Novel extreme oriental pseudomonas and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a novel extreme pseudomonas orientalis R10, which is classified and named as Pseudomonas orientalis, is preserved in the China Center for Type Culture Collection (CCTCC), and has a preservation number of CCTCC NO: M2025304. The invention also provides a biocontrol inoculant, a bacterial suspension containing the extreme Pseudomonas orientalis R10, a fermentation broth or a fermentation filtrate containing the extreme Pseudomonas orientalis R10. Meanwhile, the invention also provides an application of the extreme Pseudomonas orientalis R10 or the biocontrol inoculant in prevention and treatment of the kiwifruit canker, and a prevention and treatment method of the kiwifruit canker. The novel extreme Pseudomonas orientalis R10 is separated from kiwi fruit plant tissues and rhizosphere soil, has a good inhibition effect on kiwi fruit canker pathogens, can be used as a potential biocontrol bacterium resource, effectively prevents and treats kiwi fruit canker, and also has a good bacteriostasis effect on other plant pathogens.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a novel extreme oriental pseudomonas and application thereof. Background Art

[0002] Plant diseases have always been an important factor threatening agricultural production. Due to the high cost of agricultural prevention and control, current prevention and control measures mainly rely on chemical agents, supplemented by the breeding of disease-resistant varieties. However, the long-term use of chemical agents not only makes pathogens resistant, but also pesticide residues are harmful to humans, livestock and the environment.

[0003] As people pay more attention to healthy ecology and high-quality life, biological control has attracted more and more attention due to its safety, environmental protection, and high efficiency. Biological control is a method of using beneficial organisms or other organisms to inhibit or eliminate harmful organisms. It has the advantages of being non-toxic, harmless, pollution-free, and not easy to develop drug resistance. At the same time, plant disease biocontrol bacteria have the function of improving the environment and obtaining long-term benefits, which is in line with the development direction of plant disease control at this stage. According to relevant research reports, there are many types of microorganisms used for biological control, including bacteria, fungi, actinomycetes, and yeasts. Among them, bacteria, as a type of biocontrol bacteria widely found in soil, plants, fresh water, and the ocean, have a complex secondary metabolic system and can secrete one or more secondary metabolites with significant biological activity, which plays an important role in plant disease control. As a large class of biocontrol bacteria, most strains of Pseudomonas have antibiotic or growth-promoting effects, and some strains have both control and growth-promoting effects.

[0004] Pseudomonas colonizes in plant tissues in large quantities and produces a wide variety of secondary metabolites. For example, Pseudomonas Pf-5 and CHA0 produce pyoluteorin (Plt), pyrrolnitrin (Prn) and 2,4-diacetylphloroglucinol (DAPG) to inhibit damping-off disease and wheat take-all disease caused by Rhizoctonia solani, and also inhibit potato soft rot caused by Erwinia. At present, many antibiotics produced by Pseudomonas have been made into biocontrol agents for production, such as PCA, phenazine-1-amide, lipopeptide antibiotics, etc., which have been used as the main ingredients of biological pesticides to prevent and control wheat fusarium head blight, cucumber anthracnose, watermelon wilt, pepper blight, rice sheath blight, tobacco bacterial wilt and peanut white rot. In addition, the application of biocontrol Pseudomonas to control plant diseases is not only non-toxic, residue-free, highly specific, and harmless to non-target organisms, but also has good environmental compatibility and long-lasting control effects, and has the potential to be developed into biological pesticides.

[0005] Kiwifruit is native to China. It is rich in vitamin C, magnesium, potassium, and contains a variety of organic matter and trace elements such as carbohydrates, proteins, amino acids, etc., and is known as the "king of fruits". Compared with other fruit trees, kiwifruit has fewer diseases and pests, but kiwifruit bacterial canker caused by Pseudomonas syringae pv.actinidiae (Psa) has seriously affected the development of the kiwifruit industry. Kiwifruit canker is the most contagious and destructive disease of kiwifruit. It mainly harms the trunk, side branches, leaves and flower buds of kiwifruit plants. In severe cases, it will lead to a reduction in kiwifruit production or even a complete loss of yield.

[0006] Therefore, finding environmentally friendly biocontrol Pseudomonas to control kiwifruit canker has important practical and scientific value. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a new extreme oriental Pseudomonas and its application. The new extreme oriental Pseudomonas is isolated and screened from kiwi plant tissues and rhizosphere soil, has a good inhibitory effect on kiwi canker pathogens, can effectively prevent and treat kiwi canker disease, and also has a good antibacterial effect on other plant pathogens.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A novel extreme oriental Pseudomonas R10, with the taxonomic name Pseudomonas extremorientalis, was deposited and shown to be alive at the China Center for Type Culture Collection in Wuhan, China on February 27, 2025, with the deposit number CCTCC NO: M2025304.

[0010] As one of the preferred embodiments of the present invention, the 16S rRNA gene sequence of the extreme oriental Pseudomonas orientalis R10 is shown as SEQ ID NO:1.

[0011] As one of the preferred embodiments of the present invention, the extreme oriental Pseudomonas R10 has an inhibitory effect on the pathogenic bacteria of Pseudomonas syringae kiwifruit.

[0012] As one of the preferred embodiments of the present invention, the extreme Pseudomonas orientalis R10 has an inhibitory effect on tea ring spot pathogen, tea anthracnose pathogen, rapeseed sclerotinia pathogen, tomato canker pathogen, and rice bacterial blight pathogen.

[0013] A biocontrol agent contains the bacterial suspension, fermentation liquid or fermentation filtrate of the novel extreme oriental Pseudomonas R10.

[0014] The application of the above-mentioned novel Pseudomonas extremorientalis R10 or the above-mentioned biocontrol agent in the prevention and control of kiwifruit canker disease.

[0015] A method for preventing and controlling kiwifruit canker disease, using the above-mentioned novel Pseudomonas extremorientalis R10 or the above-mentioned biocontrol agent.

[0016] The advantages of the present invention compared with the prior art are as follows:

[0017] (1) The present invention uses the "dilution separation and plate confrontation culture method" to isolate and screen a strain R10 from kiwifruit plant tissues and rhizosphere soil, which has a strong antagonistic effect against a variety of pathogenic bacteria (such as Pseudomonas syringae pv. actinidiae, Pestalotiopsis theae, Colletotrichum camelliae, Sclerotinia sclerotiorum, Clavibacter michiganensis subsp. michiganensis, Xanthomonas oryzae pv. oryzae, etc.); through multi-gene sequence analysis, this strain is identified as Pseudomonas extremorientalis, and at the same time, the broad-spectrum antibacterial activity and the activity of the metabolites of this strain are measured, and the control effect of this strain on kiwifruit canker disease is measured through in vitro experiments, so as to be applied to the research and development of subsequent biocontrol agents for kiwifruit canker disease;

[0018] (2) The fermentation broth and fermentation filtrate of the strain R10 of the present invention can significantly inhibit the growth of Pseudomonas syringae pv. actinidiae, and the diameters of their antibacterial zones are 39.55 mm and 31.60 mm respectively; dropping the bacterial suspension of the strain R10 onto the in vitro branches inoculated with Pseudomonas syringae pv. actinidiae, after 21 days, the preventive and therapeutic control effects can reach 80.54% and 43.24% respectively, indicating that the strain R10 can be used as a potential biocontrol bacterial resource for the research and development of biocontrol agents for kiwifruit canker disease;

[0019] (3) Different from chemical control agents, the strain R10 of the present invention is environmentally friendly and has good safety. Description of the Drawings

[0020] Figure 1 It is the inhibition result of the strain R10 on Pseudomonas syringae pv. actinidiae in Example 1;

[0021] Figure 2 It is the inhibition result of the strain R10 on various pathogenic fungi in Example 2 (in the figure, the control groups of A - F are the cultures of individual pathogenic fungi, specifically A: Sclerotinia sclerotiorum, B: Fusarium graminearum, C: Pestalotiopsis theae, D: Fusarium solani f. sp. cucurbitae, E: Colletotrichum camelliae, F: Magnaporthe oryzae; the plates of a - f are the confrontation culture results corresponding to A - F);

[0022] Figure 3It is the inhibition results of strain R10 against various pathogenic bacteria in Example 2 (in the figure, Figures A - C are the inhibition results of Pseudomonas syringae pv. actinidiae from Qianshan, Yuexi, and Jinzhai respectively; Figure D is the inhibition result of Xanthomonas oryzae pv. oryzae; Figure E is the inhibition result of Clavibacter michiganensis subsp. michiganensis; Figure F is the inhibition result of Xanthomonas campestris pv. campestris).

[0023] Figure 4 It is the phylogenetic tree constructed based on multiple gene sequences for strain R10 and its related species in Example 3;

[0024] Figure 5 It is the antibacterial activity results of the fermentation broth of strain R10 against Pseudomonas syringae pv. actinidiae in Example 4 (in the figure, label "a" is the fermentation broth; label "b" is the blank medium);

[0025] Figure 6 It is the antibacterial activity results of the fermentation filtrate of strain R10 against Actinidia pathogens in Example 4 (in the figure, labels "a" and "b" are the fermentation filtrates; label "CK" is the blank medium);

[0026] Figure 7 It is the control effect and significance analysis results of Pseudomonas R10 on detached branches in Example 5 (in the figure, Figure A is the disease spot diagram of each group of detached branches; Figure B is the significance analysis result of the disease spot length; Figure C is the significance analysis result of the control effect). Detailed implementation manners

[0027] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, providing detailed implementation manners and specific operation procedures. However, the protection scope of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0028] The following are the medium formulations involved in the following examples:

[0029] NA medium (plate): Beef extract 5 g, peptone 10 g, NaCl 5 g, agar 17 g, add pure water to 1000 mL, pH 7.2.

[0030] KBA medium: Peptone 20 g, glycerol 10 g, K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, agar 15 g, add pure water to 1000 mL.

[0031] PDA medium (plate): Potato 200 g, glucose 20 g, agar 20 g, add pure water to 1000 mL.

[0032] NB medium: Beef extract 5 g, peptone 10 g, NaCl 5 g, add pure water to 1000 mL, pH 7.2.

[0033] Example 1: Isolation and screening of strain R10

[0034] (1) Isolation of strains

[0035] Healthy kiwifruit plant leaves, flowers, branches, roots and rhizosphere soil were collected. 10 g of different samples were weighed respectively. After being crushed in a sterile mortar, different tissue samples and rhizosphere soil were put into a triangular flask containing 50 mL of sterile water and shaken at 180 rpm for 10 min to obtain suspensions of different samples.

[0036] After gradient dilution of each suspension by the dilution isolation method, 100 μL was taken and spread on NA plates respectively, and then placed in an inverted culture in a constant temperature incubator at 28 °C. After single colonies of bacteria and actinomycetes grew out, representative single colonies with different morphological characteristics were picked and streaked and purified on NA plates, and then stored on a test tube slant for standby. Finally, 660 isolated strains were obtained.

[0037] (2) Screening of antagonistic bacteria

[0038] The 660 isolated strains were screened for antagonism against Pseudomonas syringae pv. actinidiae by the spot inoculation method:

[0039] After melting 150 mL of KBA medium and cooling it to 45 - 50 °C, 6 mL of Psa M228 bacterial solution (Pseudomonas syringae pv. actinidiae) with a concentration of 10 8 CFU / mL was added, mixed well, and poured into plates. The same isolated strain was inoculated 3 times on each plate with a sterile toothpick and cultured at 28 °C for 12 - 48 h. The diameter of the inhibition zone was measured by the cross method, and each experiment was repeated 3 times and the average value was taken. Finally, 60 strains with inhibitory effects on Pseudomonas syringae pv. actinidiae (inhibition zone diameter greater than 5 mm) were screened out for re-screening.

[0040] The re-screening results showed that: Strain R10 had the best antagonistic effect against Pseudomonas syringae pv. actinidiae, and its average inhibition zone diameter reached 24.22 mm ( Figure 1 ), and strain R10 with the largest inhibition zone diameter and the best inhibitory effect was selected for subsequent experiments.

[0041] Example 2: Determination of the antibacterial spectrum of strain R10

[0042] (1) Determination of the antifungal spectrum of fungi: Using the plate confrontation method, mycelial blocks of the target pathogenic fungi (Pestalotiopsis theae, Colletotrichum camelliae, Sclerotinia sclerotiorum, Trichosanthes root rot pathogen, Magnaporthe oryzae, Fusarium graminearum) were inoculated on PDA plates for activation and culture for 3 - 4 days. At the same time, the antagonistic strain R10 (obtained in Example 1) was activated on NA plates for 24 - 48 hours. After the activated strains grew well, mycelial blocks of the target pathogenic fungi (Ф = 6 mm) were respectively inoculated in the center of PDA plates, and then the activated strain R10 was point-inoculated with an inoculation needle at a distance of 2.5 cm from the pathogenic mycelial blocks. The plates were cultured in the dark at 25°C, and the colony growth of the pathogenic bacteria on each plate was observed. Calculation method of inhibition rate: Inhibition rate (%) = (control colony radius - treated colony radius) / (control colony radius - 3 mm) × 100.

[0043] (2) Determination of the antibacterial spectrum of bacteria: Using Xanthomonas euvesicatoria, Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. campestris, and Pseudomonas syringae pv. actinidiae isolated from Qianshan, Yuexi, Jinzhai and other regions as target bacteria, the point-inoculation method described in Example 1 was used to conduct confrontation culture with strain R10, and the diameter of the inhibition zone was observed and recorded.

[0044] In this example, the antifungal activity of strain R10 against six plant pathogenic fungi was determined by the plate confrontation method, and the results are as Figure 2 shown. The results show that strain R10 shows good inhibitory effects on six pathogenic fungi (Pestalotiopsis theae, Colletotrichum camelliae, Sclerotinia sclerotiorum, Trichosanthes root rot pathogen, Magnaporthe oryzae, Fusarium graminearum). Among them, the inhibitory effect on Sclerotinia sclerotiorum is the best, with an inhibition rate as high as 89.2%, and the inhibitory effect on Fusarium graminearum is the smallest, with an inhibition rate of 42.6%.

[0045] The antibacterial activity of strain R10 against pathogenic bacteria was determined by the point-inoculation method, and the results are as Figure 3 shown. The results show that strain R10 has excellent inhibitory effects on Pseudomonas syringae pv. actinidiae isolated from three regions of Qianshan, Yuexi, and Jinzhai, and the diameter of the inhibition zone is above 18 mm. Except for Pseudomonas syringae pv. actinidiae, strain R10 has different degrees of antibacterial activity against the tested pathogenic bacteria. The antibacterial effect on Xanthomonas oryzae pv. oryzae is the best, with a diameter of the inhibition zone up to 33.2 mm, and the antibacterial effect on Xanthomonas campestris pv. campestris is relatively poor, with a diameter of the inhibition zone of 15.6 mm.

[0046] Example 3. Identification of strain R10:

[0047] The genomic DNA of strain R10 was extracted by the CTAB method, and 16S rRNA (SEQ ID NO: 1) and the housekeeping gene gyrB (SEQ ID NO: 2) were selected as target genes. The systems and conditions for PCR amplification of each gene are shown in Tables 1 and 2. After amplification, 5 μL of the PCR product was taken for 1% agarose gel electrophoresis detection. The amplification products with bright, clear bands without miscellaneous bands and the same length as the amplified gene fragments were sent to a sequencing company (Sangon Biotech, Shanghai) for sequencing.

[0048] Table 1 Systems and Conditions for 16S rRNA PCR Amplification of Strain R10

[0049]

[0050]

[0051] Table 2 Systems and Conditions for gyrB Gene PCR Amplification of Strain R10

[0052]

[0053] The obtained sequences were analyzed and verified using DNAstar software, and the verified sequences were placed in NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for BLAST alignment to identify the reference strains with high homology to the test strain sequence and their species status, and the reference sequences of the species Pseudomonas and related strains were downloaded from GenBank.

[0054] The sequences of each gene of the reference strains and the self-tested strains were aligned using MEGA 11.0 software, the messy sequences at both ends were excised, and then they were connected in the order of gyrB - 16S rRNA. The maximum likelihood method was used to construct a multi-gene phylogenetic tree of representative strains of Pseudomonas to determine the taxonomic status of the antagonistic strain R10.

[0055] In this example, the housekeeping gene gyrB gene sequence and 16S rRNA of strain R10 were obtained by PCR amplification; through BLAST homology sequence analysis of the above two target genes of strain R10, it was found that the homology of the two gene sequences with the sequences of Pseudomonas extremorientalis (LT629708.1, KF979139.1) was 99.23% and 100% respectively. They were connected in the order of gyrB - 16S rRNA, and a phylogenetic tree was constructed using the maximum likelihood method ( Figure 4)。The results showed that strain R10 clustered together with the reference strain P. extremorientalis BS2774 (LT629708.1) with a bootstrap value of 99%. Finally, strain R10 was identified as Pseudomonas extremorientalis.

[0056] Meanwhile, on February 27, 2025, strain R10 was sent to the China Center for Type Culture Collection for preservation and was shown to be viable (classified as Pseudomonas extremorientalis), with the preservation number CCTCC NO: M2025304 and the preservation address: Wuhan University, Wuhan, China.

[0057] Example 4: Antibacterial activity test of strain R10:

[0058] (1) Preparation of fermentation broth and fermentation filtrate

[0059] The activated antagonistic strain was placed in an incubator and cultured at 28 °C for 2 days. Then, it was inoculated into a conical flask containing 50 mL of NB medium with a sterile toothpick and placed in a shaker and cultured at 28 °C and 180 r / min for 2 days to obtain the fermentation broth.

[0060] Take 10 mL of the fermentation broth and store it at 4 °C for later use. Meanwhile, the remaining fermentation broth was put into a centrifuge tube, placed in a high-speed centrifuge, centrifuged at 4 °C and 6000 r / min for 10 min, and then the supernatant was filtered through a 0.22 μm microporous filter to obtain the sterile fermentation filtrate.

[0061] (2) Determination of antibacterial activity of fermentation broth and fermentation filtrate

[0062] Take 150 mL of KBA medium, melt it and cool it to 45 - 50 °C, add 2 mL of Psa M228 bacterial solution with a concentration of 10 8 CFU / mL, mix well, and pour the plate; the filter paper method and the Oxford cup method were used to determine the antibacterial effects of the fermentation broth and fermentation filtrate on the kiwifruit canker pathogen. Cultivate at 28 °C for 72 h, and measure the diameter of the antibacterial circle by the cross method. Each experiment was repeated 3 times and the average value was taken.

[0063] In this example, the antibacterial activity results of the fermentation broth of strain R10 against the kiwifruit canker pathogen are as Figure 5 shown. The results showed that the fermentation broth of strain R10 had significant antagonistic activity, and the diameter of the antibacterial circle reached 39.55 mm.

[0064] The antibacterial activity results of the fermentation filtrate of strain R10 against the kiwifruit pathogen are as Figure 6As shown. The results showed that the fermentation filtrate of strain R10 had significant antagonistic activity, and the diameter of the inhibition zone reached 31.60 mm.

[0065] Example 5. Determination of the control effect of strain R10 on kiwifruit canker:

[0066] (1) Collect healthy shoots of the current-year "Hongyang" kiwifruit. First, perform surface disinfection (soak in 0.6% sodium hypochlorite solution for 20 min), then rinse 3 times with sterile water, and place them on filter paper to dry. Then cut them into 3 short shoots of appropriate length respectively, make 1 wound on each section, and treat the wound according to the grouping.

[0067] ① Treatment group with strain R10: First, add 10 μL of Psa M228 bacterial suspension (OD 600 = 0.3). After 24 h, add 10 μL of strain R10 bacterial suspension;

[0068] ② Prevention group with strain R10: First, add 10 μL of strain R10 bacterial suspension. After 4 h, add 10 μL of Psa M228 bacterial suspension (OD 600 = 0.3);

[0069] ③ Treatment group with Cu(OH)2 agent: First, add 10 μL of Psa M228 bacterial suspension (OD 600 = 0.3). After 24 h, add 10 μL of 500 μg / mL Cu(OH)2 agent;

[0070] ④ Prevention group with Cu(OH)2 agent: First, add 10 μL of 500 μg / mL Cu(OH)2 agent. After 24 h, add 10 μL of Psa M228 bacterial suspension (OD 600 = 0.3);

[0071] ⑤ M228 group (positive control): Only add 10 μL of Psa M228 bacterial suspension (OD 600 = 0.3);

[0072] ⑥ H2O group (blank control): Only add 10 μL of H2O.

[0073] (2) Place the inoculated branches of each group in an artificial climate incubator for cultivation (light cycle L / D: 16 h / 8 h; day and night temperature: 18 °C / 14 °C; relative humidity 90%). Observe and record the results.

[0074] (3) Use SPSS Statistics 21.0 software for statistical analysis. Use one-way analysis of variance (ANOVA), and use Duncan's test method for significant difference analysis, p < 0.05. Control effect % = (control lesion length - treated lesion length) / control lesion length × 100.

[0075] In this embodiment, the lesion lengths of each detached branch inoculated with Pseudomonas syringae pv. actinidiae were counted 21 days later, and according to different grouping treatment methods, the specific results were different, as Figure 7 shown.

[0076] It can be Figure 7 seen from the results that the average lesion length of the M228 group (positive control) was 18.5 mm; the average lesion length of the strain R10 treatment group was 10.5 mm, and the treatment effect was 43.24%, which was significantly better than that of the 500 μg / mL Cu(OH)2 agent treatment; the average lesion length of the strain R10 prevention group was 3.6 mm, and the prevention effect was 80.54%, which was significantly better than that of the treatment group and had no difference from the prevention effect of the 500 μg / mL Cu(OH)2 agent.

[0077] Therefore, under the condition of inoculating detached branches, the strain R10 of the present invention has a control effect on kiwifruit canker, and the prevention effect is better than the treatment effect.

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A novel extreme oriental Pseudomonas R10, characterized in that: Its classification name is Pseudomonas extremorientalis and it is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M2025304.

2. The novel extreme oriental Pseudomonas R10 according to claim 1, characterized in that The 16S rRNA gene sequence of the extreme oriental Pseudomonas orientalis R10 is shown in SEQ ID NO:

1.

3. The novel extreme oriental Pseudomonas R10 according to claim 1, characterized in that The extreme oriental Pseudomonas R10 has an inhibitory effect on the pathogenic kiwifruit bacteria of Pseudomonas syringae.

4. The novel extreme oriental Pseudomonas R10 according to claim 1, characterized in that The extreme oriental pseudomonas R10 has an inhibitory effect on tea ring spot pathogen, tea anthracnose pathogen, rapeseed sclerotinia pathogen, tomato canker pathogen and rice bacterial blight pathogen.

5. A biocontrol agent, characterized in that: A bacterial suspension, fermentation liquid or fermentation filtrate containing the novel extreme oriental Pseudomonas orientalis R10 according to any one of claims 1 to 4.

6. Use of the novel extreme oriental Pseudomonas orientalis R10 as claimed in any one of claims 1 to 4, or the biocontrol agent as claimed in claim 5 in the prevention and treatment of kiwifruit canker.

7. A method for preventing and treating kiwifruit canker, characterized in that: Utilize the novel extreme oriental Pseudomonas R10 described in any one of claims 1 to 4, or the biocontrol agent described in claim 5.

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