CN101248799A - Verticillium lecanii pesticides and uses thereof - Google Patents
Verticillium lecanii pesticides and uses thereof Download PDFInfo
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Abstract
The invention relates to a verticillium lecanii pesticide as well as the application thereof, and belongs to the plant protection and pesticide technology field. The bacterial strain provided by the invention is verticillium lecanii K1207 with CGMCC No.2326. The invention produces the bacterial strain via liquid and solid two-phase fermentation, and is characterized in that a wheat bran and bran coat culture medium is adopted as the solid culture medium. The cultured product can be added with and mixed with a wetting agent, a bulking agent and a stabilizing agent to obtain wettable powder after being parched and being crumbled. The inventive verticillium lecanii bacterial strain has high virulence, thereby having good control effects on pests such as green peach aphids, urolcueon formosanums, acyrthosiphon dirhodums, cabbage aphids, cabbage worms, diamondback moths, and greenhouse whiteflies. The materials in fermentation process are cheap and have wide sources, which is suitable for industrialized production without environmental pollution.
Description
Technical field:
The present invention relates to plant protection and technical field of pesticide, specifically be meant a kind of verticillium lecanii pesticides and application thereof.
Background technology:
Verticillium lecanii (Verticillium lecanii (zimm) viegas) is a kind of insect pathogenic fungus of Hyphomycetes.This bacterium is that Neitner found on Ceylon's coffee lecanium (Lecanii coffeae) first in 1861 in the world the earliest.About the applied basic research of this bacterium, carry out early with Hall doctor R.A. of Britain chamber crop research institute (1976～1985), and presided over the commercial research of this bacteria preparation in early 1980s.First V.lecanii aphicide product Vertalec from Hall exploitation in 1981
RSince the commercialization, so far the commodity more than 8 kinds of existing a plurality of countries are produced and sold with titles such as Vertalec, Mycotal, Thriptal, Vertisol, AgroBiocontrol Verticillium 50, Verticon, Verticillin and Mycolabs Verticillium lecanii respectively, are used for the controls of insect such as aphid, aleyrodid and thrips in places such as greenhouse.This bacteria preparation product has certain requirement because effect is slower to environment for use; (especially in the anti-application process that eliminates aphis) once was subjected to the competitive pressure influence of special-purpose chemical pesticides such as Aphox in evolution, caused occupation rate of market to descend to some extent.In recent years, because the residual contamination problem and the influence of chemical pesticide become increasingly conspicuous, under the influence and promotion of global environmental protection tide, the promotion and application of this bacteria preparation product have had development rapidly again.
Summary of the invention:
The object of the present invention is to provide a kind of verticillium lecanii pesticides and application thereof.
Verticillium lecanii provided by the invention (Verticillium lecanii) K1207, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on 01 07th, 2008; The numbering CGMCCNo.2326 that preservation is registered on the books.
Verticillium lecanii K1207 of the present invention separates on the aphid body is fallen ill in Kunming, Yunnan, and this bacterial strain has following feature: 10d colony diameter 24～27mm on PDA and CzA medium, thickness 2～3mm, bacterium colony circle shape launches, anterior view white, upright fine hair shape of aerial hyphae initial stage, the later stage is close cotton-shaped, and the edge is sparse, bacterium colony from the centre to around not obvious radial gauffer is arranged, there is tangible scrobicular ring at late stage of culture bacterium colony middle part with the edge, and the bacterium colony back is milk yellow, no soluble pigment.Aerial hyphae is colourless, has every branch, and width 1.2～2.5 μ m, the conidiophore base portion expands, and it is tapered upwards to attenuate, Dan Sheng, to giving birth to or wheel is born in aerial hyphae, colyliform bottle stalk in aerial hyphae top can reach 4～6, the few branch of conidiophore.Grow conidium from the top of stalk, be agglomerated into sphere, spore ball diameter 5～12 μ m, ball meet the hydrolysis body and discharge and be divided into spore (10～25), spore clavate, ellipse, avette, two terminal circle and symmetry, spore size 2.5～8.0 * 0.75～2.8 μ m.Strains A 1207 of the present invention is compared with existing known bacterial strain, and the temperature adaptive capacity is better, and temperature range is at 15～33 ℃, and the conidia germination rate can reach 91.78～100%.
The present invention is achieved in that the test tube cultivation of Verticillium lecanii (Verticillium lecanii) K1207 bacterial strain, the solid, liquid two-phase fermentation, be prepared into wetting powder, it is prevented and treated various aphids (black peach aphid, the red aphid of white smear, wheat do not have net Macrosiphus spp, cabbage aphid etc.), cabbage caterpillar, diamond-back moth, Trialeurodes vaporariorum Westwood.
The cultural method of Verticillium lecanii of the present invention (Verticillium lecanii) K1207 bacterial strain:
1, the test tube kind is cultivated
The test tube slant culture medium prescription is: potato 20%, glucose 1.7～2%, agar-agar 1.4～1.5%, natural pH
Verticillium lecanii K1207 is moved on on the test tube slant medium,, obtain the test tube kind at 28 ℃ of constant temperature culture 3～4d.
2, liquid culture
The liquid fermentation based formulas is improvement SDAY culture fluid: 1% peptone, 4% glucose, 0.5% yeast extract; 0.136%KH
2PO
4, 0.12%, MgSO
47H
2O; PH6.0～6.5
The test tube kind is inoculated in the 500ml triangular flask liquid nutrient medium, every bottled 200ml, in 28 ℃ of constant-temperature shaking culture 4～5d, rotating speed is 100～120rpm, with each bottle seed liquor mixing, join in the secondary culture fluid (improvement SDAY) with 20ml/ bottle amount, shaking table is cultivated 4～5d, and (100～120rpm) obtain liquid preparation.
3, solid culture
The solid culture based formulas is a wheat bran--cavings medium: 56% wheat bran, 24% thin chaff, 20% husk, 0.136%KH
2PO
4, 0.12%MgSO
47H
2O.
With liquid preparation with 3 times of aqua sterilisa dilutions after, add the solid medium in 6: 10 ratios, mix 25 ± 1 ℃ of dark culturing 6d in the solid fermentation chamber that is placed on behind the Peracetic acid suffocating sterilization thoroughly, continuous illumination 3d, acquisition solid culture.
4, the preparation of wetting powder
Product drying, pulverizing, guiding humid medium, filler and stabilizing agent that cultivation is obtained are mixed and made into wetting powder.The preferred composition and the ratio of each component of wetting powder are: conidial powder (50～60%), wetting agent (neopelex, 2～6%), stabilizing agent (glycerine, 0.5%～1%), filler (diatomite, 30～40%).
The end product quality index determining: effectively the mensuration of spore concentration, sieving rate, fineness, wetting time, suspensibility, water content, pH value physical and chemical indexes is all carried out result such as table 1 with reference to Chinese agriculture standard compilation
Every physical index of table 1 Verticillium lecanii K1207 insecticide
| Physical index | |
| Effective spore concentration sieving rate fineness wetting time suspensibility water content pH value | 3.0×10 9Individual/g crosses 300 mesh sieves 2 minutes 57 seconds 75.6% 3.0% 6～7 more than 31.2% 97% |
The spore rate of living is measured and is adopted the spore germination method to measure: get the 0.5g preparation and add the 30mL aqua sterilisa with 6 layers of filtered through gauze, get the centrifugal 1min of supernatant 500rpm behind the filtrate precipitation 1min, get the centrifugal 10min of supernatant 1500rpm again, remove supernatant, be precipitated to 10 with the dilution of 1% Glucose Liquid
4Individual spore/mL concentration is uniformly coated on this dilution on the clear water agar medium, observes the spore germination situation behind 18～24h.Preparation is made back sampling and is stored in 4 ℃ of refrigerators and the shady and cool place of room temperature respectively, 1 month and survey its spore rate of living after 8 months respectively.Test result is as follows:
Spore rate alive when preparation is made: 63.5%; Preserve the spore rate alive of preparation after 1 month under the room temperature: 60.8%; Preserve the spore rate alive of preparation after 8 months in 4 ℃ of refrigerators: 54%; Preserve the spore rate alive of preparation after 8 months under the room temperature: 46.6%.
The test of pesticide effectiveness of verticillium lecanii pesticides of the present invention
1, preparation verticillium lecanii pesticides: by the cultural method preparation test preparation of aforementioned Verticillium lecanii (Verticillium lecanii) K1207 bacterial strain.
2, Verticillium lecanii (Verticillium lecanii) K1207 liquid preparation is to the control test of various aphids (black peach aphid, the red aphid of white smear, wheat do not have net Macrosiphus spp, cabbage aphid etc.), cabbage caterpillar, diamond-back moth, Trialeurodes vaporariorum Westwood
It with the Toxicity Determination result that the spore liquid infusion process is carried out, is seen Table 2～17
Table 2 Verticillium lecanii K1207 conidium is to the LC of black peach aphid (Myzus persicae)
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 | y＝3.1063+0.2178x y＝2.9137+0.3684x y＝2.8752+0.4235x y＝3.2307+0.4132x | 0.9953 0.9718 0.9777 0.9582 | 9.27×10 6 4.60×10 5 1.04×10 5 1.91×10 4 | 10.07×10 8 8.98×10 7 1.02×10 7 2.11×10 6 |
Table 3 Verticillium lecanii K1207 conidium is to the LT of black peach aphid (Myzus persicae)
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 1.8×10 3 1.8×10 4 1.8×10 5 1.8×10 6 1.8×10 7 1.8×10 8 | y＝2.4332+2.6959x y＝2.3993+2.3711x y＝2.8122+2.8219x y＝2.1884+4.1442x y＝1.7930+5.3626x y＝1.8340+6.0774x | 0.9897 0.9924 0.9904 0.9995 0.9950 0.9961 | 9.0 8.5 6.0 4.8 4.0 3.3 | 18.4 19.2 11.9 7.6 5.7 4.6 |
According to table 2,3 measurement result, when black peach aphid being prevented and treated, be the produce effects time limit with 5～7d with Verticillium lecanii K1207 conidium, 80% is the preventive effect standard, suitable working concentration is 1.8 * 10
6Conidium/ml.
Table 4 Verticillium lecanii K1207 conidium is to the LC of the red aphid of white smear (Uroleucon formosanum)
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 | y＝2.0066+0.2745x y＝1.8057+0.4394x y＝2.9095+0.3761x y＝3.3660+0.3779x | 0.9083 0.9919 0.9973 0.9860 | 8.03×10 10 1.86×10 7 3.62×10 5 2.11×10 4 | 9.51×10 1 1.55×10 9 6.33×10 7 3.60×10 6 |
Table 5 Verticillium lecanii K1207 conidium is to the LT of the red aphid of white smear (Uroleucon formosanum)
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 2.5×10 3 2.5×10 4 2.5×10 5 2.5×10 6 2.5×10 7 2.5×10 8 | y＝0.8153+4.2370x y＝1.3060+4.0023x y＝0.8391+4.8095x y＝0.8546+5.6775x y＝0.8210+8.3440x y＝0.7498+6.6632x | 0.8953 0.9252 0.9072 0.9588 0.9935 0.9820 | 9.7 8.4 7.3 5.4 5.0 4.3 | 15.4 13.6 11.5 7.6 6.3 5.8 |
According to table 4,5 measurement result, when the red aphid of white smear being prevented and treated, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 2.5 * 10
6Conidium/ml.
Table 6 Verticillium lecanii K1207 conidium does not have the LC of net Macrosiphus spp (Acyrthosiphon dirhodum) to wheat
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 | y＝3.5590+0.2151x y＝3.6237+0.2779x y＝3.5847+0.3298x y＝3.3453+0.4543x | 0.9680 0.9603 0.9757 0.9818 | 5.00×10 6 8.96×10 4 1.96×10 4 4.39×10 3 | 4.18×10 10 9.73×10 7 7.07×10 6 3.16×10 5 |
Table 7 Verticillium lecanii K1207 conidium does not have the LT of net Macrosiphus spp (Acyrthosiphon dirhodum) to wheat
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 1.3×10 3 1.3×10 4 1.3×10 5 1.3×10 6 1.3×10 7 1.3×10 8 | y＝1.9516+3.4025x y＝2.2354+3.4840x y＝2.0157+4.5020x y＝1.8894+4.8953x y＝2.1657+5.0472x y＝2.3913+4.9647x | 0.9806 0.9884 0.9969 0.9960 0.9837 0.9903 | 7.9 6.2 4.6 4.3 3.6 3.4 | 13.9 10.9 7.1 6.4 5.4 5.0 |
According to table 6,7 measurement result, when wheat not being had the net Macrosiphus spp and prevents and treats, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 1.4 * 10
6Conidium/ml.
Table 8 Verticillium lecanii K1207 conidium is to the LC of cabbage aphid (Brevicoryne brassicae)
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 | y＝2.7488+0.2695x y＝3.4550+0.2294x y＝3.4649+0.2802x y＝3.6811+0.3147x | 0.9847 0.9943 0.9946 0.9997 | 2.26×10 8 5.43×10 6 3.01×10 5 1.55×10 4 | 3.04×10 11 2.58×10 10 3.09×10 8 7.44×10 6 |
Table 9 Verticillium lecanii K1207 conidium is to the LT of cabbage aphid (Brevicoryne brassicae)
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 1.3×10 3 1.3×10 4 1.3×10 5 1.3×10 6 1.3×10 7 1.3×10 8 | y＝1.3912+3.8370x y＝1.0350+4.7027x y＝2.0157+4.5020x y＝1.8894+4.8953x y＝2.1657+5.0472x y＝2.3913+4.9647x | 0.9790 0.9976 0.9969 0.9960 0.9837 0.9903 | 8.7 7.0 4.6 4.3 3.6 3.4 | 14.5 10.5 7.1 6.4 5.4 5.0 |
According to table 8,9 measurement result, when cabbage aphid being prevented and treated, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 1.3 * 10
5Conidium/ml.
Table 10 Verticillium lecanii K1207 conidium is to the LC of cabbage caterpillar (Artogeia rapae)
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 8 | y＝2.7275+0.2324x y＝2.8076+0.3021x y＝3.2593+0.2947x y＝3.2298+0.3452x y＝3.2590+0.3840x | 0.9797 0.9844 0.9906 0.9958 0.9982 | 6.00×10 9 1.81×10 7 8.07×10 5 1.34×10 5 3.42×10 4 | 2.056×10 13 2.96×10 12 5.88×10 8 3.73×10 7 5.38×10 6 |
Table 11 Verticillium lecanii K1207 conidium is to the LT of cabbage caterpillar (Artogeia rapae)
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 2.0×10 3 2.0×10 4 2.0×10 5 2.0×10 6 2.0×10 7 2.0×10 8 | y＝1.3936+3.5128x y＝1.3544+3.9444x y＝1.4358+4.3712x y＝1.2506+4.8540x ＝0.9420+5.7688x y＝1.6288+5.2165x | 0.9596 0.9852 0.9971 0.9996 0.9974 0.9919 | 10.6 8.4 6.5 5.9 5.0 4.4 | 18.5 13.7 10.2 8.8 7.1 6.4 |
According to table 10,11 measurement result, when cabbage caterpillar being prevented and treated, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 2.0 * 10
7Conidium/ml.
Table 12 Verticillium lecanii K1207 conidium is to the LC of diamond-back moth (Plutella xylostella) 2 instar larvaes
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 8 | y＝2.0045+0.3253x y＝2.8853+0.2958x y＝3.1601+0.3070x y＝2.9892+0.3839x y＝3.1835+0.4204x | 0.9810 0.9599 0.9908 0.9909 0.9947 | 1.62×10 9 1.41×10 7 9.84×10 5 1.73×10 5 2.09×10 4 | 6.33×10 11 1.00×10 10 5.51×10 8 2.72×10 7 2.13×10 6 |
Table 13 Verticillium lecanii K1207 conidium is to the LT of diamond-back moth (Plutella xylostella) 2 instar larvaes
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 2.2×10 3 2.2×10 4 2.2×10 5 2.2×10 6 2.2×10 7 2.2×10 8 | y＝1.2292+3.7285x y＝0.7531+4.5672x y＝0.6399+5.3313x y＝0.3879+4.1519x y＝0.0478+6.9544x y＝0.3047+7.0017x | 0.9683 0.9679 0.9841 0.9941 0.9928 0.9926 | 10.3 8.5 6.6 5.6 5.2 4.7 | 17.3 13.0 9.5 7.7 6.8 6.2 |
According to table 12,13 measurement result, when diamond-back moth being prevented and treated, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 2.2 * 10
7Conidium/ml.
Table 14 Verticillium lecanii K1207 conidium is to the LC of Trialeurodes vaporariorum Westwood (Trialeurodes vaporariorum) nymph
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 4 5 6 7 8 | y＝3.0068+0.2160x y＝3.1839+0.2636x y＝3.1709+0.3173x y＝3.1888+0.3403x y＝3.3458+0.3429x | 0.9851 0.9941 0.9990 0.9605 0.9140 | 1.69×10 9 7.76×10 6 5.82×10 5 2.10×10 5 6.67×10 4 | 1.36×10 13 1.23×10 10 2.56×10 8 6.33×10 7 1.92×10 7 |
Table 15 Verticillium lecanii K1207 conidium is to the LT of Trialeurodes vaporariorum Westwood (Trialeurodes vaporariorum) nymph
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 1.8×10 3 1.8×10 4 1.8×10 5 1.8×10 6 1.8×10 7 1.8×10 8 | y＝1.8601+3.0707x y＝1.9303+3.3668x y＝1.2291+4.6573x y＝0.9629+5.4528x y＝1.0085+5.7903x y＝1.4678+5.5597x | 0.9964 0.9547 0.9967 0.9860 0.9988 0.9996 | 10.5 8.2 6.5 5.5 4.9 4.3 | 14.6 14.5 9.8 7.9 6.8 6.1 |
According to table 14,15 measurement result, when Trialeurodes vaporariorum Westwood being prevented and treated, be the produce effects time limit with 5～7d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 1.8 * 10
7Conidium/ml.
Table 16 Verticillium lecanii K1207 is to the LC of (on the sago cycas) ceroplastes rubens (Ceroplastes rubens)
50And LC
80
| Handle back fate (d) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LC 50(conidium/ml) | LC 80(conidium/ml) |
| 7 8 9 10 11 12 | y＝2.8761+0.2258x y＝3.0904+0.2432x y＝3.2195+0.2532x y＝3.6102+0.2456x y＝3.6551+0.2801x y＝3.6323+0.3351x | 0.9794 0.9531 0.9884 0.9956 0.9983 0.9980 | 2.55×10 9 7.11×10 7 1.08×10 7 4.55×10 5 6.33×10 4 7.11×10 3 | 1.39×10 13 2.09×10 11 2.31×10 10 1.24×10 9 6.51×10 7 1.69×10 6 |
Table 17 Verticillium lecanii K1207 conidium is to the LT of (on the sago cycas) ceroplastes rubens (Ceroplastes rubens)
50And LT
80
| Concentration (conidium/ml) | Virulence regression equation (y=a+bx) | Correlation coefficient (r) | LT 50(conidium/ml) | LT 80(conidium/ml) |
| 1.76×10 3 1.76×10 4 1.76×10 5 1.76×10 6 1.76×10 7 1.76×10 8 | y＝-0.5250+4.9318x y＝-0.4559+3.1312x y＝-0.4481+5.3611x y＝-1.7369+6.9387x y＝-1.7115+7.1850x y＝-1.1757+6.9330x | 0.9833 0.9771 0.9774 0.9864 0.9844 0.9924 | 13.2 11.6 10.4 9.4 8.6 7.8 | 19.6 16.9 14.9 12.4 11.3 10.3 |
According to table 16,17 measurement result, when the ceroplastes rubens on the sago cycas being prevented and treated, be the produce effects time limit with 10～12d with K1207 bacterial strain conidium, 80% is the preventive effect standard, suitable working concentration is 1.76 * 10
6～1.76 * 10
8Conidium/ml.
Total result shows that Verticillium lecanii of the present invention (Verticillium lecanii) insecticide control black peach aphid, the red aphid of white smear, wheat do not have net Macrosiphus spp, cabbage aphid etc.), cabbage caterpillar, diamond-back moth, Trialeurodes vaporariorum Westwood effect be remarkable.The effect of its control insect pest of the plant is relevant with access times and using method, and is relevant with the kind of the insect pest that is prevented and treated simultaneously.
Embodiment:
This biological prevention and control agent can mix use with other insecticide pesticide, plant growth regulator, but must guarantee mixed medicament Verticillium lecanii (Verticillium lecanii) K1207 that the present invention adopts is had no side effect.
Verticillium lecanii of the present invention (Verticillium lecanii) does not have net Macrosiphus spp, cabbage aphid etc. to black peach aphid, the red aphid of white smear, wheat), cabbage caterpillar, diamond-back moth, Trialeurodes vaporariorum Westwood control efficiency be obvious, has characteristics such as cost is low, easy to use, the term of validity is long.
Be described in further detail the present invention with embodiment below, but content of the present invention is not limited thereto.
Embodiment one:
K1207 moves on on the test tube slant medium with Verticillium lecanii (Verticillium lecanii), and the test tube slant culture medium prescription is: potato 20%, glucose 1.7～2%, agar-agar 1.4～1.5%, natural pH.
At 28 ℃ of constant temperature culture 3～4d, obtain the test tube kind.
The test tube kind is inoculated in 500ml triangular flask (the every bottled 200ml) liquid nutrient medium, and culture medium prescription is: 1% peptone, 4% glucose, 0.5% yeast extract; 0.136%KH
2PO
4, 0.12%MgSO
47H
2O; PH6.0～6.5, in 28 ℃ of constant-temperature shaking culture 4～5d, rotating speed is 100～120rpm, joins in the secondary culture fluid (improvement SDAY) with 20ml/ bottle amount, shaking table is cultivated 4～5d, and (100～120rpm) obtain liquid preparation of the present invention.
Embodiment two:
At first obtain liquid preparation (with embodiment one), with liquid preparation with 3 times of aqua sterilisa dilutions after, add the solid medium in 6: 10 ratios, the solid culture based formulas is for being wheat bran--cavings medium: 56% wheat bran, 24% thin chaff, 20% husk, 0.136%KH
2PO
4, 0.12%MgSO
47H
2O mixes 25 ± 1 ℃ of dark culturing 6d in the solid fermentation chamber that is placed on behind the Peracetic acid suffocating sterilization thoroughly, and continuous illumination 3d takes out on the glass plate that is tiled in the fermenting cellar air-dry 5d under 35 ± 1 ℃, the 100 grades wind speed afterwards.Obtain solid pharmaceutical preparation.
Embodiment three:
At first carry out liquid-solid two-phase fermentation (with embodiment one, example two), the product drying, pulverizing, guiding humid medium, filler and the stabilizing agent that then cultivation are obtained are mixed and made into wetting powder.
The preferred composition and the ratio of each component of wetting powder are: conidial powder (50～60%), wetting agent (neopelex, 2～6%), stabilizing agent (glycerine, 0.5%～1%), filler (diatomite, 30～40%).
The end product quality index determining: effectively the mensuration of spore concentration, sieving rate, fineness, wetting time, suspensibility, water content, pH value physical and chemical indexes is all carried out result such as table 1 with reference to Chinese agriculture standard compilation
Every physical index of table 1 Verticillium lecanii K1207 insecticide
| Physical index | |
| Effective spore concentration sieving rate fineness wetting time suspensibility water content pH value | 3.0×10 9Individual/g crosses 300 mesh sieves 2 minutes 57 seconds 75.6% 3.0% 6～7 more than 31.2% 97% |
The spore rate of living is measured and is adopted the spore germination method to measure: get the 0.5g preparation and add the 30mL aqua sterilisa with 6 layers of filtered through gauze, get the centrifugal 1min of supernatant 500rpm behind the filtrate precipitation 1min, get the centrifugal 10min of supernatant 1500rpm again, remove supernatant, be precipitated to 10 with the dilution of 1% Glucose Liquid
4Individual spore/mL concentration is uniformly coated on this dilution on the clear water agar medium, observes the spore germination situation behind 18～24h.Preparation is made back sampling and is stored in 4 ℃ of refrigerators and the shady and cool place of room temperature respectively, 1 month and survey its spore rate of living after 8 months respectively.Test result is as follows:
Spore rate alive when preparation is made: 63.5%; Preserve the spore rate alive of preparation after 1 month under the room temperature: 60.8%; Preserve the spore rate alive of preparation after 8 months in 4 ℃ of refrigerators: 54%; Preserve the spore rate alive of preparation after 8 months under the room temperature: 46.6%.
Promptly get biologic product of the present invention.
Claims (3)
1, a kind of verticillium lecanii pesticides is characterized in that: the production bacterial strain of said preparation is Verticillium lecanii (Verticillium lecanii) K1207, preserving number CGMCC No.2326.
2, the cultural method of the described Verticillium lecanii of claim 1 (Verticillium lecanii) K1207 bacterial strain is characterized in that: described bacterial strain is to cultivate through liquid-solid two-phase to obtain, and its liquid and solid fermentation culture medium prescription are as follows:
(1) liquid fermentation medium prescription: 1% peptone, 4% glucose, 0.5% yeast extract; 0.136%KH
2PO
4, 0.12%MgSO
47H
2O; PH 6.0～6.5;
(2) solid fermentation culture medium prescription: 56% wheat bran, 24% thin chaff, 20% husk, 0.136%KH
2PO
4, 0.12%MgSO
47H
2O.
3, the described Verticillium lecanii of claim 1 (Verticillium lecanii) K1207 bacterial strain does not have the application in net Macrosiphus spp, cabbage aphid, cabbage caterpillar, diamond-back moth, the Trialeurodes vaporariorum Westwood insect in control black peach aphid, the red aphid of white smear, wheat.
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