CN101319193B - Verticillium lecanii strain and uses thereof - Google Patents
Verticillium lecanii strain and uses thereof Download PDFInfo
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- CN101319193B CN101319193B CN2008100291637A CN200810029163A CN101319193B CN 101319193 B CN101319193 B CN 101319193B CN 2008100291637 A CN2008100291637 A CN 2008100291637A CN 200810029163 A CN200810029163 A CN 200810029163A CN 101319193 B CN101319193 B CN 101319193B
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Abstract
The invention discloses a Verticillium lecanii strain. The strain is Verticillium lecanii VL-N6, has strain preservation No. of CCTCC No: M208086, and is preserved in China Center for Type Culture Collection on June 10th, 2008. The strain belongs to Hyphomycetales and verticillium, and is obtained by separation on a whitefly polypide which is naturally infected by entomogenous fungi in Guangzhou area; a wild strain is grafted back on the whitefly polypide and rejuvenated, and then the Verticillium lecanii strain is obtained; and a purified strain is obtained by adoption of monospore isolation of the strain. The long-term infection biological study and indoor bioassay shows that the Verticillium lecanii strain has strong effect of infection and insect disinfestation on a plurality of sucking pests such as whiteflies, aphides and so on and pests such as thrips and so on.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of verticillium lecanii strain and the application aspect pest control thereof.
Background technology
In the control of insects such as Bemisia tabaci, Trialeurodes vaporariorum Westwood, aphid and thrips, adopt traditional chemical prevention and control method more.
Verticillium lecanii (Verticillium lecanii (Zimmermann) Viegas) is a kind of insect pathogenic fungus, as a kind of living body biological agricultural chemicals, feature with the brand-new mechanism of action that is different from existing chemical insecticide, environmentally safe, noresidue, can be used as some insect that Bacillus thuringiensis (Bt) is developed immunity to drugs of control, substitute traditional chemical control medicine, to greatly develop the biological pesticide Prevention Technique.
Summary of the invention
An object of the present invention is to overcome the deficiencies in the prior art, a kind of verticillium lecanii strain is provided.
Another object of the present invention provides the application of described verticillium lecanii strain.
Purpose of the present invention realizes by following technical proposals:
A kind of verticillium lecanii strain is provided, described bacterial strain is to separate acquisition from the area, Guangzhou by the Bemisia tabaci polypide of entomogenous fungi natural infection, and tieback goes up rejuvenation in the Bemisia tabaci polypide and obtains verticillium lecanii strain, adopts monospore to separate to this bacterial strain and obtains the purifying bacterial strain.This bacterial strain is VL-N6, and the bacterial strain preserving number is: CCTCC No:M208086, on June 10th, 2008 is in China's typical culture collection center preservation.
Described verticillium lecanii strain belongs to hyphomycetales, Verticillium.
Described verticillium lecanii strain bacteria colony white is to cream-colored, and is thin cotton-shaped, and the back side is colourless to deep mixed yellow.Conidiophore is undeveloped, like vegetative hyphae, Dan Sheng is arranged on it, to the bottle stalk of giving birth to or 3～4 verticillate, and the bottle stalk is very thin, and size is because of bacterial strain and cell age is different differs greatly 8.5-40 * 0.8-2.2 μ m; Conidium is oval to cylindrical, two terminal circle, and Dan Sheng is gathered into head at bottle stalk top, and size makes a variation bigger because of bacterial strain reaches, 2.3-10 * 1-2.6 μ m.
The present invention provides the application aspect described verticillium lecanii strain prevents and treats Bemisia tabaci, Trialeurodes vaporariorum Westwood, aphid or thrips in preparation the medicine simultaneously, and Verticillium lecanii of the present invention is effective to the various pests such as Bemisia tabaci, Trialeurodes vaporariorum Westwood, aphid or thrips on the control brassicaceous vegetable.
The present invention compared with prior art, have the following advantages and beneficial effect: (1) this bacterial strain is that the Bemisia tabaci polypide of being infected from the area, GuangZhou, Guangdong Province separates acquisition, being China domestic bacterial strain, is not from external introduction, can adapt to local physical environment preferably.(2) Verticillium lecanii bacterium of the present invention is a kind of insect pathogenic fungus, as a kind of living body biological agricultural chemicals, have the feature of the brand-new mechanism of action that is different from existing chemical insecticide, environmentally safe, noresidue, adapt to the requirement that Organic food is produced.(3) Verticillium lecanii of the present invention is effective to the various pests such as Bemisia tabaci, Trialeurodes vaporariorum Westwood, aphid and thrips on the control brassicaceous vegetable.
Embodiment
Further describe the present invention below in conjunction with specific embodiment.Following embodiment is a preferred implementation of the present invention, the present invention mainly sets forth the invention thought of described bacterial strain, embodiment is not related one by one in an embodiment, but therefore do not limit the present invention, other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify, should be the substitute mode of equivalence, be included in the present invention.
Embodiment 1
1.1 material source
On the geographic vegetables cucumber in Guangzhou, collect a kind of by the Bemisia tabaci Bemisia tabaci worm corpse of worm fungal infection.
Potato dextrose agar (PDA): the 200g potato boiled get juice, in the juice of being got, add 20g glucose and 17～20g agar powder then, add entry to make cumulative volume be 1000ml and boil, through autoclaving, 121 ℃ of sterilising conditions, 30min.
Aseptic technique: all vessel and apparatus must be through autoclavings, 121 ℃ of sterilising conditions, and 30min, operations such as inoculation are all carried out in the Bechtop of the conventional regulation in laboratory.
Culture condition: place 25 ℃ of illumination (14L: 10D) thermostat container is cultivated, treat that bacterium colony forms after, transfer to the PDA inclined-plane, change 4 ℃ of freezer storages again over to.
1.2 the separation of pathogenic bacteria, evaluation
(1) isolation and purification of pathogenic bacteria
(1) separates
Bacterial isolate bacterium from the tobacco powder mite corpse sample of adopting back that is infected by entomogenous fungi.Utilize 5% chlorine bleach liquor that sample is carried out surface sterilization, the sample after the sterilization washs in aqua sterilisa 3 times, and puts into the PDA flat board, is inverted in 25 ℃ of thermostat containers and cultivates, treat that bacterium colony forms after, transfer to the PDA inclined-plane, change 4 ℃ of freezer storages again over to.
(2) rejuvenation
The bacterial strain that is separated to was cultivated 15 days on the PDA flat board, waited to produce the sterilized water that adds 0.03% tween-80 behind the conidium and collect spore, spore suspension was stirred on magnetic stirring apparatus 20 minutes, will be diluted to 1 * 10 with small-sized hand-operated atomizer
7The spore suspension of spore/ml is sprayed on the cucumber leaves that has Bemisia tabaci equably, is incubated more than 90% 24 hours.The infected Bemisia tabaci of picking is separated to the A strain isolated by foregoing method after 15 days.
(3) purifying
The A strain isolated is cultivated 15d on the PDA flat board, behind the conidium to be formed, the picking spore makes 1 * 10
3The spore suspension of spore/ml, suspension is dripped on the slide glass that is placed with cover glass, under biomicroscope, observe, will have only a conidial slide to insert on the PDA substratum in the drop, be placed in the incubator and cultivate, obtain B, C, D, E, F, G totally 6 strain isolateds.
(4) evaluation of pathogenic bacteria
Form according to cultivation proterties, mycelia, conidium and the product spore device of pathogenic bacteria is carried out preliminary evaluation.
1, colonial morphology is described
6 strain isolateds that obtain are inoculated on the PDA flat board, under 25 ℃ of conditions, cultivate.Can see the mycelia that grows white in 2～3 days, bacterium colony long wool shape, white, middle part protuberance began to produce conidium after the 7th day.The bacterium colony back side is colourless or yellow.
2, mycelia and spore shape are described
White mycelium, conidiophore is undeveloped, like vegetative hyphae, Dan Sheng, to the bottle stalk of giving birth to or 3～4 are verticillate, the bottle stalk is very thin, and size is because of bacterial strain and cell age is different differs greatly 8.5-40 * 0.8-2.2 μ m.Conidium is oval to cylindrical, two terminal circle, and Dan Sheng is gathered into head at bottle stalk top, and size makes a variation bigger because of bacterial strain reaches, 2.3-10 * 1-2.6 μ m.
(2) screening of Verticillium lecanii strain isolated
Entomogenous fungi has diversity at aspects such as heredity, ecology and biology, has the parasexuality phenomenon again, and the different strains of fungi of the same race is to the virulence significant difference of target pest, bacterial strain difference, its LD
50, LT
50Can differ several times to tens times.The screening of high yield and high quality bacterial strain and acquisition are the primary prerequisites that obtains better prevention effect.4 indexs that bacterial strain screening is commonly used are respectively virulence, sporulation quantity, spore germination rate, colony growth speed.The present invention is a foundation with these indexs, the dominant strain of screening Verticillium lecanii.
(1) processing of strains tested
Verticillium lecanii B, the C that purified back obtains, D, E, F, G be totally 6 strain isolateds, at the thermostat container (14L: 10D) in cultivate of PDA flat board in 25 ± 0.5 ℃.
(2) for examination insect and host plant
Bemisia tabaci, the Bemisia tabaci adults with raising in the solarium on cucumber is inoculated on the cucumber seedling of no worm, behind the 12h of laying eggs, removes adult, and cucumber seedling is placed 25 ± 0.5 ℃ illumination box, treats that the Bemisia tabaci growth is stand-by during nymph to 2 ages.Cucumber (Cucumis sativusL.), kind are ten thousand lucky green cucumbers, and available from academy of agricultural sciences, Guangdong Province Vegetable Research Institute, test seedling all adopts 2～3 potted plant seedlings that launch leaf.
(3) mensuration of colony growth speed and sporulation quantity
6 strain isolateds are mixed with 1 * 10 respectively
7The conidium suspension of spore/ml, get the 1ml suspension respectively and splash on the PDA substratum, smoothen, treat that it is the fresh bacterium colony of punch tool of 13mm that 2～3d grows behind the mycelia with diameter with the triangle glass stick, be inoculated on the PDA flat board, place incubator to cultivate (L: D=14: 10) then.Each bacterial strain repeats for 5 times.15d measures colony diameter and collects conidium and measure sporulation quantity with the blood counting chamber numeration.
Concrete operations are as follows: measure colony diameter with ruler, and be that the sterilization punch tool of 5mm is got the uniform bacterium piece of mycelial growth in culture dish with diameter, put into then and be added with 20ml 0.03% tween-80 sterilized water, on magnetic stirring apparatus, stirred 20 minutes, spore is fully disperseed, make spore suspension, measure sporulation quantity with the blood cell counting plate numeration.
The colony growth speed and the sporulation quantity of 6 strain isolateds see Table 1, and colony growth speed B and C difference are not remarkable, significant difference between B, C and remaining strain isolated.Grow after 15 days, strain isolated C and B, sporulation quantity difference not remarkable, the sporulation quantity significant difference between B, C and other strain isolated, wherein B, C strain isolated are more excellent strain isolated.
6 strain isolated growth velocitys of table 1 Verticillium lecanii and sporulation quantity (15d)
Annotate: represent difference not remarkable (DMRT method) with alphabetical identical person behind the column of figure in the table.
(4) mensuration of spore germination rate
After 6 strain isolateds are cultivated 15 days respectively, collect spore and make suspension, test with slide glass sprouting method with sterilized water.Spore suspension is dropped on the aseptic slide glass, places the bottom to be covered with in the culture dish of filter paper, in ware, drip 3～4 sterilized waters preserve moisture (100, RH), cultivate microscopy behind the 36h.Each handles 3 repetitions.
In 6 strain isolateds, the conidia germination rate significant difference between B, E strain isolated and other strain isolated the results are shown in Table 2, and wherein the average germination rate of the spore of B strain isolated is the highest, and the spore germination rate of D strain isolated is minimum.
The conidia germination rate (36h) of 6 strain isolateds of table 2 Verticillium lecanii
Annotate: represent difference not remarkable (DMRT method) with alphabetical identical person behind the column of figure in the table.
(5) strain isolated is to the mensuration of Bemisia tabaci nymph virulence
Behind 6 strain isolateds cultivation 15d, collect spore with sterilized water, being mixed with concentration is 1 * 10
7The spore suspension of conidium/mL.To have 2 age the Bemisia tabaci nymph cucumber leaves immerse in the suspension, contrast is immersed in the sterilized water, takes out after 20 seconds, dries naturally.Whenever be treated to 3～4 leaves, 50～100 nymphs of every leaf, 3 repetitions.Take the blade of handling, insert in the colored mud that fills sterilized water, place in the transparent crisper.Place 25 ± 0.5 ℃ illumination box (L: D=14: 10) again.Every day, microscopy and record infected mortality ratio, observed 14d continuously.Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
The virulence result of study shows that the infection rate of all strain isolateds is significant difference compared with the control.6 strain isolateds began to show at the 3rd～4 day and infect symptom, can find out that from the infection rate of 7d B, C strain isolated are significantly higher than D, F, H, G strain isolated, the infection rate significant difference of B strain isolated and other strain isolated during 14d.From in general, the virulence of strain isolated B is stronger, average out to 89.23% (14d), and the infection rate of G strain isolated is lower, is 45.22% (14d), is shown in Table 3.
6 strain isolateds of table 3 Verticillium lecanii are to the virulence of Bemisia tabaci
Annotate: represent difference not remarkable (DMRT method) with alphabetical identical person behind the column of figure in the table.
(6) screening of Verticillium lecanii strain isolated
When the good strain isolated of screening, pathogenic and sporulation quantity is important reference index, and spore germination rate and colony growth speed are taken second place.In the present invention, colony growth speed and sporulation quantity strain isolated B, C and D, E, F, G significant difference.From spore germination rate and pathogenic on, strain isolated B is comparatively good, has characteristics such as pathogenic strong, sporulation quantity height, by table 1 and table 3 as seen.The comprehensive relatively factor of each side, the B strain isolated is an optimum strain, with its called after VL-N6 bacterial strain, be accredited as Verticillium lecanii (Verticilliumlecanii (Zimmermann) Viegas) through professor Liu Aiying of Guizhou University, its bacterial strain preserving number: CCTCC No:M208086, on June 10th, 2008 is in China's typical culture collection center preservation.This Verticillium lecanii (Verticillium lecanii (Zimmermann) Viegas) bacterial strain VL-N6 belongs to hyphomycetales, Verticillium.This Verticillium lecanii bacterium colony back side is colourless or yellow, and the conidiophore list is given birth to or is gathered into coremium, 100 * 1.5～2 μ m, and wall is smooth, and is transparent, and the colyliform branch of forming whorl by living 4～6 bottles stalk is formed mostly.Bottle metulae portion sphere, or intend ellipse and expand top elongated, 5.7～8 * 1～2 μ m.The conidium cylindricality is to fusiformis, and is smooth, is clear to little incarnadine, 3～4 * 1～2 μ m, no chlamydospore.
Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
Embodiment 2 Verticillium lecanii VL-N6 bacterial strains are measured the virulence of Bemisia tabaci
Biological assay is to detect entomogenous fungi to one of the deadly degree of target pest and effective means of fatality rate, can provide important reference frame for the biological control potentiality of this fungi of comprehensive evaluation.The present invention measures the virulence of Bemisia tabaci with regard to Verticillium lecanii VL-N6 bacterial strain, to filter out optimum concn used when preventing and treating.
For trying insect and host plant: Bemisia tabaci, the Bemisia tabaci adults with raising in the greenhouse on cucumber is inoculated on the cucumber seedling of no worm, lay eggs behind the 12h, remove adult, cucumber seedling is placed 25 ± 0.5 ℃ illumination box, treat that Bemisia tabaci grows to 2 ages stand-by during nymph.Cucumber (Cucumis sativus L.), kind is ten thousand lucky green cucumbers, available from academy of agricultural sciences, Guangdong Province Vegetable Research Institute, test seedling all adopts 2～3 potted plant seedlings that launch leaf.
(1) processing of strains tested
The Verticillium lecanii VL-N6 bacterial strain that purified back obtains, adopt the PDA flat board, in 25 ± 0.5 ℃ thermostat containers (14L: cultivated 15 days 10D), the sterilized water that adds 0.3% tween-80 is collected spore, and spore suspension was stirred on magnetic stirring apparatus 20 minutes, treat sporocyst break up even after, filter decon with hospital gauze, obtain spore suspension, behind the definite spore concentration of blood cell counting plate numeration, be made into 1 * 10 again
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
85 concentration gradients of spore/ml are stand-by.
(2) strain isolated is measured the virulence of Bemisia tabaci nymph
The cucumber leaves that will have Bemisia tabaci nymph in 2 age immerses in the spore suspension of 5 gradients, and contrast is immersed and contained in the sterilized water of 0.03% tween-80, takes out after 20 seconds, dries naturally.Whenever be treated to 3～4 leaves, 50～100 nymphs of every leaf, 3 repetitions.The blade of handling is positioned over (L: D=14: 10) in 25 ± 0.5 ℃ the illumination box together with cucumber seedling.Every day, microscopy and record infected mortality ratio, observed 14d continuously.Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
(3) the Verticillium lecanii strain is to the Bemisia tabaci cumulative mortality in 2 ages
Postvaccinal the 3rd～4 day, the nymph of each treatment zone began to show disease symptom, and can be observed subsequently has the mycelia of a small amount of white to grow on the polypide, and spore appears in the polypide surface after several days, then Bemisia tabaci death.Table 4 is that Verticillium lecanii handles the cumulative mortality of back nymph 7d in 2 age, 14d, and the result shows that the mortality ratio along with 2 ages of increase Bemisia tabaci of concentration also increases, and along with the increase of time, the mortality ratio in 2 ages of Bemisia tabaci also increases under the same concentration.Wherein 1 * 10
7With 1 * 10
8Treatment zone, the cumulative mortality difference of Bemisia tabaci is not remarkable.
Table 4 Verticillium lecanii is to the cumulative mortality of Bemisia tabaci nymph in 2 age
Annotate: represent difference not remarkable (DMRT method) with alphabetical identical person behind the column of figure in the table.
Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
Embodiment 3 Verticillium lecanii VL-N6 bacterial strains are measured the virulence of aphid
For trying insect and host plant: aphid, the aphid adult with raising in the solarium on Plantula Brassicae chinensis is inoculated on the Plantula Brassicae chinensis seedling of no worm, lay eggs behind the 12h, remove adult, the Plantula Brassicae chinensis seedling is placed 25 ± 0.5 ℃ illumination box, it is stand-by during nymph to treat that aphid grew to 3 ages.Plantula Brassicae chinensis (Brassica campestris L.ssp.chinensis), available from academy of agricultural sciences, Guangdong Province Vegetable Research Institute, test seedling all adopts 6～8 potted plant seedlings that launch leaf.
(1) processing of strains tested
The Verticillium lecanii VL-N6 bacterial strain that purified back obtains, on the PDA flat board in 25 ± 0.5 ℃ thermostat container (14L: cultivated 15 days 10D), the sterilized water that adds 0.03% tween-80 is collected spore, spore suspension was stirred on magnetic stirring apparatus 20 minutes, after treating that sporocyst is broken up evenly, filter decon, obtain spore suspension with hospital gauze, behind the definite spore concentration of blood cell counting plate numeration, be made into 1 * 10 again
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
85 concentration gradients of spore/ml are stand-by.
(2) strain isolated is measured the virulence of aphid
The Plantula Brassicae chinensis blade that will have aphid nymph in 3 age immerses in the spore suspension of 5 gradients, and contrast is immersed in the sterilized water, takes out after 20 seconds, dries naturally.Whenever be treated to 3～4 leaves, 10 nymphs of every leaf, 3 repetitions.The blade of handling is put into the pallet (25cm * 35cm), and be positioned over (L: D=14: 10) in 25 ± 0.5 ℃ the illumination box of sterilization.Every day, microscopy and record infected mortality ratio, and changed fresh cabbage leaf, observed continuously 10 days.Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
(3) verticillium lecanii strain is to the aphid cumulative mortality in 3 ages
Postvaccinal the 3rd～4 day, the larva of each treatment zone began to show disease symptom, was slow in action as polypide, and can be observed subsequently has the mycelia of a small amount of white to grow on the polypide, and spore appears in the polypide surface after several days, and Bemisia tabaci is dead gradually.Table 5 is handled the cumulative mortality of 7d, 14d behind the 3 age nymphs for Verticillium lecanii, and the result shows the increase along with concentration, and the mortality ratio in 3 ages of aphid also increases, and along with the increase of time, the mortality ratio in 3 ages of aphid also increases under the same concentration.Wherein 1 * 10
7With 1 * 10
8Treatment zone, the cumulative mortality difference of aphid is not remarkable.
Table 5 Verticillium lecanii is to the cumulative mortality of aphid nymph in 3 age
Annotate: represent difference not remarkable (DMRT method) with alphabetical identical person behind the column of figure in the table.
Above-mentioned all testing datas are all finished dealing with in data processing software SAS system.
Claims (2)
1. a verticillium lecanii strain (Verticillium lecanii (Zimmermann) Viegas), it is characterized in that described bacterial strain is Verticillium lecanii VL-N6, the bacterial strain preserving number: CCTCC No:M208086, on June 10th, 2008 is in China's typical culture collection center preservation.
2. the application of the described verticillium lecanii strain of claim 1 is characterized in that being applied to prepare in the medicine of preventing and treating Bemisia tabaci, Trialeurodes vaporariorum Westwood, aphid or thrips.
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| CN101513192B (en) * | 2009-03-10 | 2011-11-16 | 华南农业大学 | Compound desinsection primary agent and pesticide consisting of verticillium lecanii and abamectin |
| CN101513194B (en) * | 2009-03-10 | 2011-08-17 | 华南农业大学 | Compound desinsection primary agent and pesticide consisting of verticillium lecanii and nimbin |
| CN102796672A (en) * | 2012-08-14 | 2012-11-28 | 黑龙江省农业科学院植物保护研究所 | Verticillium lecanii solid fermentation medium, preparation method and application |
| CN103409389B (en) * | 2013-07-13 | 2015-06-03 | 福建农林大学 | Culture medium and method for producing trehalase by fermentation of Lecanicillium lecanii |
| CN104195047B (en) * | 2013-11-13 | 2015-10-21 | 谢明 | One strain Verticillium lecanii and the application in control greenhouse insect thereof |
| CN103789218B (en) * | 2014-01-27 | 2016-02-17 | 盐城工学院 | A kind of bacterial strain producing haematochrome and the method for producing haematochrome |
| CN106244470A (en) * | 2016-09-30 | 2016-12-21 | 福建省农业科学院植物保护研究所 | A kind of Gynaikothrips uzeli prevents and treats the quick prescreening method of efficient verticillium lecanii strain |
| GB201712908D0 (en) * | 2017-08-11 | 2017-09-27 | Univ Exeter | Biopesticides |
| CN109907073B (en) * | 2018-11-06 | 2024-02-23 | 宁夏大学 | Biological pesticide for preventing and treating whiteflies and application thereof |
| CN113046249B (en) * | 2021-05-17 | 2022-08-30 | 福建省农业科学院植物保护研究所 | Verticillium lecanii LL-01 and biocontrol application thereof |
| CN115251085A (en) * | 2022-07-14 | 2022-11-01 | 山东省农业科学院 | Insecticidal fungus live spore water suspending agent and preparation method thereof |
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