Biocontrol Chryseobacterium sp. With contact sterilization activity

Abstract

The invention discloses a biocontrol Chryseobacterium sp. With'contact sterilization 'activity. The PcE1 is a biocontrol bacterium which is separated from rhizosphere soil of Chinese cabbage by utilizing a germ enrichment method and can inhibit the growth of various pathogenic bacteria in a cell contact manner. The genome of the PcE1 encodes a type VI secretion system (T6SS) gene cluster and a plurality of T6SS effect proteins with antibacterial activity. The biocontrol effect determination on plants shows that the PcE1 can inhibit the infection of pectobacterium carotovorum on carrots and also can inhibit the infection of citrus canker pathogens on citrus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the separation, identification and application of Chryseobacterium cucumerinum PcE1 which can contact and inhibit multiple horticultural crop pathogenic bacteria. Background Art

[0002] As the country attaches great importance to sustainable agricultural development and environmental protection, biological control, as an environmentally friendly, safe and effective means of plant disease control, has become increasingly important in the prevention and control of plant diseases, especially horticultural crop diseases such as vegetables and fruits. Biocontrol bacteria can inhibit the growth of pathogens and enhance plant immunity, and are the main microbial resources for biological control. Biocontrol bacteria can inhibit the growth of pathogens through various pathways, such as antimicrobial metabolites, cell wall degrading enzymes, and nutritional competition. Among them, antimicrobial metabolites are a type of secondary metabolites with antimicrobial effects produced by biocontrol bacteria, which can diffuse into the environment to inhibit the growth of pathogens. This process does not require cell contact between bacteria and is considered to be the main biological control weapon of biocontrol bacteria.

[0003] At present, the mainstream screening of biocontrol bacteria is based on the ability to produce antimicrobial metabolites as the identification standard, and most biocontrol agents are based on live bacteria or antimicrobial metabolites as the main components. However, as secondary metabolites, the synthesis of bacterial antimicrobial metabolites requires specific environmental conditions to stimulate. On the one hand, the fermentation conditions of most biocontrol metabolites are relatively special, and the fermentation cost is high; on the other hand, many live bacterial biocontrol agents cannot produce antimicrobial metabolites in the field, and lose their biocontrol activity, which limits the application of biocontrol agents based on antimicrobial metabolites. Contact sterilization is a new type of biocontrol bacterial antibacterial mechanism recently discovered by our research group. Specifically, it refers to the process in which biocontrol bacteria, in the process of contact with pathogenic bacteria, inject effector proteins into pathogenic bacteria through contact-type secretion systems such as type IV secretion system (T4SS) and type VI secretion system (T6SS), thereby killing pathogenic bacteria. Contact sterilization activity depends on the cell contact between biocontrol bacteria and pathogenic bacteria, and does not depend on antimicrobial metabolites and nutritional conditions. It is a new resource for green control of plant diseases. Summary of the invention

[0004] The invention aims to provide a strain of Chryseobacterium cucumeris PcE1 having contact sterilization effect on various horticultural crop pathogenic bacteria and application thereof.

[0005] The purpose of the present invention can be achieved by the following solutions:

[0006] The present invention provides a cucumber Chryseobacterium PcE1, which is classified and named Chryseobacterium cucumeris, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 6, 2023, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain deposit number is CGMCC NO.28367; the genome has been published in the NCBI database, and the accession number is: CP132966.1.

[0007] The cucumber Chryseobacterium PcE1 of the present invention is obtained by enrichment and separation from the rhizosphere soil of cabbage.

[0008] The invention also provides a bacterial liquid containing the Chryseobacterium cucumerinum PcE1.

[0009] The bacterial solution of the present invention can be prepared according to conventional methods in the art. In a specific embodiment, the bacterial concentration of the bacterial solution of the present invention can be adjusted according to the sterilization requirements. In some embodiments, the bacterial concentration of the bacterial solution is 1*10 7 cfu / mL~1*10 9 cfu / mL.

[0010] The invention also provides the use of the cucumber Chryseobacterium cucumerinum PcE1 or a bacterial solution containing the strain in inhibiting pathogens.

[0011] In some embodiments, the pathogens described in the present invention are one or more of Pectobacterterium carotovorum subsp. carotovorum, Pcc, Xanthomonas axonopodis pv. citri, Xac, Pseudomonas syringae pv. actinidiae, Psa, or Pseudomonas syringae pv. phaseolicola, Psp. In some more specific examples, the pathogens described in the present invention are Pectobacterterium carotovorum subsp. carotovorum, Pcc, Xanthomonas axonopodis pv. citri, Xac, Pseudomonas syringae pv. actinidiae, Psa, or Pseudomonas syringae pv. phaseolicola, Psp.

[0012] The application of the present invention specifically involves applying Chryseobacterium PcE1 or bacterial liquid directly to the location of the pathogen, so that Chryseobacterium PcE1 is in direct contact with the pathogen.

[0013] The advantages of the technical solution of the present invention over the prior art are as follows:

[0014] The cucumber Chryseobacterium cucumerinum PcE1 of the present invention can inhibit the growth of various horticultural crop pathogenic bacteria by contact sterilization. Genome sequencing combined with protein expression analysis found that the genome of PcE1 encodes a set of type VI secretion system and 4 T6SS antibacterial effector proteins. Analysis of the biocontrol activity of PcE1 on plants found that it can inhibit the infection of Pcc on carrots and Xac on citrus, and has good biocontrol activity.

[0015] Unlike conventional biocontrol bacteria that secrete metabolites to inhibit the growth of pathogens, the cucumber Chryseobacterium PcE1 described in the present invention has a contact sterilization mechanism. Compared with bacteria that synthesize antibacterial metabolites, contact sterilization does not rely on specific nutritional conditions, nor does it require specific environmental conditions to stimulate the production of metabolites. Instead, it relies on cell contact between biocontrol bacteria and pathogenic bacteria, has better environmental adaptability, and can effectively avoid the loss of antibacterial activity caused by environmental changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 PcE1 can inhibit the growth of Pcc during mixed culture;

[0017] Figure 2 PcE1 can inhibit the growth of a variety of plant pathogenic bacteria through cell contact;

[0018] Figure 3 Phylogenetic tree analysis showed that PcE1 belonged to Chryseobacterium cucumerinum;

[0019] Figure 4 The PcE1 genome encodes a T6SS gene cluster and four T6SS effector proteins with antimicrobial activity;

[0020] Figure 5 PcE1 can inhibit the infection of Pcc to carrot;

[0021] Figure 6 PcE1 can inhibit the infection of Xac to citrus leaves. DETAILED DESCRIPTION

[0022] In order to make the principle and method of the present invention more clearly understood, the present invention is further described below with specific examples, but is not limited to these examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0023] Unless otherwise specified, Pcc in the following examples refers to Pectobacterterium carotovorum subsp. carotovorumc strain PccS1, Xac refers to Xanthomonas axonopodis pv. citri strain Xac003, Psa refers to Pseudomonas syringae pv. actinidiae strain M228, and Psp refers to Pseudomonas syringae pv. phaseolicola strain 1448A. These strains are all preserved by the research group and are promised to be permanently open to the public.

[0024] Example 1: PcE1 can inhibit the growth of Pectobacterium carotovora in mixed culture

[0025] The cucumber Chryseobacterium PcE1 provided by the present invention is isolated from the rhizosphere soil of Chinese cabbage soft rot (the pathogen is Pcc). The specific source is to culture Pcc in LB overnight, centrifuge to remove the culture medium, and resuspend the cells in sterile water to OD 600 =1.0. 20 μL of Pcc cell suspension was added to 20 mL of 100-fold diluted soil suspension and cultured at 28°C and 150 rpm for 2 days. The culture was then spread on a PVDF membrane with a pore size of 0.22 μm to simulate the contact between soil bacteria and Pcc cells, and the PVDF membrane was placed on an LB plate containing 100 μg / mL X-gal and cultured for 2 days. Pcc naturally has β-galactosidase activity, and the colonies are blue. The colonies around the blue colonies are picked as potential biocontrol bacteria for purification. The present invention preliminarily identified the isolated strains by sequencing the 16S rRNA gene sequence and comparing it in the NCBI database, and obtained a strain of Chryseobacterium cucumeris, named PcE1, and classified as Chryseobacterium cucumeris. The strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), with the strain deposit number CGMCC NO.28367; the genome has been sequenced and published in the NCBI database, with the accession number CP132966.1. Pcc and PcE1 strains were cultured in LB medium at 28°C overnight, centrifuged at 6000 rpm for 3 minutes to remove the medium, and then resuspended with sterile water to adjust the OD 600 =1.0(about 1*10 8cfu / mL). PcE1 strain was mixed with Pcc at a volume ratio of 1:15, 1:5, 1:1, 5:1, and 15:1, and then the mixed bacterial solution was inoculated on LA medium containing X-gal and cultured at 28°C for 2 days. Escherichia coli Top10 and Pseudomonas biocontrol Pf-5 were used as negative and positive controls, respectively, and mixed with Pcc at a volume ratio of 1:15, 1:5, 1:1, 5:1, and 15:1. Pcc naturally has β-galactosidase activity. If the mixed colony is blue, it means that Pcc is alive. The results show that PcE1 can inhibit the growth of Pcc ( Figure 1 ).

[0026] Example 2 PcE1 can inhibit the growth of various pathogenic bacteria through cell contact

[0027] The contact bactericidal spectrum of PcE1 was analyzed using GFP-labeled horticultural crop pathogens (Pcc, Xac, Psa, and Psp) stored in the laboratory. PcE1 and different pathogenic bacteria were cultured overnight in LB, centrifuged at 6000 rpm for 3 minutes to remove the culture medium, and then resuspended in sterile water to adjust the OD 600 =1.0. PcE1 and pathogenic bacteria were mixed in a 1:1 ratio, and PcE1, pathogenic bacteria, and PcE1 / pathogen mixed bacterial solution were inoculated on the same LA plate; at the same time, a sterile filter membrane with a pore size of 0.22 μm cut to an appropriate size was inserted into the culture medium, and PcE1 and pathogenic bacteria were inoculated on both sides of the filter membrane as a non-cell contact control. After 1 day of culture, the GFP fluorescence signal of the colony was detected using a biomacromolecule analyzer (VilberFusion Fx7), and it was found that Pcc, Xac, Psa, and Psp cultured alone all had GFP signals, indicating that these pathogenic bacteria survived normally; while the mixed colonies of these pathogenic bacteria and PcE1 had no GFP signals, indicating that PcE1 can kill these pathogenic bacteria during the mixed culture process. When PcE1 and pathogenic bacteria were separated by the filter membrane, they lost their bactericidal ability, indicating that the bactericidal activity of PcE1 against these pathogenic bacteria is dependent on cell contact ( Figure 2 ).

[0028] Example 3 PcE1 genome encodes four antibacterial T6SS effector proteins

[0029] The contact bactericidal activity of bacteria is usually mediated by contact protein secretion systems (such as T4SS and T6SS). The present invention sequenced the genome of PcE1 and found that the genome size of PcE1 was 5.02Mb, and it was predicted to encode 4666 genes. The phylogenetic tree analysis of PcE1 based on the whole genome was performed using the TYGS (https: / / tygs.dsmz.de / ) platform, and it was found that PcE1 was most closely related to Chryseobacterium cucumerinum GSE06, indicating that PcE1 belongs to Chryseobacterium cucumerinum ( Figure 3 ). The protein secretion system encoded by the PcE1 genome was predicted using the TXSEDB (https: / / tool2-mml.sjtu.edu.cn / TXSEDB / ) platform, and it was found that the PcE1 genome contained a T6SS gene cluster ( Figure 4 A); The prediction of T6SS effector proteins revealed that in addition to VgrG1, VgrG2 and PAAR1 located in the T6SS gene cluster, two possible T6SS effector proteins (T6SE1 and T6SE2) were predicted in the genome of PcE1 ( Figure 4 B). The five T6SS effector proteins were analyzed for their bactericidal activity using the E. coli inducible expression system. The effector protein genes were constructed into the IPTG (isopropylthiogalactoside)-inducible periplasmic expression vector pET22b(+)-PldB, and the constructed vectors were transferred into the E. coli expression strain BL21. The BL21 transformants were cultured overnight in LB, centrifuged at 6000 rpm for 3 minutes to remove the culture medium, and then resuspended in sterile water to adjust the OD 600 =1.0. The bacterial solution was then diluted 10-fold and inoculated on LB (non-induced) and LB+0.1mM IPTG (induced) plates, with BL21 transformed with an empty vector (EV) as a negative control and BL21 transformed with pldB (antibacterial T6SS effector protein in Pseudomonas aeruginosa PA01) as a positive control. The cells were observed after 1 day of culture. The results showed that VgrG1, VgrG2, PAAR1 and T6SE1 could inhibit the growth of Escherichia coli ( Figure 4 C), indicating that PcE1 may inject antimicrobial effector proteins into pathogenic bacteria through T6SS to achieve contact sterilization.

[0030] Table 1 Effector protein expression primers used in this patent

[0031]

[0032] Example 4 PcE1 can inhibit the pathogenicity of Pectobacterium carotovora

[0033] In this example, whether PcE1 can inhibit the pathogenicity of Pcc was tested. Carrots were sliced ​​and the surface of the cut surface was disinfected with 70% ethanol. After the ethanol evaporated, 100 μL of PcE1 bacterial solution (LB cultured to OD 600 =1.0), LB was used as negative control, and the carrot slices were incubated at 28°C for 6 hours after drying. Then, a wound was punctured on the surface of the carrot with a syringe, and 20 μL of Pcc bacterial solution (LB cultured to OD 600 =1.0) was inoculated at the wound, cultured at 28°C for 2 days, and the lesions were observed. The carrots were weighed, the rotten parts were removed, and weighed again to calculate the weight of the rotten parts. The results showed that PcE1 treatment could inhibit the infection of Pcc on carrots ( Figure 5 ).

[0034] Example 5 PcE1 can inhibit the pathogenicity of citrus canker bacteria

[0035] PcE1 and Xac were cultured in LB until OD 600 =1.0, then PcE1 and Xac were mixed at a volume ratio of 1:1, and the LB / Xac mixture was used as a negative control. Fresh citrus leaves were taken, and wounds were punctured on the surface of the leaves with a syringe. The mixed bacterial liquid was then inoculated into the wounds, and the lesions were observed at 28°C for 7 days. The degree of disease was calculated by measuring the diameter of the lesions. The results showed that PcE1 treatment could significantly inhibit the infection of Xac on citrus leaves ( Figure 6 ).

Claims

1. A strain of Chryseobacterium cucumeris PcE1, classified and named Chryseobacterium cucumeris, with a strain collection number of CGMCC NO.28367.

2. Use of the Chryseobacterium spp. PcE1 according to claim 1 in inhibiting pathogens.

3. The use according to claim 2, characterized in that: The pathogens are one or more of carrot soft rot pectinobacterium, citrus canker bacteria, kiwi canker bacteria or bean blight bacteria.

4. The use according to claim 2, characterized in that: The pathogen is carrot soft rot and / or citrus canker.

5. The use according to any one of claims 2 to 4, characterized in that: Specifically, Chryseobacterium PcE1 is directly applied to the location of the pathogen, so that Chryseobacterium PcE1 is in direct contact with the pathogen.

6. A bacterial liquid containing the Chryseobacterium tumefaciens PcE1 according to claim 1.

7. The bacterial solution according to claim 1, characterized in that The bacterial concentration of the bacterial solution is 1*10 7 cfu / mL~1*10 9 cfu / mL.

8. Use of the bacterial solution according to claim 5 or 6 in inhibiting pathogens.

9. The use according to claim 8, characterized in that: The pathogen is one or more of Pectobacterium carotovora, Pseudomonas aeruginosa, Pseudomonas aeruginosa or Pseudomonas aeruginosa, preferably Pseudomonas aeruginosa and / or Pseudomonas aeruginosa.

10. The use according to claim 8 or 9, characterized in that: Specifically, the bacterial solution is directly applied to the location of the pathogen, so that Chryseobacterium PcE1 is in direct contact with the pathogen.

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