CN105002111B - It is a kind of prevent and treat cucumber downy mildew bacterial strain TDJN1 and its application - Google Patents
It is a kind of prevent and treat cucumber downy mildew bacterial strain TDJN1 and its application Download PDFInfo
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Abstract
The present invention disclose it is a kind of prevent and treat cucumber downy mildew bacterial strain TDJN1, classification naming be germ oligotrophy unit cell (Stenotrophomonas maltophilia), it is preserved in China General Microbiological culture presevation administrative center, preservation date on May 19th, 2015, deposit number is CGMCC No.10827.The invention also discloses a kind of biocontrol agent, which be prepared by the bacterial strain TDJN1 of prevention and treatment cucumber downy mildew.The invention also discloses application of the above-mentioned biocontrol agent in terms of preventing and treating cucumber downy mildew.The present invention has a degree of control efficiency to cucumber downy mildew using biocontrol agent TDJN1 in the greenhouse, greenhouse preventive effect reaches 69.04%, field efficacy reaches 42.38%, the content of cucumber vitamin C, soluble protein, soluble sugar etc. can be improved in TDJN1 bacterial strain preparation, and effect of increasing production reaches 34.73%.
Description
Technical field
The invention belongs to crops Prevention Technique fields, and in particular to it is a kind of prevent and treat cucumber downy mildew bacterial strain TDJN1 and
It is applied, and the green organic prevention and treatment of cucumber downy mildew is exclusively used in.
Background technique
Cucumber downy mildew is a kind of gas biography as caused by Cuba artificial downy mildew [Pseudoperonospora cubensis]
Disease is a kind of destructive disease on one of the important leaf diseases in cucumber production area, the world and dependent territory cucumber, there is 70
This disease successively occurred for multiple countries.In recent years, being continuously increased with dependent territory cucumber cultivated area, cucumber downy mildew
Harm aggravate year by year, become restrict greenhouse cucumber yield important factor.
Currently, prevention and treatment cucumber downy mildew relies primarily on breeding resistant variety, Agro-chemicals control, such as hundred bacterium are followed by selected
Clearly, aliette, metalaxyl, Mancozeb, Propamocarb metalaxyl etc.；But the breeding for disease resistance duty cycle is long, chemical pesticide
Reusability is also easy to produce drug resistance and pollution environment.Once the disease can foreshorten to 3-4 using the interval of chemical agent prevalence
It, Pesticide Residue is extremely prominent.Therefore, of increasing concern using biological control strategy prevention and treatment cucumber downy mildew, biology
Control strategy becomes the new direction of this disease control research.
In recent years, also increasingly prosperous for preventing and treating the biological and ecological methods to prevent plant disease, pests, and erosion product of cucumber downy mildew, mainly have: plant extracts, micro- life
Species, bioprotein class, volatile organic matter etc., their biocontrol mechanism mainly pass through competition, antibiosis and induced resistance
Etc. carry out disease prevention and treatment；Existing numerous studies, which are reported, can be improved the disease-resistant, degeneration-resistant of plant for biocontrol microorganisms introduced plant
Property.Due to broad spectrum activity, high efficiency and safety of the biocontrol microorganisms in terms of inhibiting plant disease, becomes pesticide market at present and develop
Trend, most potential developing direction of being known as.
Because still lacking ideal high resisting high-yield product kind and effective agent so far, and chemical prevention environmental pollution
The problem of become increasingly conspicuous, so in today of emphasis on development No-harmful apple orchard technology, biological control is as prevention and treatment cucumber downy mildew
A kind of new approaches, will obtain further pay attention to and development.Therefore, new, highly efficient and safe microorganism system is developed
Agent control cucumber downy mildew is the needs for improving output, while even more agricultural safety and sustainable development
It needs.
Summary of the invention
Goal of the invention: for cucumber downy mildew not compared with the biocontrol bacterial strain preparation status of high preventive effect, of the invention first
Purpose, which is to provide, develops a kind of single bacterial strain for preventing and treating cucumber downy mildew.
A second object of the present invention is to provide the bacterial strain TDJN1 of prevention and treatment cucumber downy mildew in prevention and treatment greenhouse cucumber downy mildew
The application of aspect.
Third object of the present invention is to provide biocontrol agents, are exclusively used in prevention and treatment cucumber downy mildew, and the preventive effect of bacterial strain is high, and
And there is preferable volume increase function.
Fourth object of the present invention is to provide the preparation method of above-mentioned biocontrol agent.
Fifth object of the present invention is to provide application of the above-mentioned biocontrol agent in terms of preventing and treating cucumber downy mildew.
Technical solution: in order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is as follows: a kind of prevention and treatment cucumber cream
The bacterial strain TDJN1 of mildew, classification naming are germ oligotrophy unit cell (Stenotrophomonas maltophilia), should
Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 19th, 2015, address: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number CGMCC
No.10827。
Application of the bacterial strain TDJN1 of above-mentioned prevention and treatment cucumber downy mildew in terms of preventing and treating greenhouse cucumber downy mildew.
A kind of biocontrol agent, above-mentioned biocontrol agent are prepared by the bacterial strain TDJN1 of prevention and treatment cucumber downy mildew.
The preparation method of above-mentioned biocontrol agent, comprising the following steps: bacterial strain TDJN1 is crossed on LB plate, 28 DEG C
14-16h is cultivated in biochemical cultivation case, after growing single colonie, picking single colonie accesses the test tube of the LB culture solution containing 5mL
In, it is placed in 28 DEG C, cultivates in the shaking table of 200r/min, seed liquor is made；Seed liquor is inoculated in 1% inoculum concentration and is equipped with
In the triangular flask of the LB culture solution of 500mL, it is placed in 28 DEG C, cultivates 48h in the shaking table of 200r/min, reaches to bacterial concentration
109It is diluted when CFU/mL for use, as prepared TDJN1 biocontrol agent.
Application of the above-mentioned biocontrol agent in terms of preventing and treating cucumber downy mildew.
Its application method, which is that bacterial strain preparation is spraying before crop transplanting, to be used, and also can be made compost use, or after dilution
In transplanting, pouring root is used.
The utility model has the advantages that the present invention is compared with existing cucumber downy mildew Prevention Technique, advantage and good effect are shown: this
Invention be specific to cucumber downy mildew exploitation biocontrol bacterial strain, thus in terms of prevent and treat cucumber downy mildew at present other wide spectrums
Biological bactericide is significantly improved compared to control efficiency.Due to being Biocontrol microorganism preparation, absolutely not because of the use of chemical pesticide
And a series of problems is brought, thus be conducive to the No-harmful apple orchard of crop, peasant can not have to or reduce other prevention and treatment cucumber creams
The dosage of mildew chemical pesticide, this can not only reduce expenses for peasant, but also be conducive to the outlet of crop.Meanwhile biocontrol bacterial strain
Preparation also increases production function, can increase income for peasant.
TDJN1 bacterial strain preparation can be sprayed in fertilizer or on blade, before greenhouse transplanting then with fertilizer in site preparation process
In bury in soil, can also when planting seedling pouring root use.After planting seedling, bacterial strain is worked by a variety of Biocontrol Mechanisms, to reach
The effect for preventing and treating cucumber downy mildew.Experiments have shown that being had to a certain degree using TDJN1 bacterial strain preparation to cucumber downy mildew in the greenhouse
Control efficiency, greenhouse preventive effect reaches 69.04%, and field efficacy, which reaches 42.38%, TDJN1 bacterial strain preparation, can be improved cucumber
The content of vitamin C, soluble protein, soluble sugar etc., there are also a degree of effect of increasing production, effect of increasing production reaches
34.73%.
Detailed description of the invention
Fig. 1 is for biocontrol agent TDJN1 in field experiment to the control efficiency of cucumber downy mildew after sowing 55 days；
Note a: left side-CK, the right side-TDJN1.
Specific embodiment
Below by specific embodiment, the present invention is further described, it is noted that for the ordinary skill of this field
For personnel, without departing from the principle of the present invention, several variations and modifications can also be made, these also should be regarded as belonging to
Protection scope of the present invention.
The screening and identification of 1 bacterial strain of embodiment
In November, 2012, in Taicang City, Jiangsu Province, the serious plot of greenhouse morbidity acquires sample.It is adopted using 5 points
Sample method, every interval at least 10m.The plant of every three plants of healthy plants and severe disease is collected together together with rhizosphere soil.After acquisition
Sample be quickly charged in the polybag for finish writing number and take back laboratory and separated.
Bacterial strain TDJN1 is isolated out of morbidity plants stems.Its separation method are as follows: each 3g of root, stem and leaf is cut off respectively,
With 1% sodium hypochlorite 1mL soak 5min, 70% alcohol soak 2min, sterile water wash 3 times.(the solution for taking last time to clean
0.1mL applies R2A plate, or takes 0.1mL last time cleaning solution that R2A culture solution, 30 DEG C of shaken cultivations, if sterile after 48h are added
Growth then thinks that surface sterilization is clean).The good root, stem and leaf of surface sterilization is ground respectively with sterilizing Yan Portland, adds 3mL sterile water, nothing
The filtering of bacterium cotton dilutes 10 times with sterile water, 0.1mL is taken to apply LB plate, and each point is repeated 3 times, 28 DEG C of culture 48h, is counted, is chosen
All bacterium colonies on plate of making even isolate and purify, and -70 DEG C, 40% glycerol saves backup.
By morphological feature, bio-chemical characteristics and 16SrDNA gene sequencing identify the bacterial strain.
16SrDNA gene magnification and sequence analysis:
Bacterial strain TDJN1 is in R2It is cultivated to logarithmic phase for 28 DEG C in A culture medium, thallus is collected with 12000r/min centrifugation 5min,
Using the genomic DNA rapidly extracting kit of Shanghai SBS Genetech gene technology Co., Ltd, the genomic DNA of bacterium is extracted,
Using the DNA product of extraction as template, with bacterial 16 S rDNA gene magnification universal primer U8-27 (5 '-AGAGTTTGATC (AC)
TGGCTCAG-3’)、L1494-1514(5’-CTACGG(AG)TACCTTGTTACGAC-3’)
16S rDNA genetic fragment is amplified from genomic DNA.PCR product serves Hai Shenggong bioengineering Co., Ltd
It is sequenced.Tetraploid rice is carried out to measurement 16S rDNA gene order by BLAST software, is as a result accredited as the bacterial strain
Stenotrophomonas category (Stenotrophomonas sp.).
Bacterial strain TDJN1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation is compiled
Number be CGMCC No.10827.
Embodiment 2: the preparation of biocontrol agent
Bacterial strain TDJN1 is crossed on LB plate, cultivates 14-16h in 28 DEG C of biochemical cultivation cases, after growing single colonie,
Picking single colonie accesses in the test tube of the LB culture solution containing 5mL, is placed in 28 DEG C, cultivates in the shaking table of 200r/min, kind is made
Sub- liquid；Seed liquor is inoculated in the triangular flask of the LB culture solution equipped with 500mL with 1% inoculum concentration, is placed in 28 DEG C, 200r/
48h is cultivated in the shaking table of min, reaches 10 to bacterial concentration9It is diluted when CFU/mL for use, as prepared TDJN1 biocontrol microorganisms
Agent.
Embodiment 3: the experiment of greenhouse preventive effect and the test of greenhouse growth-promoting
1, greenhouse preventive effect is tested:
Pouring root and spraying treatment are carried out with biocontrol microorganisms when greenhouse cucumber seedling grows 4 true leaves.Root irrigation bacterial concentration
It is 1 × 108CFU/ml, every young plant dosage are 25ml, are slowly poured in Cucumber root.Spraying treatment bacterial concentration be 5 ×
107Surface active agent tween 20 is added according to 0.01% final concentration in CFU/ml, and even spraying is in cucumber leaves.Spraying mark
Standard is: having misty liquid droplets distribution on every leaf, is subject to and does not fall down, cucumber leaves front and back sides will spray application to, control treatment
It is handled using clear water.Biocontrol microorganisms handle spray inoculation pathogen after 5d, investigate disease severity after being inoculated with pathogen 20d and calculate
Biocontrol effect.Disease severity grade scale: 0 grade, no illness；1 grade, vaccination has slight scab, and diameter is less than 0.5cm；3
Grade, 0.5~1.3cm of lesion diameter；5 grades, vaccination yellow area accounts for cotyledon area 1/2 hereinafter, necrosis area accounts for 1/3 or less；7
Grade, necrotic plaque area account for leaf area 1/3~2/3；9 grades, necrotic plaque area accounts for 2/3 or more leaf area or complete stool is withered.
Disease severity=∑ (diseased plant numbers at different levels × grade value)/(investigation total strain number × superlative degree value) × 100%；
Control efficiency=(control group disease severity-processing group disease severity)/control group disease severity ×
100%.
Biocontrol agent TDJN1 handle 25 days after investigation result show, life of the biocontrol agent TDJN1 to cucumber downy mildew
Object control efficiency (hereinafter referred to as preventive effect) reaches 53.76% (69.04%) (table 1).
1 biocontrol agent TDJN1 of table use 20 days after to the greenhouse preventive effect of cucumber downy mildew
Note: this result is that biocontrol microorganisms handle the preventive effect statistical result after 25 days (pathogen is inoculated with 20 days).
2, greenhouse growth-promoting is tested
In growth-promoting test, using root irrigation and spraying treatment, root irrigation bacterial concentration is 1 × 108CFU/ml, often
Young plant dosage is 25ml, is slowly poured in Cucumber root.Spraying treatment bacterial concentration is 5 × 107CFU/ml, according to 0.01%
Surface active agent tween 20 is added in final concentration, and even spraying is in cucumber leaves.After biocontrol microorganisms handle 30d, cucumber plant height, leaf are measured
The indexs such as area and measurement fresh weight, dry weight and biomass increase.
The increase calculation formula of biomass is as follows:
Under greenhouse experiment, after biocontrol agent TDJN1 handles 30d, the cucumber seedling of multiple processing is in plant height, leaf area, fresh weight
And there is significant difference between compareing in the indexs such as dry weight, biomass increases up to 27.94% (table 2).
30 days greenhouse growth-promoting effects to cucumber downy mildew after 2 biocontrol agent TDJN1 of table processing
Note: numerical value is average value ± standard error, between different letter expression processing under the level of signifiance of P=0.05, difference
Property is significant.
The experiment of 3 crop field preventive effect of embodiment
The sun light green house in the modern agriculture garden of Taicang City, Jiangsu Province of the second half year in 2013 carries out crop field efficiency test.Plantation side
Formula is live streaming, is planted in large-scale basin alms bowl, and the Nutrition Soil used is import matrix.Test use RANDOMIZED BLOCK DESIGN, cell with it is small
Protection row is equipped between area.The 1st processing is carried out when cucumber grows 5~6 true leaves.Spray biocontrol agent TDJN130mL/
Strain, control group is spraying in the same way with equivalent clear water, and final concentration of 0.01% surfactant is added when spraying
Tween-20.Field experiment uses the field management mode of standard, and does not use other fungicide.Biocontrol agent TDJN1
It is spaced 10d to be administered 1 time, in being throughout the growing season administered 3 times altogether for cucumber；7,17 and 27d investigation disease after spraying for the first time is simultaneously
Record.
Cucumber downy mildew grade scale (GB/T17980.26-2000.): 0 grade in the disease survey of crop field, disease-free spot；1 grade,
Lesion area accounts for 5% or less entire leaf area；3 grades, lesion area accounts for the 6%~10% of entire leaf area；5 grades, scab face
Product accounts for the 11%~25% of entire leaf area；7 grades, lesion area accounts for the 26%~50% of entire leaf area；9 grades, lesion area
Account for 50% or more of entire leaf area.Spraying every blade of plant is investigated.
Disease severity=∑ (diseased plant numbers at different levels × grade value)/(investigation total strain number × superlative degree value) × 100%；
Control efficiency=(control group disease severity-processing group disease severity)/control group disease severity ×
100%；
Single plant yield=single fruit weight × fruiting number；
Rate of growth=(∑ processing group single plant yield-∑ control group single plant yield)/∑ control group single plant yield × 100%.
The 7th, the 17 and 27d after biocontrol agent TDJN1 is spraying for the first time, the disease severity of biocontrol agent TDJN1 processing are equal
It is compareed lower than clear water.The preventive effect of biocontrol agent TDJN1 respectively reaches 29.39%, 53.03%, 42.38%, and is significantly higher than
Control group and Chemical treatment group (table 3).
Crop field preventive effect of the 3 biocontrol agent TDJN1 of table to cucumber downy mildew
Note: numerical value is average value ± standard error, between different letter expression processing under the level of signifiance of P=0.05, difference
Property is significant.
After biocontrol agent TDJN1 handles cucumber plant 60d, cucumber single plant yield and nutritional ingredient are measured, finds biocontrol microorganisms
The cucumber of agent TDJN1 processing, single fruit weight improve (table 4) compared with clear water control group, and effect of increasing production reaches
34.73%.Wherein, treated that single fruit weight reaches 266.15g by biocontrol agent TDJN1, is significantly higher than clear water control group.
4 cucumber fruits nutrient component determining of table
Note: numerical value is average value ± standard error, between different letter expression processing under the level of signifiance of P=0.05, difference
Property is significant.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (5)
1. a kind of bacterial strain TDJN1 for preventing and treating cucumber downy mildew, which is characterized in that its classification naming is germ oligotrophy unit cell
(Stenotrophomonas maltophilia), which has been preserved in Chinese microorganism strain guarantor on May 19th, 2015
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.10827.
2. application of the bacterial strain TDJN1 of prevention and treatment cucumber downy mildew described in claim 1 in terms of preventing and treating greenhouse cucumber downy mildew.
3. a kind of biocontrol agent, which is characterized in that the biocontrol agent is by preventing and treating cucumber downy mildew as described in claim 1
Bacterial strain TDJN1 be prepared.
4. the preparation method of biocontrol agent as claimed in claim 3, which comprises the following steps: bacterial strain TDJN1 exists
The flat lining out of LB cultivates 14-16h in 28 DEG C of biochemical cultivation cases, and after growing single colonie, the access of picking single colonie contains 5mL
LB culture solution test tube in, be placed in 28 DEG C, cultivate in the shaking table of 200r/min, seed liquor is made；With 1% inoculum concentration
Seed liquor is inoculated in the triangular flask of the LB culture solution equipped with 500mL, is placed in 28 DEG C, cultivates 48 in the shaking table of 200r/min
H reaches 10 to bacterial concentration9It is diluted when CFU/mL for use, as prepared TDJN1 biocontrol agent.
5. application of the biocontrol agent as claimed in claim 3 in terms of preventing and treating 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 |
| CN113151118A (en) * | 2021-06-02 | 2021-07-23 | 湖北省农业科学院中药材研究所 | Stenotrophomonas maltophilia for preventing and treating gray mold of paris polyphylla and application of stenotrophomonas maltophilia |
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| CN102146351A (en) * | 2011-01-12 | 2011-08-10 | 南京农业大学 | Biocontrol strain 2JW6 for preventing and controlling bacterial wilt of ginger |
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| CN102851225A (en) * | 2012-03-15 | 2013-01-02 | 王彩霞 | Stenotrophomonas acidaminiphila and application in control of apple tree canker thereof |
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| CN102146351A (en) * | 2011-01-12 | 2011-08-10 | 南京农业大学 | Biocontrol strain 2JW6 for preventing and controlling bacterial wilt of ginger |
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