CN103756913B - A kind of rose dark brown Isaria bacterial strain - Google Patents
A kind of rose dark brown Isaria bacterial strain Download PDFInfo
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- CN103756913B CN103756913B CN201310721109.XA CN201310721109A CN103756913B CN 103756913 B CN103756913 B CN 103756913B CN 201310721109 A CN201310721109 A CN 201310721109A CN 103756913 B CN103756913 B CN 103756913B
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Abstract
The invention discloses a kind of rose dark brown Isaria bacterial strain (<i>Isaria</i><i>Fumosorosea</i>). This bacterial strain is to separate from Nature Reserve, Jiangxi Province by the coptotermes formosanus polypide of entomogenous fungi natural infection the wild strain obtaining; wild strain tieback is obtained to rose dark brown Isaria bacterial strain of the present invention in the upper rejuvenation of coptotermes formosanus polypide, adopt monospore to separate to this bacterial strain and obtain purifying bacterial strain. This purifying bacterial strain is rose dark brown Isaria bacterial strain SCAU-IFCF02, is preserved in Chinese Typical Representative culture collection center on November 1st, 2013, does is deposit number CCTCC? NO:M? 2013526. Through long-term biological study and the indoor bioassay of infecting, show that this bacterial strain has very strong application potential in diamondback moth and coptotermes formosanus biological control.
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, account for entomopathogen kind 60% withOn. A large amount of exploitation of microbial pesticide can effectively reduce and eliminate ubiquitous residual, the toxicity of current chemical pesticide andMany negative effects such as the resistance to the action of a drug. Rose dark brown Isaria (Isariafumosorosea) is that a kind of important Insect Pathogenic is trueBacterium, is distributed widely in the torrid zone, subtropical zone and Temperate Region in China, can comprise Homoptera, Diptera, Lepidoptera, Semiptera, elytrum by parasitismThe various vegetables such as order and Hymenoptera, fruit tree and Pests of Tea-Plants. Rose dark brown Isaria is to pest efficient, environmentally friendly, difficult producingThe advantages such as the raw resistance to the action of a drug, therefore become the focus that domestic and international biological control is studied. With fungi microbe pesticide control phytopathy wormEvil is one of important means in the sustainable production of non-polluted farm product, has broad application prospects.
Rose dark brown Isaria is applied to control of insect as a kind of common insect pathogenic fungus, its grow be subject to multipleEcological factor impact, to the pathogenic course complexity of host insect, and relates to the regulation and control of several genes and product thereof, and application in practice is anti-Imitate unstablely, limited its application. Though strain improvement can improve virulence and expand its range of application, but still exist much to be solvedOr unknown problem.
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 preventive effectDefect and the technical deficiency such as unstable, provide a kind of rose dark brown Isaria bacterial strain.
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 is rose dark brown Isaria (IsariaFumosorosea (Wize) Brown&Smith) bacterial strain SCAU-IFCF02, during this bacterial strain was preserved on November 7th, 2013State's typical case's culture collection center, deposit number is CCTCCNO:M2013526, preservation address is that Wuhan, Wuhan, China city is largeLearn.
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 yellow fractionLook, center is to interior many mound shapes protuberances of edge radius 15mm circle.
Mycelia and spore shape: by microexamination, can see that conidiophore raw on vegetative hyphae. Mycelia is dividedEvery, transparent, smooth, wide 1.5～2.5 μ m. Coremium, 100 × 1.5～100 × 2.0 μ are given birth to or be gathered into conidiophore listM, wall is smooth, transparent, and the colyliform branch that mostly forms verticil by raw 4～6 bottles stalk forms. Bottle metulae portion is spherical, or intendsEllipse expands, top elongated, 5.7～8.0 × 1.0～2.0 μ m. Conidium is spherical to nearly fusiformis, smooth, is clear to micro-lightRedness, 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 of entomogenous fungi natural infection from Nature Reserve, Jiangxi Province;
S2. separate: bacterial isolate bacterium from the coptotermes formosanus worm corpse sample being infected by entomogenous fungi of adopting back. Utilize 5%Liquor natrii hypochloritis carries out surface sterilization to sample, and the sample after sterilization washs 3 times in aqua sterilisa, and puts into potato grapeIn sugar agar medium (PDA) flat board, be placed in 25 ± 1 DEG C of constant incubators and cultivate, after bacterium colony forms, transfer to PDA obliqueFace, then proceed to preservation in 4 DEG C of refrigerators;
S3. rejuvenation: the bacterial strain being separated to is cultivated 15 days on PDA flat board, added and contain 0.03% after bacterium colony to be formedSpore under the aseptic washing of Tween-80, then moves in beaker, spore suspension stirred on magnetic stirring apparatus 30 minutes,Dilution is 1 × 107The spore suspension of individual spore/mL, is evenly sprayed on coptotermes formosanus worker ant body surface, moisturizing with small manual sprayerThe coptotermes formosanus that after 90%～100%, 7d, picking infects, separates and obtains A separated strain according to foregoing method;
S4. purifying: A separated strain is cultivated 15d on PDA flat board, after rose dark brown spore to be formed, picking is made by conidium1×103The spore suspension of individual spore/mL in being placed with on the slide of cover glass, is seen hanging drop under biomicroscopeExamine, by only having a conidial slide to insert on PDA culture medium in a drop, be placed in incubator and cultivate, acquisition B,C, D, E, F be totally 5 separated strains;
S5. qualification: carry out Preliminary Identification according to the form of the cultivation shape of pathogen, mycelia, conidium and product spore device;
S6. screening: measure respectively pathogenicity, bacterial strain sporulation quantity, spore germination rate and the bacterium of 5 separated strains to target pestThe growth rate that falls, comparative result filters out active strong, best to insect preventive effect rose dark brown Isaria bacterial strain. Through United States Department of AgricultureBiological control laboratory insect pathology expert GuyMecardier in Europe is accredited as Isariafumosorosea (Wize)Brown&Smith, bacterial strain called after SCAU-IFCF02, was preserved in Chinese Typical Representative culture collection on November 7th, 2013The heart, deposit number is CCTCCNO:M2013526, preservation address is Wuhan University of Wuhan, China city.
The present invention's potato dextrose agar used (PDA) formula is: 200g potato, and 17～20g agar,20g glucose, 1000mL water, packing, 121 DEG C of sterilizing 30min of high-pressure sterilizing pot.
Entomogenous fungi has diversity aspect heredity, ecology and biological characteristics, and the different strains of fungi of the same race is to orderThe pathogenicity of mark insect exists significant difference, bacterial strain difference, its LD50、LT50Can differ even decades of times of several times. Screening andObtaining the high and virulent quality strains of high sporulation quantity, spore germination rate, is prerequisite and the necessity that obtains good prevention effectLink. Reported to the more intense disease fungus of diamondback moth virulence in, beauveria bassiana CS-1 is 1 in concentration for the treatment of ×105When spore/ml, the LT to diamondback moth 2 instar larvaes under greenhouse experiment50Be 3.61 days (Yoonetal., 1999); Green deadlockBacterium is 1 × 10 in concentration for the treatment of8When spore/ml, the LT of diamondback moth 2 instar larvaes50It is 4.97 days, to the LC of diamondback moth 2 instar larvaes50Be 2.03 × 104Spore/ml (Ma, 2000).
4 indexs that bacterial strain screening is conventional are respectively pathogenicity, bacterial strain sporulation quantity, the spore germination of bacterial strain to target pestRate and colony growth speed. The present invention is taking These parameters as foundation, and screening obtains the strain excellent SCAU-of rose dark brown IsariaIFCF02. After inoculation rose dark brown Isaria bacterial strain SCAU-IFCF02 the 2nd day, in each processing, 2 instar larvaes all started Mortality,Show that described bacterial strain has very strong pathogenicity to diamondback moth. At maximum concentration 1 × 107When individual spore/mL, diamondback moth 2,3Be respectively 100%, 98.93%, 87.23% with the cumulative correction death rate of 4 instar larvaes. In diamondback moth 2,3 and 4 instar larvae inoculationsLatter the 7th day, lethal concentration of 50 LC50 was respectively 7.63 × 103Individual spore/mL, 1.05 × 104Individual spore/mL and 2.40 × 104IndividualSpore/mL. After inoculation rose dark brown Isaria bacterial strain SCAU-IFCF02 the LC50 of diamondback moth 2,3 and 4 instar larvaes be respectively 7.63 ×103、1.05×104、2.40×104Individual spore/mL. Be 1 × 10 in concentration for the treatment of7When individual spore/mL, 2,3 and 4 instar larvaesLT50 is respectively 1.61 days, 1.66 days and 1.80 days. Than current conventional rose dark brown Isaria bacterial strain, rose of the present inventionDark brown Isaria SCAU-IFCF02 bacterial strain has significant highly pathogenicity to diamondback moth larvae, and this is lethal to diamondback moth from itMiddle concentration and the lethal times of 50 two aspect all can show. Rose dark brown Isaria SCAU-IFCF02 bacterial strain is institute so farThe insect pathogenic fungus to the minimum lethasl concentration of diamondback moth larvae tool and the fastest lethal speed of report, shows this bacterial strainIn Biological Control of Plutella xylostella, there is 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 Cruciferae vegetablesDish insect, especially the prevention effect highly significant to coptotermes formosanus and diamondback moth, be reported so far to diamondback moth childrenMinimum and the lethal fastest insect pathogenic fungus of worm lethasl concentration has stronger in coptotermes formosanus and Biological Control of Plutella xylostellaApplication 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 agricultureMedicine, has the brand-new mechanism of action that is different from existing chemical insecticide, and environmentally safe, noresidue, to the mankind and domestic animalSafety, has adapted to the requirement of organic foodstuff production; And this bacterial strain is infected from Nature Reserve, Jiangxi ProvinceThe upper separation of coptotermes formosanus polypide obtains, and is China domestic bacterial strain, not from external introduction, can adapt to local natural environment. ?In the biological control of the Cruciferous Vegetable Pests such as coptotermes formosanus and diamondback moth, there is very strong application value.
Detailed description of the invention
Further illustrate the present invention below in conjunction with specific embodiment, but that embodiment does not do the present invention is any type ofLimit. 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 conventional method preparation or commercial, rose dark brown Isaria bacterial strainCultivate and all cultivate according to this area conventional 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) collect a kind of coptotermes formosanus of being infected by entomogenous fungi in Nature Reserve, Jiangxi Province(Coptotermesformosanus) worm corpse.
(2) potato dextrose agar (PDA): 200g potato, 17～20g agar, 20g glucose,1000mL water, packing, 121 DEG C of sterilizing 30min of high-pressure sterilizing pot.
(3) aseptic technique: all vessel and apparatus must be through 121 DEG C of sterilizing 30min of high-pressure sterilizing pot, the behaviour such as inoculationDo all in superclean bench, to carry out.
(4) strain culturing condition: be placed in 25 ± 1 DEG C of illumination insulating boxs and cultivate, the photoperiod of illumination insulating box is 14L:10D, after bacterium colony forms, transfers to PDA inclined-plane, then proceeds to preservation in 4 DEG C of refrigerators.
2, the separation of pathogen and qualification
(1) separate: bacterial isolate bacterium from the coptotermes formosanus worm corpse sample being infected by entomogenous fungi of adopting back. Utilize 5%Liquor natrii hypochloritis carries out surface sterilization to sample, and the sample after sterilization washs 3 times in aqua sterilisa, and puts into PDA flat board,Be placed in 25 ± 1 DEG C of constant incubators and cultivate, after bacterium colony forms, transfer to PDA inclined-plane, then proceed to preservation in 4 DEG C of refrigerators.
(2) rejuvenation: the bacterial strain being separated to is cultivated 15 days on PDA flat board, told with containing 0.03% after bacterium colony to be formedSpore under the aseptic washing of temperature-80, obtains spore suspension, moves in beaker, and spore suspension is stirred on magnetic stirring apparatusMix 30 minutes, dilution is 1 × 107The spore suspension of individual spore/mL, is evenly sprayed on coptotermes formosanus worker ant with small manual sprayerBody surface, moisturizing 90%～100%, the coptotermes formosanus that after 7d, picking infects, separates and obtains A separated strain according to the method (1) Suo Shu.
(3) purifying: A separated strain is cultivated to 15d respectively on PDA flat board, after rose dark brown spore to be formed, the mitogenetic spore of pickingSon makes 1 × 103The spore suspension of individual spore/mL, by hanging drop in being placed with on the slide of cover glass, at biology microscopeMicroscopic observation, by only having a conidial slide to insert on PDA culture medium in a drop, is placed in incubator and cultivates,Obtain B, C, D, E, F totally 5 separated strains.
(4) qualification: carry out Preliminary Identification according to the form of the cultivation shape of pathogen, mycelia, conidium and product spore device.
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; The back sideMilk yellow, center is to interior many mound shapes protuberances of edge radius 15mm circle.
Mycelia and spore shape are described: by microexamination, can see that conidiophore raw on vegetative hyphae. BacteriumSilk is separated, transparent, smooth, wide 1.5～2.5 μ m. Coremium is given birth to or be gathered into conidiophore list, 100 × 1.5～100 ×2.0 μ m, wall is smooth, transparent, and the colyliform branch that mostly forms verticil by raw 4～6 bottles stalk forms. Bottle metulae portion is spherical,Or intend ellipse and expand, top elongated, 5.7～8.0 × 1.0～2.0 μ m. Conidium is spherical to nearly fusiformis, smooth, is clear toMicro-pale red, 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 is to orderThe pathogenicity of mark insect exists significant difference, bacterial strain difference, its LD50、LT50Can differ even decades of times of several times. Screening andObtaining the high and virulent quality strains of high sporulation quantity, spore germination rate, is prerequisite and the necessity that obtains good prevention effectLink. 4 indexs that bacterial strain screening is conventional are respectively pathogenicity, bacterial strain sporulation quantity, the spore germination rate of bacterial strain to target pestWith colony growth speed. The present invention, taking These parameters as foundation, screens the strain excellent of rose dark brown Isaria.
(1) processing of strains tested
The rose dark brown Isaria B of purified rear acquisition, C, D, E, F totally 5 separated strains, on PDA flat board in 25 ± 1 DEG CIn insulating box (periodicity of illumination is illumination: dark=14:10), cultivate.
(2) for examination worm source
Coptotermes formosanus is picked up from Campus of South China Agricultural University, and the indoor pine piece that utilizes is raised worker ant and termite, experiment picking healthWorker ant for Toxicity Determination.
(3) mensuration of colony growth speed and sporulation quantity
5 separated strains are mixed with respectively to 1 × 107The conidial suspension of individual spore/mL, gets respectively 500 μ L and suspendsDrop enters on PDA culture medium, smoothens with triangular glass rod, after 2～3d grows mycelia, drills through newly with the card punch that diameter is 5mmFresh bacterium colony, and be inoculated on PDA flat board, being then placed in incubator and cultivating, the photoperiod of illumination insulating box is 14L:10D. Often5 repetitions of bacterial strain. The increment that orientation measurement colony diameter is determined in timing in every 2 days, surveys 4 times altogether, calculates growth rate, growth rateUnit be mm/ days. Continue to be cultured to 15d, measure the sporulation quantity of each bacterial strain. Concrete operations are as follows: with going out of diameter 13mmBacterium card punch is in the 1/2 place punching sampling of bacterium colony center to edge, and sample is put into the sterilized water that 10mL contains 0.03% Tween-80In, on magnetic stirring apparatus, stir 20 minutes, spore is uniformly dispersed, make spore suspension, with the survey of blood counting chamber countingDetermine sporulation quantity. Result is as shown in table 1.
(4) mensuration of spore germination rate
5 separated strains are cultivated respectively after 15d, used the sterilized water that contains 0.03% Tween-80 collect spore and make outstandingSupernatant liquid, the concentration of suspension miospore is: in every visual field, have 100 left and right conidiums. With the test of slide sprouting method, by sporeSub-suspension directly drops on aseptic slide, is placed in bottom and is covered with in the culture dish of sterilizing filter paper, drips 4～5 in wareSterilized water to be to keep 100% relative humidity (RH), cultivates microscopy after 18h. 5 repetitions of each processing. Result is as shown in table 1.
(5) Pathogenic Tests of separated strain to coptotermes formosanus worker ant.
Prepare the spore suspension of each strains tested: each strains tested is inoculated on PDA flat board and cultivates 15d, with containingSpore under the aseptic washing of 0.03% Tween-80, uses magnetic stirrer 30min, and dual-layer sterilization gauze elimination mycelia, makesSpore suspension, and with blood counting chamber adjust spore concentration be 1 × 107Individual spore/mL.
Examination worm processing: adopt dip method to measure the pathogenicity of each separated strain to coptotermes formosanus worker ant. Choose healthy worker ant, useSoft tweezers will be chosen in the dilution of each separated strain spore suspension for trial work ant, and described dilution is to tell with containing 0.03%Temperature-80 sterilized water dilutes gained, after dipping 10s, chooses, and worker ant is placed on the filter paper of sterilizing and blots after excessive moisture,Move to bottom and be lined with in the diameter 9cm culture dish of moistening filter paper, culture dish seals with preservative film, pricks hole ventilation on film, every20 worker ants of individual processing, repeat 5 times, 100 worker ants totally. Separately with the sterilized water that contains 0.03% Tween-80 in contrast, operationMethod is the same, repeats 5 times. After inoculation, each processing is placed in artificial intelligence case, 25 ± 1 DEG C, relative humidity 90%, illuminationCycle is illumination: dark=14:10. After 12 hours, start to observe, raise the worker ant of processing with filter paper, taken food depending on filter paper in wareSituation is changed fresh filter paper. Record the susceptible symptom of worker ant and dead borer population, record altogether 7 days. The statistical correction death rate. Result asShown in table 1.
(6) screening of rose dark brown Isaria separated strain
In the time of the good separated strain of screening, pathogenic and sporulation quantity is important reference index, and spore germination rate and bacterium colony are flatAll growth rate is taken 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 bacteriumStrain, 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 in 5 points of bacterial strainsHigh, reach 2.71 × 107Individual spore/mL. It is 2.32 × 10 that bacterial strain D takes second place7Individual spore/mL. Aspect spore germination rate, eachSeparated strain spore germination rate significant difference, is wherein up to 92.11% and 90.39% with bacterial strain B and C spore germination rate. Raw surveyResult shows: the corrected mortality that bacterial strain B infects coptotermes formosanus is the highest, reaches 100%, demonstrates superpower pathogenicity.
Table 1 Biological Characteristics of Strain and the corrected mortality to coptotermes formosanus 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, above-mentioned all test datas are all finished dealing with in data processing software DPS system.
The factor of each side in comprehensive comparison sheet 1, B separated strain is optimum strain, through United States Department of Agriculture's Europe biological controlLaboratory insect pathology expert GuyMecardier is accredited as Isariafumosorosea (Wize) Brown&Smith,Bacterial strain called after SCAU-IFCF02, is preserved in Chinese Typical Representative culture collection center on November 7th, 2013, and deposit number isCCTCCNO:M2013526, preservation address is Wuhan University of Wuhan, China city.
This rose dark brown Isaria Isariafumosorosea (Wize) Brown&Smith bacterial strain SCAU-IFCF02, belongs to silkSpore order, Isaria belongs to. This bacterial strain bacterium colony front fine hair shape, just white, produces after spore and is light rose dark brown, concavo-convex fluffy bisque;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 μM. Conidiophore list is raw or be gathered into coremium, 100 × 1.5～100 × 2.0 μ m, and wall is smooth, transparent, mostly by raw 4The colyliform branch composition of～6 bottle stalk composition verticils. 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-pale red, 3.0～4.0 × 1.0～2.0 μM, without chlamydospore.
The Pathogenic Tests of embodiment 2 rose dark brown Isaria SCAU-IFCF02 bacterial strains to diamondback moth
Biologicall test is one of technology very important in entomomycete and insect pathology theory and application study, isDetecting one of entomogenous fungi important means to target pest killing extent and fatality rate, can be overall merit rose dark brown clusterThe Biocontrol Potential of spore SCAU-IFCF02 bacterial strain provides important reference frame. The present invention is with regard to rose dark brown Isaria SCAU-IFCF02Bacterial strain is measured the pathogenicity of diamondback moth, infects optium concentration and the best length of time of diamondback moth to determine this bacterial strain, withInstruct the application of this bacterial strain in control Field Pests.
1, for examination material
(1) for examination insect: diamondback moth Plutellaxylostella (L.) mature larva picks up from Suburbs of Guangzhou Agriculture In South ChinaNear vegetables cabbage mustard ground university, indoor cabbage mustard seedling subculture was raised to the 5th generation, and experiment is picking diamondback moth Plutella respectivelyXylostella (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, in the people of 25 ± 1 DEG CIn work intelligent box, cultivate 15d, the photoperiod of described artificial intelligence case is 14L:10D. With the sterilized water that contains 0.03% Tween-80Wash lower spore, on magnetic stirring apparatus, stir 30 minutes, after spore is uniformly dispersed, with the double-deck hospital gauze filtration of sterilizing, obtainSpore suspension, determine the concentration of spore suspension with blood counting chamber, then be diluted to 1 × 10 with sterilized water7、1×106、1×105、1×104、1×103Individual spore/mL5 concentration is stand-by.
(4) Pathogenic Tests of rose dark brown Isaria SCAU-IFCF02 bacterial strain to diamondback moth larvae
Adopt dip method to measure rose dark brown Isaria SCAU-IFCF02 bacterial strain causing a disease to diamondback moth 2,3 and 4 instar larvaesPower. Choose Individual Size, the consistent each instar larvae of diamondback moth of vigor, with banister bruss will for examination larva choose into above-mentioned spore dilutionIn, dipping is chosen after 10s, and larva is placed on the filter paper of sterilizing and blots after excessive moisture, then larva is moved on blade, puts into the endPortion is lined with in the diameter 9cm culture dish of moistening filter paper, and culture dish seals moisturizing with preservative film, pricks hole ventilation on film. EachProcess 20 larvas, repeat 5 times, totally 100 larvas. Separately with the sterilized water that contains 0.03% Tween-80 in contrast, operation sideMethod is the same, repeats 5 times. After inoculation, each processing is placed in to artificial intelligence case, 25 ± 1 DEG C, relative humidity 90%, periodicity of illuminationFor illumination: dark=14:10. After 24 hours, start observe, optionally change fresh cabbage leaves, record every day susceptible symptom andDead borer population, Continuous Observation 7 days, and the worm of checkmating is placed with in the culture dish of wet filter paper at the bottom of choosing ware, 25 ± 1 DEG C of moisturizings are cultivated, and seeExamine mycelial growth situation on worm corpse, to distinguish whether as the lethal worm of rose dark brown Isaria SCAU-IFCF02 bacterial strain.
The death rate (%)=dead borer population/each concentration is processed examination worm sum × 100
Corrected mortality (%)=(processing the death rate-contrast death rate)/(1-contrasts the death rate) × 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, being placed in 0.1% mercuric chloride solution soaks 1～2min, then uses aseptic water washing 3 times, and switching, on SDAY culture medium flat plate, is cultivated after 3 days, on culture medium flat plate for 25 ± 1 DEG CPolypide grow mycelia, 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, H201L, stirs and evenly mixs, then packing, and sterilizing, for subsequent use.
2, results and analysis
(1) the cumulative correction death rate comparison in diamondback moth each length of time
After variable concentrations rose dark brown Isaria SCAU-IFCF02 bacterial strain is processed, each instar larvae accumulative total school of the 7th dayThe positive death rate is as shown in table 2. Latter the 2nd day of inoculation, in each processing, each instar larvae all starts Mortality, shows rose dark brown IsariaSCAU-IFCF02 bacterial strain has very strong lethal effect to diamondback moth. In surveyed concentration range 1 × 103～1×107In, with denseIts pathogenicity of raising of degree obviously strengthens. At maximum concentration 1 × 107Individual spore/mL, the accumulative total school of diamondback moth 2,3 and 4 instar larvaesThe positive death rate is respectively 100%, 98.93%, 87.23%. The larva of susceptible death shows the lethal symptom of typical entomogenous fungi,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 it is low that contrast also hasIn 10% larva death, but cause death rate is too low, and none shows the lethal symptom of fungi.
Table 2 diamondback moth infects after rose dark brown Isaria the cumulative correction death rate of 7 days each length of time
(2) the toxicity test result of rose dark brown Isaria SCAU-IFCF02 bacterial strain to the each instar larvae of diamondback moth
Rose dark brown Isaria SCAU-IFCF02 bacterial strain is as shown in table 3 to the lethal concentration of 50 result of the each instar larvae of diamondback moth,2, the LC of the 7th day after 3 and 4 instar larvae inoculations50Be respectively 7.63 × 103Individual spore/mL, 1.05 × 104Individual spore/mL and 2.40×104Individual spore/mL.
The virulence of rose dark brown Isaria SCAU-IFCF02 bacterial strain to the each instar larvae of diamondback moth in table 37 day
Rose dark brown Isaria SCAU-IFCF02 bacterial strain is as shown in table 4 to the lethal time of 50 of the each instar larvae of diamondback moth, along with allergenicThe increase of sub-suspension concentration, for the LT of the each instar larvae of examination50Value is successively decreased, 1 × 105～1×107In the scope of individual spore/mL,The LT of 2 instar larvaes50Dropped to 1.61 days from 2.86 days, the LT of 3 instar larvaes50Dropped to 1.66 days from 3.21 days, the LT of 4 instar larvaes50FromWithin 3.4 days, drop to 1.80 days. 2 instar larvaes are 1 × 107When the high concentration of individual spore/mL, LT90It is 3.24 days. As can be seen here, 2 age childrenThe lethal required shortest time of worm, is secondly 3 instar larvaes, and 4 lethal required times of instar larvae are the longest.
The LT of table 4 rose dark brown Isaria SCAU-IFCF02 bacterial strain to the each instar larvae of diamondback moth50And LT90
This experimental result shows, rose dark brown Isaria SCAU-IFCF02 bacterial strain has very strong pathogenicity to diamondback moth larvae,From it, the lethal concentration of 50 to diamondback moth and the lethal times of 50 two aspect all can show for this. Inoculation rose dark brown Isaria bacterial strainThe LC of diamondback moth 2,3 and 4 instar larvaes after SCAU-IFCF0250Be respectively 7.63 × 103、1.05×104、2.40×104Individual spore/ML. Be 1 × 10 in concentration for the treatment of7When individual spore/mL, the LT of 2,3 and 4 instar larvaes50Be respectively 1.61 days, 1.66 days and 1.80My god. Rose dark brown Isaria SCAU-IFCF02 bacterial strain of the present invention has significant highly pathogenicity to diamondback moth larvae.
Claims (3)
1. a rose dark brown Isaria bacterial strain (Isariafumosorosea), is characterized in that, described bacterial strain is rose dark brown rodBundle spore bacterial strain SCAU-IFCF02, this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 1st, 2013, and preservation is compiledNumber be CCTCCNO:M2013526.
2. rose dark brown Isaria bacterial strain according to claim 1 (Isariafumosorosea), is characterized in that, described inRose dark brown Isaria bacterial strain SCAU-IFCF02 belong to hyphomycetales, Isaria and belong to.
3. rose dark brown Isaria bacterial strain according to claim 1 (Isariafumosorosea), is characterized in that, described inThe morphological feature of rose dark brown Isaria bacterial strain SCAU-IFCF02 be 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, center is to edgeIn the circle of radius 15mm, there are many mound shape protuberances;
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, wall is smooth, transparent, mostly byThe colyliform branch composition of raw 4～6 bottles stalk composition verticil; Bottle metulae portion is spherical, or intends ellipse and expand, top elongated, 5.7～8.0×1.0～2.0μm；
Conidium is spherical to nearly fusiformis, smooth, is clear to micro-pale red, and 3.0～4.0 × 1.0～2.0 μ m, without chlamydospore.
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| CN108865900B (en) * | 2018-07-16 | 2021-07-02 | 海南大学 | Isaria javanicus strain and application thereof |
| 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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