CN103215203A - 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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- CN103215203A CN103215203A CN2013100974818A CN201310097481A CN103215203A CN 103215203 A CN103215203 A CN 103215203A CN 2013100974818 A CN2013100974818 A CN 2013100974818A CN 201310097481 A CN201310097481 A CN 201310097481A CN 103215203 A CN103215203 A CN 103215203A
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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 the biocontrol strain BF6 and the application thereof of control tomato yellow leaf curl virus disease.
Background technology
The tomato yellow leaf curl virus disease is caused by the tomato yellow leaf curl virus (TYLCV) that geminivirus infection section (Geminiviridae) bean golden mosaic virus belongs to (Begomovirus).One band is found TYLCV in the Israel Jordan River early than nineteen thirty-nine, and in 1964 by definite designation.After 30 in the period of, this virus is diffused into numerous countries and regions such as the Middle East, Mediterranean Sea bank, Africa, Europe, America, Asia and Australia rapidly.Compare with other virus diseases, the tomato yellow leaf curl virus disease has that speed of mutation is fast, expansion rapidly, strong, the big characteristics of treatment difficulty of hazardness, be called as plant " cancer ", be a kind of destructive disease on the tomato production.
From the nineties in 20th century, on the various crop such as tobacco, tomato and pumpkin on ground such as Yunnan Province of China, Guangxi, Guangdong, Hainan, take place in succession.In recent years, this disease progressively spreads from south to north rapidly in China.Remove the Hunan now, Qinghai, Heilungkiang, minority provinces such as Gansu do not find outside this virus disease that all there is generation all the other various places, have caused extremely serious loss for local tomato production.According to incompletely statistics, the year of tomato in China tomato yellow leaf curl China viral disease area taking place surpasses 6.7 ten thousand hm at present
2, financial loss is above 2,000,000,000 yuan.
The main methods such as agricultural measures and chemical prevention that adopt are prevented and treated in the production, and cost height, effect are not ideal enough, even come negative impact to environment.Though cultivate disease-resistant product are preferred approach of control tomato yellow leaf curl virus disease, because the genetics of resistance of tomato yellow leaf curl virus disease is complicated, add that the resistant material with commercial value is limited, are difficult to select the stable breeding strain of resistance.And existing disease-resistant variety major part has region, is not easy to promote on a large scale.
In sum, what develop new, more efficient and safe microbial preparation control tomato yellow leaf curl virus disease is the needs that improve the social production total amount, simultaneously the needs of agriculture especially safety and Sustainable development.
Summary of the invention
The objective of the invention is above-mentioned deficiency, the biocontrol strain BF6 of control tomato yellow leaf curl virus disease is provided at prior art.
Another object of the present invention provides the microbial inoculum of this bacterial strain preparation.
Another purpose of the present invention provides the application of this bacterial strain.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of control tomato yellow leaf curl virus diease occurrence is prevented bacterial strain BF6, through identifying that this Pseudomonas is in pseudomonas (Pseudomonas sp.), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 31st, 2013, culture presevation number is CGMCC NO.7238.
Biocontrol fungicide with the anti-bacterial strain BF6 preparation of described control tomato yellow leaf curl virus diease occurrence.
The preparation method of described biocontrol fungicide: with the anti-bacterial strain BF6 30 ℃ of 200rpm shaking culture 24h in the LB nutrient solution of described control tomato yellow leaf curl virus diease occurrence, the centrifugal 10min of 6000rpm then, dilute with aqua sterilisa and to make microbial inoculum, the viable bacteria total concn is 1 * 10 in the finished product microbial inoculum
9-1 * 10
10CFU/mL.
Certainly, utilize the anti-bacterial strain BF6 of described control tomato yellow leaf curl virus diease occurrence to prepare biocontrol fungicide and be not limited to above method.Everyly can cultivate BF6 in a large number, and keep the active method of its anti-tomato yellow leaf curl virus 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 at the sick biological prevention and control agent of developing of tomato yellow leaf curl virus.Owing to be biotechnological formulation, there are not a series of problems that use brought fully because of chemical pesticide, thereby help the green non-pollution production of tomato, the peasant can or reduce the consumption of other chemical pesticides, this not only can be the peasant and reduces expenses, and reduce the residual of chemical agent, benefit the nation and the people.
The greenhouse experiment shows: the sick effect of pseudomonas BF6 control tomato yellow leaf curl virus is remarkable, can reach 59.51%.Simultaneously the 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., be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on January 31st, 2013, the preservation address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and culture presevation number is CGMCC NO.7238.
Embodiment
Embodiment 1
Screening method: pseudomonas BF6 is obtained by tomato rhizosphere soil separation in Gaoyou town, the Yangzhou Gaoyou City tomato booth.Brush the soil of getting attached to the tomato root table gently with banister brush, take by weighing 3g and add among the 0.85%NaCl of 27ml sterilization, 180rmp shaking table vibration 0.5h leaves standstill 5min, obtains the 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 the LB flat board, and 28 ° of C cultivate 48h, and picking list colony inoculation obtains the purifying bacterial strain to fresh LB solid medium through transferring repeatedly.Bacterial strain behind the 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, and no pod membrane has the end flagellum.
The major physiological biochemical character:
Table 1
16S rRNA gene amplification and sequential analysis:
Pseudomonas BF6 in the 2A substratum 28 ℃ be cultured to logarithmic phase, collect thalline 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, DNA product with extraction is a template, and universal primer U8-27 (5 '-AGAGTTTGATC (AC) TGGCTCAG-3 '), L1494-1514 (5 '-CTACGG (AG) TACCTTGTTACGAC-3 ') amplify 16S rDNA gene fragment from genomic dna with bacterial 16 S rRNA amplification.The PCR product is served sea living worker's biotechnology company limited and is checked order.Carry out homology relatively by BLAST software to measuring 16S rRNA gene order, therefore abridged table, is pseudomonas (Pseudomonas sp.) with this identification of strains as a result.
The preparation of embodiment 2BF6 biocontrol fungicide
With pseudomonas BF6(CGMCC NO.7238) be inoculated in the LB nutrient solution 30 ℃ of 200rpm shaking culture 16h after, take a sample in Bechtop every 2h and to survey it in the OD at 600nm place value, when the OD value finishes during at 0.5-0.8 to cultivate, this bacterium liquid is as kind of a daughter bacteria liquid.Be seeded to fermentation culture in the LB fermentation culture planting daughter bacteria liquid with the 1:500 volume ratio, 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.The centrifugal 10min of 6000rpm then gets precipitation and dilutes with aqua sterilisa and make microbial inoculum, and the viable bacteria total concn is 1 * 10 in the finished product microbial inoculum
9-1 * 10
10CFU/mL.
The preventive effect of BF6 to the tomato yellow leaf curl virus disease measured in embodiment 3 greenhouse tests
Tomato variety is " cooperation 903 ", transplants during the phase at 4 leaves, transplants the processing of spraying after 5 days.Get the microbial inoculum of embodiment 2 preparations, being diluted with water to bacterium liquid final concentration is 5 * 10
7CFU/mL, the spraying standard is that vaporific droplet distribution is arranged on the every leaf, is as the criterion not fall down.Biocontrol fungicide is handled the 7th day inoculating tomato yellow leaf curl virus in back.Handle for the 1st time and carry out the 2nd second phase after 15 days with handling.Not carrying out the processing that biocontrol microorganisms sprays with independent virus inoculation handles in contrast.Each treatment group 24 strain tomato, three repetitions.It is 28 ℃ that tomato is placed temperature, and illumination is cultivated down for the 12h/12h greenhouse experiment, investigates disease progression when to be fallen ill, and calculates disease severity and preventive effect.
State of an illness grade scale according to M Jos ' e D ' iez and Fernando Nuez1997 propose calculates disease severity and preventive effect.
The sick state of an illness grade scale of tomato yellow leaf curl virus is:
0 grade: asymptomatic.
1 grade: the slight yellow of top vane, limb edge are slightly curled.Florescence flower variation.
2 grades: the yellow of top vane moderate, the limb edge moderate fold that curls.Florescence flower variation, the slight underproduction of fruiting period output.
3 grades: the serious yellow of blade, curl 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 downgrades.Florescence, Hua Yandu came off,
The serious underproduction of fruiting period output 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%
Investigation result after biocontrol fungicide BF6 handles 30 days the 1st time shows that biocontrol fungicide BF6 reaches 59.51%(table 3 to the preventive effect of tomato yellow leaf curl virus disease).
Table 3 biocontrol fungicide BF6 is to the biocontrol effect of tomato yellow leaf curl virus disease
Annotate: this result handles 30 days preventive effect statisticses after (control group morbidity 15 days) for BF6.
Embodiment 4 sun light green house fields checking biocontrol fungicide BF6 is to the preventive effect and the production-increasing function of tomato yellow leaf curl virus disease
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 handled and the clear water control treatment.3 sub-districts are established in every processing, 60 seedlings in each sub-district, and arrange each sub-district district's group at random, isolates with guard rows between the sub-district.Handle for the 1st time and carry out the 2nd second phase after 15 days with handling.Application method is spraying.The spraying standard is: vaporific droplet distribution is arranged on the every leaf, be as the criterion not fall down.Spraying concentration is 5 * 10
7CFU/mL.
Data presentation, tomato are transplanted biocontrol fungicide BF6 after 45 days the preventive effect of tomato yellow leaf curl virus disease are reached 50.79%(table 4).
Table 4 biocontrol fungicide BF6 is to the biocontrol effect of tomato yellow leaf curl virus disease
Annotate: this result handles 45 days preventive effect statisticses after (control group morbidity 15 days) for BF6.
Simultaneously, we have carried out the statistics of output.The result shows that biocontrol fungicide BF6 is 35.89% to the effect of increasing production of tomato compared with the control, and single fruit weight is significantly higher than control group (table 5).
Table 5 biocontrol fungicide BF6 is to the influence of 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 handle and the clear water control treatment.3 sub-districts are established in each processing, 50 seedlings in each sub-district, and arrange each sub-district district's group at random, isolates with guard rows between the sub-district.Handle for the 1st time and carry out the 2nd second phase after 15 days with handling.Application method is spraying.The spraying standard is: vaporific droplet distribution is arranged on the every leaf, be as the criterion not fall down.Spraying concentration is 5 * 10
7CFU/mL.
Data presentation, tomato are transplanted biocontrol fungicide BF6 after 40 days the preventive effect of tomato yellow leaf curl virus disease are reached 53.92%(table 6).
Table 6 biocontrol fungicide BF6 is to the biocontrol effect of tomato yellow leaf curl virus disease
Annotate: this result handles 40 days preventive effect statisticses after (control group morbidity 15 days) for BF6.
Simultaneously, we add up tomato yield.The result shows, comparing the effect of increasing production that biocontrol fungicide BF6 handles tomato with control treatment is 28.34%, and single fruit weight is significantly higher than control group (table 7).
Table 7 biocontrol fungicide BF6 is to the influence of tomato yield
Claims (5)
1. a control tomato yellow leaf curl virus diease occurrence is prevented bacterial strain BF6, through identifying that this Pseudomonas is in pseudomonas (Pseudomonas sp.), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 31st, 2013, culture presevation number is CGMCC NO.7238.
2. prevent the biocontrol fungicide of bacterial strain BF6 preparation with the described control tomato yellow leaf curl virus of claim 1 diease occurrence.
3. the preparation method of the described biocontrol fungicide of claim 2 is characterized in that:
With the anti-bacterial strain BF6 30 ℃ of 200rpm shaking culture 24h in the LB nutrient solution of the described control tomato yellow leaf curl virus of claim 1 diease occurrence, the centrifugal 10min of 6000rpm then dilutes with aqua sterilisa and to make microbial inoculum, and the viable bacteria total concn is 1 * 10 in the finished product microbial inoculum
9-1 * 10
10CFU/mL.
4. the application of the described biocontrol strain BF6 of claim 1 aspect control tomato yellow leaf curl virus disease and volume increase.
5. the application of the described biocontrol fungicide of claim 2 aspect control tomato yellow leaf curl virus disease and volume increase.
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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 (2)
| Title |
|---|
| CHOWDHURY, A. K.: "Effect of different biotic and abiotic agents for the management of tomato leaf curl virus disease", 《JOURNAL OF MYCOPATHOLOGICAL RESEARCH》 * |
| 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》 * |
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Address after: 211225 Lishui County, Nanjing City, white horse town national agricultural science and Technology Park, Nanjing Agricultural University, Applicant after: Nanjing Agricultural University Address before: Weigang Xuanwu District of Nanjing Jiangsu province 210095 No. 1 Applicant before: Nanjing Agricultural University |
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