CN114015589B

CN114015589B - Biocontrol microbial agent containing campylobacter TA-12 and application thereof

Info

Publication number: CN114015589B
Application number: CN202011456895.1A
Authority: CN
Inventors: 张倩; 陈义伦; 吴澎; 郗良卿; 杜海云; 辛力; 邹曼; 杨娟侠; 陈雨诗
Original assignee: Shandong Institute of Pomology
Current assignee: Shandong Institute of Pomology
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2022-07-12
Anticipated expiration: 2040-12-10
Also published as: CN114015589A

Abstract

The invention provides a biocontrol microbial agent which contains bacillus curvatus TA-12, wherein the bacillus curvatus TA-12 has broad-spectrum antibacterial activity on sweet cherries, and has obvious antibacterial effects on pathogenic bacteria of the sweet cherries, namely, Colletotrichum arenicola, Rhizopus stolonifer and Aspergillus tubingensis.

Description

Biocontrol microbial agent containing campylobacter TA-12 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a biocontrol microbial agent containing Bacillus curvatus TA-12 and application thereof.

Background

Sweet cherry is also called large cherry and cherry, and is plant of Rosaceae (Rosaceae), Prunus, and Prunus (Cerasus). The sweet cherry has rich nutritive value, soluble protein content of 589mg/100g, more Vc and multiple trace elements, rich iron content of 8mg/100g, and anticancer, antitumor and hemoglobin regeneration promoting effects. The sweet cherry pulp is rich in various phenolic compounds including hydroxycinnamic acid, procyanidine, flavone aldehyde and anthocyanin, has strong antioxidant capacity, and plays an important role in preventing diseases and human health.

The putrefaction caused by fungal pathogenic microorganisms is a great problem in the production of cherry industry, and the rotting loss caused by the putrefaction can reach more than 50 percent of the total loss. Currently, the main postharvest diseases of domestic sweet cherries include Penicillium, gray mold, root rot, brown rot, soft rot, black spot, and the like, and fungal pathogens causing the diseases include Penicillium expansum (Penicillium expansum), Monilinia fructicola (Monilinia fructicola), Botrytis cinerea (Botrytis cinerea), Rhizopus (Rhizopus sp.), Mucor (Mucor sp.), Alternaria alternata (Alternaria alternata) and anthrax (Colletotrichum sp.), Rhizopus stolonifer (Rhizopus stolonifer), Aspergillus niger (Aspergillus niger), Aspergillus niger (Aspergillus tubingensis), and the like.

Chinese patent document CN105557756A (application No. 201610082878.3) discloses a plant bacteriostasis method implemented by utilizing bacillus methylotrophicus HKG-1, and a biological bactericide prepared by utilizing a bacterial liquid of bacillus methylotrophicus HKG-1 has obvious inhibiting effect on botrytis cinerea, botryococcus, mesochitis, botrytis cinerea, gluconococcus vinosus, megatheca oryzae, colletotrichum capsici, cucurbitaceae colletotrichum, rhizopus stolonifer, fusarium graminearum, fusarium oxysporum, trichoderma viride, phomopsis asparagi, cornplanosporium, alternaria alternata, oomycete trichotheca, pseudomonas solani, pseudomonas solanacearum, mallow mutant of carpet grass and the like; but Aspergillus tubingensis is not involved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a biocontrol microbial agent containing Bacillus curvatus TA-12 and application thereof.

The microbial agent contains campylobacter TA-12, which can effectively inhibit three pathogenic bacteria of Colletotrichum aescinagma, Rhizopus stolonifer and Aspergillus tubingensis, wherein the Colletotrichum aescina is one of anthrax.

Technical scheme related to the invention

A biocontrol microbial agent, said microbial agent comprising Bacillus flexus (Bacillus flexus) TA-12.

According to the invention, preferably, the Bacillus flexus TA-12 is preserved in China general microbiological culture Collection center in 11 months and 5 days in 2020 at the preservation address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC No. 21107.

The biocontrol microbial agent is applied to the prevention and treatment of microbial diseases of sweet cherries.

Preferably, the sweet cherry microbial disease is a fungal disease; more preferably, the fungal disease is caused by one or more of Colletotrichum versipellis (Colletotrichum aenigma), Rhizopus stolonifer (Rhizopus stolonifer), and Aspergillus tubingensis (Aspergillus tubingensis).

According to the invention, the concentration of the Bacillus flexus TA-12 is ≧ 1 × 10⁹CFU/mL

Advantageous effects

The invention provides a biocontrol microbial agent which contains Bacillus flexus TA-12, wherein the Bacillus flexus TA-12 has broad-spectrum antibacterial activity on sweet cherries, and has obvious antibacterial effects on pathogenic bacteria of the sweet cherries, namely, Colletotrichum aenigma, Rhizopus stolonifer and Aspergillus tubingensis.

Drawings

FIG. 1 is a microscopic photograph of Sphaerotheca fuliginosus (Colletotrichum aenigma);

FIG. 2 is a microscopic photograph of Rhizopus stolonifer;

FIG. 3 is a photomicrograph of an Aspergillus tubingensis (Aspergillus tubingensis) microscope;

FIG. 4 disease development of cherries after inoculation with Colletotrichum aestivum (Colletotrichum aenigma);

FIG. 5 disease development of cherries after inoculation with Rhizopus stolonifer (Rhizopus stolonifer);

FIG. 6 pathogenesis of cherry after inoculation with Aspergillus tubingensis (Aspergillus tubingensis);

FIG. 7 is a phylogenetic tree constructed from the 16S rDNA gene sequence of Bacillus curvatus TA-12;

FIG. 8 is a photograph of an experimental plate of Bacillus flexus TA-12 inhibiting Colletotrichum crypthecium (Colletotrichum aenigma);

FIG. 9 is a photograph of an experimental plate showing that Bacillus curvatus TA-12 inhibits Rhizopus stolonifer (Rhizopus stolonifer);

FIG. 10 is a photograph of an experiment in which Bacillus curvatus TA-12 inhibits Aspergillus tubingensis (Aspergillus tubingensis);

FIG. 11 is a graph showing the growth of Bacillus flexus strain TA-12.

Detailed Description

The technical solution of the present invention is further described below with reference to the following examples and the accompanying drawings, but the scope of the present invention is not limited thereto. Reagents and medicines involved in the examples are all common commercial products unless otherwise specified; the experimental procedures referred to in the examples are those conventional in the art unless otherwise specified.

The microorganisms referred to in the examples:

bacillus flexus (Bacillus flexus) is preserved in China general microbiological culture collection center in 11 months and 5 days in 2020, with the preservation address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC No. 21107; (hereinafter abbreviated as Bacillus flexus TA-12).

The Aphanizomenon crypthecelloides (Colletotrichum aenigma), Rhizopus stolonifer (Rhizopus stolonifer) and Aspergillus tubingensis (Aspergillus tubingensis) are purchased from the common market or separated and screened from sweet cherries, and the latter are obtained in the invention.

Media referred to in the examples:

PDA culture medium: peeling potato 200g, glucose 20g, agar 20g, supplementing water 1000mL, sterilizing at 115 deg.C for 30 min. The PDB culture medium is obtained without adding agar.

LB culture medium: 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 20g of agar and 1000mL of deionized water are boiled, the pH value is adjusted to 7.0, and the mixture is sterilized at 121 ℃ for 20 min.

The cherries referred to in example 1 were purchased from Dalian "Samituo" greenhouse cherries.

The cherries referred to in example 2 were purchased from "meizao" cherries in the taianman hua garden greenhouse.

Example 1

Separation identification and pathogenicity verification of pathogenic bacteria

1.1 isolation and identification of pathogenic bacteria

The method comprises the steps of disinfecting the surface of a rotten sweet cherry sample by using 75% medical alcohol, cleaning the surface for 3 times by using sterile water, naturally drying the surface, cutting a small amount of rotten fruit tissues by using a sterile blade and tweezers, placing the rotten fruit tissues in the center of a resistance (streptomycin and penicillin) PDA (potato dextrose agar) culture medium, and culturing at the constant temperature of 28 ℃ for 3-4 days. After the plate bacterial colony grows out, picking the marginal bacterial block and placing the marginal bacterial block in another PDA culture medium, culturing at the constant temperature of 28 ℃, and purifying for multiple times until the single bacterial colony is formed. A single colony was inoculated onto a test tube slant and stored in a refrigerator at 4 ℃ for future use. The above operations are all carried out in a sterile environment.

Inserting a sterilized cover glass into the center of a PDA culture medium at an angle of 45 degrees, picking a single separated and purified colony by using an aseptic inoculating loop, placing the single colony at the junction of the cover glass and the PDA culture medium, culturing at a constant temperature of 28 ℃ for 5d, lightly taking out the cover glass by using sterilized tweezers, lightly wiping the other surface of the cover glass by using an alcohol cotton ball, contacting one surface with vigorous hypha growth with a glass slide on which 1 drop of a gossypol blue staining solution is dropped, and observing the growth conditions of the hypha and the spores under a microscope, wherein the figure is shown in figure 1, figure 2 and figure 3.

1.2 detection of pathogenicity of pathogenic bacteria

According to the verification of Koehz's law, the separated and purified pathogenic bacteria are put into a PDB test tube, cultured for 48 hours at the temperature of 28 ℃ and at the speed of 180r/min to prepare the pathogenic bacteria with the concentration of 1 multiplied by 10⁵CFU/mL bacterial suspension. Soaking greenhouse cherry with healthy surface without any damage in 75% medical alcohol for 20s, and soaking in the above solutionCleaning with sterile water for 3 times, air drying under sterile condition, pricking 3mm deep wound at equator of fruit with sterilized yellow gun head, sucking 10 μ L of 1 × 10⁵Placing the CFU/mL bacterial suspension at the wound, placing the fruit in a sealed bag after the fruit is naturally sucked, culturing at the constant temperature of 28 ℃ for 3d, repeating for 3 times, and observing the morbidity degree of 24h, 36h, 48h, 60h and 72h after the fruiting.

The disease condition of cherries after inoculation of Colletotrichum erythraeum (Colletotrichum aenigma) is shown in fig. 4; as can be seen from FIG. 4, a larger area of decay occurred on the fruit surface 72h after inoculation with the pathogenic bacterium Colletotrichum cryptum (Colletotrichum aenigma).

The incidence of cherries after inoculation with Rhizopus stolonifer (Rhizopus stolonifer), see fig. 5; as can be seen from FIG. 5, a large area of rot appeared on the surface of the fruit 36 hours after inoculation of Rhizopus stolonifer (Rhizopus stolonifer), and the fruit was completely rotten 48 hours after inoculation.

The incidence of cherries after inoculation with Aspergillus tubingensis (Aspergillus tubingensis), see fig. 6; as can be seen from FIG. 6, a large area of rot appeared on the fruit surface 36 hours after inoculation with Aspergillus tubingensis (Aspergillus tubingensis) pathogen, and the fruit was completely rotten 48 hours after inoculation.

1.3 biological identification of pathogenic bacteria

Inoculating the separated and purified pathogenic bacteria into a PDB culture medium, and culturing at 28 ℃ for 48h at 180r/min to prepare a bacterial suspension. 100mg of mycelium pellets are picked and ground by liquid nitrogen for 3 times, DNA is extracted according to a fungal genome kit method, PCR amplification is carried out by adopting fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and PCR products are purified by Rui Boxing science biotechnology, Inc. and then sequenced. Performing Blast comparison on the sequencing result on an NCBI website, performing homology comparison in a GenBank database, selecting a strain sequence with higher homology, and constructing a phylogenetic tree by using MEGA7.0 software.

Biological identification three pathogens were identified as Colletotrichum crypthecellum (Colletotrichum aenigma), Rhizopus stolonifer (Rhizopus stolonifer), Aspergillus tubingensis (Aspergillus tubingensis) in combination with morphological characteristics.

Example 2

Separation, screening and identification of antagonistic bacteria

2.1 isolation of bacteria

Grinding and crushing 30 healthy 'Meizao' cherries, weighing 1.0g of pulp, adding 9mL of sterile water, mixing uniformly to prepare a stock solution, and standing for 20 min. Adopting a gradient dilution method to obtain four solutions with dilution gradients, namely stock solution and 10^-1、10^-2、10^-3(ii) a mu.L of each of the obtained solutions was applied to LB medium in 3-parallel gradient format, and incubated at 37 ℃ for 24 hours. Inoculating the grown colony to another LB culture medium for three-zone streaking, and culturing at 37 ℃ until a single colony is grown. Picking single colony to LB inclined plane, and preserving at 4 deg.c after colony grows out.

2.1 DNA identification of bacteria

Extracting bacterial DNA according to a bacterial genome DNA kit method, carrying out PCR (polymerase chain reaction) amplification sequence and sequencing, building a tree according to MEGA software, analyzing a phylogenetic tree of the obtained strain, and analyzing and identifying a plurality of bacteria, wherein the phylogenetic tree of the Campylobacter TA-12 is shown as 12 in figure 7.

2.2 bacteriostatic experiment of Bacillus flexus TA-12

Measuring the TA-12 antibacterial effect of Bacillus flexus by plate opposition method, inoculating pathogenic bacteria at the center of PDA plate, and inoculating the bacteria at one side of the center of the plate with concentration of 1 × 10⁹Culturing CFU/mL Bacillus flexus TA-12 fermentation liquid at a constant temperature of 28 ℃ to obtain the change condition of the bacterial colony of the process plate; FIG. 8 shows photographs of the plates of Bacillus flexus TA-12 inhibiting Colletotrichum odoratum (Colletotrichum aenigma); the photograph of the experimental plate for inhibiting Rhizopus stolonifer (Rhizopus stolonifer) is shown in FIG. 9; a photograph of an experiment plate for inhibiting Aspergillus tubingensis (Aspergillus tubingensis) is shown in FIG. 10; the bacillus curvatus TA-12 has obvious transparent circles generated by the inhibition effect on the three pathogenic bacteria, and has the best inhibition effect on the Colletotrichum cryptum (Colletotrichum aenigma).

The method for culturing the point-inoculated curvibacillus flexus TA-12 fermentation broth in the bacteriostasis experiment comprises the following steps:

preparation of LB medium: 10g of peptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of deionized water are boiled, the pH value is adjusted to 7.0 (LB culture medium can be directly purchased), 50mL of LB culture medium is added into a 250mL conical flask per bottle, 8 layers of gauze are sealed, and the mixture is sterilized for 20min at 121 ℃.

Preparing a seed solution: inoculating the activated strain into a sterilized LB culture medium by using an inoculating loop, and performing shaking culture at a constant temperature of 37 ℃ and 180r/min for 24 hours to prepare a seed solution.

Preparing fermentation liquor: inoculating the shaken seed solution into a sterilized LB culture medium, inoculating 1mL (using a sterilized 1mL gun head) of the seed solution into each bottle, and placing the bottle in a constant-temperature shaking table at 37 ℃ and 180r/min for shaking culture for 24 hours to obtain the Bacillus flexus TA-12 fermentation liquor.

Example 3

Growth curve of Bacillus curvatus TA-12 strain

Respectively inoculating the activated liquid of the strain into LB liquid culture medium, and carrying out shake cultivation at 37 ℃ and 150 r/min. Sampling and detecting 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 24h and 26h after inoculation, taking a blank LB liquid culture medium without inoculated strains as a blank reference, taking a bacterial liquid in a 1cm cuvette, measuring the absorbance at 600nm by using a spectrophotometer, and drawing a growth curve of the strain as shown in figure 11, wherein the curve 11 can show that the Bacillus flexus TA-12 enters a stationary phase at 12h and has the highest OD value at 12 h.

Claims

1. A biocontrol microbial agent comprising Bacillus flexus: (A), (B), (C), and (C)Bacillus flexus）TA-12；

(ii) the Bacillus flexus: (A)Bacillus flexus) TA-12, deposited in China general microbiological culture Collection center at 11/5/2020, with the deposition address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC number 21107.

2. The biocontrol microbial agent of claim 1 is applied to the control of sweet cherry fungal diseases;

the fungal diseases are caused by plectrospora crypthecelloides (A) and (B)Colletotrichum aenigma) Rhizopus stolonifer (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Rhizopus stolonifer) Aspergillus tubingensis（Aspergillus tubingensis) One or more than two.

3. The use according to claim 2, wherein Bacillus flexus (B) is Bacillus flexus (A), (B), (C) and (C)Bacillus flexus) Concentration of TA-12 ≧ 1 × 10⁹CFU/mL。