CN111197016A - Screening method of kiwifruit canker antagonistic strain - Google Patents
Screening method of kiwifruit canker antagonistic strain Download PDFInfo
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Abstract
The invention discloses a screening method of kiwifruit canker antagonistic strains, which comprises the following steps: preparing a strain; preparing strain fermentation liquor: preparation of active crude extract, etc. The invention aims to provide a screening method of kiwi canker antagonistic strains, which is rapid and easy to implement, good in screening effect, wide in raw material source, economical and low in cost.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a strain screening method, in particular to a screening method of kiwifruit canker antagonistic strains.
Background
The kiwi fruit is also called kiwi fruit, the pulp of the kiwi fruit is rich in sugar, various vitamins, amino acids, organic acids, K +, Mg2+, pigment and other components, the kiwi fruit can be eaten fresh or processed into preserved fruit, cans, various beverages and the like in large quantity, and kiwi fruit seeds, leaves, branches and flowers have further processing and developing values. Therefore, the kiwi fruit has wide market development prospect and good research value.
However, bacterial canker caused by Pseudomonas syringae kiwi fruit pathopoiesia (Pseudomonas syringae pv. Actinidiae, Psa) is the largest disease faced in kiwi fruit production, and the kiwifruit canker mainly infects trunks, branches, leaves and flowers, causing branch canker or wilting of branches and leaves. Since the kiwi fruit canker is reported in Japan for the first time in 1980, the kiwi fruit canker is widely distributed in a worldwide kiwi fruit cultivation area, is seriously damaged, can reduce the yield and the quality of fruits at the same time, and causes huge economic loss.
At present, chemical pesticide is still the main prevention and treatment means for kiwifruit canker, but long-term excessive use of chemical pesticide can cause pathogenic bacteria to generate drug resistance, so that the prevention effect is reduced; and the high residue of chemical pesticide has great damage to environment and is harmful to human health. Therefore, the development of a novel pesticide with high efficiency, safety and low residue is urgently needed, and the development and utilization of biological control of antagonistic microorganisms is one of effective hands for controlling the kiwifruit canker.
Disclosure of Invention
The invention aims to solve the problems, provides a screening method of an antagonistic strain of kiwifruit canker, screens out endophytic actinomycetes of leaves and branches of kiwifruit, and provides support for the prevention and treatment of bacterial canker of kiwifruit from the perspective of biopesticides.
In order to realize the purpose, the invention adopts the technical scheme that:
a screening method of kiwifruit canker antagonistic strains comprises the following steps:
(1) preparation of the strain: preparing kiwi fruit leaves or branches into tissue blocks, sterilizing the surfaces of the tissue blocks, pasting the tissue blocks with sterilized surfaces into a flat plate made of a starch casein culture medium and/or a Gao's I synthetic culture medium under an aseptic condition, placing the flat plate in an incubator at 26-29 ℃, culturing for 5-7 days, purifying strains after bacterial colonies grow out, and inoculating the purified strains into a slant culture medium for storage and standby;
(2) preparing strain fermentation liquor: filling millet culture medium into a culture bottle, sterilizing and cooling; inoculating the purified strain into a culture bottle, placing the culture bottle into a shaking table at 26-29 ℃, culturing for 4-6d at the rotating speed of 190r/min, filtering and collecting filtrate, and preserving at low temperature for later use;
(3) preparation of active crude extract: enriching the filtrate with adsorption resin, eluting with methanol water, collecting methanol eluate, and concentrating to 1/5 of methanol water elution volume as active crude extract.
Preferably, 4 tissue blocks are attached to each plate in the step (1), the plates are placed in an incubator at 28 ℃, the culture is carried out for 6 days, and after colonies grow out, the strains are classified and purified according to differences of forms and colors.
Preferably, in the step (2), the culture flask inoculated with the purified strain is placed in a shaker at 28 ℃ and cultured for 5 days at a rotating speed of 180r/min, and the filtrate is filtered and collected and stored at 4 ℃ for later use.
Preferably, in the step (3), methanol water with the concentration of 40%, 60% and 100% is used for gradient elution, methanol elution is collected, and active crude extract is obtained by fraction concentration.
Preferably, the step (2) of testing the bacteriostatic activity of the strain fermentation liquor comprises the following substeps:
① the kiwifruit canker bacteria is prepared to concentration of 1 × 106Taking 1mL of the bacterial suspension and 9mL of beef extract peptone culture medium, and fully and uniformly mixing to prepare 8 groups of bacteria-carrying flat plates;
② placing 5 Oxford cups at equal distance on each bacterium-carrying plate in the step ①;
③ adding 200 μ L of strain fermentation liquid with corresponding number into each Oxford cup, culturing at 37 deg.C for 24h, and measuring the diameter of zone of inhibition;
④ repeating the step ③ 2 times, and taking the average diameter of the zone of inhibition.
Preferably, the beef extract peptone medium comprises the following components: 3.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 15.0g of agar and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2.
Preferably, the starch casein culture medium in the step (1) comprises the following components: 10.0g of soluble starch, 2.0g of dipotassium phosphate, 2.0g of potassium nitrate, 2.0g of sodium chloride, 0.3g of casein, 0.05g of magnesium sulfate heptahydrate, 0.02g of calcium carbonate, 0.01g of ferrous sulfate heptahydrate, 15.0g of agar powder and 1000mL of distilled water.
Preferably, the medium for synthesizing the culture medium of Gauss I in the step (1) comprises the following components: 20g of soluble starch, 0.5g of sodium chloride, 1.0g of dipotassium hydrogen phosphate, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate heptahydrate, 1.0g of potassium nitrate, 15.0g of agar powder, 1000mL of distilled water and pH 7.2-7.4.
Preferably, the medium for fermenting the millet in the step (2) comprises the following components: 10.0g of glucose, 10.0g of millet, 2.5g of sodium chloride, 3.0g of peptone, 1.0g of calcium carbonate, 1.0g of ammonium sulfate, 1000mL of distilled water and pH 7.2.
The method has the characteristics of quick and easy screening and good screening effect, and the obtained bacterial strain can be used for effectively preventing and treating the kiwifruit canker, so that the survival rate and the fruit yield of the kiwifruit tree are improved; according to the invention, the kiwi fruit branches and leaves are used as the initial raw material for screening, the obtaining way is wide, the method is economical and cheap, and the obtained bacterial strain and the prepared microbial inoculum can be directly applied to kiwi fruit control work.
Detailed Description
The present invention is described in detail below for the purpose of better understanding technical solutions of the present invention, and the description of the present section is only exemplary and explanatory and should not be construed as limiting the scope of the present invention in any way.
Sample preparation:
collecting 3 kiwi fruit plantation districts of Shanxi province Weinan city Shangwei village, Gao Li village and Huazhou district, and randomly collecting branches and leaves of healthy kiwi fruit plants in 2018 in 4-5 months, wherein 8 leaf samples are obtained, and 6 leaves are provided for example 1; 8 portions of branch samples, 6 branches per portion, are provided for example 2; leaf and shoot mixed samples 8, 6 leaves per leaf and 6 shoots for use in example 3.
Preparation of pathogenic bacteria: the kiwifruit canker is a conventional pathogen and is provided by a plant pathology research laboratory of northwest agriculture and forestry science and technology university, other similar pathogens can be adopted, and a biological storage sample is not required to be provided here.
Preparation of a culture medium:
starch casein culture medium: 10.0g of soluble starch, 2.0g of dipotassium phosphate, 2.0g of potassium nitrate, 2.0g of sodium chloride, 0.3g of casein, 0.05g of magnesium sulfate heptahydrate, 0.02g of calcium carbonate, 0.01g of ferrous sulfate heptahydrate, 15.0g of agar powder and 1000mL of distilled water.
High-count one-synthesis medium: 20g of soluble starch, 0.5g of sodium chloride, 1.0g of dipotassium hydrogen phosphate, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate heptahydrate, 1.0g of potassium nitrate, 15.0g of agar powder, 1000mL of distilled water and pH 7.2-7.4.
Beef extract peptone medium: 3.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 15.0g of agar and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2.
Millet fermentation medium: 10.0g of glucose, 10.0g of millet, 2.5g of sodium chloride, 3.0g of peptone, 1.0g of calcium carbonate, 1.0g of ammonium sulfate, 1000mL of distilled water and pH 7.2.
Example 1
Strain screening:
(1) preparation of the strain: cutting leaves into small round pieces with the diameter of 0.6cm by a puncher to prepare tissue blocks, sterilizing the surfaces of the tissue blocks, namely washing the tissue blocks by using 75% alcohol for 1 time → washing the tissue blocks by using 3.5% sodium hypochlorite for 2 times → washing the tissue blocks by using 75% alcohol for 1 time, pasting the tissue blocks with the sterilized surfaces in flat plates prepared from a starch casein culture medium and a Gao's I synthetic culture medium under the aseptic condition, pasting 4 tissue blocks on each flat plate, placing the flat plates in an incubator at 28 ℃, culturing for 6d, classifying and purifying strains according to the difference of forms and colors after colonies grow out, and inoculating the purified strains in a slant culture medium for storage and later use.
After the tissue block is disinfected, whether the disinfected tissue block is disinfected completely or not needs to be detected, and the specific detection method comprises the following steps: immediately cleaning the tissue blocks with sterile water for 3 times after the tissue blocks are disinfected, adding 1000 mu L of cleaning solution into a flat plate made of a starch casein culture medium and a Gao's first synthetic culture medium, uniformly coating, placing the flat plate in an incubator at 28 ℃, culturing for 3d, observing whether bacterial colonies grow out on the surface, if the bacterial colonies grow out, the disinfection is thorough, and if the bacterial colonies grow out, the disinfection is incomplete, and repeating the disinfection treatment.
(2) Preparing strain fermentation liquor: 50mL of millet culture medium is filled into a 250mL triangular flask, and the millet culture medium is sterilized and cooled; inoculating each purified strain into a culture bottle, placing into a shaking table at 28 ℃, culturing for 5d at the rotating speed of 180r/min, filtering, collecting filtrate, and preserving at 4 ℃ for later use.
After the steps are finished, the antibacterial activity of the strain fermentation liquor is measured by adopting a tube-disc method, and the tube-disc method comprises the following steps:
① the kiwifruit canker bacteria is prepared to concentration of 1 × 106Taking 1mL of the bacterial suspension and 9mL of beef extract peptone culture medium, and fully and uniformly mixing to prepare a bacterium-carrying flat plate;
② placing 5 Oxford cups with equal distance on the bacterium carrying flat plate in the step ①;
③ adding 200 μ L fermentation liquid into each Oxford cup, culturing at 37 deg.C for 24h, and measuring the diameter of zone;
④ repeating the step ③ 2 times, and taking the average diameter of the zone of inhibition.
The activity determination result can be used for ensuring that the strain fermentation liquor contains the strain, and can also be used for screening the strain fermentation liquor with the best antibacterial effect for use in the step (3).
(3) Preparation of active crude extract: enriching the filtrate by HPD100 adsorption resin, gradient eluting with 40%, 60%, 100% methanol water respectively, collecting methanol eluate, and concentrating the eluate to 1/5 of methanol water elution volume to obtain active crude extract.
Example 2
Strain screening:
(1) preparation of the strain: cutting branches into small sections with incisions at two ends of 0.6cm by using a branch scissors subjected to aseptic treatment to prepare tissue blocks, sterilizing the surfaces of the tissue blocks, namely washing the tissue blocks by using 75% alcohol for 3 times → 3.5% sodium hypochlorite for 4 times → 75% alcohol for 3 times, pasting the tissue blocks with sterilized surfaces in flat plates prepared from a starch casein culture medium and a Gao's I synthetic culture medium under an aseptic condition, pasting 4 tissue blocks on each flat plate, placing the flat plates in an incubator at 28 ℃, culturing for 6d, classifying and purifying strains according to differences of forms and colors after bacterial colonies grow out, and inoculating the purified strains in a slant culture medium for storage for later use;
after the tissue block is disinfected, whether the disinfected tissue block is disinfected completely or not needs to be detected, and the specific detection method comprises the following steps: immediately cleaning the tissue blocks with sterile water for 3 times after the tissue blocks are disinfected, adding 1000 mu L of cleaning solution into a flat plate made of a starch casein culture medium and a Gao's first synthetic culture medium, uniformly coating, placing the flat plate in an incubator at 28 ℃, culturing for 3d, observing whether bacterial colonies grow out on the surface, if the bacterial colonies grow out, the disinfection is thorough, and if the bacterial colonies grow out, the disinfection is incomplete, and repeating the disinfection treatment.
(2) Preparing strain fermentation liquor: 50mL of millet culture medium is filled into a 250mL triangular flask, and the millet culture medium is sterilized and cooled; inoculating each purified strain into a culture bottle, placing into a shaking table at 28 ℃, culturing for 5d at the rotating speed of 180r/min, filtering, collecting filtrate, and preserving at 4 ℃ for later use.
After the steps are finished, the antibacterial activity of the strain fermentation liquor is measured by adopting a tube-disc method, and the tube-disc method comprises the following steps:
① the kiwifruit canker bacteria is prepared to concentration of 1 × 106Taking 1mL of the bacterial suspension and 9mL of beef extract peptone culture medium, and fully and uniformly mixing to prepare a bacterium-carrying flat plate;
② placing 5 Oxford cups with equal distance on the bacterium carrying flat plate in the step ①;
③ adding 200 μ L fermentation liquid into each Oxford cup, culturing at 37 deg.C for 24h, and measuring the diameter of zone;
④ repeating the step ③ 2 times, and taking the average diameter of the zone of inhibition.
The activity determination result can be used for ensuring that the strain fermentation liquor contains the strain, and can also be used for screening the strain fermentation liquor with the best antibacterial effect for use in the step (3).
(3) Preparation of active crude extract: enriching the filtrate by HPD100 adsorption resin, gradient eluting with 40%, 60%, 100% methanol water respectively, collecting methanol eluate, and concentrating the eluate to 1/5 of methanol water elution volume to obtain active crude extract.
Example 3
Strain screening:
(1) preparation of the strain: cutting leaves into small round pieces with the diameter of 0.6cm by using a puncher, shearing branches into small sections with notches at two ends of 0.6cm by using a branch shear subjected to aseptic treatment to prepare tissue blocks, and sterilizing the surfaces of the tissue blocks, namely flushing the branches by using 75% alcohol for 3 times → 3.5% sodium hypochlorite for 4 times → 75% alcohol for 3 times; washing the leaves with 75% alcohol for 1 time → 3.5% sodium hypochlorite for 2 times → 75% alcohol for 1 time, pasting tissue blocks with disinfected surfaces on flat plates made of a starch casein culture medium and a Gao's first synthetic culture medium under an aseptic condition, pasting 4 tissue blocks on each flat plate, placing the flat plates in an incubator at 28 ℃, culturing for 6d, classifying and purifying strains according to differences of forms and colors after colonies grow out, obtaining 37 strains after purification, inoculating the purified strains in a slant culture medium for storage, and numbering WN1-WN37 for each strain;
after the tissue block is disinfected, whether the disinfected tissue block is disinfected completely or not needs to be detected, and the specific detection method comprises the following steps: immediately cleaning the tissue blocks with sterile water for 3 times after the tissue blocks are disinfected, adding 1000 mu L of cleaning solution into a flat plate made of a starch casein culture medium and a Gao's first synthetic culture medium, uniformly coating, placing the flat plate in an incubator at 28 ℃, culturing for 3d, observing whether bacterial colonies grow out on the surface, if the bacterial colonies grow out, the disinfection is thorough, and if the bacterial colonies grow out, the disinfection is incomplete, and repeating the disinfection treatment.
(2) Preparing strain fermentation liquor: 50mL of millet culture medium is filled into a 250mL triangular flask, and the millet culture medium is sterilized and cooled; inoculating each purified strain into a culture bottle, placing the culture bottle into a shaking table at 28 ℃, culturing for 5d at the rotating speed of 180r/min, filtering and collecting 37 parts of filtrate, and preserving at 4 ℃ for later use.
After the steps are finished, the antibacterial activity of the strain fermentation liquor is measured by adopting a tube-disc method, and the tube-disc method comprises the following steps:
① the kiwifruit canker bacteria is prepared to concentration of 1 × 106Taking 1mL of the bacterial suspension and 9mL of beef extract peptone culture medium, and fully and uniformly mixing to prepare 8 groups of bacteria-carrying flat plates;
② placing 5 Oxford cups at equal distance on each bacterium-carrying plate in the step ①;
③ adding 200 μ L of strain fermentation liquid with corresponding number into each Oxford cup, culturing at 37 deg.C for 24h, and measuring the diameter of zone of inhibition;
④ repeating the step ③ 2 times, and taking the average diameter of the zone of inhibition.
The activity determination result can be used for ensuring that the strain fermentation liquor contains the strain, and can also be used for screening the strain fermentation liquor with the best antibacterial effect for use in the step (3).
(3) Preparation of active crude extract: enriching the filtrate in each number by HPD100 adsorption resin, gradient eluting with 40%, 60% and 100% methanol water respectively, collecting methanol eluate, and concentrating the eluate to 1/5 of methanol water eluate volume to obtain active crude extract.
The experimental result shows that the last cleaning solution of all the tissue blocks of the sample is added into a flat plate made of starch casein culture medium and Gao's I synthetic culture medium, the flat plate is uniformly coated and placed in an incubator at 28 ℃ for culture for 3 days, no bacterial colony is observed on the surface, and the tissue block surface disinfection effect is good.
37 endophytic actinomycetes are separated from tissues such as branches and leaves of collected kiwi fruit plants, 8 endophytic actinomycetes are separated from the branch tissues, and 29 endophytic actinomycetes are separated from the leaf tissues.
The bacteriostatic activity of each strain fermentation liquor on the kiwifruit canker pathogen is determined by a tube-plate method, and the experimental results are shown in the following table 1:
as can be seen from the above table 1, the fermentation broth of 37 strains of endophytic actinomycetes, wherein the fermentation broth of 8 strains of endophytic actinomycetes has a strong inhibitory effect on kiwifruit canker pathogenic bacteria, wherein the fermentation broth of the strain numbered WN34 has the strongest inhibitory activity, and the diameter of the inhibition zone reaches 21 mm.
The crude active extract of the strain WN34 was used for field experiments
The field disease control experiment is carried out in a Weinan city near Weiwei region kiwi fruit garden in 7 th of 2018, the control effect experiment is carried out by a method of spraying the whole plant and coating branches, and the tested kiwi fruit variety has slow fragrance.
1. Spraying with WN34 strain active crude extract diluent (concentration is original strain fermentation liquid concentration), spraying on the whole plant, treating 10 diseased plants, spraying 1 time at 7d intervals, continuously spraying 4 times, investigating the healing condition of diseased spots on each treated branch at 7d, 14d, 21d and 28d, calculating relative prevention effect, and taking 45% amobam 150-fold diluent and clear water as controls.
2. The smearing method comprises scraping the bark of the diseased part of the plant with a knife before applying the medicine, smearing the diluted solution of the active crude extract of strain WN34 (the concentration is the concentration of the original strain fermentation liquid), treating 10 diseased plants, and comparing with 45% diluted solution 150 times of amobam and clear water.
The healing condition of the scab is investigated after 6 months of smearing the medicament, the healing tissue generated at the scab is taken as the basis for healing the scab, and the prevention and treatment effect is calculated by the following specific formula:
the healing rate of the disease spot is (the healing number of the disease spot after prevention/the total number of the disease spot before prevention) multiplied by 100 percent
The prevention and treatment effect is [ (the healing rate of the lesion in the treatment area-the healing rate of the lesion in the control area)/(the healing rate of the lesion in the 1-the control area) ] × 100%
1. The results of the field test using the spray method are shown in table 2 below:
as can be seen from the above table 2, the WN34 strain active crude extract has better treatment effect on kiwifruit canker within the spraying time of 14d, 21d and 28d, the prevention effect is better than that of 45% ambam which is a positive control, and the prevention effect is improved to a certain extent along with the time, and after 28d of spraying, the prevention effect of the WN34 strain active crude extract on kiwifruit canker reaches 66.0%.
2. The results of the field trials using the spreading method are given in table 3 below:
| treatment of | Lesion healing rate (%) | Control effect (%) |
| Active crude extract diluent | 78.5 | 78.2 |
| 45% amobam 150-fold diluent | 67.2 | 66.7 |
| CK | 1.52 | - |
As can be seen from the above table 3, after the medicament is applied, the control effect of the active crude extract of the WN34 strain on the kiwifruit canker is 78.2%, which is superior to that of 45% amobam of the positive control, and in the experiment, it is observed that the scab is healed 6 months after the medicament is applied, and a healed layer is formed at the scab, which indicates that the active crude extract of the WN34 strain has a better control effect on the kiwifruit canker.
The field test result shows that: by adopting a spraying method, the WN34 strain active crude extract has a good treatment effect on kiwifruit canker, and the prevention effect reaches 66.0 percent after spraying 28 days. By adopting a smearing method, the control effect of the WN34 strain active crude extract on kiwifruit canker is 78.2 percent, which is superior to that of positive control 45 percent ambam; and the healing phenomenon of the scab is observed and found in the experiment after the medicine is smeared for 6 months, and a healing layer is formed at the scab; the WN34 strain active crude extract has better control effect on kiwifruit canker.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. The foregoing is only a preferred embodiment of the present invention, and it should be noted that there are objectively infinite specific structures due to the limited character expressions, and it will be apparent to those skilled in the art that a plurality of modifications, decorations or changes may be made without departing from the principle of the present invention, and the technical features described above may be combined in a suitable manner; such modifications, variations, combinations, or adaptations of the invention using its spirit and scope, as defined by the claims, may be directed to other uses and embodiments.
Claims (9)
1. A screening method of kiwifruit canker antagonistic strains is characterized by comprising the following steps:
(1) preparation of the strain: preparing kiwi fruit leaves or branches into tissue blocks, sterilizing the surfaces of the tissue blocks, pasting the tissue blocks with sterilized surfaces into a flat plate made of a starch casein culture medium and/or a Gaoshi-I synthetic culture medium under an aseptic condition, placing the flat plate in an incubator at 26-29 ℃, culturing for 5-7 days, purifying strains after colonies grow out, and inoculating the purified strains into a slant culture medium for storage and standby;
(2) preparing strain fermentation liquor: filling millet culture medium into a culture bottle, sterilizing and cooling; inoculating the purified strain into a culture bottle, placing the culture bottle into a shaking table at 26-29 ℃, culturing for 4-6d at the rotating speed of 190r/min, filtering and collecting filtrate, and preserving at low temperature for later use;
(3) preparation of active crude extract: enriching the filtrate with adsorption resin, eluting with methanol water, collecting methanol eluate, and concentrating to 1/5 of methanol water elution volume as active crude extract.
2. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein in step (1), 4 tissue blocks are attached to each plate, the plates are placed in an incubator at 28 ℃ for 6d, and after colonies grow out, the strains are classified and purified according to the difference of morphology and color.
3. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein in the step (2), the culture bottle inoculated with the purified strain is placed in a shaker at 28 ℃ and cultured for 5d at a rotation speed of 180r/min, and the filtrate is filtered and collected and stored at 4 ℃ for later use.
4. The screening method of kiwifruit canker antagonistic strain according to claim 1, characterized in that in step (3), methanol water with concentration of 40%, 60%, 100% is used for gradient elution, methanol is collected for washing and dehydration, and active crude extract is obtained by fraction concentration.
5. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein the step (2) of measuring the bacteriostatic activity of the strain fermentation liquor comprises the following substeps:
① the kiwifruit canker bacteria is prepared to concentration of 1 × 106Taking 1mL of the bacterial suspension and 9mL of beef extract peptone culture medium, and fully and uniformly mixing to prepare 8 groups of bacteria-carrying flat plates;
② placing 5 Oxford cups at equal distance on each bacterium-carrying plate in the step ①;
③ adding 200 μ L of strain fermentation liquid with corresponding number into each Oxford cup, culturing at 37 deg.C for 24h, and measuring the diameter of zone of inhibition;
④ repeating the step ③ 2 times, and taking the average diameter of the zone of inhibition.
6. The screening method of kiwifruit canker antagonistic strain according to claim 5, wherein the beef extract peptone medium comprises the following components: 3.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride, 15.0g of agar and 1000mL of distilled water, wherein the pH value is 7.2 +/-0.2.
7. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein the starch casein medium in the step (1) comprises the following components: 10.0g of soluble starch, 2.0g of dipotassium phosphate, 2.0g of potassium nitrate, 2.0g of sodium chloride, 0.3g of casein, 0.05g of magnesium sulfate heptahydrate, 0.02g of calcium carbonate, 0.01g of ferrous sulfate heptahydrate, 15.0g of agar powder and 1000mL of distilled water.
8. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein the synthetic culture medium of crow's first in the step (1) comprises the following components: 20g of soluble starch, 0.5g of sodium chloride, 1.0g of dipotassium hydrogen phosphate, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate heptahydrate, 1.0g of potassium nitrate, 15.0g of agar powder, 1000mL of distilled water and pH 7.2-7.4.
9. The screening method of kiwifruit canker antagonistic strain according to claim 1, wherein the components of the millet fermentation medium in the step (2) are as follows: 10.0g of glucose, 10.0g of millet, 2.5g of sodium chloride, 3.0g of peptone, 1.0g of calcium carbonate, 1.0g of ammonium sulfate, 1000mL of distilled water and pH 7.2.
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