (such as a high-speed blender). Filter through 25μm gauze again to remove impurities, and obtain the clubroot spore suspension.

[0038] 2. Test materials: Healthy Chinese cabbage seedlings (cultured for 7 days).

[0039] 3. Group Design: Pk-5 strain: Three concentration groups were set up, namely T1 group (1.00×10⁻⁶). 6 CFU / mL), T2 group (1.00×10 5 CFU / mL), T3 group (1.00×10 4 (CFU / mL) Positive control (T4 group): Bacillus cereus (CGMCC No. 21141; 1.00×10⁻⁶) 6 (CFU / mL) Blank control (T5 group): Equal volume of sterile water.

[0040] 4. Inoculation treatment: All groups were inoculated with 5 mL of clubroot spore suspension; The experimental groups (T1-T3) were simultaneously inoculated with the corresponding concentration of Pk-5 bacterial suspension, the T4 group was inoculated with Bacillus cereus (CGMCC No. 21141) bacterial suspension, and the T5 group was inoculated with sterile water; each group consisted of 10 pots, and the experiment was repeated 3 times independently.

[0041] Samples were taken on day 21 (i.e., day 28) after vaccination: Figure 4 The effect of Pseudomonas Pk-5 treatment on the fresh weight of Chinese cabbage roots; Figure 5 The effect of Pseudomonas Pk-5 treatment on the morphology of Chinese cabbage; Figure 6 The effects of Pseudomonas Pk-5 treatment on plant leaf area, aboveground fresh weight, number of leaves, and aboveground dry weight.

[0042] In terms of the number of blades ( Figure 6 T1 and T3 both have 12.75 pieces, T2 has 12 pieces, while T4 and T5 have lower values ​​of 10.75 pieces and 10 pieces, respectively.

[0043] The results of the stem diameter showed ( Figure 6The stem diameter of T1 was the highest at 6.69 mm, which was significantly different from the other treatment groups, while the stem diameters of T3 and T4 were relatively low, indicating that the inoculation concentration had a certain impact on stem development.

[0044] In terms of fresh weight ( Figure 6 The fresh weight of T1 reached 61.31 g, which was significantly higher than that of other groups, especially T5 at 22.99 g, showing that the treatment with clubroot fungus severely inhibited plant growth.

[0045] The results for aboveground fresh weight and dry weight were similar to those for fresh weight. Figure 6 The aboveground fresh weight of T1 was 51.08 g, which was significantly better than other treatments, especially T5 at 24.55 g, further confirming the adverse effects of clubroot disease on plant growth.

[0046] In terms of root fresh weight ( Figure 6 The fresh root weight of T1 was 8.58 g, which was significantly higher than that of other treatments, especially T5 at 4.81 g, demonstrating the effectiveness of strain Pk5.

[0047] Overall, inoculation with the Pk5 strain, especially at 1.00 × 10⁻⁶, was effective. 6 At a concentration of CFU / mL, the biomass of Chinese cabbage was significantly increased, demonstrating its potential in plant growth and clubroot disease control. It can improve plant biomass under diseased conditions. The fresh and dry weight of plants co-inoculated with strain Pk5 and clubroot pathogen increased by 108.06% and 116.37% compared to plants inoculated only with clubroot pathogen. As a positive control, the fresh and dry weight of plants co-inoculated with strain Pk5 and clubroot pathogen were 22.84% and 28.91% higher than those co-inoculated with Bacillus cereus (CGMCC No. 21141) and clubroot pathogen. Furthermore, the results indicate that strain Pk5 is more effective than Bacillus cereus (CGMCC No. 21141) in inhibiting clubroot disease.

[0048] It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in these embodiments can be arbitrarily combined with each other.

Claims

1. A type of Pseudomonas, characterized in that, The Pseudomonas species is *Pseudomonas endophyta*. Pseudomonas kilonensis Pk-5 Its accession number is CGMCC No. 32570.

2. The use of the Pseudomonas as described in claim 1 as a plant biocontrol agent.

3. The use as described in claim 2, characterized in that, The biocontrol agent is used to control clubroot disease in plants.

4. The use as described in claim 3, characterized in that, The clubroot disease is caused by the fungus *Cladophora brassicae*. Plasmodiophora brassicae Woronin cause.

5. The use as described in claim 2, characterized in that, The plant in question is a cruciferous vegetable.

6. The use of the Pseudomonas as described in claim 1 in promoting plant growth.

7. The use as described in claim 6, characterized in that, The growth-promoting effect is manifested in increasing biomass; preferably, the biomass includes at least one of root fresh weight, aboveground dry weight, aboveground fresh weight, number of leaves, and leaf area.

8. The use as described in claim 6, characterized in that, The plant in question is a cruciferous vegetable infected with clubroot fungus.

9. A method for promoting plant growth, characterized in that, This includes applying the Pseudomonas aeruginosa as described in claim 1 to plants; preferably, the application amount is 1.00 × 10⁻⁶. 5 CFU / mL ~1.00×10 7 CFU / mL.

10. A method for promoting plant growth, characterized in that, This includes applying the Pseudomonas and clubroot pathogens as described in claim 1 to plants.

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