CN103387942B - Metarrhizum anisopliae for preventing and treating palm insect rhynchophorus ferrugineus and application thereof - Google Patents
Metarrhizum anisopliae for preventing and treating palm insect rhynchophorus ferrugineus and application thereof Download PDFInfo
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- CN103387942B CN103387942B CN201310364795.XA CN201310364795A CN103387942B CN 103387942 B CN103387942 B CN 103387942B CN 201310364795 A CN201310364795 A CN 201310364795A CN 103387942 B CN103387942 B CN 103387942B
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Abstract
The invention relates to metarrhizum anisopliae for preventing and controlling palm insect rhynchophorus ferrugineus and application thereof. The strain is named metarrhizum anisopliae WENCHANG-LV2 is preserved in the China General Microbiological Culture Collection Center on June 9th, 2013 with the preservation number of CGMCC NO.7692. The metarrhizum anisopliae WENCHANG-LV2 provided by the invention can be applied to rhynchophorus ferrugineus prevention and control. Direct application testing indicates that the metarrhizum anisopliae WENCHANG-LV2 has a good insecticide effect, can be used for effectively controlling the number of species of rhynchophorus ferrugineus, is a biocontrol strain which has a potential meaning in preventing and treating rhynchophorus ferrugineus, and favors the development of palm industry in China.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of Metarhizium Strains and application thereof, specifically, relate to green muscardine fungus and the application of a strain control palm insect red plam weevil.
Background technology
Red plam weevil (Rhynchophorus ferrugineus Oliv.) belongs to Coleoptera (Coleoptera) Culculionidae (Curculionidae) palm and resembles genus Rhynchophorus sp., English by name Red Palm Weevil (being called for short RPW), claims again that Asia palm weevil, rust palm resemble, coconut beetle, the hidden beak of coconut resemble, India's red plam weevil etc.Red plam weevil is mainly distributed in the ground such as Taiwan, Fujian, Hong Kong, Hainan, Guangdong, Guangxi, Shanghai, Yunnan, Tibet (Motuo) at home.Red plam weevil is carried out to the demonstration of pest risk analysis result, and it belongs to excessive risk harmful organism, in a lot of areas of China, easily surely grows, very large to palm crops threats such as the oil palm of China, betel nut, coconuts, in China, has serious economic hazardness.
Known Metarhizium fungi (Metarhizum anisopliae var.anisopliae) can extensively parasitize about 8 orders, 30Ge section at present, altogether in 200 various insects, nematode and mite class body, green muscardine fungus is in biological control, to use the insect pathogenic fungus that is only second to muscardine, and with Metarhizium anisopliae, be most widely used in Metarhizium, in various habitats, the whole world, use widely, in forest, agricultural crops and public health insect, all with Metarhizium anisopliae, carried out the assessment of effect.The people such as green muscardine fungus fungi biological prevention and control agent is used at first some insects such as prevention and control locust, leafhopper, Scholte E J are in the also discovery of some researchs in Africa, and Metarhizium anisopliae has fairly obvious control effect to the main propagation insect mosquito of malaria.Green muscardine fungus is as most important a kind of in biocontrol fungi, and it has in the use, and parasitic species are many, strong adaptability, the cycle of causing a disease is short, specificity strong, to the mankind and livestock, can not cause murder by poisoning, and environmentally friendly feature.Through retrieval, utilize at present fungi to control red plam weevil and there is not yet relevant report.
Summary of the invention
The object of this invention is to provide green muscardine fungus and the application of a strain control palm insect red plam weevil.
One strain Metarhizium Strains, called after Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2, China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on June 9th, 2013, have been preserved in: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Deposit number: CGMCC NO.7692.
Metarhizium anisopliae provided by the present invention (Metarhizum anisopliae) WENCHANG-LV2 bacterial strain, be located away from the red plam weevil bombys batryticatus of natural infection, through morphology, cultivation proterties, conventional Physiology and biochemistry and DNA sequence dna, identify, this bacterial strain is Metarhizium anisopliae var. Anisopliae, a new bacterial strain for Metarhizium fungi, called after Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2.
The Basic Biological Character of Metarhizium anisopliae provided by the present invention (Metarhizum anisopliae) WENCHANG-LV2 is:
Bacterial strain streak culture rear mycelium on dull and stereotyped SDAY substratum is transparence and has barrier film; On SDAY substratum, the initial stage is white or faint yellow excessive to yellow, is shaped as cotton-shaped or fine hair shape projection, and the later stage occurs that green is the color of the adjacent chromatogram of basic tone; Conidiophore is bottleneck shape, has bifurcated, and single raw or many raw growths that intersect, are wound in fence shape mutually.Each size of conidium is 4.8～9.3 μ m * 2.2～3.1 μ m, oblong, and bar-shaped, the spore multiple aggregation that two terminal circle slippage falls becomes lumps.
The purposes of Metarhizium anisopliae provided by the present invention (Metarhizum anisopliae) WENCHANG-LV2 is its application in control red plam weevil.
The biotechnological formulation that Metarhizium anisopliae provided by the present invention (Metarhizum anisopliae) WENCHANG-LV2 of take is activeconstituents also belongs to protection scope of the present invention.When needing, said preparation can comprise common carrier and auxiliary material prepared by biotechnological formulation.
The present invention is by directly smearing the insecticidal activity of method research Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 to red plam weevil of administration, and experimental result shows that Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 has good insecticidal action to red plam weevil.Show that this bacterial strain is that a strain has the biocontrol strain of potential significance on control red plam weevil.
The present invention is by the screening and separating to microorganism in the red plam weevil bombys batryticatus of natural infection, obtain Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2, through insecticidal test, show that this bacterial strain has good insecticidal action to red plam weevil, can effectively control red plam weevil population quantity, reduce the loss, that a strain has the biocontrol strain of potential significance on control red plam weevil, there is the features such as insecticidal toxicity is strong, good disinsection effect, for the development of China's Palmae industry is offered help.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.
Screening and the evaluation thereof of embodiment mono-bacterial strain
1, the screening of bacterial strain
Get the red plam weevil bombys batryticatus sample being collected, first use 70% alcohol disinfecting one minute, after sterile water wash, the mercury chloride with 0.1% carries out surface sterilization 30s to susceptible sample again, sterile water wash 3 times, a little bombys batryticatus tissue of picking is inoculated in separated SDAY substratum (4% glucose, 1% peptone, 1% yeast powder, 2% agar and 500 μ g/mL Streptomycin sulphates), be inverted in 28 ± 1 ℃ of constant temperature and be inverted cultivation 3-4d, choose monospore and be inoculated in purifying SDAY substratum (4% glucose, 1% peptone, 1% yeast powder and 2% agar), every ware 3 points, be triangularly arranged, after monospore purifying, obtain the bacterial strain with insecticidal action, called after Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2, on June 9th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number: CGMCC NO.7692.
2, strain morphology is identified
Streak culture on the dull and stereotyped SDAY substratum that the bacterial strain that separation and purification is obtained is 90mm at diameter, to put into 28 ℃ of incubators and cultivate after 4-5 days, the form of mycelia and the conidiophore of bacterial strain is examined under a microscope in the tender white hypha film-making of picking children; The conidium film-making of picking maturation the sizes and shape of paired observation spore again after 10-12 days; The mode of appearance feature of 14 days left and right observed and recorded bacterium colonies.Bacterial strain was white or faint yellow excessive to yellow produce spore on SDAY substratum before, was shaped as cotton-shaped or fine hair shape projection, As time goes on started to occur the color of the green adjacent chromatogram for basic tone.Mycelium is transparence and has barrier film.Infect dead bombys batryticatus with it the general initial stage become white fine hair to cover, slowly by bottle-green spore, substituted; Conidiophore is bottleneck shape, has bifurcated, and single raw or many raw growths that intersect, are wound in fence shape mutually, are gathered in a large number closely sporodochium.Each size of conidium is 4.8-9.3 μ m * 2.2-3.1 μ m, oblong, and bar-shaped, the spore multiple aggregation that two terminal circle slippage falls becomes lumps.
3, ITS sequence amplification, sequencing and the molecular classification of bacterial strain
Under 28 ℃, 200rpm/min condition, constant-temperature table is cultivated 72h respectively, with gauze suction filtration, collects mycelia, uses 65 ℃ of water-bath preheating 2%CTAB extraction buffers.Scrape the green muscardine fungus mycelia that takes a morsel (about 1g) and be placed in mortar, with liquid nitrogen, be milled to white powder; In ground powder, add the preheated 2%CTAB extraction buffer of 700ul, slowly stir; Mixed solution is transferred in the sterilizing centrifuge tube of 1.5ml, noticed that the volume of mixed solution does not surpass 2/3rds of centrifuge tube; Be positioned in the shaking bath of 60 ℃, 100rpm/min shake take out after 1h standby; After taking out, after the cooling 2min of normal temperature, in centrifuge tube, add the about 0.5ml of chloroform-primary isoamyl alcohol (24:1), slowly shake up both are mixed; With liquid-transfering gun, draw supernatant, proceed in the sterilizing centrifuge tube that contains 600 μ l Virahols, centrifuge tube is slowly shaken to 30sec, can not acutely rock, control damages DNA makes water layer fully mix with Virahol, until can see DNA floss suspended substance; Continue after the centrifugal 1min of 10000rpm, slowly sucking-off clear liquid body is outwelled and is retained precipitation, notices that white precipitate is that DNA is sure not to pour out, and centrifuge tube is stood upside down to be positioned on the paper handkerchief spreading out and draw remaining liq; After 1min, upright centrifuge tube, adds the sodium-acetate of 80 μ l5M and adds 75% the ethanol of 720 μ l, rocks gently, at the bottom of finger bomb tube, the DNA suspended substance of managing the end and precipitation is floated in liquid; Place 30min, the impurity of DNA suspended substance is dissolved gradually; Again, after the centrifugal 30sec of 10000rpm, outwell supernatant liquor, then add 75% ethanol, throw out is washed to 30min again; After the centrifugal 30sec of 10000rpm, outwell immediately supernatant liquor, seasoning DNA precipitates (super clean bench blowing); DNA is precipitated and dissolved in 1ml sterilized water, adds RNA enzyme (final concentration 50 μ g/ml), DNA is fully dissolved ,-20 ℃ save backup.
Adopt the ITS1-5.8s-ITS2rDNA section of fungi Internal Transcribed Spacer (ITS) universal primer ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') ITS5 (5 '-GGAAGTAAAAGTCGTAACAAGG-3 ') amplification pathogenic bacterium, synthetic by Shenzhen Hua Da genome company.PCR instrument: takara company.PCR reaction system (50 μ L): 50ng DNA profiling, 10 * PCR buffer5 μ L, each 1 μ L of 10pmol/L primer I TS4 and ITS5,10mmol/L dNTPs1 μ L, 4U/ μ L Taq archaeal dna polymerase 1 μ L, adds ddH2O and complements to 50 μ L.PCR reaction conditions: 94 ℃ of 5min, 1 circulation; 94 ℃ of 30sec, 52 ℃ of 30sec, 72 ℃ of 30sec, 29 circulations; 72 ℃ are extended 10min, 1 circulation; PCR product is served Hai Sheng work biotech firm and is checked order.Adopt the Multiple Sequence Alignment of DNAMAN5.2 software to carry out rDNA-ITS homology analysis, and the configuration of phylogenetic tree construction growth tree and stability DNAMAN5.2 software sampling analysis 1000 times, carry out Bootstrap value and analyze and evaluate.Molecular biology identification result shows that the similarity that the ITS sequence of this bacterial strain and Metarhizium anisopliae belong to is the highest, is 99%.
4, bacterial strain produces spore characteristic
By concentration, be 1 * 10
8the WENCHANG-LV2 spore suspension of cfu/ml is seeded in (100 μ l/ ware) on SDAY plate culture medium, sporulation quantity cultivate 15 days under 20 ℃ of differing tempss, the culture condition of 22 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 35 ℃ after on analytical unit area, determines that WENCHANG-LV2 bacterial strain produces spore amount for best under 28 ℃ of conditions.
Embodiment bis-bacterial strains are to the determination of activity of red plam weevil indoor insecticidal
Get the Metarhizium anisopliae that isolation and purification culture is good (Metarhizum anisopliae) WENCHANG-LV2 bacterial strain, conidial powder is packed in the aseptic centrifuge tube of 10ml, add after 5mL0.1% tween 80 sterilized water, with the vibration of vortex vibrator, 15min mixes, then with blood counting chamber, measure every milliliter of suspension miospore number, by suitable adjustment, it is 1.0 * 10 that bacterial strain is configured to spore concentration
8individual/mL, is stored in 4 ℃ of refrigerators and saves backup.The spore of Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 bacterial strain is made into the finish of 5 concentration with the method for gradient dilution, concentration is respectively 1.0 * 10
8, 5 * 10
7, 2.5 * 10
7, 1.25 * 10
7, 6.25 * 10
6individual/ml.Spore oil suspension preparation method is: scraping bacterial strain conidial powder, ratio with plam oil and cochin oil 10:1 adds miscible oil, with vortex vibrator vibration 15min, be mixed into the spore oil suspension of high density, with blood counting chamber, measure spore content, redilution becomes the finish of serial spore content.With 1ml liquid-transfering gun, draw 1ml spore oil suspension and be applied in after larva belly, then they are transferred in feed, be placed in 28 ℃, relative humidity 80% growth cabinet and raise.15 of every processing examination worms, repeat for 3 times.After processing, every 24h observes 1 time, records dead borer population, until 15d after processing, with pure miscible oil, being treated to equally contrast raises, and dead worm is carried out to moisturizing cultivation, the feature mycelia growing according to worm corpse surface determines whether examination worm is that fungal infection is lethal, the results are shown in Table 1.By laboratory experiment result, can be found out, WENCHANG-LV2 bacterial strain has lethal effect to red plam weevil, and its toxic limit medium dose is 6.05 * 10
6cfu/ml.
Table 1 bacterial strain different concns biological activity determination to red plam weevil larva after 15 days
The outdoor prevention effect of embodiment tri-bacterial strain
The finish of Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 bacterial strain is brought to field, with mixing oil, be diluted to containing conidium 1 * 10
8individual/mL, and stir, ultra low volume spray.
Start spraying control on May 6th, 2013, became a fine day at that time, temperature 34, ℃ gentle breeze, during spraying, crosswind angle is 50 °, droplet above distributes more and even near tree head (vegetative point), the average 23.8/cm of coverage density
3.Every weekly check once, is chosen the 3 strain coconut palms that the shape of being injured is identical at every turn and is dissected, and statistics worm alive and the dead borer population amount of infection, the results are shown in Table 2.By result, found out after processing 15 days, in trunk, the survival rate of larva starts remarkable decline, after 40 days, survival rate is only 41.25%, show that Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 has stronger insecticidal toxicity, and good disinsection effect, can effectively control red plam weevil population quantity, reduce the loss, be that a strain has the biocontrol strain of potential significance on control red plam weevil.
The biological activity determination of table 2. bacterial strain field to red plam weevil
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. a strain Metarhizium Strains, called after Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2, China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC NO.7692 on June 9th, 2013, have been preserved in.
2. the application of Metarhizium anisopliae claimed in claim 1 (Metarhizum anisopliae) WENCHANG-LV2 in control red plam weevil.
3. prevent and treat a biotechnological formulation for red plam weevil, its activeconstituents is that deposit number claimed in claim 1 is Metarhizium anisopliae (Metarhizum anisopliae) WENCHANG-LV2 of CGMCC NO.7692.
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| CN109221237A (en) * | 2018-11-06 | 2019-01-18 | 云南浩绿实业集团有限公司 | A kind of control method of babassu red plam weevil pest |
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| Title |
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| 张晶 等.金龟子绿僵菌对红棕象甲的室内致病力测定.《热带作物学报》.2012,第33卷(第5期), * |
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