CN114480227B

CN114480227B - Pseudomonas aeruginosa and application thereof in preventing and treating bacterial soft rot of plants

Info

Publication number: CN114480227B
Application number: CN202210346599.9A
Authority: CN
Inventors: 周佳暖; 刘帆; 胡明; 王思; 张炼辉
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2023-07-07
Anticipated expiration: 2042-04-02
Also published as: CN114480227A

Abstract

The invention discloses pseudomonas aeruginosa and application thereof in preventing and treating bacterial soft rot of plants. The Pseudomonas aeruginosa L5 has the preservation number of GDMCCNO.62059, has good quenching effect on the VFM colony induction signals generated by soft rot fungi of Dickeya, can obviously reduce the occurrence of bacterial soft rot of various crops, and has good control effect in potting experiments; in addition, the pseudomonas aeruginosa L5 has no pathogenicity, can be used for developing biocontrol agents aiming at crop bacterial soft rot, and provides a new thought and a new material for biologically controlling the crop bacterial soft rot.

Description

Pseudomonas aeruginosa and application thereof in preventing and treating bacterial soft rot of plants

Technical Field

The invention belongs to the technical field of plant disease biocontrol, and in particular relates to pseudomonas aeruginosa and application thereof in preventing and treating bacterial soft rot of plants.

Background

Quorum Sensing (QS) is a process of intercellular communication that enables bacteria to collectively change behavior in response to changes in cell density and environment of surrounding microbial communities. The quorum sensing system among bacteria becomes a research hotspot in the field of microorganisms in the last ten years, not only reveals a regulation network affecting important life processes of microorganisms, but also brings new hopes for research and treatment of microbial infectious diseases. The group quenching (QQ) refers to blocking signal communication among bacteria by interfering or destroying a group induction system under the condition of not generating drug resistance and not affecting microorganism growth, thereby achieving the aim of effectively inhibiting virulence gene expression, being a novel environment-friendly pathogen control strategy and having good development prospect.

Soft rot Enterobacteriaceae (Soft-rot Enterobacteriaceae, SRE) is mainly composed of Pectobacterium genus and Dickeya genus, and is a kind of gram-negative pathogenic bacteria causing destructive diseases of various grains, vegetables and ornamental plants worldwide. Currently, 12 species are known from Dickeya to produce two types of Quorum Sensing (QS) signals AHL (Acyl homoserine lactone) and VFM (Virulence Factor Modulating) that regulate different biological phenotypes of pathogenic bacteria. Early-stage researches show that AHL signals mainly regulate the motility and biofilm formation of pathogenic bacteria, and have little influence on the pathogenicity of the pathogenic bacteria; and VFM is a novel QS signal, mainly controls the generation of virulence factors of pathogenic bacteria, and obviously controls the pathogenicity of the pathogenic bacteria. Therefore, the screening of the QQ biocontrol strain through the targeted VFM signal has great significance for the safe and efficient biocontrol of bacterial soft rot. Currently, the chemical structure of the VFM colony induction signals is unknown, so that the VFM colony induction signals become one of barriers for the development of the Dickeya soft rot fungus QQ technology.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the existing crop bacterial soft rot control technology, provides a biocontrol bacterium-pseudomonas aeruginosa L5 with excellent quenching effect on the VFM quorum sensing signals of the Dickeya crop soft rot bacteria, provides a new development resource for replacing chemical synthesis bactericides with microorganisms, and can be used as biological pesticides for development and utilization.

The invention utilizes a VFM quorum sensing signal reporting system VR2 (disclosed in patent application CN 202110772560.9) constructed in advance to screen a pseudomonas aeruginosa Pseudomonas chlororaphis L from the environment, and can remarkably reduce pathogenicity of various crop soft rot fungi by quenching the VFM quorum sensing signal on the premise of not influencing the growth of the reporting system VR2, thereby effectively preventing and controlling the bacterial soft rot of the crops.

The invention discovers that the pseudomonas aeruginosa L5 strain can efficiently quench the VFM colony induction signals of Dickeya, does not influence the growth of bananas, rice, taros and potatoes in a greenhouse potting test, and can effectively inhibit the infection of banana bacterial soft rot fungi D.zeae MS2 on banana and rice bacterial basal rot fungi D.oryzae EC1 on rice and potato soft rot fungi D.dadantii 3937 on potato and taro soft rot fungi D.fangzhongdai CL3 on taros. Illustrating that the pseudomonas aeruginosa L5 of the present invention can be used to develop biopesticides against bacterial soft rot of crops. The discovery of the strain is beneficial to alleviating the problem of abuse of chemical agents and provides resources for preventing and controlling bacterial soft rot of crops by utilizing a group quenching technology.

Accordingly, it is a first object of the present invention to provide a Pseudomonas aeruginosa L5 having accession number GDMCC No.62059.

The invention also provides application of the pseudomonas aeruginosa L5 in preventing and treating plant bacterial soft rot.

Preferably, the plant bacterial soft rot is a bacterial disease caused by bacteria of the genus Dickeya and mediated by a sensing signal molecule dependent on the VFM population.

More preferably, the plant bacterial soft rot is banana bacterial soft rot, rice bacterial basal rot, potato soft rot and taro soft rot.

Preferably, the use comprises the step of inoculating Pseudomonas aeruginosa L5 and/or a culture comprising Pseudomonas aeruginosa L5 to the plant body.

Preferably, root injury bacteria-filling inoculation method is adopted for inoculation.

Preferably, the inoculation is performed by injection inoculation.

Preferably, the inoculation adopts a stabbing inoculation method.

The invention also provides a biocontrol agent for plant bacterial soft rot, which contains pseudomonas aeruginosa L5 and/or a culture containing pseudomonas aeruginosa L5 as an active ingredient.

Preferably, the biocontrol agent is a bacterial suspension of pseudomonas aeruginosa L5 with the concentration of OD ₆₀₀ 1.0～1.5。

The invention has the following beneficial effects:

the result of the invention shows that the pseudomonas aeruginosa L5 can efficiently quench the VFM quorum sensing signals generated by the bacteria of the genus Dickeya; in the inoculation test, the pseudomonas aeruginosa L5 is not pathogenic to bananas, rice, potatoes and taros, but can well inhibit infection of pathogenic bacteria D.zeae MS2 to bananas, D.oryzae EC1 to rice, D.dadantii 3937 to potatoes and D.fangzhhongdai CL3 to taros, and reduce the incidence of bacterial soft rot of the taros. In addition, the pseudomonas aeruginosa L5 has no pathogenicity, can be used for developing biopesticide aiming at crop bacterial diseases, and provides a new thought and new strain resources for biologically preventing and treating crop bacterial soft rot.

The invention provides pseudomonas aeruginosa L5 and proves that the pseudomonas aeruginosa L5 has good quenching effect on the VFM colony induction signals of crop bacterial soft rot, obviously reduces the occurrence of various crop bacterial soft rot and has excellent control effect in potting experiments. In addition, the pseudomonas aeruginosa L5 is self-avirulent, can be used for developing biocontrol agents aiming at crop bacterial soft rot, and provides new ideas and materials for biocontrol of crop bacterial soft rot.

Preservation description:

pseudomonas chlororaphis L5 (Pseudomonas aeruginosa L5) of the invention was deposited at the Guangdong province microbiological bacterial deposit center (GDMCC) at 11/12 of 2021, accession number: GDMCC No.62059, deposit address: building 5, guangzhou city, first, middle road 100, university, 59, university of Guangdong, academy of sciences of China, and microbiological study.

Drawings

FIG. 1 is a graph showing the antagonistic effect of strain L5 on Dickeya zeae MS2, a banana bacterial soft rot fungus.

FIG. 2 is a graph showing the effect of flow cytometry on the quenching of VFM signals in VR2 reporter system by strain L5.

FIG. 3 is a cluster map of strain L5 based on the 16S rDNA gene sequence.

FIG. 4 is a graph of a joint phylogenetic cluster of strain L5 based on rpoB and rpoD gene sequences.

Fig. 5 shows that strain L5 significantly reduced the occurrence of bacterial soft rot symptoms on bananas and rice.

Fig. 6 shows that strain L5 significantly reduced the occurrence of bacterial soft rot symptoms on potatoes and taros.

Detailed Description

The following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1: isolation and screening of Strain L5

1. Isolation of strains

Sample collection: collecting field soil samples in the Hubei Xiaozhong paddy field, bagging and preserving, and carrying the soil samples back to a laboratory for strain separation.

Isolation of strains: the soil samples were subjected to strain isolation using a dilution-spread plate method. Specifically, after the collected field soil samples are fully and evenly mixed, weighing 5.0g of the mixture to be placed in a sterilization centrifuge tube filled with 15mL of fresh LB liquid medium, shake culturing the mixture for 15min at 28 ℃ and 200rpm, standing the mixture until soil particles sink to the bottom of the centrifuge tube, taking supernatant to be diluted with sterile water in a gradient manner, and sucking 100 mu L of 10 ^-5 、10 ^-6 、10 ^-7 The diluted solution is coated on an LB plate, and the single colony obtained after the dilution is inversely cultured in a constant temperature incubator at 28 ℃ for 24 hours is streaked and purified. Single colony on the purified flat plate is picked, marked and placed in a 2mL centrifuge tube containing 1mL of liquid LB, and shake-cultured at 28 ℃ and 200rpm shaking table overnight to be used as bacterial liquid to be screened for later use.

2. Strain screening: and (5) high-throughput screening by using an enzyme-labeled instrument. Specifically, VR2 reporter strain (disclosed in patent application CN202110772560.9, which is a VFM colony induction signal reporter strain obtained by genetically engineering banana bacterial soft rot fungus Dickeya zeae MS 2) is streaked and activated by LB+Kan solid culture medium, single colony is selected and placed in LB+Kan liquid culture medium, and shake-cultured at 28 ℃ and 200rpm until the bacterial liquid OD ₆₀₀ The preparation method comprises the steps of adding (1.0) the components into LB liquid culture medium according to a ratio of 1:100 (inoculum size), adding 200 mu L of the components into a 96-well culture plate after uniformly mixing, inoculating the prepared bacteria liquid to be screened into the inoculated components with 0.1%, repeating each sample treatment three times, taking the sterile LB liquid culture medium as a blank control, and taking E.coli DH5 alpha with 0.1% inoculum size as a negative control. After inoculation, sealing film is stuck, and a plate cover is covered, and the mixture is put into an enzyme-labeled instrument for continuous measurement for 24 hours, and OD is measured every three hours ₆₀₀ And OD (optical density) ₄₈₅ /OD ₅₂₈ . Preliminary screening for VFM quorum sensing of pathogenic bacteriaStrains whose signal has a quenching effect.

3. Bacterial strain bacteriostasis assay: and (5) performing bacteriostasis circle experiments. Specifically, the strains obtained by preliminary screening by using an enzyme-labeled instrument are picked up as single colonies in a 50mL centrifuge tube containing 15mL of LB culture solution, and shake culture is carried out at 28 ℃ and 200rpm for later use. Pouring 15mL of melted solid LB culture medium into a square culture dish with the length of 10 multiplied by 10cm, drying, pouring 15mL of 1% agarose (firstly cooling to 50-60 ℃ and then adding 150 mu L of bacterial soft rot fungus Dickeya zeae MS2 bacterial liquid) into the square culture dish paved with LB, and drying. Punching with a puncher with the diameter of 5mm, sterilizing the toothpick, picking the culture medium, injecting 20 mu L of the bacteria liquid to be tested cultured overnight into the hole, drying, and sealing. The plates are placed in a 28 ℃ incubator for 16-18 hours, and whether transparent inhibition zones exist or not is observed. A biocontrol strain which has quenching effect on the quorum sensing signals of pathogenic bacteria VFM and has no antibacterial effect is obtained and is named as strain L5 (figure 1).

Example 2: flow cytometry verifies the quenching effect of strain L5 on VFM

Marking and activating VR2 report strain with LB+Kan solid culture medium, picking single colony, placing into LB+Kan liquid culture medium, shake culturing at 28deg.C with 200rpm shaking table until bacterial liquid OD ₆₀₀ Equal amounts of reporter strain VR2 and strain L5 (quenched) were inoculated and mixed in culture, and the sample fluorescence intensity MFI was measured every four hours with access to only reporter strain VR2 as a control. As can be seen from FIG. 2, strain L5 significantly quenched the VFM signal produced by VR2 when the strain was cultured for 4-8 hours, and the VR2 self-quenched gene degraded the VFM signal in the system when the strain was cultured for 12 hours, with and without strain L5, and the value of the VFM signal responsive to the reporting system was drastically reduced compared with the original value.

Example 3: identification of Strain L5

1. Morphological identification

Strain L5 is gram negative; the bacterial colony on the LB nutrient medium is orange-yellow, round, convex, smooth in surface, sticky, easy to pick up and neat in edge.

2. Molecular characterization

To clarify the taxonomic status of strain L5 obtained in example 1, we identified strain L5 by a method of multi-site sequence analysis (Multilocus sequences analysis, MLSA) in combination with phylogenetic tree analysis (Phylogenetic analysis).

The 16S rDNA sequence analysis is a molecular biological identification means for bacteria identification, a phylogenetic tree is constructed by analyzing a 16S rDNA sequence (the nucleotide sequence is shown as SEQ ID NO. 1) and combining a conserved housekeeping gene rpoB (the nucleotide sequence is shown as SEQ ID NO. 2) and a rpoD sequence (the nucleotide sequence is shown as SEQ ID NO. 3) and adopting a Neighbor-joining method by using MEGA 6.0 software, the evolution relation of the strain L5 is comprehensively analyzed, and researches show that the 16S rDNA sequence of the strain L5 and the strains of pseudomonas aeruginosa Pseudomonas chlororaphis LMG 21630 and Lzh-T5 reach the highest 99.93 percent, the evolution coefficient is most similar to that of the strain P.chlorofis LMG 21630 (figure 3), and the strain L5 is found to be most similar to that of the strain P.chlorofis ATCC 13985 and PCLRTO2 by analyzing the rpoD gene sequences (figure 4).

In summary, by combining phylogenetic analyses of a plurality of housekeeping genes, the biocontrol strain is identified as Pseudomonas chlororaphis of pseudomonas and named as pseudomonas aeruginosa Pseudomonas chlororaphis L; and the strain was deposited with the Guangdong province microorganism strain collection (GDMCC) at 11 and 12 days 2021, accession number: GDMCC No.62059.

Example 4: determination of the control effect of Strain L5 on bacterial Soft rot

The pathogenic bacteria banana bacterial soft rot fungi D.zeae MS2, rice bacterial basal rot fungi D.oryzae EC1, potato soft rot fungi D.dadantiii 3937, taro soft rot fungi D.fangzhongdai CL3 (hereinafter abbreviated as strains MS2, EC1, 3937 and CL3 respectively) and (biocontrol) strain L5 are activated on an LB solid medium, and are inverted in an incubator at 28 ℃ for overnight culture for later use.

1. Washing the bacterial cells on the MS2 flat plate and the L5 flat plate respectively by PBS solution, and washing the MS2 bacterial suspension and the L5 bacterial suspension OD ₆₀₀ Adjusting to 0.5; taking 1mL of MS2 bacterial suspension and an equal amount of PBS solution to mix (MS2) as a positive control; 1mL of L5 bacterial suspension was mixed with an equal amount of MS2 bacterial suspension (L5+Ms2); 1mL of L5 bacteria suspension was mixed with an equal amount of PBS solution (L5) asNegative control; 2mL of PBS solution (PBS) was used as a blank.

The bananas are inoculated by an injection inoculation method. 12 banana seedlings with similar growth vigor are selected, 200 mu L of MS2, L5+MS2, L5 and PBS are respectively taken by a 1mL injector to be injected into the pseudostems of the bananas, 3 repeats are treated each time, and the growth condition of the banana seedlings is observed every day to record.

The experimental result shows that the control rate of the bacterial soft rot of the banana by the strain L5 reaches 100% after 7 days of inoculation (figure 5A).

2. Washing the bacterial cells on the strain EC1 plate and the strain L5 plate respectively by PBS solution, and washing the EC1 bacterial suspension and the L5 bacterial suspension OD ₆₀₀ Adjusting to 0.5; 10mL of EC1 bacterial suspension and equal amount of PBS solution are mixed (EC1) to serve as positive control; 10mL of L5 bacterial suspension is mixed with the same amount of EC1 bacterial suspension (L5+EC1); 10mL of L5 bacterial suspension and an equal amount of PBS solution are mixed (L5) to serve as negative control; 20mL of PBS solution (PBS) was used as a blank. After the volume of all bacterial solutions is fixed to 100mL by PBS, the bacterial solutions are inoculated into the soil of a rice pot by adopting a root injury bacteria filling method (the root of the rice is treated by stabbing), each pot is provided with 12 rice seedlings with equal growth vigor, each treatment is repeated for 3 times, and the growth condition of inoculated plants is observed every day to record.

Experimental results show that after two days of inoculation, the prevention and control rate of the bacterial basal rot of rice by the strain L5 reaches 100% (figure 5B).

3. Single colonies of strain 3937, CL3 and strain L5 were picked separately in 50mL centrifuge tubes containing 10mL LB and placed in a shaker at 28℃and 200rpm overnight. 1mL OD ₆₀₀ 3937 or CL3 bacterial liquid of about 1.0 is added into the equivalent LB liquid culture medium to be mixed and used as positive control respectively; 1mL OD ₆₀₀ 3937 or CL3 bacterial liquid with the concentration of about 1.0 is added into the L5 bacterial liquid with the same amount and mixed; 1mL OD ₆₀₀ About 1.0L 5 broth was mixed with an equal amount of LB broth as a negative control, and LB broth as a blank control.

The potato and taro were inoculated separately using the stab inoculation method, 3 replicates per treatment. After potato and taro slices are processed, 2 mu L of 3937 bacteria liquid and LB mixed liquid (3937), 3937 bacteria liquid and L5 bacteria liquid mixed liquid (L5+3937), L5 bacteria liquid and LB mixed liquid (L5) and LB liquid culture medium (LB) are respectively inoculated to the potato slices subjected to stab wound processing; and respectively inoculating 2 mu L of CL3 bacterial liquid and LB mixed liquid (CL 3), CL3 bacterial liquid and L5 bacterial liquid mixed liquid (L5+CL3), L5 bacterial liquid and LB mixed liquid (L5) and LB liquid culture medium (LB) onto the stabbed taro slices, observing the disease condition of the inoculated slices, and recording.

Experimental results show that strain L5 significantly reduced the onset of bacterial soft rot on potato and taro 24 hours after inoculation (FIG. 6).

Sequence listing

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<120> Pseudomonas aeruginosa strain and application thereof in preventing and treating bacterial soft rot of plants

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atcctgtttg ctccccacgc tttcgcacct cagtgtcagt atcagtccag gtggtcgcct 780

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Claims

1. Pseudomonas aeruginosa L5, characterized in that it has deposit number GDMCCNO.62059.

2. Use of pseudomonas aeruginosa L5 according to claim 1 for controlling plant bacterial soft rot caused by Dickeya bacteria and relying on VFM colony inducing signal molecules to mediate pathogenic banana bacterial soft rot, rice bacterial basal rot, potato soft rot and taro soft rot.

3. Use according to claim 2, characterized in that it comprises the step of inoculating pseudomonas aeruginosa L5 and/or a bacterial liquid comprising pseudomonas aeruginosa L5 to the plant body.

4. The use according to claim 3, wherein said inoculation is by root-injured bacteria inoculation.

5. The use according to claim 3, wherein said inoculation is by injection inoculation.

6. The use according to claim 3, wherein said inoculation is by a stab inoculation method.

7. A biocontrol agent for plant bacterial soft rot characterized by comprising the Pseudomonas aeruginosa L5 according to claim 1 and/or a bacterial liquid containing the Pseudomonas aeruginosa L5 according to claim 1 as an active ingredient.

8. The biocontrol formulation of claim 7, wherein the biocontrol formulation is a bacterial suspension of pseudomonas aeruginosa L5 at OD concentration ₆₀₀ 1.0～1.5。