CN111286477B - Pseudomonas syringae and application thereof in prevention and treatment of apple ring rot - Google Patents
Pseudomonas syringae and application thereof in prevention and treatment of apple ring rot Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a Pseudomonas syringae (Pseudomonas syringae) strain B-1, wherein the strain B-1 is preserved in the following parts: china center for type culture Collection, accession number: CCTCC NO: M2015813. The pseudomonas syringae B-1 has a remarkable prevention and treatment effect on apple ring spot, and the study of the embodiment shows that the strain B-1 can remarkably inhibit the growth of ring spot pathogen hypha, has a wide antibacterial spectrum, and has remarkable antagonistic action on various fruit and vegetable pathogenic fungi such as botrytis cinerea, penicillium, apple tree rot, pear tree rot, apple anthracnose blight, apple heart rot, peach brown rot and the like after fruits and vegetables are picked besides the apple ring spot; the prevention and treatment effects of the bacterial suspension and the fermentation supernatant of the bacterial strain B-1 on apple tree branch and fruit ring spot are more than 70%, and the method has the characteristics of stable prevention and treatment effect, environmental friendliness and the like, and is simple in fermentation process and low in production cost in large-scale production.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to pseudomonas syringae and application thereof.
Background
Apple ring rot is one of the important diseases of apple branches and fruits and is caused by plasmodiophora dothidea (Botryosphaeria dothidea). Apple ring rot germs can infect branches and trunks of apple trees all the year round to form ring rot tumors, dry rot disease spots or saddle disease spots, so that tree vigor is seriously weakened, and dead branches and even dead trees are caused; the germs infect fruits, causing ring rot. Since the grape-vine lochia can invade the young apple fruits from 5 months, a large amount of grape-vine lochia occurs in the near-mature period, and even a large amount of rotten fruits can be caused in the storage and transportation process of the apples.
At present, the national survey shows that the incidence rate of apple branch ring spot is up to 77.6%; the incidence rate of the fruit ring rot disease in general years is 5-20%, and the rotten fruit rate in the years of large occurrence is more than 80%. In 2013, in apple growing areas of Shandong, the ring rot rate of the bagged cultured apples is 20% -30%, and the rot rate of heavily-ill orchards is up to more than 70%. Effectively control the ring spot of the apple and have important significance for ensuring the healthy and sustainable development of the apple industry in China.
At present, the prevention and treatment of the apple ring spot are mainly protected by chemical agents, and the chemical bactericide has the advantages of high efficiency, low cost and simple and convenient operation, but has potential risks and influences on the environment and human health while preventing and treating diseases. The biocontrol microbial inoculum for preventing and treating the ring rot of the apple is environment-friendly, and can avoid a series of problems caused by chemical prevention and treatment. The biocontrol microbial inoculum can reduce or replace the use of chemical agents, thereby achieving the purpose of preventing and treating apple branch and fruit ring rot. No report of adopting Pseudomonas syringae (Pseudomonas syringae) for preventing and treating apple ring rot exists in the prior art. How to solve the technical problems is a technical problem to be solved in the technical field of microorganisms at present.
Disclosure of Invention
The Pseudomonas syringae (Pseudomonas syringae) B-1 provided by the invention is preserved in the following steps in 2015 at 12 and 31 days: china center for type culture Collection, accession number: m2015813, the preservation address is as follows: wuhan city, Hubei province, the flood mountain area eight ways.
The biocontrol microbial inoculum prepared by the Pseudomonas syringae (Pseudomonas syringae) strain B-1 is fermentation supernatant of the strain B-1 or suspension of the strain B-1.
The biocontrol microbial inoculum provided by the invention is used for preventing and treating fruit and vegetable diseases.
The biocontrol microbial inoculum provided by the invention is used for preventing and treating apple ring rot.
The preparation method of the biocontrol microbial inoculum provided by the invention comprises the following steps:
(1) inoculating the strain B-1 in an NA culture medium in a streak manner, and performing activation culture at constant temperature of 30 ℃ for 24h to obtain an activated strain B-1 single colony A;
(2) inoculating the activated strain B-1 single colony A to NB culture in a bottling amount of 80mL/250mLIn nutrient medium, 180 r.min-1Carrying out shake culture at constant temperature of 30 ℃ for 12h to obtain a strain B-1 fermentation seed solution B;
(3) inoculating the fermentation seed liquid B into an NB culture medium according to the inoculation amount of 1:100 for amplification culture, and performing shake culture at constant temperature of 30 ℃ for 24h to obtain 109cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 109cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain strain B-1 fermentation supernatant as biocontrol microbial inoculum; resuspending the pellet in sterile water to obtain 109cfu·mL-1The bacterial suspension is the biological control agent.
The NA culture medium provided by the invention is prepared by the following method: taking 10g of peptone, 3g of beef extract, 5g of NaCl and 20g of agar powder, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, and sterilizing for 20min by high-pressure steam at 121 ℃.
The NB culture medium provided by the invention is prepared by the following method: taking 10g of peptone, 3g of beef extract and 5g of NaCl, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, subpackaging in 250mL triangular bottles with the bottling amount of 80mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
The pesticide compound agent for preventing and treating the apple ring rot provided by the invention has the effective components of 2, 6-di-tert-butyl-p-cresol (BHT) and pseudomonas syringae B-1 bacterial suspension.
The invention provides a pesticide compound agent for preventing and treating apple ring rot, which is prepared from the following active ingredients in parts by volume: 0.1 mmol. L -11 part of 2, 6-di-tert-butyl-p-cresol solution and 10 parts of5cfu·mL -11 part of pseudomonas syringae B-1 bacterial suspension.
The pesticide compound agent for preventing and treating the apple ring spot is used for preventing and treating the apple ring spot.
The invention has the beneficial effects that: the Pseudomonas syringae (Pseudomonas syringae) B-1 has a remarkable prevention and treatment effect on apple ring rot, and the study of the embodiment shows that the strain B-1 can remarkably inhibit the growth of ring rot hyphae, has a wide antibacterial spectrum, and has remarkable antagonistic action on various fruit and vegetable pathogenic fungi such as botrytis cinerea, penicillium wilt of fruits and vegetables after fruits and vegetables are picked, apple rot pathogen, pear rot pathogen, apple anthracnose blight, apple heart pathogen, peach brown rot pathogen and the like besides the apple ring rot; the prevention and treatment effects of the bacterial suspension and the fermentation supernatant of the bacterial strain B-1 on apple tree branch and fruit ring spot are more than 70%, and the method has the characteristics of stable prevention and treatment effect, environmental friendliness and the like, and is simple in fermentation process and low in production cost in large-scale production.
Drawings
FIG. 1 is a schematic diagram of PCR amplification of 16S rDNA sequence of DNA extracted from strain B-1;
wherein, M in the figure is nucleic acid Marker DL 2000; FIG. 1 shows the result of amplification of the 16S rDNA sequence of the genomic DNA of strain B-1;
FIG. 2 shows the inhibitory effect of the strain B-1 on the growth of hyphae of ring rot apple;
wherein, A in the figure is a colony of the apple ring rot pathogen cultured for 5 days on PDA without adding the strain B-1; b, C, D, E shows that the bacterial concentration of the ring rot apple added with B-1 is 105、106、107And 108cfu·mL-1Culturing the colonies of 5d on the PDA;
FIG. 3 shows the control effect of the supernatant fermented by the strain B-1 on ring spot of apple fruits;
wherein, A in the figure is a control of inoculating ring rot germ after inoculating NB culture medium; in the figure, B is the control effect of the supernatant liquid fermented by the strain B-1 on the ring spot of the apple fruit;
FIG. 4 shows the prevention and treatment effect of the bacterial suspension of the strain B-1 on the ring spot of apple fruits;
wherein, A in the figure is a control of inoculating the ring rot fungi after inoculating the sterile water; b, C, D in the figure are respectively strain B-1 bacterial suspension 105、107And 109cfu·mL-1Preventing and treating effect on apple ring spot.
Detailed Description
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 only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preparation method of the biocontrol microbial inoculum provided by the invention comprises the following steps:
(1) inoculating the strain B-1 in an NA culture medium in a streak manner, and performing activation culture at constant temperature of 30 ℃ for 24h to obtain an activated strain B-1 single colony A;
(2) inoculating the activated strain B-1 single colony A into NB culture medium with bottling amount of 80mL/250mL for 180r min-1Carrying out shake culture at constant temperature of 30 ℃ for 12h to obtain a strain B-1 fermentation seed solution B;
(3) inoculating the fermentation seed liquid B into an NB culture medium according to the inoculation amount of 1:100 for amplification culture, and performing shake culture at constant temperature of 30 ℃ for 24h to obtain 109cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 109cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, collecting supernatant, and filtering with 0.22 μm microporous membrane to obtain strain B-1 fermentation supernatant as biocontrol microbial inoculum; resuspending the pellet in sterile water to obtain 109cfu·mL-1The bacterial suspension is the biological control agent.
In addition, the above-mentioned reference numeral 109cfu·mL-1The bacterial suspension is generally diluted 0-10000 times.
The NA culture medium provided by the invention is prepared by the following method: taking 10g of peptone, 3g of beef extract, 5g of NaCl and 20g of agar powder, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, and sterilizing for 20min by high-pressure steam at 121 ℃.
The NB culture medium provided by the invention is prepared by the following method: taking 10g of peptone, 3g of beef extract and 5g of NaCl, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, subpackaging in 250mL triangular bottles with the bottling amount of 80mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
Example 1
Separation and screening of Pseudomonas syringae (Pseudomonas syringae) B-1 and strain identification
1. Isolation and selection of strains
Mature and healthy fruits are collected from a Shandong tobacco terrace commercialized Fuji apple orchard, fruit tissues are ground after surface disinfection, sterile water is added, a conventional gradient dilution coating separation method is adopted, NA culture media are respectively used for culture at 28 ℃, colonies with obvious colony morphology differences are picked, the colonies are purified and stored on the NA culture media, primary screening and repeated screening of antagonistic bacteria are carried out by taking apple ring rot bacteria as target pathogenic bacteria, and finally a bacterial strain with strong antibacterial activity is obtained and named as B-1.
2. Identification of strains
(1) Morphological characteristics: after the bacterial strain B-1 is cultured on the NA culture medium for 24 hours, the bacterial colony is milky white, round or nearly round, the surface is smooth, the middle is convex, the quality of the bacterial colony is uniform, the growth is fast, and the bacterial body is rod-shaped in microscopic examination and is a gram-negative bacterium.
(2) Physiological and biochemical characteristics: the strain B-1 can utilize glucose, fructose and D-xylose as carbon sources, but can not utilize sucrose, maltose, lactose and the like as carbon sources; the oxidase reaction is negative, the polymer particles are negative, starch is not hydrolyzed, H2S is not produced, indole and arginine are not produced, and the catalase reaction is positive; can liquefy and obviously teach, has positive VP reaction, and the characteristics accord with the description of Pseudomonas syringae (Pseudomonas syringae) in a common bacteria identification manual.
(3)16S rDNA sequence analysis: extracting bacterial genome DNA by a CTAB method, and performing amplification by using a universal Primer F: 5'-AGAGTTTGATCCTGGCTCAG-3', Primer R: 5'-AAGGAGGTGATCCAGCCGCA-3' performing PCR amplification on the DNA template; PCR amplification reaction (25. mu.L): 2.5 μ L10 XTaq enzyme buffer, 0.2 μmol/L primer, 2 μ L200 mmol/L dNTPs, 20ng DNA template, 1U Taq enzyme (TaKaRa), 17 μ L ddH 2O. PCR amplification procedure: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s, annealing at 54 ℃ for 60s, extension at 72 ℃ for 2min, 35 cycles, and extension at 72 ℃ for 5 min. The amplification product (8. mu.L) was detected by electrophoresis on a 1% agarose gel. The PCR amplification results are shown in FIG. 1.
After being cut and recovered, the PCR amplification product is directly delivered to Shanghai bioengineering GmbH for sequence determination. The analysis result of the determined sequence shows that the length of the ITS sequence amplification fragment is 1402bp, and the sequence is shown as the sequence table SEQ ID NO. 1. The resulting sequence was aligned with the nucleic acid sequence in GenBank using BLAST software in NCBI database (http:// www.ncbi.nlm.nih.gov) to show 100% homology with the 16S rDNA sequence of Pseudomonas syringae (Pseudomonas syringae) IPPBC-R30 strain (Accession No: HQ 840766.1).
The strain B-1 is identified as Pseudomonas syringae (Pseudomonas syringae) by combining the 3 points, morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis of genome.
Example 2
Effect of Pseudomonas syringae (Pseudomonas syringae) B-1 on mycelial growth of ring rot apple
Preparing PDA culture medium, adding bacterial suspension of strain B-1 to make its final concentration be 105、106、107And 108cfu·mL-1The activated apple ring rot pathogen was inoculated into the PDA medium as a control without addition of the strain B-1. The cells were incubated at 25 ℃ for 5 days in the dark at a constant temperature, and the colony diameter was measured.
The PDA culture medium comprises the following components in percentage by weight: peeling potatoes, weighing 200g, cutting into small pieces, boiling in water for 15-20 min, filtering with four layers of gauze, adding 20g of glucose and 15g of agar powder, fixing the volume to 1000mL, keeping the pH value natural, and sterilizing with high-pressure steam at 121 ℃ for 20 min.
The result shows that the addition of the bacterial strains B-1 with different concentrations has obvious inhibition effect on the ring rot of apple, and when the bacterial strains B-1 are not added, the diameter of the bacterial colony is 8.52 cm; the bacterial concentration of the bacterial strain B-1 is 105cfu·mL-1When the number of the colonies is larger, the diameter of the colonies is only 3.98cm, and hyphae are sparse; cell B-1 at a cell concentration of 106～108cfu·mL-1When the number of colonies was less than 0.5cm (FIG. 2).
Example 3
Prevention and treatment effect of fermentation supernatant of Pseudomonas syringae (Pseudomonas syringae) B-1 on apple ring rot
Inoculating seed fermentation liquid of Pseudomonas syringae (Pseudomonas syringae) B-1 in NB culture medium, shake culturing at 30 deg.C for 24 hr to obtain cell culture liquid of B-1 strain, and adjusting concentration to 109cfu·mL-1And centrifuging and filtering to obtain fermentation supernatant. And (3) performing activated culture on the apple ring rot on the PDA for 3d, and beating a fungus cake with the diameter of 5mm at the edge of a colony for later use.
The prevention and treatment effect on the fruit ring spot is as follows: selecting Fuji apples with uniform size, sterilizing the surface of 75% alcohol, air drying at room temperature, and making 3 wounds with sterile puncher at the equator position of the apple, wherein each wound has a diameter of 3mm and a depth of 3 mm. Inoculating 30 μ L of Pseudomonas syringae (Pseudomonas syringae) B-1 fermentation supernatant to the fruit wound, air drying at room temperature, inoculating the activated ring rot apple cake, inoculating NB culture medium, and inoculating ring rot apple as control group.
The diameter of the disease spot is observed and measured 5 days after inoculation, and the control effect is calculated according to the area of the disease spot, wherein the control effect is (the area of the disease spot of a control group-the area of the disease spot of a treatment group)/the area of the disease spot of the control group multiplied by 100 percent.
The diseased spots of the fruits in the control group are rapidly expanded after the fruits are inoculated with the ring rot apple germs, while the diseased spots of the treatment group inoculated with the fermentation supernatant of the Pseudomonas syringae (Pseudomonas syringae) B-1 are slowly expanded, and the area of the diseased spots is obviously smaller than that of the control group. The lesion area of the control group is 9.42cm 5 days after inoculation2And the lesion area after the B-1 supernatant treatment is only 1.10cm2The prevention and treatment effect reaches 88.32 percent (figure 3).
The prevention and treatment effect on the ring spot of the branch is as follows: selecting 1-2 year-old healthy Fuji branches with consistent growth vigor, scalding with an electric iron, wherein the length of a wound is about 3mm, the depth of the wound is 1mm, inoculating 20 mu L of Pseudomonas syringae (Pseudomonas syringae) B-1 fermentation supernatant to each wound, airing at room temperature, inoculating the activated ring rot apple fungus cake, and inoculating NB culture medium and then inoculating ring rot apple as a control group.
And observing and measuring the length of the disease spot 7d after inoculation, wherein the disease spot is rapidly expanded after the branches of the control group are inoculated with the ring rot apple germs, the average length of the disease spot is 4.38cm and the width is 1.59cm at 7d, while the disease spot is slowly expanded in the treatment group inoculated with the fermentation supernatant of the Pseudomonas syringae (Pseudomonas syringae) B-1, the length of the disease spot is only 1.52cm and the width is 1.04cm, and the prevention and treatment effect is 77.31%.
Example 4
Prevention and treatment effect of Pseudomonas syringae (Pseudomonas syringae) B-1 bacterial suspension with different concentrations on apple ring rot
Preparation of Pseudomonas syringae (Pseudomonas syringae) B-1 suspensions of various concentrations, 105cfu·mL-1，107cfu·mL-1And 109cfu·mL-1. Selecting Fuji apples with uniform size, sterilizing the surface of 75% alcohol, air drying at room temperature, and making 3 wounds with sterile puncher at the equator position of the apple, wherein each wound has a diameter of 5mm and a depth of 3 mm. Inoculating 30 μ L of Pseudomonas syringae (Pseudomonas syringae) B-1 bacterial suspension to the wound of fruit, air drying at room temperature, inoculating the activated ring rot apple cake, and inoculating sterile water and ring rot apple as control. The control effect is calculated according to the area of the disease spots, and the control effect is (the area of the disease spots of the control group-the area of the disease spots of the treatment group)/the area of the disease spots of the control group multiplied by 100 percent.
The lesion area of the control group is 8.48cm 5 days after inoculation2And the lesion area is obviously reduced after the B-1 bacterial suspension treatment, 105cfu·mL-1After the bacterial suspension is treated, the area of the lesion spot is 1.26cm2The control effect is 85.14%; 107cfu·mL-1After the bacterial suspension is treated, the area of the lesion spot is 0.72cm2The control effect reaches 91.51%; 109cfu·mL-1After the bacterial suspension treatment, the fruits do not get ill, and the control effect is as high as 100% (figure 4).
Selecting 1-2 year-old healthy Fuji branches with consistent growth, scalding with electric iron to obtain wound with length of 3mm and depth of 1mm, inoculating 20 μ L Pseudomonas syringae B-1 bacterial suspension 10 per wound5cfu·mL-1，107cfu·mL-1And 109cfu·mL-1And after being dried at room temperature, the activated ring rot apple fungus cakes are inoculated, and the treatment of inoculating sterile water and then inoculating the ring rot apple is used as a control.
And respectively observing and measuring the length of the lesion spots 5d and 10d after inoculation, and calculating the area and the prevention and treatment effect of the lesion spots, wherein the prevention and treatment effect is more than 70% (table 1).
TABLE 1 prevention and treatment effect of Pseudomonas syringae (Pseudomonas syringae) B-1 suspension with different concentrations on ring rot
Example 5
Inhibition of Pseudomonas syringae (Pseudomonas syringae) B-1 on various plant pathogenic fungi
Pseudomonas syringae (Pseudomonas syringae) B-1 has obvious inhibiting effect on fruit and vegetable botrytis cinerea, fruit and vegetable penicillium, apple tree rot, apple anthracnose leaf blight, apple moldy heart, peach brown rot and other main fruit and vegetable pathogenic fungi, shows good broad-spectrum antibacterial property, and the antibacterial rate is shown in Table 2 when the fruit and vegetable botrytis cinerea is cultured for 5 days. The inhibition ratio is (control colony diameter-treatment colony diameter)/control colony diameter × 100%.
TABLE 2 inhibitory Effect of Pseudomonas syringae (Pseudomonas syringae) B-1 on major pathogenic fungi of fruits and vegetables
Example 6
Prevention and treatment effect of pesticide mixture on apple ring spot
The solution was disposed at 0.1 mmol. multidot.L-12, 6-di-tert-butyl-p-cresol solution to prepare pseudomonas syringae B-1 bacterial suspension 105cfu·mL-1And mixing the components in equal volume to prepare the pesticide compound agent for preventing and treating the ring rot of the apples.
According to the method of the above example 4, healthy apple fruits and branches were selected and wounds were made, 20. mu.L of the pesticidal compound was inoculated to each wound, and after drying at room temperature, activated cake of Verticillium bacteria was inoculated to inoculate 0.1 mmol.L alone-12,6 ofSolution of di-tert-butyl-p-cresol 105cfu·mL-1The pseudomonas syringae B-1 bacterial suspension and sterile water are inoculated with the ring rot germ cake for comparison.
And (5) observing and measuring the length of the disease spot 5d after inoculation, and calculating the disease spot area and the prevention and treatment effect, wherein the prevention and treatment effect is (the disease spot area of the control group-the disease spot area of the treatment group)/the disease spot area of the control group multiplied by 100%.
As can be seen in Table 3, the pesticide mixture can obviously improve the control effect of pseudomonas syringae B-1 on apple ring spot alone, the control effect of fruit ring spot can be improved to 100% from 85.15%, and the control effect of stem ring spot can be improved to 94.33% from 70.69%.
TABLE 32, 6-Di-tert-butyl-p-cresol for improving the control effect of pseudomonas syringae B-1 on apple ring rot
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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<120> pseudomonas syringae and application thereof in prevention and treatment of apple ring rot
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gtccccccga aggttagact agctacttct ggtgcaaccc actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgcgacattc tgattcgcga ttactagcga 120
ttccgacttc acgcagtcga gttgcagact gcgatccgga ctacgatcgg ttttgtgaga 180
ttagctccac ctcgcggctt ggcaaccctc tgtaccgacc attgtagcac gtgtgtagcc 240
caggccgtaa gggccatgat gacttgacgt catccccacc ttcctccggt ttgtcaccgg 300
cagtctcctt agagtgccca ccataatgtg ctggtaacta aggacaaggg ttgcgctcgt 360
tacgggactt aacccaacat ctcacgacac gagctgacga cagccatgca gcacctgtct 420
caatgttccc gaaggcacca atccatctct ggaaagttca ttggatgtca aggcctggta 480
aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcatttg agttttaacc ttgcggccgt actccccagg cggtcaactt aatgcgttag 600
ctgcgccact aagagctcaa ggctcccaac ggctagttga catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgca cctcagtgtc agtatcagtc 720
caggtggtcg ccttcgccac tggtgttcct tcctatatct acgcatttca ccgctacaca 780
ggaaattcca ccaccctcta ccatactcta gcttgccagt tttggatgca gttcccaggt 840
tgagcccggg gatttcacat ccaacttaac aaaccaccta cgcgcgcttt acgcccagta 900
attccgatta acgcttgcac cctctgtatt accgcggctg ctggcacaga gttagccggt 960
gcttattctg tcggtaacgt caaaacaatc acgtattagg taactgccct tcctcccaac 1020
ttaaagtgct ttacaatccg aagaccttct tcacacacgc ggcatggctg gatcaggctt 1080
tcgcccattg tccaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1140
ttccagtgtg actgatcatc ctctcagacc agttacggat cgtcgccttg gtgagccatt 1200
acctcaccaa ctagctaatc cgacctaggc tcatctgata gcgcaaggcc cgaaggtccc 1260
ctgctttctc ccgtaggacg tatgcggtat tagcgtccgt ttccgagcgt tatcccccac 1320
taccaggcag attcctaggc attactcacc cgtccgccgc tcgccaccag gtacaagtac 1380
ccgtgctgcc gctcgactgc at 1402
Claims (10)
1. Pseudomonas syringae (A. syringae)Pseudomonas syringae) Strain B-1, said strain B-1 being deposited with: china center for type culture Collection, accession number: CCTCC NO: M2015813.
2. Pseudomonas syringae (Pseudomonas syringae) according to claim 1Pseudomonas syringae) The biocontrol microbial inoculum prepared by the strain B-1 is characterized in that: the biocontrol microbial inoculum is strain B-1 fermentation supernatant or strain B-1 bacterial suspension.
3. The use of the biocontrol microbial inoculum of claim 2, which is characterized in that: the bio-control microbial inoculum is used for preventing and controlling fruit and vegetable diseases.
4. The use of the biocontrol microbial inoculum according to claim 3, wherein: the biocontrol microbial inoculum is used for preventing and treating apple ring rot.
5. A method for preparing the biocontrol microbial inoculum of claim 2, which is characterized by comprising the following steps: the method comprises the following steps:
(1) inoculating the strain B-1 in an NA culture medium in a streak manner, and performing activation culture at constant temperature of 30 ℃ for 24h to obtain an activated strain B-1 single colony A;
(2) inoculating the activated strain B-1 single colony A into NB culture medium with bottling amount of 80mL/250mL for 180r min-1Carrying out shake culture at constant temperature of 30 ℃ for 12h to obtain a strain B-1 fermentation seed solution B;
(3) inoculating the fermentation seed liquid B into an NB culture medium according to the inoculation amount of 1:100 for amplification culture, and performing shake culture at constant temperature of 30 ℃ for 24h to obtain 109cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 109cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, and filtering the collected supernatant through a 0.22 mu m microporous filter membrane to obtain a strain B-1 fermentation supernatant, namely the biocontrol microbial inoculum; resuspending the pellet in sterile water to obtain 109cfu·mL-1The bacterial suspension is the biological control agent.
6. The method for preparing a biocontrol microbial inoculum according to claim 5, which is characterized by comprising the following steps: the NA culture medium is prepared by the following method: taking 10g of peptone, 3g of beef extract, 5g of NaCl and 20g of agar powder, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, and sterilizing for 20min by high-pressure steam at 121 ℃.
7. The method for preparing a biocontrol microbial inoculum according to claim 5, which is characterized by comprising the following steps: the NB medium was prepared by the following method: taking 10g of peptone, 3g of beef extract and 5g of NaCl, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, subpackaging in 250mL triangular bottles with the bottling amount of 80mL, and sterilizing for 20min by high-pressure steam at 121 ℃.
8. A pesticide mixture for preventing and treating apple ring rot is characterized in that: the pesticide compound agent comprises the effective components of 2, 6-di-tert-butyl-p-cresol (BHT) and the pseudomonas syringae B-1 bacterial suspension as claimed in claim 1.
9. The pesticidal combination according to claim 8, wherein: the pesticide compound agent is prepared from the following active ingredients in parts by volume: 0.1 mmol. L-11 part of 2, 6-di-tert-butyl-p-cresol solution and 10 parts of5 cfu·mL-11 part of pseudomonas syringae B-1 bacterial suspension.
10. Use of a pesticidal combination according to claim 8 or 9, wherein: the pesticide compound agent is used for preventing and treating apple ring rot.
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| CN103525725B (en) * | 2013-09-25 | 2015-03-25 | 东北农业大学 | Pseudomonas syringae and application thereof in prevention of tomato gray mold |
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