CN110468060B

CN110468060B - Pantoea strain and application thereof in biological control

Info

Publication number: CN110468060B
Application number: CN201810463490.7A
Authority: CN
Inventors: 周江鸿; 夏菲; 车少臣
Original assignee: BEIJING INSTITUTE OF LANDSCAPE ARCHITECTURE
Current assignee: BEIJING INSTITUTE OF LANDSCAPE ARCHITECTURE
Priority date: 2018-05-15
Filing date: 2018-05-15
Publication date: 2021-05-07
Anticipated expiration: 2038-05-15
Also published as: CN110468060A

Abstract

The invention provides a Pantoea strain and application thereof in biological control. The strain preservation number is CGMCC NO. 14361. The invention also provides a strain preparation containing the strain or a biological control preparation containing the strain culture. The invention also provides application of the strain in inhibiting diseases or pollution caused by pathogenic bacteria such as verticillium dahliae and the like. The strain of the invention is adopted to biologically prevent and treat the smoke tree blight caused by verticillium dahliae, and the disease index of the smoke tree blight can be effectively reduced by more than 20 percent compared with a control.

Description

Pantoea strain and application thereof in biological control

Technical Field

The invention belongs to the field of landscape plant disease control, and relates to a bacterial strain for biological control and application thereof, in particular to application in biological control of cotinus coggygria wilt.

Background

Cotinus coggygria (Cotinus coggygria) is a main red-leaf tree species, and has an irreplaceable role in inheritance of red-leaf culture.

Take Beijing Xiangshan park as an example. Since the 80 s of the last century, the smoke tree forest in Beijing Xiangshan park has a large-area withering phenomenon due to blight, and the annual withered smoke tree reaches thousands of plants, which is one of the problems to be solved in the ecological system construction of Beijing urban gardens (Song Li Zhou, Du Wan Guang, Li Wei. Xiangshan park smoke tree blight prevention and treatment technical research [ J ]. Beijing gardens, 2011, 27 (2): 51-56; Ku Yu Henan, Zhou Shi hong, Liuyang, Wang Liangsheng. Huang smoke tree belongs to resources, cultivation and propagation, chemical components and leaf color regulation and control research progress.

The disease is a systemic infection disease caused by Verticillium dahlia, pathogenic bacteria of the disease have wide host range and are widely distributed in soil and plant bodies, more than 40 plants including crops, vegetables, fruits, ornamental flowers, fibers, oil crops, various woody plants and the like can be infected, the thorough removal of the disease by using chemical bactericides is difficult to realize, and the use of excessive chemical pesticides causes serious damage to the soil micro-ecological environment, so that the plants are weakened, and the severity of the occurrence of the disease is further increased.

At present, specific control measures for the disease mainly focus on the following aspects: (1) emphasizes prevention as the main, strictly controls disease diffusion, strengthens quarantine supervision, and strictly controls diseased seedlings and soil to enter disease-free areas. (2) In the aspect of physical control, two measures, namely a soil solarization method and a biological soil fumigation method, are usually adopted, but the two physical control methods have slow effect. (3) In chemical prevention and control, methyl bromide, trichloronitromethane, methyl isothiocyanate and the like are commonly used for soil fumigation, or a plant growth regulator is used for relieving symptoms, or benzimidazole, benomyl, thiophanate methyl and the like are directly injected into branches and trunks for prevention and control (unlegared, the influence of soil factors on the occurrence of the smoke tree blight and preliminary research on disease prevention and control [ D ]. Beijing: Beijing university of forestry, 2008.). Through medicament screening tests, Hanjing and other people prove that 300 times of fusarium wilt and 400 times of carbendazim liquid have the best control effect on cotinus coggygria wilt (Hanjing, 2009). However, the application strength is gradually controversial with serious consequences of environmental pollution, pesticide residue and the like caused by the use of chemical pesticides. (4) The biological prevention and control by applying antagonistic bacteria brings a new hope for the comprehensive prevention and control of cotinus coggygria fusarium wilt (a high gem, face paste, Songzhou and the like) [ J ] comprehensive prevention and control strategy of cotinus coggygria fusarium wilt [ J ] China urban forestry, 2007, 5 (3): 43-44.; Wangjian Mei, study of pathogenic bacteria and pathogenesis of cotinus coggygria fusarium wilt [ D ]. Beijing university of forestry, 2009 ]), but currently, antagonistic bacteria screening is mainly concentrated on crops, and reports on antagonistic bacteria screening of the cotinus coggygria fusarium wilt are less (Wang. interaction process of cotinus coggygria and verticillium dahliae and quantitative study of pathogenic bacteria [ D ]. Beijing university of forestry, 2012.). Therefore, the screened bacterial strain with the inhibiting effect on the Cotinus coggygria fusarium oxysporum has important application value in the aspect of biological prevention and control of landscape plant diseases.

Disclosure of Invention

Technical problem to be solved

The present invention is directed to a method for effectively biologically controlling cotinus coggygria blight to overcome the above-mentioned deficiencies of the prior art. The main technical problem at present is that no strain which can effectively inhibit the smoke tree fusarium wilt (namely verticillium dahliae) and can be used for biological control is found, so that the smoke tree fusarium wilt cannot be effectively and biologically controlled.

(II) technical scheme

The present inventors screened an endophytic bacterial strain CCBC3-3-1 (hereinafter sometimes referred to as the present strain) from the branches of cotinus coggygria, and unexpectedly found that the strain has a strong inhibitory effect on a wide variety of species including verticillium dahliae, and then deposited the strain and cultured the strain to produce a bacterial agent for biological control of diseases caused by pathogenic bacteria including verticillium dahliae, such as cotinus coggygria blight, thereby completing the present invention.

Thus, the present invention provides, in a first aspect, a strain for biocontrol having a accession number of CGMCC NO.14361 (hereinafter sometimes referred to as the strain of the invention and used interchangeably with strain CCBC 3-3-1).

In a second aspect, the present invention provides a bacterial seed preparation comprising a viable strain of the first aspect of the invention, i.e. a strain of the invention.

In a third aspect, the present invention provides a biological control bacterial agent comprising a culture grown from the strain of the first aspect of the invention.

In a fourth aspect, the present invention provides the use of a strain according to the first aspect of the invention for inhibiting at least one pathogenic bacterium selected from the group consisting of Verticillium dahlia (Verticillium dahlia), Staphylum botrytis (Botryosphaeria dothidea), Rhizoctonia solani (Rhizoctonia solani), Rhizopus solani (Valsa sordida) and Populus sordida (Valsa sordida).

In a fifth aspect, the present invention provides the use of the strain according to the first aspect of the present invention for controlling diseases caused by at least one pathogenic bacterium of the group consisting of Verticillium dahlia, botryococcus dothieida, Rhizoctonia solani (Rhizoctonia solani), willow rot (Valsa sorda) and poplar rot (Valsa sorda).

In a sixth aspect, the invention provides a method for isolating and identifying the strain of the first aspect of the invention.

(1) Preparing a solid LB culture medium and preparing a plate: the formula is as follows: comprises 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract powder and 8g/L agar, the pH value is adjusted to 7.2, the mixture is heated, dissolved and sterilized, and then the mixture is poured into a culture dish to prepare an LB flat plate;

(2) strain separation: sterilizing the cotinus coggygria branch section by using 70% ethanol, washing the cotinus coggygria branch section by using sterile water, putting the cotinus coggygria branch section into a 5% sodium hypochlorite solution for sterilization, washing the cotinus coggygria branch section by using the sterile water again, inoculating the obtained sterilized cotinus coggygria branch section on an LB (LB) flat plate, and culturing at 28 ℃; after bacterial colonies grow out, selecting bacteria and transferring the bacteria to another LB flat plate, and after single bacterial colonies grow out, selecting the single bacterial colonies and continuously culturing the single bacterial colonies into pure bacterial strains.

(3) Identification of strains

Extracting DNA of the pure strain, performing PCR amplification by using the obtained DNA as a template and using a universal primer 27F with a sequence shown in SEQ ID NO.1 and a universal primer 1495R with a sequence shown in SEQ ID NO.2 to obtain an amplification product and sequencing, and confirming that the similarity of the sequence and the sequence shown in SEQ ID NO.3 reaches more than 90, preferably more than 95%, more preferably more than 99%, most preferably 100%, and the obtained strain is the strain of the invention.

(III) advantageous effects

The bacterial strain of the invention separated from the branches and the stems of the cotinus coggygria has strong inhibiting effect on Verticillium dahlia (Verticillium dahlia), effectively reduces the disease index of the cotinus coggygria wilt, for example, and reduces the disease index by more than 20 percent compared with a control, thereby being used for biological control of the cotinus coggygria wilt. Furthermore, the strain of the present invention also has a significant inhibitory effect on, for example, Staphylococus viticola (Botryosphaeria dothidea), Rhizoctonia solani (Rhizoctonia solani), Rhizopus salmonida (Valsa sordida) and Poplar rot (Valsa sordida), and therefore the culture of the strain can also be made into a broad-spectrum biocontrol microbial inoculum such as a powder or a suspension for biocontrol of these germs.

Drawings

FIG. 1 shows the colony morphology of the bacterium CCBC3-3-1 when cultured on PDA medium for 10 days.

FIG. 2 shows the cell morphology of bacterium CCBC3-3-1 under a scanning electron microscope (10500X).

FIG. 3 shows a 1510bp band amplified by bacterial CCBC3-3-1 using bacterial universal primers. In the figure, M: nucleic acid molecular weight D2000; 1-5: CCBC 3-3-1; 6: negative control (double distilled water).

FIG. 4 shows a CCBC3-3-1 phylogenetic tree constructed based on the 16S rDNA sequence of CCBC 3-3-1.

FIG. 5 shows the colony morphology of Cotinus coggygria (i.e., Verticillium dahliae, used interchangeably herein with Verticillium dahliae) when cultured against the bacterium CCBC3-3-1 for 21 days (Cotinus coggygria on the left and CCBC3-3-1 on the right).

FIG. 6 shows the colony morphology of Cotinus coggygria Fusarium cultured on PDA culture plate for 21 days.

FIG. 7 shows the colony morphology of Cotinus coggygria Fusarium cultured on PDA medium + bacterium CCBC3-3-1 on sterile fermentation broth plate for 21 days.

FIG. 8 shows the colony morphology of Staphylococus viticola when cultured with CCBC3-3-1 for 6 days (the left side is Staphylococus viticola, and the right side is CCBC 3-3-1).

FIG. 9 shows the colony morphology of Rhizoctonia solani cultured on the bacterial CCBC3-3-1 for 6 days (Rhizoctonia solani on the left and bacterial CCBC3-3-1 on the right).

FIG. 10 shows the colony morphology of Cylindera aggregata cultured on the opposite side of the bacterium CCBC3-3-1 for 6 days (Cylindera aggregata on the left side and the bacterium CCBC3-3-1 on the right side)

FIG. 11 shows the colony morphology of the Poplar rotting pathogen when cultured on the bacterial CCBC3-3-1 for 6 days (the left side is Poplar rotting pathogen, and the right side is bacterial CCBC 3-3-1).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

As described above, the present invention provides in a first aspect a strain for biocontrol, said strain having a accession number of CGMCC NO. 14361.

The strain is numbered CCBC3-3-1, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms at 27.6.2017, has the address of No.3 of the West Lu No.1 of the Kyoho, Beijing City, and the microorganism institute of the Chinese academy of sciences, has the strain preservation number of CGMCC NO.14361, and is classified and named as Pantoea SP.

Morphological identification of the strains: the potato is cultured on a PDA culture medium (the formula is that 200g of peeled fresh potatoes are taken, 1000ml of water is added to the peeled fresh potatoes, the peeled fresh potatoes are boiled for 20min, 15g of glucose and 8g of agar are added after double-layer gauze filtration, a proper amount of water is supplemented to 1000ml, the mixture is fully shaken up and then is put into a triangular flask, the mixture is sterilized for 30min at 121 ℃, and is poured into a sterilization culture dish to be prepared into a flat plate, the PDA culture medium used in the following examples adopts the formula), the culture medium is cultured for 10d at 30 ℃, the diameter of a colony reaches 18mm, the colony is light yellow, the surface is raised, moist and smooth (shown in figure 1), and the colony is detected to. The cells were observed by scanning electron microscopy to be straight rods of 0.5-1.5 μm (as shown in FIG. 2).

Molecular identification of the strains: extracting DNA of CCBC3-3-1 by using a magnetic bead method bacterial genome DNA extraction kit (Shanghai bioengineering Co., Ltd.), and performing PCR amplification by using a 16S rDNA universal primer 27F (shown as SEQ ID NO. 1) and a universal primer 1495R (shown as SEQ ID NO. 2) to obtain a 1510bp PCR product (see FIG. 3); and connecting the obtained PCR product to a T vector by using a T-vector PCR product cloning kit (Shanghai bioengineering Co., Ltd.), then carrying out heat shock transformation, picking out positive clones, sending the positive clones to the Shanghai bioengineering Co., Ltd for sequencing to obtain a 1510bp 16S rDNA sequence (shown as SEQ ID NO. 3) of the strain, and carrying out BLAST comparison on the sequence in an www.ncbi.nlm.nlh.gov database. As a result of the comparison, the similarity of the 16S rDNA sequence of the bacterium CCBC3-3-1 to three species of Pantoea agglomerans (Pantoea agglomerans), Pantoea ananatis (Pantoea ananatic) and Pantoea vagans known in the Pantoea was 98%, and it was confirmed that the bacterium CCBC3-3-1 was a variant Pantoea sp. The result of the phylogenetic Tree analysis using Neighbor-joining Tree in MEGA software shows that the bacterium CCBC3-3-1 has a close relationship with Pantoea agglomerans (Pantoea ananatic) (as shown in FIG. 4).

In some preferred embodiments the biocontrol is the use of the strain to inhibit or eliminate disease caused by or contamination by at least one pathogenic bacterium selected from the group consisting of Verticillium dahlia (Verticillium dahlia), Staphylum botryococcus (Botryosphaeria dothidea), Rhizoctonia solani (Rhizoctonia solani), Rhizopus solani (Valsa sordida) and Populus sordida (Valsa sordida).

In some preferred embodiments, said biocontrol is the biological control of cotinus coggygria blight using said strain to inhibit verticillium dahliae.

In a second aspect, the invention provides a bacterial preparation comprising a viable strain of the invention. The strain preparation may be used, for example, for the production of a biocontrol microbial inoculum comprising a culture grown from a strain according to the first aspect of the invention.

Thus, in a third aspect, the present invention provides a biocontrol bacterial agent comprising a culture grown from a strain of the invention.

In some preferred embodiments, the microbial agent is a suspending agent and the culture is a fermentation broth; preferably, the fermentation broth is cultured by: inoculating the strain on an LB flat plate for activation to obtain an activated strain, then inoculating the activated strain into an LB liquid culture medium, and performing shake culture at 30 ℃ for 7 days to obtain the fermentation liquor.

In some preferred embodiments, the biological control agents further comprise adjuvants and stabilizers. The adjuvant is not particularly limited in the present invention, but preferably, the adjuvant is selected from the group consisting of polyethylene glycol (PEG)8000, polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), tea saponin, and gum arabic. The stabilizer is not particularly limited in the present invention, but preferably, the stabilizer is selected from the group consisting of calcium carbonate, potassium phosphate, dipotassium hydrogen phosphate and sodium carboxymethylcellulose.

In some preferred embodiments, the biocontrol microbial inoculum is a powder and the culture is a solid culture; preferably, the solid culture is obtained by: inoculating the strain on a solid culture medium such as wheat bran, culturing at 30 deg.C in dark for 15 days to obtain a solid culture, and pulverizing the solid culture to obtain the powder.

The biological control microbial inoculum can be conveniently injected into the branches and the stems of the cotinus coggygria or spread around rhizosphere when the cotinus coggygria is transplanted in spring, so that the morbidity of the cotinus coggygria wilt can be effectively reduced.

In a sixth aspect, the present invention provides a method for isolating a strain according to the first aspect of the invention.

(1) Preparing a solid LB culture medium and preparing a plate: the formula is as follows: 10g/L sodium chloride, 10g/L tryptone, 5g/L yeast extract powder and 8g/L agar, adjusting the pH to 7.2, heating to melt, sterilizing, and pouring into a culture dish to obtain an LB plate;

(3) Identification of strains

The invention will be further illustrated by the following examples. However, these examples are for illustrative purposes only. The scope of protection of the invention is not limited to these examples.

Examples

Example 1: separation, purification and identification of bacteria CCBC3-3-1

(1) The formula of the solid LB culture medium is as follows: 10g/L of sodium chloride, 10g/L of trypsin, 5g/L of yeast extract powder and 8g/L of agar (solid LB culture medium used in other embodiments is prepared according to the method, and the liquid LB culture medium is compared with the solid LB culture medium except that the agar is not added), the pH value is adjusted to 7.2, the mixture is heated and fully dissolved, then the mixture is filled into a triangular flask, sterilized at 121 ℃ for 30min, and poured into a sterilized culture dish to prepare an LB plate.

(2) Separation and purification of CCBC 3-3-1: cutting fresh cotinus coggygria branches into 0.5cm small sections, disinfecting the small sections with 70% ethanol for 5min, washing the small sections with sterile water for 1 time, then putting the small sections into a 5% sodium hypochlorite solution for disinfecting the small sections for 30min, washing the small sections with the sterile water for 3 times, inoculating the small sections on an LB (LB) plate, culturing the small sections at the temperature of 28 ℃, picking a small number of bacteria to streak and transfer the bacteria to a new LB plate after bacterial colonies grow out, and picking single bacterial colonies to continue culturing the pure bacterial strains after single bacterial colonies grow out.

(2) Identification of the bacterium CCBC 3-3-1: scanning electron microscope samples were prepared and morphology observation was performed (results are shown in fig. 2). In addition, DNA of the obtained pure strain is extracted, bacterial universal primer 27F (shown as SEQ ID NO. 1) and universal primer 1495R (shown as SEQ ID NO. 2) are utilized to carry out PCR amplification, the sequencing result of a PCR product shows that a 1510bp 16s rDNA sequence (shown as SEQ ID NO. 3) is obtained, the sequence alignment is carried out on NCBI, and then Neighbor-join Tree in MEGA software is utilized to carry out evolutionary Tree analysis, and the result shows that CCBC3-3-1 has a closer relationship with Pantoea agglomerans (Pantoea ananatic).

TABLE 1 sequences of Universal primer 27F and Universal primer 1495R and amplification product AP.

Example 2: culture of bacteria CCBC3-3-1 and Cotinus coggygria Fusarium in opposition

Inoculating a hypha block of a cotinus coggygria fusarium oxysporum strain CCW2-4-2 on a PDA (personal digital assistant) plate, placing the plate in an incubator at 25 ℃ for 7 days, then inoculating bacteria CCBC3-3-1, keeping a distance of 3cm from the colony edge of the cotinus coggygria fusarium oxysporum strain CCW2-4-2, placing the plate in the incubator at 25 ℃ for cultivation, measuring the size of a bacteriostatic zone by using a vertical cross method, measuring for 1 time every 7 days, continuously measuring for 3 times, and recording data. As a result, the bacteria CCBC3-3-1 and cotinus coggygria wilt are found to form an obvious inhibition zone when being oppositely cultured on a PDA culture medium, and the diameter of the inhibition zone reaches 3.2cm when being cultured for 21 days (as shown in figure 5).

Example 4: culture of bacteria CCBC3-3-1 against Staphylococus (Botryosphaeria dothidea), Rhizoctonia solani (Rhizoctonia solani), Rhizopus variabilis (Valsa sordida) and Poplar rot (Valsa sordida)

Inoculating hypha blocks of staphylococcus aureus (Botryosphaeria dothidea), Rhizoctonia solani (Rhizoctonia solani), willow rotting pathogen (Valsa sordida) and poplar rotting pathogen (Valsa sordida) on a PDA plate respectively, placing the PDA plate in an incubator at 25 ℃ for culturing for 7 days, then inoculating Pantoea CCBC3-3-1, culturing the PDA plate in the incubator at 25 ℃ at a distance of 3cm from the edge of a colony inoculated for the first time, measuring the size of a bacteriostatic ring by using a vertical cross method, and recording data. The confrontation culture experiment shows that the bacteria CCBC3-3-1 have weak inhibition effect on staphylococcus aureus (Botryosphaeria dothidea) and Rhizoctonia solani (Rhizoctonia solani), and the diameters of inhibition zones are 2.5cm and 2.8cm respectively when the bacteria are cultured for 6 days in confrontation culture (FIG. 8 and FIG. 9); has strong inhibiting effect on willow rotting pathogen (Valsa sordida) and poplar rotting pathogen (Valsa sordida), and the diameter of the inhibition zone reaches 3.2cm when the willow rotting pathogen and poplar rotting pathogen are cultured for 6 days (figures 10 and 11).

Example 4: inhibition of bacteria CCBC3-3-1 aseptic fermentation liquor on smoke tree blight germ

Inoculating bacteria CCBC3-3-1 in LB liquid culture medium, culturing at 28 deg.C under shaking at 200rpm for 5 days, filtering the fermentation liquid with 0.22 μm bacteria filter to obtain sterile fermentation liquid, culturing with PDA 1: 1, preparing a plate after uniformly mixing, inoculating cotinus coggygria fusarium oxysporum (figure 7), taking a PDA plate added with the same volume of sterile water as a control (figure 6), then placing the plate in an incubator at 25 ℃ for culture, measuring the diameter of a colony by a vertical cross method, measuring for 1 time every 7 days, continuously measuring for 3 times, and recording data. The result shows that the bacteria CCBC3-3-1 sterile fermentation liquor has obvious inhibiting effect on the Cotinus coggygria fusarium oxysporum (see figure 6 and figure 7), and compared with the control, the bacteriostasis rate is 63.37% when the bacteria are cultured for 21 days.

Example 5: preparation of bacterial CCBC3-3-1 biological control microbial inoculum (suspending agent)

Bacterial CCBC3-3-1 strain is inoculated on an LB plate for activation, the activated strain is inoculated in an LB liquid culture medium (compared with a solid LB culture medium, the difference is that agar is not added), and 10g of polyvinyl alcohol (PVA) serving as an auxiliary agent and 2g of sodium hydroxymethyl cellulose serving as a stabilizing agent are added into 100mL of fermentation liquor for 7 days after shaking culture at 30 ℃ to be processed into an aqueous solution.

Example 6: preparation of bacterial CCBC3-3-1 biological control microbial inoculum (suspending agent)

Inoculating bacteria CCBC3-3-1 strain on sterilized wheat bran, dark culturing at 30 deg.C for 15 days, pulverizing the solid culture (particle size 300-.

Example 7: use of bacterial CCBC3-3-1 biological control microbial inoculum suspending agent

Diluting CCBC3-3-1 biological control microbial inoculum suspending agent with water to obtain 105/mL liquid medicine, injecting the diluted liquid medicine into the xylem of the cotinus coggygria by adopting a trunk injection method before 3 months cotinus coggygria new leaves are unfolded, and taking injection clear water as a control. The incidence and severity of Cotinus coggygria wilt disease was investigated in months 6 and disease indices were calculated.

(1) The incidence of diseases is as follows: the number of diseased plants was examined for a given number of plants in units of the whole plant, and the disease incidence was calculated.

The disease incidence is the number of diseased plants/the total number of investigated plants multiplied by 100%

(2) Severity: the grade of disease occurrence was investigated according to the following classification standards, using the whole plant as a unit.

Table 2 grading criteria for disease severity.

Grade	Grading standards
		0	The whole leaf has no wilting symptom
I	1 branch and leaf has wilting symptom
		II	2-3 branches and leaves have wilting symptom
III	More than half of the plant leaves will wither and some leaves will fall off
		IV	All plants wither or die

(3) Disease index:

disease index is the sum of disease strains at each stage multiplied by the corresponding stage number/total number of investigated strains multiplied by the highest stage number multiplied by 100%

Results show that compared with a control, after CCBC3-3-1 biological control microbial inoculum suspending agent is injected, the disease index of the smoke tree blight can be reduced by 30%.

Example 8: use of bacterial CCBC3-3-1 biological control microbial inoculum powder

CCBC3-3-1 biological control microbial inoculum powder is uniformly spread on the rhizosphere when 3 months common cotinus coggygria is planted, the dosage of each tree hole is 250g, and then soil is covered. The incidence and severity of Cotinus coggygria wilt disease were investigated in 6 months of the next year, and disease index was calculated, using the treatment without biocontrol agent as a control.

(2) Severity: the disease occurrence rating was examined on the whole plant basis according to the rating scale shown in Table 2.

(3) Disease index:

Results show that compared with a control, the disease index of the smoke tree blight can be reduced by 20 percent after CCBC3-3-1 biological control microbial inoculum powder is applied to the rhizosphere in the planting process for 1 year.

In view of the above, it is desirable to provide,

finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

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<120> a Pantoea strain and application thereof in biological control

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cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 360

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gtaaagtact ttcagcgggg aggaaggtgt tgaggttaat aacctcagca attgacgtta 480

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agcgttaatt cggaattact gggcgtaaag cgcacgcagg cggtctgtca agtcagatgt 600

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ggggggtaga attccaggtg tagcggtgaa atgcgtagag atctggagga ataccggtgg 720

cgaaggcggc cccctggaca aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag 780

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taaccttcgg gagggcgctt accactttgt gattcatgac tggggtgaag tcgtaacaag 1500

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Claims

1. A strain pantoea (B) for biological controlPantoeasp.) CCBC3-3-1, characterized in that the strain has the preservation number of CGMCC NO. 14361.

2. A bacterial preparation comprising a viable strain according to claim 1.

3. A biological control bacterial agent comprising a culture obtained by culturing the strain according to claim 1.

4. The biocontrol microbial inoculant of claim 3 wherein the biocontrol microbial inoculant is a suspension agent and the culture is a fermentation broth.

5. The biocontrol microbial inoculum of claim 4 wherein the fermentation broth is cultured by: inoculating the strain on an LB flat plate for activation to obtain an activated strain, then inoculating the activated strain into an LB liquid culture medium, and performing shaking culture at 30 ℃ for 7 days to obtain the fermentation liquor.

6. The biocontrol microbial inoculum of claim 4 or 5 further comprising an adjuvant and a stabilizer.

7. The biocontrol microbial inoculum of claim 6 wherein the adjuvant is selected from the group consisting of polyethylene glycol 8000, polyvinyl alcohol, sodium lauryl sulfate, tea saponin and gum arabic; the stabilizer is selected from the group consisting of calcium carbonate, potassium phosphate, dipotassium hydrogen phosphate and sodium carboxymethylcellulose.

8. The biocontrol microbial inoculum of claim 3 wherein the biocontrol microbial inoculum is a dust and the culture is a solid culture.

9. The biocontrol bacterial agent of claim 8, wherein said solid culture is obtained by: inoculating the strain on a solid culture medium, performing dark culture at 30 ℃ for 15 days to obtain a solid culture, and then performing crushing processing on the solid culture to obtain the powder.

10. The biocontrol microbial inoculant of claim 9 wherein the solid media is wheat bran.

11. The strain of claim 1 for inhibiting a microorganism selected from the group consisting of Verticillium dahliae (A), (B), (CVerticillium dahlia) Willow rot pathogen: (Valsa sordida) And Poplar rot disease bacteria: (Valsa sordida) Use in at least one pathogenic bacterium of the group consisting of.

12. The use of the strain of claim 11, wherein the strain of claim 1 is used for the biological control of cotinus coggygria wilt disease by inhibiting verticillium dahliae.