韭蛆肠道细菌分离鉴定及其抗生素敏感性分析

doi:10.16409/j.cnki.2095-039x.2021.07.019

摘要/Abstract

摘要： 为探究韭菜迟眼蕈蚊Bradysia odoriphaga（韭蛆）幼虫肠道可培养细菌的菌群结构及抗生素敏感性。本研究对韭蛆3龄幼虫肠道细菌进行分离培养和16S rDNA鉴定后，采用23种抗生素对其分别进行药敏试验。研究结果表明，经分离鉴定后的6种肠道细菌属于氧化微杆菌Microbacterium oxydans HQ202813.1，葡萄球菌Staphylococcus vitulinus MN640907.1，蜡样芽胞杆菌Bacillus cereus CP045606.2，芽胞杆菌Bacillus sp. MG914019.1，沙雷氏菌属菌株Serratia fonticola MH379711.1，沙福芽胞杆菌Bacillus safensis MN704552.1，并分别命名为B.o1、B.o2、B.o3、B.o4、B.o5、B.o6。对6种可培养细菌的抗生素敏感性进行测试，结果表明，B.o1对21种抗生素都敏感，其中对多粘菌素和磷霉素表现为耐药；B.o2对21种抗生素都敏感，其中对乙酰螺旋素、林可霉素表现为耐药；B.o3对19种抗生素敏感，对青霉素、氨苄西林和多粘菌素耐药；B.o4对19种抗生素敏感，对青霉素、氨苄西林、林可霉素、多粘菌素耐药；B.o5对15种抗生素敏感，对青霉素、氨苄西林、庆大霉素、萘替米星、红霉素、阿奇霉素、麦迪霉素和多粘菌素耐药；B.o6对氨苄西林、磷霉素耐药，对其余19种抗生素敏感。综上所述，韭蛆幼虫肠道细菌菌群具有多样性，存在天然耐药的现象，本研究为进一步开展韭蛆幼虫肠道细菌微生物区系和功能细菌的研究提供了基础。

关键词: 韭菜迟眼蕈蚊, 肠道, 细菌, 抗生素敏感性

Abstract: To investigate the structure of intestinal culturable bacteria and antibiotic sensitivity of Bradysia odoriphaga larvae the intestinal bacteria of the third-instar B.odoriphaga larvae were isolated and cultured and identified by 16S rDNA, and were tested for drug sensitivity with 23 kinds of antibiotics. The results showed that the six types of intestinal bacteria isolated and identified belonged to Microbacilli Oxydans HQ202813.1, Staphylococcus vitulinus MN640907.1, Bacillus cereus CP045606.2, Bacillus sp. Mg914019.1, Serratia fonticola MH379711.1, Bacillus safensis MN704552.1 was named as B.o1, B.o2, B.o3, B.o4, B.o5 and B.o6. The antibiotic sensitivity test of six culturable bacteria showed that B.o1 was sensitive to 21 kinds of antibiotics, among which, it was resistant to polymyxin and fosfomycin. B.o2 was sensitive to 21 antibiotics, and showed resistance to acetylspirin and lincomycin. B.o3 was sensitive to 19 antibiotics and resistant to penicillin, ampicillin and polymyxin. B.o4 was sensitive to 19 antibiotics and resistant to penicillin, ampicillin, lincomycin and polymyxin. B.o5 was sensitive to 15 antibiotics and resistant to penicillin, ampicillin, gentamicin, naphthimidine, erythromycin, azithromycin, medemycin and polymyxin. B.o6 was resistant to ampicillin and fosfomycin, and sensitive to the other 19 antibiotics. To sum up, the intestinal bacterial flora of leek maggot larvae is diverse and has the phenomenon of natural drug resistance. This study provides a basis for further research on intestinal microbial flora and functional bacteria of B. odoriphaga larvae.

Key words: Bradysia odoriphaga, gut, bacteria, antibiotic susceptibility

中图分类号:

S476.1

宋健, 张海剑, 刘莉, 柳健虎, 曹伟平. 韭蛆肠道细菌分离鉴定及其抗生素敏感性分析[J]. 中国生物防治学报, 2023, 39(3): 690-696.

SONG Jian, ZHANG Haijian, LIU Li, LIU Jianhu, CAO Weiping. Isolation and Identification of Intestinal Bacteria from Leeks and Analysis of Antibiotic Sensitivity[J]. Chinese Journal of Biological Control, 2023, 39(3): 690-696.

参考文献

[1] Li H J, He X K, Zeng A J, et al. Bradysia odoriphaga copulatory behavior and evidence of a female sex pheromone[J]. Journal of Agricultural and Urban Entomology, 2007, 24(1):27-34.
[2] Yabuki Y, Mukaida Y, Saito Y, et al. Characterisation of volatile sulphur-containing compounds generated in crushed leaves of Chinese chive (Allium tuberosum Rottler)[J]. Food Chemistry, 2010, 120:343-348.
[3] Misawa T, Kuninaga S. First report of while leaf rot on Chinese chives caused by Rhizoctonia solani AG-2-1[J]. Journal of General Plant Pathology, 2013, 79, 280-283.
[4] 党志红, 董建臻, 高占林, 等. 不同种植方式下韭菜迟眼蕈蚊发生为害规律的研究[J]. 河北农业大学学报, 2001, 24(4):65-68.
[5] Li W X, Yang Y T, Xie W, et al. Effects of temperature on the age-stage, two-sex life table of Bradysia odoriphaga (Diptera:Sciaridae)[J]. Journal of Economic Entomology, 2015, 108, 126-134.
[6] Zhang P, Liu F, Mu W, et al. Life table study of the effects of sublethal concentrations of thiamethoxam on Bradysia odoriphage Yang and Zhang[J]. Pesticide Biochemistry and Physiology, 2014, 111:31-37.
[7] Chen C, Shi X, Desneux, N, et al. Detection of insecticide resistance in Bradysia odoriphaga Yang et Zhang (Diptera:Sciaridae) in China[J]. Ecotoxicology, 2017, 26:868-875.
[8] Arrington A E, Kennedy G G, Abney M R. Applying insecticides through drip irrigation to reduce wireworm (Coleoptera:Elateridae) feeding damage in sweet potato[J]. Pest Manage Science, 2016, 72:1133-1140.
[9] Kumar U, Berliner J, Adak T, et al. Non-target effect of continuous application of chlorpyrifos on soil microbes, nematodes and its persistence under sub-humid tropical rice-rice cropping system[J]. Ecotoxicology Environmental Safety, 2017, 135:225-235.
[10] 宋健, 曹伟平, 冯书亮, 等. 对韭菜迟眼蕈蚊幼虫高毒力Bt资源筛选及效果评价[J]. 应用昆虫学报, 2016, 53(6):1217-1224.
[11] 吴青君, 于毅, 谷希树, 等. 韭菜根蛆的发生危害及综合防治技术研究——公益性行业(农业)科研专项"作物根蛆类害虫综合防治技术研究与示范"进展[J]. 应用昆虫学报, 2016, 53(6):1165-1173.
[12] 张治军, 李文香, 何敏, 等. 韭菜迟眼蕈蚊雌成虫对其不同虫态气味的行为反应[J]. 应用昆虫学报, 2016, 53(6):1198-1204.
[13] 周仙红, 赵楠, 陈浩, 等. 防虫网对韭菜迟眼蕈蚊隔离效果和对韭菜生长的影响[J]. 应用昆虫学报, 2016, 53(6):1211-1216.
[14] Sharon G, Segal D, Ringo J M, et al. Commensal bacteria play a role in mating preference of Drosophila melanogaster[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(46):20051-20056.
[15] McLean A H C, van Asch M, Ferrari J, et al. Effects of bacterial secondary symbionts on host plant use in pea aphids[J]. Proceedings of the Royal Society B Biological Science, 2011, 278(1706):760-766.
[16] Behar A, Yuval B, Jurkevitch E. Gut bacterial communities in the Mediterranean fruit fly (Ceratitis capitata) and their impact on host longevity[J]. Journal of Insect Physiology, 2008, 54(9):1377-1383.
[17] Dillon R C, Vennard A, Buckling A, et al. Diversity of locust gut bacteria protects against pathogen invasion[J]. Ecology Letters, 2005, 8:1291-1298.
[18] 王天召, 王正亮, 朱杭烽, 等. 基于高通量测序的褐飞虱肠道微生物多样性分析[J]. 昆虫学报, 2019, 62(3):323-333.
[19] Kikuchi Y, Hosokawa T, Fukatsu T. Insect-microbe mutualism without vertical transmission:a stinkbug acquires a beneficial gut symbiont from the environment every generation[J]. Applied Environmental Microbiology, 2007, 73:4308-4316.
[20] Mason C J, Raffa K F. Acquisition and structuring of midgut bacterial communities in gypsy moth (Lepidoptera:Erebidae) larvae[J]. Environmental Entomology, 2014, 43:595-604.
[21] Hooper L V, Gordon J I. Commensal host-bacterial relationships in the gut[J]. Science, 2001, 292:1115-1118.
[22] Kümmerer K. Antibiotics in the aquatic environment- A review- Part I[J]. Chemosphere, 2009, 75(4):417-434.
[23] Kumar K, Thompson A, Singh A K, et al. Enzyme-linked immunosorbent assay for ultratrace determination of antibiotics in aqueous samples[J]. Journal of Environment Quality, 2004, 33(1):250-256.
[24] Rooklidge S J. Environmental antimicrobial contamination from terraccumulation and diffuse pollution pathways[J]. Science of the Total Environment, 2004, 325:1-13.
[25] Robinson C J, Schloss P, Ramos Y, et al. Robustness of the bacterial community in the cabbage white butterfly larval midgut[J]. Microbiology and Ecology, 2010, 59(2):199-211.
[26] Lundgren J G, Lehman R M. Bacterial gut symbionts contribute to seed digestion in an omnivorous beetle[J]. PLoS ONE, 2010, 5(5):e10831.
[27] Edlund A, Ek K, Breitholtz M, et al. Antibiotic-induced change of bacterial communities associated with the copepod Nitocra spinipes[J]. PLoS ONE, 2012, 7(3):e33107.
[28] Gaio A de O, Gusmão D S, Santos A V, et al. Contribution of midgut bacteria to blood digestion and egg production in Aedes aegypti (diptera:culicidae) (L.)[J]. Parasites & Vectors, 2011, 4:105.
[29] Rosengaus R B, Zecher C N, Schultheis K F, et al. Disruption of the termite gut microbiota and its prolonged consequences for fitness[J]. Applied and Environmental Microbiology, 2011, 77(13):4303-4312.
[30] Edlund A, Ek K, Breitholtz M, et al. Antibiotic-induced change of bacterial communities associated with the copepod Nitocra spinipes[J]. PLoS ONE, 2012, 7(3):e33107.
[31] Kafil M, Bandani A R, Kaltenpoth M, et al. Role of symbiotic bacteria in the growth and development of the Sunn pest, Eurygaster integriceps[J]. Journal of Insect Science, 2013, 13(99):1-12.
[32] Kopecky J, Nesvorna M, Mareckova-Sagova M, et al. The effect of antibiotics on associated bacterial community of stored product mites[J]. PLoS ONE, 2014, 9(11):e112919.
[33] Lin X L, Kang Z W, Pan Q J, et al. Evaluation of five antibiotics on larval gut bacterial diversity of Plutella xylostella (Lepidoptera:Plutellidae)[J]. Insect Science, 2015, 22(5):619-628.
[34] Visweshwar R, Sharma H C, Akbar S M D, et al. Elimination of gut microbes with antibiotics confers resistance to Bacillus thuringiensis toxin proteins in Helicoverpa armigera (Hübner)[J]. Applied Biochemistry and Biotechnology, 2015, 177(8):1621-1637.
[35] 柳健虎, 张康, 藏金萍, 等. 禾谷镰孢菌过氧化物酶家族基因的表达规律分析[J]. 农业生物技术学报, 2019, 6:1090-1100.
[36] Mariana R D, Lilha M B dos Santos, Ana C P V, et al. Effects of environment, dietary regime and ageing on the dengue vector microbiota:evidence of a core microbiota throughout Aedes aegypti lifespan[J]. Memrias do instituto Oswaldo Cruz, 2016, 111(9):577-87.
[37] 宋建, 郑方强, 李照会, 等. 韭菜迟眼蕈蚊幼虫消化系统的解剖学和组织学[J]. 华东昆虫学报, 2004, 13(1):42-47.
[38] Ashok B H, Chandra S P, Snehal C C, et al. Diversity of hacterial communities in the midgut of bactrocera cucurbitae (Diptera:Tephritidae) populations and their potential use as attractants[J]. Pest Management Science, 2016, 72(6):1222-1230.