昆虫病原线虫异小杆属新种Heterorhabditis beicherriana共生菌的分离及致病性

doi:10.16409/j.cnki.2095-039x.2018.01.016

摘要/Abstract

摘要： 昆虫病原线虫共生菌在农业及医药领域已成为一种重要的生物活性资源。本研究通过致死的昆虫血腔和侵染期线虫直接分离线虫Heterorhabditis beicherriana的共生细菌，根据形态学特征、生理生化特性及16S rDNA序列初步鉴定为Photorhabdus luminescens，系统发育分析显示为发光杆菌属新亚种菌株。采用共生菌细菌体稀释液注射接种进行致病性测定，结果显示，每头大蜡螟幼虫接种菌量为600 cfu/mL，72 h时死亡率达到100%；24 h时LC₅₀为213.13 cfu/mL，48 h时LC₅₀为46.87 cfu/mL。这为研究该线虫H.beicherriana的致病机制奠定基础，以期发掘新的具有重要价值的活性资源。

关键词: 异小杆属线虫, 共生细菌, 形态学特征, 16S rDNA, 致病性

Abstract: The symbiotic bacteria associated with entomopathogenic nematodes are potential biological resources in the field of agriculture and medicine. In this study, symbiotic bacteria were isolated directly through infective juveniles or the blood chamber of Galleria mellonella larvae which were infected by Heterorhabditis beicherriana. The symbiotic bacterium was identified as Photorhabdus luminescens based on morphology, physiological and biochemical characteristics and sequence analysis of 16S rDNA; phylogenetic analysis demonstrated the strain was shown to be part of the species of P. luminescens, but it was clearly sperated with clear separation from currently recognized subspecies. It is proposed to be a new subspecies. G. mellonella larvae injected with the cell diluent were used to test pathogenicity of the symbiotic bacterium. The strain C1 exhibited strong pathogenicity to the G. mellonella larvae. Mortality reached 100% at 72 h when one wax moth was inoculated with the concentration of 600 cfu/mL moth. The LC₅₀ was 213.13 cfu/mL at 24 h and 46.87 cfu/mL at 48 h. Our study will lay the foundation for the study of the pathogenic mechanism of H. beicherriana, thus leading to explore a new bacterial strain with important value.

Key words: Heterorhabditis beicherriana, symbiotic bacteria, morphological and physiological characteristics, 16S rDNA, pathogenicity

中图分类号:

S476

付俊瑞, 刘奇志, 李星月. 昆虫病原线虫异小杆属新种Heterorhabditis beicherriana共生菌的分离及致病性[J]. 中国生物防治学报, 2018, 34(1): 133-140.

FU Junrui, LIU Qizhi, LI Xingyue. Isolation and Pathogenicity of Symbiotic Bacteriua associated with the Entomopathogenic Nematode Heterorhabditis beicherriana[J]. journal1, 2018, 34(1): 133-140.

参考文献

[1] Poinar G O J,Gaugler R,Kaya H K. Taxonomy and biology of Steinernematidae and Heterorhabditidae[M]//Gaugler R, Kaya H Keds. Entomopathogenic Nematodes in Biological Control. Boca Raton, 1990, 23-61.
[2] Grewal P S, Lewis E E, Gaugler R. Response of infective stage parasites (Nematoda:Steinernematidae) to volatile cues from infected hosts[J]. Journal of Chemical Ecology, 1997, 23(2):503-515.
[3] Bowen D J, Ensign J C. Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium photorhabdus luminescens[J]. Applied and Environmental Microbiology, 1998, 64(8):3029-3035.
[4] Fischer-Le S M, Viallard V, Brunel B, et al. Polyphasic classification of the genus Photorhabdus and proposal of new taxa:P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P. asymbiotica sp. nov.[J]. International Journal of Systematic and Evolutionary Microbiology, 1999, 49(4):1645-1656.
[5] Akhurst R J, Boemare N E, Janssen P H, et al. Taxonomy of Australian clinical isolates of the genus Photorhabdus and proposal of Photorhabdus asymbiotica subsp asymbiotica subsp nov and P. asymbiotica subsp australis subsp nov.[J]. International Journal of Systematic and Evolutionary Microbiology, 2004, 54(4):1301-1310.
[6] Ferreira T, van Reenen C A, Endo A, et al. Photorhabdus heterorhabditis sp. nov. a symbiont of the entomopathogenic nematode Heterorhabditis zealandica[J]. International Journal of Systematic and Evolutionary Microbiology, 2014, 64(5):1540-1545.
[7] Tailliez P, Laroui C, Ginibre N, et al. Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. Proposal of new taxa:X. vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov[J]. International Journal of Systematic and Evolutionary Microbiology, 2010, 60(8):1921-1937.
[8] An R, Grewal P S. Erratum to:Photorhabdus luminescens, subsp. kleinii, subsp. nov. (Enterobacteriales:Enterobacteriaceae)[J]. Current Microbiology, 2011, 62(2):539-543.
[9] Hazir S, Stackebrandt E, Lang E, et al. Two new subspecies of Photorhabdus luminescens, isolated from Heterorhabditis bacteriophora (Nematoda:Heterorhabditidae):Photorhabdus luminescens subsp. kayaii subsp. nov. and Photorhabdus luminescens subsp. thracensis subsp. nov.[J]. Systematic and Applied Microbiology, 2004, 27(1):36-42.
[10] Ferreira T, Van R C, Pagès S, et al. Photorhabdus luminescens subsp. noenieputensis subsp. nov. a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica[J]. International Journal of Systematic and Evolutionary Microbiology, 2013, 63(5):1853.
[11] Orozco R A, Hill T, Stock S P. Characterization and phylogenetic relationships of Photorhabdus luminescens subsp. sonorensis (γ-Proteobacteria:Enterobacteriaceae), the bacterial symbiont of the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda:Heterorhabditidae)[J]. Current Microbiology, 2013, 66(1):30-39.
[12] Glaeser S P, Tobias N J, Thanwisai A, et al. Photorhabdus luminescens subsp. namnaonensis subsp. nov. isolated from Heterorhabditis baujardi nematodes in Nam Nao district of central Thailand[J]. International Journal of Systematic and Evolutionary Microbiology, 2016, 67(4):1046-1051.
[13] Crouse J, Amorese D. Ethanol precipitation:ammonium acetate as an alternative to sodium acetate[J]. Focus, 1987, 9(2):3-5.
[14] Ffrenchconstant R, Waterfield N, Daborn P, et al. Photorhabdus:towards a functional genomic analysis of a symbiont and pathogen[J]. FEMS Microbiology Reviews, 2003, 26(5):433-456.
[15] Grewal P S, Ehlers R U, Shapiroilan D I. Nematodes as Biocontrol Agents[M]. CABI Publishing, CAB International, Oxon, UK, 2005.
[16] Yang X F, Zhang Z M, Yang H W, et al. Inhibition of metabolites from Xenorhabdus nematophilus against Phytophora infestans[J]. Journal of Agricultural University of Hebei, 2001, 24(2):65-68.
[17] Paik S, Park Y H, Suh S. Unusual cytotoxic phenethyl amides from Xenorhabdus nematophilus[J]. Bulletin of the Korean Chemical Society, 2001, 22(4):372-374.
[18] Li X Y, Liu Q Z, Nermu J, et al. Heterorhabditis beicherriana n. sp. (Nematoda:Heterorhabditidae), a new entomopathogenic nematode from the Shunyi district of Beijing, China[J]. Zootaxa, 2012, 3569:25-40.
[19] 刘奇志, 陈贤群, 曹景富, 等. 樱桃异小杆线虫在防治核桃云斑天牛中的应用[P]. 中国发明专利, 2015, CN104813877A.
[20] Dutky S R, Thompson J V, Cantwell G E. A technique for the mass propagation of the DD-136 nematode[J]. Journal of Insect Physiology, 1964, 6:417-422.
[21] 东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京:科学出版社, 2002.
[22] George M G, Julia A B, Timothy G L. Bergey's Manual of Systematic Bacteriology[M]. East Lansing:Michigan State University, 2004, 651-654.
[23] Akhurst R J. Morphological and functional dimorphism in Xenorhabdus spp. bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis[J]. Microbiology, 1980, 121(2):303-309.
[24] Ffrenchconstant R, Waterfield N, Daborn P, et al. Photorhabdus:towards a functional genomic analysis of a symbiont and pathogen[J]. FEMS Microbiology Reviews, 2003, 26(5):433-456.
[25] Zhao R, Han R, Qiu X, et al. Cloning and heterologous expression of insecticidal-protein-encoding genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for termite control[J]. Applied and Environmental Microbiology, 2008, 74(23):7219-26.
[26] Hinchliffe S J, Hares M C, Dowling A J, et al. Insecticidal toxins from the Photorhabdus and Xenorhabdus bacteria[J]. Open Toxinology Journal, 2010, 3(1):101-118.
[27] 刘峥, 简恒, 杨秀芬, 等. 昆虫病原线虫共生细菌发酵液对棉铃虫和菜青虫的口服毒性[J]. 植物保护学报, 2003, 30(1):19-24.
[28] Mohan S, Raman R, Gaur H S. Foliar application of Photorhabdus luminescens, symbiotic bacteria from entomopathogenic nematode, Heterorhabditis indica, to kill cabbage butterfly, Pieris brassicae[J]. Current Science, 2006, 84(11):1392.