CN1542123A - Metarhizium anisopliae - Google Patents
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- CN1542123A CN1542123A CNA2003101109018A CN200310110901A CN1542123A CN 1542123 A CN1542123 A CN 1542123A CN A2003101109018 A CNA2003101109018 A CN A2003101109018A CN 200310110901 A CN200310110901 A CN 200310110901A CN 1542123 A CN1542123 A CN 1542123A
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The present invention is one new strain of locust killing fungus of green muscardine, named Metarhizium anisopliae CQMA102 and with conservation number of CGMCC No. 0877. Compared with other locust killing pesticide, the biological pesticide produced with the strain has high toxicity on locust and similar pests, no harm to no-target life forms, heat resistance, powerful contamination tolerance, high spore yield and other excellent characteristics.
Description
Technical field
The present invention relates to the novel metarhizium anisopliae of a strain, belong to field of biological pesticide.With natural screening method only bring out to the locustamigratoria insect show high virulence, high temperature resistant, antipollution power by force, do not influence environmental organism, spore production rate high kill the new bacterial strain of locust, belong to green muscardine fungus and belong to.The oil content powder preparation that the dried cryptogam of the liquid-solid fermentative production of this bacterial strain and vegetables oil mix is the biotic pesticide that are applicable to control locustamigratoria agriculture and forestry injurious insect, and it is to person poultry harmless, spore production rate height, anti-living contaminants, stable performance.Claimed this bacterial strain.
Prior art
The plague of locusts is a kind of worldwide biological epidemics that agriculture production had crushing blow, also be the significant threat of China's husbandry safety in production and the important hidden danger of social stability, crop loss 5-18% is caused, nearly 10,000,000,000 dollars of financial loss because of the plague of locusts in the annual whole world.The main applied chemistry agricultural chemicals of China's control plague of locusts have a small amount of microsporidium biotechnological formulation to enter locust control field in recent years, but usable floor area is little at present, and result of use is stable inadequately.The chemical pesticide used in a large number of locust control midium or long term both serious environment pollution, threaten people's health, also kill and wound pest natural enemy in a large number, destroy the eubiosis.Six academician's suggestions such as calendar year 2001 Qiu Shibang add the dynamics that mcroorganism is eliminated locusts; The Ministry of Agriculture has formulated " the sustainable resolution of national locust disaster ", and with the main means of biological control technology as the sustainable control of the plague of locusts, claiming during "the 10th five-years", the biological control area reaches more than 30%.Therefore safe and effective, as not kill and wound nontarget organism locust biological pesticide extremely is badly in need of in locust control.
Fungi is plant insect, germ and the careless topmost natural enemy of evil, plays an important role in the control of plant disease pest and weed.To the existing in the world many successful examples of the research and development of Mycophyta biological pesticide.Aspect the entomogenous fungi preparation, only the registered in the world product of green muscardine fungus and muscardine preparation promptly has more than 20, produces beauveria bassiana preparation Mycontrol RWP as nineteen ninety-five Environmental Protection Agency's approval Mycotech company, is used to prevent and treat vegetables pest; The green muscardine fungus finish of international biological control research institute of the later stage eighties (Britain) development, this product has been transferred the possession of France and South Africa two companies, in Africa migratory locusts and grassland grasshopper preventive effect is reached more than 90%.But because external selected green muscardine fungus bacterial strain spore production rate is low, non-target (environment other) biology is had lethal effect, the green muscardine fungus that makes with it kills locust preparation cost height, therefore, is not extensively utilized in China.
Domestic research to the fungi biological prevention and control agent starts from the fifties, some of them have had suitable scale, reach several ten million mu as muscardine control pine moth at the annual area of using of China, the liquid-solid two-phase fungal spore production technology of China development the approval of abroad going together, but because the bacterial strain virulence instability of using, a little less than the antipollution power, reasons such as production technique backwardness and fundamental research difference, before the present invention, still none commodity fungus insecticide is registered.Though there are last hundred tame plant produced muscardines, green muscardine fungus in China at present, but all exist product spore efficient low in various degree, the defective that production cycle is long does not meet the basic demand of commercial scale production, becomes the fungal organism agricultural chemicals and fails a commercial major reason in China.
In sum, in the process of locust biological control, the problem of most critical is to obtain that the target insect is had the ability of enough killing, and can realize the microorganism strains of commercial scale production.
Summary of the invention
The objective of the invention is by natural method for screening, obtain the novel metarhizium anisopliae of a plant height virulence, strong specialization, that utilizes this bacterial strain production kills the locust biological pesticide to environment insect and biological no injury effect.
Green muscardine fungus bacterial strain of the present invention is in Beijing China Committee for Culture Collection of Microorganisms common micro-organisms culture presevation administrative center (CGMCC) preservation, and preservation date is on January 10th, 2003, preserving number CGMCC No.0877; Particular content is: Metarhizium anisopliae CQMA102, Metarhiziumanisopliae.CQMA102, CGMCC No.0877.
Technical solution of the present invention is to gather the natural susceptible locust → antibacterial dull and stereotyped inducing culture of the cultivation bombys batryticatus → separation and purification bacterial strain → lower concentration → biological characteristics comparison → production traits comparison → production bacterial strain of preserving moisture to determine → liquid-solid two-phase fermentation → separated and collected xerospore powder → finish preparation → drop inoculation locust → bacterial strain toxicity test.Through repeated screening, select that a strain anti-soil is assorted, high virulence, high yield spore kill locust Metarhizium anisopliae bacterial strain, be numbered the CQM102 bacterium.Now finished between the interior open gene of this bacterial strain region nucleotide sequence and measured and analyze, therefrom determined the molecule marker of this bacterial strain, can provide appraisal basis with arbitration law for the molecule checking and the lawsuit of this bacterial strain.This strain classification status; Green muscardine fungus belongs to Metarizium, Metarhizium anisopliae Metarizium anisopliae.
The bacterium colony cultural characteristic: the fine and soft shape of bacterium colony initial stage white on sabouraud medium, greyish-green grows the green mitogenetic sorus of clump from inside to outside to olive-green when producing spore.The conidiophore list is given birth to or is assembled or closely arrangement broom shape branch or whorl.Later stage culture bacterium colony is shelly.Bacterial strain can be on SDA 28 ℃ of one weeks of growth, diameter reaches 3.5cm; Colony growth speed is slack-off in the time of 35 ℃, and all colony diameters are 2.4cm.
The strain morphology feature: mycelia tool branch is separated, thick 1.4-2.1um.Bottle stalk type conidiogenous cell, magnitude of size is 2.1-2.9 * 7.1-7.5 μ m; Produce to the sophisticated conidia chain of base from bottle stalk top; Spore chain tie point obliquity is little; Conidium is unicellular, and oblong is justified shape to ovum, the two ends blunt round, and an end is thin slightly, and spore size luffing is bigger, 1.9-3.1 * 5.5-9.5um.Conidium also singly is born in mycelia branch falx end sometimes.
Utilize the locust biological pesticide that kills of bacterial strain production of the present invention not only to have to locustamigratoria insect virulence height,, good characteristics such as antipollution power is strong, spore production rate height harmless, high temperature resistant to non-target organism, and can under the low environment humidity condition, effectively prevent and treat locust, and can stablize and store more than 1 year.
Green muscardine fungus bacterial strain biological characteristics is measured
One, the insecticidal toxicity of CQMA102 bacterial strain and host range
Collect the xerospore powder (water content is below 5%) of the green muscardine fungus CQMA102 of liquid-solid biphasic fermentation production, disperse with salad oil, being configured to final concentration is 1 * 10
7The oily spore suspension of spores/ml is inoculated the various for the examination locust of age in days basically identical.Oily spore liquid point is dropped in under the examination locust pronotum (5ul/ head), and every kind of locust inoculates 50, and the rearing-box artificial breeding of putting into 60cm * 60cm * 40cm after the inoculation regulates the room temperature to 30 ℃, humidity 40～50%, light, dark cycle 16h/8h.Each is handled and establishes a blank, replaces the muscardine spore inoculation with edible oil in the control treatment.Yeast powder=4: 1) and fresh wheat seeding regularly observe every day, changes feed (wheat bran:.Write down dead borer population every day, obtain LT50 (median lethal time), LT90 (terminal hour causes death) through the analysis of DPS2000 software statistics.
Table 1CQMA102 is to the virulence of different locusts
Locust LT50 (P=0.05) LT90 (P=0.05)
Asiatic migrotory locust 3.66 ± 0.12 4.88+0.19
Ceracris kiangsui 3.32 ± 0.15 4.23 ± 0.13
Chinese rice grasshopper 3.45 ± 0.11 4.72 ± 0.24
Acrida cinerea 3.27 ± 0.16 4.76 ± 0.20
Cotton locust 4.25 ± 0.22 5.87 ± 0.27
Asia dolly locust 3.02 ± 0.15 403 ± 0.13
CQMA102 has tangible prevention effect to 6 kinds of locusts such as Asiatic migrotory locust, Ceracris kiangsui, Chinese rice grasshopper, Acrida cinerea, Asia dolly locust and cotton locusts as can be seen from Table 1, the LT90 of several locusts is all less than 6 days, 4,5 days is dead peak period (cotton locust is big because of polypide, and dead peak period is 5-6 days).
Two, the CQMA102 bacterial strain is to the tolerance of sterilant
Contain 10,30 respectively, on 1/4SDA (10% glucose, 2.5% peptone, the 5% yeast extract) solid medium of 50ppm F-1991, inoculation 5 * 10
6The CQMA102 spore suspension of/mL is coated on the above-mentioned culture medium flat plate equably, cultivates in 25 ± 1 ℃ of thermostat containers, observes growth every day and produces the spore situation.Produce behind the spore to beat at random and get bacterium piece (3/ware), put into the 50ml triangular flask that the 20mL0.05% tween-80 is housed, on magnetic stirring apparatus, fully stir evenly the back with blood counting chamber mensuration green muscardine fungus spore content with diameter 9mm punch tool.Making not, 1/4 SDA substratum of adding of germicide is contrast.As can be seen from Figure 1: lower concentration such as 10ppm F-1991 do not have the obvious suppression effect to CQMA102 strain growth and product spore, compare the processing of 10ppm sterilant and the dull and stereotyped spore production rate no significant difference of contrast with contrast.Can be subjected in various degree inhibition and grow up and produce spore, show as the sparse or sporulation quantity of mycelia and obviously descend at the above concentration bacterial strain of 30ppm.This shows that the CQM102 bacterial strain has certain tolerance to sterilant.
Three, CQMA102 bacterial strain thermotolerance
CQMA102 and control strain ME1 xerospore powder mixed being formed in the salad oil respectively, be configured to 1 * 10
10The finish of individual/ml concentration, be positioned over 25 ± 1 ℃ after 16 hours, be sub-packed in the Eppendorf tube, in being set to 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ grads PCR instrument, take out after being incubated 1h, 2h, 4h respectively, spread upon and be placed with on the 1/4SDAY flat board that diameter is a 9mm glassine paper disk, every 3h gets a slice glassine paper disk cotton blue dyeing back microscopy, count 200-300 spore, the spore count that record is sprouted calculates germination rate.Three repetitions are established in each processing.
As can be seen from Figure 2 the CQMA102 bacterial strain does not have noticeable change handling the back germination rate below 60 ℃, and the Temperature Treatment spore germination rate sharply descends more than 60 ℃, and the germination rate after 70 ℃ of processing is divided into 16%, slightly is better than the spore germination rate of ME1 bacterial strain.24 hours germination rates of two bacterial strains do not have significant difference under other temperature.The high temperature tolerance ability that shows CQMA102 bacterial strain spore is better than control strain.
Four, CQMA102 bacterial strain total protease activity
According to the active principle that is proportionate with strain pathogenic strength of fungal bacterial strain total protease, measure the bacterial strain virulence by the active prescreening method of simple and easy total protease fast.With spore concentration 10
8The CQM102 bacterial strain of individual/ml is inoculated in respectively in the inducing culture liquid that contains cockroach body wall powder with other contrast green muscardine fungus inoculation liquid, and (it is standby to get supernatant liquor for 150 commentaries on classics/min) cultivate 48h, the centrifugal 10min of 6 000g in 26 ℃ of vibrations.Get 30ml 0.5% casein solution, with 70ml 0.05mol/L, the phosphoric acid buffer mixing of pH7.5 adds the 1g agar powder again, and pouring diameter after dissolving into is in the 6cm plastic culture dish, the 6mL/ ware.Punch tool with diameter 4mm punches in substratum, the different strains supernatant liquor of getting 10 μ l inducing culture adds in the hand-hole, preserve moisture for 26 ℃ and spend the night, in culture dish, add 5ml 20% Tricholroacetic Acid liquid, the active circle of total protease will show as transparent circle, judge the activity of bacterial strain total protease with the transparent circle size, transparent circle is big more, and total protease activity or bacterial strain virulence are also stronger.Detected result to be finding out, compares the transparent circle maximum of CQMA102 bacterial strain with control strain, and the total enzyme activity that shows this bacterial strain is higher than other bacterial strain for examination.
Five, the CQMA102 bacterial strain is to ultraviolet tolerance
According to the otherness of green muscardine fungus different strains to the tolerance of envrionment conditions such as high temperature, ultraviolet radiation etc., with respectively with ME1 and CQMA102 make 1 * 10
10After the oily spore of/mL green muscardine fungus finish and thinner mixed by 1: 1,26 ℃ of thermostat containers spend the night, spread upon respectively with transfering loop on the 1/4SDAY flat board of diameter 9mm glassine paper disk, after placing local vertical irradiation 60S apart from the ultraviolet lamp 35cm of 30w, 120S, 240S, after taking-up is put into 26 ℃ of thermostat containers and cultivated 24h, get a slice glassine paper disk with cotton blue dyeing back microscopy, several 200～300 spores, the spore count that record is sprouted calculates germination rate.
Can find out that from table 2 CQMA102 bacterial strain and ME1 are stronger to ultraviolet resistivity, the 24h germination rate all reaches more than 90%, does not have significant difference with untreated control.Show that the CQMA102 bacterial strain is consistent with reference culture to the tolerance of uviolizing.
The influence that table 2. uviolizing is sprouted muscardine spore
Irradiation time (s) ME1 CQMA102
60 96％ 97％
120 95％ 96％
240 94％ 95％
CK 98％ 99％
Six, the spore production rate of CQMA102 bacterial strain
To cultivate the material rice and contain 2% yeast powder, 5% SODIUMNITRATE and copper, boron, soak in the nutritive medium of trace elements such as zinc in the air-permeable anti-bacterial composite solid fermentation bag of packing into behind the 2h (during second stage employ, in fermentation materials: nutritive medium is that 2: 1 ratio adds 4% yeast powder, 8% SODIUMNITRATE and copper, boron, the nutritive medium of trace element such as zinc), mix back 121 ℃ of high pressure moist heat sterilization 30min thoroughly, behind the naturally cooling in cultivating material and bacterium liquid=10～15: 1 ratio, inoculation green muscardine fungus CQMA102 bacterium liquid is also mixed thoroughly evenly, places 26 ± 1 ℃ the static fermentation of proving room drying (30 ℃～34 ℃) to the material moisture that heats up after 10～12 days to be lower than 5%.Randomly draw the material fermented sample, get the sample about 5g behind the mixing, put into the 50ml triangular flask that 20mL 0.05%Tween-80 is housed, fully measure the spore content of product behind the mixing with blood counting chamber.Fermentation is by 500 * 10 for the second time
8Individual/g and rice converse the solid fermentation spore production rate with back loss 15% for the first time.The total spore production rate of culture material＞5% after the fermenting twice.
Seven, the CQMA102 bacterial strain is to the influence of non-target organism in the environment
Honeybee is put into the diameter 25cm that honey is housed, in the plastics string bag of high 30cm, every string bag 100.Only; 60 ants are put into 40cm * 50cm * 30cm plastic tub that the basin mouth scribbles Vaseline all around; Tadpole, crucian are put into the plastic tub (55 tadpole/basins, 15 crucian/basins) that fills 10 premium on currency, and river prawn is put into plastic tub (20 shrimp/basins), use the oxygenator oxygenation in the basin; Chicken, duckling are put into the plastic tub of 40cm * 50cm, 10 in every basin, feed 250g.Kill locust green muscardine fungus finish and fungal farm chemicals diluent (patent applied in 1: 2 ratio with 20%, application number 02134002.1) after the mixing, put into 26～28 ℃ indoor environment, throw in feed (chicken, duckling use the feed behind the spray medicine to raise) every day, and record supplies to try the biological death number respectively.Each establishes 3 repetitions for the examination biology, establishes a blank.After mulberry leaf were sprayed the green muscardine fungus finish in order to last method, the silkworm 2-5 instar larvae of feeding respectively (each 30 of every processing are established 3 repetitions, a contrast) was put down in writing larval mortality day by day.
Table 3 green muscardine fungus finish is measured the influence of non-target organism
Table 4 green muscardine fungus finish is measured the influence of silkworm
From table 3,4 as can be seen: the green muscardine fungus finish is handled back honeybee, ant, tadpole, crucian, river prawn, chicken, duckling and silkworm mortality ratio and contrast no significant difference, illustrate that the various biological deaths after green muscardine fungus is handled belong to natural death, green muscardine fungus CQMA102 bacterial strain does not have detrimentally affect to above non-target organism.
Description of drawings
Fig. 1 is the tolerance figure of CQMA102 bacterial strain to sterilant.
Fig. 2 is that the thermotolerance of bacterial strain CQMA102 and ME1 bacterial strain compares (24h) figure.
Embodiment
Best implementation: actication of culture-first order seed fermentation-secondary seed fermentation-liquid fermenting-inoculation material-static fermentation-spore drying-quality examination-packing is sealed (-preparation processing-product) up for safekeeping.
Claims (1)
1. the novel metarhizium anisopliae of a strain is characterized in that this bacterial strain belongs to green muscardine fungus and belongs to Metarhizium anisopliae CQMA102, Metarhizium anisopliae.CQMA102, CGMCC No.0877.
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| CN100339472C (en) * | 2005-09-30 | 2007-09-26 | 高小文 | Metarrhizium anisopliae deltoid moths subspecies and its disinsection culture and use method |
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| CN102776130B (en) * | 2012-08-03 | 2014-05-28 | 中国热带农业科学院环境与植物保护研究所 | Metarhizium anisopliae and application thereof |
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| CN109401981B (en) * | 2018-10-11 | 2020-05-22 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHBHC-7 and application thereof |
| CN114196556A (en) * | 2021-12-31 | 2022-03-18 | 重庆大学 | Metarhizium anisopliae strain with high locust toxicity and construction method thereof |
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