A novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus indica sp. nov., symbiotically associated with entomopathogenic nematode Steinernema thermophilum Ganguly and Singh, 2000

In the search for novel Xenorhabdus strains in a recently described nematode species, Steinernema thermophilum, three strains (strain 28(T) = DSM 17382(T), strain 42 = DSM 17383 and strain 43 = DSM 17384) were isolated from three independent isolation approaches from crushed mixture of infective juveniles. 16S rRNA gene sequence comparison of strains 28(T) and DSM 17383 indicated identity and the phylogenetic position pointed towards an individual taxon within the phylogenetic dendrogram of Xenorhabdus type strains. The nearest phylogenetic relatives of strain 28(T) were Xenorhabdus poinarii and Xenorhabdus szentirmaii (97.7% each). The three isolates were almost identical in reaction towards the API and BIOLOG substrate panels but differed in their reactions from those of the established type strains of the genus Xenorhabdus. These clear genomic and metabolic differences let us propose a new species, Xenorhabdus indica sp. nov. for the three clones. The type strain is strain 28(T), DSM 17382(T), CIP 108830(T).     

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342^T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337^T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336^T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338^T, isolated from Steinernema rarum.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

以23S rDNA一个多变区作为分子标记对致病杆菌属细菌进行分类鉴定

【目的】致病杆菌属(Xenorhabdus)细菌是一类重要的生物杀虫剂,斯氏属昆虫病原线虫的共生菌,建立快速准确的分类鉴定方法,对研究开发这类细菌至关重要。【方法】本研究PCR扩增测序了本室保藏的26株,含20种已定名致病杆菌属细菌的一段845 bp的23S rDNA序列,构建了基于这段序列的致病杆菌属系统树并与基于几乎全长16S rDNA序列的相应系统树进行比较,分析了两者作为致病杆菌属细菌分类鉴定分子标记的优缺点。【结果】结果表明,与全长16S rDNA序列相比,所选择的23S rDNA序列片段所含可变位点、简约信息位点比例更高,遗传距离数值跨度大。【结论】上述结果显示该序列片段可用于致病杆菌属细菌进行分类鉴定,特别适用于对野外资源调查中采集到的大量菌株进行快速鉴定。     

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.     

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).     

The Xenorhabdus nematophila nilABC genes confer the ability of Xenorhabdus spp. to colonize Steinernema carpocapsae nematodes

Members of the Steinernema genus of nematodes are colonized mutualistically by members of the Xenorhabdus genus of bacteria. In nature, Steinernema carpocapsae nematodes are always found in association with Xenorhabdus nematophila bacteria. Thus, this interaction, like many microbe-host associations, appears to be species specific. X. nematophila requires the nilA, nilB, and nilC genes to colonize S. carpocapsae. In this work, we showed that of all the Xenorhabdus species examined, only X. nematophila has the nilA, nilB, and nilC genes. By exposing S. carpocapsae to other Xenorhabdus spp., we established that only X. nematophila is able to colonize S. carpocapsae; therefore, the S. carpocapsae-X. nematophila interaction is species specific. Further, we showed that introduction of the nilA, nilB, and nilC genes into other Xenorhabdus species enables them to colonize the same S. carpocapsae host tissue that is normally colonized by X. nematophila. Finally, sequence analysis supported the idea that the nil genes were horizontally acquired. Our findings indicate that a single genetic locus determines host specificity in this bacteria-animal mutualism and that host range expansion can occur through the acquisition of a small genetic element.     

Identification of Carnobacterium spp. and Leuconostoc spp. in meat by genus-specific 16S rRNA probes

Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases. The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat. Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested. The probes were also hybridized with nucleic acids from unknown strains of LAB. The identification was consistent with the results of biochemical tests used to characterize the two genera.     

拟双角斯氏线虫共生细菌的分离与鉴定

从我国东北地区采集的拟双角斯氏线虫(Steinernema ceratophorum)肠道内分离到1株具有较强生物活性功能的致病杆菌菌株CB43。形态特征及生理生化特征测定结果表明,CB43菌株与致病杆菌属(Xe-norhabdus)的基本特性相似;对寄主线虫的产量有明显的促进作用,其代谢物对细菌和真菌均有较强的抑制活性,符合线虫共生菌的基本特性和功能。16S rRNA序列及根据16S rRNA序列构建的系统发育树中,CB43菌株与Xenorhabdus budapestensis序列同源性最高,形成一个类群。但CB43菌株不能产生吲哚,在麦芽糖、海藻糖、D-葡萄糖酸和乙酸利用等生化特征与X.budapestensis存在一定的差异,可能是由于菌株的生态差异造成的。根据形态及生理生化特征,结合16S rRNA序列分析,CB43菌株属于Xenorhabdus budapestensis。     

一株高毒力致病杆菌CB6的鉴定

从北京郊区果园采集的小卷蛾斯氏线虫（Steinernema carpocapsae）肠道内分离到一株具有较强杀虫和抑菌活性的致病杆菌菌株CB6。形态特征及生理生化特征测定结果表明,CB6菌株与致病杆菌属（Xenorhabdus）中的嗜线虫致病杆菌（X. nematophila）种的特征基本一致。测定了该菌株的16S rRNA序列并根据16S rRNA序列构建了系统发育树;在系统发育树中,CB6菌株与嗜线虫致病杆菌其他4个菌株形成一个类群,序列同源性大于99%。但CB6菌株的酪氨酸酶、脂酶（蛋黄）的产生、核糖产酸等生化特征与嗜线虫致病杆菌种内的其他菌株存在一定的差异,且具有更强的杀虫和抑菌活性。因此认为CB6菌株是嗜线虫致病杆菌的一个变种,命名为嗜线虫致病杆菌北京变种（X. nematophila var. pekingensis）。     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Morphology and ultrastructure of the bacterial receptacle in Steinernema nematodes (Nematoda: Steinernematidae)

Infective juveniles of entomopathogenic nematodes in the genus Steinernema harbor symbiotic bacteria, Xenorhabdus spp., in a discrete structure located in the anterior portion of the intestine known as the 'bacterial receptacle' (formerly known as the bacterial or intestinal vesicle). The receptacle itself is a structured environment in which the bacteria are spatially restricted. Inside this receptacle, bacterial symbionts are protected from the environment and grow to fill the receptacle. Until now, no comparative study across different Steinernema spp. has been undertaken to investigate if morphological variation in this structure exists at the interspecific level. In this study, we examined the bacterial receptacles of 25 Steinernema spp. representatives of the currently accepted five evolutionary clades. Our observations confirmed the bacterial receptacle is a modification of the two most anterior cells of the ventricular portion of the intestine. Size of the bacterial receptacle varied across the examined species. Steinernema monticolum (clade II) had the largest receptacle of all examined species (average: 46×17 μm) and S. rarum (no clade affiliation) was noted as the species with the smallest observed receptacle (average: 8×5 μm). At the morphological level, species can be grouped into two categories based on the presence or absence of vesicle within the receptacle. The receptacles of all examined species harbored an intravesicular structure (IVS) with variable morphology. All examined taxa members of the 'feltiae' (clade III) and 'intermedium' (clade II) clades were characterized by having a vesicle. This structure was also observed in S. diaprepesi (clade V), S. riobrave (clade IV) and S. monticolum (clade I).     

Gnotobiological study of infective juveniles and symbionts of Steinernema scapterisci: A model to clarify the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes.

Gnotobiology of Steinernema scapterisci and bacteriological study of its symbiont confirmed that this nematode harbors a symbiotic species of Xenorhabdus, as do other Steinermena species. Based on phenotypic and 16S rDNA data, this Xenorhabdus strain UY61 could be distinguished from other Xenorhabdus species. Bacteria reported previously as being associated with this nematode and belonging to several other genera were probably contaminating bacteria located in the intercuticular space of the infective juveniles (IJs). These bacteria were detrimental to nematode reproduction in Galleria mellonella. Axenic S. scapterisci and its symbiont Xenorhabdus strain UY61 alone were not pathogenic to G. mellonella. The combination of both partners reestablished the pathogenicity of the complex toward G. mellonella. This combination also gave the best yields of IJs when produced in this insect and in vitro production on artificial diet.     

Neoaplectana species: Specificity of association with bacteria of the genus Xenorhabdus

Each of five Neoaplectana (Nematoda: Steinernematidae) species was cultured monoxenically with various Xenorhabdus (Eubacteriales: Enterobacteriaceae) isolates. The nematodes were usually able to reproduce when cultured with the bacterial symbiont of any one of the five Neoaplectana spp. but never with Xenorhabdus luminescens, symbiotic with Heterorhabditis spp., or with the Xenorhabdus sp. isolated from an undescribed steinernematid species. Only Neoaplectana bibionis could be cultured with the Xenorhabdus symbiont of Steinernema kraussei. A high proportion of infectives were able to carry within their intestine X. nematophilus isolated from other strains of the same nematode species; a small proportion of infectives were able to carry X. nematophilus isolated from other nematode species.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Identification, typing, and insecticidal activity of Xenorhabdus isolates from entomopathogenic nematodes in United Kingdom soil and characterization of the xpt toxin loci

Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Divergence in the chloroplast genome and nuclear rDNA of the rare western australian plant lambertia orbifolia gardner (Proteaceae)

The population genetic structure of the Australian plant Lambertia orbifolia was investigated for chloroplast DNA (cpDNA) and rDNA based on restriction fragment length polymorphism. Variation was assessed in 14-20 individuals from six populations with probes covering the majority of the chloroplast genome and the whole rRNA gene unit. For cpDNA, eight mutations were detected which were distributed over five haplotypes. Nucleotide diversity in the species was high and the majority of this diversity was distributed between populations with diversity within populations restricted to a single population. There was significant differentiation between the two regions in the species distribution with the Narrikup region being distinguished by a single haplotype that was characterized by six unique mutations. Variation in rDNA was detected with three gene length variants present in most individuals. However, the Narrikup region was characterized by homogenization of the gene unit to a single length variant in all individuals. The divergence of the Narrikup region suggests that the disjunction in the species distribution has been present for a long time and the two regions represent separate evolutionary lineages.