WO2022217475A1 - Method for improving resistance of bombyx mori to phoxim pesticides - Google Patents
Method for improving resistance of bombyx mori to phoxim pesticides Download PDFInfo
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- WO2022217475A1 WO2022217475A1 PCT/CN2021/087085 CN2021087085W WO2022217475A1 WO 2022217475 A1 WO2022217475 A1 WO 2022217475A1 CN 2021087085 W CN2021087085 W CN 2021087085W WO 2022217475 A1 WO2022217475 A1 WO 2022217475A1
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- spp
- phoxim
- resistance
- sphingomonas
- pesticides
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 87
- ATROHALUCMTWTB-OWBHPGMISA-N phoxim Chemical compound CCOP(=S)(OCC)O\N=C(\C#N)C1=CC=CC=C1 ATROHALUCMTWTB-OWBHPGMISA-N 0.000 title claims abstract description 56
- 229950001664 phoxim Drugs 0.000 title claims abstract description 55
- 239000000575 pesticide Substances 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 27
- 235000008708 Morus alba Nutrition 0.000 claims abstract description 43
- 240000000249 Morus alba Species 0.000 claims abstract description 43
- 241000894006 Bacteria Species 0.000 claims abstract description 35
- 230000001580 bacterial effect Effects 0.000 claims abstract description 18
- 238000005507 spraying Methods 0.000 claims abstract 2
- 239000007788 liquid Substances 0.000 claims description 40
- 241001467578 Microbacterium Species 0.000 claims description 28
- 241000589291 Acinetobacter Species 0.000 claims description 27
- 241000607715 Serratia marcescens Species 0.000 claims description 25
- 241001524188 Glutamicibacter nicotianae Species 0.000 claims description 19
- 241001135759 Sphingomonas sp. Species 0.000 claims description 19
- 241000588748 Klebsiella Species 0.000 claims description 17
- 241000192097 Staphylococcus sciuri Species 0.000 claims description 16
- 241000736131 Sphingomonas Species 0.000 claims description 14
- 230000003287 optical effect Effects 0.000 claims description 12
- 241000191940 Staphylococcus Species 0.000 claims description 9
- 241000186063 Arthrobacter Species 0.000 claims description 7
- 241000697618 Klebsiella michiganensis Species 0.000 claims description 6
- 241000555745 Sciuridae Species 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 claims 1
- 238000001035 drying Methods 0.000 claims 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 abstract description 12
- 239000002671 adjuvant Substances 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 30
- 244000005700 microbiome Species 0.000 description 7
- 241000238631 Hexapoda Species 0.000 description 6
- 108010022752 Acetylcholinesterase Proteins 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 4
- 239000007921 spray Substances 0.000 description 4
- 102000012440 Acetylcholinesterase Human genes 0.000 description 3
- 229940022698 acetylcholinesterase Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000007654 immersion Methods 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000255777 Lepidoptera Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000009366 sericulture Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000255794 Bombyx mandarina Species 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008560 physiological behavior Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000009394 selective breeding Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
Definitions
- the invention relates to a method for improving the resistance of silkworms to phoxim pesticides, in particular to a method for improving silkworms' resistance to phoxim pesticides by feeding mixed microbial inoculants.
- the silkworm ( Bombyx mori ) belongs to the family Lepidoptera, and originated from the ancient wild silkworm in China. Bombyx mori is a model insect of Lepidoptera and an important economic insect. Under the long-term artificial selection, the silk production performance of silkworm has been greatly improved. At the same time, due to the long-term indoor breeding, the chance of exposure to chemical pesticides and the bad environment in the wild is relatively small, and its resistance to the bad environment is weak. Phoxim pesticides have the characteristics of high efficiency, spectrum and low toxicity, and are widely used in the control of agricultural and forestry pests. Due to the long residual period of phoxim pesticides, silkworms often eat mulberry leaves with phoxim drugs and cause poisoning in production.
- the applicant has disclosed a method for reducing the sensitivity of silkworm type 1 acetylcholinesterase to phoxim, which specifically includes the following steps: (1) cloning of silkworm type 1 acetylcholinesterase gene (Bmace1); Site-directed mutagenesis, cloned and sequenced the product, and obtained mutated Bmace1; (3) The silkworm type 1 acetylcholinesterase with reduced sensitivity to phoxim was obtained by expressing the mutated Bmace1 using a eukaryotic expression system. In this method, three important sites in Bmace1 are mutated to change the encoded amino acids, so that the translated protein is less sensitive to phoxim pesticides.
- the novel silkworm type 1 acetylcholinesterase obtained by this method is: Transgenic breeding of new silkworm varieties resistant to phoxim pesticides provides material. But this method is too complicated.
- the insect gut is a complex system in which many microorganisms live. These microorganisms play important roles in insect physiological activities and behaviors, such as nutrient absorption, immune response, pathogen defense, mating preference, etc.
- the purpose of the present invention is to provide a method for improving the resistance of silkworms to phoxim pesticides, so as to promote the healthy development of silk industry.
- a method for improving the resistance of silkworms to phoxim pesticides comprising the following steps: feeding silkworms with mulberry leaves with combined bacteria to improve the silkworms' resistance to phoxim pesticides.
- the invention discloses the application of the combined bacteria in improving the resistance of silkworms to phoxim pesticides; or the application of the combined bacteria in preparing feed for improving the silkworms' resistance to phoxim pesticides.
- the combined bacteria are Sphingomonas spp., Staphylococcus sciuri spp., Microbacterium spp., Klebsiella spp.
- Serratia marcescens spp. Acinetobacter spp.
- Arthrobacter nicotianae spp. Several combinations of Serratia marcescens spp., Acinetobacter spp., Arthrobacter nicotianae spp.; preferably, the combination is Sphingomonas spp. , Staphylococcus sciuri spp., Microbacterium spp., Klebsiella michiganensis spp., Serratia marcescens spp., Acinetobacter spp .) and Arthrobacter nicotianae spp..
- Sphingomonas spp. is Sphingomonas A28241 bacteria ( Sphingomonas spp. ); Staphylococcus sciuri spp. is Staphylococcus squirrels LH-T5 ( Staphylococcus sciuri spp. ); Microbacterium spp. is Microbacterium HBUM178903 ( Microbacterium spp. ); Klebsiella spp. is Klebsiella michiganensis spp. ; Serratia marcescens spp. is Serratia marcescens N1.8 ( Serratia marcescens spp. ); Acinetobacter spp. is Acinetobacter IILSFSC204 ( Acinetobacter spp. ); Arthrobacter nicotianae spp.) is Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ).
- the combined bacteria solution is sprayed on suitable mature mulberry leaves to obtain mulberry leaves with combined bacteria; wherein the optical density (OD 600 ) of the combined bacteria solution is 0.3-0.5, preferably, the optical density (OD 600 ) is 0.4 .
- the dosage of 0.4-0.6 L is added according to 50 kg of mulberry leaves, and the combined bacteria liquid is sprayed on the suitable mature mulberry leaves, and then air-dried naturally to obtain the mulberry leaves with the combined bacteria.
- the Sphingomonas ( Sphingomonas spp.) liquid, the Staphylococcus ( Sciuri spp.) liquid, the Microbacterium ( Microbacterium spp.) liquid, the Klebsiella ( Klebsiella spp.) liquid, Serratia marcescens spp. liquid, Acinetobacter spp. liquid and Arthrobacter nicotianae spp. liquid are mixed to obtain a combined bacterial liquid; preferably, Sphingomonas ( Sphingomonas ) spp.) solution, Staphylococcus sciuri spp. solution, Microbacterium spp. solution, Klebsiella spp.
- the optical density (OD 600 ) of the Acinetobacter ( Acinetobacter spp.) solution and the Arthrobacter nicotianae spp. solution is 0.3-0.5, preferably, the optical density (OD 600 ) is 0.4.
- the volume ratio of Serratia marcescens spp., Acinetobacter spp., and Arthrobacter nicotianae spp. is 1: (0.9-1.1): (0.4-0.6): (0.9- 1.1): (0.4-0.6): (0.4-0.6): (0.5-0.7), preferably 1:1:0.5:1:0.5:0.5:0.6.
- the invention develops microbial adjuvants for silkworms, exerts the biological functions of various intestinal microorganisms, and improves the resistance of silkworms to phoxim pesticides in combination with the structural characteristics of the intestinal flora of silkworms; the combined bacterial solution is fully mixed before use.
- Evenly spray the appropriate mature leaves the small amount of microbial adjuvant will affect the use effect, and the large amount will increase the use cost and affect the resistance of silkworms; after the mulberry leaves are naturally dried and fed to silkworms, it will not affect the digestion and absorption of silkworm feed.
- the feeding amount of mulberry leaves is the same as the feeding amount under normal circumstances, which does not affect the use effect. For example, feeding silkworms for 2 to 5 days can significantly improve the resistance of silkworms to phoxim pesticides.
- the invention adopts the immersion method to detect the resistance of silkworms to phoxim pesticides, and counts the survival rate of the silkworms of the test group and the control group after feeding the mulberry leaves treated with sub-lethal dose of phoxim.
- the lethal concentration (LC 50 ) of phoxim on day 3 (24 h) of the 5th instar silkworm was 7.86 ⁇ g/mL (Peng GD, Wang JM, Ma L, Wang YH, Cao YQ, Shen WD, Li B. Transcriptional characteristics of acetylcholinesterase genes in domestic silkworms ( Bombyx mori ) exposed to phoxim. Pesticide Biochemistry and Physiology, 2011, 101: 154-158.), the concentration of phoxim in the immersion method of the present invention is 2ug/mL.
- the present invention has the following advantages.
- the present invention utilizes microbial bacteria as an adjuvant to be sprayed on mulberry leaves to feed silkworms, which can significantly improve the resistance of silkworms to phoxim pesticides, and the effect is remarkable.
- Microbial auxiliaries are suitable for all ages of silkworm and are versatile and innovative.
- the microbial adjuvant used in the method disclosed in the present invention has the advantages of low dosage, low cost, environmental protection and safety; no need to change the production law, convenient operation, and easy popularization and use.
- the method for improving silkworm resistance to phoxim pesticide disclosed in the present invention is as follows.
- (1) Configuration of the combined bacterial solution the Sphingomonas ( Sphingomonas spp.) solution, the Staphylococcus ( Sciuri spp.) solution, the Microbacterium ( Microbacterium spp.) solution, the Klebsiella ( Klebsiella ) solution spp.) solution, Serratia marcescens ( Serratia marcescens spp.) solution, Acinetobacter ( Acinetobacter spp.) solution, and Arthrobacter nicotianae spp. solution were diluted with water to an optical density (OD 600 ) of 0.3 ⁇ 0.5; and then mixed to obtain a combined bacterial solution, whose optical density (OD 600 ) was also 0.3 to 0.5.
- OD 600 optical density
- Feeding silkworms add 0.4-0.6L of the combined bacterial solution according to 50 kg of mulberry leaves, spray the combined bacterial solution prepared in step (1) on the mature leaves, and feed the silkworms after the mulberry leaves are naturally dried. Improve silkworm resistance to phoxim pesticides.
- All the bacteria in the invention are conventional microorganisms, and the resistance of silkworms to phoxim pesticides can be effectively improved without other reagents and without changing the feeding rule.
- the present invention will be further described below in conjunction with examples.
- the test method of optical density (OD 600 ) is to detect the absorbance (OD 600 ) of the bacterial liquid at a wavelength of 600 nm using an ultraviolet spectrophotometer.
- Embodiment 1 a method for improving silkworm resistance to phoxim pesticides.
- Bombyx mori variety Suxiu ⁇ Chunfeng, which was bred and preserved by the Sericulture Research Institute of Soochow University.
- Sphingomonas A28241 Sphingomonas spp.
- Staphylococcus sciuri LH-T5 Staphylococcus sciuri spp.
- Microbacterium HBUM178903 Microbacterium spp.
- Klebsiella michiganensis spp . Serratia marcescens N1.8
- Serratia marcescens spp. Acinetobacter IILSSFSC204 ( Acinetobacter spp. ), Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ) are stored in the resource bank of the Sericulture Research Institute of Soochow University.
- the cryopreserved bacteria were recovered and cultured in LB liquid medium (Shanghai Shenggong) for 12 h (30 °C, 200 rpm), and then diluted with water to an optical density (OD 600 ) of 0.4.
- the above seven strains were operated respectively to obtain seven groups of bacterial liquids with an optical density (OD 600 ) of 0.4, and then the seven bacterial liquids were prepared according to Sphingomonas A28241 ( Sphingomonas spp. ) liquid, Staphylococcus squirrel LH-T5 ( Staphylococcus sciuri spp. ) liquid, Microbacterium HBUM178903 ( Microbacterium spp.
- Phoxim pesticide (CAS number: 14816-18-3), a product of Sigma company.
- the combined bacterial liquid was shaken well before use, and 0.5 L dose was added per 50 kg of mulberry leaves to spray suitable mature leaves of the 5th instar.
- the mulberry variety was Yu 711, and the mulberry leaves with the combined bacteria were obtained after the mulberry leaves were naturally dried.
- the amount of mulberry given is the same as that used for normal feeding silkworms.
- the 5th instar silkworm larvae were tested for drug resistance on the 3rd day by liquid immersion method. There were 30 silkworms in each experimental group, with three replicates in each group, totaling 90 silkworms.
- the specific test method is.
- the mature leaves of the 5th instar stage used in the experiment were not treated with any reagents. They were washed and dried, and then treated with bacteria or phoxim, or only sprayed with the same amount of water.
- test group The mulberry leaves with the combined bacteria were fed to the 5th instar silkworm, and the mulberry amount was the same as that of the normal silkworm feeding, for a total of 3 days. A total of 4 days, that is, feeding to 5 years of age and 7 days.
- a comparison group Use a bacterial solution (optical density (OD 600 ) of 0.4) to spray the 5-year-old suitable mature leaves at a dose of 0.5 L per 50 kg of mulberry leaves.
- the mulberry variety is Yu 711. After the mulberry leaves are naturally dried, a The mulberry leaves of single bacteria were fed to the 5th instar silkworm, and the amount of mulberry was the same as that of the normal feeding silkworm, for a total of 3 days. days, that is, feeding to 5 years of age for 7 days.
- Ck control group
- Sp Sphingomonas A28241
- Ss Staphylococcus squirrel LH-T5
- Km Klebsiella Michigan
- Mi Microbacterium HBUM178903
- Sm Serratia marcescens N1 .8
- Ac Acinetobacter IILSFSC204
- An Arthrobacter Dc-06
- Mb Combination bacteria (Sp: Ss: Km: Mi: Sm: Ac: An volume ratio of 1: 1: 0.5: 1: 0.5: 0.5:0.6).
- Table 1 show that the silkworms fed Sphingomonas, Staphylococcus squirrel, Klebsiella Michigan, and mulberry leaves combined with bacterial liquid were fed phoxim-treated mulberry leaves, and the 24 h, 48 h, The survival rates at 72 h and 96 h were significantly higher than those in the control group. Among them, the survival rate of the combined bacteria solution group at 96 h was significantly higher than that of the single bacteria solution treatment group and the control group. In particular, only one of the 90 silkworms died.
- the example of feeding microbial inoculants to silkworms shows that 50 kg of mulberry leaves added with 0.4-0.6L doses of mulberry leaves sprayed with mixed inoculants can significantly improve the silkworm's resistance to phoxim after feeding silkworms.
- Sphingomonas A28241 Sphingomonas spp. liquid
- Staphylococcus squirrel LH-T5 Staphylococcus sciuri spp.
- Microbacterium HBUM178903 Microbacterium spp.
- Serratia marcescens N1.8 Serratia marcescens spp.
- Acinetobacter IILSFSC204 Acinetobacter spp.
- Arthrobacter Dc-06 Arthrobacter nicotianae spp.
- the invention utilizes the function of intestinal microorganisms to degrade and metabolize pesticides, and develops a low-cost, environmentally friendly and effective method for improving the resistance of silkworms to phoxim pesticides.
- the microorganisms in the intestinal tract can help host insects degrade and metabolize pesticides. Thereby increasing the resistance of host insects to pesticides.
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Abstract
A method for improving the resistance of Bombyx mori to phoxim pesticides, which employs the feeding and spraying of a mixed microbial adjuvant, so that the survival rate of Bombyx mori when low-dose phoxim pesticides are applied may be improved. An application of a combination of bacteria in improving the resistance of Bombyx mori to phoxim pesticides, wherein mulberry leaves with the combination of bacteria are used to feed Bombyx mori, so that the resistance of Bombyx mori to phoxim pesticides may be improved. An application of a combination of bacteria in preparing a feed for improving the resistance of Bombyx mori to phoxim pesticides, wherein mulberry leaves sprayed with a microbial mixed bacterial agent are used as a feed to feed Bombyx mori. The microbial adjuvant used in the foregoing manner has a small dosage, is low cost and environmentally friendly and safe without changing production rules, is easy to operate, and is suitable for popularization and application in silkworm areas in China.
Description
本发明涉及一种提高家蚕对辛硫磷农药抗性的方法，具体涉及一种通过添食混合微生物菌剂提高家蚕对辛硫磷农药抗性的方法。The invention relates to a method for improving the resistance of silkworms to phoxim pesticides, in particular to a method for improving silkworms' resistance to phoxim pesticides by feeding mixed microbial inoculants.
家蚕（
Bombyx mori）属于鳞翅目蚕蛾科昆虫，起源于中国的古野桑蚕。家蚕是鳞翅目模式昆虫，也是重要的经济昆虫。在漫长的人工选择下，家蚕的产丝性能在得到大幅度提高的同时，由于长期在室内饲养，接触到化学农药及野外不良环境的机会相对较少，其对不良环境的抗性较弱。辛硫磷农药具有高效、光谱和低毒的特征，广泛应用于防治农业、林业害虫。由于辛硫磷农药残毒期较长，生产上经常出现家蚕误食带有辛硫磷药的桑叶而引起中毒的现象。家蚕接触辛硫磷，其正常的生长发育和生理代谢受到影响，家蚕的营养利用效率和经济性状也因此受到影响。所以生产上迫切需要采取必要措施提高家蚕对辛硫磷药的抗性，实现我国蚕丝健康发展。
The silkworm ( Bombyx mori ) belongs to the family Lepidoptera, and originated from the ancient wild silkworm in China. Bombyx mori is a model insect of Lepidoptera and an important economic insect. Under the long-term artificial selection, the silk production performance of silkworm has been greatly improved. At the same time, due to the long-term indoor breeding, the chance of exposure to chemical pesticides and the bad environment in the wild is relatively small, and its resistance to the bad environment is weak. Phoxim pesticides have the characteristics of high efficiency, spectrum and low toxicity, and are widely used in the control of agricultural and forestry pests. Due to the long residual period of phoxim pesticides, silkworms often eat mulberry leaves with phoxim drugs and cause poisoning in production. Bombyx mori exposed to phoxim, its normal growth and development and physiological metabolism were affected, and the nutrient utilization efficiency and economic traits of silkworm were also affected. Therefore, it is urgent to take necessary measures in production to improve the resistance of silkworms to phoxim, and to realize the healthy development of silk in my country.
申请人已经公开了一种降低家蚕1型乙酰胆碱酯酶对辛硫磷敏感性的方法，具体包括以下步骤：（1）家蚕1型乙酰胆碱酯酶基因(Bmace1)的克隆；（2）对Bmace1进行定点突变，对产物克隆与测序验证，获得突变的Bmace1；（3）利用真核表达系统对突变的Bmace1进行表达获得对辛硫磷敏感性降低的家蚕1型乙酰胆碱酯酶。该方法通过对Bmace1中3个重要位点进行突变，使其编码的氨基酸发生改变，使其翻译的蛋白对辛硫磷农药的敏感性降低，由此方法获得的新型家蚕1型乙酰胆碱酯酶为转基因培育抗辛硫磷农药家蚕新品种提供了素材。但是该方法过于复杂。昆虫肠道是一个复杂的系统，其中生活着许多微生物。这些微生物在昆虫的生理活动和行为中起着重要的作用，例如营养吸收，免疫反应，病原体防御，交配偏好等。但是现有技术未见微生物与家蚕对辛硫磷农药抗性之间关系的报道。The applicant has disclosed a method for reducing the sensitivity of silkworm type 1 acetylcholinesterase to phoxim, which specifically includes the following steps: (1) cloning of silkworm type 1 acetylcholinesterase gene (Bmace1); Site-directed mutagenesis, cloned and sequenced the product, and obtained mutated Bmace1; (3) The silkworm type 1 acetylcholinesterase with reduced sensitivity to phoxim was obtained by expressing the mutated Bmace1 using a eukaryotic expression system. In this method, three important sites in Bmace1 are mutated to change the encoded amino acids, so that the translated protein is less sensitive to phoxim pesticides. The novel silkworm type 1 acetylcholinesterase obtained by this method is: Transgenic breeding of new silkworm varieties resistant to phoxim pesticides provides material. But this method is too complicated. The insect gut is a complex system in which many microorganisms live. These microorganisms play important roles in insect physiological activities and behaviors, such as nutrient absorption, immune response, pathogen defense, mating preference, etc. However, there is no report on the relationship between microorganisms and silkworm resistance to phoxim pesticides in the prior art.
本发明的目的是提供一种提高家蚕对辛硫磷农药抗性的方法，以促进蚕丝业的健康发展。The purpose of the present invention is to provide a method for improving the resistance of silkworms to phoxim pesticides, so as to promote the healthy development of silk industry.
为达到上述发明的目的，本发明采用的技术方案是。In order to achieve the purpose of the above invention, the technical solution adopted in the present invention is:
一种提高家蚕对辛硫磷农药抗性的方法，包括以下步骤，将带有组合菌的桑叶添食家蚕，提高家蚕对辛硫磷农药的抗性。A method for improving the resistance of silkworms to phoxim pesticides, comprising the following steps: feeding silkworms with mulberry leaves with combined bacteria to improve the silkworms' resistance to phoxim pesticides.
本发明公开了组合菌在提高家蚕对辛硫磷农药的抗性中的应用；或者组合菌在制备提高家蚕对辛硫磷农药抗性的饲料中的应用。The invention discloses the application of the combined bacteria in improving the resistance of silkworms to phoxim pesticides; or the application of the combined bacteria in preparing feed for improving the silkworms' resistance to phoxim pesticides.
本发明中，组合菌为鞘氨醇单胞菌（
Sphingomonas spp.）、松鼠葡萄球菌（
Staphylococcus sciuri spp.）、微杆菌（
Microbacterium spp.）、克雷伯氏菌（
Klebsiellaspp.）、粘质沙雷菌（
Serratia marcescens spp.）、不动杆菌（
Acinetobacter spp.）、节杆菌（
Arthrobacter nicotianae spp.）中的几种组合；优选的，组合菌为鞘氨醇单胞菌（
Sphingomonas spp.）、松鼠葡萄球菌（
Staphylococcus sciuri spp.）、微杆菌（
Microbacterium spp.）、密歇根克雷伯氏菌（
Klebsiella michiganensis spp.）、粘质沙雷菌（
Serratia marcescens spp.）、不动杆菌（
Acinetobacter spp.）和节杆菌（
Arthrobacter nicotianae spp.）。
In the present invention, the combined bacteria are Sphingomonas spp., Staphylococcus sciuri spp., Microbacterium spp., Klebsiella spp. Several combinations of Serratia marcescens spp., Acinetobacter spp., Arthrobacter nicotianae spp.; preferably, the combination is Sphingomonas spp. , Staphylococcus sciuri spp., Microbacterium spp., Klebsiella michiganensis spp., Serratia marcescens spp., Acinetobacter spp .) and Arthrobacter nicotianae spp..
进一步的，本发明的菌种具体如下：鞘氨醇单胞菌（
Sphingomonas spp.）为鞘氨醇单胞菌A28241菌（
Sphingomonas
spp.）；松鼠葡萄球菌（
Staphylococcus sciuri spp.）为松鼠葡萄球菌LH-T5（
Staphylococcus
sciuri spp.）；微杆菌（
Microbacterium spp.）为微杆菌HBUM178903（
Microbacterium
spp.）；克雷伯氏菌（
Klebsiellaspp.）为密歇根克雷伯氏菌（
Klebsiella michiganensis spp.）；粘质沙雷菌（
Serratia marcescens spp.）为粘质沙雷菌N1.8（
Serratia
marcescens spp.）；不动杆菌（
Acinetobacter
spp.）为不动杆菌IILSFSC204（
Acinetobacter
spp.）；节杆菌（
Arthrobacter nicotianae spp.）为节杆菌Dc-06（
Arthrobacter
nicotianae spp.）。
Further, the bacterial species of the present invention are specifically as follows: Sphingomonas spp. is Sphingomonas A28241 bacteria ( Sphingomonas spp. ); Staphylococcus sciuri spp. is Staphylococcus squirrels LH-T5 ( Staphylococcus sciuri spp. ); Microbacterium spp. is Microbacterium HBUM178903 ( Microbacterium spp. ); Klebsiella spp. is Klebsiella michiganensis spp. ; Serratia marcescens spp. is Serratia marcescens N1.8 ( Serratia marcescens spp. ); Acinetobacter spp. is Acinetobacter IILSFSC204 ( Acinetobacter spp. ); Arthrobacter nicotianae spp.) is Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ).
本发明中，将组合菌液喷洒适熟桑叶，得到带有组合菌的桑叶；其中组合菌液的光密度（OD
600）为0.3～0.5，优选的，光密度（OD
600）为0.4。
In the present invention, the combined bacteria solution is sprayed on suitable mature mulberry leaves to obtain mulberry leaves with combined bacteria; wherein the optical density (OD 600 ) of the combined bacteria solution is 0.3-0.5, preferably, the optical density (OD 600 ) is 0.4 .
本发明中，按照50kg桑叶添加0.4～0.6L剂量，将组合菌液喷洒适熟桑叶，然后自然晾干，得到带有组合菌的桑叶。In the present invention, the dosage of 0.4-0.6 L is added according to 50 kg of mulberry leaves, and the combined bacteria liquid is sprayed on the suitable mature mulberry leaves, and then air-dried naturally to obtain the mulberry leaves with the combined bacteria.
本发明中，将鞘氨醇单胞菌（
Sphingomonas spp.）液、松鼠葡萄球菌（
Staphylococcus sciuri spp.）液、微杆菌（
Microbacterium spp.）液、克雷伯氏菌（
Klebsiellaspp.）液、粘质沙雷菌（
Serratia marcescens spp.）液、不动杆菌（
Acinetobacter spp.）液和节杆菌（
Arthrobacter nicotianae spp.）液混合，得到组合菌液；优选的，鞘氨醇单胞菌（
Sphingomonas spp.）液、松鼠葡萄球菌（
Staphylococcus sciuri spp.）液、微杆菌（
Microbacterium spp.）液、克雷伯氏菌（
Klebsiellaspp.）液、粘质沙雷菌（
Serratia marcescens spp.）液、不动杆菌（
Acinetobacter spp.）液、节杆菌（
Arthrobacter nicotianae spp.）液的光密度（OD
600）为0.3～0.5，优选的，光密度（OD
600）为0.4。优选的，鞘氨醇单胞菌（
Sphingomonas spp.）液、松鼠葡萄球菌（
Staphylococcus sciuri spp.）液、微杆菌（
Microbacterium spp.）液、克雷伯氏菌（
Klebsiellaspp.）液、粘质沙雷菌（
Serratia marcescens spp.）液、不动杆菌（
Acinetobacter spp.）液、节杆菌（
Arthrobacter nicotianae spp.）液的体积比1∶（0.9～1.1）∶（0.4～0.6）∶（0.9～1.1）∶（0.4～0.6）∶（0.4～0.6）∶（0.5～0.7），优选为1∶1∶0.5∶1∶0.5∶0.5∶0.6。
In the present invention, the Sphingomonas ( Sphingomonas spp.) liquid, the Staphylococcus ( Sciuri spp.) liquid, the Microbacterium ( Microbacterium spp.) liquid, the Klebsiella ( Klebsiella spp.) liquid, Serratia marcescens spp. liquid, Acinetobacter spp. liquid and Arthrobacter nicotianae spp. liquid are mixed to obtain a combined bacterial liquid; preferably, Sphingomonas ( Sphingomonas ) spp.) solution, Staphylococcus sciuri spp. solution, Microbacterium spp. solution, Klebsiella spp. solution, Serratia marcescens spp. solution, The optical density (OD 600 ) of the Acinetobacter ( Acinetobacter spp.) solution and the Arthrobacter nicotianae spp. solution is 0.3-0.5, preferably, the optical density (OD 600 ) is 0.4. Preferably, Sphingomonas ( Sphingomonas spp.) liquid, Staphylococcus ( Sciuri spp.) liquid, Microbacterium ( Microbacterium spp.) liquid, Klebsiella ( Klebsiella spp.) liquid, mucilage The volume ratio of Serratia marcescens spp., Acinetobacter spp., and Arthrobacter nicotianae spp. is 1: (0.9-1.1): (0.4-0.6): (0.9- 1.1): (0.4-0.6): (0.4-0.6): (0.5-0.7), preferably 1:1:0.5:1:0.5:0.5:0.6.
本发明开发了蚕用微生物助剂，发挥各肠道微生物的生物学功能，结合家蚕肠道菌群结构特征，提高家蚕对辛硫磷农药的抗性；将组合菌液在使用前充分混匀，均匀喷洒适熟叶，微生物助剂量少会影响使用效果，量多会增加使用成本且影响家蚕抗性；待桑叶自然晾干后饲喂家蚕，不影响家蚕饲料的消化和吸收。桑叶饲喂量和正常情况下饲喂量一样，不影响使用效果，比如添食家蚕2～5天，即可显著提高家蚕对辛硫磷农药的抗性。The invention develops microbial adjuvants for silkworms, exerts the biological functions of various intestinal microorganisms, and improves the resistance of silkworms to phoxim pesticides in combination with the structural characteristics of the intestinal flora of silkworms; the combined bacterial solution is fully mixed before use. , Evenly spray the appropriate mature leaves, the small amount of microbial adjuvant will affect the use effect, and the large amount will increase the use cost and affect the resistance of silkworms; after the mulberry leaves are naturally dried and fed to silkworms, it will not affect the digestion and absorption of silkworm feed. The feeding amount of mulberry leaves is the same as the feeding amount under normal circumstances, which does not affect the use effect. For example, feeding silkworms for 2 to 5 days can significantly improve the resistance of silkworms to phoxim pesticides.
本发明采用浸液法检测家蚕对辛硫磷农药的抗性，统计试验组和对照组家蚕在添食亚致死剂量辛硫磷处理的桑叶后存活率。辛硫磷对5龄第3天（24 h）家蚕的半致死浓度（LC
50）是7.86 μg/mL（Peng G D, Wang J
M, Ma L, Wang Y H, Cao Y Q, Shen W D, Li B. Transcriptional characteristics of
acetylcholinesterase genes in domestic silkworms (
Bombyx mori) exposed
to phoxim. Pesticide Biochemistry and Physiology, 2011, 101: 154-158.），本发明浸液法中辛硫磷浓度为2ug/mL。
The invention adopts the immersion method to detect the resistance of silkworms to phoxim pesticides, and counts the survival rate of the silkworms of the test group and the control group after feeding the mulberry leaves treated with sub-lethal dose of phoxim. The lethal concentration (LC 50 ) of phoxim on day 3 (24 h) of the 5th instar silkworm was 7.86 μg/mL (Peng GD, Wang JM, Ma L, Wang YH, Cao YQ, Shen WD, Li B. Transcriptional characteristics of acetylcholinesterase genes in domestic silkworms ( Bombyx mori ) exposed to phoxim. Pesticide Biochemistry and Physiology, 2011, 101: 154-158.), the concentration of phoxim in the immersion method of the present invention is 2ug/mL.
由于上述方案的运用，本发明具有以下优点。Due to the application of the above scheme, the present invention has the following advantages.
1、本发明利用微生物菌作为助剂喷洒到桑叶上饲喂家蚕，可以显著提高家蚕对辛硫磷农药的抗性，效果显著。微生物助剂适用家蚕所有龄期，具有通用性和创新性。1. The present invention utilizes microbial bacteria as an adjuvant to be sprayed on mulberry leaves to feed silkworms, which can significantly improve the resistance of silkworms to phoxim pesticides, and the effect is remarkable. Microbial auxiliaries are suitable for all ages of silkworm and are versatile and innovative.
2、本发明公开的方法中使用的微生物助剂用量少、成本低、环保安全；无需改变生产规律，操作方便，易于推广使用。2. The microbial adjuvant used in the method disclosed in the present invention has the advantages of low dosage, low cost, environmental protection and safety; no need to change the production law, convenient operation, and easy popularization and use.
本发明公开的提高家蚕对辛硫磷农药抗性的方法如下。The method for improving silkworm resistance to phoxim pesticide disclosed in the present invention is as follows.
（1）组合菌液的配置：将鞘氨醇单胞菌（
Sphingomonas spp.）液、松鼠葡萄球菌（
Staphylococcus sciuri spp.）液、微杆菌（
Microbacterium spp.）液、克雷伯氏菌（
Klebsiellaspp.）液、粘质沙雷菌（
Serratia marcescens spp.）液、不动杆菌（
Acinetobacter spp.）液、节杆菌（
Arthrobacter nicotianae spp.）液分别用水稀释至光密度（OD
600）为0.3～0.5；然后混合，得到组合菌液，其光密度（OD
600）也为0.3～0.5。
(1) Configuration of the combined bacterial solution: the Sphingomonas ( Sphingomonas spp.) solution, the Staphylococcus ( Sciuri spp.) solution, the Microbacterium ( Microbacterium spp.) solution, the Klebsiella ( Klebsiella ) solution spp.) solution, Serratia marcescens ( Serratia marcescens spp.) solution, Acinetobacter ( Acinetobacter spp.) solution, and Arthrobacter nicotianae spp. solution were diluted with water to an optical density (OD 600 ) of 0.3～ 0.5; and then mixed to obtain a combined bacterial solution, whose optical density (OD 600 ) was also 0.3 to 0.5.
（2）添食家蚕：按照50kg桑叶添加0.4～0.6L的组合菌液，将步骤（1）中制备的组合菌液喷洒适熟叶，待桑叶自然晾干后饲喂家蚕，即可提高家蚕对辛硫磷农药的抗性。(2) Feeding silkworms: add 0.4-0.6L of the combined bacterial solution according to 50 kg of mulberry leaves, spray the combined bacterial solution prepared in step (1) on the mature leaves, and feed the silkworms after the mulberry leaves are naturally dried. Improve silkworm resistance to phoxim pesticides.
本发明所有菌都为常规微生物，无需其他试剂，不改变添食规律，即可有效提高家蚕对辛硫磷农药的抗性。下面结合实例对本发明作进一步描述。All the bacteria in the invention are conventional microorganisms, and the resistance of silkworms to phoxim pesticides can be effectively improved without other reagents and without changing the feeding rule. The present invention will be further described below in conjunction with examples.
光密度（OD
600）的测试方法为利用紫外分光光度计检测波长600
nm下的菌液吸光值（OD
600）。
The test method of optical density (OD 600 ) is to detect the absorbance (OD 600 ) of the bacterial liquid at a wavelength of 600 nm using an ultraviolet spectrophotometer.
实施例一：一种提高家蚕对辛硫磷农药抗性的方法。Embodiment 1: a method for improving silkworm resistance to phoxim pesticides.
（1）实验材料和药品。(1) Experimental materials and medicines.
家蚕品种：苏秀×春丰，为苏州大学蚕桑研究所育成和保存品种，该品种已江苏省蚕品种审定委员会审定（审定号：201001），具有家蚕一般品种的普遍性特征。Bombyx mori variety: Suxiu×Chunfeng, which was bred and preserved by the Sericulture Research Institute of Soochow University.
微生物助剂：鞘氨醇单胞菌A28241菌（
Sphingomonas
spp.）、松鼠葡萄球菌LH-T5（
Staphylococcus
sciuri spp.）、微杆菌HBUM178903（
Microbacterium spp.）、密歇根克雷伯氏菌（
Klebsiella
michiganensis spp.）、粘质沙雷菌N1.8（
Serratia marcescens spp.）、不动杆菌IILSFSC204（
Acinetobacter
spp.）、节杆菌Dc-06（
Arthrobacter
nicotianae spp.）为苏州大学蚕桑研究所资源库保存。根据常规方法，将冻存菌复苏后在LB液体培养基（上海生工）中培养12 h（30℃，200 rpm），然后用水稀释，至光密度（OD
600）为0.4。以上七个菌种分别操作，得到光密度（OD
600）为0.4的七组菌液，然后七种菌液按鞘氨醇单胞菌A28241菌（
Sphingomonas spp.）液、松鼠葡萄球菌LH-T5（
Staphylococcus sciuri spp.）液、微杆菌HBUM178903（
Microbacterium
spp.）液、密歇根克雷伯氏菌（
Klebsiella michiganensis spp.）液、粘质沙雷菌N1.8（
Serratia
marcescens spp.）液、不动杆菌IILSFSC204（
Acinetobacter spp.）液、节杆菌Dc-06（
Arthrobacter nicotianae spp.）液体积比例1∶1∶0.5∶1∶0.5∶0.5∶0.6混合，得到组合菌液，测试光密度（OD
600）为0.4。
Microbial additives: Sphingomonas A28241 ( Sphingomonas spp. ), Staphylococcus sciuri LH-T5 ( Staphylococcus sciuri spp. ), Microbacterium HBUM178903 ( Microbacterium spp. ), Klebsiella michiganensis spp .), Serratia marcescens N1.8 ( Serratia marcescens spp. ), Acinetobacter IILSSFSC204 ( Acinetobacter spp. ), Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ) are stored in the resource bank of the Sericulture Research Institute of Soochow University. According to the conventional method, the cryopreserved bacteria were recovered and cultured in LB liquid medium (Shanghai Shenggong) for 12 h (30 °C, 200 rpm), and then diluted with water to an optical density (OD 600 ) of 0.4. The above seven strains were operated respectively to obtain seven groups of bacterial liquids with an optical density (OD 600 ) of 0.4, and then the seven bacterial liquids were prepared according to Sphingomonas A28241 ( Sphingomonas spp. ) liquid, Staphylococcus squirrel LH-T5 ( Staphylococcus sciuri spp. ) liquid, Microbacterium HBUM178903 ( Microbacterium spp. ) liquid, Klebsiella michiganensis spp. liquid, Serratia marcescens spp. liquid, immobile Bacillus IILSFSC204 ( Acinetobacter spp. ) solution and Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ) solution were mixed in a volume ratio of 1:1:0.5:1:0.5:0.5:0.6 to obtain a combined bacterial solution, and the optical density (OD 600 ) was tested. is 0.4.
辛硫磷农药（CAS号：14816-18-3），Sigma公司产品。Phoxim pesticide (CAS number: 14816-18-3), a product of Sigma company.
（2）微生物菌剂的使用。(2) The use of microbial inoculants.
组合菌液使用前常规摇匀，按照每50kg桑叶添加0.5L剂量进行喷洒5龄期的适熟叶，桑树品种为育711，待桑叶自然晾干后得到带有组合菌的桑叶，饲喂家蚕，给桑量与正常饲喂家蚕使用量相同。The combined bacterial liquid was shaken well before use, and 0.5 L dose was added per 50 kg of mulberry leaves to spray suitable mature leaves of the 5th instar. The mulberry variety was Yu 711, and the mulberry leaves with the combined bacteria were obtained after the mulberry leaves were naturally dried. For feeding silkworms, the amount of mulberry given is the same as that used for normal feeding silkworms.
（3）抗药性检测。(3) Drug resistance detection.
采用浸液法，对5龄第3天家蚕幼虫进行抗药性检测，每个试验组30头家蚕，每组三个重复，共90头。The 5th instar silkworm larvae were tested for drug resistance on the 3rd day by liquid immersion method. There were 30 silkworms in each experimental group, with three replicates in each group, totaling 90 silkworms.
具体试验方法为。The specific test method is.
试验所用5龄期的适熟叶未经任何试剂处理，清洗后晾干，然后进行菌处理或者辛硫磷处理，或者仅做喷洒等量水处理。The mature leaves of the 5th instar stage used in the experiment were not treated with any reagents. They were washed and dried, and then treated with bacteria or phoxim, or only sprayed with the same amount of water.
将5龄期的适熟叶浸入辛硫磷水溶液（2ug/mL）中30s，没入即可，取出后自然晾干，得到辛硫磷处理的桑叶。Immerse the mature leaves of the 5th instar in an aqueous solution of phoxim (2ug/mL) for 30s, then submerge them in, and then take them out and dry them naturally to obtain phoxim-treated mulberry leaves.
实验组。带有组合菌的桑叶，饲喂5龄起蚕，给桑量与正常饲喂家蚕使用量相同，一共3天，第4天（5龄4天）饲喂辛硫磷处理的桑叶，一共4天，即饲喂至5龄7天。test group. The mulberry leaves with the combined bacteria were fed to the 5th instar silkworm, and the mulberry amount was the same as that of the normal silkworm feeding, for a total of 3 days. A total of 4 days, that is, feeding to 5 years of age and 7 days.
对照组。仅喷洒水处理的5龄期的适熟叶饲喂5龄起蚕，给桑量与正常饲喂家蚕使用量相同，一共3天，第4天（5龄4天）饲喂辛硫磷处理的桑叶，一共4天，即饲喂至5龄7天。control group. Only the appropriate mature leaves of the 5th instar treated with water were fed to the 5th instar silkworm, and the amount of mulberry was the same as that of the normal feeding silkworm, for a total of 3 days, and the 4th day (4 days of the 5th instar) was fed with phoxim. mulberry leaves, a total of 4 days, that is, fed to 5 years of age for 7 days.
对比组。用一种菌液（光密度（OD
600）为0.4）按照每50kg桑叶添加0.5L剂量进行喷洒5龄期的适熟叶，桑树品种为育711，待桑叶自然晾干后得到带有单菌的桑叶，饲喂5龄起蚕，给桑量与正常饲喂家蚕使用量相同，一共3天，第4天（5龄4天）饲喂辛硫磷处理的桑叶，一共4天，即饲喂至5龄7天。
comparison group. Use a bacterial solution (optical density (OD 600 ) of 0.4) to spray the 5-year-old suitable mature leaves at a dose of 0.5 L per 50 kg of mulberry leaves. The mulberry variety is Yu 711. After the mulberry leaves are naturally dried, a The mulberry leaves of single bacteria were fed to the 5th instar silkworm, and the amount of mulberry was the same as that of the normal feeding silkworm, for a total of 3 days. days, that is, feeding to 5 years of age for 7 days.
实验组和对照组（喷洒等量水）、对比组家蚕的抗性进行检测，结果如表1所示。The resistance of silkworms in the experimental group and the control group (sprayed with the same amount of water) and the control group were tested, and the results are shown in Table 1.
备注：Ck：对照组；Sp：鞘氨醇单胞菌A28241菌；Ss：松鼠葡萄球菌LH-T5；Km：密歇根克雷伯氏菌；Mi：微杆菌HBUM178903；Sm：粘质沙雷菌N1.8；Ac：不动杆菌IILSFSC204；An：节杆菌Dc-06；Mb：组合菌（Sp：Ss：Km：Mi：Sm：Ac ：An的体积比为1：1：0.5：1：0.5：0.5：0.6）。Remarks: Ck: control group; Sp: Sphingomonas A28241; Ss: Staphylococcus squirrel LH-T5; Km: Klebsiella Michigan; Mi: Microbacterium HBUM178903; Sm: Serratia marcescens N1 .8; Ac: Acinetobacter IILSFSC204; An: Arthrobacter Dc-06; Mb: Combination bacteria (Sp: Ss: Km: Mi: Sm: Ac: An volume ratio of 1: 1: 0.5: 1: 0.5: 0.5:0.6).
表1结果显示：添食鞘氨醇单胞菌、松鼠葡萄球菌、密歇根克雷伯氏菌，以及组合菌液桑叶的家蚕饲喂辛硫磷处理的桑叶后，24 h、48 h、72 h和96 h的存活率显著高于对照组。其中，组合菌液组96 h的存活率显著高于单独菌液处理组和对照组，特别的，90头家蚕仅死亡1头。The results in Table 1 show that the silkworms fed Sphingomonas, Staphylococcus squirrel, Klebsiella Michigan, and mulberry leaves combined with bacterial liquid were fed phoxim-treated mulberry leaves, and the 24 h, 48 h, The survival rates at 72 h and 96 h were significantly higher than those in the control group. Among them, the survival rate of the combined bacteria solution group at 96 h was significantly higher than that of the single bacteria solution treatment group and the control group. In particular, only one of the 90 silkworms died.
给家蚕添食微生物菌剂的实施例表明，50 kg桑叶添加0.4～0.6L剂量混合菌剂喷洒的桑叶饲喂家蚕后能显著提高家蚕对辛硫磷的抗性。The example of feeding microbial inoculants to silkworms shows that 50 kg of mulberry leaves added with 0.4-0.6L doses of mulberry leaves sprayed with mixed inoculants can significantly improve the silkworm's resistance to phoxim after feeding silkworms.
实施例二。Example two.
在实施例一的基础上，鞘氨醇单胞菌A28241菌（
Sphingomonas
spp.）液、松鼠葡萄球菌LH-T5（
Staphylococcus
sciuri spp.）液、微杆菌HBUM178903（
Microbacterium spp.）液、密歇根克雷伯氏菌（
Klebsiella
michiganensis spp.）液、粘质沙雷菌N1.8（
Serratia marcescens spp.）液、不动杆菌IILSFSC204（
Acinetobacter
spp.）液、节杆菌Dc-06（
Arthrobacter
nicotianae spp.）液体积比例调整为1∶0.5∶1∶1∶0.5∶1∶0.5，其余不变，同样的方法得到带有组合菌的桑叶，饲喂5龄起蚕，给桑量与正常饲喂家蚕使用量相同，一共3天，第4天（5龄4天）饲喂辛硫磷处理的桑叶，一共4天，即饲喂至5龄7天。存活率统计（%）为94.44%（24h）、91.11%（48h）、86.67%(72h)、81.11%(96h)。
On the basis of Example 1, Sphingomonas A28241 ( Sphingomonas spp. ) liquid, Staphylococcus squirrel LH-T5 ( Staphylococcus sciuri spp. ) liquid, Microbacterium HBUM178903 ( Microbacterium spp. ) liquid, Michigan Cray Klebsiella michiganensis spp. liquid, Serratia marcescens N1.8 ( Serratia marcescens spp. ) liquid, Acinetobacter IILSFSC204 ( Acinetobacter spp. ) liquid, Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ) liquid The volume ratio was adjusted to 1: 0.5: 1: 1: 0.5: 1: 0.5, and the rest remained unchanged. The same method was used to obtain mulberry leaves with combined bacteria, and the 5th instar silkworms were fed. The same amount of mulberry leaves was fed for a total of 3 days on the 4th day (4 days of age 5) for a total of 4 days, that is, 7 days of feeding to the age of 5. The survival rate statistics (%) were 94.44% (24h), 91.11% (48h), 86.67% (72h), and 81.11% (96h).
本发明利用肠道微生物降解代谢杀虫剂的功能，开发出一种低成本、环保、有效的提高家蚕对辛硫磷农药抗性的方法，肠道内微生物可以帮助宿主昆虫降解代谢杀虫剂，从而提高宿主昆虫对杀虫剂的抗性。The invention utilizes the function of intestinal microorganisms to degrade and metabolize pesticides, and develops a low-cost, environmentally friendly and effective method for improving the resistance of silkworms to phoxim pesticides. The microorganisms in the intestinal tract can help host insects degrade and metabolize pesticides. Thereby increasing the resistance of host insects to pesticides.
Claims (10)
- 一种提高家蚕对辛硫磷农药抗性的方法，其特征在于，包括以下步骤，将带有组合菌的桑叶添食家蚕，提高家蚕对辛硫磷农药的抗性；组合菌为鞘氨醇单胞菌（ Sphingomonas spp.）、松鼠葡萄球菌（ Staphylococcus sciuri spp.）、微杆菌（ Microbacterium spp.）、克雷伯氏菌（ Klebsiella spp.）、粘质沙雷菌（ Serratia marcescens spp.）、不动杆菌（ Acinetobacter spp.）、节杆菌（ Arthrobacter nicotianae spp.）中的几种组合。 A method for improving the resistance of silkworms to phoxim pesticides, comprising the following steps: adding mulberry leaves with combined bacteria to feed silkworms to improve the silkworms' resistance to phoxim pesticides; the combined bacteria is sphingosine Sphingomonas spp., Staphylococcus sciuri spp., Microbacterium spp., Klebsiella spp., Serratia marcescens spp. , Acinetobacter ( Acinetobacter spp.), Arthrobacter ( Arthrobacter nicotianae spp.) in several combinations.
- 根据权利要求1所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，鞘氨醇单胞菌（ Sphingomonas spp.）为鞘氨醇单胞菌A28241菌（ Sphingomonas spp.）；松鼠葡萄球菌（ Staphylococcus sciuri spp.）为松鼠葡萄球菌LH-T5（ Staphylococcus sciuri spp.）；微杆菌（ Microbacterium spp.）为微杆菌HBUM178903（ Microbacterium spp.）；克雷伯氏菌（ Klebsiella spp.）为密歇根克雷伯氏菌（ Klebsiella michiganensis spp.）；粘质沙雷菌（ Serratia marcescens spp.）为粘质沙雷菌N1.8（ Serratia marcescens spp.）；不动杆菌（ Acinetobacter spp.）为不动杆菌IILSFSC204（ Acinetobacter spp.）；节杆菌（ Arthrobacter nicotianae spp.）为节杆菌Dc-06（ Arthrobacter nicotianae spp.）。 The method for improving silkworm resistance to phoxim pesticide according to claim 1, is characterized in that, Sphingomonas spp. ( Sphingomonas spp.) is Sphingomonas A28241 bacteria ( Sphingomonas spp. ); Staphylococcus sciuri spp. is Staphylococcus squirrel LH-T5 ( Staphylococcus sciuri spp. ); Microbacterium spp. is Microbacterium HBUM178903 ( Microbacterium spp. ); Klebsiella spp. is Michigan Klebsiella michiganensis spp.; Serratia marcescens spp. N1.8 ( Serratia marcescens spp. ); Acinetobacter spp. Bacillus IILSFSC204 ( Acinetobacter spp. ); Arthrobacter nicotianae spp. is Arthrobacter Dc-06 ( Arthrobacter nicotianae spp. ).
- 根据权利要求1所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，组合菌为鞘氨醇单胞菌（ Sphingomonas spp.）、松鼠葡萄球菌（ Staphylococcus sciuri spp.）、微杆菌（ Microbacterium spp.）、克雷伯氏菌（ Klebsiella spp.）、粘质沙雷菌（ Serratia marcescens spp.）、不动杆菌（ Acinetobacter spp.）和节杆菌（ Arthrobacter nicotianae spp.）。 The method for improving silkworm resistance to phoxim pesticide according to claim 1, wherein the combined bacteria are Sphingomonas ( Sphingomonas spp.), Staphylococcus sciuri spp., Microbacterium (Sphingomonas spp.) Microbacterium spp.), Klebsiella spp., Serratia marcescens spp., Acinetobacter spp. and Arthrobacter nicotianae spp.
- 根据权利要求1所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，将组合菌液喷洒适熟桑叶，得到带有组合菌的桑叶。The method for improving silkworm resistance to phoxim pesticide according to claim 1, characterized in that, the combined bacteria liquid is sprayed on suitable mature mulberry leaves to obtain mulberry leaves with combined bacteria.
- 根据权利要求4所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，组合菌液的光密度（OD 600）为0.3～0.5。 The method for improving silkworm resistance to phoxim pesticide according to claim 4, wherein the optical density (OD 600 ) of the combined bacterial solution is 0.3-0.5.
- 根据权利要求4所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，将鞘氨醇单胞菌（ Sphingomonas spp.）液、松鼠葡萄球菌（ Staphylococcus sciuri spp.）液、微杆菌（ Microbacterium spp.）液、克雷伯氏菌（ Klebsiella spp.）液、粘质沙雷菌（ Serratia marcescens spp.）液、不动杆菌（ Acinetobacter spp.）液和节杆菌（ Arthrobacter nicotianae spp.）液混合，得到组合菌液；鞘氨醇单胞菌（ Sphingomonas spp.）液、松鼠葡萄球菌（ Staphylococcus sciuri spp.）液、微杆菌（ Microbacterium spp.）液、克雷伯氏菌（ Klebsiella spp.）液、粘质沙雷菌（ Serratia marcescens spp.）液、不动杆菌（ Acinetobacter spp.）液、节杆菌（ Arthrobacter nicotianae spp.）液的体积比1∶（0.9～1.1）∶（0.4～0.6）∶（0.9～1.1）∶（0.4～0.6）∶（0.4～0.6）∶（0.5～0.7）。 The method for improving the resistance of silkworm to phoxim pesticide according to claim 4, wherein the sphingomonas ( Sphingomonas spp.) liquid, the Staphylococcus sciuri spp. Microbacterium spp., Klebsiella spp., Serratia marcescens spp., Acinetobacter spp. and Arthrobacter nicotianae spp. Mixed to obtain the combined bacterial liquid ; The volume ratio of liquid, Serratia marcescens ( Serratia marcescens spp.) liquid, Acinetobacter ( Acinetobacter spp.) liquid, Arthrobacter nicotianae spp. liquid is 1:(0.9～1.1):(0.4～0.6) : (0.9 to 1.1): (0.4 to 0.6): (0.4 to 0.6): (0.5 to 0.7).
- 根据权利要求4所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，按照50kg桑叶添加0.4～0.6L剂量，将组合菌液喷洒适熟桑叶，然后自然晾干，得到带有组合菌的桑叶。The method for improving silkworm resistance to phoxim pesticide according to claim 4, characterized in that, adding 0.4-0.6L dose according to 50kg of mulberry leaves, spraying the combined bacterial liquid on suitable mature mulberry leaves, and then naturally drying to obtain a Mulberry leaves with assemblage.
- 根据权利要求1所述提高家蚕对辛硫磷农药抗性的方法，其特征在于，将带有组合菌的桑叶添食家蚕2～5天，提高家蚕对辛硫磷农药的抗性。The method for improving the resistance of silkworms to phoxim pesticides according to claim 1, wherein the mulberry leaves with the combined bacteria are fed to silkworms for 2 to 5 days to improve the silkworms' resistance to phoxim pesticides.
- 组合菌在提高家蚕对辛硫磷农药的抗性中的应用；组合菌为鞘氨醇单胞菌（ Sphingomonas spp.）、松鼠葡萄球菌（ Staphylococcus sciuri spp.）、微杆菌（ Microbacterium spp.）、克雷伯氏菌（ Klebsiella spp.）、粘质沙雷菌（ Serratia marcescens spp.）、不动杆菌（ Acinetobacter spp.）、节杆菌（ Arthrobacter nicotianae spp.）中的几种组合。 The application of combined bacteria in improving the resistance of silkworm to phoxim pesticides; the combined bacteria are Sphingomonas spp., Staphylococcus sciuri spp., Microbacterium spp., Several combinations of Klebsiella spp., Serratia marcescens spp., Acinetobacter spp., Arthrobacter nicotianae spp.
- 组合菌在制备提高家蚕对辛硫磷农药抗性的饲料中的应用；组合菌为鞘氨醇单胞菌（ Sphingomonas spp.）、松鼠葡萄球菌（ Staphylococcus sciuri spp.）、微杆菌（ Microbacterium spp.）、克雷伯氏菌（ Klebsiella spp.）、粘质沙雷菌（ Serratia marcescens spp.）、不动杆菌（ Acinetobacter spp.）、节杆菌（ Arthrobacter nicotianae spp.）中的几种组合。 Application of combined bacteria in preparing feed for improving silkworm resistance to phoxim pesticides; combined bacteria are Sphingomonas spp., Staphylococcus sciuri spp., Microbacterium spp. ), Klebsiella spp., Serratia marcescens spp., Acinetobacter spp., Arthrobacter nicotianae spp.
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| CN105534988A (en) * | 2015-12-21 | 2016-05-04 | 山东省蚕业研究所 | A medicine for increasing yields and preventing and treating bacterial diseases for silkworms and applications thereof |
| WO2020254592A1 (en) * | 2019-06-19 | 2020-12-24 | Universität Für Bodenkultur Wien | Method for biotransformation of trichothecenes |
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| CN102823547A (en) * | 2012-08-22 | 2012-12-19 | 福建省农业科学院植物保护研究所 | Drug-resistant neoseiulus amblyseius cucumeris strain |
| CN105534988A (en) * | 2015-12-21 | 2016-05-04 | 山东省蚕业研究所 | A medicine for increasing yields and preventing and treating bacterial diseases for silkworms and applications thereof |
| WO2020254592A1 (en) * | 2019-06-19 | 2020-12-24 | Universität Für Bodenkultur Wien | Method for biotransformation of trichothecenes |
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