Pseudomonas aeruginosa and application thereof

Abstract

The application discloses a Pseudomonas aeruginosa, which is Pseudomonas aeruginosa EB-Q03-02, and the preservation number is CGMCC No.30779; and the application of the Pseudomonas aeruginosa in preventing and treating Graymold, Pythium aphanidermatum and tobacco blight. The Pseudomonas aeruginosa EB-Q03-02 is separated from the root system of healthy passion fruit, has antagonistic effect on the passion fruit blight, and has obvious antagonistic effect on the passion fruit blight caused by tobacco blight. Further, the Pseudomonas aeruginosa EB-Q03-02 can effectively inhibit the Graymold, Pythium aphanidermatum and tobacco blight, and the inhibition rate is as high as 81% or more.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Pseudomonas aeruginosa and its applications. Background Technology

[0002] Passion fruit blight is caused by a lower fungus, and cool, humid conditions favor its development. Both leaves and fruits can be affected. On leaves, the disease often starts at the leaf tip, appearing as water-soaked lesions. As the disease progresses, the lesions enlarge, spreading across the entire leaf, eventually causing the leaf surface to become translucent. On fruits, initial lesions appear as water-soaked, grayish-green spots. Without timely treatment, the lesions gradually spread to the entire fruit, eventually causing fruit drop. Passion fruit blight has occurred in passion fruit producing areas such as Guangdong, Guangxi, Hainan, Fujian, Yunnan, and Taiwan, significantly impacting passion fruit yield and quality. The earliest report of passion fruit blight was in 2022; it is a relatively new disease affecting passion fruit.

[0003] Currently, the control of passion fruit blight mainly relies on chemical control, which is costly and ineffective. Long-term, repeated, and excessive use of chemical pesticides causes soil, water, and air pollution, increases pesticide residues, and severely disrupts the ecological balance. Biological control refers to the use of beneficial organisms or their metabolites to control plant diseases and pests. It has advantages such as safety for humans and animals, no environmental pollution, and low likelihood of developing pesticide resistance. Therefore, there is an urgent need in production to utilize antagonistic bacteria for the biological control of passion fruit blight. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a strain of Pseudomonas aeruginosa EB-Q03-02 isolated from the roots of healthy passion fruit, which has an antagonistic effect on passion fruit blight. This antagonistic effect is used to achieve safe, effective, and environmentally friendly biological control of passion fruit blight.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A strain of *Pseudomonas aeruginosa*, named *Pseudomonas aeruginosa* EB-Q03-02, with accession number CGMCC No. 30779, was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The 16S rRNA sequence of *Pseudomonas aeruginosa* EB-Q03-02 is 1436 bp in length (as shown in the third sequence in the sequence listing).

[0007] The above describes the application of Pseudomonas aeruginosa EB-Q03-02 in the control of Pyrrosia lingua, Helicobacter pylori, and Phytophthora cirrhosa.

[0008] The application of Pseudomonas aeruginosa EB-Q03-02 in the prevention and control of passion fruit blight, as described above.

[0009] Preferably, the application of the aseptic fermentation filtrate and volatile products of Pseudomonas aeruginosa EB-Q03-02 in the prevention and control of passion fruit blight.

[0010] Preferably, the aseptic fermentation filtrate of Pseudomonas aeruginosa EB-Q03-02 is prepared by inoculating the Pseudomonas aeruginosa EB-Q03-02 strain into YSP medium at a volume ratio of 1%, incubating at 28°C and 200 rpm for 5 to 7 days, centrifuging, collecting the supernatant, and filtering it through a 0.22 μm microporous membrane to remove bacteria, thereby obtaining the aseptic fermentation broth. The obtained fermentation filtrate is stored at 4°C for later use.

[0011] The above describes the application of Pseudomonas aeruginosa EB-Q03-02 in the prevention and control of passion fruit blight caused by Phytophthora indicum.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The *Pseudomonas aeruginosa* EB-Q03-02 strain of this invention was isolated from the roots of healthy passion fruit and has an antagonistic effect on passion fruit blight, especially on the prevention and control of passion fruit blight caused by *Phytophthora indicum*. Furthermore, the *Pseudomonas aeruginosa* EB-Q03-02 strain of this invention can effectively inhibit *Pseudomonas aeruginosa*, *Helicobacter pylori*, and *Phytophthora indicum*, with an inhibition rate of over 81%.

[0014] Preservation Information

[0015] Pseudomonas aeruginosa EB-Q03-02 was deposited on May 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30779. Attached Figure Description

[0016] Figure 1 This is the electrophoresis result of the 16S rRNA gene of Pseudomonas aeruginosa EB-Q03-02.

[0017] Figure 2This is a phylogenetic tree of 16S rRNA from Pseudomonas aeruginosa EB-Q03-02.

[0018] Figure 3 The inhibitory effect of Pseudomonas aeruginosa EB-Q03-02 on various plant pathogens.

[0019] Figure 4 The study investigated the inhibitory effect of aseptic fermentation filtrate of Pseudomonas aeruginosa EB-Q03-02 on the mycelium of Phytophthora infestans of passion fruit.

[0020] Figure 5 The volatile product of Pseudomonas aeruginosa EB-Q03-02 has an inhibitory effect on the mycelium of Phytophthora infestans of passion fruit.

[0021] Figure 6 The inhibitory effect of Pseudomonas aeruginosa EB-Q03-02 on the formation of zoosporangia of Phytophthora in passion fruit is shown.

[0022] Figure 7 The study investigated the in vitro control efficacy of Pseudomonas aeruginosa strain EB-Q03-02 against passion fruit blight.

[0023] Figure 8 It is Pseudomonas aeruginosa strain EB-Q03-02. Detailed Implementation

[0024] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available. Figure 3-5 , Figure 7-8 In the accompanying description, "front" refers to a top view of the petri dish showing the test results or inhibition effect, and "back" refers to a bottom view of the petri dish showing the test results or inhibition effect.

[0025] The causal agent of passion fruit blight was isolated and purified from diseased plants collected from a passion fruit base in Nama Town, Liangqing District, Nanning City, Guangxi Province. Koch's postulates confirmed its identity as *Phytophthora indicum*. It was further identified as belonging to *Phytophthora indicum* and is preserved at -80℃ in the State Key Laboratory of Subtropical Agricultural Biological Resources for future use. All subsequent experiments using this *Phytophthora indicum* strain will utilize this fungus.

[0026] The culture medium used in the examples is as follows:

[0027] LA medium: Weigh 10g tryptone, 5g yeast extract, 10g NaCl, and 15g agar powder, add deionized water to a final volume of 1000ml, and autoclave at 121℃ for 25min. (LB medium does not contain agar powder.)

[0028] PDA medium: Weigh 20g glucose, 6g potato starch, and 20g agar powder, add deionized water to a final volume of 1000ml, and autoclave at 121℃ for 25min.

[0029] LB medium: Weigh 10g tryptone, 5g yeast extract, and 10g NaCl. Add deionized water to a final volume of 1000ml and autoclave at 121℃ for 25min. (LB medium does not contain agar powder.)

[0030] PDB medium: Weigh 20g glucose and 6g potato starch, add deionized water to a final volume of 1000ml, and autoclave at 121℃ for 25min.

[0031] YESPA medium: 10g yeast extract, 20g sucrose, 20g peptone, 15g agar powder, add deionized water to a final volume of 1000ml, autoclave at 121℃ for 25min (YESP medium does not contain agar powder).

[0032] YSP medium: 5g yeast extract powder, 20g sucrose, 10g peptone, add deionized water to a final volume of 1000ml, autoclave at 121℃ for 25min.

[0033] Sporulation-inducing solution for Phytophthora tobaccois: 0.004 mol / L MgSO4·7H2O, 0.05 mol / L KNO3, 1 mL Fe-EDTA, and diluted to 1000 mL with deionized water; wherein the Fe-EDTA formulation is 14.86 g EDTA-Na2 and 24.9 g FeSO4·7H2O, diluted to 1000 mL with deionized water.

[0034] Example 1

[0035] Isolation and purification of antagonistic strains

[0036] Under aseptic conditions, healthy passion fruit roots collected from Luowei Town, Wuming District, Nanning City, Guangxi Province, were cut into 5mm x 5mm pieces. These pieces were then immersed in 75% alcohol for 30 seconds for disinfection, rinsed in 4% sodium hypochlorite solution for 10 minutes, and finally rinsed 4-5 times with sterile water. The roots were then chopped and diluted to a 10% solution. -3 10 -4 10 -5 Diluted to 10 times the volume, take 10 drops-3 10 -4 10 -5 100 μL of each dilution was spread onto LA medium plates, with each concentration repeated three times. The plates were incubated at 28°C for 3–5 days. Single colonies with significant differences in colony morphology were selected based on colony size, color, and surface morphology and purified by streak plating. The purified colonies were then transferred to fresh LA medium and named EB-Q03-02.

[0037] Identification of strains

[0038] EB-Q03-02 colonies on LA medium are nearly round, moist, non-protruding, easily picked up, ginger-yellow in color, with smooth, transparent edges. Figure 8 .

[0039] Total DNA was extracted from the purified strain EB-Q03-02, and the 16S rRNA sequence was amplified using primers 27F / 1492R. The total reaction volume was 25 μL, including 12.5 μL of 2×Rapid Taq Master Mix, 1 μL each of forward and reverse primers, and 19 μL of ddH2O. The reaction program was: 95℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 54℃ (16S rRNA) annealing for 30 s, 72℃ extension for 45 s, for 33 cycles; and 72℃ extension for 10 min. The PCR products obtained from the above reactions were purified by 1.0% agarose gel electrophoresis. The target fragment was cloned using the pEASY-T1 Cloning Kit. Following the manufacturer's instructions, the target fragment and pEASY-T1 vector were mixed at a volume ratio of 1:4 and incubated at 25°C for 25 min. Then, 10 μL of the ligation product was gently added to 100 μL of *Escherichia coli* DH5α competent cells. The transformed *E. coli* DH5α cells were plated on LA medium containing 50 mg / L ampicillin and incubated at 37°C for 12 h. Single colonies were then picked for colony PCR identification. The correctly identified strains were inoculated into 1 mL of LB medium and incubated at 37°C and 200 rpm for 1 h. The cultured bacterial solution was then sent to Aoke Dingsheng Biotechnology Co., Ltd. for sequencing. The final representative strain sequences were compared with the NCBI database using BLASTn (https: / / blast.ncbi.nlm.nih.gov). The nucleotide sequences of standard strains with high similarity were downloaded and analyzed with MAGE11.0 and SNAPGENE software. A phylogenetic tree was constructed using the neighbor-joining method.

[0040] Molecular identification results of strain EB-Q03-02

[0041] 16S rRNA gene amplification and sequencing

[0042] Using genomic DNA from strain EB-Q03-02 as a template, the 16S rRNA gene was amplified using 27F / 1492R specific primers (Table 1). Electrophoresis results showed that a 16S rRNA gene fragment of approximately 1500 bp was obtained. Figure 1 M: Marker, 1: EB-Q03-02). The target PCR product was recovered, cloned, and sequenced. The sequencing results showed that the 16S rRNA sequence of the bacteria was 1436 bp in length (as shown in the third sequence in the sequence listing).

[0043] Table 1 27F / 1492R sequence

[0044]

[0045] Analysis of 16S rRNA gene sequence and construction of phylogenetic tree

[0046] The sequencing results were compared with the 16S rRNA sequence of strain EB-Q03-02 using BLASTn. The two sequences with the highest similarity to 16S rRNA were both Pseudomonas aeruginosa, with accession numbers JX090597.1 and GQ375800.1, respectively, with 100% similarity. A phylogenetic tree was constructed using the 16S rDNA gene sequence of strain EB-Q03-02 and its homologous similar sequences. Figure 2 The results showed that strain EB-Q03-02 and P. aeruginosa were on the same branch. Based on the morphological characteristics of the colony, strain EB-Q03-02 was identified as Pseudomonas aeruginosa and named Pseudomonas aeruginosa EB-Q03-02. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30779.

[0047] Example 2

[0048] Pseudomonas aeruginosa EB-Q03-02 Antibacterial Spectrum

[0049] In a clean bench, the plate confrontation method was used to determine the antibacterial activity of activated Rhizoctonia solani, Anthracnose sclerotium, Sclerotinia sclerotiorum, Fusarium solani, Helicobacter pylori, Phytophthora tobaccoii, and Pyrrosia lingua (Rhizoctonia solani, Anthracnose sclerotiorum, Sclerotinia sclerotiorum, Fusarium solani, Helicobacter pylori, Phytophthora tobaccoii, and Pyrrosia lingua were all isolated and purified from samples with typical symptoms, identified by Koch's postulates, and preserved). A mycelial cake was made at the edge of the test bacterial colony using a 6mm diameter punch. A sterile toothpick was used to transfer the mycelial cake to the center of a PDA agar plate. Pseudomonas aeruginosa EB-Q03-02 was streaked on both sides of the PDA plate 20mm away from the test bacteria. A PDA agar plate containing only the test bacterial mycelial cake served as a blank control. Each treatment was repeated in triplicate. Incubate in a 28℃ constant temperature incubator. When the blank control has covered the entire culture dish, measure the colony diameter of the test bacteria to calculate the antagonistic effect.

[0050] Antibacterial inhibition rate calculation: Antibacterial inhibition rate = (colon diameter of control group - colony diameter of treatment group) / (colon diameter of control group) × 100

[0051] The results showed that EB-Q03-02 had a certain inhibitory effect on *Rhizoctonia solani*, *Anthracis citrinum*, *Sclerotinia sclerotiorum*, *Fusarium solani*, *Pyrrosia lingua*, *Helicobacter pylori*, and *Phytophthora nicotineae*. The inhibition rates for *Pyrrosia lingua*, *Helicobacter pylori*, and *Phytophthora nicotineae* all exceeded 80%. The inhibition rates are shown in Table 2. Specific details are as follows: Figure 3 As shown.

[0052] Table 2 Inhibition rates of EB-Q03-02 against various plant pathogens

[0053]

[0054] Example 3

[0055] Inhibitory effect of EB-Q03-02 aseptic fermentation filtrate on mycelial growth of Phytophthora blight of passion fruit

[0056] The EB-Q03-02 strain was inoculated into YSP medium at a volume ratio of 1%, and cultured at 28℃ with shaking at 200 rpm for 3, 5, and 7 days. After centrifugation at 10000 rpm for 10 min, the supernatant was collected. The supernatant was filtered through a 0.22 μm microporous membrane for sterilization, and the resulting fermentation filtrate was stored at 4℃ for later use. 1.5 mL of the fermentation filtrate was mixed with 13.5 mL of melted PDA medium to prepare a plate. A 6 mm diameter mycelial cake of passion fruit blight pathogen was inoculated in the center of the plate. An equal volume of sterile water was used as a control (CK). Each treatment was repeated three times. After incubation at 28℃ for 10 days, the colony diameter was measured.

[0057] Inhibition rate (%) = {(Control colony diameter - Mycelial cake diameter) - (Treatment colony diameter - Mycelial cake diameter)} / (Control colony diameter - Mycelial cake diameter) × 100

[0058] The results showed that the aseptic fermentation broth of EB-Q03-02 had a significant inhibitory effect on the pathogen of passion fruit blight, such as... Figure 4 As shown in Table 3, the fermentation broth fermented for 7 days showed the most significant inhibitory effect on the pathogen, followed by 5 days, and the worst effect was observed after 3 days. The OD600 of the bacterial broth was inversely proportional to the fermentation time. This indicates that the fermentation product of strain EB-Q03-02 has an inhibitory effect on the passion fruit blight pathogen, and that the longer the fermentation time (less than 7 days), the better the effect.

[0059] Table 3 Inhibition rate of EB-Q03-04 aseptic fermentation filtrate against passion fruit blight pathogen.

[0060]

[0061] Example 4

[0062] Inhibitory effect of EB-Q03-02 volatile products on passion fruit blight pathogen

[0063] In a clean bench, using separate petri dishes, one side was inoculated with PDA medium and the other with LA medium. The PDA side was inoculated with a 6mm diameter mycelial cake of *Passiflora thuringiensis*, and the LA side was inoculated with antagonistic bacteria. The plate with one 6mm diameter mycelial cake of *Passiflora thuringiensis* inoculated on the PDA side and a blank LA medium plate on the other side was designated as the control (CK). The CK plate contained no antagonistic bacteria and produced no volatile substances; the pathogen grew naturally. When the CK plate was fully colonized by the pathogen, the radius of the pathogen on the plate was measured, and its inhibition rate was calculated.

[0064] Inhibition rate (%) = {(Control colony diameter - Mycelial cake diameter) - (Treatment colony diameter - Mycelial cake diameter)} / (Control colony diameter - Mycelial cake diameter) × 100

[0065] Experiments showed that EB-Q03-02 had a significant inhibitory effect on the mycelium of the pathogen causing passion fruit blight, with an inhibition rate of 63.20% (e.g., ...). Figure 5 ).

[0066] Example 5

[0067] Determination of the inhibitory effect of EB-Q03-02 on zoosporangium formation of Phytophthora infestans of passion fruit

[0068] 1 cm diameter mycelial cakes were collected from the edges of passion fruit blight pathogen colonies cultured for 14 days. EB-Q03-02 culture solutions with OD600 = 0.8 and passion fruit blight pathogen sporulation induction solution were prepared at volume concentrations of 1%, 2%, 3%, 4%, 5%, and 10% as treatment groups, with pure sporulation induction solution as the control group (ck). 10 mL of each treatment solution was measured into 50 mL Corning tubes, and two passion fruit blight pathogen mycelial cakes were placed inside. The tubes were then incubated at 28℃ under light. After 72 hours, the number of sporangia produced by passion fruit blight pathogens at each concentration was observed and recorded under a microscope. The average number of zoosporangia in three *400 fields of view was calculated for each concentration, and photographs were taken and saved.

[0069] The results showed that different concentrations of EB-Q03-02 bacterial suspension had varying effects on the production of zoosporangia by *Phytophthora infestans*, the causal agent of passion fruit blight. A concentration of EB-Q03-02 not exceeding 3% significantly promoted zoosporangia production, presumably because EB-Q03-02 created a certain adverse environment for the growth of *Phytophthora infestans*, thereby promoting mycelial differentiation and zoosporangia production. When the concentration of EB-Q03-02 reached 4%, it inhibited zoosporangia production, with a sporulation rate of only 27.50%. At a concentration of 5%, the sporulation rate was only 6.70%, and at a concentration of 10%, no sporulation occurred. Specific details are shown in Table 4. (The text then repeats the description of zoosporangia growth.) Figure 6 As shown.

[0070] Table 4. Effects of EB-Q03-02 on zoosporangium formation of Phytophthora in passion fruit.

[0071]

[0072] Example 6

[0073] Determination of the in vitro control effect of strain EB-Q03-02 against passion fruit blight

[0074] Select strain EB-Q03-02 and inoculate it into a 50ml Corning tube containing 20mL LB medium. Incubate in a shaker at 28℃ and 200r / min. After 6h, measure the OD600 value of the bacterial solution using a UV spectrophotometer and adjust its OD value to 0.6 for later use.

[0075] Passion fruit leaves of uniform maturity were evenly sprayed with EB-Q03-02 bacterial solution. The leaf tissue was then punctured with a sterile plum blossom needle. One day later, 6 mm diameter passion fruit blight fungal cakes were inoculated at the wound sites. After spraying with EB-Q03-02 bacterial solution again, the leaves were placed in a humidified box at 28℃. LB medium was used as a control (CK). Three inoculation points were performed on each leaf, with each treatment replicated three times. The diameter of the lesions was measured using the cross-sectional method after 5 days.

[0076] Control efficacy (%) = [(Control lesion area - Treatment lesion area) / Control lesion area] × 100% Results showed that the average size of lesions on the three leaves of the control group was 39.1 mm, while no obvious lesions appeared on the three leaves inoculated with EB-Q03-02, achieving a control efficacy of 100%. Details are as follows... Figure 7 As shown, EB-Q03-02 has a good in vitro control effect against passion fruit blight and can inhibit the infection of pathogens.

[0077] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), characterized in that: The *Pseudomonas aeruginosa* mentioned is *Pseudomonas aeruginosa* (… Pseudomonas aeruginosa )EB-Q03-02, with accession number CGMCC No.30779.

2. The application of Pseudomonas aeruginosa as described in claim 1 in the prevention and control of Pyrrosia lingua, Helicobacter pylori, and Phytophthora nicotineae.

3. The application of the fermentation filtrate of *Pseudomonas aeruginosa* as described in claim 1 in the prevention and control of passion fruit blight caused by *Phytophthora indicum*. in, The fermentation filtrate is prepared by inoculating the *Pseudomonas aeruginosa* onto YSP medium, incubating at 28°C and 200 r / min for 5-7 days, centrifuging, collecting the supernatant, and filtering it through a microporous membrane to remove bacteria, thus obtaining sterile fermentation broth.

4. The application according to claim 3, characterized in that: The *Pseudomonas aeruginosa* was inoculated into YSP medium at a volume ratio of 1%.

5. The application of Pseudomonas aeruginosa as described in claim 1 in the prevention and control of passion fruit blight caused by Phytophthora indicum.

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