CN112760267B - An Enzyme-producing Lysobacter CX06 against Xanthomonas campestris and its Application - Google Patents

Abstract

The invention discloses an lysobacter enzymogenes CX06 for antagonizing xanthomonas campestris and application thereof, wherein the lysobacter enzymogenes CX06 has been preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 07-08 days in 2020, and the preservation number is CGMCC No.20321. The invention also discloses application of the lysobacter enzymogenes CX06 in inhibiting pathogenic bacteria. In the bacteriostatic spectrum determination test, the strain CX06 shows broad-spectrum resistance and has antagonistic effect on 7 pathogenic fungi and 5 pathogenic bacteria. In a greenhouse pot culture control effect test, the control effect of the strain CX06 on cabbage black rot is as high as 90.7%, the control effect on cabbage stem basal rot is 62.8%, and the control effect on cabbage bacterial black rot is 79.98%, which shows that the strain CX06 has a good application prospect and provides a certain theoretical basis and technical support for further development and utilization.

Description

An Enzyme-producing Lysobacterium CX06 Antagonizing Xanthomonas campestris and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an enzyme-producing lysobacterium CX06 strain antagonizing Xanthomonas campestris and its application.

Background technique

At present, the control measures against Xanthomonas campestris in production mainly rely on disease-resistant breeding and chemical control. Pesticides commonly used in chemical control include some agricultural antibiotics (kasugamycin), copper preparations (copper quinolate), etc. Generally speaking, there are fewer types of pesticides used to prevent and control Xanthomonas campestris disease, and the use of some pesticides will be inhibited, and some control effects are mediocre. Therefore, there is an urgent need to develop more new, efficient and safe control measures in production . Among them, biological control is a control method that uses beneficial microorganisms to inhibit pathogenic bacteria, reduce the number of pathogenic bacteria, prevent the infection of pathogenic bacteria, and reduce the occurrence of diseases. Bacterial disease control has great development prospects.

Lysobacter enzymogenes belong to the family Xanthomonadaceae, order Xanthomonadales, class Gammaproteobacteria, and phylum Proteobacteria. Lysobacterium enzymogenes has high-efficiency antagonistic activity and potential for biocontrol of plant diseases, and is a new type of biocontrol bacteria for plant diseases. Lysobacterium enzymogenes has strong chitinase, protease, and cellulase activities, and can dissolve Gram-positive and negative bacteria, filamentous fungi, yeast, and algae. Its disease prevention mechanism is: (1) rich in extracellular enzymes; (2) secreting biosurfactants; (3) producing antibiotics; (4) better colonization ability. Therefore, the isolation and identification of zymolysobacterium strains that have antagonism to Xanthomonas campestris, from which antibacterial active substances can be obtained, is beneficial to the development of zymolysobacterium strains with safe, high-efficiency, and broad-spectrum biocontrol potential, as well as biocontrol resources. It is of great significance to build, protect the ecological environment and reduce the use of chemical pesticides.

Contents of the invention

The object of the present invention is to provide an enzyme-producing lysobacterium CX06 strain antagonizing Xanthomonas campestris and its application.

In order to achieve the above object, the present invention firstly provides Lysobacter enzymogenes CX06, whose preservation number is CGMCC No.20321.

The Lysobacter enzymogenes CX06 provided by the present invention has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures (CGMCC for short) on July 08, 2020; address: Courtyard 3, No. 1 Beichen West Road, Chaoyang District, Beijing No., Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101), and the deposit number is CGMCCNo.20321.

In order to achieve the above object, the present invention further provides the bacterial suspension or culture broth or fermentation broth or fermentation product of the above-mentioned Lysobacter enzymogenes CX06 or a microbial agent containing it.

In order to achieve the above object, the present invention also provides the application of the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture liquid or fermentation liquid or fermentation product or a bacterial agent containing it in inhibiting pathogenic bacteria.

The present invention also provides the application of the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture liquid or fermented liquid or fermented product or the bacterial agent containing it in the preparation of products for inhibiting pathogenic bacteria.

The present invention also provides the application of the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture liquid or fermented liquid or fermented product or the bacterial agent containing it in the prevention and treatment of plant diseases caused by pathogenic bacteria.

The present invention also provides the application of the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture liquid or fermentation liquid or fermentation product or the bacterial agent containing it in the preparation of products for preventing and controlling plant diseases caused by pathogenic bacteria .

The present invention also provides the above-mentioned Lysobacter enzymogenes (Lysobacter enzymogenes) CX06 or its bacterium suspension or culture liquid or fermentation liquid or fermentation product or the bacterium agent containing it in preventing and treating cabbage black rot and/or cabbage stalk rot and / or application in cabbage bacterial black rot.

The present invention also provides the above-mentioned Lysobacter enzymogenes (Lysobacter enzymogenes) CX06 or its bacterial suspension or culture liquid or fermentation liquid or fermentation product or the bacterial agent containing it in the preparation and control of cabbage black rot and/or cabbage stalk rot And/or application in products of cabbage bacterial black rot.

In order to achieve the above object, the present invention also provides a product whose active ingredient is the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture broth or fermentation broth or fermentation product or a microbial agent containing it;

The product has any one of the following functions a1)-a3):

a1) inhibit pathogenic bacteria;

a2) Prevent and control plant diseases caused by pathogenic bacteria;

a3) Controlling cabbage black rot and/or cabbage stalk rot and/or cabbage bacterial black rot.

In order to achieve the above object, the present invention finally provides any method in the following b1)-b3):

b1) A method for inhibiting pathogenic bacteria, comprising the steps of: treating pathogenic bacteria with the above-mentioned Lysobacterenzymogenes CX06 or its bacterial suspension or culture fluid or fermentation broth or fermentation product or a microbial agent containing it;

b2) A method for preventing and treating plant diseases caused by pathogenic bacteria, comprising the steps of: using the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or culture liquid or fermentation liquid or fermentation product or a bacterial agent containing it handling plants;

b3) a method for preventing and treating cabbage black rot and/or cabbage stalk rot and/or cabbage bacterial black rot, comprising the steps of: using the above-mentioned Lysobacter enzymogenes CX06 or its bacterial suspension or Brassica oleracea or Chinese cabbage treated with culture liquid or fermented liquid or fermented product or bacterial agent containing it.

Any of the above-mentioned pathogenic bacteria is a plant pathogenic bacteria. The phytopathogenic bacteria can be pathogenic fungi or pathogenic bacteria.

Described pathogenic fungus specifically can comprise Corynespora cassiicola, Fusarium oxysporum, Phytophthora capsici, Botrytiscinerea, Rhizoctonia solani ( Rhizoctonia solani), Stemphylium solani, Ascochyta citrullina.

The pathogenic bacteria may specifically include Xanthomonas campestris pv. campestris, Clavibacter michiganensis subsp. sepedonicus, Pseudomonas syringae Pseudomonassyringae pv. lachryrnans, Agrobacterium vitis, Clavibacter michiganensis subsp. michiganensis.

Any of the above-mentioned plant diseases include black rot and foot rot. The black rot may specifically be black rot of cabbage or bacterial black rot of cabbage. Specifically, the base rot may be root rot of cabbage stems.

Any one of the plants mentioned above can specifically be cabbage or Chinese cabbage.

The bacterial concentration of Lysobacter enzymogenes CX06 in any of the above-mentioned bacterial suspensions or culture broths or fermentation broths or fermentation products or bacterial agents can be 1×10 ⁷ cfu/mL-1×10 ⁹ cfu/mL mL, specifically 1×10 ⁸ cfu/mL.

In a specific embodiment, the specific preparation method of the bacterial suspension or culture fluid or fermentation broth or fermentation product or bacterial agent may include the following steps: inoculating Lysobacter enzymogenes CX06 in LB liquid medium , 28°C, 180r/min shaking culture to a concentration of 1×10 ⁸ cfu/mL.

In another specific embodiment, the specific preparation method of the bacterial suspension or culture fluid or fermentation broth or fermentation product or microbial agent may include the following steps: inoculating Lysobacter enzymogenes CX06 in LB liquid culture medium medium, 28°C, 180r/min shaking culture to a concentration of 1×10 ⁸ cfu/mL, and then diluted with distilled water to 100 times the volume.

The present invention isolates a strain CX06 which has obvious antagonistic effect on the pathogenic variety of xanthomonas oleifera from crop rhizosphere soil. The strain CX06 was preliminarily identified as Lysobacter enzymogenes after analyzing the strain's morphological characteristics, Biolog detection, electron microscope scanning, physiological and biochemical indicators and multigene phylogenetic tree. On different media, strain CX06 grows very fast. On NA medium, LB medium, and TSA medium, the colonies are pale yellow, with prominent surfaces, moist and shiny bacteria, and are not easy to provoke; strain CX06 is resistant to pathogenic fungi Mycelium has a certain influence, which will cause mycelium to expand, twist, deform and affect the normal growth and reproduction of mycelium. The enzyme activity test of the strain CX06 was carried out, and the metabolites of the strain CX06 were determined to contain cellulase, chitinase and protease. In the antibacterial spectrum test, the strain CX06 showed broad-spectrum resistance, and had antagonistic effects on 7 kinds of pathogenic fungi and 5 kinds of pathogenic bacteria. In the greenhouse potted control effect test, the control effect of strain CX06 on cabbage black rot was as high as 90.7%, the control effect on cabbage stalk rot was 62.8%, and the control effect on cabbage bacterial black rot was 79.98%. It shows that the strain CX06 has a good application prospect, which provides a certain theoretical basis and technical support for further development and utilization.

Description of drawings

Figure 1 shows the 8 strains obtained by isolation and screening of soil samples. A: CX01; B: CX02; C: CX03; D: CX04; E: CX05; F: CX06; G: CX07; H: CX08.

Figure 2 shows the morphological characteristics of strain CX06 on different media. A: LB medium; B: TSA medium; C: NA medium; D: 10% TSA medium.

Figure 3 is a scanning electron micrograph of strain CX06.

Fig. 4 is the phylogenetic tree of strain CX06 based on polygene sequence.

Figure 5 is the fungal inhibition spectrum analysis of the strain CX06 and its effect on the hyphae of pathogenic fungi. Figure 5a is the fungal inhibition spectrum analysis of strain CX06, wherein, A: Fusarium oxysporum (Fusarium oxysporum); B: Botrytis cinerea (Botrytiscinerea); C: Rhizoctonia solani (Rhizoctonia solani); Corynesporacassiicola; E: Diaporthe batatas; F: Stemphylium solani; G: Phytophthora capsici. Figure 5b is the effect of strain CX06 on the hyphae of pathogenic fungi.

Fig. 6 is the bacterial inhibition spectrum analysis of bacterial strain CX06. A: Clavibacter michiganensis subsp.sepedonicus; B: Clavibacter michiganensis subsp.michiganensis; C: Agrobacterium vitis ); D: Xanthomonas campestrispv. campestris; E: Pseudomonas syringaepv. lachryrnans.

Figure 7 is the determination of enzyme activity of strain CX06. A: Celluase; B: Chitinase; C: Protease.

Figure 8 is the growth curve of strain CX06.

Fig. 9 is the pH value change curve of strain CX06.

Figure 10 is a pot test of strain CX06 on cabbage black rot. A: strain CX06; B: kasugamycin 1000 times solution; C: blank control (distilled water); D: healthy control.

Figure 11 is a pot test of strain CX06 on cabbage stalk rot. A: strain CX06; B: 500-fold solution of 4% Jinggangmycin; C: blank control (distilled water).

Figure 12 is a comparison test of the control effect of strain CX06 and other agents. A: CX06; B: Polyoxin; C: Paenibacillus polymyxa; D: Bacillus cereus; E: Pseudomonas fluorescens; F: Bacillus firmus; G: Bacillus licheniformis; H: Mesozoic Bacterin; I: copper hydroxide; J: Bacillus subtilis; K: kasugamycin; L: thiazole zinc; M: acetoallin; N: copper succinate; O: copper quinolate; Copper; Q: tetramycin; R: blank control (distilled water).

Preservation instructions

Chinese name: Lysobacterium enzymogenes

Latin name: Lysobacter enzymogenes

Strain number: CX06

Preservation institution: General Microbiology Center of China Committee for the Collection of Microorganisms

Depository institution abbreviation: CGMCC

Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Preservation date: July 08, 2020

Registration number of the collection center: CGMCC No.20321

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. The medium in the following examples, unless otherwise specified, is at natural pH. Unless otherwise specified, the quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The test medium and the test solution involved in the following examples are as follows:

The PDA medium was used for the antagonistic effect test, and the specific formula of the PDA medium was as follows: 200 g of potatoes, 20 g of glucose, 15 g of agar, and 1000 mL of distilled water.

CMC medium is used for the determination of cellulase, and the specific formula of CMC medium is as follows: Magnesium sulfate 0.1g, ammonium sulfate 1g, 10 * phosphate buffer (dipotassium hydrogen phosphate 70g, potassium dihydrogen phosphate 20g, distilled water 1000mL) 100mL, Yeast extract 5g, glycerin 2mL, sodium carboxymethylcellulose 1g, agar 8g, distilled water 900mL.

LB medium, NB medium, and WA medium are used for the determination of bacterial inhibition spectrum. The specific formula of LB liquid medium is as follows: tryptone 10g, yeast extract 5g, NaCl 10g, distilled water 1000mL. The specific formula of NB medium is as follows: peptone 10g, beef powder 3g, NaCl 5g, distilled water 1000mL, pH7.0. The specific formula of WA medium is as follows: 5g of agar, 1000mL of distilled water, divided into test tubes, each 5mL.

The protease medium is used for the determination of protease. The specific formula of the protease medium is as follows: NA medium (peptone 10g, beef powder 3g, NaCl 5g, agar 15g, distilled water 980mL, pH7.0), 20mL skimmed milk is heated to a high temperature and boiled, then added to melt in the subsequent NA medium.

The chitinase medium is used for the determination of chitinase, and the specific formula of the chitinase medium is as follows: 5 g of caseinase (without vitamins), 3 g of chitin salt, 7 mL of 5% dipotassium hydrogen phosphate aqueous solution, MgSO ₄ ·7H ₂ O (1:10000) 5mL, agar 15g, add water to make up to 1000mL, pH 7.0, 116°C, autoclave for 15min.

Congo red solution: 0.2% (w/v). Congo red powder 2g, distilled water 1000mL. A small amount of alcohol can be added to help dissolve evenly.

NaCl solution: 1M. NaCl 58.5g, distilled water 1000mL.

Embodiment 1, isolation, identification and preservation of bacterial strain CX06

1. Isolation of strain CX06

1. Strain isolation

Take rhizosphere soil samples from different crops, weigh 10g of each soil sample, add to 90mL sterilized water, and place in a constant temperature shaker at 28°C for 30min. Dilute the mixed soil samples in a gradient from 10 ^-1 to 10 ^-8 , apply the soil samples diluted to gradients 10 ^-6 , 10 ^-7 , and 10 ^-8 , draw 100 μL for each gradient, and coat the LB plate On each plate, 3 plates were applied and cultured at 28°C for 2 days. Different types of colonies were picked and purified on LB plates. After purification three times, they were stored in -80°C cryopreservation tubes. Finally, 8 bacterial strains were isolated from the rhizosphere soil of different crops, named CX01-CX08 respectively (Fig. 1).

2. Strain screening

The 8 strains obtained in step 1 were used as test strains, Xanthomonas campestris pv. Strain CX06.

The specific method is as follows: pick a single colony of the strain and inoculate it in LB liquid medium, culture it with shaking at 28°C and 180 r/min until the concentration is 1×10 ⁸ cfu/mL, inoculate 5 μL of the suspension of the strain to be tested in the center of the PDA plate, 28 After culturing at ℃ for 24 hours, the petri dishes were inverted, and 3 mL of chloroform was added to each petri dish in a fume hood, and stood for 12 hours to inactivate the antagonistic bacteria. The pathogenic bacteria were inoculated in NB medium, cultured with shaking at 28°C and 180r/min for 36h, and 100μL of bacterial suspension was added to 4mL of 5% (m/v) WA medium, mixed and poured into a PDA plate as the upper layer. Cultivate in the incubator for 24 hours, observe and measure the size of the inhibition zone.

2. Identification of strain CX06

1. Physiological and biochemical identification

Referring to the method of "Common Bacteria System Identification Manual", the following physiological and biochemical tests were carried out on the strain CX06: Gram staining test, growth temperature test, salt tolerance test, motility test, contact enzyme test, V-P test, starch hydrolysis Test, citrate utilization test, gelatin liquefaction test.

The results of physiological and biochemical tests are shown in Table 1. The results show that the strain CX06 is a Gram-negative bacterium with an optimum growth temperature of 28-37°C. It grows normally in 1% NaCl, inhibits its growth in 4% NaCl, and has motility. It can hydrolyze chitin, gelatin, and Tween 20, but cannot hydrolyze starch and cellulose, and can use citrate as a carbon source.

Table 1. Analytical results of physiological and biochemical characteristics of strain CX06

Note: + indicates that the test result is positive; - indicates that the test result is negative.

2. Biolog detection

Pick a single colony of strain CX06 and inoculate it on the slant of the NA medium test tube, and incubate at 28°C for 24h. The China Agricultural Microorganism Culture Collection Center used the BIOLOG GENⅢ kit (operated according to the kit instructions) to measure the utilization of the sole carbon source of the strain CX06.

The Biolog test results are shown in Table 2. The results show that the strain CX06 can utilize D-maltose, α-D-lactose, D-melibiose, D-mannose, D-trehalose and L-rhamnose, but cannot utilize Dextran, stachyose, D-mannitol, and minocycline. The strain CX06 was preliminarily identified as Lysobacter.

Table 2. Determination of the sole carbon source utilization of strain CX06 by using BIOLOG GENIII reagent strips

Note: +, positive; -, negative; w, weakly positive

3. Morphological identification

1) Morphological observation

Take 3 μL of the CX06 bacterial solution and place them in the center of 10% TSA, TSA, NA, and LB medium respectively, culture at 28°C for 2-4 days, and observe the morphology.

The results are shown in Figure 2, and the results show that the colony morphology of bacterial strain CX06 on NA medium, LB medium, and TSA medium is light yellow, the surface of the colony is prominent, the thalline is moist and shiny, mucus-like, and is not easy to provoke; On the 10% TSA medium, the colony appears milky white, the surface is flat, and it is not easy to provoke.

2) Electron microscope observation

Strain CX06 was streaked on the NA medium, and the colony was washed with sterilized water at the place where the colony was fresh, and the medium was slightly shaken, washed repeatedly, and prepared into a bacterial suspension. Add 1-2 drops of fresh PTA counterstain solution on the paraffin plate, then take a new copper mesh to absorb the bacterial suspension, and place it on a clean filter paper to dry. When the bacterial suspension on the copper mesh is dry, touch the side of the copper mesh that absorbs the bacterial suspension to the negative stain solution, and float and stain for 15 seconds. Take out the copper mesh from the negative dye solution, blot the negative dye solution with filter paper, and let it dry under the fluorescent lamp for 45 seconds. The samples were placed under a transmission electron microscope at 80 kV to observe the cell morphology. The result is shown in Figure 3.

4. Molecular identification

After a small amount of genomic DNA of strain CX06 was extracted with a common bacterial genome extraction kit, the 16S rDNA sequence of strain CX06 was detected using the universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′ of bacterial 16S rDNA Perform PCR amplification.

PCR operation program: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, a total of 34 cycles; extension at 72°C for 10 min. The obtained product was sequenced by Biomed Biological Company, and the sequencing results showed that the 16S rDNA gene sequence of strain CX06 is shown in sequence 1 in the sequence listing.

The remaining sequences were downloaded from the NCBI website, and a phylogenetic tree was constructed using the maximum likelihood method using MEGA7.0 to analyze their genetic relationship.

The results are shown in Figure 4, the strain and Lysobacter enzymogenes YC36 clustered together, and the strain CX06 was determined to be Lysobacter enzymogenes.

Combined with the results of morphological identification, physiological and biochemical identification, Biolog determination and molecular identification, it was determined that the strain CX06 belonged to Lysobacter enzymogenes.

3. Preservation of bacterial strain CX06

Lysobacter enzymogenes CX06 was deposited in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short) on July 08, 2020; address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology; Zip Code: 100101), the deposit number is CGMCC No.20321.

Embodiment 2, the mensuration of bacterial strain CX06 antibacterial spectrum

1. Determination of fungal inhibition spectrum and the effect of strain CX06 on pathogenic fungal hyphae

1. Determination of fungal inhibition spectrum

The plate confrontation method was used to determine the antibacterial spectrum of fungi.

Pathogenic fungi: Corynespora cassiicola, Fusarium oxysporum, Phytophthora capsici, Botrytis cinerea, Rhizoctonia solani, Solanum solani Stemphylium solani, Ascochyta citrullina.

Corynespora cassiicola has been described in the literature "Gao Wei, Li Baoju, Shi Yanxia, Xie Xuewen. Differentiation of pathogenicity of Corynespora on cucumber, tomato and eggplant hosts. Journal of Horticultural Science, 2011, 38(3) : 465-470.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

Fusarium oxysporum has been reported in the literature "Shi Yanxia, Zhang Xiaohui, Xu Yufang, Xie Xuewen, Chai Ali, Li Baoju. Pyrazolopyrimidine derivative BDO-1 induces cucumber resistance to Fusarium wilt. Acta Horticultural Science, 2019, 46(05): 877-890.", the public can obtain it from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and it cannot be used for other purposes.

Phytophthora capsici has been published in the literature "Cheng Yingchao, Kang Huajun, Shi Yanxia, Chai Ali, Zhang Hongjie, Xie Xuewen, Li Baoju. Establishment and application of RT-PCR detection technology for Phytophthora capsici. Journal of Horticultural Science, 2018, 45(05) : 997-1006.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

Stemphylium solani has been published in the literature "Li Xinyu, Li Lei, Chen Lida, Shi Yanxia, Chai Ali, Xie Xuewen, Li Baoju. Screening and identification of antagonistic bacteria to tomato Stemphylium solani. Acta Horticultural Science, 2020, 47(04 ): 741-748.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

Ascochyta citrullina has been published in the literature "Liu Shulin, Gu Xingfang, Shi Yanxia, Li Baoju, Miao Han, Wang Min, Wang Ye, Zhang Shengping. Research Overview of Melon Vegetable Blight. Chinese Vegetables, 2013 (18): 1 -10.", the public can obtain it from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and it cannot be used for other purposes.

Botrytis cinerea has been published in the literature "Shi Yanxia, Tang Ming, Jin Zhiwen, Xie Xuewen, Chai Ali, Li Baoju. Evaluation of the resistance of Botrytis cinerea to different types of fungicides in vegetable crops. Chinese Vegetables, 2016, ( 03): 60-65.", the public can obtain from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and cannot be used for other purposes.

Rhizoctonia solani (Rhizoctonia solani) has been described in the literature "Gao Wei, Li Baoju, Sun Junde, Shi Yanxia, Jindan. Antagonistic effect of Trichoderma viride on cucumber Rhizoctonia solani and Fusarium oxysporum. Chinese Vegetables, 2008, ( 6): 9-12.", the public can obtain from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and cannot be used for other purposes.

The specific method is as follows: Inoculate 5 mm of the selected fungal target bacteria in the center of a 90 mm PDA plate, and culture at 28 ° C for 2 days. The strain was inoculated in liquid LB medium, shaken at 28°C and 180r/min for 16h, and the concentration of the bacterial suspension was adjusted to 10 ⁸ cfu/mL. Inoculate 5 μL of bacterial suspension at 4 points 10 mm away from the edge of the petri dish, use inoculated liquid LB medium as a blank control, measure the control growth (colony radius) and treatment growth of the target bacteria after culturing at 28°C for 5 days (Growth radius after inoculation of bacteria), represented by the bacteriostatic rate. Bacterial inhibition rate (%)=(control growth amount-treatment growth amount)/control growth amount×100.

The results are shown in Table 3 and Figure 5a. The results showed that the strain CX06 could effectively inhibit Corynemycetes polybasicum, Phytophthora capsici, Fusarium oxysporum, Botrytis cinerea, Phytophthora solani, S. Mycelial growth of Rhizoctonia solani. The inhibition rate against Rhizoctonia solani is over 50%, and the inhibition rate against other pathogenic fungi reaches over 20%.

Table 3. Fungal inhibition rate

Note: The data are mean ± standard error, and different small letters indicate significant differences at the 0.05 level.

2. The effect of strain CX06 on the hyphae of pathogenic fungi

Pick the hyphae at the boundary of the colonies on the confrontation plate in the determination of the fungal antibacterial spectrum, make milk phenol oil slides, observe them under an optical microscope, and take pictures.

The results are shown in Figure 5b. The results showed that: compared with normal mycelia, the mycelia of strain CX06 after confrontation with pathogenic fungi had abnormal morphology, distortion, deformity, thickened cell wall at the top, local cell enlargement, and intracellular mycelium containing Uneven distribution of substances, disordered growth, thickening of the wall and increase of abnormal branches, etc.

2. Determination of Bacterial Inhibition Spectrum

Bacterial inhibition spectrum was determined by double-layer culture method.

Pathogenic bacteria: Xanthomonas campestrispv. campestris, Clavibacter michiganensis subsp. sepedonicus, Pseudomonas syringaepv lachryrnans), Agrobacterium vitis, Clavibacter michiganensis subsp. michiganensis.

Xanthomonas campestris pv. campestris has been published in the literature "Zhang Yang, Li Jinping, Zhou Huimin, Li Baoju. Occurrence and control of bacterial black rot in cruciferous vegetables. Chinese Vegetables, 2011, 17 : 23-25.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

Clavibacter michiganensis subsp.sepedonicus has been published in the literature "Li Lei, Zhao Yurong, Zheng Fei, Shi Yanxia, Chai Ali, Xie Xuewen, Li Baoju. Screening and control effect of biocontrol bacteria for potato black mole disease .Acta Phytopathology, 2020, https://doi.org/10.13926/j.cnki.apps.000357.”, the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used as other purpose use.

Pseudomonas syringae pv. lachryrnans has been published in the literature "Li Huanling, Li Baoju. Dr. Li Baoju's Diagnosis Notes (53) Symptom Diversity and Comprehensive Control of Cucumber Bacterial Keratosis. Chinese Vegetables , 2012, (21): 23-25.", the public can obtain from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and can not be used for other purposes.

Agrobacterium vitis has been published in the literature "Zhao Yurong, Li Lei, Xie Xuewen, Shi Yanxia, Chai Ali, Sun Guangyu, Li Baoju. The control effect of Bacillus Velez ZF2 on multi-primary corynesporium. Chinese Journal of Biological Control, 2019, 35 (02): 217-225.", the public can obtain from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and cannot be used for other purposes.

The pathogenic strain of Corynebacterium michiganensis subsp.michiganensis has been published in the literature "Li Lei, Zhao Yurong, Zheng Fei, Shi Yanxia, Chai Ali, Xie Xuewen, Li Baoju. Screening and control effects of antagonistic bacillus against celery soft rot. Chinese Journal of Biological Control, 2020, 36(03): 388-395.", the public can obtain it from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and cannot be used for other purposes.

The specific method is as follows: the strain was inoculated in liquid LB medium, cultured with shaking at 28°C and 180r/min for 16h, and the concentration of the bacterial suspension was adjusted to 10 ⁸ cfu/mL. Inoculate 5 μL of bacterial suspension in the center of a 90 mm PDA plate, and inoculate liquid LB medium as a blank control. After culturing at 28°C for 24 hours, add 3 mL of chloroform to the inverted Petri dish in a fume hood, and let it stand for 12 hours. The pathogenic bacteria were inoculated in NB medium, cultured with shaking at 28°C for 36 hours, and the concentration of the bacterial suspension was adjusted to 10 ⁸ cfu/mL. Add 100 μL of pathogenic bacteria suspension to 4 mL of 5% (m/v) WA medium, mix well and pour it into a PDA plate as the upper layer. Cultivate in the incubator for 48 hours, observe and measure the size of the inhibition zone. Bacterial inhibition rate (%)=(control growth amount-treatment growth amount)/control growth amount×100.

The results are shown in Table 4 and Figure 6. The results show that the bacterial strain CX06 also has a good inhibitory effect on pathogenic bacteria. bacillus, Pseudomonas syringae pv. lachryma, and Corynebacterium michiganii pv. potato ring rot were all above 1.70 cm in diameter, and the antibacterial rate against xanthomonas pv. More than 30%.

Table 4. Bacterial inhibition rate

Note: The data are mean ± standard error, and different small letters indicate significant differences at the 0.05 level.

Embodiment 3, bacterial strain CX06 enzyme activity detection

1. Detection of protease

Pick a single colony of strain CX06 and inoculate it in LB liquid medium, and culture it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL. The bacterial suspension of strain CX06 was inoculated at the center of the protease medium, and each treatment was repeated 3 times. After culturing at 28°C for 24 hours, the digestion circle was observed.

Second, the detection of cellulase

Pick a single colony of strain CX06 and inoculate it in LB liquid medium, and culture it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL. Inoculate the bacterial suspension of strain CX06 in the center of the CMC medium, culture at 28°C for 24 hours, stain with 3 mL of Congo red dye for 30 minutes, and decolorize with 5 mL of 1mol/L NaCl solution for 15 minutes. Each treatment was repeated 3 times, and the digestion circle was observed.

3. Chitinase assay

Pick a single colony of strain CX06 and inoculate it in LB liquid medium, and culture it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL. The strain CX06 bacterial suspension was inoculated at the center of the chitinase medium, each treatment was repeated 3 times, and the digestion circle was observed after incubation at 28°C for 24 hours.

The results are shown in Figure 7, and the results show that: bacterial strain CX06 has obvious transparent circles in the protease medium, and after staining and decolorization in the CMC medium, a yellow transparent circle is formed. A distinct transparent circle is produced. It shows that the strain CX06 has the ability to produce protease, cellulase and chitinase.

Embodiment 4, bacterial strain CX06 growth curve and pH value measurement

Pick a single colony of the strain and inoculate it in LB liquid medium, culture it with shaking at 28°C and 180r/min for 16h, and adjust the concentration of the bacterial suspension to 1×10 ⁸ cfu/mL. The bacterial suspension was added to LB liquid medium at a ratio of 1:1000, and cultured with shaking at 28°C. Once every 4 hours, the OD ₆₀₀ value, the number of viable bacteria and the pH value of the strains were continuously measured for 48 hours, and repeated three times.

The results are shown in Figure 8 and Figure 9, and the results show that: under the condition of 28°C, the number of bacteria strain CX06 rapidly increased at the initial stage of culture, and reached the maximum after 30 hours, the _OD600 value was 2.12, and the number of colonies on the plate was 2.7×10 ¹² /mL; then the number of bacteria gradually stabilized. In addition, with the extension of culture time, the pH value of strain CX06 gradually increased, and the pH value was 8.14 after 48 hours.

Example 5, detection of the effect of bacterial strain CX06 on controlling cabbage black rot

The control effect of cabbage black rot was determined by spray inoculation method.

The specific method is as follows: Pick a single colony of Xanthomonas campestrispv. campestris and inoculate it in NB medium, culture it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL, The seedlings of cabbage (Zhonggan No. 21) were pretreated by spray inoculation. Pick a single colony of strain CX06 and inoculate it in LB liquid medium, culture it with shaking at 28°C and 180 r/min to a concentration of 1×10 ⁸ cfu/mL, dilute it 100 times, and spray it on the cabbage seedlings 24 hours after inoculation with pathogenic bacteria. 10 plants per treatment were repeated 3 times. Thereafter, the incidence of cabbage was observed every day. Calculate the incidence rate, disease index and control effect. Three treatments were set up in the test: (1) 100-fold solution of bacterial strain CX06 (1×10 ⁸ cfu/mL); (2) 1000-fold solution of 4% kasugamycin; (3) blank control (distilled water); (4 ) healthy control.

Xanthomonas campestris pv. campestris has been published in the literature "Zhang Yang, Li Jinping, Zhou Huimin, Li Baoju. Occurrence and control of bacterial black rot in cruciferous vegetables. Chinese Vegetables, 2011, 17 : 23-25.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

4% kasugamycin wettable powder (Shandong Province Rushan Hanwei Biotechnology Co., Ltd., registration number: PD20100329).

Incidence rate (%)=100×total number of diseased leaves/total number of investigated leaves.

Disease index = 100 × Σ (number of diseased leaves at all levels × value of relative level) / (total number of leaves under investigation × 9). Disease classification standard: 0: no lesion; 1: less than 5% of the total leaf area; 2: 6% to 25% of the entire leaf area; 3: lesion area Accounting for 26% to 50% of the entire leaf area; Grade 4: the lesion area accounts for 51% to 75% of the entire leaf area; Grade 5: the lesion area accounts for more than 75% of the entire leaf area.

Control effect (%)=100×(control disease index-treatment disease index)/control disease index.

The results are shown in Table 5 and Figure 10. The results show that the strain CX06 has a good control effect on cabbage black rot caused by Pv. xanthomonas in wild rape under greenhouse conditions. The disease index of cabbage plants inoculated with strain CX06 was 8.4, which was 82.2 lower than that of the control group inoculated with Xanthomonas oleracea. In terms of control effect, the control effect of strain CX06 on cabbage black rot was 90.7%, which was higher than that of kasugamycin.

Table 5. Pot control effect of strain CX06 on cabbage black rot

deal with concentration Disease index Control effect (%) CX06 1×10⁸cfu/mL 8.4 90.7 Kasugamycin 1000 times solution 14.4 84.1 Control CK - 90.6 -

Example 6, detection of the effect of bacterial strain CX06 on preventing and treating stem rot of cabbage

The control effect of cabbage stalk rot was determined by wheat kernel inoculation method.

The specific method is as follows: the stem base of cabbage (Zhonggan No. 21) is wounded by acupuncture, and then inoculated with wheat grains with Rhizoctonia solani hyphae. After 1 day of treatment, the root irrigation method is used to pick a single colony of strain CX06 Inoculate in LB liquid medium, shake culture at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL, use 10 mL for each plant for root irrigation treatment, 10 plants for each treatment, and then observe the incidence of cabbage every day. Calculate the incidence rate, disease index and control effect. Three treatments were set up in the experiment: (1) strain CX06 (1×10 ⁸ cfu/mL); (2) 4% Jinggangmycin 500 times solution; (3) blank control (distilled water).

Rhizoctonia solani (Rhizoctonia solani) has been described in the literature "Gao Wei, Li Baoju, Sun Junde, Shi Yanxia, Jindan. Antagonistic effect of Trichoderma viride on cucumber Rhizoctonia solani and Fusarium oxysporum. Chinese Vegetables, 2008, ( 6): 9-12.", the public can obtain from the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences to repeat the experiment of this application, and cannot be used for other purposes.

4% Jinggangmycin aqueous solution (Jining Tongda Chemical Factory, Shandong Province, PD20142295).

Incidence rate (%)=100×total number of diseased leaves/total number of investigated leaves.

Disease index = 100 × Σ (number of diseased leaves at all levels × value of relative level) / (total number of leaves under investigation × 9). Disease classification standard: 0: no lesion; 1: less than 5% of the total leaf area; 2: 6% to 25% of the entire leaf area; 3: lesion area Accounting for 26% to 50% of the entire leaf area; Grade 4: the lesion area accounts for 51% to 75% of the entire leaf area; Grade 5: the lesion area accounts for more than 75% of the entire leaf area.

Control effect (%)=100×(control disease index-treatment disease index)/control disease index.

The results are shown in Table 6 and Figure 11. The results show that the strain CX06 has a good control effect on the stem rot of cabbage caused by Rhizoctonia solani under greenhouse conditions. The disease index of cabbage plants inoculated with strain CX06 was 31.25, which was 53.45 lower than that of the control group inoculated with Rhizoctonia solani. From the perspective of control effect, the control effect of strain CX06 on cabbage stalk rot was 62.8%, which was higher than the control effect of 4% Jinggangmycin.

Table 6. Pot control effect of strain CX06 on stem rot of cabbage

deal with concentration Disease index Control effect (%) CX06 1×10⁸cfu/mL 31.25 62.8 4% Jinggangmycin aqueous solution 500 times solution 44.4 47.2 Control CK - 84.1 -

Embodiment 7, Parallel test of Lysobacterium enzymogenes CX06 and other medicament control effect comparison

The in vivo control effect of strain CX06 and other agents on cabbage bacterial black rot was determined by spraying method.

The specific method is as follows: Pick a single colony of Xanthomonas campestrispv. campestris and inoculate it in NB medium, culture it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL, Spray inoculation on cabbage seedlings. 24 hours after the inoculation of the pathogenic bacteria, the control agent and the CX06 bacterial suspension were diluted 100 times, respectively, and then sprayed and inoculated on the cabbage seedlings, and cultured normally. After the plants in the blank control group were completely infected, the disease index and control effect of the other treatment groups were investigated. Distilled water was used as the blank control (CK) of the test sample. 15 plants per treatment, repeated 3 times. The preparation method of the CX06 bacterial suspension is as follows: Pick a single colony of the strain CX06 and inoculate it in LB liquid medium, and cultivate it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL.

The contrast agents were: 1 billion CFU/gram Paenibacillus polymyxa wettable powder (Guangdong Gudifeng Biotechnology Co., Ltd., registration number: PD20181264) 350 times liquid, 100 billion spores/gram Bacillus subtilis wettable powder ( Wuhan Kenuo Biotechnology Co., Ltd., registration certificate number: PD20140209) 1400 times liquid, 800 million/g Bacillus cereus wettable powder (Shandong Tainuo Pharmaceutical Co., Ltd., registration certificate number: PD20094534) 100 times liquid, 10 billion spores/gram Bacillus firmus wettable powder (Jiangxi Shunquan Biotechnology Co., Ltd., registration number: PD20184023) 100 times liquid, 8 billion live spores/ml Bacillus licheniformis water agent (Guangxi Jinyanzi Pesticide Co., Ltd., Registration certificate number: PD20140122) 90 times liquid, 500 million spores/gram Pseudomonas fluorescens wettable powder (Shandong Tainuo Pharmaceutical Co., Ltd., registration certificate number: PD20140874) 50 times liquid, 3% Zhongshengmycin wettable Powder (Fujian Kaili Biological Products Co., Ltd., registration certificate number: PD20110113) 650 times liquid, 4% kasugamycin wettable powder (Shandong Province Rushan Hanwei Biotechnology Co., Ltd., registration certificate number: PD20100329) 1000 times liquid, 80 % allicin EC (Kaifeng Dadi Agrochemical Biotechnology Co., Ltd., registration number: PD20101285) 2200 times liquid, 0.3% tetramycin aqueous solution (Liaoning Weike Bioengineering Co., Ltd., registration number: PD20160345) 1100 times Liquid, 10% polyoxin wettable powder (Shandong Dezhou Xianglong Biochemical Co., Ltd., registration number: PD20100857) 1000 times liquid, 30% succinic acid copper wettable powder (Heilongjiang Qiqihar Siyou Chemical Co., Ltd., Registration number: PD20097367) 275 times liquid, 46% copper hydroxide water dispersible granule (U.S. DuPont Company, registration number: PD20110053) 1800 times liquid, 33.5% quinoline copper suspension concentrate (Shandong Aosheng Biotechnology Co., Ltd., Registration certificate number: PD20200743) 1500 times liquid, 1.6% thiamethoxam smear (Shaanxi Xidahuate Technology Industrial Co., Ltd., registration certificate number: PD20120375) 700 times liquid, 20% thiazole zinc suspension concentrate (Zhejiang Xinnong Chemical Co., Ltd. Co., Ltd., registration number: PD20096932) 1000 times liquid.

Disease classification standard: 0: no lesion; 1: less than 5% of the total leaf area; 2: 6% to 25% of the entire leaf area; 3: lesion area Accounting for 26% to 50% of the entire leaf area; Grade 4: the lesion area accounts for 51% to 75% of the entire leaf area; Grade 5: the lesion area accounts for more than 75% of the entire leaf area. Incidence rate (%)=100×total number of diseased leaves/total number of investigated leaves. Disease index = 100 × Σ (number of diseased leaves at all levels × value of relative level) / (total number of leaves under investigation × 9). Control effect (%)=100×(control disease index-treatment disease index)/control disease index.

Xanthomonas campestris pv. campestris has been published in the literature "Zhang Yang, Li Jinping, Zhou Huimin, Li Baoju. Occurrence and control of bacterial black rot in cruciferous vegetables. Chinese Vegetables, 2011, 17 : 23-25.", and the public can obtain it from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, to repeat the experiment of this application, and cannot be used for other purposes.

The results are shown in Table 7 and Figure 12. The results showed that the control effect of strain CX06 on bacterial black rot of cabbage was 79.98%, significantly higher than other agents.

Table 7. Comparative results of the control effects of Lysobacter zymogenes CX06 and other agents

Embodiment 7, bacterial strain CX06 is to the tolerance level of antibiotic

Select 10 antibiotics: ampicillin, carbenicillin, kanamycin, chloramphenicol, streptomycin, tetracycline, gentamicin, rifampicin, vancomycin, spectinomycin, and prepare 50 μg/mL, 60 μg /mL, 70μg/mL three kinds of antibiotic gradient plates, inoculated with CX06 bacterial suspension, observed the growth, repeated 3 times. The preparation method of the CX06 bacterial suspension is as follows: Pick a single colony of the strain CX06 and inoculate it in LB liquid medium, and cultivate it with shaking at 28°C and 180r/min until the concentration is 1×10 ⁸ cfu/mL.

The results are shown in Table 8. The results show that: the strain CX06 has a high tolerance to various antibiotics, and is resistant to ampicillin, carbenzyl, chloramphenicol, kanamycin, streptomycin, gentamicin, spectinomycin The tolerance level of the antibiotics reached above 50 μg/mL, the tolerance level of tetracycline reached 30 μg/mL, and the tolerance level of rifampicin and vancomycin was below 50 μg/mL, indicating that the strain CX06 was only resistant to rifampicin and vancomycin Mycomycin sensitive, the other 8 kinds of antibiotics are not sensitive.

Table 8, bacterial strain CX06 is to antibiotic resistance level (μg/mL)

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

sequence listing

<110> Vegetable and Flower Research Institute, Chinese Academy of Agricultural Sciences

<120> An Enzyme-producing Lysobacterium CX06 Antagonizing Xanthomonas campestris and Its Application

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1326

<212>DNA

<213> Artificial Sequence

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tagtaagcca gtttccaatg ccattcccag gttgagccca gggctttcac atcagactta 840

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cccgtc 1326

Claims (9)

1. Lysobacter enzymogenes CX06, the preservation number of which is CGMCC No.20321. 2. The bacterial suspension or fermentation liquid of Lysobacter enzymogenes CX06 described in claim 1 or the microbial agent containing said Lysobacter enzymogenes CX06. 3. The application of Lysobacter enzymogenes CX06 or its bacterial suspension or fermentation liquid or the bacterial agent containing said Lysobacter enzymogenes CX06 in inhibiting plant pathogenic bacteria according to claim 1; The plant pathogenic bacteria are pathogenic fungi or pathogenic bacteria; The pathogenic fungi are Corynespora cassiicola , Fusarium oxysporum , Phytophthora capsici , Botrytis cinerea , Rhizoctonia solani solani ), Stephylium solani , Ascochyta citrullina ; The pathogenic bacteria are Xanthomonas campestris pv. campestris , Clavibacter michiganensis subsp. sepedonicus , Pseudomonas syringae Pseudomonas syringae pv. lachryrnans , Agrobacterium vitis , Clavibacter michiganensis subsp. michiganensis . 4. The application of Lysobacter enzymogenes CX06 or its bacterial suspension or fermentation liquid or the bacterial agent containing said Lysobacter enzymogenes CX06 in the preparation of products for inhibiting plant pathogenic bacteria according to claim 1; The plant pathogenic bacteria are pathogenic fungi or pathogenic bacteria; The pathogenic fungi are Corynespora cassiicola , Fusarium oxysporum , Phytophthora capsici , Botrytis cinerea , Rhizoctonia solani solani ), Stephylium solani , Ascochyta citrullina ; The pathogenic bacteria are Xanthomonas campestris pv. campestris , Clavibacter michiganensis subsp. sepedonicus , Pseudomonas syringae Pseudomonas syringae pv. lachryrnans , Agrobacterium vitis , Clavibacter michiganensis subsp. michiganensis . 5. The application of Lysobacter enzymogenes CX06 or its bacterial suspension or fermentation broth or the bacterial agent containing said Lysobacter enzymogenes CX06 in the prevention and treatment of plant diseases caused by plant pathogenic bacteria according to claim 1; The plant pathogenic bacteria are pathogenic fungi or pathogenic bacteria; The pathogenic fungi are Corynespora cassiicola , Fusarium oxysporum , Phytophthora capsici , Botrytis cinerea , Rhizoctonia solani solani ), Stephylium solani , Ascochyta citrullina ; The pathogenic bacteria are Xanthomonas campestris pv. campestris , Clavibacter michiganensis subsp. sepedonicus , Pseudomonas syringae Pseudomonas syringae pv. lachryrnans , Agrobacterium vitis , Clavibacter michiganensis subsp. michiganensis . 6. Lysobacter enzymogenes ( Lysobacter enzymogenes ) CX06 as claimed in claim 1 or its bacterial suspension or fermentation liquid or the inoculant containing said Lysobacter enzymogenes CX06 is used in the preparation of products for the prevention and treatment of plant diseases caused by plant pathogenic bacteria Applications; The plant pathogenic bacteria are pathogenic fungi or pathogenic bacteria; The pathogenic fungi are Corynespora cassiicola , Fusarium oxysporum , Phytophthora capsici , Botrytis cinerea , Rhizoctonia solani solani ), Stephylium solani , Ascochyta citrullina ; The pathogenic bacteria are Xanthomonas campestris pv. campestris , Clavibacter michiganensis subsp. sepedonicus , Pseudomonas syringae Pseudomonas syringae pv. lachryrnans , Agrobacterium vitis , Clavibacter michiganensis subsp. michiganensis . 7. Lysobacter enzymogenes ( Lysobacter enzymogenes ) CX06 or its bacterium suspension or fermented liquid or the inoculant containing said Lysobacter enzymogenes CX06 described in claim 1 is effective in preventing and treating cabbage black rot and/or cabbage stalk rot And/or application in cabbage bacterial black rot. 8. Lysobacter enzymogenes ( Lysobacter enzymogenes ) CX06 as claimed in claim 1 or its bacterial suspension or fermented liquid or the inoculant containing said Lysobacter enzymogenes CX06 is used in the preparation and treatment of cabbage black rot and/or cabbage stalk rot disease and/or bacterial black rot of cabbage. 9. Either of the following b1)-b3) methods: b1) A method for inhibiting plant pathogenic bacteria, comprising the following steps: using Lysobacter enzymogenes CX06 according to claim 1 or its bacterial suspension or fermentation broth or bacteria containing said Lysobacter enzymogenes CX06 agent treatment of plant pathogens; b2) A method for preventing and treating plant diseases caused by phytopathogenic bacteria, comprising the following steps: using the Lysobacter enzymogenes CX06 of claim 1 or its bacterial suspension or fermentation liquid or containing the lysobacter enzymogenes Bacillus CX06 bacteria agent treatment plant; b3) A method for preventing and treating cabbage black rot and/or cabbage stalk rot and/or cabbage bacterial black rot, comprising the following steps: using the enzyme-producing lysobacterium ( Lysobacter enzymogenes ) CX06 or its Bacterial suspension or fermented liquid or bacterial agent containing said zymolytic Lysobacterium CX06 to treat cabbage or Chinese cabbage; Wherein, the phytopathogenic bacteria mentioned in b1) or b2) are pathogenic fungi or pathogenic bacteria; The pathogenic fungi are Corynespora cassiicola , Fusarium oxysporum , Phytophthora capsici , Botrytis cinerea , Rhizoctonia solani solani ), Stephylium solani , Ascochyta citrullina ; The pathogenic bacteria are Xanthomonas campestris pv. campestris , Clavibacter michiganensis subsp. sepedonicus , Pseudomonas syringae Pseudomonas syringae pv. lachryrnans , Agrobacterium vitis , Clavibacter michiganensis subsp. michiganensis .

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