CN103981249B - A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target - Google Patents
A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target Download PDFInfo
- Publication number
- CN103981249B CN103981249B CN201410247409.3A CN201410247409A CN103981249B CN 103981249 B CN103981249 B CN 103981249B CN 201410247409 A CN201410247409 A CN 201410247409A CN 103981249 B CN103981249 B CN 103981249B
- Authority
- CN
- China
- Prior art keywords
- seed
- grams
- zoospore
- nutrition liquid
- epidemic disease
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses a kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target, 30 grams of haydites are put into tissue culture bottle, add plant nutrition liquid 40mL to just not having haydite, the pepper seed field planting of the vernalization of being crossed by biocontrol microorganisms seed soaking is on haydite, treat that capsicum grows to four true leaves, below nutrition liquid level, inoculate 5mL adjustment concentration is 10
5mL
-1zoospore suspension.Zoospore suspension by phytophthora blight of pepper on CA substratum intermittent illumination process after 6 ~ 8 days low temperature induction obtain.Range of application is the screening etc. of the biocontrol microorganisms taking capsicum epidemic disease as target.Method of the present invention is simple to operate, short from nursery to transfer period, and zoospore inoculation is convenient to implement and observe, and has actual directive significance in the biological control research work of Plant diseases.
Description
Technical field
The invention belongs to agriculture plant protection technical field, relate to the screening method of multiple kinds of crops pestilence disease, the biocontrol strain high-efficiency screening method of to be a kind of in particular with capsicum epidemic disease be target.
Background technology
Capsicum epidemic disease be by phytophthora blight of pepper (
phytophthora capsicileonian) the worldwide soil-borne disease of the one caused, all has generation in each pepper producing area.Its pathogenic bacteria, except harm capsicum, also can infect 20 various crop such as summer squash, cucumber, pumpkin, eggplant, tomato.This disease now generally occurs in the whole world first at New Mexico report for 1918.The sixties in 20th century, China to find that this is sick by larger silk ribbon for holding a jade seal through its nose at first in Jiangsu, so far capsicum epidemic disease in Xinjiang, Hebei, Heilungkiang, Guizhou, Guangzhou, Gansu, Yunnan, Shanghai and Shaanxi etc. economize (city) occurrence injury.Except China, all there is generation in states such as France, Australia, Korea S and India.In recent years along with the continuous cropping plantation in each main producing region of the increase of protecting field pepper planting area and capsicum, capsicum epidemic disease have the trend increased the weight of year by year.Because capsicum epidemic disease is popular, open country and protecting field all occur, and often cause plant death loss in blocks serious, general field strain sickness rate 20% ~ 30%, and morbidity field reaches more than 70%, seriously have impact on the output of capsicum, causes huge loss to peasant.
Phytophthora blight of pepper (
phytophthora capsicileonian) mainly survive the winter in plant invalid body, soil or seed with oospore, chlamydospore, can survive 2 ~ 3 years, become the primary source of infection of disease in the coming year.Germ after surviving the winter is backwash and pass on stem of Bush Redpepper base portion or blade, fruit and cause morbidity through rainwater or when watering, and especially after heavy rain, weather clears up suddenly, and temperature sharply rising disease is happening and prevelence very easily.Pathogen growth grows thermophilic 30 DEG C, the highest 38 DEG C, minimum 8 DEG C.General field temperature 25 ~ 28 DEG C, relative humidity higher than 85% when be beneficial to infecting of this bacterium most.Germ is from invading morbidity, and in only 3 ~ 4 days incubation period, the sporocyst that sick leaf produces passes to each position of host with pour water, air-flow etc., can propagate again and cause harm, constantly cause and infect, cause the popular of disease.There is no the disease-resistant variety of effectively control capsicum epidemic disease at present, in production, control is mainly based on chemical agent.But due to life-time service chemical agent, P.
capsiciresistance is in various degree created to most of common pesticides, due to adsorption by soil and microbiological deterioration, use Agro-chemicals control epidemic disease poor effect, and a large amount of, the high frequency use of chemical pesticide bring serious environment and Residual Pesticides in Farm Produce problem.It is reported, China has multiple area and utilizes nature beneficial microorganism to carry out biological control to Plant diseases, and prevention effect is better.For a long time, the biological control work progress of capsicum epidemic disease is slow, and wherein the screening difficulty of capsicum epidemic disease biocontrol microorganisms is one of bottleneck of restriction Study on Phytophthora Blight of Pepper.In order to carry out the research of capsicum epidemic disease biological control smoothly, key seeks one for the efficient screening method of capsicum epidemic disease biocontrol microorganisms.Therefore, study a kind of efficient, easy, feasible be that the biocontrol strain high-efficiency screening method of target becomes capsicum epidemic disease biological control and plants the important leverage of the sick screening operation of farm crop pestilence with capsicum epidemic disease.
Summary of the invention
What common biocontrol strain screening adopted is indoor pot experiment or field plot trial two kinds of methods, and these two kinds of methods are higher from the viewpoint of test preparation, test design and test enforcement three requirement, and the cycle is long.The present invention take capsicum epidemic disease as the biocontrol strain screening method of target, adopts soilless culture method, haydite (also known as ceramic nutrient soil) as matrix, tissue culture bottle do chamber test articles for use cheap, be convenient to buy and reusable, saved experimentation cost; Planting requirement is simple, and zoospore inoculation is convenient to implement and observe, and the cycle is short, and screening is efficient.
For achieving the above object, the invention discloses following technical scheme:
Take capsicum epidemic disease as a biocontrol strain high-efficiency screening method for target, it is characterized in that, comprise the steps:
(1) seed disinfection: by requisite number object pepper seed aseptic water washing one time, then successively 30s and 5min is processed respectively with the alcohol of 75% and the NaClO of 5%, seed selection capsicum variety is improvement 8819 line green pepper, is provided (externally on sale) by treasure standing grain vegetable breeding center, Qing County.
(2) presprouting of seeds: the seed of disinfecting is soaked 4h in sterilized water, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally;
(3) matrix sterilizing: 30 grams of haydites are put into tissue culture bottle is highly 1/3 of tissue culture bottle height, adds plant nutrition liquid 40mL to just not having haydite, seals with envelope bottle film.Moist heat sterilization, 121 DEG C, 30min; Wherein, haydite diameter 2 ~ 8 ㎜, buys in Cao village flowers market.High 100 ㎜ of tissue culture bottle, bore 50 ㎜, external diameter 60 ㎜, buys in Tianjin, Tianjin hundred along experimental installation company limited;
The preparation of described plant nutrition liquid: add each 0.16 gram of 0.22 gram, ammonium phosphate, 1.05 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.01 gram, ferrous sulfate in 1 liter of deionized water, mixing is nutritive medium;
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mL LB inoculums are housed, in 37 DEG C, 150rmin
-1concussion cultivation 18 h makes Antagonistic Fungi liquid, and (adjustment Antagonistic Fungi liquid bacterial content is 1.0 × 10
8cfumL
-1), the seed of vernalization is immersed Antagonistic Fungi immersion kind 30 min; After described Antagonistic bacteria strains refers to and separates from vegetables rhizosphere soil by dull and stereotyped antagonistic experiment screen to the good bacterial strain of Phytophthora capsici antagonistic effect, source: by Tianjin Plant Protection Institute agricultural fermentable research department provide (externally can provide free).LB inoculum refers to: the preparation of LB inoculum: add peptone 10 grams in 1 liter of deionized water, yeast extract 5 grams, NaCl 5 grams.
(5) field planting: treated seeds tweezers are are evenly clicked and entered in sterilization matrix, every bottle of point 8 seeds, seal up bottle film, and put into growth cabinet and cultivate, growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h; Sterilization matrix refers to the haydite in step (3);
(6) interpolation of nutritive medium: the Expenditure Levels of every weekly check nutritive medium, the interpolation (referring to plant nutrition liquid above-mentioned) of nutritive medium was not there to be seed to be advisable;
(7) zoospore preparation: phytophthora blight of pepper is seeded on CA substratum (Radix Dauci Sativae substratum), intermittent illumination process is after 6 ~ 8 days, microscopy sporocyst quantity, when there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, 1h is placed in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then media surface is scrubbed with sterilization writing brush, four layers of sterile gauze filter, collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1;
Described phytophthora blight of pepper refers to: provided (externally can provide free) by Tianjin Plant Protection Institute's disease flocking biocontrol room.
Described CA substratum refers to Radix Dauci Sativae substratum.Preparation method is: by 200 grams of Radix Dauci Sativae sections, add 500 mL deionized waters, refiner will be smashed, and double gauze filters, and add deionized water and mend to 1 liter, add 20 grams of agar, every bottle of 100 mL packing, sterilizing is for subsequent use.
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension, in order to be beneficial to the generation of capsicum epidemic disease, inoculation after growth cabinet optimum configurations be: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h.3rd, 5 day investigation incidence after inoculation.
The present invention is more detailed to be described below:
Take capsicum epidemic disease as a biocontrol strain high-efficiency screening method for target, comprise the steps:
(1) seed disinfection: by requisite number object pepper seed, the capsicum variety that such as seed selection is provided by treasure standing grain vegetable breeding center, Qing County is that improvement 8819 line green pepper is as pepper seed.With aseptic water washing one time, then successively process 30s and 5min respectively with the alcohol of 75% and the NaClO of 5%.
(2) presprouting of seeds: the seed of disinfecting is soaked 4h in sterilized water, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally.
(3) matrix sterilizing: 30 grams of haydites are put into tissue culture bottle, is highly approximately 1/3 of tissue culture bottle height, adds plant nutrition liquid 40mL to there just be not haydite, seals with envelope bottle film.Moist heat sterilization, 121 DEG C, 30min.Wherein, haydite diameter 2 ~ 8 ㎜, buys in Cao village flowers market.High 100 ㎜ of tissue culture bottle, bore 50 ㎜, external diameter 60 ㎜, buys in Tianjin, Tianjin hundred along experimental installation company limited.The preparation of plant nutrition liquid: add each 0.16 gram of 0.22 gram, ammonium phosphate, 1.05 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.01 gram, ferrous sulfate in 1 liter of deionized water, mixing is nutritive medium.
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mLLB inoculums are housed, in 37 DEG C, 150rmin
-1concussion cultivation 18 h makes Antagonistic Fungi liquid, and (adjustment bacterial content is 1.0 × 10
8cfumL
-1), the seed of vernalization is immersed Antagonistic Fungi immersion kind 30 min.
(5) field planting: evenly clicked and entered in sterilization matrix by treated seeds tweezers, every bottle of point 8 seeds, seal up bottle film, put into growth cabinet and cultivate.Growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h.
(6) interpolation of nutritive medium: the Expenditure Levels of every weekly check nutritive medium, the interpolation of nutritive medium was advisable there not to be seed.
(7) zoospore preparation: be seeded in by phytophthora blight of pepper on CA substratum (Radix Dauci Sativae substratum), intermittent illumination process is after 6 ~ 8 days, microscopy sporocyst quantity.When there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, 1h is placed in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then scrub media surface with sterilization writing brush, four layers of sterile gauze filter, and collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1.
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension.In order to be beneficial to the generation of capsicum epidemic disease, after inoculation, growth cabinet optimum configurations is: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h.3rd, 5 day investigation incidence after inoculation.
Disclosed by the invention with capsicum epidemic disease for the positively effect that the biocontrol strain high-efficiency screening method of target is compared with prior art had is:
(1) cost-saving, environmental protection.Tissue culture bottle used in the present invention and haydite all can infinitely repeatedly use, and pollute few, protection of the environment.
(2) breakneck acceleration is fast.Phytophthora Capsici Zoospores inoculation ambient moisture is 100%, and haydite space is large, is conducive to zoospore and infects, and accelerates the generation of capsicum epidemic disease, improves screening efficiency.
(3) screening efficiency is high.Test space involved in the present invention is little, and soilless culture chili growth is fast, and capsicum epidemic disease occurs fast, and every batch of screening can doing multiple biocontrol strain, substantially increases the screening efficiency of biocontrol microorganisms.
Embodiment:
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments should be understood only be not used in for illustration of the present invention and limit the scope of the invention.After Antagonistic bacteria strains refers to and separates from vegetables rhizosphere soil by dull and stereotyped antagonistic experiment screen to the good bacterial strain of Phytophthora capsici antagonistic effect, source: by Tianjin Plant Protection Institute agricultural fermentable research department provide (externally can provide free); Haydite, improve 8819 line greens pepper, tissue culture bottle all has commercially available.
Embodiment 1:
A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target:
(1) seed disinfection: by requisite number object pepper seed aseptic water washing one time, then successively process 30s and 5min respectively with the alcohol of 75% and the NaClO of 5%, seed selection capsicum variety is improvement 8819 line green pepper;
(2) presprouting of seeds: the seed of disinfecting is soaked 4h in sterilized water, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally;
(3) matrix sterilizing: 30 grams of diameter 2 ㎜ haydites are put into tissue culture bottle, and haydite height is 1/3 of tissue culture bottle height, adds plant nutrition liquid 40mL, seals with envelope bottle film; Moist heat sterilization, 121 DEG C, 30min; High 100 ㎜ of tissue culture bottle, bore 50 ㎜, external diameter 60 ㎜;
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mL LB inoculums are housed, in 37 DEG C, 150rmin
-1it is 1.0 × 10 that concussion cultivation 18 h makes content
8cfumL
-1antagonistic Fungi liquid, immerses Antagonistic Fungi immersion kind 30 min by the seed of vernalization; Described LB inoculum refers to: add peptone 10 grams in 1 liter of deionized water, yeast extract 5 grams, NaCl 5 grams;
(5) field planting: treated seeds tweezers are are evenly clicked and entered in step (3) sterilization matrix, every bottle of point 8 seeds, seal up envelope bottle film, put into growth cabinet to cultivate, growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h;
(6) interpolation of plant nutrition liquid: the Expenditure Levels of every weekly check nutritive medium, the interpolation of plant nutrition liquid was advisable there not to be seed;
(7) zoospore preparation: phytophthora blight of pepper is seeded on CA substratum, intermittent illumination process is after 6 days, and microscopy sporocyst quantity, when there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, place 1h in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then scrubs media surface with sterilization writing brush, four layers of sterile gauze filter, collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1;
Described CA substratum refers to Radix Dauci Sativae substratum, and its formula is: by 200 grams of Radix Dauci Sativae sections, add 500 mL deionized waters, refiner will be smashed, and double gauze filters, and add deionized water and mend to 1 liter, add 20 grams of agar, every bottle of 100 mL packing, sterilizing is for subsequent use;
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension, inoculation after growth cabinet optimum configurations be: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h, inoculation after the 3rd, 5 day investigation incidence.The preparation of described plant nutrition liquid: add each 0.16 gram of 0.22 gram, ammonium phosphate, 1.05 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.01 gram, ferrous sulfate in 1 liter of deionized water.
Embodiment 2
A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target:
(1) seed disinfection: by requisite number object pepper seed aseptic water washing one time, then successively process 30s and 5min respectively with the alcohol of 75% and the NaClO of 5%, seed selection capsicum variety is improvement 8819 line green pepper;
(2) presprouting of seeds: the seed of disinfecting is soaked 4h in sterilized water, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally;
(3) matrix sterilizing: 30 grams of diameter 8 ㎜ haydites are put into tissue culture bottle, high 100 ㎜ of tissue culture bottle, bore 50 ㎜, external diameter 60 ㎜.Haydite height is 1/3 of tissue culture bottle height, adds plant nutrition liquid 40mL, seals with envelope bottle film; Moist heat sterilization, 121 DEG C, 30min; The preparation of described plant nutrition liquid: add each 0.45 gram of 0.45 gram, ammonium phosphate, 1.25 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.02 gram, ferrous sulfate in 1 liter of deionized water.
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mL LB inoculums are housed, in 37 DEG C, 150rmin
-1it is 1.0 × 10 that concussion cultivation 18 h makes content
8cfumL
-1antagonistic Fungi liquid, immerses Antagonistic Fungi immersion kind 30 min by the seed of vernalization; Described LB inoculum refers to: add peptone 10 grams in 1 liter of deionized water, yeast extract 5 grams, NaCl 5 grams;
(5) field planting: treated seeds tweezers are are evenly clicked and entered in step (3) sterilization matrix, every bottle of point 8 seeds, seal up envelope bottle film, put into growth cabinet to cultivate, growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h;
(6) interpolation of plant nutrition liquid: the Expenditure Levels of every weekly check nutritive medium, the interpolation of plant nutrition liquid was advisable there not to be seed;
(7) zoospore preparation: phytophthora blight of pepper is seeded on CA substratum, intermittent illumination process is after 6 ~ 8 days, and microscopy sporocyst quantity, when there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, place 1h in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then scrubs media surface with sterilization writing brush, four layers of sterile gauze filter, collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1;
Described CA substratum refers to Radix Dauci Sativae substratum, and its formula is: by 200 grams of Radix Dauci Sativae sections, add 500 mL deionized waters, carrot slice is smashed by refiner, and double gauze filters, and adds deionized water and mends to 1 liter, add 20 grams of agar;
Every bottle of 100 mL packing, sterilizing is for subsequent use;
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension, inoculation after growth cabinet optimum configurations be: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h, inoculation after the 3rd, 5 day investigation incidence.
Embodiment 3
Practical situations
Indoor measurement 7 strain is from sieving biocontrol microorganisms to capsicum epidemic disease prevention effect.
Phytophthora blight of pepper (
phytophthora capsici), dry thread Pyrenomycetes (
rhizoctonia solani), tomato gray mould bacterium (
botrytis cinerea) and scraping and printing unit (
pythium aphanidermatum) four kinds of soil-borne disease pathogenic bacterias provide (externally can provide free) by Tianjin Plant Protection Institute's disease flocking biocontrol room.
7 from sieving bacterial strain (TB1309, TB1312, TB1317, TB1321, TB1335, TB1340, TB1348)
Preparation method as follows:
(1) separation of bacterial more than 10,000 strain from the ground vegetables rhizosphere soils such as Tianjin, Hebei, Shandong, Shanxi, Fujian, Henan and Jiangsu;
(2) filter out to phytophthora blight of pepper (
phytophthora capsici) there is bacterium 802 strain of obvious antagonistic action;
(3) use further dry thread Pyrenomycetes (
rhizoctonia solani), tomato gray mould bacterium (
botrytis cinerea) and scraping and printing unit (
pythium aphanidermatum) go out bacterial strain 62 strain above pathogenic fungi being had to better antagonistic action for target sieving;
(4) from 62 strain bacterial strains, 7 strains all good to four kinds of pathogenic bacteria antagonistic effects are selected: TB1309, TB1312, TB1317, TB1321, TB1335, TB1340, TB1348.
Test choose to phytophthora blight of pepper (
phytophthora capsici), dry thread Pyrenomycetes (
rhizoctonia solani), tomato gray mould bacterium (
botrytis cinerea) and scraping and printing unit (
pythium aphanidermatum) four kinds of all good 7 of soil-borne disease pathogenic bacteria antagonistic effects carry out capsicum epidemic disease prevention effect mensuration from sieving bacterial strain (TB1309, TB1312, TB1317, TB1321, TB1335, TB1340, TB1348).
Control strain chooses Biocontrol Bacillus subtilis B579, is provided (externally can provide free) by disease flocking biocontrol research department of Tianjin Plant Protection Institute;
CK1: neither inoculate biocontrol microorganisms and also do not inoculate pathogenic bacteria;
Positive control CK2: only inoculate pathogenic bacteria, does not inoculate biocontrol microorganisms.Each process 10 bottles of pepper seedlings.
Concrete steps:
(1) seed disinfection: 800 improvement 8819 line green pepper seeds (being provided by treasure standing grain vegetable breeding center, Qing County) are used aseptic water washing one time, then successively processes 30s and 5min respectively with the alcohol of 75% and the NaClO of 5%.
(2) presprouting of seeds: the seed of disinfecting is placed in sterilized water and soaks 4h, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally.
(3) matrix sterilizing: prepare 100 tissue culture bottles, puts into 30 grams of haydites, is highly approximately 1/3 of tissue culture bottle height for every bottle, adds plant nutrition liquid 40mL to just not having haydite, and envelope bottle film is sealed.Moist heat sterilization, 121 DEG C, 30min.Wherein, haydite diameter 2 ~ 8 ㎜ not etc., does not buy in village flowers market, business section, Tianjin.High 100 ㎜ of tissue culture bottle, bore 50 ㎜, external diameter 60 ㎜, buys in Tianjin, Tianjin hundred along experimental installation company limited.The preparation of nutritive medium: add each 0.16 gram of 0.22 gram, ammonium phosphate, 1.05 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.01 gram, ferrous sulfate in 1 liter of deionized water, mixing is nutritive medium.
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mLLB inoculums are housed, in 37 DEG C, 150rmin
-1concussion cultivation 18 h makes Antagonistic Fungi liquid, and (adjustment bacterial content is 1.0 × 10
8cfumL
-1), the seed of vernalization is immersed Antagonistic Fungi immersion kind 30 min.
(5) field planting: evenly click and enter in sterilized matrix by processed pepper seed tweezers, every bottle of point 8 seeds, seal up bottle film, put into growth cabinet and cultivate.Growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h.Field planting treats after 28 days that capsicum grows four true leaves, can do pathogenic bacteria inoculation test.
(6) interpolation of plant nutrition liquid: the Expenditure Levels of every weekly check plant nutrition liquid, the interpolation of nutritive medium was advisable there not to be seed.
(7) zoospore preparation: be seeded in by phytophthora blight of pepper on CA substratum (Radix Dauci Sativae substratum), intermittent illumination process is after 6 ~ 8 days, microscopy sporocyst quantity.When there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, 1h is placed in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then scrub media surface with sterilization writing brush, four layers of sterile gauze filter, and collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1.
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, every bottle of Hot Pepper Seedling choosing 5 growing ways consistent does efficiency test.Liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension.In order to be beneficial to the generation of capsicum epidemic disease, after inoculation, growth cabinet optimum configurations is: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h.3rd, 5 day investigation incidence after inoculation.
Table 1 antagonistic strain is to the potted plant prevention effect of capsicum epidemic disease
Conclusion:
The result that after pathogenic bacteria, 3 d and 5 d investigate plant incidence shows: also do not inoculate (CK1) in the process of biocontrol microorganisms and do not have plant to fall ill neither inoculating pathogenic bacteria; Only connecing pathogenic bacteria, do not inoculate (CK2) in the process of biocontrol microorganisms, plant all falls ill; Inoculation pathogenic bacteria and each process of biocontrol microorganisms are compared with CK2 at the same time, its sickness rate is all showing downward trend in various degree, and after 5 d, bacterial strain TB1309, TB1312, TB1317, TB1321, TB1335, TB1340, TB1348 prevent and treat efficiency relative to B579 bacterial strain is respectively 48%, 45%, 70%, 65%, 86%, 82%, 75% and 50%.Result shows, 7 strains all show from sieve antagonistic strain and B579 bacterial strain has preventive effect effect in various degree to capsicum epidemic disease, and wherein TB1335 bacterial strain preventive effect is best.So this method is easy, quick, feasible in the biocontrol strain screening operation taking capsicum epidemic disease as target.
Claims (2)
1. be a biocontrol strain high-efficiency screening method for target with capsicum epidemic disease, it is characterized in that being undertaken by following step:
(1) seed disinfection: by requisite number object pepper seed aseptic water washing one time, then successively process 30s and 5min respectively with the alcohol of 75% and the NaClO of 5%, seed selection capsicum variety is improvement 8819 line green pepper;
(2) presprouting of seeds: the seed of disinfecting is soaked 4h in sterilized water, after put into the culture dish being covered with wet gauze, in the incubator of 25 DEG C, lucifuge 48h vernalization is to showing money or valuables one carries unintentionally;
(3) matrix sterilizing: 30 grams of diameter 2 ~ 8 ㎜ haydites are put into tissue culture bottle, and haydite height is 1/3 of tissue culture bottle height, adds plant nutrition liquid 40mL, seals with envelope bottle film; Moist heat sterilization, 121 DEG C, 30min; The preparation of described plant nutrition liquid: add each 0.16 ~ 0.45 gram of 0.22 ~ 0.45 gram, ammonium phosphate, 1.05 ~ 1.25 grams, saltpetre, ammonium sulfate and ammonium nitrate, 0.01 ~ 0.02 gram, ferrous sulfate in 1 liter of deionized water;
(4) biocontrol microorganisms seed soaking: with the single bacterium colony choosing pin picking Antagonistic bacteria strains, be transferred in the triangular flask that 50 mL LB inoculums are housed, in 37 DEG C, 150rmin
-1it is 1.0 × 10 that concussion cultivation 18 h makes content
8cfumL
-1antagonistic Fungi liquid, immerses Antagonistic Fungi immersion kind 30 min by the seed of vernalization; Described LB inoculum refers to: add peptone 10 grams in 1 liter of deionized water, yeast extract 5 grams, NaCl 5 grams;
(5) field planting: treated seeds tweezers are are evenly clicked and entered in step (3) sterilization matrix, every bottle of point 8 seeds, seal up envelope bottle film, put into growth cabinet to cultivate, growth cabinet optimum configurations is: temperature is 26 DEG C, and humidity is 50%, illumination 16h, dark 8h;
(6) interpolation of plant nutrition liquid: the Expenditure Levels of every weekly check plant nutrition liquid, the interpolation of plant nutrition liquid was advisable there not to be seed;
(7) zoospore preparation: phytophthora blight of pepper is seeded on CA substratum, intermittent illumination process is after 6 ~ 8 days, and microscopy sporocyst quantity, when there being a large amount of sporocyst in the microscopy visual field, 10 mL aseptic waters are added in culture dish, place 1h in 4 DEG C, light at room temperature induces Zoospore liberation according to 1h subsequently, then scrubs media surface with sterilization writing brush, four layers of sterile gauze filter, collect zoospore suspension, microscopy zoospore quantity, adjustment inoculum density is 10
5mL
-1; Described CA substratum refers to Radix Dauci Sativae substratum, and its formula is: by 200 grams of Radix Dauci Sativae sections, add 500 mL deionized waters, carrot slice is smashed by refiner, and double gauze filters, and adds deionized water and mends to 1 liter, add 20 grams of agar, every bottle of 100 mL packing, sterilizing is for subsequent use;
(8) pathogenic bacteria inoculation: field planting treats after 28 days that capsicum grows four true leaves, liquid transfer gun head is inserted below nutrition liquid level inoculate 5 mL adjust concentration be 10
5mL
-1zoospore suspension, inoculation after growth cabinet optimum configurations be: temperature is 28 DEG C, and humidity is 70%, illumination 8h, dark 16h, inoculation after the 3rd, 5 day investigation incidence.
2. be the biocontrol strain high-efficiency screening method of target with capsicum epidemic disease described in claim 1, high 100 ㎜ of the tissue culture bottle wherein described in step (3), bore 50 ㎜, external diameter 60 ㎜.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410247409.3A CN103981249B (en) | 2014-06-06 | 2014-06-06 | A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410247409.3A CN103981249B (en) | 2014-06-06 | 2014-06-06 | A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103981249A CN103981249A (en) | 2014-08-13 |
| CN103981249B true CN103981249B (en) | 2015-09-30 |
Family
ID=51273420
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410247409.3A Expired - Fee Related CN103981249B (en) | 2014-06-06 | 2014-06-06 | A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103981249B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447966B (en) * | 2014-12-01 | 2017-05-10 | 山东农业大学 | Phytophthora capsici cell division protein as well as encoding gene and application thereof |
| CN105985164A (en) * | 2015-03-02 | 2016-10-05 | 天津市植物保护研究所 | Microbial seedling raising medium formula |
| CN104726390A (en) * | 2015-03-17 | 2015-06-24 | 内蒙古农业大学 | Culture medium for prompting Phytophthora capsici Leonian to generate zoosporangium |
| CN106929561A (en) * | 2017-02-21 | 2017-07-07 | 中国农业科学院植物保护研究所 | A kind of screening technique of biocontrol of plant disease bacterial strain |
| CN108034629B (en) * | 2018-01-19 | 2021-05-25 | 北京农学院 | Application of CA culture medium in culturing strawberry brown leaf spot pathogen sporulation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543801A (en) * | 2003-11-22 | 2004-11-10 | 西北农林科技大学 | Preparation for biological prevention and cure of hot pepper epidemic disease and method for producing the same |
-
2014
- 2014-06-06 CN CN201410247409.3A patent/CN103981249B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543801A (en) * | 2003-11-22 | 2004-11-10 | 西北农林科技大学 | Preparation for biological prevention and cure of hot pepper epidemic disease and method for producing the same |
Non-Patent Citations (1)
| Title |
|---|
| 内生芽孢杆菌TB2防治辣椒疫病效果及其机理初探;邱思鑫等;《植物病理学报》;20041231;第34卷(第2期);第173－179页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103981249A (en) | 2014-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107446847B (en) | Bacillus belgii GT11 and application thereof | |
| CN107254427B (en) | Bacillus belgii strain JN5 and application thereof | |
| CN101473853B (en) | Application of Bacillus cereus CMCC63305 in agricultural field | |
| CN103981249B (en) | A kind of take capsicum epidemic disease as the biocontrol strain high-efficiency screening method of target | |
| CN101575574A (en) | Trichoderma harzianum composite bacteria culture and application of trichoderma harzianum composite bacteria culture in aspect of plant protection | |
| CN106172507B (en) | Applications of the bacillus subtilis HMB19198 in prevention powdery mildew of cucumber | |
| CN104531545A (en) | Bacillus atrophaeus strain DPPG-28 and application in crop disease control | |
| CN107475134B (en) | One plant of Fusarium solani and its application in prevention and treatment dendrobium candidum southern blight | |
| CN105018385A (en) | Bacillus amyloliquefaciens and application thereof | |
| CN105432381B (en) | A kind of preventing control method of capsicum epidemic disease | |
| WO2010135893A1 (en) | Microbial agent ps and psx for preventing and curing soil-borne disease of plants and preparation method and use thereof | |
| CN102533603B (en) | Pseudomonas sp. 841P-3 for preventing cotton verticillium wilt and use thereof | |
| CN106754567B (en) | A kind of biological and ecological methods to prevent plant disease, pests, and erosion composite bacteria agent LAS of efficiently prevention various crop droop | |
| CN108913621B (en) | Bacillus methylotrophicus YH-18 capable of effectively preventing and treating oriental cherry root cancer and application thereof | |
| CN109169712B (en) | Composite biological control agent and preparation method and application thereof | |
| CN111363691B (en) | Paenibacillus polymyxa and application thereof | |
| CN102443559B (en) | Baclillus subtilis used for controlling cotton verticillium wilt and application thereof | |
| CN102321559B (en) | Bio-control strain CS23 used for controlling downy mildew of greenhouse cucumbers, and application thereof | |
| CN102191198B (en) | Biocontrol strain JH21 for controlling rice sheath blight and rice blast and biocontrol bacterial agent thereof | |
| CN103299811B (en) | Method for enhancing soil waterflooding effect to control pepper phytophthora blight | |
| CN105820969A (en) | Bacillus amyliquefaciens strain and application thereof | |
| CN105624047A (en) | Coix lacroyma-jobi L.var.ma-yuen (Roman.) Stapf endophytic fungus and application thereof | |
| CN111471627B (en) | Serratia marcescens BSZ and application thereof | |
| CN103013853A (en) | Biocontrol strain KMXU22 capable of preventing and treating soft rot of konjak and antibiological inoculant of biocontrol strain KMXU22 | |
| CN111909863B (en) | Bacillus amyloliquefaciens and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150930 Termination date: 20160606 |