【作者】 张崇星；

【导师】 赖仞； 张克云；

【作者基本信息】 南京农业大学 ， 动物学， 2009， 博士

【摘要】 昆虫病原线虫(Entomopathogenic nematodes,EPN)是20世纪发展起来的一种有潜能的、安全的生物防治因子,由于昆虫病原线虫寄主范围广泛,对土栖性及钻蛀性害虫与隐蔽性害虫有特殊防效,对人畜安全,不会污染环境,亦不会产生抗性,且能以低成本大规模生产、使用方便、杀虫速度快、并能与化学杀虫剂混用,因此已广泛应用于防治农林及牧草等害虫,受到众多学者及商业部门的高度重视。现已描述的昆虫病原线虫共有约77种,分别是异小杆科(Heterorhabditidae)中唯一的属异小杆属(Heterorhabditis)14种,斯氏科(Steinernematidae)的斯氏属(Steinernema)61种与新斯氏属(Neosteinernema)1种以及小杆科(Rhabditidae)拟异小杆线虫属(Heterorhabdoides)1种,所有描述的昆虫病原线虫都属于小杆目。在其肠道内分别与肠杆菌科(Enterobacteriaceae)的致病杆菌属(Xenorhabdus)细菌、发光杆菌属(Photorhabdus)细菌以及沙雷氏属(Serratia)细菌相互共生。其中拟异小杆属崇明拟异小杆线虫(Heterorhabditidoides chongmingensis)及其共生菌嗜线虫沙雷氏菌(Serratia nematodiphila)是本研究描述的昆虫病原线虫及其共生菌新种嗜线虫沙雷氏菌。利用大蜡螟幼虫诱捕的方法从上海崇明岛采集的土样中分离到一个昆虫病原线虫新品系,标记为DZ0503CMFT。线虫DZ0503CMFT的体长：0.8-2.2 mm；线虫的表皮有环纹；口大约是头宽的1-1.5倍,口有厚壁,角皮化；唇口腔壁角皮化；存在较长的食管领,有中间食道球；雄成虫：交合刺长=51(37-68)μm；D%=107；SW=1.95；雄成虫粘液囊蝴蝶状,上面有9对生殖乳突,排列方式为1、2、3、3,在雄虫末端的排列方式及数目不变；交合刺不融合,稍向腹面弯曲；雌成虫：生殖腺成对；阴道外没有表皮覆盖,尾部通常有1-6个感受器；雌成虫尾部圆锥形或稍尖；侵染期线虫：体长=428(395-474)μm；EP=90(80-105)μm；ES=104(92-120)μm；带鞘尾长111(89-159)μm；a=19.1(15-21)；E％=83(67-97)；线虫DZ0503CMFT形态结构特征与小杆科的小杆线虫属、小杆属、秀丽小杆属、幽灵杆属、大杆属有明显区别,与已描述的昆虫病原线虫异小杆科异小杆属也有明显区别。18S rDNA和ITSrDNA基因系统发育树证明线虫DZ0503CMFT是单系群,是小杆科已描述其它属的姊妹群。线虫DZ0503 CMFT核酸中ITS1区域的A,T,C和G的百分比与异小杆属和小杆科已描述属有显著不同,结合昆虫病原线虫DZ0503CMFT不同发育时期的形态结构与分子系统进化特性分析,最终确定为小杆科中一个新属,根据国际动物命名原则,将昆虫病原线虫DZ0503CMFT命名为崇明拟异小杆线虫(Heterorhabditidoides chongmingensis)。通过碾碎100条表面消毒的侵染期崇明拟异小杆线虫或是取被崇明拟异小杆线虫侵染了24-48小时的大蜡螟血淋巴涂布于营养溴汾蓝平板鉴别培养基上,从崇明拟异小杆线虫肠道中分离到一株细胞和菌落形态以及保守性16S rDNA和rpoB基因与它相邻的模式菌株具有差别的新菌株,标记为DZ0503SBS1T。新分离菌株DZ0503SBS1T在营养琼脂上呈红色,革兰氏染色反应呈阴性,能运动,有荧光,菌体短杆状,单鞭毛。通过16S rDNA以及rpoB基因分析显示细菌为沙雷氏属,16SrDNA与日本朔沙雷菌JCM 11315T,粘质沙雷菌DSM 30121T,尿素水解沙雷菌LMG 22860T序列相似性分别为99.8%、99.5%、98.3%；rpoB基因与以上菌株的相似性分别为98%,97.4%和98.3%。菌株DZ0503SBS1T与日本朔沙雷菌JCM11315T,粘质沙雷菌DSM 30121T,尿素水解沙雷菌LMG 22860T DNA-DNA杂交结果分别为68.2%,65.1%,53%。DZ0503SBS1T主要脂肪酸为十六碳饱和脂肪酸(34.76%),环式十七碳饱和脂肪酸(20.03%)和ω8c-环式十九碳饱和脂肪酸(17.24%)。通过对新分离菌株DZ0503SBS1T形态学、细胞化学成分、生理生化和16S rDNA以及rpoB基因系统发育等多相指标分析,最终确定新分离菌株DZ0503SBS1T的分类学地位为沙雷氏菌属中一个新种,将其命名为嗜线虫沙雷菌(Serratia nematodiphila)。用荧光显微镜对活体崇明拟异小杆线虫进行观察,发现其前端的中肠作为共生细菌储存的特殊器官。崇明拟异小杆线虫侵染大蜡螟幼虫后,可以在昆虫宿主的血淋巴内分离到共生菌嗜线虫沙雷氏菌,嗜线虫沙雷氏菌在昆虫宿主的死亡过程中起着重要的作用,具有较强的致病性,大约300个细菌就可以致死大蜡螟幼虫,并且嗜线虫沙雷氏菌对于崇明拟异小杆线虫的繁殖、生长、发育都有明显促进作用。更多还原

【Abstract】 Entomopathogenic nematodes (EPN) are promising biological control agents since the early of 20th century. Entomopathogenic nematodes are obligate and lethal to a wide range of insect pests, especially to soil-inhabiting insects, plant-boring insects and cryptic pests. They are safety for human and animals, no contamination and resistance production, kill their hosts quickly, can be easily mass-produced and applied with conventional pesticide equipment, tolerate chemical pesticides. So entomopathogenic nematodes have been widely used to control pest insects in agriculture, forest, turgrass and attached importance to many researchers and commercial departments. Up to today, there are about 77 valid EPN species: 14 species have been reported in the only genus Heterorhabditis of family Heterorhabditidae and 61 valid species in the Steinernema and 1 species in the Neosteinernema of family Steinernematidae and 1 species in the genus Heterorhabditidoid.es of Rhabditidae. All of the described species of EPN belong to Rhabditida. Steinernematids and Heterorhabditids are characterized by carrying specific symbiotic bacteria of the genus Xenorhabdus and Photorhabdus in their intestine. And Heterorhabditidoides chongmingensis is entomopathogenic nematode that described in this study, the symbiotic bacterium of Heterorhabditidoides chongmingensis is Serratia nematodiphila.The new species of entomopathogenic nematode was obtained from a soil sample collected from Chongming Island in the north eastern area of Shanghai city using the insect baiting method, designated DZ0503CMFT. Body length:0.8-2.2 mm, cuticle annulated, stoma about 1-1.5 times as long as head width, stomal with thick walls, cuticularized, cheilorhabdions cuticularized, esophagus collar present and long with 2 to 5 warts; median bulb present; For males:Spicule length= 51 (37-68)μm; D%= 107; SW= 1.95; Bursa peloderan, well developed with nine pairs of genital papillae, the papillae formula of bursa is 1,2,3,3. with constant papillae number in the terminal group; Spicules not fused, slightly curved ventrally. Female:gonads paired, tail variable, conoid, sharply pointed, vulva without cuticular flaps to protected, with 1-6 phasmids on the tail; Infective juveniles, body length= 428 (395-474)μm EP= 90 (80-105)μm, ES= 104 (92-120)μm, tail length with sheath=111 (89-159)μm, a= 19.1 (15-21); E%= 83 (67-97); Morphological characteristics of DZ0503CMFT can be different from Pellioditis, Caenorhabditis, Phasmarhabditis, Dolichorhabditis, Oscheius of Rhabditidae and Heterorhabditis of Heterorhabditidae. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae. The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of Heterorhabditids and other rhabditids in Rhabditidae. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, according to the International Code of Zoological Nomenclature, and described as Heterorhabditidoides chongmingensis.A novel symboitic bacterium strain, DZ0503SBS1T, isolated from the intestine of Heterorhabditidoides chongmingensis by crush ca 100 surface-disinfected infective juveniles or plate a drop of hemolymph harvested from insects parasitized for 24-48 h by Heterorhabditidoides chongmingensis on nutrient bromothymol blue agar plates. The strain was red-pigmented, Gram-negative, motile, fluorescent, rod-shaped, with a solo lateral flagellum. Comparative 16S rDNA and rpoB gene sequences analysis indicated that the bacteria is a members of the genus Serratia, sharing 16S rDNA sequence similarity with the type strain of S. marcescens subsp. sakuensis JCM 11315T (99.8%), S. marcescens subsp. marcescens DSM 30121T (99.5%) and S. ureilytica LMG 22860T (98.3%), similarity of with these strains rpoB is 98%,97.4% and 98.3%, respectively. Strain DZ0503SBS1T exhibited 68.2%,65.1%,53% similarity to S. marcescens subsp. sakuensis JCM 11315T, S. marcescens subsp. marcescens DSM 30121T and S. ureilytica LMG 22860T by DNA-DNA hybridization, respectively. The predominantly fatty acids were C16:o (34.76 %), cyclo-C17:0 (20.03%), cyclo-C19:0ω8c (17.24%). The phenotypic, biochemical characteristics,16S rDNA and rpoB gene sequences analysis showed that strain DZ0503SBS1T represents a novel species, for which the name Serratia nematodiphila is proposed.Examined live Heterorhabditidoides chongmingensis under fluorescence microscope can observe anterior midgut as specialized symbiotic organ. When Galleria mellonella was infected by Heterorhabditidoides chongmingensis can insolated Serratia nematodiphila from the hemocoel of it. The symbiont Serratia nematodiphila is essential for Heterorhabditidoides chongmingensis as a pathogen of insects. Injection of about 300 cells of bacteria was necessary to reach the LD50. The bacterial symbiont was lethal to G. mellonella. Bacterial symbiont Serratia nematodiphila facilitate reproduction, growth and development of Heterorhabditidoides chongmingensis.更多还原