CN113142245A - Synergistic prevention and treatment of common thrips by Beauveria bassiana SB038 and spinetoram - Google Patents
Synergistic prevention and treatment of common thrips by Beauveria bassiana SB038 and spinetoram Download PDFInfo
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Abstract
The beauveria bassiana SB038 and the spinetoram in the pesticide provided by the invention generate a remarkable synergistic effect on the poisoning effect of common thrips. Through long-term infection biological research and indoor bioassay, the combined use of the beauveria bassiana and the spinetoram has good prevention and treatment effects on common thrips, has very strong application potential in biological prevention and treatment, can reduce the use amount of chemical pesticides, has the characteristics of low toxicity and low drug resistance, has no pollution and no residue on the environment, and is beneficial to delaying the occurrence and development of drug resistance of thrips pests.
Description
Technical Field
The invention belongs to the technical field of biological control. In particular to biological control of common thrips prevention and treatment, and more particularly to synergistic control of beauveria bassiana and spinetoram on common thrips.
Background
Chemical pesticides play a great role in pest control, and the usage amount and yield of pesticides in China are the first place in the world for a long time. However, according to statistics, the effective utilization rate of global pesticides is less than 30%, the annual chemical pesticide utilization area in China is more than 2.8 hundred million hm2, the application amount reaches 50-60 ten thousand tons, and about 80% of chemical pesticides directly enter the environment (Liliang, Zhang Ying, Sun Hui Ying, and the like, pesticide hazard and green plant protection technology discussion [ J ]. Hubei plant protection, 2018, (4): 63-64). With the long-term use of a large amount of chemical pesticides, the problems of '3R', namely pest Resistance (Resistance), pesticide Residue (Resistance) and pest regressions (Resistance) and environmental pollution, are more and more serious, and the natural enemies of many pests are mistakenly killed in the control process (Mengyu. measures for protecting the natural enemies of the pests in the chemical control process of the pests [ J ] Hebei agriculture, 2012, (03): 35-37.). With the rapid development of modern green food production, traditional pest control patterns need to be changed, and sustainable green management approaches are urgently sought (Chen X, Li L, HuQ, et al.. Expression of dsRNA in recombinant Isarialysosea strain the TLR7 gene in Beimisatababi [ J ]. Bmc Biotechnology,2015,15 (64)).
Common Cirsium japonicum Megalluthrips usatus (Bagrall), also known as Strychnos faberi and Strychnos stigmatis, is a Thysanoptera thrips subfamily Thripina (Stephens) Priesner Cirsium Megalluthrips insect (Han Takayao, China economic insect Institution fifty-five book: Thysanoptera [ M ]. Beijing: scientific Press, 1997: 39-59.). Common thistle belongs to a parasitic insect, the host plants include 28 species, and belong to 9 families, 16 of which are leguminous crops (Aliakbarbour H, Rawi C S M. the species composition of pests in fungi: thraustaine) inhibiting microorganisms in pulupiang, malaysia [ J ]. Tropical Life Sciences Research,2012,23(1): 45.). The common Cirsium japonicum mainly occurs from 10 months to the next 5 months, has strong concealment, is only observed at the blooming moment of blooming, is hidden and eaten in flowers in most of the rest time, has high pest amount of 35 flowers and large pest amount, and has great control difficulty (Tangliand, Hanyun, Wujianhui, etc.. bean Cirsium japonicum indoor chemotactic response to different colors and light waves [ J ] plant protection, 2015(6):169 and 172.). The characteristics of strong concealment and high propagation speed of the thrips bring great difficulty to prevention and control, the damaged part of the plant is white at first, then gradually withers and turns brown, when the heart leaves are damaged, the damaged part shrinks and curls to deform until the plant dies, the damage to the seedling stage is light, the damaged part is mainly cowpea flowers and fruits, blackheads are caused, and the blacktails seriously influence the appearance quality and the nutritive value of the commodity. At present, the prevention and treatment of thrips also mainly depends on chemical prevention and treatment means. Due to the unreasonable use of a large amount of chemical agents, various problems of pest drug resistance, natural enemies killing, pesticide residue, environmental pollution and the like are caused, and the quality and safety of products are threatened. Therefore, the search for environment-friendly control strategies is extremely urgent today, and biological control has wide application prospects in the control work of thrips pests.
Beauveria bassiana belongs to Deuteromycotina, Moniliales, Moniliaceae and Beavinia, and is an important biocontrol strain. Beauveria bassiana is found on Melanopiusmexicanus Mexicanus for the first time, and is successfully applied to Western fimbristylis formosanus as a biocontrol bacterium in the early 70 s, thereby attracting wide attention. For example, Yuanhongensis et al (2011 and 2013) isolated from the larvae of Argyria argentea to obtain beauveria bassiana MZ050724 strain with high virulence against Frankliniella occidentalis, and beauveria bassiana Bb080717 strain and MZ050724 strain with high virulence against Frankliniella auricularia indica (university of southwest, environmental insect school newspaper, Chinese forest pest); two strains of beauveria bassiana (CN 104212724A and CN 104263655A) with good control effect on Frankliniella occidentalis are separated from corn borer by Wanghaihong, etc.; beauveria bassiana (CN 106135295A) with good control effect on alfalfa thrips is found by daubing the soldier and the like (2016), and the like. Among them, the biocontrol effect of beauveria bassiana on thrips was most studied.
The entomogenous fungi is environment-friendly, can parasitize various pests, and is the best choice in biological control. However, the entomogenous fungi has slow insecticidal effect (the effect is achieved within 3 days); poor control (< 50%); short shelf life (<6 months) of the formulation; the defects of difficult industrialized mass production (strain degeneration) and the like are obvious, and in order to better prevent and treat the strain, a pesticide compounding technology or a mixed application technology can be adopted. How to realize high-efficiency, pollution-free and residue-free prevention and control when the common thrips are prevented and controlled is the key for solving the practical problems at present and the key for developing the pesticide compound agent.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing common thrips prevention and treatment technology and provide an efficient, pollution-free and residue-free pesticide combining beauveria bassiana and spinetoram and having a synergistic effect. According to the invention, through researching the compounding effect of beauveria bassiana and spinetoram, the insecticide can show a remarkable synergistic effect after compounding, the control effect is improved, the use dosage of a chemical pesticide single agent can be remarkably reduced, the environmental pollution is reduced, and the problems that the existing pesticide is easy to generate resistance, the pesticide effect is not remarkable and the like are effectively solved.
Based on the fact that multiple beauveria bassiana strains have certain biological control effect on common thistle, in order to further seek better control effect, a large number of researches and exploration experiments show that the combination of the beauveria bassiana strain SB038 and spinetoram can generate obvious synergistic interaction effect, and the effect of preventing and controlling pests such as thrips is obvious, and finally the invention is completed.
Therefore, the invention firstly provides a combined pesticide of beauveria bassiana and spinetoram, wherein the beauveria bassiana is beauveria bassiana strain SB038 with the preservation number of GDMCC NO: 61303, respectively; the pesticide is used for preventing and treating common thrips. The strain SB038, originally isolated from soil, is a native strain of China and can be well adapted to local natural environment.
Preferably, in the above insecticide, the beauveria bassiana is beauveria bassiana spores, and the beauveria bassiana spores may be suspension or spore powder.
More preferably, the using ratio of the beauveria bassiana to the spinetoram is as follows: the final concentration of Beauveria bassiana is 1 × 104～1×108conidia/mL, preferably 1X 104～1×107Conidium per mL, and the final concentration of spinetoram is 0.625-10 mg/L, preferably 1.25-10 mg/L; more preferably Beauveria bassiana and BThe ratio of spinosad is 1 × 104～1×107conidia/mL: 1.25-10 mg/L, more preferably the ratio of beauveria bassiana to spinetoram is 1 x 105～1×107conidia/mL: 2.5-10 mg/L, and more preferably the ratio of beauveria bassiana to spinetoram is 1 x 105～1×107conidia/mL: 2.5-5 mg/L. Most preferably, the ratio of beauveria bassiana to spinetoram is 1 × 105conidia/mL: 2.5 mg/L.
The invention further provides application of the pesticide in preventing and treating common Cirsium japonicum pests.
Compared with the prior art, the invention has the following beneficial effects: the beauveria bassiana SB038 and the spinetoram in the pesticide provided by the invention generate obvious synergistic effect on the poisoning effect of common thrips, long-term infection biological research and indoor bioassay show that the combined use of the beauveria bassiana and the spinetoram has good control effect on the common thrips, and the growth influence of the spinetoram on the beauveria bassiana SB038 is small, and the compatibility is good, so that the pesticide has very strong application potential in biological control of thrips pests. The pesticide has good control effect, so the pesticide has the characteristics of low toxicity and low drug resistance, can reduce the usage amount of chemical pesticides, meets the requirements of organic food production, has no pollution and residue to the environment, and is favorable for delaying the occurrence and development of drug resistance of thrips pests.
The Beauveria bassiana (Beauveria basssaana) SB038 is preserved in Guangdong province microbial strain preservation center (address: No. 59 building 5 of Michelia Tokoro No. 100 Mditerrand Guangzhou, Guangdong, China) at 11 and 20 days in 2020, and the preservation number is GDMCC No: 61303.
drawings
FIG. 1 SB038 colony morphology. Wherein, the left and right images are front and back images of SB038 colony.
FIG. 2 SB038 spore morphology.
FIG. 3 shows the electrophoresis of PCR amplification products in molecular assay.
FIG. 4 molecular systems Tree.
FIG. 5 Effect of spinetoram on colony growth of strain SB 038.
FIG. 6 Effect of acetamiprid on colony growth of strain SB 038.
FIG. 7 Effect of spinetoram on spore germination rate of strain SB 038.
FIG. 8 effect of acetamiprid on spore germination rate of strain SB 038.
Detailed Description
The invention is further illustrated by the following specific examples. The following examples are preferred embodiments of the present invention, but are not intended to limit the scope of the present invention in any manner. The present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are intended to be included in the scope of the present invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Example 1 screening of highly virulent Strain
1.1 test insects
Common thrips is collected from cowpea field in Zhu village in Zengzhou city, Guangdong province in 2017, collected and then raised with cowpea pod in RXZ-500C type intelligent artificial climate box (Ningbo Jiangnan Instrument factory) under the conditions of temperature (26 +/-5) deg.C, illumination period of 12L:12D and relative humidity (70 +/-5)%.
1.2 test strains
The strains listed for screening all belong to Beauveria bassiana (Beauveria bassiana) and are all preserved in the engineering research center of department of biological control and education of southern China university of agriculture, and the specific steps are as follows: SP433, SB036, SB062, SB037, SB015, SB063, SB051, SB041, SB004, SB043, SP016, SB038, SB050, SB 006.
Among them, biological material deposits for patent procedures were also made for SB063 and SB 038. Among these, the disclosure relating to the strain SB063 will be the subject of another patent application.
1.3 Experimental methods
1.3.1 preparation of spore suspensions
Culturing at 26 + -1 deg.C on PDA plate for 7 days, eluting conidia of entomogenous fungi with 0.05% Tween-80 sterile water, stirring with magnetic stirrer, shaking at 25 deg.C in shaking table at 180rpm for 25min, filtering with double-layer mirror paper, counting with blood count plate, measuring mother liquor concentration, and making into 1 × 108spores/mL of spore suspension.
1.3.2 determination of virulence of entomogenous fungi on common thrips
Placing the prepared spore suspension in a 15ml centrifuge tube, picking 20 heads of female thrips into the immersed bacteria liquid for 10s, then placing the immersed bacteria liquid in a 35mm culture dish with 1cm beans (two ends without holes), sealing the preservative film, pricking holes, placing the culture dish in a climatic chamber, taking 0.05% Tween-80 sterile water as a blank control, repeating the treatment for 4 times, and continuously recording the death rate for 7 days.
The screened high-toxicity strain is prepared into 1 × 10 with 0.05% Tween-80 sterile water8、1×107、1×106、1×105、1×104spores/mL of spore suspension, and their mortality was determined in the same bioassay method.
1.3.3 data processing
SPSS19.0 software is used for experimental treatment analysis, single-factor analysis of variance is used for analyzing each result, and Tukey is used for detecting the significance of difference.
1.2 results of the experiment
The experimental results show that the pathogenicity of different strain spore suspensions to common thrips is obviously different, the strain SB038 and the strain SB063 are obviously higher than other strains (Table 1), the death rate of the common thrips female imagoes is increased along with the increase of the concentration and the treatment time, when the concentration is 1 multiplied by 108The cumulative mortality at spore/mL for SB038 and SB063 at 7d was 91, respectively.07% and 83.33%, significantly higher than other concentrations.
The results show that the strains SB038 and SB063 have good lethal effects on common thrips, and belong to potential excellent biocontrol. The two strains were tested for virulence at different concentrations and different treatment times, and the results are shown in tables 2 and 3, respectively.
TABLE 1 corrected mortality of different strains on common thrips (%)
Note that different lower case letters in the same column indicate significant pathogenicity difference between different strains (P < 0.05) by Tukey test.
TABLE 2 corrected mortality of different concentrations of the SB063 strain on common thrips (%)
Note that different lower case letters in the same column indicate significant pathogenicity difference between different strains (P < 0.05) by Tukey test.
Table 3 corrected mortality of SB038 Strain at different concentrations on common thrips (%)
Note that different lower case letters in the same column indicate significant pathogenicity difference among different strains through Tukey test (P < 0.05)
Example 2 identification of Strain SB038
(1) Collection of Strain SB038
The strain SB038 was originally isolated from the soil inlaying the white flag in the union of Sinomonas Guo in the inner Mongolia autonomous region in 2009, and was a Chinese indigenous strain.
And (3) collecting a soil sample, taking 100g of soil 10-15 cm below surface soil during sampling, packaging the soil sample by using a plastic bag, and then bringing the soil sample back to a laboratory for treatment. Sieving a soil sample to remove stone grains and impurities, suspending 10g of clean soil in 90mL of 0.1% Tween-80 solution, shaking uniformly, standing for 15min, diluting 2mL of supernatant in 8mL of 0.05% Tween-80, and preparing into a soil suspension. Inoculating 0.1mL of soil suspension on a Bengal agar medium plate, uniformly pushing the suspension below the surface of the plate by using a triangular glass scraper, culturing for 3-7 days at 25 ℃, then cutting a single colony by using an inoculating loop, inoculating on a PDA plate, and continuously culturing to obtain the beauveria bassiana strain SB 038. The cut mycelium blocks were transplanted on PDA slant, cultured continuously, and stored in a refrigerator at 4 ℃.
(2) Morphological identification
The strain SB038 is on PDA plate at 25 deg.C for 10 days, the diameter of colony is 50.17mm, the aerial hypha in the center of colony is protruded, the middle of late stage has protruded thick powder, concentric circles, hairy edge, white or milky white surface, the culture medium plate has no color change, and has liquid drop (figure 1). The base of the spore-forming cell of the strain SB038 expands, the spore-forming axis is bent in a Z shape, the conidium has a spherical shape and an oval shape, and the diameter of the conidium is 1.7-2.6 μm (figure 2).
(3) Molecular biological identification
The DNA is extracted by a CTAB method, which comprises the following steps:
(a) culturing the strain SB038 on a PDA plate for a week, carefully scraping the mycelium into a mortar, adding liquid nitrogen, and quickly and fully grinding;
(b) rapidly transferring a proper amount of ground mycelium into a centrifuge tube, adding 300 mu L of DNA extract lysate preheated at 65 ℃, fully and uniformly mixing, adding chloroform/isoamylol (24/1) mixed solution with the same volume, uniformly mixing, carrying out water bath at 65 ℃ for 1h, slightly shaking for about 10min, and centrifuging at 12000rpm for 5min at normal temperature;
(c) slowly sucking the supernatant into another new centrifuge tube, slowly adding chloroform/isoamylol (24/1) mixed solution with the same volume for re-extraction, and centrifuging at 12000rpm for 5min at normal temperature;
(d) slowly sucking the supernatant into another new centrifuge tube, adding 1/10 volumes of sodium acetate and equal volume of precooled isopropanol, and standing at room temperature for 15 min;
(e) adding 70% ethanol, washing and precipitating for 2 times;
(f) the liquid transferring gun is tightly attached to the pipe wall to slowly suck out ethanol, and the liquid transferring gun is placed in an ultra-clean workbench to be dried;
(g) the resulting precipitate was dissolved in 50. mu.L of TE;
(h) detecting with 1% agarose gel electrophoresis, and storing the rest extract at-20 deg.C.
The primers used for PCR of fungal DNA are universal primers for the Bloc gene: B5.1F: 5'-cgacccggccaactactttga-3' and B3.1R: 5'-gtcttccagtaccactacgcc-3' are provided. Amplification conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s for 35 cycles; extension at 72 ℃ for 10 min.
The 50 μ L reaction was as follows:
and (3) carrying out 1% agarose gel electrophoresis (containing EB) on the PCR product, wherein the buffer solution is 1 XTAE, the voltage is 150V, the current is 220mA, and Marker DL2000 is used as a molecular weight standard reference substance, and after the electrophoresis is finished, detecting on a gel imager and photographing for recording.
The specific PCR product was sequenced by Shanghai Meiji Biopharmaceutical science and technology Co. Sequence alignment and development tree construction: the sequencing results were searched for related sequences with higher homology using BLAST, and sequence alignment and multiple sequence alignment were performed using ClusterW in MEGA5.1 software. And constructing a Neighbour-join (NJ) phylogenetic tree in the software.
Identification results
(1) As shown in FIG. 3, the electrophoretic band of each lane is clear, and the gene fragment of the strain can be accurately judged to be about 600bp according to the migration distance of the PCR product, wherein only the strains numbered 1 to 24 are shown, and the strain numbered 14 is SB 038.
(2) The Bloc sequence of the obtained genotype of the strain and the related sequence of 3 beauveria bassiana strains downloaded from GenBank are combined to construct a molecular system tree, and the result is shown in FIG. 4, wherein the SB038 strain belongs to Beauveria bassiana and is beauveria bassiana.
The result of the identification by combining morphological characteristics and physiological and biochemical characteristics shows that the strain belongs to the beauveria bassiana, is named as SB038, and is preserved in the Guangdong province microorganism strain preservation center.
Example 3 determination of the virulence of different pesticides on common thrips
Preparing pesticides with different concentration gradients by adopting a pesticide film method, soaking a centrifuge tube (with a hole) of 5cm in a liquid medicine for 2 hours, soaking beans (with no holes at two ends) of 1cm in the liquid medicine for 10 seconds, naturally drying, then picking 20 heads of the common female thrips into the centrifuge tube with the beans, repeatedly feeding the adult thrips in an incubator for 4 times at each concentration, observing for 2 days, and recording the death rate of the adult thrips.
SPSS19.0 software is used for experimental data processing and analysis, single-factor variance analysis is adopted for analyzing toxicity results of biological/chemical pesticides on thrips, Tukey is used for detecting difference significance, and high-toxicity pesticides are screened.
The results show that: after the common thrips is treated by the 5 biochemical pesticides with different concentrations, the corrected mortality rate of the pesticide increases along with the increase of the concentration, wherein when the concentration of the spinetoram is 20mg/L, the mortality rate of the pesticide is up to 91.25 percent, which is far higher than the mortality rates of other 4 medicaments. When the concentration of the lambda-cyhalothrin is 500mg/L, the mortality rate is only 31.25 percent, the toxicity difference of the 5 biochemical pesticides to common thrips is obvious, and the LC of the spinetoram is seen from the lethal middle concentration of the lambda-cyhalothrin to the common thrips (shown in the table)50The value is lower, is 4.2935mg/L, has higher toxicity, and is acetamiprid secondly. Therefore, the spinetoram, the acetamiprid and the beauveria bassiana are selected for compounding subsequently.
Lethal middle concentration (LC) of different agents on common thrips50)
Example 4 compatibility study of pesticides and Beauveria bassiana SB038
1 materials and methods
1.1 test strains
Beauveria bassiana SB 038.
1.2 test Agents
Spinetoram and acetamiprid.
1.3 test Medium
Sabouraud dextrose agar, SDA): 10g of peptone, 20g of agar and 40g of glucose, adding distilled water to reach the constant volume of 1000mL, and sterilizing by high-pressure steam at 121 ℃ for 30 min.
1.4 preparation of spore suspensions of different concentrations
Inoculating Beauveria bassiana SB038 strain on PDA culture medium, activating at 26 deg.C for 10d in biochemical incubator, selecting strong active conidia, inoculating on PDA plate, performing propagation culture in biochemical incubator for 7d, eluting with 0.05% Tween-80 sterile water after mass spore production, stirring with magnetic stirrer, filtering with sterilized double-layer absorbent gauze, examining under biological microscope, counting with blood count plate, and making into 1 × 108spore/mL mother liquor spore suspension, then diluted to 1X 107、1×106、1×105、1×104spore/mL of suspension was ready for use.
1.5 Effect of pesticides on growth of Beauveria bassiana SB038 Strain colonies
Adding spinetoram or acetamiprid into the prepared PDA culture medium, pouring the plate, placing the plate on an ultraclean workbench for ultraviolet irradiation for 2 hours, and preparing the PDA culture medium plate containing the medicine with a proper concentration gradient. Taking a dish of a high-toxicity strain with uniform hypha growth after activation, punching the dish into a fungus cake with the thickness of 5mm by a puncher, placing the fungus cake in the middle of a PDA culture medium plate containing the medicine for inoculation, culturing the fungus cake in a biochemical incubator at the temperature of 26 ℃, measuring the diameter of a bacterial colony by a cross method every day, and measuring for 7 days. 10 replicates were set for each treatment, with medium supplemented with sterile water as a blank.
1.6 Effect of pesticides on spore germination Rate of Beauveria bassiana SB038 Strain
Adding spinetoram or acetamiprid into prepared SDA liquid culture medium and placing into super-clean chamberUltraviolet irradiation is carried out for 2h to prepare a drug-containing SDA liquid culture medium with a proper concentration gradient. Preparing the activated high-toxicity bacterial strain into 1.0 multiplied by 106Adding 2mL of spore suspension into 50mL of liquid culture medium containing SDA with different concentrations, placing in a shaking table at 180rpm and 25 ℃ for shaking culture for 3d, sampling in an ultra-clean workbench every day, performing microscopic examination on spore germination conditions of the strain on a blood counting cell plate, and calculating the spore germination rate. 3 replicates were set for each treatment, with medium supplemented with sterile water as a blank.
1.7 data statistics and analysis
Experimental data analysis was performed using SPSS19.0 software and Tukey was used to check for significance of difference. Graphs were plotted using GraphPad Prism 6 and Excel. 9
2 results and analysis
2.1 Effect of pesticides on growth of Beauveria bassiana SB038 Strain colonies
The results show that after treatment of beauveria bassiana SB038 with different concentrations of spinetoram, the strain diameter decreased with increasing concentration, and the difference was significant (fig. 5). After 3d treatment, the colony diameters of the strains after the high-concentration medicament treatment are smaller than those of a blank control (16.0mm), and the colony diameters of the white muscardine fungi SB038 treated by the spinetoram at the concentrations of 0.625, 1.25, 2.5, 5 and 10mg/L are respectively 14.5, 14, 15 and 12 mm. After 7 days of treatment, the colony diameters of Spinetoram SB038 treated with spinetoram at concentrations of 0.625, 1.25, 2.5, 5 and 10mg/L were 31.5, 31, 30.5, 30, 29.5mm, respectively, lower than that of the blank control (34 mm).
After treating beauveria bassiana SB038 with different concentrations of acetamiprid, the strain diameter decreased with increasing concentration, and the difference was significant (FIG. 6). After 3d treatment, the colony diameters of the strains after treatment with different concentrations of the medicaments are smaller than those of a blank control (16.5mm), and the colony diameters of the acetamiprid-treated beauveria bassiana SB038 at concentrations of 12.5, 25, 50, 100 and 200mg/L are respectively 15.5, 15, 14.5, 14 and 13 mm. After 7 days of treatment, 12.5, 25, 50, 100 and 200mg/L acetamiprid-treated Beauveria bassiana SB038 had colony diameters of 28.5, 28, 27, 24.5, 25mm, respectively, which were lower than the blank (31 mm).
2.2 Effect of spinetoram on spore germination Rate of Beauveria bassiana SB038 Strain
The test result shows that the spore germination rate of the strain after the spinetoram treats the beauveria bassiana SB038 is small and has no significant difference with a blank control (figure 7). After 3 days of treatment, the strains of beauveria bassiana SB038 treated with spinetoram at concentrations of 0.625, 1.25, 2.5, 5 and 10mg/L had spore germination rates of 92.65%, 92.92%, 92.06%, 92.57% and 91.06%, respectively, with no significant difference (94.80%) from the blank control.
The spore germination rate of the acetamiprid-treated beauveria bassiana SB038 strain is small and has no significant difference with that of a blank control (figure 8). After 3 days of treatment, the spore germination rates of the acetamiprid-treated beauveria bassiana SB038 at the concentrations of 12.5, 25, 50, 100 and 200mg/L are respectively 95.83%, 95.93%, 94.78%, 93.33% and 88%, the low-concentration treatment and the blank control have no significant difference (96.02%), and the high-concentration 200mg/L and the blank control have significant difference.
In summary, the following steps: spinetoram has less influence on the strain than acetamiprid, no matter whether the bacterial strain grows in colony or spore germination rate. In this experiment, after 2d of treatment, there was no significant difference in spore germination of spinetoram at each concentration (0.625, 1.25, 2.5, 5 and 10mg/L) for beauveria sp 038 compared to the blank control; after treatment of the acetamiprid at each concentration, the difference of spore germination of beauveria bassiana SB038 and the blank control is obvious, and after treatment for 3d, the difference of the high concentration of 200mg/L and the blank control is obvious. After the bacterial colony growth of the bacterial strain is treated for 7 days, the bacterial colony growth of spinetoram to beauveria bassiana SB038 at various concentrations (0.625, 1.25, 2.5, 5 and 10mg/L) is different from that of a blank control, but the difference is not large, the bacterial colony growth is slowed down by 13% in the highest concentration treatment, and the bacterial colony growth is slowed down by only 7% in the low concentration treatment; while the growth of the high concentration colonies treated with the pyridine 10 acetamiprid was reduced by 21%. Therefore, spinetoram was selected for subsequent testing.
Example 5 synergistic Effect of Beauveria bassiana SB038 and spinetoram on the control of Cisii vulgaris
2.1 materials and methods
2.1.1 sources of test insects
Common thrips is 1.1.
2.1.2 test strains
SB038。
2.1.3 determination of toxicity of insecticides on common thrips
Preparing pesticides with different concentration gradients by adopting a pesticide film method, soaking a centrifuge tube (with a hole) of 5cm in a liquid medicine for 2 hours, soaking beans (with no holes at two ends) of 1cm in the liquid medicine for 10 seconds, naturally drying, then picking 20 heads of the common female thrips into the centrifuge tube with the beans, repeatedly feeding the adult thrips in an incubator for 4 times at each concentration, observing for 2 days, and recording the death rate of the adult thrips.
2.1.4 method for determining toxicity of two compounded agents of high toxicity bacterial strain and spinetoram to common thrips
0.625, 1.25, 2.5, 5, 10mg/L of spinetoram and 1 × 10 are respectively prepared4、1×105、1×106、1×107、1×108spore/mL of high-toxicity bacterial strain spore suspension is reserved, and prepared spinetoram with various concentrations is combined with bacterial strains with various concentrations in pairs respectively for toxicity determination. The virulence determination method is as in 1.3.2.
2.1.5 data statistics and analysis
SPSS19.0 software is used for processing and analyzing test data, single-factor analysis of variance is used for analyzing results of common thistle mortality, and Tukey is used for testing the significance of difference. The data calculation formula refers to huang angetal (2013), and the specific formula is as follows:
Me＝Ma+Mb×(1-Ma)
in the formula, the terms have the following meanings:
and Ma: actual corrected mortality of spinetoram alone on common thrips;
mb: actual corrected mortality of highly virulent strains alone against common thrips;
and Mab: the actual corrected mortality rate of the compound agent of the fungus medicine to the common thrips;
me: the expected mortality rate of the bactericide compounding agent to the common thrips;
according to the formula, the χ 2 value of the microbial medicine compound agent is respectively calculated, and the P value corresponding to df being 1 is searched in the χ 2 table and is 3.841.
When the calculated chi 2 is less than 3.841, the compound of the bacterial strain and the spinetoram shows antagonistic action;
when the calculated Chi 2 is more than 3.841, the combination of the bacterial strain and the spinetoram shows a synergistic effect.
2.2 results of the experiment
The toxicity result of the synergistic effect of beauveria bassiana SB038 and spinetoram on common thrips: when different concentrations of spinetoram (0.625, 1.25, 2.5, 5 and 10mg/L) are used for treating the common thrips, the corrected mortality rate of the 2d is 11.25%, 28.75%, 37.5%, 45% and 66.25% respectively; beauveria bassiana (1X 10) with different concentrations is used alone4、1×105、1×106、1×107、1×108spores/mL) treated common thrips, the corrected mortality for 7d was 15%, 26.67%, 40%, 68.33%, 91.07%, respectively.
The lethality of the spinetoram with different concentrations and beauveria bassiana with different concentrations in combination on common thrips is obviously higher than that of each single dose (table 3). In the toxicity determination of the combined action of the spinetoram and the beauveria bassiana SB038 on the common thrips, the spinetoram and low concentration (1 × 10) are respectively added at the later stage of treatment5、1×104spore/mL) beauveria brookfield SB038 combination showed synergistic effect. After 7 days of treatment, the highest mortality combination was 2.5mg/L of spinetoram with 1X 10 spinetoram in synergistic combination5spores/mL Beauveria bassiana SB038, 96.67%.
TABLE 3 virulence of Beauveria bassiana SB038 and spinetoram on thrips vulgaris
Note: the data in the table are mean values +/-standard errors, through Tukey test, different lower case letters indicate that the difference between different combined concentrations at the same time is obvious (P is less than 0.05), and numerical values in brackets indicate that chi-square values chi are obtained through calculation2Denotes a combination having a synergistic effect.
Claims (7)
1. A combined insecticide containing spinetoram and beauveria bassiana as effective components, wherein the beauveria bassiana is beauveria bassiana strain SB038 with the preservation number of GDMCC NO: 61303, respectively; the pesticide is used for preventing and treating common thrips.
2. The pesticide of claim 1, wherein said beauveria bassiana is in the form of a suspension or a spore powder of spores of beauveria bassiana.
3. The pesticide of claim 2, wherein the beauveria bassiana and the spinetoram are used in a ratio of: the final concentration of Beauveria bassiana is 1 × 104~1×108conidia/mL, preferably 1X 104~1×107Conidium per mL, and the final concentration of spinetoram is 0.625-10 mg/L, preferably 1.25-10 mg/L.
4. The pesticide of claim 3, wherein said beauveria bassiana and spinetoram are in a ratio of 1 x 104~1×107conidia/mL: 1.25-10 mg/L, preferably the ratio of beauveria bassiana to spinetoram is 1 multiplied by 105~1×107conidia/mL: 2.5-10 mg/L.
5. The pesticide of claim 4, wherein said beauveria bassiana and spinetoram are in a ratio of 1 x 105~1×107conidia/mL: 2.5-5 mg/L.
6. The pesticide of claim 3, wherein said beauveria bassiana and spinetoram are in a ratio of 1 x 105conidia/mL: 2.5 mg/L.
7. Use of the pesticide according to any one of claims 1 to 4 for controlling common thistle pests.
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