CN103756913A - Isaria fumosorosea - Google Patents
Isaria fumosorosea Download PDFInfo
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- CN103756913A CN103756913A CN201310721109.XA CN201310721109A CN103756913A CN 103756913 A CN103756913 A CN 103756913A CN 201310721109 A CN201310721109 A CN 201310721109A CN 103756913 A CN103756913 A CN 103756913A
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Abstract
The invention discloses an isaria fumosorosea. The isaria fumosorosea is a wild strain obtained from a coptotermes body naturally soaked in entomogenous fungi in a separating manner from the national nature conservation area of Jiulianshan in Jiangxi province. The wild strain is grafted back to the coptotermes body so as to be rejuvenated, thereby obtaining the isaria fumosorosea. A purification strain is obtained by performing monospore separation on the isaria fumosorosea. The purification strain is an isaria fumosorosea SCAU-IFCF02 which is collected in CCTCC (China Center for Type Culture Collection) on November 1st, 2013 and has the collection number of CCTCCNO:M2013526. According to a long-term infection biology study and indoor bioassay, the isaria fumosorosea has very strong application potential for the biological prevention and control of diamond black moths and coptotermes.
Description
Technical field
The invention belongs to microbial technology field.More specifically, relate to a kind of rose dark brown Isaria bacterial strain.
Background technology
Insect pathogenic fungus is the important component part of insecticidal microorganism, accounts for the more than 60% of entomopathogen kind.A large amount of exploitation of microbial pesticide can effectively reduce and eliminate many negative effects such as ubiquitous residual, the toxicity of current chemical pesticide and resistance.Rose dark brown Isaria (
isaria fumosorosea) be a kind of important insect pathogenic fungus, be distributed widely in the torrid zone, subtropics and Temperate Region in China, can comprise various vegetables, fruit tree and the Pests of Tea-Plants such as Homoptera, Diptera, lepidopteran, Hemiptera, Coleoptera and Hymenoptera by parasitism.Rose dark brown Isaria, to pest efficient, environmentally friendly, the advantage such as be difficult for developing immunity to drugs, therefore becomes the focus of domestic and international biological control research.With fungi microbe pesticide control plant pest, be one of important means in non-polluted farm product Sustainable Production, have broad application prospects.
Rose dark brown Isaria is applied to pest control as a kind of common insect pathogenic fungus, it grows and affected by multiple ecological factor, complicated to the pathogenic course of host insect, and relates to the regulation and control of several genes and product thereof, practical application preventive effect is unstable, has limited its application.Though strain improvement can improve virulence and expand its range of application, but still there are a lot of to be solved or unknown problems.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing rose dark brown Isaria bacterial strain pest control weak effect and defect and the technical deficiency such as preventive effect is unstable, and a kind of rose dark brown Isaria bacterial strain is provided.
The object of the invention is to provide above-mentioned rose dark brown Isaria bacterial strain and cultural method thereof.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The invention provides a kind of rose dark brown Isaria bacterial strain, described bacterial strain be rose dark brown Isaria (
isaria fumosorosea(Wize) Brown & Smith) bacterial strain SCAU-IFCF02, this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 1st, 2013, deposit number is CCTCC NO:M 2013526, and preservation address is Wuhan, China city Wuhan University.
Described rose dark brown Isaria bacterial strain SCAU-IFCF02 belongs to hyphomycetales, Isaria belongs to.
The morphological feature of described rose dark brown Isaria bacterial strain SCAU-IFCF02 is described below:
Colonial morphology: bacterium colony front fine hair shape, just white, is light rose dark brown, concavo-convex fluffy bisque after product spore; Back side milk yellow, center is to interior many mound shapes protuberances of edge radius 15mm circle.
Mycelia and spore shape: by microscopic examination, can see that conidiophore raw on vegetative hyphae.Mycelia is separated, transparent, smooth, wide 1.5～2.5 μ m.Conidiophore list is raw or be gathered into coremium, 100 * 1.5～100 * 2.0 μ m, and wall is smooth, transparent, and the colyliform branch that mostly forms whorl by raw 4～6 bottles stalk forms.Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0 * 1.0～2.0 μ m.Conidium is spherical to nearly fusiformis, smooth, is clear to micro-incarnadine, and 3.0～4.0 * 1.0～2.0 μ m, without chlamydospore.
The step that the present invention obtains rose dark brown Isaria bacterial strain SCAU-IFCF02 is as follows:
S1. sampling: take by the Coptotermes formosanus Shtrari． of entomogenous fungi natural infection from Nature Reserve, Jiangxi Province;
S2. separated: bacterial isolate bacterium from the Coptotermes formosanus Shtrari． worm corpse sample being infected by entomogenous fungi of adopting back.Utilize 5% chlorine bleach liquor to carry out surface sterilization to sample, sample after sterilization washs 3 times in aqua sterilisa, and put into potato dextrose agar (PDA) flat board, being placed in 25 ± 1 ℃ of constant incubators cultivates, after bacterium colony forms, transfer to PDA inclined-plane, then proceed to preservation in 4 ℃ of refrigerators;
S3. rejuvenation: the bacterial strain being separated to is cultivated 15 days on PDA flat board, added spore under the aseptic washing that contains 0.03% tween-80 after bacterium colony to be formed, then move in beaker, spore suspension is stirred on magnetic stirring apparatus 30 minutes, dilution is 1 * 10
7the spore suspension of individual spore/mL, is evenly sprayed on Coptotermes formosanus Shtrari． worker ant body surface with small manual sprayer, moisturizing 90%～100%, and the Coptotermes formosanus Shtrari． that after 7 d, picking infects, obtains A strain isolated according to foregoing method separation;
S4. purifying: A strain isolated is cultivated 15 d on PDA flat board, and after rose dark brown spore to be formed, picking conidium makes 1 * 10
3the spore suspension of individual spore/mL, in being placed with on the slide glass of cover glass, at biology microscope Microscopic observation, will in a drop, only have a conidial slide to insert on PDA substratum hanging drop, be placed in incubator and cultivate, obtain B, C, D, E, F totally 5 strain isolateds;
S5. identify: according to the form of the cultivation shape of pathogenic bacteria, mycelia, conidium and product spore device, carry out preliminary evaluation;
S6. screening: measure respectively 5 strain isolateds to the virulence of target pest, bacterial strain sporulation quantity, spore germination rate and colony growth speed, comparative result filters out active strong, best to insect preventive effect rose dark brown Isaria bacterial strain.Through USDA Europe biological control, laboratory insect pathology expert Guy Mecardier is accredited as
isaria fumosorosea(Wize) Brown & Smith, bacterial strain called after SCAU-IFCF02, on November 1st, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2013526, and preservation address is Wuhan, China city Wuhan University.
The present invention's potato dextrose agar used (PDA) formula is: 200g potato, and 17～20g agar, 20g glucose, 1000mL water, packing, high-pressure sterilizing pot sterilizing (121 ℃, 30min).
Entomogenous fungi has diversity aspect heredity, ecology and biological characteristics, and the different strains of fungi of the same race exists significant difference to the virulence of target pest, and bacterial strain is different, its LD
50, LT
50can differ even decades of times of several times.Screening and obtain high sporulation quantity, spore germination rate is high and virulent quality strains, is prerequisite and the necessary links of obtaining good prevention effect.Reported to the more intense pathogenic fungi of small cabbage moth virulence in, beauveria bassiana CS-1 is 1 * 10 in concentration for the treatment of
5during spore/ml, the LT to small cabbage moth 2 instar larvaes under greenhouse experiment
50be 3.61 days (Yoon
et al., 1999); Green muscardine fungus is 1 * 10 in concentration for the treatment of
8during spore/ml, the LT of small cabbage moth 2 instar larvaes
50be 4.97 days, the LC to small cabbage moth 2 instar larvaes
50be 2.03 * 10
4spore/ml(Ma, 2000).
4 indexs that bacterial strain screening is conventional are respectively that bacterial strain is to the virulence of target pest, bacterial strain sporulation quantity, spore germination rate and colony growth speed.The present invention be take These parameters as foundation, and screening obtains the strain excellent SCAU-IFCF02 of rose dark brown Isaria.After inoculation rose dark brown Isaria bacterial strain SCAU-IFCF02 the 2nd day, each process in 2 instar larvaes all start Mortality, show that described bacterial strain has very strong virulence to small cabbage moth.At maximum concentration 1 * 10
7during individual spore/mL, the cumulative correction mortality ratio of small cabbage moth 2,3 and 4 instar larvaes is respectively 100%, 98.93%, 87.23%.Small cabbage moth 2,3 and 4 instar larvaes, inoculate latter the 7th day, median lethal concentration(LC&-{50}) LC50 is respectively 7.63 * 10
3individual spore/mL, 1.05 * 10
4individual spore/mL and 2.40 * 10
4individual spore/mL.After inoculation rose dark brown Isaria bacterial strain SCAU-IFCF02, the LC50 of small cabbage moth 2,3 and 4 instar larvaes is respectively 7.63 * 10
3, 1.05 * 10
4, 2.40 * 10
4individual spore/mL.In concentration for the treatment of, be 1 * 10
7during individual spore/mL, the LT50 of 2,3 and 4 instar larvaes is respectively 1.61 days, 1.66 days and 1.80 days.Than current conventional rose dark brown Isaria bacterial strain, rose dark brown Isaria SCAU-IFCF02 bacterial strain of the present invention has significant highly pathogenicity to diamondback moth larvae, and this all can show the median lethal concentration(LC&-{50}) of small cabbage moth and median lethal time two aspect from it.Rose dark brown Isaria SCAU-IFCF02 bacterial strain is the insect pathogenic fungus to the minimum lethal concentration of diamondback moth larvae tool and the fastest lethal speed reported so far, shows this bacterial strain and in Biological Control of Plutella xylostella, has stronger application potential.
The present invention has following beneficial effect:
The invention provides a kind of rose dark brown Isaria bacterial strain SCAU-IFCF02, this bacterial strain can be prevented and treated Cruciferous Vegetable Pests, especially the prevention effect highly significant to Coptotermes formosanus Shtrari． and small cabbage moth, be the minimum and lethal fastest insect pathogenic fungus to diamondback moth larvae lethal concentration reported so far, in Coptotermes formosanus Shtrari． and Biological Control of Plutella xylostella, there is stronger application potential.
In addition, rose dark brown Isaria bacterial strain SCAU-IFCF02 is a kind of insect pathogenic fungus, as a kind of living body biological agricultural chemicals, there is the brand-new mechanism of action that is different from existing chemical insecticide, environmentally safe, noresidue, to the mankind and safety of livestock, adapted to the requirement of organic foodstuff production; And upper separated acquisition of the Coptotermes formosanus Shtrari． polypide that this bacterial strain Shi Cong Jiangxi Province is infected Nature Reserve, is China domestic bacterial strain, not from external introduction, can adapt to local physical environment.In the biological control of the Cruciferous Vegetable Pests such as Coptotermes formosanus Shtrari． and small cabbage moth, there is very strong application value.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention, but embodiment does not limit in any form to the present invention.Unless stated otherwise, reagent, the method and apparatus that the present invention adopts is the conventional reagent of the art, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention is ordinary method preparation or commercial, and the cultivation of rose dark brown Isaria bacterial strain is all cultivated according to this area ordinary method.
The used sterilized water that contains 0.03% tween-80 in embodiment, described 0.03% refers to volume ratio.
the acquisition of embodiment 1 rose dark brown Isaria bacterial strain SCAU-IFCF02
1, experiment material and condition
(1) in Nature Reserve, Jiangxi Province, collect a kind of Coptotermes formosanus Shtrari． of being infected by entomogenous fungi (
coptotermes formosanus) worm corpse.
(2) potato dextrose agar (PDA): 200g potato, 17～20g agar, 20g glucose, 1000mL water, packing, high-pressure sterilizing pot sterilizing (121 ℃, 30min).
(3) aseptic technique: all vessel and apparatus must (121 ℃, 30min), the operations such as inoculation be all carried out in Bechtop through high-pressure sterilizing pot sterilizing.
(4) strain culturing condition: be placed in 25 ± 1 ℃ of illumination thermostat containers (14L:10D) and cultivate, after bacterium colony forms, transfer to PDA inclined-plane, then proceed to preservation in 4 ℃ of refrigerators.
2, the separation of pathogenic bacteria and evaluation
(1) separation: bacterial isolate bacterium from the Coptotermes formosanus Shtrari． worm corpse sample being infected by entomogenous fungi of adopting back.Utilize 5% chlorine bleach liquor to carry out surface sterilization to sample, the sample after sterilization washs 3 times in aqua sterilisa, and puts into PDA flat board, being placed in 25 ± 1 ℃ of constant incubators cultivates, after bacterium colony forms, transfer to PDA inclined-plane, then proceed to preservation in 4 ℃ of refrigerators.
(2) rejuvenation: the bacterial strain being separated to is cultivated 15 days on PDA flat board, after bacterium colony to be formed, with spore under the aseptic washing that contains 0.03% tween-80, obtain spore suspension, move in beaker, spore suspension is stirred on magnetic stirring apparatus 30 minutes, dilution is 1 * 10
7the spore suspension of individual spore/mL, is evenly sprayed on Coptotermes formosanus Shtrari． worker ant body surface with small manual sprayer, moisturizing 90%～100%, and the Coptotermes formosanus Shtrari． that after 7d, picking infects, obtains A strain isolated according to the method separation (1) Suo Shu.
(3) purifying: A strain isolated is cultivated to 15d respectively on PDA flat board, and after rose dark brown spore to be formed, picking conidium makes 1 * 10
3the spore suspension of individual spore/mL, in being placed with on the slide glass of cover glass, at biology microscope Microscopic observation, will in a drop, only have a conidial slide to insert on PDA substratum hanging drop, be placed in incubator and cultivate, obtain B, C, D, E, F totally 5 strain isolateds.
(4) identify: according to the form of the cultivation shape of pathogenic bacteria, mycelia, conidium and product spore device, carry out preliminary evaluation.
Colonial morphology is described: bacterium colony front fine hair shape, and just white, is light rose dark brown, concavo-convex fluffy bisque after product spore; Back side milk yellow, center is to interior many mound shapes protuberances of edge radius 15mm circle.
Mycelia and spore shape are described: by microscopic examination, can see that conidiophore raw on vegetative hyphae.Mycelia is separated, transparent, smooth, wide 1.5～2.5 μ m.Conidiophore list is raw or be gathered into coremium, 100 * 1.5～100 * 2.0 μ m, and wall is smooth, transparent, and the colyliform branch that mostly forms whorl by raw 4～6 bottles stalk forms.Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0 * 1.0～2.0 μ m.Conidium is spherical to nearly fusiformis, smooth, is clear to micro-incarnadine, and 3.0～4.0 * 1.0～2.0 μ m, without chlamydospore.
3, the screening of rose dark brown Isaria bacterial strain SCAU-IFCF02
Entomogenous fungi has diversity aspect heredity, ecology and biological characteristics, and the different strains of fungi of the same race exists significant difference to the virulence of target pest, and bacterial strain is different, its LD
50, LT
50can differ even decades of times of several times.Screening and obtain high sporulation quantity, spore germination rate is high and virulent quality strains, is prerequisite and the necessary links of obtaining good prevention effect.4 indexs that bacterial strain screening is conventional are respectively that bacterial strain is to the virulence of target pest, bacterial strain sporulation quantity, spore germination rate and colony growth speed.The present invention be take These parameters as foundation, the strain excellent of screening rose dark brown Isaria.
(1) processing of strains tested
The rose dark brown Isaria B of purified rear acquisition, C, D, E, F be totally 5 strain isolateds, and on PDA flat board, in the thermostat container of 25 ± 1 ℃, (periodicity of illumination is illumination: dark=14:10), cultivate.
(2) for examination worm source
Coptotermes formosanus Shtrari． is picked up from Campus of South China Agricultural University, and the indoor pine piece that utilizes is raised worker ant and termite, and the worker ant of experiment picking health is for Toxicity Determination.
(3) mensuration of colony growth speed and sporulation quantity
5 strain isolateds are mixed with respectively to 1 * 10
7the conidial suspension of individual spore/mL, get respectively 500 μ L suspension and splash on PDA substratum, with triangular glass rod, smoothen, after 2～3d grows mycelia, with the punch tool that diameter is 5mm, drill through fresh bacterium colony, and be inoculated on PDA flat board, be then placed in incubator and cultivate (14L:10D).5 repetitions of every bacterial strain.The increment of regularly determining directional survey colony diameter in every 2 days, surveys 4 times altogether, calculates growth velocity (mm/ days).Continue to be cultured to 15d, measure the sporulation quantity of each bacterial strain.Concrete operations are as follows: with sterilizing punch tool 1/2 place punching sampling at bacterium colony center to edge of diameter 13mm, sample is put into the sterilized water that 10mL contains 0.03% tween-80, on magnetic stirring apparatus, stir 20 minutes, spore is uniformly dispersed, make spore suspension, with blood counting chamber counting, measure sporulation quantity.Result is as shown in table 1.
(4) mensuration of spore germination rate
5 strain isolateds are cultivated respectively after 15d, with the sterilized water that contains 0.03% tween-80, collect spore and make suspension (100 left and right conidiums in every visual field), by slide glass sprouting method, test, spore suspension is directly dropped on aseptic slide glass, being placed in bottom is covered with in the culture dish of sterilizing filter paper, in ware, drip 4～5 sterilized waters to keep 100% relative humidity (RH), microscopy after cultivation 18h.Each processes 5 repetitions.Result is as shown in table 1.
(5) Pathogenic Tests of strain isolated to Coptotermes formosanus Shtrari． worker ant.
The spore suspension of preparing each strains tested: each strains tested is inoculated on PDA flat board and cultivates 15d, with spore under the aseptic washing that contains 0.03% tween-80, use magnetic stirrer 30min, dual-layer sterilization gauze elimination mycelia, make spore suspension, and be 1 * 10 with blood counting chamber adjustment spore concentration
7individual spore/mL.
Worm is processed in examination: adopt dip method to measure the virulence of each strain isolated to Coptotermes formosanus Shtrari． worker ant.Choose healthy worker ant, with soft tweezers, will for trial work ant, choose in the diluent (sterilized water that contains 0.03% tween-80 dilutes) of each strain isolated spore suspension, after dipping 10s, choose, worker ant is placed on the filter paper of sterilizing and blots after excessive moisture, move to bottom and be lined with in the culture dish (diameter is 9cm) of moistening filter paper, culture dish seals with preservative film, on film, prick hole ventilation, each processes 20 worker ants, repeats 5 times, totally 100 worker ants.Separately with the sterilized water that contains 0.03% tween-80 in contrast, working method is the same, repeats 5 times.After inoculation, each processing is placed in artificial intelligence case, 25 ± 1 ℃, relative humidity 90%, periodicity of illumination is illumination: dark=14:10.After 12 hours, start to observe, with filter paper, raise the worker ant of processing, depending on filter paper in ware, by the situation of taking food, changed fresh filter paper.Record the susceptible symptom of worker ant and dead borer population, record altogether 7 days.Statistical correction mortality ratio.Result is as shown in table 1.
(6) screening of rose dark brown Isaria strain isolated
When the good strain isolated of screening, pathogenic and sporulation quantity is important reference index, and spore germination rate and bacterium colony average growth rate take second place.In the present invention, as can be seen from Table 1, the bacterium colony average growth rate of bacterial strain B is significantly higher than all the other bacterial strains, and the colony growth speed of B bacterial strain is the fastest, is 15.79mm/ days, and the sporulation quantity of bacterial strain B is also the highest in 5 minutes bacterial strains, reaches 2.71 * 10
7individual spore/mL.It is 2.32 * 10 that bacterial strain D takes second place
7individual spore/mL.Aspect spore germination rate, each strain isolated spore germination rate significant difference, is wherein up to 92.11% and 90.39% with bacterial strain B and C spore germination rate.The raw result of surveying shows: the corrected mortality that bacterial strain B infects Coptotermes formosanus Shtrari． is the highest, reaches 100%, demonstrates superpower virulence.
Table 1 Biological Characteristics of Strain and the corrected mortality to Coptotermes formosanus Shtrari． worker ant thereof
In table, data are Mean ± S.E., and after same column data, different lowercase alphabets show significant difference (Duncan method, P < 0.05), and above-mentioned all testing datas are all finished dealing with in data processing software DPS system.
The factor of each side in comprehensive comparison sheet 1, B strain isolated is optimum strain, through USDA Europe biological control, laboratory insect pathology expert Guy Mecardier is accredited as
isaria fumosorosea(Wize) Brown & Smith, bacterial strain called after SCAU-IFCF02, on November 1st, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2013526, and preservation address is Wuhan, China city Wuhan University.
This rose dark brown Isaria
isaria fumosorosea(Wize) Brown & Smith bacterial strain SCAU-IFCF02, belongs to hyphomycetales, and Isaria belongs to.This bacterial strain bacterium colony front fine hair shape, first white, is light rose dark brown, concavo-convex fluffy bisque after producing spore; Back side milk yellow, center is to interior many mound shapes protuberances of edge radius 15mm circle.Mycelia is separated, transparent, smooth, wide 1.5～2.5 μ m.Conidiophore list is raw or be gathered into coremium, 100 * 1.5～100 * 2.0 μ m, and wall is smooth, transparent, and the colyliform branch that mostly forms whorl by raw 4～6 bottles stalk forms.Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0 * 1.0～2.0 μ m.Conidium is spherical to nearly fusiformis, smooth, is clear to micro-incarnadine, and 3.0～4.0 * 1.0～2.0 μ m, without chlamydospore.
the Pathogenic Tests of embodiment 2 rose dark brown Isaria SCAU-IFCF02 bacterial strains to small cabbage moth
Biological assay is one of technology very important in entomomycete and insect pathology theory and applied research, be to detect entomogenous fungi to one of important means of target pest killing extent and fatality rate, can provide important reference frame for the Biocontrol Potential of comprehensive evaluation rose dark brown Isaria SCAU-IFCF02 bacterial strain.The present invention measures the virulence of small cabbage moth with regard to rose dark brown Isaria SCAU-IFCF02 bacterial strain, infects optimum concn and the best length of time of small cabbage moth to determine this bacterial strain, to instruct the application of this bacterial strain in control Field Pests.
1, for examination material
(1) for examination insect: small cabbage moth
plutella xylostella(L.) mature larva picks up near vegetables cabbage mustard ground Suburbs of Guangzhou Agricultural University Of South China, and indoor cabbage mustard seedling subculture was raised to the 5th generation, and experiment is picking small cabbage moth respectively
plutella xylostella(L.) 2,3 and 4 instar larvaes are for Bioactivity.
(2) for studying thing: cabbage mustard (
b. alboglabrabailey)
(3) processing of strains tested
The rose dark brown Isaria SCAU-IFCF02 bacterial strain of purified rear acquisition, coats PDA flat board, cultivates 15d in the artificial intelligence case (14L:10D) of 25 ± 1 ℃.With spore under the aseptic washing that contains 0.03% tween-80, on magnetic stirring apparatus, stir 30 minutes, after spore is uniformly dispersed, with the double-deck hospital gauze of sterilizing, filter, obtain spore suspension, with blood counting chamber, determine the concentration of spore suspension, then be diluted to 1 * 10 with sterilized water
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
35 concentration of individual spore/mL are stand-by.
(4) Pathogenic Tests of rose dark brown Isaria SCAU-IFCF02 bacterial strain to diamondback moth larvae
Adopt dip method to measure the virulence of rose dark brown Isaria SCAU-IFCF02 bacterial strain to small cabbage moth 2,3 and 4 instar larvaes.Choose Individual Size, consistent each instar larvae of small cabbage moth of vigor, with banister bruss, will choose in above-mentioned spore diluent for examination larva, after dipping 10s, choose, larva is placed on the filter paper of sterilizing and blots after excessive moisture, again larva is moved on blade, put into the culture dish (diameter is 9cm) that bottom is lined with moistening filter paper, culture dish seals moisturizing with preservative film, pricks hole ventilation on film.Each processes 20 larvas, repeats 5 times, totally 100 larvas.Separately with the sterilized water that contains 0.03% tween-80 in contrast, working method is the same, repeats 5 times.After inoculation, each processing is placed in to artificial intelligence case, 25 ± 1 ℃, relative humidity 90%, periodicity of illumination is illumination: dark=14:10.After 24 hours, start to observe, optionally change fresh cabbage leaves, record susceptible symptom and dead borer population every day, Continuous Observation 7 days, and the worm of checkmating is chosen in the culture dish that is placed with wet filter paper at the bottom of ware, 25 ± 1 ℃ of moisturizings are cultivated, and observe mycelial growth situation on worm corpse, take whether distinguish be the lethal worm of rose dark brown Isaria SCAU-IFCF02 bacterial strain.
Mortality ratio (%)=dead borer population/each concentration is processed examination worm sum * 100
Corrected mortality (%)=(processing mortality ratio-contrast mortality ratio)/(1-contrasts mortality ratio) * 100
The analysis of all data all adopts DPS software to complete.
Picking dead and the with it long larva that has rose dark brown mycelia, be placed in 0.1% mercuric chloride solution and soak 1～2min, then use aseptic water washing 3 times, switching is on SDAY culture medium flat plate, cultivate after 3 days for 25 ± 1 ℃, polypide on culture medium flat plate grows mycelia, and last film-making is observed, and identifies whether the mycelia that picking obtains is rose dark brown Isaria.
Described SDAY solid medium compound method is as follows: glucose 40g/L, peptone 10g/L, yeast extract 20g/L, agar 15～20g/L, H
20 1L, stirring and evenly mixing, then packing, sterilizing, standby.
2, results and analysis
(1) the cumulative correction mortality ratio comparison in small cabbage moth each length of time
After different concns rose dark brown Isaria SCAU-IFCF02 bacterial strain is processed, each instar larvae cumulative correction mortality ratio of the 7th day is as shown in table 2.Latter the 2nd day of inoculation, in each processing, each instar larvae all starts Mortality, shows that rose dark brown Isaria SCAU-IFCF02 bacterial strain has very strong lethal effect to small cabbage moth.In surveyed concentration range 1 * 10
3～1 * 10
7in, with its virulence of raising of concentration, obviously strengthen.At maximum concentration 1 * 10
7individual spore/mL, the cumulative correction mortality ratio of small cabbage moth 2,3 and 4 instar larvaes is respectively 100%, 98.93%, 87.23%.The larva of susceptible death shows the lethal symptom of typical entomogenous fungi, and the moisturizing of worm corpse starts to grow mycelia from body surface after cultivating on the 2nd day, produces subsequently the spore of a large amount of rose dark brown.Although contrast also has the larva of a small amount of (being usually less than 10%) dead, none shows the lethal symptom of fungi.
Table 2 small cabbage moth infects the cumulative correction mortality ratio (7 days) after rose dark brown Isaria each length of time
(2) the toxicity test result of rose dark brown Isaria SCAU-IFCF02 bacterial strain to each instar larvae of small cabbage moth
Rose dark brown Isaria SCAU-IFCF02 bacterial strain is as shown in table 3 to the median lethal concentration(LC&-{50}) result of each instar larvae of small cabbage moth, the LC of the 7th day after 2,3 and 4 instar larvae inoculations
50be respectively 7.63 * 10
3individual spore/mL, 1.05 * 10
4individual spore/mL and 2.40 * 10
4individual spore/mL.
Table 3 rose dark brown Isaria SCAU-IFCF02 bacterial strain is to the virulence of each instar larvae of small cabbage moth (7 days)
The LT of table 4 rose dark brown Isaria SCAU-IFCF02 bacterial strain to each instar larvae of small cabbage moth
50and LT
90
This experimental result shows, rose dark brown Isaria SCAU-IFCF02 bacterial strain has very strong virulence to diamondback moth larvae, and this all can show the median lethal concentration(LC&-{50}) of small cabbage moth and median lethal time two aspect from it.The LC of small cabbage moth 2,3 and 4 instar larvaes after inoculation rose dark brown Isaria bacterial strain SCAU-IFCF02
50be respectively 7.63 * 10
3, 1.05 * 10
4, 2.40 * 10
4individual spore/mL.In concentration for the treatment of, be 1 * 10
7during individual spore/mL, the LT of 2,3 and 4 instar larvaes
50be respectively 1.61 days, 1.66 days and 1.80 days.Rose dark brown Isaria SCAU-IFCF02 bacterial strain of the present invention has significant highly pathogenicity to diamondback moth larvae.
Claims (5)
- A rose dark brown Isaria bacterial strain ( isaria fumosorosea), it is characterized in that, described bacterial strain is rose dark brown Isaria bacterial strain SCAU-IFCF02, and this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 1st, 2013, and deposit number is CCTCC NO:M 2013526.
- Rose dark brown Isaria bacterial strain according to claim 1 ( isaria fumosorosea), it is characterized in that, described rose dark brown Isaria bacterial strain SCAU-IFCF02 belongs to hyphomycetales, Isaria belongs to.
- Rose dark brown Isaria bacterial strain according to claim 1 ( isaria fumosorosea), it is characterized in that, the morphological feature of described rose dark brown Isaria bacterial strain SCAU-IFCF02 is described below:Bacterium colony front fine hair shape, just white, is light rose dark brown, concavo-convex fluffy bisque after product spore; Back side milk yellow, there are many mound shape protuberances at center to the circle of edge radius 15mm;Mycelia is separated, transparent, smooth, wide 1.5～2.5 μ m;Conidiophore list is raw or be gathered into coremium, 100 * 1.5～100 * 2.0 μ m, and wall is smooth, transparent, and the colyliform branch that mostly forms whorl by raw 4～6 bottles stalk forms; Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0 * 1.0～2.0 μ m;Conidium is spherical to nearly fusiformis, smooth, is clear to micro-incarnadine, and 3.0～4.0 * 1.0～2.0 μ m, without chlamydospore.
- 4. the separation method of the rose dark brown Isaria bacterial strain described in claim 1,2 or 3, is characterized in that, comprises the following steps:S1. sampling: take by the Coptotermes formosanus Shtrari． of entomogenous fungi natural infection from Nature Reserve, Jiangxi Province;S2. separated: bacterial isolate bacterium from the Coptotermes formosanus Shtrari． worm corpse sample being infected by entomogenous fungi of adopting back, utilize 5% chlorine bleach liquor to carry out surface sterilization to sample, sample after sterilization washs in aqua sterilisa, and put into potato dextrose agar flat board, being placed in 25 ℃ of constant incubators cultivates, after bacterium colony forms, transfer to potato dextrose agar panel bevel, stored refrigerated;S3. rejuvenation: the bacterial strain being separated to is cultivated on potato dextrose agar flat board, after bacterium colony to be formed, add spore under the aseptic washing that contains 0.03% tween-80 to obtain spore suspension, spore suspension is stirred on magnetic stirring apparatus after 30 minutes, dilution is 1 * 10 7the spore suspension of individual spore/mL, is evenly sprayed on Coptotermes formosanus Shtrari． worker ant body surface, moisturizing 90%～100%, and the Coptotermes formosanus Shtrari． that after 7 d, picking infects, obtains A strain isolated according to the method separation described in S2;S4. purifying: A strain isolated is cultivated on potato dextrose agar flat board, and after rose dark brown spore to be formed, picking conidium makes 1 * 10 3the spore suspension of individual spore/mL, by hanging drop in being placed with on the slide glass of cover glass, at biology microscope Microscopic observation, to in a drop, only have a conidial slide to insert on potato dextrose agar, be placed in incubator and cultivate, obtain B, C, D, E, F totally 5 strain isolateds;S5. identify: according to the form of the cultivation shape of pathogenic bacteria, mycelia, conidium and product spore device, carry out preliminary evaluation;S6. screening: measure respectively 5 strain isolateds to the virulence of target pest, bacterial strain sporulation quantity, spore germination rate and colony growth speed, comparative result filters out active strong, best to insect preventive effect rose dark brown Isaria bacterial strain, i.e. rose dark brown Isaria bacterial strain SCAU-IFCF02.
- 5. separation method according to claim 4, is characterized in that, the compound method of described potato dextrose agar is: the potato of 0.2g/mL, the agar of 0.017～0.02g/mL, the glucose of 0.02g/mL, adds water constant volume, packing, 121 ℃ of sterilizings.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108865896A (en) * | 2018-06-15 | 2018-11-23 | 广东省农业科学院果树研究所 | A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation |
| CN108865900A (en) * | 2018-07-16 | 2018-11-23 | 海南大学 | A kind of Java Isaria bacterial strain and its application |
| KR20200074288A (en) * | 2018-12-11 | 2020-06-25 | 대한민국(농촌진흥청장) | Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media |
| CN112301087A (en) * | 2020-11-05 | 2021-02-02 | 江苏省农业科学院 | Method for scientifically evaluating quality of fungal insecticide product by combining fungal spore germination and biological activity measurement |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182466A (en) * | 2007-11-20 | 2008-05-21 | 华南农业大学 | Paecilomyces Fumosoroseus strain and uses thereof |
| CN103451113A (en) * | 2013-09-02 | 2013-12-18 | 山西农业大学 | Separation and cultivation method for isaria fumosorosea |
-
2013
- 2013-12-24 CN CN201310721109.XA patent/CN103756913B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182466A (en) * | 2007-11-20 | 2008-05-21 | 华南农业大学 | Paecilomyces Fumosoroseus strain and uses thereof |
| CN103451113A (en) * | 2013-09-02 | 2013-12-18 | 山西农业大学 | Separation and cultivation method for isaria fumosorosea |
Non-Patent Citations (5)
| Title |
|---|
| YAYAN SANJAYA ETAL: "Selection of entomopathogenic fungi against the red spider mite Tetranychus kanzawai (Kishida) (Tetranychidae: Acarina)", 《ARTHROPODS》, vol. 2, no. 4, 30 April 2013 (2013-04-30), pages 208 - 215 * |
| 吕利华等: "玫烟色拟青霉对小菜蛾致病力的时间-剂量-死亡率模型模拟", 《昆虫学报》, vol. 50, no. 6, 30 June 2007 (2007-06-30), pages 564 * |
| 张伟等: "环境因子及常用农药对玫烟色棒束孢SCAU-IFCF01孢子萌发的影响", 《中国生物防治学报》, vol. 29, no. 1, 28 February 2013 (2013-02-28), pages 50 - 2 * |
| 王宏民等: "玫烟色拟青霉对小菜蛾幼虫的侵染过程及接菌方法对其致病力的影响", 《中国生态农业学》, vol. 17, no. 4, 31 July 2009 (2009-07-31), pages 704 - 708 * |
| 雷妍圆等: "玫烟色棒束孢的生物学特性与其对小菜蛾致病力的相关性", 《植物保护学报》, vol. 37, no. 3, 30 June 2010 (2010-06-30), pages 217 - 221 * |
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| CN108865896A (en) * | 2018-06-15 | 2018-11-23 | 广东省农业科学院果树研究所 | A kind of method of Fusarium oxysporum Cuba specialized form bacterial strain rejuvenation |
| CN108865900A (en) * | 2018-07-16 | 2018-11-23 | 海南大学 | A kind of Java Isaria bacterial strain and its application |
| CN108865900B (en) * | 2018-07-16 | 2021-07-02 | 海南大学 | Isaria javanicus strain and application thereof |
| KR20200074288A (en) * | 2018-12-11 | 2020-06-25 | 대한민국(농촌진흥청장) | Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media |
| KR102145620B1 (en) | 2018-12-11 | 2020-08-20 | 대한민국 | Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media |
| CN112301087A (en) * | 2020-11-05 | 2021-02-02 | 江苏省农业科学院 | Method for scientifically evaluating quality of fungal insecticide product by combining fungal spore germination and biological activity measurement |
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