CN103215203B - Biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof - Google Patents
Biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof Download PDFInfo
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- CN103215203B CN103215203B CN201310097481.8A CN201310097481A CN103215203B CN 103215203 B CN103215203 B CN 103215203B CN 201310097481 A CN201310097481 A CN 201310097481A CN 103215203 B CN103215203 B CN 103215203B
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Abstract
The invention belongs to the technical field of agricultural microbe, and relates to a biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus and application thereof. The biocontrol strain BF 6 is identified to be pseudomonas sp. The biocontrol strain BF 6 for preventing and treating tomato yellow leaf curl virus is preserved in common microorganism center of China Microbial Strain Preserving Management Committee on 27th, January, 2013, the strain preserving number is CGMCC No.7238. The strain can significantly control the tomato yellow leaf curl virus, reaching up to 59.51%. Field experiments show that the biocontrol strain BF 6 can significantly reduce the disease severity of the tomato yellow leaf curl virus disease with preventing efficiency of 50.79%-53.72%. The yield-increasing effect is more than 28%. The biocontrol strain BF 6 and especially prepared fungicide can be applied in the tomato yellow leaf curl virus disease control and yield increasing aspects.
Description
Technical field
The invention belongs to agriculture microorganism field, relate to biocontrol strain BF6 and the application thereof of control tomato yellow leaf curl virus disease.
Background technology
The tomato yellow leaf curl virus (TYLCV) of tomato yellow leaf curl virus Bing You geminivirus infection section (Geminiviridae) Begomovirus (Begomovirus) causes.Early than nineteen thirty-nine, in the Israel Jordan River, one band is found TYLCV, and in 1964 by definite designation.After 30 years between, this virus is diffused into rapidly numerous countries and regions such as the Middle East, Mediterranean, Africa, Europe, America, Asia and Australia.Compare with other virus diseases, tomato yellow leaf curl virus disease has that speed of mutation is fast, expansion rapidly, strong, the large feature for the treatment of difficulty of hazardness, be called as plant " cancer ", be a kind of destructive disease in tomato production.
From the nineties in 20th century, in the various crop such as tobacco, tomato and pumpkin on the ground such as Yunnan Province of China, Guangxi, Guangdong, Hainan, in succession occur.In recent years, Gai Bing China is progressively spread from south to north rapidly.Now except Hunan, Qinghai, Heilungkiang, the minority provinces such as Gansu do not find outside this virus disease, all there is generation all the other various places, have caused extremely serious loss to local tomato production.According to incompletely statistics, the year of tomato in China tomato yellow leaf curl China viral disease there is area at present and surpass 6.7 ten thousand hm
2, financial loss is over 2,000,000,000 yuan.
In production, the main methods such as agricultural measures and chemical prevention that adopt are prevented and treated, and cost is high, effect is not ideal enough, even to environment, carrys out negative impact.Although cultivate disease-resistant product and be the preferred approach of control tomato yellow leaf curl virus disease, because the genetics of resistance of tomato yellow leaf curl virus disease is more complicated, add that to have the resistant material of commercial value limited, be difficult to select the breeding strain that resistance is stable.And existing disease-resistant variety major part has region, be not easy to promote on a large scale.
In sum, what develop that new, more efficient and safe microbial preparation controls tomato yellow leaf curl virus disease is the needs that improve output, simultaneously the needs of agricultural safety and Sustainable development especially.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the biocontrol strain BF6 of control tomato yellow leaf curl virus disease is provided.
Another object of the present invention is to provide microbial inoculum prepared by this bacterial strain.
Another object of the present invention is to provide the application of this bacterial strain.
Object of the present invention can be achieved through the following technical solutions:
The sick biocontrol strain BF6 of a kind of control tomato yellow leaf curl virus, through identifying that this Pseudomonas is in pseudomonas (Pseudomonas sp.), on January 31st, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.7238.
The biocontrol fungicide of preparing with the sick biocontrol strain BF6 of described control tomato yellow leaf curl virus.
The preparation method of described biocontrol fungicide: by the sick biocontrol strain BF6 of described control tomato yellow leaf curl virus 30 ℃ of 200rpm shaking culture 24h in LB nutrient solution, then the centrifugal 10min of 6000rpm, with aqua sterilisa, dilute and make microbial inoculum, in finished product microbial inoculum, viable bacteria total concn is 1 * 10
9-1 * 10
10cFU/mL.
Certainly, utilize the sick biocontrol strain BF6 of described control tomato yellow leaf curl virus to prepare biocontrol fungicide and be not limited to above method.Everyly can cultivate in a large number BF6, and keep the method for its anti-tomato yellow leaf curl virus activity all to can be used for preparing the biocontrol fungicide that the present invention asks for protection.
The application of described biocontrol strain BF6 aspect control tomato yellow leaf curl virus disease and volume increase.
The application of described biocontrol fungicide aspect control tomato yellow leaf curl virus disease and volume increase.
Beneficial effect:
The present invention is specially for the sick biological prevention and control agent of developing of tomato yellow leaf curl virus.Owing to being biotechnological formulation, the series of problems bringing less than the use because of chemical pesticide completely, thereby the green non-pollution that is conducive to tomato is produced, peasant can or reduce the consumption of other chemical pesticides, this not only can be peasant and reduces expenses, and reduce the residual of chemical agent, benefit the nation and the people.
Greenhouse experiment shows: the sick effect of pseudomonas BF6 control tomato yellow leaf curl virus is remarkable, can reach 59.51%.Simultaneously field experiment result shows, biocontrol fungicide BF6 can significantly reduce the disease severity of tomato yellow leaf curl virus disease, preventive effect between 50.79%-53.72%, this microbial inoculum to the effect of increasing production of tomato more than 28%.
Biological sample preservation information
BF6, pseudomonas Pseudomonas sp., on January 31st, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and culture presevation number is CGMCC NO.7238.
Embodiment
Embodiment 1
Screening method: in pseudomonas BF6You Yangzhou Gaoyou City Gaoyou town tomato booth, the separation of tomato rhizosphere soil obtains.With banister brush, brush gently and get the soil that is attached to tomato root table, take in the 0.85%NaCl that 3g adds 27ml sterilizing, 180rmp shaking table vibration 0.5h, standing 5min, obtains 10-1 diluent, draws supernatant liquor 0.1mL and dilutes successively, gets 10
-4, 10
-5, 10
-6each 0.1mL of the diluent of three gradients is applied on LB flat board, and 28 ° of C cultivate 48h, and picking list colony inoculation is to fresh LB solid medium, through repeatedly transferring and obtain purifying bacterial strain.Bacterial strain after purifying is stored in-70 ° of C Ultralow Temperature Freezers with 40% glycerine.
Authentication method: by morphological specificity, Physiology and biochemistry experiment and 16S rDNA sequential analysis are identified this bacterial strain.
Morphological feature: gram negative bacillus, do not produce gemma, bacterium colony is smooth, without pod membrane, has end flagellum.
Major physiological biochemical character:
Table 1
16S rRNA gene amplification and sequential analysis:
Pseudomonas BF6 in 2A substratum 28 ℃ be cultured to logarithmic phase, with the centrifugal 5min of 12000r/min, collect thalline, adopt the genomic dna rapid extraction test kit of match Parkson, Shanghai gene engineering company limited, extract the genomic dna of bacterium, the DNA product extracting of take is template, and with bacterial 16 S rRNA amplification, universal primer U8-27 (5 '-AGAGTTTGATC (AC) TGGCTCAG-3 '), L1494-1514 (5 '-CTACGG (AG) TACCTTGTTACGAC-3 ') amplify 16S rDNA gene fragment from genomic dna.PCR product is served Hai Shenggong biotechnology company limited and is checked order.By BLAST software, to measuring 16S rRNA gene order, carry out homology comparison, result abridged table, therefore, is pseudomonas (Pseudomonas sp.) by this identification of strains.
The preparation of embodiment 2BF6 biocontrol fungicide
By pseudomonas BF6(CGMCC NO.7238) be inoculated in LB nutrient solution after 30 ℃ of 200rpm shaking culture 16h, every 2h, in Bechtop, sample and survey it in the OD at 600nm place value, when OD value finishes during at 0.5-0.8 to cultivate, this bacterium liquid is as kind of a daughter bacteria liquid.Planting daughter bacteria liquid, with 1:500 volume ratio, be seeded to fermentation culture in LB fermentation culture, 30 ℃, 200rpm are cultivated 24h, and fermented liquid pH value maintains 7.0, dissolved oxygen amount 20%, air flow 5-7m
3/ hour, tank pressure 0.05-0.1KPa.Then the centrifugal 10min of 6000rpm, gets precipitation and dilutes and make microbial inoculum with aqua sterilisa, and in finished product microbial inoculum, viable bacteria total concn is 1 * 10
9-1 * 10
10cFU/mL.
The preventive effect of BF6 to tomato yellow leaf curl virus disease measured in embodiment 3 greenhouse tests
Tomato variety is " cooperation 903 ", at 4 leaves, during the phase, transplants, and transplants the processing of spraying after 5 days.The microbial inoculum of getting embodiment 2 preparations, being diluted with water to bacterium liquid final concentration is 5 * 10
7cFU/mL, spraying standard is on every leaf, to have vaporific droplet distribution, not fall down and to be as the criterion.Biocontrol fungicide is processed rear the 7th day inoculating tomato yellow leaf curl virus.Process after 15 days for the 1st time and carry out same treatment the 2nd time.With independent virus inoculation, not carrying out the processing that biocontrol microorganisms sprays processes in contrast.Each treatment group 24 strain tomato, repeats for three times.It is 28 ℃ that tomato is placed in to temperature, and illumination is to cultivate under 12h/12h greenhouse experiment, investigates disease progression when falling ill, and calculates disease severity and preventive effect.
The severity Scaling standard proposing according to M Jos ' e D ' iez and Fernando Nuez1997, calculates disease severity and preventive effect.
The sick severity Scaling standard of tomato yellow leaf curl virus is:
0 grade: asymptomatic.
1 grade: yellow that top vane is slight, limb edge is slightly curling.Florescence flower variation.
2 grades: the yellow of top vane moderate, the curling fold of limb edge moderate.Florescence flower variation, the underproduction that fruiting period output is slight.
3 grades: the serious yellow of blade, curling, fold.Florescence flower moderate comes off, the underproduction of fruiting period output moderate.
4 grades: big area blade severe deformities is dwindled, and plant strain growth is slow, obviously downgrade.Florescence, Hua Yandu came off,
Fruiting period output Severe Reduction or total crop failure.
Disease severity %=[∑ (disease plant number * sick progression)/(total plant number * the highest sick progression)] * 100%,
Biological control effect %=[(contrast disease severity-processing disease severity)/contrast disease severity] * 100%
At biocontrol fungicide BF6, process the investigation result demonstration after 30 days for the 1st time, biocontrol fungicide BF6 reaches 59.51%(table 3 to the preventive effect of tomato yellow leaf curl virus disease).
The biocontrol effect of table 3 biocontrol fungicide BF6 to tomato yellow leaf curl virus disease
Note: this result is that BF6 processes 30 days preventive effect statisticses after (control group morbidity 15 days).
Embodiment 4 sun light green house field checking biocontrol fungicide BF6 are to the preventive effect of tomato yellow leaf curl virus disease and production-increasing function
Experiment place: Shahe town, Ganyu County, Lianyungang tomato sun light green house
Tomato variety is " Ge Ruisi ".Field experiment is established 2 treatment group altogether, and the BF6 biocontrol fungicide that is respectively embodiment 2 preparations is processed and clear water control treatment.3 communities are established in every processing, 60 of each communities seedling, and random district, each community group is arranged, and between community, to protect to go, isolates.Process after 15 days for the 1st time and carry out same treatment the 2nd time.Application method is spraying.Spraying standard is: on every leaf, have vaporific droplet distribution, not fall down and to be as the criterion.Spraying concentration is 5 * 10
7cFU/mL.
Data presentation, tomato is transplanted biocontrol fungicide BF6 after 45 days the preventive effect of tomato yellow leaf curl virus disease is reached to 50.79%(table 4).
The biocontrol effect of table 4 biocontrol fungicide BF6 to tomato yellow leaf curl virus disease
Note: this result is that BF6 processes 45 days preventive effect statisticses after (control group morbidity 15 days).
Meanwhile, we have carried out product quantitative statistics.Result demonstration, biocontrol fungicide BF6 is 35.89% to the effect of increasing production of tomato compared with the control, single fruit weight is significantly higher than control group (table 5).
The impact of table 5 biocontrol fungicide BF6 on tomato yield
Experiment place: Yancheng City Dongtai City sun light green house
Tomato variety is " No. 9, Soviet Union's powder ".2 processing are established in test altogether, are respectively biocontrol fungicide BF6 and process and clear water control treatment.3 communities are established in each processing, 50 of each communities seedling, and random district, each community group is arranged, and between community, to protect to go, isolates.Process after 15 days for the 1st time and carry out same treatment the 2nd time.Application method is spraying.Spraying standard is: on every leaf, have vaporific droplet distribution, not fall down and to be as the criterion.Spraying concentration is 5 * 10
7cFU/mL.
Data presentation, tomato is transplanted biocontrol fungicide BF6 after 40 days the preventive effect of tomato yellow leaf curl virus disease is reached to 53.92%(table 6).
The biocontrol effect of table 6 biocontrol fungicide BF6 to tomato yellow leaf curl virus disease
Note: this result is that BF6 processes 40 days preventive effect statisticses after (control group morbidity 15 days).
Meanwhile, we add up tomato yield.Result demonstration, comparing biocontrol fungicide BF6 processing is 28.34% with control treatment to the effect of increasing production of tomato, single fruit weight is significantly higher than control group (table 7).
The impact of table 7 biocontrol fungicide BF6 on tomato yield
Claims (5)
1. prevent and treat the sick biocontrol strain BF6 of tomato yellow leaf curl virus for one kind, through identifying that this Pseudomonas is in pseudomonas (Pseudomonas sp.), on January 31st, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.7238.
2. the biocontrol fungicide of preparing with the sick biocontrol strain BF6 of control tomato yellow leaf curl virus claimed in claim 1.
3. the preparation method of biocontrol fungicide claimed in claim 2, is characterized in that:
By the sick biocontrol strain BF6 of control tomato yellow leaf curl virus claimed in claim 1 30 ℃ of 200rpm shaking culture 24h in LB nutrient solution, then the centrifugal 10min of 6000rpm, dilutes and makes microbial inoculum with aqua sterilisa, and in finished product microbial inoculum, viable bacteria total concn is 1 * 10
9-1 * 10
10cFU/mL.
4. the application of biocontrol strain BF6 aspect control tomato yellow leaf curl virus disease and volume increase described in claim 1.
5. the application of biocontrol fungicide aspect control tomato yellow leaf curl virus disease and volume increase described in claim 2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1568709A (en) * | 2004-05-13 | 2005-01-26 | 南京农业大学 | Composite fungus agent for preventing blight of vegetable in greenhouse |
| CN102239247A (en) * | 2008-12-02 | 2011-11-09 | 李容溱 | Effective control of viral plant disease with strains of pseudomonas oleovorans |
-
2013
- 2013-03-25 CN CN201310097481.8A patent/CN103215203B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1568709A (en) * | 2004-05-13 | 2005-01-26 | 南京农业大学 | Composite fungus agent for preventing blight of vegetable in greenhouse |
| CN102239247A (en) * | 2008-12-02 | 2011-11-09 | 李容溱 | Effective control of viral plant disease with strains of pseudomonas oleovorans |
Non-Patent Citations (4)
| Title |
|---|
| Chowdhury, A. K..Effect of different biotic and abiotic agents for the management of tomato leaf curl virus disease.《Journal of Mycopathological Research》.2010,第48卷(第2期),403-405. |
| Effect of different biotic and abiotic agents for the management of tomato leaf curl virus disease;Chowdhury, A. K.;《Journal of Mycopathological Research》;20101231;第48卷(第2期);403-405 * |
| Induced systemic resistance to tomato leaf curl virus and increased yield in tomato by plant growth promoting rhizobacteria under field conditions;Vasanthi, V. J.;《Archives of Phytopathology and Plant Protection》;20101231;第43卷(第15期);1463-1472 * |
| Vasanthi, V. J..Induced systemic resistance to tomato leaf curl virus and increased yield in tomato by plant growth promoting rhizobacteria under field conditions.《Archives of Phytopathology and Plant Protection》.2010,第43卷(第15期),1463-1472. |
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