CN105002111A - Bacterial strain TDJN1 for preventing and treating cucumber downy mildew and use thereof - Google Patents
Bacterial strain TDJN1 for preventing and treating cucumber downy mildew and use thereof Download PDFInfo
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- CN105002111A CN105002111A CN201510409223.8A CN201510409223A CN105002111A CN 105002111 A CN105002111 A CN 105002111A CN 201510409223 A CN201510409223 A CN 201510409223A CN 105002111 A CN105002111 A CN 105002111A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The invention discloses a bacterial strain TDJN1 for preventing and treating cucumber downy mildew. The bacterial strain TDJN1 has a class name of Stenotrophomonas maltophilia, is preserved in the China general microbiological culture collection center On May 19, 2015 and has a preservation number of CGMCC No. 10827. The invention discloses a biological antifungal agent. The biological antifungal agent is prepared from the bacterial strain TDJN1 for preventing and treating cucumber downy mildew. The invention also discloses a use of the biological antifungal agent in prevention and treatment on cucumber downy mildew. The biological antifungal agent produces a certain prevention and treatment effect on cucumber downy mildew in a greenhouse by the bacterial strain TDJN1 and has greenhouse prevention efficiency of 69.04% and field prevention efficiency of 42.38%. The bacterial strain TDJN1 can improve vitamin C, soluble protein and soluble sugar contents of cucumber and realizes a yield increasing rate of 34.73%.
Description
Technical field
The invention belongs to farm crop Prevention Technique field, be specifically related to a kind of the bacterial strain TDJN1 and the application thereof that prevent and treat cucumber downy mildew, be exclusively used in the green organic control of cucumber downy mildew.
Background technology
Cucumber downy mildew is a kind of air infection diseases caused by Cuba artificial downy mildew [Pseudoperonospora cubensis]; it is one of the important leaf diseases in cucumber production district of the world; also be a kind of destructive disease on dependent territory cucumber, have more than 70 country successively this disease to occur.In recent years, along with the continuous increase of dependent territory cucumber cultivated area, the harm of cucumber downy mildew increases the weight of year by year, has now become the important factor of restriction warmhouse booth cucumber yield.
At present, control cucumber downy mildew mainly relies on seed selection resistant variety, is secondly select Agro-chemicals control, as m-tetrachlorophthalodinitrile, phosethyl Al, metaxanin, zinc manganese ethylenebisdithiocarbamate, Propamocarb metaxanin etc.; But the breeding for disease resistance work period is long, the Reusability of chemical pesticide easily develops immunity to drugs and contaminate environment.This disease, once popular, uses to foreshorten to 3-4 days the interval of chemical agent, and Pesticide Residue is abnormal outstanding.Therefore, utilize biological control strategy to prevent and treat cucumber downy mildew and day by day receive publicity, biological control strategy becomes the new direction of this disease control research.
In recent years, biological and ecological methods to prevent plant disease, pests, and erosion product for preventing and treating cucumber downy mildew is also day by day prosperous, mainly contain: plant milk extract, microbiology class, bioprotein class, volatile organic matter etc., their biocontrol mechanism mainly carries out the control of disease by aspects such as competition, antibiosis and induced resistance; Existing large quantity research reports disease-resistant, the resistance that biocontrol microorganisms introduced plant can be improved plant.Because biocontrol microorganisms is suppressing the broad spectrum in Plant diseases, high efficiency and security, becoming the trend of pesticide market development at present, most potential developing direction of being known as.
Because still lack desirable high resisting high-yield product kind and beneficial agents up to now, and the problem of chemical prevention environmental pollution becomes increasingly conspicuous, so in today of emphasis on development No-harmful apple orchard technology, biological control, as a kind of new approaches of control cucumber downy mildew, will obtain further paying attention to and development.Therefore, what develop that new, more efficient and safe microbial preparation controls cucumber downy mildew is improve the needs of output, the needs of agricultural safety and Sustainable development especially simultaneously.
Summary of the invention
Goal of the invention: the biocontrol strain preparation present situation not having higher preventive effect for cucumber downy mildew, first object of the present invention is to provide a kind of single bacterial strain preventing and treating cucumber downy mildew of exploitation.
Second object of the present invention is to provide the application of bacterial strain TDJN1 in control greenhouse cucumber oidium of control cucumber downy mildew.
3rd object of the present invention is to provide biocontrol fungicide, and be exclusively used in control cucumber downy mildew, the preventive effect of bacterial strain is high, and has and increase production function preferably.
4th object of the present invention is to provide the preparation method of above-mentioned biocontrol fungicide.
5th object of the present invention is to provide the application of above-mentioned biocontrol fungicide in control cucumber downy mildew.
Technical scheme: in order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of bacterial strain TDJN1 preventing and treating cucumber downy mildew, its Classification And Nomenclature is germ oligotrophy unit cell (Stenotrophomonas maltophilia), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 19th, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is CGMCC No.10827.
The application of bacterial strain TDJN1 in control greenhouse cucumber oidium of above-mentioned control cucumber downy mildew.
A kind of biocontrol fungicide, above-mentioned biocontrol fungicide is prepared from by the bacterial strain TDJN1 of control cucumber downy mildew.
The preparation method of above-mentioned biocontrol fungicide, comprise the following steps: by bacterial strain TDJN1 at the flat lining out of LB, 14-16h is cultivated in 28 DEG C of biochemical cultivation cases, after growing single bacterium colony, in the test tube of the LB nutrient solution of picking list bacterium colony access containing 5mL, be placed in 28 DEG C, the shaking table of 200r/min cultivates, make seed liquor; Seed liquor is inoculated in the triangular flask of the LB nutrient solution that 500mL is housed by the inoculum size with 1%, is placed in 28 DEG C, the shaking table of 200r/min cultivates 48h, treat that bacterial concentration reaches 10
9dilute stand-by during CFU/mL, be prepared TDJN1 biocontrol fungicide.
The application of above-mentioned biocontrol fungicide in control cucumber downy mildew.
Its application method is, bacterial strain preparation is spraying use before crop is transplanted, and also can make compost and use, or fills with root use when transplanting after dilution.
Beneficial effect: the present invention is compared with existing cucumber downy mildew Prevention Technique, its advantage and positively effect show: the present invention is specially for the biocontrol strain of cucumber downy mildew exploitation, and thus in control cucumber downy mildew, prevention effect significantly improves compared with other broad spectrum biological disinfection agent at present.Owing to being Biocontrol microorganism preparation, do not bring series of problems because of the use of chemical pesticide completely, thus the No-harmful apple orchard of crop is conducive to, peasant or can reduce the consumption of other control cucumber downy mildew chemical pesticides, this not only can be peasant and reduces expenses, and is conducive to the outlet of crop.Meanwhile, biocontrol strain preparation increases production function in addition, can be peasant and increases income.
TDJN1 bacterial strain preparation before greenhouse is transplanted, can be sprayed in fertilizer or on blade, then buries in soil with fertilizer in whole ground process, also can fill with root when planting seedling and use.After planting seedling, bacterial strain is worked by multiple Biocontrol Mechanism, thus reaches the effect of control cucumber downy mildew.Test shows, in greenhouse, use TDJN1 bacterial strain preparation to have prevention effect to a certain degree to cucumber downy mildew, greenhouse preventive effect reaches 69.04%, field efficacy reaches 42.38%, TDJN1 bacterial strain preparation can improve the content of cucumber vitamins C, soluble proteins, soluble sugar etc., also have effect of increasing production to a certain degree, its effect of increasing production reaches 34.73%.
Accompanying drawing explanation
Fig. 1 be in field experiment biocontrol fungicide TDJN1 in sowing prevention effect to cucumber downy mildew after 55 days;
Note a: left side-CK, the right side-TDJN1.
Embodiment
Below by specific embodiment, the present invention is further described; it should be pointed out that for the person of ordinary skill of the art, under the premise without departing from the principles of the invention; can also make some modification and improvement, these also should be considered as belonging to protection scope of the present invention.
The selection systems of embodiment 1 bacterial strain
In November, 2012, in the plot collected specimens that Taicang City, Jiangsu Province warmhouse booth is fallen ill serious.Adopt five point sampling methods, often some interval at least 10m.Often three strain healthy plants are collected together with rhizosphere soil in the lump with the plant that morbidity weighs.Sample after collection loads rapidly to be taken back laboratory in the plastics bag finishing writing numbering and is separated.
Bacterial strain TDJN1 is separated to obtain in the plants stems of morbidity.Its separation method is: cut off each 3g of root, stem and leaf respectively, and the clorox 1mL with 1% soaks 5min, 70% alcohol leaching 2min, sterile water wash 3 times.(it is dull and stereotyped that the solution 0.1mL getting last cleaning is coated with R2A, or get the last scavenging solution of 0.1mL and add R2A nutrient solution, 30 DEG C of shaking culture, if asepsis growth after 48h, thinks that surface sterilization is clean).The good root, stem and leaf of surface sterilization is ground respectively with sterilizing Yan Portland, add 3mL sterilized water, aseptic cotton filters, dilute 10 times with sterilized water, get 0.1mL and be coated with LB flat board, each some repetition 3 times, cultivate 48h for 28 DEG C, counting, bacterium colony separation and purification all on picking flat board ,-70 DEG C, 40% glycerine saves backup.
By morphological specificity, bio-chemical characteristics and 16SrDNA gene sequencing are identified this bacterial strain.
16SrDNA gene amplification and sequential analysis:
Bacterial strain TDJN1 is at R
2in A substratum, 28 DEG C are cultured to logarithmic phase, thalline is collected with the centrifugal 5min of 12000r/min, adopt the genomic dna rapid extraction test kit of match Parkson, Shanghai gene engineering company limited, extract the genomic dna of bacterium, with the DNA product extracted for template, with bacterial 16 S rDNA gene amplification universal primer U8-27 (5 '-AGAGTTTGATC (AC) TGGCTCAG-3 '), L1494-1514 (5 '-CTACGG (AG) TACCTTGTTACGAC-3 ')
16S rDNA gene fragment is amplified from genomic dna.PCR primer is served Hai Shenggong biotechnology company limited and is checked order.Carry out tetraploid rice by BLAST software to mensuration 16S rDNA gene order, this identification of strains is that Stenotrophomonas belongs to (Stenotrophomonas sp.) by result.
This bacterial strain TDJN1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.10827.
Embodiment 2: the preparation of biocontrol fungicide
By bacterial strain TDJN1 at the flat lining out of LB, in 28 DEG C of biochemical cultivation cases, cultivate 14-16h, after growing single bacterium colony, in the test tube of the LB nutrient solution of picking list bacterium colony access containing 5mL, be placed in 28 DEG C, the shaking table of 200r/min cultivates, make seed liquor; Seed liquor is inoculated in the triangular flask of the LB nutrient solution that 500mL is housed by the inoculum size with 1%, is placed in 28 DEG C, the shaking table of 200r/min cultivates 48h, treat that bacterial concentration reaches 10
9dilute stand-by during CFU/mL, be prepared TDJN1 biocontrol fungicide.
Embodiment 3: the experiment of greenhouse preventive effect and the test of greenhouse growth-promoting
1, greenhouse preventive effect experiment:
Filling root and spraying process is carried out with biocontrol microorganisms when greenhouse cucumber seedling grows 4 true leaves.Root irrigation bacterial concentration is 1 × 10
8cFU/ml, every young plant consumption is 25ml, slowly waters in Cucumber root.Spraying process bacterial concentration is 5 × 10
7cFU/ml, the final concentration according to 0.01% adds surface active agent tween 20, and even spraying is in cucumber leaves.The standard of spraying is: every sheet leaf has misty liquid droplets to distribute, and be as the criterion not fall down, cucumber leaves pros and cons all will spray application to, and control treatment adopts clear water process.Spray inoculation pathogenic bacteria after biocontrol microorganisms process 5d, inoculation pathogenic bacteria 20d " Invest, Then Investigate " disease severity also calculates biocontrol effect.Disease severity grade scale: 0 grade, without illness; 1 grade, vaccination has slight scab, and its diameter is less than 0.5cm; 3 grades, lesion diameter 0.5 ~ 1.3cm; 5 grades, vaccination yellow area accounts for cotyledon area less than 1/2, and necrosis area accounts for less than 1/3; 7 grades, necrotic plaque area accounts for leaf area 1/3 ~ 2/3; 9 grades, necrotic plaque area account for leaf area more than 2/3 or complete stool withered.
Disease severity=∑ (diseased plant number × level value at different levels)/(investigating total strain number × superlative degree value) × 100%;
Prevention effect=(control group disease severity-treatment group disease severity)/control group disease severity × 100%.
In the investigation result display of biocontrol fungicide TDJN1 process after 25 days, the biological control effect (hereinafter referred to as preventive effect) of biocontrol fungicide TDJN1 to cucumber downy mildew reaches 53.76% (69.04%) (table 1).
Table 1 biocontrol fungicide TDJN1 uses the greenhouse preventive effect to cucumber downy mildew after 20 days
Note: this result is the preventive effect statistics behind biocontrol microorganisms process 25 days (pathogenic bacteria inoculates 20 days).
2, greenhouse growth-promoting test
In growth-promoting test, adopt root irrigation and spraying process, root irrigation bacterial concentration is 1 × 10
8cFU/ml, every young plant consumption is 25ml, slowly waters in Cucumber root.Spraying process bacterial concentration is 5 × 10
7cFU/ml, the final concentration according to 0.01% adds surface active agent tween 20, and even spraying is in cucumber leaves.After biocontrol microorganisms process 30d, measure cucumber plant height, leaf area and measure the indexs such as fresh weight, dry weight and biomass increase.
The increase calculation formula of biomass is as follows:
Under greenhouse experiment, after biocontrol fungicide TDJN1 process 30d, the cucumber seedling of multiple process has significant difference in the indexs such as plant height, leaf area, fresh weight and dry weight with between contrasting, and the increase of biomass reaches 27.94% (table 2).
The table 2 biocontrol fungicide TDJN1 process latter 30 days greenhouse growth-promoting effects to cucumber downy mildew
Note: numerical value is mean value ± standard error, between different letter representation process under the conspicuous level of P=0.05, otherness is remarkable.
The preventive effect experiment of embodiment 3 land for growing field crops
The second half year in 2013, sun light green house carried out land for growing field crops efficiency test in modern agriculture garden, Taicang City, Jiangsu Province.Planting patterns is live, plants in large-scale basin alms bowl, and the Nutrition Soil of use is import matrix.Test adopts randomized block design, is equipped with protection row between community and community.The 1st process is carried out when cucumber grows 5 ~ 6 true leaves.Spray biocontrol fungicide TDJN130mL/ strain, control group equivalent clear water is sprayed in the same way, all adds the tensio-active agent Tween-20 that final concentration is 0.01% when spraying.Field experiment adopts the field management pattern of standard, and does not re-use other sterilant.Biocontrol fungicide TDJN1 interval 10d dispenser 1 time, in the throughout the growing season altogether dispenser 3 times of cucumber; After first spraying 7,17 and 27d investigate disease record.
Cucumber downy mildew grade scale (GB/T17980.26-2000.) in the disease survey of land for growing field crops: 0 grade, without scab; 1 grade, lesion area accounts for less than 5% of whole leaf area; 3 grades, lesion area accounts for 6% ~ 10% of whole leaf area; 5 grades, lesion area accounts for 11% ~ 25% of whole leaf area; 7 grades, lesion area accounts for 26% ~ 50% of whole leaf area; 9 grades, lesion area accounts for more than 50% of whole leaf area.The plant of spraying often opens blade and all investigates.
Disease severity=∑ (diseased plant number × level value at different levels)/(investigating total strain number × superlative degree value) × 100%;
Prevention effect=(control group disease severity-treatment group disease severity)/control group disease severity × 100%;
Single plant yield=single fruit weight × number of bearing fruit;
Stimulation ratio=(∑ treatment group single plant yield-∑ control group single plant yield)/∑ control group single plant yield × 100%.
After biocontrol fungicide TDJN1 sprays for the first time the 7th, 17 and the disease severity of 27d, biocontrol fungicide TDJN1 process all contrast lower than clear water.The preventive effect of biocontrol fungicide TDJN1 reaches 29.39%, 53.03%, 42.38% respectively, and is significantly higher than control group and Chemical treatment group (table 3).
Table 3 biocontrol fungicide TDJN1 is to the land for growing field crops preventive effect of cucumber downy mildew
Note: numerical value is mean value ± standard error, between different letter representation process under the conspicuous level of P=0.05, otherness is remarkable.
After biocontrol fungicide TDJN1 process cucumber plant 60d, measure cucumber single plant yield and nutritive ingredient, find the cucumber of biocontrol fungicide TDJN1 process, single fruit weight all improves (table 4) compared with clear water control group, and effect of increasing production reaches 34.73%.Wherein, the single fruit weight after biocontrol fungicide TDJN1 process reaches 266.15g, is significantly higher than clear water control group.
Table 4 cucumber fruits nutrient component determining
Note: numerical value is mean value ± standard error, between different letter representation process under the conspicuous level of P=0.05, otherness is remarkable.
Specific description of embodiments of the present invention does not above limit the present invention, and those skilled in the art can make various change or distortion according to the present invention, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (5)
1. prevent and treat a bacterial strain TDJN1 for cucumber downy mildew, it is characterized in that, its Classification And Nomenclature be germ oligotrophy unit cell (
stenotrophomonas maltophilia), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 19th, 2015, and deposit number is CGMCC No.10827.
2. the application of bacterial strain TDJN1 in control greenhouse cucumber oidium of control cucumber downy mildew according to claim 1.
3. a biocontrol fungicide, is characterized in that, described biocontrol fungicide is prepared from by the bacterial strain TDJN1 of control cucumber downy mildew.
4. the preparation method of biocontrol fungicide according to claim 3, it is characterized in that, comprise the following steps: by bacterial strain TDJN1 at the flat lining out of LB, 14-16h is cultivated in 28 DEG C of biochemical cultivation cases, after growing single bacterium colony, in the test tube of the LB nutrient solution of picking list bacterium colony access containing 5mL, be placed in 28 DEG C, the shaking table of 200r/min cultivates, make seed liquor; Seed liquor is inoculated in the triangular flask of the LB nutrient solution that 500mL is housed by the inoculum size with 1%, is placed in 28 DEG C, the shaking table of 200r/min cultivates 48 h, treat that bacterial concentration reaches 10
9dilute stand-by during CFU/mL, be prepared TDJN1 biocontrol fungicide.
5. the application of biocontrol fungicide according to claim 3 in control cucumber downy mildew.
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| CN110972895A (en) * | 2019-12-10 | 2020-04-10 | 太仓绿丰农业资源开发有限公司 | Novel seedling culture substrate for resisting cucumber downy mildew |
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