Pseudomonas chlororaphis and application thereof in plant disease control

Abstract

The invention belongs to the technical field of microorganisms, and relates to pseudomonas chlororaphis and application thereof in plant disease control. The invention provides pseudomonas chlororaphis, the pseudomonas chlororaphis is Pseudomonas chlororaphis, the pseudomonas chlororaphis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the pseudomonas chlororaphis is CGMCC No. 32517. The strain can inhibit the growth of various fungal pathogenic bacteria such as lycium barbarum root rot, schisandra chinensis root rot and alternaria alternata, has wide antibacterial ability, can induce the expression of defensive genes such as NPR1 and WRKY22 of plants, and improves the disease resistance of the plants. Pot experiments show that the strain can reduce the morbidity of the wolfberry root rot, and the relative control effect on the wolfberry root rot can reach 72.73%. The strain can be used as an effective biological control resource for plant diseases (especially lycium barbarum root rot).

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a species of Pseudomonas aeruginosa and its application in the prevention and control of plant diseases. Background Technology

[0002] my country's agricultural development is severely impacted by pests and diseases, and current control of these pests and diseases still relies primarily on chemical methods. The long-term use of chemical pesticides has led to a series of problems, including environmental pollution, increased pathogen resistance, and pesticide residues in agricultural products. Therefore, developing safe, efficient, and environmentally friendly control methods is of great significance. Microbial inoculants, as alternatives to chemical pesticides, have become a research hotspot in the field of plant protection in recent years. Although extensive research has been conducted both domestically and internationally, the availability of high-quality microbial strains for practical application remains relatively limited, necessitating the discovery of new, highly effective biological control strains.

[0003] Goji berries ( Lycium barbarum Goji berries are an important ecological and economic crop in Northwest my country, possessing both medicinal and edible value. The goji berry industry is also a significant pillar industry in Qinghai and Ningxia. However, with increasing planting years, continuous cropping and improper field management in the Qaidam Basin of Qinghai have led to persistently high levels of pathogens in the soil, resulting in widespread goji berry root rot, which severely restricts the healthy development of the goji berry industry. Fusarium spp . ) and Rhizoctonia solani ( Rhizoctonia spp . Soil-borne diseases caused by various pathogenic fungi, such as Bacillus subtilis, can lead to root rot and plant death through root wounds or direct invasion of vascular tissue. These diseases are characterized by their high latency and difficulty in control. Currently, chemical pesticides remain the primary method of control, but long-term use has resulted in increased pathogen resistance, soil degradation, and excessive pesticide residues, posing a threat to the ecological environment and food safety. Against this backdrop, biological control, as an environmentally friendly and sustainable management strategy, shows broad application prospects. Compared to chemical pesticides, biological control agents can work synergistically through multiple mechanisms: on the one hand, such as Bacillus subtilis... Bacillus Trichoderma () Trichoderma Antagonistic microorganisms, such as lycopene and chloramphenicol, can directly inhibit the growth of pathogens through competition for nutrients and space, and by secreting antibacterial substances (such as ituronidin and chloramphenicol). On the other hand, the chitinases and other hydrolytic enzymes they produce can degrade the cell walls of pathogens and induce systemic resistance (ISR) in plants, thus enhancing the plant's own immunity. Furthermore, some biocontrol bacteria can improve the rhizosphere microecological environment, promote plant growth, and achieve a dual effect of disease prevention and growth promotion.

[0004] Currently, research on the biological control of root rot in wolfberry has made some progress. For example, Aspergillus terreus (…) was isolated from the rhizosphere of healthy wolfberry plants… Aspergillus terreus ), Bacillus belesi ( Bacillus velezensis Trichoderma longifolia ( T. longibra-chiatum Strains such as [list of strains] have shown significant antagonistic activity against Fusarium. However, existing biocontrol agents generally suffer from problems such as a single mechanism of action and unstable field efficacy. There is still a shortage of multifunctional strains that possess broad-spectrum disease resistance, efficient colonization, and the ability to induce plant resistance. Therefore, it is urgent to screen for biocontrol strains with highly efficient antibacterial and disease-inducing properties to provide new solutions for the green control of wolfberry root rot and promote high-quality green agricultural development. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a *Pseudomonas aeruginosa* strain and its application in the control of plant diseases. The *Pseudomonas aeruginosa* strain of this invention was isolated from the soil surrounding the roots of *Lycium barbarum* plants at Nuomuhong Farm in Qinghai Province. This *Pseudomonas aeruginosa* strain exhibits good control effects against root rot in *Lycium barbarum*.

[0006] On the one hand, the present invention provides a *Pseudomonas aeruginosa* strain, the preservation information of which is as follows: Strain name: H2Q4; Category Naming: Pseudomonas chlororaphis ; The collection center received the document on November 6, 2024. Date of issuance of preservation certificate: November 18, 2024; Preservation institution: China General Microbiological Culture Collection Center (CGMCC); Accession number: CGMCC No. 32517; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0007] It should be noted that the strain name of Pseudomonas aeruginosa can be abbreviated as H2.

[0008] Preferably, the nucleotide sequence of the 16S rDNA of *Pseudomonas aeruginosa* is shown in SEQ ID NO:1, and is as follows:

[0009] Preferably, the P. chlororaphis is capable of inhibiting the growth of L. lycopersici, P. cubensis, B. cinerea, A. solani, E. amylovora, V. albo-atrum and F. oxysporum.

[0010] Preferably, the P. chlororaphis is capable of producing protease and amylase.

[0011] Preferably, the P. chlororaphis is capable of inducing the expression of plant-related defense genes.

[0012] More preferably, the plant-related defense genes include at least one of RBOHD (C) , ERF1, NPR1 and WRKY22 genes.

[0013] Preferably, the P. chlororaphis is capable of inducing the expression of Lycium chinense RBOHD (C) , ERF1, NPR1 and WRKY22 genes to be enhanced.

[0014] In another aspect, a microbial agent containing the P. chlororaphis, the fermentation broth of the P. chlororaphis and / or the dried substance of the P. chlororaphis according to the present application is provided.

[0015] In another aspect, the use of the P. chlororaphis according to the present application or the microbial agent according to the present application in the prevention of plant diseases is provided.

[0016] Preferably, the plant disease is Lycium barbarum root rot.

[0017] It should be noted that the P. chlororaphis and the fermentation broth according to the present application are preferred embodiments of the microbial agent, which can be processed into an agriculturally acceptable preparation for application. The microbial agent provided by the present application is generally used in agriculture in the form of a preparation (such as wettable powder, suspension, paste-like coating agent, etc.), which, in addition to the microbial agent containing the P. chlororaphis strain, the fermentation broth (fermentation product) and the like, also includes an agriculturally acceptable carrier or adjuvant.

[0018] Compared with the prior art, the technical solution provided by the present application at least has the following beneficial effects or advantages: The P. chlororaphis with the preservation number of CGMCC No. 32517 obtained by the present application can utilize a variety of nutrients; has the ability to produce protease and amylase; has a wide antibacterial spectrum and can inhibit the growth of L. lycopersici, P. cubensis, B. cinerea, A. solani, E. amylovora, V. albo-atrum and F. oxysporum.

[0019] In addition, the application of the fermentation broth of the P. chlororaphis can induce the expression of Lycium chinense RBOHD(C)、 ERF1 、 NPR1 、 WRKY22 The expression level of the Pseudomonas chlororaphis strain H2 is shown in Table 1, and the pot culture prevention effect test result of the Pseudomonas chlororaphis strain H2 shows that the Pseudomonas chlororaphis has a good prevention effect on the root rot of the Lycium barbarum, and can effectively reduce the incidence of the root rot and the harm to the plant. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0021] Figure 1 Figure 1 is a graph of in-dish streaking of the strain H2 of the present application; Figure 2 Figure 2 is a graph of plate confrontation of the strain H2 of the present application; Figure 3 Figure 3 is a graph of mycelial inhibition rate of the strain H2 of the present application; Figure 4 Figure 4 is a graph of colony morphology and gram staining of the strain H2 of the present application; Figure 5 Figure 5 is a graph of phylogenetic tree of the strain H2 of the present application; Figure 6 Figure 6 is a graph of antibacterial spectrum of the strain H2 of the present application; Figure 7 Figure 7 is a graph of detection of siderophore production ability of the strain H2 of the present application; Figure 8 Figure 8 is a graph of detection of cellulase production ability of the strain H2 of the present application; Figure 9 Figure 9 is a graph of detection of protease production ability of the strain H2 of the present application; Figure 10 Figure 10 is a graph of detection of amylase production ability of the strain H2 of the present application; Figure 11 Figure 11 is a graph of detection of induced resistance ability of the strain H2 of the present application; Figure 12 Figure 12 is a graph of pot culture prevention effect of the strain H2 of the present application on Lycium barbarum. DETAILED DESCRIPTION

[0022] In the following, the technical solutions of the present application will be described in conjunction with the embodiments, but the present application is not limited to the following embodiments. The experimental methods and detection methods described in the embodiments are all conventional methods unless otherwise specified; and the reagents and materials described are all commercially available unless otherwise specified.

[0023] The components and preparation of the culture medium involved in the embodiments are shown as follows: Culture medium formula: KB medium: 20.0 g peptone, 1.5 g K2HPO4, 1.5 g MgSO4, 15.0 mL glycerol, 15-20 g agar, add water to 1000 mL, autoclave at 121℃ for 20 min.

[0024] PDA medium: 200.0g potato (peeled) heated to boiling for 30min and filtered through gauze, 20.0g glucose, 15.0g agar, add distilled water to 1000mL, adjust pH to 7.0, autoclave at 121℃ for 20min.

[0025] LB medium: 10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride (NaCl), 15.0g agar, add distilled water to 1000mL, adjust pH to 7.0, autoclave at 121℃ for 20min.

[0026] Example 1 This embodiment provides the screening of strains for the biological control of root rot in wolfberry.

[0027] 1. Activation of the strain The biocontrol strain stored at -80℃ was taken out and activated by streak plating on KB medium. Figure 1 After activation twice, subsequent experiments were conducted.

[0028] 2. Antagonistic ability test of biological control bacteria The target pathogen is *Fusarium oxysporum*, the pathogen causing root rot of wolfberry, isolated by the applicant. Fusarium oxysporum H0. A 5mm diameter mycelial cake was collected from activated Lycium barbarum root rot pathogens. The cake was inverted and placed in the center of a PDA medium. Pseudomonas aeruginosa grown on the plate was then added. Pseudomonas chlororaphis Cut H2 into long strips and inoculate them 2.5cm apart on both sides of the mycelial cake containing the Lycium barbarum root rot pathogen, leaving the rest uninoculated. Pseudomonas chlororaphis The H2 treatment was used as the control group (CK). After sealing, the culture dish was placed at 28℃ for 7 days. After the mycelium of the control group had fully grown in the culture dish, the antibacterial ability of the antagonistic strain was statistically calculated using the growth rate method.

[0029] The experimental results showed that *Pseudomonas aeruginosa* exhibited strong antibacterial activity in the in-plate test. Pseudomonas chlororaphis H2 (H2) exhibits a significant inhibitory effect on the pathogen causing root rot in wolfberry, with an inhibition zone width reaching approximately 10 mm. Based on the cross-multiplication method, the amount of *Pseudomonas aeruginosa* was calculated. Pseudomonas chlororaphis H2 inhibited the pathogen of wolfberry root rot by more than 60%. Figure 2 ).

[0030] Example 2 This embodiment provides *Pseudomonas aeruginosa*.Pseudomonas chlororaphis H2 sterile fermentation broth inhibits the mycelium growth ability of the root rot pathogen of wolfberry.

[0031] Preparation of sterile fermentation broth: pick Pseudomonas chlororaphis strain H2 Pseudomonas chlororaphis H2 single colony was inoculated in LB liquid medium, 28℃, 150 rpm shake flask culture for 48 h to obtain Pseudomonas chlororaphis strain H2 Pseudomonas chlororaphis H2 fermentation broth, after removing the bacterial bodies by centrifugation of the fermentation broth at 12000 rpm for 10 min, the obtained fermentation broth supernatant was filtered by 0.22 μm filter membrane to obtain Pseudomonas chlororaphis strain H2 Pseudomonas chlororaphis H2 sterile fermentation broth.

[0032] The obtained Pseudomonas chlororaphis strain H2 Pseudomonas chlororaphis H2 sterile fermentation broth was mixed with PDA medium at about 40℃ to prepare PDA medium plate containing 5% sterile fermentation broth. A 5 mm diameter bacterial cake was prepared from the edge mycelium of activated Fusarium oxysporum, and the bacterial cake was inverted and placed in the center of the mixed PDA medium as the experimental group (H2 treatment). The PDA medium inoculated with Fusarium oxysporum mycelium was used as the control group (CK). After sealing, it was placed in a 28℃ incubator. After the CK group of root rot pathogens of wolfberry grew on the culture dish, observation was made. The diameters of the bacterial cakes of the CK group and the H2 treatment group were measured by cross intersection method, and the pathogen inhibition rate was calculated. The pathogen inhibition rate calculation formula is as follows: Pathogen inhibition rate = (CK group bacterial cake diameter - H2 treatment bacterial cake diameter) / CK group bacterial cake diameter × 100% The test results are shown in Figure 3 The sterile fermentation broth of Pseudomonas chlororaphis strain H2 Pseudomonas chlororaphis H2 has a certain inhibitory effect on the mycelium growth of Fusarium oxysporum, and the inhibition rate of the root rot pathogen of wolfberry reaches 26.14%.

[0033] Example 3 This example provides the identification of strain H2.

[0034] 1. Morphological identification of strain: After culturing strain H2 on solid LB medium at 37℃ for 24 h, the colony morphological characteristics were observed. After culturing for 48 h, gram staining was performed. As shown in Figure 4 (left), after culturing strain H2 on solid LB medium at 37℃ for 24 h, the colony was light yellow, the edge was neat, the surface of the colony was smooth, the center was slightly raised, the colony was very sticky when picked up, and there was transparent sticky substance around the colony. As shown in Figure 4 (right), the gram staining of strain H2 was red, indicating that the strain was a gram-negative bacterium, and the bacterial body was slender rod-shaped.

[0035] 2. Physicochemical property identification: After activating strain H2 on solid LB medium, single colonies were picked and inoculated into liquid LB medium and cultured at 37°C for 24 h. The cell suspension concentration was then adjusted to OD. 600 =0.1 for backup. The nitrate reduction ability, fructose, xylose, glucose, maltose, galactose, cellobiose, sorbitol, mannitol, urea, glycerol, gelatin liquefaction, nitrite gas production, and nitrite reduction ability of strain H2 were tested using ordinary bacterial identification tubes. The operating procedures were performed according to the instruction manual for single-box biochemical identification tubes from Guangdong Huankai Microbial Technology Co., Ltd. The identification results are shown in Table 1. The results indicate that strain H2 can decompose sugars such as fructose, glucose, cellobiose, maltose, and galactose, and can also utilize substances such as nitrite, but lacks the ability to utilize substances such as sorbitol, mannitol, and glycerol.

[0036] Table 1 Results of physiological and biochemical properties determination of strain H2

[0037] Note: + indicates positive, - indicates negative.

[0038] 3. Biological identification: DNA was extracted from strain H2 using a bacterial genomic DNA extraction kit. The 16S rRNA gene sequence of strain H2 was amplified by PCR using universal bacterial primers.

[0039] PCR reaction mixture (25 μL): 12.5 μL 2×Taq MasterMix, 1 μL each of forward and reverse primers, 1 μL template, and 9.5 μL ddH2O. PCR reaction program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min 30 s, and 72℃ final extension for 2 min, for 35 cycles.

[0040] The PCR products obtained from the above amplification were detected by 1% agarose gel electrophoresis. The PCR products with correctly identified target bands were recovered and sent to Beijing Qingke Biotechnology Co., Ltd. for purification and sequencing. The sequencing results were aligned to the NCBI database. A phylogenetic tree was constructed using MAGEX software and the neighbor-joining method. The phylogenetic tree is shown below. Figure 5 As shown. The results showed that strain H2 was similar to *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis subsp. aur eofaciens The similarity to strain ATCC 13985 NR_114473.1 was 99.65%, therefore the target strain was identified as *Pseudomonas aeruginosa*. Pseudomonas chlororaphis It was named Pseudomonas aeruginosa. Pseudomonaschlororaphis H2, with accession number CGMCC No. 32517, has a nucleotide sequence of 16S rDNA as shown in SEQ ID NO:1.

[0041] Example 4 This embodiment provides *Pseudomonas aeruginosa*. Pseudomonas chlororaphis Evaluation of the comprehensive biocontrol potential of strain H2.

[0042] 1. *Pseudomonas aeruginosa* Pseudomonas chlororaphis H2 strain antibacterial spectrum test The experiment selected the fungus causing root rot of Schisandra chinensis ( Fusarium oxysporum Tobacco star bacterium (Alternaria alternata) Apple anthracnose bacteria ( Colletotrichum gloeosporioides ), apple rot pathogen ( Valsa mali ), tomato gray mold ( Botrytis cinerea ) and cotton damping-off fungus ( Rhizoctonia solani The target pathogens (all strains were isolated and preserved in the applicant's laboratory) were identified using the plate confrontation method with *Pseudomonas aeruginosa*. Pseudomonas chlororaphis H2 was used in a confrontation test to detect *Pseudomonas aeruginosa*. Pseudomonas chlororaphis H2's antagonistic ability against various pathogens.

[0043] Detection of Pseudomonas aeruginosa by in-plate confrontation test Pseudomonas chlororaphis H2's broad-spectrum antibacterial ability, such as Figure 6 As shown, CK represents the control group not inoculated with H2, and H2 represents the treatment group inoculated with H2. The results indicate that *Pseudomonas aeruginosa*... Pseudomonas chlororaphis H2 exhibits varying degrees of antagonistic activity against the tested pathogens. *Pseudomonas aeruginosa* Pseudomonas chlororaphis H2 exhibits particularly strong antagonistic effects against *Pseudomonas aeruginosa*, *Schisandra chinensis* root rot pathogen, and *Botrytis cinerea*, with inhibition bands exceeding 10 mm in all cases; it also shows a significant antagonistic effect against *Pseudomonas aeruginosa*. These results indicate that *Pseudomonas aeruginosa*... Pseudomonas chlororaphis H2 has good broad-spectrum antibacterial ability.

[0044] 2. *Pseudomonas aeruginosa* Pseudomonas chlororaphis H2 enzyme production capacity assay The culture medium components involved in this embodiment are as follows: LB liquid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, add distilled water to 1000 mL, and adjust the pH to 7.0 at the same time; Casein culture medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 15 g agar, 10 g casein, add distilled water to 1000 mL, and adjust the pH to 7.2. Siderogenic medium (CAS): 1 mL 20% sucrose solution, 3 mL 10% acid-hydrolyzed casein, 100 μL 1 mmol / L CaCl2, 2 mL 1 mmol / L MgSO4, 5 mL 10×MM9 salt solution, 5 mL CAS staining solution, 1.8 g / L agar, 83.9 mL ddH2O; Cellulase screening medium: 20 g sodium carboxymethyl cellulose, 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 15 g agar, and distilled water added to 1000 mL; Amylase screening medium: 10.0 g / L soluble starch, 5.0 g / L peptone, 5.0 g / L NaCl, 5.0 g / L beef extract, 1000 mL distilled water, 20 g / L agar, pH adjusted to 7.0; PDA medium: 200.0 g potato (peeled) heated to boiling for 30 min and filtered through gauze, 20.0 g glucose, 15.0 g agar, add distilled water to 1000 mL, and adjust pH to 7.0.

[0045] (1) Pseudomonas aeruginosa Pseudomonas chlororaphis H2 iron-producing capacity test Pick strains Pseudomonas chlororaphis H2 single colonies were inoculated into LB liquid medium and incubated at 150 rpm and 37°C for 24 h to produce OD. 600 A bacterial suspension of 0.1 mg / L was inoculated into the center of CAS solid medium, with 5 μL of the suspension applied. Each treatment was repeated three times. The cultures were incubated at 28°C for 2 days. The siderophore production capacity of the strain was assessed by observing whether a yellow halo formed around the colony. The experimental results are as follows: Figure 7 As shown, the absence of a yellow halo around the colony indicates that *Pseudomonas aeruginosa* is present. Pseudomonas chlororaphis H2 does not have the ability to produce iron carriers.

[0046] (2) Pseudomonas aeruginosa Pseudomonas chlororaphis H2 cellulase production capacity assay Five μL of H2 bacterial suspension was symmetrically inoculated into the center of the cellulase selection medium. Each treatment was repeated three times. After incubation at 28°C for 3 days, the medium was stained with Congo red for 30 min, destained three times with physiological saline, and the presence or absence of a clear zone was observed. The experimental results are as follows: Figure 8 As shown, no clear zone was formed around the strain, indicating that it is *Pseudomonas aeruginosa*. Pseudomonas chlororaphisH2 does not have cellulase production ability.

[0047] (3) Pseudomonas chlororahs Pseudomonas chlororaphis H2 protease production ability detection 5 μL H2 bacterial suspension was symmetrically inoculated in the central of caseinase culture medium, each treatment was repeated 3 times, incubated at 28℃ for 3 days, the detection method was the same as cellulase production ability detection, and whether transparent circle was observed. The test results are shown in Table 3. Figure 9 As shown in Table 3, a larger transparent circle was produced around the strain, and the strain grew rapidly on the detection medium, indicating that Pseudomonas chlororahs Pseudomonas chlororaphis H2 has very good protease production ability.

[0048] (4) Pseudomonas chlororahs Pseudomonas chlororaphis H2 amylase production ability detection 5 μL H2 bacterial suspension was symmetrically inoculated in the central of amylase production screening medium, each treatment was repeated 3 times, incubated at 28℃ for 3 days, and stained with Gores iodine solution as a staining agent for 20 min in the dark. If a transparent circle is produced, it indicates that the strain has the ability to secrete amylase. The test results are shown in Table 4. Figure 10 As shown in Table 4, a larger transparent circle was produced around the strain, indicating that Pseudomonas chlororahs Pseudomonas chlororaphis H2 has very good amylase production ability.

[0049] Based on the above test results, it is shown that Pseudomonas chlororahs Pseudomonas chlororaphis H2 does not have the ability to produce siderophores and cellulase, but has good protease production ability and amylase production ability.

[0050] Example 5 This example is an induction of disease resistance of Pseudomonas chlororahs Pseudomonas chlororaphis H2 to wolfberry.

[0051] Preparation of biocontrol bacterial fermentation broth: Pseudomonas chlororahs Pseudomonas chlororaphis H2 single colony was inoculated in LB liquid medium, and 28℃, 150 rpm shake flask culture for 48 h to obtain H2 fermentation broth.

[0052] The test material was selected as one-year-old wolfberry seedlings, and LB liquid medium was set as a blank control (CK) for root irrigation of wolfberry. The 10-fold diluted liquid was used for root irrigation as a treatment group, and each group of treatment was set with 3 pots. After treatment, 1-2 g of root sample was taken from each pot at 0 h, 3 h, 6 h, 12 h and 24 h, repeated three times, and immediately put into liquid nitrogen after each sampling, and stored in a-80℃ refrigerator after quick freezing. The sample was subjected to RNA extraction by using plant RNA extraction kit (Huaiyueyang), and cDNA was obtained by reverse transcription, and then fluorescence quantitative PCR was used to detect defense-related genes NPR1, ERF1, RBOHD (C), WRKY22 The expression level was determined. Primers for gene quantification are shown in Table 2.

[0053] Table 2 Primer sequence list for defense gene detection

[0054] Detection of salicylic acid pathway marker genes by real-time PCR NPR1 Jasmonic acid pathway marker genes WRKY22 Lycium barbarum ethylene (ET) pathway marker gene ERF1, respiratory burst oxidase pathway marker genes RBOHD The expression of (C) and other factors was detected using wolfberry. EF-1 a The gene is an internal reference gene. The test results are as follows: Figure 11 As shown, the results indicate that *Pseudomonas aeruginosa* Pseudomonas chlororaphis H2 had varying degrees of influence on the expression of all tested wolfberry resistance genes, including the gene... RBOHD (C) , ERF1, NPR1 Expression levels were significantly upregulated at 24 h of treatment, with a relative expression level approximately 6 times that of the control, while jasmonic acid pathway genes... WRKY22 It is significantly upregulated at 3 hours. ， Indicates Pseudomonas aeruginosa in root irrigation Pseudomonas chlororaphis H2 fermentation broth effectively induced the expression of defense-related genes in wolfberry plants, improving plant disease resistance, which is consistent with the results of pot experiments.

[0055] Example 6 This embodiment uses *Pseudomonas aeruginosa*. Pseudomonas chlororaphis H2 fermentation broth potted plant efficacy test.

[0056] This embodiment relates to the components of the culture medium: PDB medium: 200.0 g potato (peeled) heated to boiling for 30 min and filtered through gauze, 20.0 g glucose, add distilled water to 1000 mL, and adjust pH to 7.0; LB liquid medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, 1000 mL distilled water, pH adjusted to 7.0.

[0057] Goji berry seedlings were cultivated in a greenhouse. When the above-ground parts reached about 40 cm in height, plants with similar growth conditions were selected for root irrigation, with 6 pots per group. Sterile water was used to regulate *Pseudomonas aeruginosa*. Pseudomonas chlororaphis The H2 cell concentration was adjusted to 6 × 10⁻⁶. 8 After applying CFU / mL solution, the roots were irrigated with 100 mL of diluted bacterial solution per wolfberry plant. Plants irrigated with sterile water served as the control (CK).

[0058] The activated Fusarium oxysporum was inoculated in PDB medium and cultured at 25℃. After sporulation, a spore suspension with a concentration of 1×10 6 The spore suspension was diluted and irrigated on the soil of the roots of the wolfberry plants, 50 mL per plant. The treated wolfberry plants were placed in a greenhouse with ventilation and light. The temperature was maintained above 30℃ during the disease process. The water was supplemented as appropriate. After 20 days of culture, the disease index and control effect were calculated and counted.

[0059] The grading standard of root rot of wolfberry in the pot experiment is as follows: 0: No root rot, healthy state.

[0060] 1: The root rot area is not more than 1 / 4, and the aboveground part has no obvious symptoms or only slight leaf yellowing, and the main root is slightly damaged.

[0061] 2: The rotting part accounts for 1 / 4-1 / 2 of the total root area, the main root disease begins to appear, the plant growth is slow, and the lower leaves are yellow and wilting.

[0062] 3: The rotting area reaches 1 / 2-3 / 4, the main root disease is more obvious, the plant is obviously dwarfed, the leaves are yellow and shed in large areas, and some branches are dead.

[0063] 4: More than 3 / 4 of the root is rotten, the main root is severely damaged, the plant is dying or completely dead, and loses economic value.

[0064] The calculation formula is as follows: Disease incidence (%) = number of diseased plants / total number of plants investigated × 100; Disease index = [∑(number of plants at each level × representative value of that level) / (total number of plants examined × representative value of the highest level)] × 100; Control effect = [(disease index of the control area - disease index of the treated area) / disease index of the control area] × 100%.

[0065] Table 3: Results of wolfberry pot experiment

[0066] The test results are shown in Table 3, and the results of the wolfberry pot experiment are shown in Table 3. The results show that the Pseudomonas viridiflava H2 bacterial agent can significantly reduce the incidence and disease index of wolfberry root rot, significantly reduce the wilting symptoms of the aboveground part of the plant, and significantly reduce the root rot rate and rotting area. The incidence is reduced to 66.67%, and the relative control effect can reach 72.73%. Figure 12 Pseudomonas chlororaphis

[0067] ​​The above-described embodiments are merely some of the embodiments of the present application, but not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. All other embodiments obtained by persons of ordinary skill in the art based on the concept of the present application, without making creative labor, are within the scope of the present application.

Claims

1. A type of *Pseudomonas aeruginosa*, characterized in that, The *Pseudomonas aeruginosa* is... Pseudomonas chlororaphis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32517.

2. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of *Pseudomonas aeruginosa* is shown in SEQ ID NO:

1.

3. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The *Pseudomonas aeruginosa* strain described above can inhibit the growth of *Lycium barbarum* root rot fungus, *Schisandra chinensis* root rot fungus, *Gyromitra esculenta* fungus, *Tomato* gray mold fungus, *Tobacco* red spot fungus, *Apple* anthracnose fungus, *Apple* rot fungus, and *Cotton* damping-off fungus.

4. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The *Pseudomonas aeruginosa* species can produce proteases and amylases.

5. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The *Pseudomonas aeruginosa* strain described above can induce the expression of plant-related defense genes.

6. The *Pseudomonas aeruginosa* according to claim 5, characterized in that, The plant-related defense genes include RBOHD (C) ERF1, NPR1 and WRKY22 At least one of the genes.

7. The *Pseudomonas aeruginosa* according to claim 1, characterized in that, The *Pseudomonas aeruginosa* can induce *Lycium barbarum* RBOHD (C) ERF1, NPR1 and WRKY22 Enhanced gene expression.

8. An inoculum containing the *Pseudomonas aeruginosa* as described in claim 1, the fermentation broth of *Pseudomonas aeruginosa*, and / or dried *Pseudomonas aeruginosa*.

9. The application of the *Pseudomonas aeruginosa* of claim 1 or the fungal agent of claim 8 in the prevention and control of plant diseases.

10. The application according to claim 9, characterized in that, The plant disease mentioned is wolfberry root rot.

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