Phylogenetic Taxonomy Of Entomopathogenic Nematode Heterorhabditidoides Chongmingensis Gen.Nov., Sp. Nov. And Its Symbiotic Bactreium Serratia Nematodiphila Sp. Nov.

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.