CN101985604A - Method for efficiently separating Phytophthora capsici from aging disease tissue - Google Patents
Method for efficiently separating Phytophthora capsici from aging disease tissue Download PDFInfo
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Abstract
The invention relates to a method for efficiently separating and purifying bacterial strains of Phytophthora capsici from a diseased tissue of aging Phytophthora capsici Leonian, the method includes steps of acquisition of the diseased tissue of Phytophthora capsici Leonian, surface sterilization, preparation of Cucumis sativus, surface sterilization of the Cucumis sativus, culturing the diseased tissue and the Cucumis sativus, making a selective culture medium, and separation, purification and storage of the Phytophthora capsici. The method ensures separating Phytophthora capsici from the aging disease tissue, and the method is simple, easy, efficient, free from Miscellaneous bacteria pollution and adapted to separation of mass bacterial strains.
Description
Technical field
The present invention relates to the phytopathogen separation technology field, be specifically related to the efficient separation method of phytophthora blight of pepper in the aging diseased tissues.
Background technology
Capsicum is the conventional industries of China, since the reform and opening-up, along with the propelling of market economy, China's capsicum industry every year is with 7% speed development, China's capsicum outlet is also in continuous growth, and Korea S, Japan, Australia, the U.S. and South East Asia etc. have become the long-term importer of China capsicum.Because the adaptability of capsicum is strong, cultural area is extensive, economic benefit and the social benefit of plantation capsicum are all very high, and significant for the year-round supply in China's Vegetable market, capsicum has become the good assistant who shakes off poverty and sets out on the road to prosperity in the poor mountain area of China's part, the staple vegetable of city vegetable basket.Phytophthora blight of pepper (
Phytophthora capsiciLeon) be a kind of important pathogen of plant, it can infect capsicum and cause root, stem and fruit rot, and under suitable onset condition, phytophthora blight of pepper infects capsicum can cause destructive disease, causes the host crop total crop failure.In recent years, capsicum epidemic disease is caused harm serious day by day in the many provinces of China, and grave illness field seedling mortality reaches 30-100%, and becoming influences the significant obstacle that China capsicum produces.Except phytophthora blight of pepper, sickle-like bacteria, Ralstonia solanacearum also can cause that capsicum withers and bacterial wilt causes root-rot mashed, anthrax-bacilus, botrytis cinerea can cause that also capsicum anthrax and gray mold cause stem and fruit rot, and the symptom of these diseases is very approaching in plant growth later stage or morbidity later stage and the microbial eqpidemic disease of Phytophthora capsici.At present, the method of separating pepper phytophthora mainly contains the diseased tissues direct method of isolation, carry disease germs soil or water sample trapping partition method etc., wherein the diseased tissues direct method of isolation requires can only be separated to phytophthora blight of pepper from obvious, the fresh sick sample of disease symptom than higher to the sick sample of gathering; Soil or the water sample trapping law of segregation that carries disease germs is subject to the influence of other assorted bacterium to soil and the water sample, and separation efficiency is lower.Aging, other assorted bacterium of the serious multiplicity of infection of septic diseased tissues, separation and purification phytophthora blight of pepper wherein is very difficult, and the research of relevant this respect is also seldom.Therefore, the method of necessary foundation high efficiency separation phytophthora blight of pepper from aging diseased tissues, be intended to lay the foundation, provide theoretical foundation and basis for setting up effective pest control method for the population genetic research of carrying out phytophthora blight of pepper and propagation and the pests occurrence rule that discloses disease.
Summary of the invention
In order to address the above problem, the objective of the invention is to propose a kind of by method by healthy cucumber fruits separating pepper phytophthora from aging diseased tissues, this method has alleviated Study on Phytophthora Blight of Pepper personnel's workload, reduce the difficulty of capsicum epidemic disease collection of specimens, thereby realized the high efficiency separation in enormous quantities of phytophthora blight of pepper.
For achieving the above object, the present invention is achieved by the following technical programs.
A kind of from aging diseased tissues the method for high efficiency separation phytophthora blight of pepper, it is characterized in that the step is poly-as follows:
(1)The preparation of the aging diseased tissues of capsicum epidemic disease: gather aging diseased tissuess such as capsicum epidemic disease root, stem, fruit;
(2)Aging diseased tissues surface sterilization is handled: step (1) each several part tissue is rinsed the soil and the foreign material on surface well with tap water, in aseptic super clean bench, with aseptic water washing 3 times of each several part tissue, adopt 70-80% ethanol to carry out surface sterilization, again behind the aseptic water washing 3 times, be positioned in the good culture dish of sterilization, in Bechtop, surface-moisture dried up;
(3)The preparation of cucumber fruit: the cucumber fruit of preparing healthy no scab;
(4)Cucumber fruit surface sterilization: the cucumber fruit of preparing in the step (3) is rinsed well surface irregularities under tap water, soak 0.1-1min in the 70-80% ethanol, take out the back and on the clear lights flame of wine, the ethanol of remained on surface dryouied and get final product;
(5)Aging diseased tissues and cucumber are really cultivated altogether: the ready cucumber of step (4) is really cut a suitably big or small opening with aseptic art designing's blade at the base of fruit position, the aging diseased tissues of step (2) is sandwiched in step poly-(4) cucumber fruit opening 20-30 ℃ of cultivation 4-6d that preserves moisture under the aseptic condition;
(6)The preparation of selective medium: get 50g rye seed and soaked 36 hours in the 200ml deionized water, stamp mill is smashed to pieces, 60 ℃ of water-baths 2 hours, remove slag through 2 layers of filtered through gauze, the filtered liquid moisturizing adds agar powder 15g to 1L, 121 ℃ of high pressure steam sterilization 15min are the rye substratum.Treat that the rye substratum is cooled to 50 ℃, adding the Rifampin mother liquor is that 100-300mg/L, acillin mother liquor to final concentration are that 100-300mg/L and quintozene mother liquor to final concentration are 100-300mg/L to final concentration, mixing, the flat board that falls is selective medium.
(7)The separation and purification of phytophthora blight of pepper: after treating that step (5) cucumber surface grows the white mould layer, directly the mould layer of picking is on step (6) selective medium, cultivate 2-4d down in 20-30 ℃ of dark condition, after treating that bacterium colony forms, the a small amount of mycelia of picking is observed the mycelia form under low-power microscope, after determining that isolating bacterium colony is phytophthora blight of pepper, the a small amount of mycelia of the isolated phytophthora blight of pepper of picking, be seeded on step (6) selective medium, 15-25 ℃ of dark condition cultivated 1-3d down, use aseptic operation blade cuts colony edge to be the tip of radial single mycelia, downcut together with substratum, place rye slant medium flat board, the bacterium colony that grows out is phytophthora blight of pepper.
(8)The preservation of phytophthora blight of pepper: the phytophthora blight of pepper that step (7) is grown out changes that (test tube slant should be not long in the test tube of rye medium slant 15 * 150mm over to, 1/3 of test tube length is advisable), 20-30 ℃ of dark condition cultivated 4-6d down to covering with the inclined-plane, perfusion is higher than the sterilization paraffin oil of test tube slant 1.5cm, 13 ℃ of dark conditions are preserved down, and 1 year activation tube once.
The described aging diseased tissues of step of the present invention (2) should be made some necessary processing before with the tap water flushing: root and stem should use art designing's blade with the epidermis scraped clean earlier; The part that aging diseased tissues is adopted in sterilization should be not excessive, and the big or small as follows of diseased tissues each several part wears out: root, stem's diameter surpass will cutting away half earlier of 0.5cm, and length is no longer than 2cm, and fruit partly cuts the pulp area and is not more than 2 * 2cm; As follows to aging diseased tissues each several part disinfecting time: root, stem are to soak 1-2min in the 70-80% ethanol, and aseptic water washing 3 times, fruit are to soak 0.5-1.5min in the 70-80% ethanol, aseptic water washing 3 times.
The cucumber fruit that the described cucumber fruit of step of the present invention (3) can be a shagreen also can be that the cucumber fruit of white skin and the cucumber variety of being planted do not have dependency, as long as employed cucumber really is healthy no scab.
The cultivation of preserving moisture under the described aseptic condition of step of the present invention (5) is meant that the cucumber that will clip aging tissue really places the crisper of the bacterium of having gone out, shop, crisper bottom one layer thickness is no more than the bacterium absorbent cotton that gone out of 1cm, with sterilized water with the abundant humidification of absorbent cotton, be put in 25 ℃ of biochemical incubators after fresh-keeping lid is tight, the 12h alternation of light and darkness is cultivated.
The described Rifampin of step of the present invention (5), the compound method of acillin and quintozene mother liquor is as follows: take by weighing the former powder of the acillin that contains effective composition 0.5000g (accurately to 0.0002g) respectively, the former powder of Rifampin (accurately to 0.0002g) that contains effective composition 0.5000g, the former powder of quintozene (accurately to 0.0002g) that contains effective composition 0.5000g, place the 50ml sterilization volumetric flask that has label respectively, the former powder of acillin is with dissolved in distilled water and be diluted to scale, after quintozene dissolves with an amount of toluene, be diluted to scale with the 0.1% tween 80 aqueous solution, the former powder of Rifampin is with the dissolving of anhydrous alcohol and be diluted to scale, and the medicament mother liquid concentration that is made into is 2 * 10
4Mg/L.
Remarkable advantage of the present invention
Separation of Plant diseases pathogenic bacteria and purifying are the bases of the former flora body of the sick plant of understanding hereditary feature, the in good time effective prophylactico-therapeutic measures of formulation Plant diseases.Exist many compound assorted bacterium of infecting in the aging incidence tissue of plant, make that the separation and purification of purpose bacterial strain is very difficult, separation efficiency is low.The aging diseased tissues co-culture system of cucumber fruits that the present invention adopts and capsicum epidemic disease makes the phytophthora blight of pepper in the aging diseased tissues infect the cucumber fruit, really goes up separating pepper phytophthora aimed strain from the cucumber of being infected, thereby has got rid of the pollution of the assorted bacterium of non-target.The present invention have draw materials easily, cost is low, operation steps is simple, the separation efficiency advantages of higher.The present invention provides technical guarantee for the research of Plant diseases capsicum epidemic disease.
Description of drawings
Aging cane tissue of Fig. 1 capsicum epidemic disease and cucumber fruits are cultivated body altogether;
The colonial morphology of Fig. 2 phytophthora blight of pepper on the rye substratum;
Fig. 3,4 phytophthora blight of pepper sporocyst form features.
Embodiment
Below in conjunction with example to the present invention a kind of from aging diseased tissues high efficiency separation phytophthora blight of pepper method further describe.
Embodiment 1:
Select for use and pick up from Xin Dian town, Fuzhou City, Fujian Province vegetables base capsicum epidemic disease morbidity later stage root sample, carry out phytophthora blight of pepper by following program and separate:
(1) the aging root of capsicum epidemic disease is taken back the laboratory, with tap water the soil and the foreign material on surface are rinsed well, with root epidermis scraped clean, be cut into the tissue that length is 2cm with cutter, if base diameter surpasses 0.5cm, then need vertically on average to cut two halves open along xylem, in aseptic super clean bench, the tissue that cuts aseptic water washing 3 times, soak 1.5min in 75% ethanol, behind the aseptic water washing 3 times, be positioned in the good culture dish of sterilization, in Bechtop, surface-moisture dried up;
(2) ready cucumber fruit is rinsed well surface irregularities under tap water, soak 0.5min in 75% ethanol, taking out the back dryouies the ethanol of remained on surface on the clear lights flame of wine, cut a suitably big or small opening at cucumber base of fruit position with aseptic art designing's blade, the ready tissue block of step (1) is sandwiched in the cucumber fruit opening, is made into the cucumber fruit and cultivates body altogether with aging diseased tissues;
(3) cultivate the crisper that body places the bacterium of having gone out altogether with step (2) is ready, shop, crisper bottom one layer thickness is no more than the bacterium absorbent cotton that gone out of 1cm,, be put in 25 ℃ of biochemical incubators after fresh-keeping lid is tight the abundant humidification of absorbent cotton with sterilized water, the 12h alternation of light and darkness is cultivated 5d;
(4) preparation of selective medium: get 50g rye seed and soaked 36 hours in the 200ml deionized water, stamp mill is smashed to pieces, 60 ℃ of water-baths 2 hours, remove slag through 2 layers of filtered through gauze, the filtered liquid moisturizing adds agar powder 15g to 1L, 121 ℃ of high pressure steam sterilization 15min are the rye substratum; Treat that the rye substratum is cooled to 50 ℃, adding the Rifampin mother liquor is that 200mg/L, acillin mother liquor to concentration are that 200mg/L and quintozene mother liquor to concentration are 200mg/L to concentration, mixing, and the flat board that falls is selective medium;
(5) treat that step (3) cucumber surface grows the mould layer of white after, directly the mould layer of picking is on selectivity rye substratum, cultivate 3d down in 25 ℃ of dark conditions, after treating that bacterium colony forms, the a small amount of mycelia of picking is observed mycelia and sporocyst form (as Fig. 3 under low-power microscope, shown in 4), after determining that isolating bacterium colony is phytophthora blight of pepper, the a small amount of mycelia of the isolated phytophthora blight of pepper of picking, be inoculated on the selectivity rye substratum, 20 ℃ of dark conditions are cultivated 2d down, use aseptic operation blade cuts colony edge to be the tip of radial single mycelia, downcut together with substratum, place rye slant medium flat board, the bacterium colony that grows out is phytophthora blight of pepper.
(6) result shows: adopt the aging root tissue of cucumber and capsicum epidemic disease to cultivate altogether, re-use selective medium separating pepper phytophthora from cucumber is really organized, has very high separation rate, being separated into power is 83.6%, has got rid of the influence of other assorted bacterium to phytophthora blight of pepper separation and purification in the aging root tissue of falling ill effectively.
Embodiment 2:
Select for use and pick up from Xin Dian town, Fuzhou City, Fujian Province vegetables base capsicum epidemic disease morbidity later stage stipe part sample, carry out phytophthora blight of pepper by following program and separate:
(1) the aging stipe part sample of capsicum epidemic disease is taken back the laboratory, with tap water surface irregularities is rinsed well, with the epidermis scraped clean, being cut into length is the cane tissue of 2cm with cutter, if the stipe part diameter surpasses 0.5cm, then need vertically on average to cut two halves open along xylem, in aseptic super clean bench, cane aseptic water washing 3 times, soak 1.5min in 70% ethanol, behind the aseptic water washing 3 times, be positioned in the good culture dish of sterilization, in Bechtop, surface-moisture dried up;
(2) ready cucumber fruit is rinsed well surface irregularities under tap water, soak 1.0min in 70% ethanol, taking out the back dryouies the ethanol of remained on surface on the clear lights flame of wine, cut a suitably big or small opening at cucumber base of fruit position with aseptic art designing's blade, the ready cane tissue of step (1) is sandwiched in the cucumber fruit opening, is made into the cucumber fruit and organizes with cane and cultivate body (as shown in Figure 1) altogether;
(3) cultivate the crisper that body places the bacterium of having gone out altogether with step (2) is ready, shop, crisper bottom one layer thickness is no more than the bacterium absorbent cotton that gone out of 1cm,, be put in 25 ℃ of biochemical incubators after fresh-keeping lid is tight the abundant humidification of absorbent cotton with sterilized water, the 12h alternation of light and darkness is cultivated 5d;
(4) preparation of selective medium: get 50g rye seed and soaked 36 hours in the 200ml deionized water, stamp mill is smashed to pieces, 60 ℃ of water-baths 2 hours, remove slag through 2 layers of filtered through gauze, the filtered liquid moisturizing adds agar powder 15g to 1L, 121 ℃ of high pressure steam sterilization 15min are the rye substratum; Treat that the rye substratum is cooled to 50 ℃, adding the Rifampin mother liquor is that 100mg/L, acillin mother liquor to concentration are that 300mg/L and quintozene mother liquor to concentration are 100mg/L to concentration, mixing, and the flat board that falls is selective medium;
(5) treat that step (3) cucumber surface grows the mould layer of white after, directly the mould layer of picking is on selectivity rye substratum, cultivate 4d down in 20 ℃ of dark conditions, after treating that bacterium colony forms, the a small amount of mycelia of picking is observed mycelia and sporocyst form (as Fig. 3 under low-power microscope, shown in 4), after determining that isolating bacterium colony is phytophthora blight of pepper, the a small amount of mycelia of the isolated phytophthora blight of pepper of picking, be inoculated on the selectivity rye substratum, 15 ℃ of dark conditions are cultivated 3d down, use aseptic operation blade cuts colony edge to be the tip of radial single mycelia, downcut together with substratum, place rye slant medium flat board, the bacterium colony that grows out is phytophthora blight of pepper.
(6) result shows: adopt the aging stipe part tissue of cucumber and capsicum epidemic disease to cultivate altogether, re-using selective medium is separating pepper phytophthora the fruit tissue from cucumber, has very high separation rate, being separated into power is 91.2%, has got rid of the influence of other assorted bacterium to phytophthora blight of pepper separation and purification in the aging stipe part tissue of falling ill effectively.
Embodiment 3:
Select for use and pick up from Nan Kouzhen vegetables base, Sha County, Fujian Province capsicum epidemic disease morbidity later stage pepper fruit, carry out phytophthora blight of pepper by following program and separate:
(1) the aging sick fruit sample of capsicum epidemic disease is taken back the laboratory, with tap water surface irregularities is rinsed well, be cut into area and be not more than 2 * 2cm pulp organization, in aseptic super clean bench, pulp organization soaks 1min with aseptic water washing 3 times in 80% ethanol, behind the aseptic water washing 3 times, be positioned in the good culture dish of sterilization, in Bechtop, surface-moisture dried up;
(2) ready cucumber fruit is rinsed well surface irregularities under tap water, soak 0.5min in 75% ethanol, taking out the back dryouies the ethanol of remained on surface on the clear lights flame of wine, cut a suitably big or small opening at cucumber base of fruit position with aseptic art designing's blade, the ready fruit tissue of step (1) is sandwiched in the cucumber fruit opening, is made into the cucumber fruit and cultivates body altogether with the aging pulp organization of capsicum epidemic disease;
(3) cultivate the crisper that body places the bacterium of having gone out altogether with step (2) is ready, shop, crisper bottom one layer thickness is no more than the bacterium absorbent cotton that gone out of 1cm,, be put in 30 ℃ of biochemical incubators after fresh-keeping lid is tight the abundant humidification of absorbent cotton with sterilized water, the 12h alternation of light and darkness is cultivated 4d;
(4) preparation of selective medium: get 50g rye seed and soaked 36 hours in the 200ml deionized water, stamp mill is smashed to pieces, 60 ℃ of water-baths 2 hours, remove slag through 2 layers of filtered through gauze, the filtered liquid moisturizing adds agar powder 15g to 1L, 121 ℃ of high pressure steam sterilization 15min are the rye substratum; Treat that the rye substratum is cooled to 50 ℃, adding the Rifampin mother liquor is that 300mg/L, acillin mother liquor to concentration are that 100mg/L and quintozene mother liquor to concentration are 300mg/L to concentration, mixing, and the flat board that falls is selective medium;
(5) treat that step (3) cucumber surface grows the white mould layer after, directly the mould layer of picking is on selectivity rye substratum, cultivate 2d down in 30 ℃ of dark conditions, after treating that bacterium colony forms, the a small amount of mycelia of picking is observed mycelia and sporocyst form (as Fig. 3 under low-power microscope, shown in 4), after determining that isolating bacterium colony is phytophthora blight of pepper, the a small amount of mycelia of the isolated phytophthora blight of pepper of picking, be inoculated on the selectivity rye substratum, 25 ℃ of dark conditions are cultivated 1d down, use aseptic operation blade cuts colony edge to be the tip of radial single mycelia, downcut together with substratum, place rye slant medium flat board, the bacterium colony that grows out is phytophthora blight of pepper (as shown in Figure 2).
(6) result shows: the common cultivation body that adopts cucumber fruit and capsicum epidemic disease aged fruit tissue to make, re-use selective medium separating pepper phytophthora from the cucumber tissue, has very high separation rate, being separated into power is 95.8%, has got rid of the influence of other assorted bacterium to phytophthora blight of pepper separation and purification in the capsicum epidemic disease morbidity aged fruit tissue effectively.
The above only is preferred embodiment of the present invention, and all equalizations of being done according to the present patent application claim change and modify, and all should belong to covering scope of the present invention.
Claims (5)
1. the method for a high efficiency separation phytophthora blight of pepper from aging diseased tissues, it is characterized in that: the concrete step of described method is poly-as follows:
(1)The preparation of the aging diseased tissues of capsicum epidemic disease: the aging diseased tissues of gathering capsicum epidemic disease root, stem, fruit;
(2)Aging diseased tissues surface sterilization is handled: step (1) each several part tissue is rinsed the soil and the foreign material on surface well with tap water, in aseptic super clean bench, with aseptic water washing 3 times of each several part tissue, adopt 70-80% ethanol to carry out surface sterilization, after using aseptic water washing 3 times again, be positioned in the good culture dish of sterilization, in Bechtop, surface-moisture dried up;
(3)The preparation of cucumber fruit: the cucumber fruit of preparing healthy no scab;
(4)Cucumber fruit surface sterilization: the cucumber fruit of preparing in the step (3) is rinsed well surface irregularities under tap water, soak 0.1-1min in the 70-80% ethanol, take out the back and on the clear lights flame of wine, the alcohol of remained on surface dryouied and get final product;
(5)Aging diseased tissues and cucumber are really cultivated altogether: the ready cucumber of step (4) is really cut a suitably big or small opening with aseptic art designing's blade at the base of fruit position, the aging diseased tissues of step (2) is sandwiched in step poly-(4) cucumber fruit opening 20-30 ℃ of cultivation 4-6d that preserves moisture under the aseptic condition;
(6)The preparation of selective medium: get 50g rye seed and soaked 36 hours in the 200ml deionized water, stamp mill is smashed to pieces, 60 ℃ of water-baths 2 hours, remove slag through 2 layers of filtered through gauze, the filtered liquid moisturizing adds agar powder 15g to 1L, 121 ℃ of high pressure steam sterilization 15min are the rye substratum; Treat that the rye substratum is cooled to 50 ℃, adding the Rifampin mother liquor is that 100-300mg/L, acillin mother liquor to concentration are that 100-300mg/L and quintozene mother liquor to concentration are 100-300mg/L to concentration, mixing, and the flat board that falls is selective medium;
(7)The separation and purification of phytophthora blight of pepper: after treating that step (5) cucumber surface grows the white mould layer, directly the mould layer of picking is on step (6) selective medium, cultivate 2-4d down in 20-30 ℃ of dark condition, after treating that bacterium colony forms, the a small amount of mycelia of picking is observed the mycelia form under low-power microscope, after determining that isolating bacterium colony is phytophthora blight of pepper, the a small amount of mycelia of the isolated phytophthora blight of pepper of picking, be seeded on step (6) selective medium, 15-25 ℃ of dark condition cultivated 1-3d down, use aseptic operation blade cuts colony edge to be the tip of radial single mycelia, downcut together with substratum, place rye slant medium flat board, the bacterium colony that grows out is phytophthora blight of pepper;
(8)The preservation of phytophthora blight of pepper: the phytophthora blight of pepper that step (7) is grown out changes in the rye medium slant test tube, 20-30 ℃ of dark condition cultivated 4-6d down to covering with the inclined-plane, perfusion sterilization paraffin oil, 13 ℃ of dark conditions are preserved down, and 1 year activation tube is once.
2. according to claim 1 a kind of from aging diseased tissues the method for high efficiency separation phytophthora blight of pepper, it is characterized in that the described aging diseased tissues of step (2) should make some necessary processing before with the tap water flushing: root and stem should be earlier with art designing's blade with the epidermis scraped clean; The part of aging diseased tissues usefulness of taking in sterilization should be not excessive, adopts the big or small as follows of aging diseased tissues each several part: root, stem's diameter surpass will cutting away half earlier of 0.5cm, and length is no longer than 2cm, and fruit partly cuts the pulp area and is not more than 2 * 2cm; As follows to aging diseased tissues each several part disinfecting time: root, stem are 70-80% alcohol immersion 1-2min, and aseptic water washing 3 times, fruit are 70-80% alcohol immersion 0.5-1.5min, aseptic water washing 3 times.
3. according to claim 1 a kind of from aging diseased tissues the method for high efficiency separation phytophthora blight of pepper, it is characterized in that the cucumber fruit that the described cucumber fruit of step (3) can be a shagreen also can be the cucumber fruit of white skin, there is not dependency with the cucumber variety of being planted, as long as employed cucumber really is healthy no scab.
4. according to claim 1 a kind of from aging diseased tissues the method for high efficiency separation phytophthora blight of pepper, the cultivation that it is characterized in that preserving moisture under the described aseptic condition of step (5) is meant that the cucumber that will clip aging tissue really places the crisper of the bacterium of having gone out, shop, crisper bottom one layer thickness is no more than the bacterium absorbent cotton that gone out of 1cm, with sterilized water with the abundant humidification of absorbent cotton, be put in 25 ℃ of biochemical incubators after fresh-keeping lid is tight, the 12h alternation of light and darkness is cultivated.
5. according to claim 1 a kind of from aging diseased tissues the method for high efficiency separation phytophthora blight of pepper, it is characterized in that the described Rifampin of step (5), the compound method of acillin and quintozene mother liquor is as follows: take by weighing the former powder of the acillin that contains effective composition 0.5000g respectively, the former powder of Rifampin that contains effective composition 0.5000g, the former powder of quintozene that contains effective composition 0.5000g, place the 50ml sterilization reagent bottle that has label respectively, the former powder of acillin is with dissolved in distilled water and be diluted to scale, after quintozene dissolves with an amount of toluene, be diluted to scale with the 0.1% tween 80 aqueous solution, the former powder of Rifampin is with the dissolving of anhydrous alcohol and be diluted to scale, and the medicament mother liquid concentration that is made into is 2 * 10
4Mg/L.
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| CN102763661A (en) * | 2012-05-31 | 2012-11-07 | 中国农业大学 | Medium for separating Phytophthora capsici |
| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
| CN105695333A (en) * | 2014-11-27 | 2016-06-22 | 山东棉花研究中心 | Method for separating cottonbollblight pathogenic bacteria in soil |
| CN108384725A (en) * | 2018-04-25 | 2018-08-10 | 河南科技大学 | A kind of separation method of black root of tobacco bacterium |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN108865815A (en) * | 2018-09-10 | 2018-11-23 | 保定市稀成生物科技有限公司 | Portable mould efficiently divides pure stick |
| CN113637588A (en) * | 2021-07-22 | 2021-11-12 | 海南大学 | Method for simply, conveniently and rapidly separating peronophythora litchi |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810952A (en) * | 2006-02-16 | 2006-08-02 | 苏州农业职业技术学院 | Microbe fermenting culture liquid and its prepn process |
| CN101250573A (en) * | 2008-04-11 | 2008-08-27 | 云南省农业科学院生物技术与种质资源研究所 | Method for inducing phytophthora infestans to produce pathogenic secretory protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810952A (en) * | 2006-02-16 | 2006-08-02 | 苏州农业职业技术学院 | Microbe fermenting culture liquid and its prepn process |
| CN101250573A (en) * | 2008-04-11 | 2008-08-27 | 云南省农业科学院生物技术与种质资源研究所 | Method for inducing phytophthora infestans to produce pathogenic secretory protein |
Non-Patent Citations (2)
| Title |
|---|
| 《Plant Dis》 19921231 Kim F S ET AL Virulence to Korean pepper cultivars of isolates of Phytophthora capsici from different geographic areas 486-489 1-5 第76卷, 2 * |
| 《应用生态学报》 20101031 梅新兰等 辣椒疫病拮抗菌株筛选、鉴定及其防效 2652－2658 1-5 第21卷, 第10期 2 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102763661A (en) * | 2012-05-31 | 2012-11-07 | 中国农业大学 | Medium for separating Phytophthora capsici |
| CN102763661B (en) * | 2012-05-31 | 2014-05-28 | 中国农业大学 | Medium for separating Phytophthora capsici |
| CN103525913A (en) * | 2013-09-22 | 2014-01-22 | 福建省农业科学院植物保护研究所 | Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof |
| CN105695333A (en) * | 2014-11-27 | 2016-06-22 | 山东棉花研究中心 | Method for separating cottonbollblight pathogenic bacteria in soil |
| CN108384725A (en) * | 2018-04-25 | 2018-08-10 | 河南科技大学 | A kind of separation method of black root of tobacco bacterium |
| CN108410793A (en) * | 2018-04-27 | 2018-08-17 | 江西省农业科学院植物保护研究所 | A kind of induction phytophthora blight of pepper production culture medium of spore, preparation method and application |
| CN108410793B (en) * | 2018-04-27 | 2020-11-06 | 江西省农业科学院植物保护研究所 | Culture medium for inducing phytophthora capsici to produce spores, preparation method and application |
| CN108865815A (en) * | 2018-09-10 | 2018-11-23 | 保定市稀成生物科技有限公司 | Portable mould efficiently divides pure stick |
| CN113637588A (en) * | 2021-07-22 | 2021-11-12 | 海南大学 | Method for simply, conveniently and rapidly separating peronophythora litchi |
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| CN101985604B (en) | 2012-08-08 |
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