CN111280183A - Apple anthracnose leaf blight biocontrol microbial inoculum and preparation method and application thereof - Google Patents
Apple anthracnose leaf blight biocontrol microbial inoculum and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
- A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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 an apple anthracnose leaf blight biocontrol microbial inoculum which is a bacterial suspension of Pseudomonas syringae (Pseudomonas syringae) strain B-1. The Pseudomonas syringae (Pseudomonas syringae) B-1 has a remarkable control effect on apple anthracnose leaf blight, and researches of the embodiments show that the strain B-1 can remarkably inhibit the growth of hypha of the apple anthracnose leaf blight, and the control effects of the bacterial suspension of the strain B-1 on the apple anthracnose leaf blight are all over 60 percent; the apple anthracnose leaf blight biocontrol microbial inoculum is environment-friendly, simple in fermentation process in large-scale production and low in production cost.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to an apple anthracnose leaf blight biocontrol microbial inoculum, and a preparation method and application thereof.
Background
The apple contains abundant carbohydrate, vitamins, inorganic salt and other substances, and is an important nutrient source and material basis for people. The apple is taken as an important economic crop and export fruit in China, the cultivation area and the yield are at the top of the world, the apple is one of important post industries for increasing the income of farmers in the northern China, but the healthy development of the apple industry in China is severely restricted by diseases.
Apple anthracnose leaf blight (GLS) caused by colletotrichum species (glomeriella cingulata) is a newly discovered disease in apples in China in recent years, and mainly damages apple leaves and fruits, so that the leaves are scorched and a large amount of leaves fall off in advance. After the apple fruits are infected, black disease spots with the diameter of 1-2mm are formed on the surfaces of the apples, so that the yield and the quality of the apples are directly reduced, serious economic loss is caused, the tree vigor is seriously weakened, the disease resistance of trees is reduced, and the harm of branch diseases and root diseases is induced. Apple anthracnose leaf blight disease is first reported in China in 2012, and has spread to most apple main producing areas in 2014. Wherein, such as: gala, Jinguan, Qinhuan and other varieties are highly susceptible to diseases, and Fuji, Hongxing and other varieties have strong resistance.
The breeding by utilizing the disease-resistant gene is an ideal strategy for overcoming the anthracnose and leaf blight of the apples, but related researches have not made breakthrough progress yet, and the method has a long distance from the application to production. At present, the control of the anthracnose leaf blight of the apples mainly depends on chemical bactericides, and frequent use of the chemical bactericides can not only deteriorate the ecological environment of an orchard, but also bring great hidden danger to food safety; moreover, the anthracnose and leaf blight of the apple is rapidly attacked, the incubation period is only 2-4 days, and once pathogenic bacteria invade host tissues, almost no drug control time is available. If the disease cannot be effectively controlled, good varieties with diseases such as Gala, golden crown, Qinhuan and the like face the risk of being eliminated in China. The wide use of chemical bactericides brings serious threats to human health and environmental safety, and further the problem of drug resistance is gradually revealed. Therefore, the search for safe and effective control measures capable of replacing chemical bactericides has become a problem to be solved urgently in apple production and disease control.
The method for preventing and controlling the anthracnose and leaf blight of the apples by adopting the bio-control fungicide is environment-friendly, and can avoid a series of problems caused by chemical prevention and control. The biocontrol microbial inoculum can reduce or replace the use of chemical agents, thereby achieving the purpose of preventing and controlling the anthracnose and leaf blight of apples. No report that a Pseudomonas syringae (Pseudomonas syringae) is adopted to prepare an apple anthracnose leaf blight biocontrol microbial inoculum for controlling apple anthracnose leaf blight 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 apple anthracnose leaf blight biocontrol microbial inoculum provided by the invention is pseudomonas syringae (Pseudomonas aeruginosa) strain B-1 bacterial suspension.
The apple anthracnose leaf blight biocontrol microbial inoculum provided by the invention has the concentration of 103-107cfu·mL-1A strain of Pseudomonas syringae (Pseudomonas syringae) strain B-1.
The invention provides a concentration of 107cfu·mL-1A strain of Pseudomonas syringae (Pseudomonas syringae) strain B-1.
The biocontrol microbial inoculum for the apple anthracnose leaf blight provided by the invention is used for controlling the apple anthracnose leaf blight.
The preparation method of the apple anthracnose leaf blight 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 medium with bottling amount of 80mL/250mL，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 12h to obtain 107cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 107cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, collecting thallus precipitate, and resuspending with sterile water to obtain 107cfu·mL-1The bacterial suspension is the apple anthracnose leaf blight biocontrol bacterial 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 anthracnose leaf blight provided by the invention has the effective components of 2, 6-ditert-butyl-p-cresol (BHT) and pseudomonas syringae B-1 bacterial suspension.
The pesticide compound agent for preventing and treating the apple anthracnose leaf blight provided by the invention 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 anthracnose leaf blight provided by the invention is used for preventing and treating the apple anthracnose leaf blight.
The invention has the beneficial effects that: the Pseudomonas syringae (Pseudomonas syringae) B-1 has a remarkable control effect on apple anthracnose leaf blight, and researches of the embodiments show that the strain B-1 can remarkably inhibit the growth of hypha of the apple anthracnose leaf blight, and the control effects of the bacterial suspension of the strain B-1 on the apple anthracnose leaf blight are all over 60 percent; the apple anthracnose leaf blight biocontrol microbial inoculum is environment-friendly, simple in fermentation process in large-scale production and low in production cost.
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 different concentrations of the strain B-1 on the hypha growth of apple colletotrichum gloeosporioides;
FIG. 3 shows the control effect of different concentrations of the bacterial strain B-1 on apple anthracnose leaf blight;
wherein A is the influence of the strains B-1 with different concentrations on the disease index of the apple anthracnose leaf blight, and B is the influence of the strains B-1 with different concentrations on the disease index scab number of the apple anthracnose leaf blight.
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 12h to obtain 107cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 107cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, collecting thallus precipitate, and resuspending with sterile water to obtain 107cfu·mL-1The bacterial suspension is the apple anthracnose leaf blight biocontrol bacterial agent.
In addition, the above-mentioned reference numeral 107cfu·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 L200mmol/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 products (8. mu.L) were detected by electrophoresis on a 1% agarose gel (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 3
Effect of Pseudomonas syringae (Pseudomonas syringae) B-1 on hypha growth of Aphyllophora pomifera
Inoculating seed fermentation liquid of Pseudomonas syringae (Pseudomonas syringae) B-1 into NB culture medium, shake culturing at constant temperature of 30 ℃ for 12h to obtain cell culture liquid of the strain B-1, centrifuging and collecting the strain B-1.
PDA medium was prepared, and the cells of the strain B-1 were added to the medium to give a final concentration of 104、105、106And 107cfu·mL-1And inoculating activated apple colletotrichum gloeosporioides to the PDA culture medium by taking the PDA culture medium without adding the strain B-1 as a control. The cells were incubated at 25 ℃ for 3d in the dark at constant temperature, and the colony diameter was measured.
The PDA culture medium comprises the following components in percentage by weight: peeling potato, weighing 200g, cutting into small pieces, boiling in water for 15-20min, filtering with four layers of gauze, adding glucose 20g and agar powder 15g, diluting to 1000mL, adjusting pH to natural, and sterilizing with high pressure steam at 121 deg.C for 20 min.
The results show that the addition of the bacterial strains B-1 with different concentrations has obvious inhibition effect on the apple colletotrichum gloeosporioides, and the bacterial suspension concentration of the bacterial strain B-1 is 104cfu·mL-1When the bacteria is detected, the bacteriostasis rate is 45.25 percent; b-1 at a cell concentration of 105cfu·mL-1When the bacteria is detected, the bacteriostasis rate is 68.98%; b-1 at a cell concentration of 106cfu·mL-1-107cfu·mL-1The bacteriostasis rate is over 85 percent (figure 2).
Example 4
Control effect of Pseudomonas syringae (Pseudomonas syringae) B-1 bacterial suspension on apple anthracnose leaf blight
Preparing spore suspension of apple colletotrichum gloeosporioides, culturing pathogenic bacteria on PDA at constant temperature in dark, scraping off aerial hyphae with inoculating ring when hyphae grow to 2/3 flat dish, allowing generation of orange conidia after 2-3 days, configuring conidia suspension, and adjusting concentration to 5 × 104each.mL-1。
Preparation of suspensions of Strain B-1 at different concentrations, 103cfu·mL-1，105cfu·mL-1And 107cfu·mL-1. And selecting the gala apple branches with consistent growth vigor and completely unfolded leaves for one to two years, respectively and uniformly spraying the bacterial suspension of the bacterial strains B-1 with different concentrations until water drops on the leaves, and taking sterile water as a reference. Treating the B-1 bacterial suspension with different concentrations for 3d, and spraying to inoculate the prepared apple anthrax leavesAnd (4) carrying out field inoculation on the fusarium oxysporum spore suspension, then moisturizing for 24h by using a plastic bag, and regularly observing and recording the disease condition of the leaves.
Grading according to GB/T17980.124-2004 'test criteria for pesticide field efficacy (II)', and calculating disease index.
Level 0: no disease spots; level 1: the lesion area accounts for less than 10% of the whole leaf area; and 3, level: the area of the lesion spots accounts for 11-30% of the area of the leaves; and 5, stage: the area of the lesion spots accounts for 30-50% of the area of the leaves; and 7, stage: the lesion area accounts for 51 percent of the leaf area; and 9, stage: and (4) falling leaves.
Calculating the disease index as 100 x (the number of each disease leaf multiplied by the disease value)/(the total number of investigated leaves multiplied by the highest value); the control effect is (contrast disease index-strain B-1 suspension treatment disease index)/contrast disease index is multiplied by 100 percent
The control effect of the bacterial suspension of the bacterial strains B-1 with different concentrations on the apple anthracnose leaf blight is shown in figure 3, after the bacterial suspension of the bacterial strains B-1 with different concentrations is treated, the number of spots and disease index of the apple anthracnose leaf blight are both obviously reduced, and the bacterial concentration of B-1 is 103cfu·mL-1The control effect is 60.95 percent, and the concentration of B-1 bacteria is 105cfu·mL-1And 107cfu·mL-1The prevention and treatment effects respectively reach 78.05% and 80.73%.
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 pathogenic fungi such as fruit and vegetable botrytis cinerea, fruit and vegetable penicillium, apple tree rot, apple ring rot, apple pith and peach brown rot, and shows good broad-spectrum antibacterial property, and the antibacterial rate is shown in Table 1 when the fruit and vegetable pathogenic fungi are cultured for 5 days. The inhibition ratio is (control colony diameter-treatment colony diameter)/control colony diameter × 100%.
TABLE 1 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 compound agent on apple anthracnose leaf blight
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 anthracnose and leaf blight of the apples.
Preparing spore suspension of apple colletotrichum gloeosporioides, culturing pathogenic bacteria on PDA at constant temperature in dark, scraping off aerial hyphae with inoculating ring when hyphae grow to 2/3 flat dish, allowing generation of orange conidia after 2-3 days, configuring conidia suspension, and adjusting concentration to 5 × 104each.mL-1。
Selecting Gala apple branches with completely developed leaves of one to two years of consistent growth vigor, uniformly spraying the pesticide compound agent until water drops on the leaves drop, and independently inoculating 0.1 mmol.L-12, 6-di-tert-butyl-p-cresol solution of (5), 105cfu·mL-1The suspension of Pseudomonas syringae B-1 strain and sterile water were used as controls.
Inoculating the spore suspension of the apple colletotrichum gloeosporioides 3 days after the different treatments are sprayed, moisturizing the inoculated apple colletotrichum gloeosporioides in a plastic bag for 24 hours after the field inoculation, and regularly observing and recording the disease condition of the leaves. Calculating disease index and prevention and treatment effect according to disease grading standard, wherein the disease index is 100 multiplied by (the number of each grade of diseased leaves multiplied by the disease value)/(the number of total investigated leaves multiplied by the highest value); the control effect is (control disease index-strain B-1 suspension treatment disease index)/control disease index is multiplied by 100%.
As can be seen in Table 2, the pesticide compound agent can remarkably improve the control effect of the pseudomonas syringae B-1 on the apple anthracnose leaf blight alone, and the control effect is improved from 78.11% to 92.89%.
TABLE 22, 6-Di-tert-butyl-p-cresol for improving the control effect of pseudomonas syringae B-1 on anthracnose leaf blight
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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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 (8)
1. A biocontrol microbial inoculum for apple anthracnose leaf blight is characterized in that: the apple anthracnose leaf blight biocontrol microbial inoculum is a suspension of Pseudomonas syringae (Pseudomonas syringae) strain B-1.
2. The biocontrol bacterial agent for anthracnose leaf blight of apple as claimed in claim 1, which is characterized in that: the apple anthracnose leaf blight biocontrol microbial inoculum is 10 in concentration3-107cfu·mL-1A strain of Pseudomonas syringae (Pseudomonas syringae) strain B-1.
3. The apple of claim 1The fruit anthracnose leaf blight biocontrol microbial inoculum is characterized in that: the apple anthracnose leaf blight biocontrol microbial inoculum is 10 in concentration7cfu·mL-1A strain of Pseudomonas syringae (Pseudomonas syringae) strain B-1.
4. The application of the biocontrol bacterial agent for the anthracnose leaf blight of apples as defined in any one of claims 1 to 3, which is characterized in that: the apple anthracnose leaf blight biocontrol microbial inoculum is used for controlling apple anthracnose leaf blight.
5. The preparation method of the biocontrol microbial inoculum for the apple anthracnose leaf blight of the claim 1, which is characterized by comprising the following steps of: 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 12h to obtain 107cfu·mL-1Cell culture solution C of B-1 strain;
(4) will 107cfu·mL-1Cell culture solution C of B-1 strain at 4 deg.C for 12000 r.min-1Centrifuging for 15min, collecting thallus precipitate, and resuspending with sterile water to obtain 107cfu·mL-1The bacterial suspension is the apple anthracnose leaf blight biocontrol bacterial agent.
6. A pesticide compound agent for preventing and controlling apple anthracnose leaf blight is characterized in that: the active ingredients in the pesticide compound agent are 2, 6-di-tert-butyl-p-cresol (BHT) and pseudomonas syringae B-1 bacterial suspension.
7. The pesticidal combination according to claim 6, 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 of5cfu·mL-11 part of pseudomonas syringae B-1 bacterial suspension.
8. Use of a pesticidal combination according to claim 6 or 7, wherein: the pesticide compound agent is used for preventing and controlling apple anthracnose leaf blight.
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| CN112175869A (en) * | 2020-09-29 | 2021-01-05 | 烟台市林业科学研究所 | Pseudomonas and application thereof |
| CN112772657A (en) * | 2021-01-12 | 2021-05-11 | 青岛农业大学 | Application of pipecolic acid as plant disease-resistant activator in preventing and treating apple leaf diseases |
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| CN112772657B (en) * | 2021-01-12 | 2021-10-01 | 青岛农业大学 | Application of pipecolic acid as plant disease-resistant activator in preventing and treating apple leaf diseases |
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