Symbionts Of Entomopathogenic Nematodes Research Articles

Antifungal Effect of Metabolites from Bacterial Symbionts of Entomopathogenic Nematodes on Fusarium Head Blight of Wheat.

Fungal diseases such as Fusarium head blight (FHB) are significant biotic stressors, negatively affecting wheat production and quality. This study explored the antifungal activity of the metabolites produced by the bacterial symbionts of entomopathogenic nematodes (EPNs) against FHB-causing Fusarium sp. Fusarium graminearum. To achieve this, the symbiotic bacteria of nine EPN isolates from the EPN collection at the Agricultural Research Council-Small Grains (ARC-SG) were isolated from the cadavers of Galleria mellonella (Lepidoptera: Pyralidae) larvae after infection with EPNs. Broth cultures (crude) and their supernatants (filtered and autoclaved) of each bacterial isolate were used as bacterial metabolite treatments to test their inhibitory effect on the mycelial growth and spore germination of F. graminearum. Mycelial growth inhibition rates varied among both bacterial isolates and treatments. Crude metabolite treatments proved to be more effective than filtered and autoclaved metabolite treatments, with an overall inhibition rate of 75.25% compared to 23.93% and 13.32%, respectively. From the crude metabolite treatments, the Xenorhabdus khoisanae SGI 197 bacterial isolate from Steinernema beitlechemi SGI 197 had the highest mean inhibition rate of 96.25%, followed by Photorhabdus luminescens SGI 170 bacteria isolated from Heterorhabditis bacteriophora SGI 170 with a 95.79% mean inhibition rate. The filtered metabolite treatments of all bacterial isolates were tested for their inhibitory activity against Fusarium graminearum spore germination. Mean spore germination inhibition rates from Xenorhabdus spp. bacterial isolates were higher (83.91 to 96.29%) than those from Photorhabdus spp. (6.05 to 14.74%). The results obtained from this study suggest that EPN symbiotic bacterial metabolites have potential use as biological control agents of FHB. Although field efficacy against FHB was not studied, the significant inhibition of mycelial growth and spore germination suggest that the application of these metabolites at the flowering stage may provide protection to plants against infection with or spread of F. graminearum. These metabolites have the potential to be employed as part of integrated pest management (IPM) to inhibit/delay conidia germination until the anthesis (flowering stage) of wheat seedlings has passed.

Discovery of a new highly pathogenic toxin involved in insect sepsis.

Insect sepsis is a severe consequence that arises from the invasion of the hemocoel by symbionts of entomopathogenic nematodes and bacteria. In the present study, we unveiled the heightened virulence of the entomopathogenic nematode Steinernema feltiae and the entomopathogenic bacteria Xenorhabdus bovienii, which operate symbiotically, against the wax moth Galleria mellonella. Maximum mortality was observed at 25°C while the optimal infestation efficiency was 20 nematodes per host. After infestation, G. mellonella displayed rapid darkening and softening, accompanied by an escalated esterase activity at 9 h. The X. bovienii, released by S. feltiae, underwent substantial proliferation and discharged toxins that attacked hemocytes, thus triggering extensive hemolysis and sepsis. The host G. mellonella was usually killed within 24 h due to disseminated septicemia. Additionally, X. bovienii infestation led to the upregulation of metabolites like 3-hydroxyanthranilic acid. Strikingly, we identified the perilous actinomycin D, generated through kynurenine metabolites, representing a novel biomarker of insect sepsis. Furthermore, a comprehensive transcriptomic analysis unveiled a noteworthy upregulation of gene expression associated with actinomycin D. Overall, X. bovienii induced apoptosis and sepsis through actinomycin D production, indicating its pivotal role in infestation activity. These findings open up new avenues for studying the mechanism of sepsis and developing innovative biotic pesticides. IMPORTANCE As a current biocontrol resource, entomopathogenic nematodes and their symbiotic bacterium can produce many toxin factors to trigger insect sepsis, having the potential to promote sustainable pest management. In this study, we found Steinernema feltiae and Xenorhabdus bovienii were highly virulent against the insects. After infective juvenile injection, Galleria mellonella quickly turned black and softened with increasing esterase activity. Simultaneously, X. bovienii attacked hemocytes and released toxic components, resulting in extensive hemolysis and sepsis. Then, we applied high-resolution mass spectrometry-based metabolomics and found multiple substances were upregulated in the host hemolymph. We found extremely hazardous actinomycin D produced via 3-hydroxyanthranilic acid metabolites. Moreover, a combined transcriptomic analysis revealed that gene expression of proteins associated with actinomycin D was upregulated. Our research revealed actinomycin D might be responsible for the infestation activity of X. bovienii, indicating a new direction for exploring the sepsis mechanism and developing novel biotic pesticides.

Toxicity of Photorhabdus luminescens and Xenorhabdus bovienii bacterial metabolites to pecan aphids (Hemiptera: Aphididae) and the lady beetle Harmonia axyridis (Coleoptera: Coccinellidae)

Toxicity of the metabolites of two bacteria, Photorhabdus luminescens and Xenorhabdus bovienii, symbionts of entomopathogenic nematodes, were tested in the laboratory against the multicolored Asian lady beetle, Harmonia axyridis, the black pecan aphid, Melanocallis caryaefoliae, and the blackmargined aphid, Monellia caryella. Bacterial broth prepared from both P. luminescens and X. bovienii demonstrated high levels of toxicity equivalent to the pyrethroid insecticide bifenthrin and caused higher insect mortality than tryptic soy broth plus yeast extract (TSY) (blank control) against M. caryella; broth culture of P. luminescens was more effective than TSY against M. caryaefoliae. At the levels tested, the metabolites were not toxic to H. axyridis.

Comparative Genomics and Pathogenicity Analysis of Two Bacterial Symbionts of Entomopathogenic Nematodes: The Role of the GroEL Protein in Virulence.

Bacteria of the genera Xenorhabdus and Photorhabdus are symbionts of entomopathogenic nematodes. Despite their close phylogenetic relationship, they show differences in their pathogenicity and virulence mechanisms in target insects. These differences were explored by the analysis of the pangenome, as it provides a framework for characterizing and defining the gene repertoire. We performed the first pangenome analysis of 91 strains of Xenorhabdus and Photorhabdus; the analysis showed that the Photorhabdus genus has a higher number of genes associated with pathogenicity. However, biological tests showed that whole cells of X. nematophila SC 0516 were more virulent than those of P. luminescens HIM3 when both were injected into G. mellonella larvae. In addition, we cloned and expressed the GroEL proteins of both bacteria, as this protein has been previously indicated to show insecticidal activity in the genus Xenorhabdus. Among these proteins, Cpn60-Xn was found to be the most toxic at all concentrations tested, with an LC50 value of 102.34 ng/larva. Sequence analysis suggested that the Cpn60-Xn toxin was homologous to Cpn60-Pl; however, Cpn60-Xn contained thirty-five differentially substituted amino acid residues that could be responsible for its insecticidal activity.

Screening and Molecular Identification of Bacteria from the Midgut of Amphimallon solstitiale Larvae Exhibiting Antagonistic Activity against Bacterial Symbionts of Entomopathogenic Nematodes.

Entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) are a group of organisms capable of infecting larvae of insects living in soil, including representatives of the family Scarabaeidae. Their insecticidal activity is related to the presence of symbiotic bacteria Xenorhabdus spp. or Photorhabdus spp. in the alimentary tract, which are released into the insect body, leading to its death caused by bacterial toxins and septicemia. Although the antibacterial activities of symbionts of entomopathogenic nematodes have been well described, there is insufficient knowledge of the interactions between these bacteria and microorganisms that naturally inhabit the alimentary tract of insects infested by nematodes. In this study, 900 bacterial strains isolated from midgut samples of Amphimallon solstitiale larvae were tested for their antagonistic activity against the selected five Xenorhabdus and Photorhabdus species. Cross-streak tests showed significant antibacterial activity of 20 isolates. These bacteria were identified as Bacillus [Brevibacterium] frigoritolerans, Bacillus toyonensis, Bacillus wiedmannii, Chryseobacterium lathyri, Chryseobacterium sp., Citrobacter murliniae, Enterococcus malodoratus, Paenibacillus sp., Serratia marcescens and Serratia sp. Since some representatives of the intestinal microbiota of A. solstitiale are able to inhibit the growth of Xenorhabdus and Photorhrhabdus bacteria in vitro, it can be assumed that this type of bacterial interaction may occur at certain stages of insect infection by Steinernema or Heterorhabditis nematodes.

Photorhabdus spp.: An Overview of the Beneficial Aspects of Mutualistic Bacteria of Insecticidal Nematodes.

The current approaches to sustainable agricultural development aspire to use safer means to control pests and pathogens. Photorhabdus bacteria that are insecticidal symbionts of entomopathogenic nematodes in the genus Heterorhabditis can provide such a service with a treasure trove of insecticidal compounds and an ability to cope with the insect immune system. This review highlights the need of Photorhabdus-derived insecticidal, fungicidal, pharmaceutical, parasiticidal, antimicrobial, and toxic materials to fit into current, or emerging, holistic strategies, mainly for managing plant pests and pathogens. The widespread use of these bacteria, however, has been slow, due to cost, natural presence within the uneven distribution of their nematode partners, and problems with trait stability during in vitro culture. Yet, progress has been made, showing an ability to overcome these obstacles via offering affordable mass production and mastered genome sequencing, while detecting more of their beneficial bacterial species/strains. Their high pathogenicity to a wide range of arthropods, efficiency against diseases, and versatility, suggest future promising industrial products. The many useful properties of these bacteria can facilitate their integration with other pest/disease management tactics for crop protection.

Bacteria from the Midgut of Common Cockchafer (Melolontha melolontha L.) Larvae Exhibiting Antagonistic Activity Against Bacterial Symbionts of Entomopathogenic Nematodes: Isolation and Molecular Identification.

The mechanisms of action of the complex including entomopathogenic nematodes of the genera Steinernema and Heterorhabditis and their mutualistic partners, i.e., bacteria Xenorhabdus and Photorhabdus, have been well explained, and the nematodes have been commercialized as biological control agents against many soil insect pests. However, little is known regarding the nature of the relationships between these bacteria and the gut microbiota of infected insects. In the present study, 900 bacterial isolates that were obtained from the midgut samples of Melolontha melolontha larvae were screened for their antagonistic activity against the selected species of the genera Xenorhabdus and Photorhabdus. Twelve strains exhibited significant antibacterial activity in the applied tests. They were identified based on 16S rRNA and rpoB, rpoD, or recA gene sequences as Pseudomonas chlororaphis, Citrobacter murliniae, Acinetobacter calcoaceticus, Chryseobacterium lathyri, Chryseobacterium sp., Serratia liquefaciens, and Serratia sp. The culture filtrate of the isolate P. chlororaphis MMC3 L3 04 exerted the strongest inhibitory effect on the tested bacteria. The results of the preliminary study that are presented here, which focused on interactions between the insect gut microbiota and mutualistic bacteria of entomopathogenic nematodes, show that bacteria inhabiting the gut of insects might play a key role in insect resistance to entomopathogenic nematode pressure.

NgrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Trade-off between reproductive and anti-competitor abilities in an insect-parasitic nematode-bacteria symbiosis.

Mutualistic symbionts can provide diverse benefits to their hosts and often supply key trait variation for host adaptation. The bacterial symbionts of entomopathogenic nematodes play a crucial role in successful colonization of and reproduction in the insect host. Additionally, these symbionts can produce a diverse array of antimicrobial compounds to deter within‐host competitors. Natural isolates of the symbiont, Xenorhabdus bovienii, show considerable variation in their ability to target sympatric competitors via bacteriocins, which can inhibit the growth of sensitive Xenorhabdus strains. Both the bacteria and its nematode partner have been shown to benefit from bacteriocin production when within‐host competition with a sensitive competitor occurs. Despite this benefit, several isolates of Xenorhabdus do not inhibit sympatric strains. To understand how this variation in allelopathy could be maintained, we tested the hypothesis that inhibiting isolates face a reproductive cost in the absence of competition. We tested this hypothesis by examining the reproductive success of inhibiting and non‐inhibiting isolates coupled with their natural nematode host in a non‐competitive context. We found that nematodes carrying non‐inhibitors killed the insect host more rapidly and were more likely to successfully reproduce than nematodes carrying inhibitors. Lower reproductive success of inhibiting isolates was repeatable across nematode generations and across insect host species. However, no difference in insect mortality was observed between inhibiting and non‐inhibiting isolates when bacteria were injected into insects without their nematode partners. Our results indicate a trade‐off between the competitive and reproductive roles of symbionts, such that inhibiting isolates, which are better in the face of within‐host competition, pay a reproductive cost in the absence of competition. Furthermore, our results support the hypothesis that symbiont variation within populations can be maintained through context‐dependent fitness benefits conferred to their hosts. As such, our study offers novel insights into the selective forces maintaining variation within a single host–symbiont population and highlights the role of competition in mutualism evolution.

Characterization Physiology and Molecular Bacteria Symbiont- Entomopathogenic Nematodes based of Gene Sequences Encoding the 16S rRNA District of Bromo Probolinggo


 
 
 This study aims to identify entomopathogenic nematodes symbiotic bacteria phenotypically and based on the gene encoding 16S rRNA sequences. Bacterial symbionts of entomopathogenic nematodes, obtained from isolates from the area Wonokerto (WN01) and isolates Sukapura (SP01), Bromo, Probolinggo, two symbiont bacteria was found in entomopathogenic nematodes Steinernema sp. The method used in this study are: the isolation of entomopathogenic nematodes Steinernema sp. and bacterial symbionts conventionally for the identification of phenotypically, after the characterization of bacterial isolates, the isolation of genomic DNA, 16S rRNA PCR, DNA purification and DNA sequence analysis. The results based on phenotypic characterization showed that isolates WN01 and SP01, yellowish white, gram positive, negative bioluminenscene, catalase positive, can not hydrolyze urea, and also can not produce H2S. The results of the gene encoding 16S rRNA sequence can be deduced WN01 isolates have in common with the bacteria Bacillus strain toyonensis BCT 7112, while the SP01 isolates have in common with the bacteria Bacillus strain cereus ATCC 14 579.
 
 

Relative potency of culture supernatants of Xenorhabdus and Photorhabdus spp. on growth of some fungal phytopathogens

In previous research, concentrated metabolites produced by bacteria of the genera Xenorhabdus and Photorhabdus (which are symbionts of entomopathogenic nematodes) were reported to be highly suppressive to fungal and oomycete plant pathogens. Conceivably, application of non-concentrated bacterial filtrates would be more economically feasible compared to using concentrated metabolites. We evaluated the potency of 10 % v/v cell-free supernatants of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), F. effusum (pecan scab), Monilinia fructicola (brown rot), Glomerella cingulata (anthracnose) and Armillaria tabescens (root rot). A bioactive compound derived from Photorhabdus bacteria, trans-cinnamic acid (TCA), was also compared with the bacterial filtrates. Fungal colony size based on manual measurements was compared for accuracy to measurements taken by image analysis. Supernatants of Xenorhabdus spp. exhibited stronger suppressive effects on spore germination and vegetative growth when compared with Photorhabdus spp. Overall, TCA was the most effective treatment; vegetative growth was completely inhibited by TCA (1.27 mg/ml). TCA treatments also suppressed spore germination of F. carpophylium and F. effussum by approximately 90 %. The efficacy of supernatants varied among Xenorhabdus species depending on the species tested, but X. szentirmaii filtrates tended to cause greater inhibition relative to the other bacteria supernatants. Manual measurement of colony diameter required at least two replicate estimates of the colony to avoid a type II error. Area measurements were slightly overestimated based on ruler measurements, but did not affect the outcome of the analysis. Supernatants of Xenorhabdus spp., Photorhabdus spp., or TCA, did not cause any phytotoxic effects when applied to various plant species in the greenhouse. Our results indicate the potential of using TCA or Xenorhabdus cell free supernatants as bio-fungicides. Such a product, based on bacterial culture supernatants, would be economically viable, marketable and easily applicable by the end-users in many situations.

Diversity of Xenorhabdus and Photorhabdus spp. and their symbiotic entomopathogenic nematodes from Thailand.

Xenorhabdus and Photorhabdus spp. are bacterial symbionts of entomopathogenic nematodes (EPNs). In this study, we isolated and characterized Xenorhabdus and Photorhabdus spp. from across Thailand together with their associated nematode symbionts, and characterized their phylogenetic diversity. EPNs were isolated from soil samples using a Galleria-baiting technique. Bacteria from EPNs were cultured and genotyped based on recA sequence. The nematodes were identified based on sequences of 28S rDNA and internal transcribed spacer regions. A total of 795 soil samples were collected from 159 sites in 13 provinces across Thailand. A total of 126 EPNs isolated from samples taken from 10 provinces were positive for Xenorhabdus (n = 69) or Photorhabdus spp. (n = 57). Phylogenetic analysis separated the 69 Xenorhabdus isolates into 4 groups. Groups 1, 2 and 3 consisting of 52, 13 and 1 isolates related to X. stockiae, and group 4 consisting of 3 isolates related to X. miraniensis. The EPN host for isolates related to X. stockiae was S. websteri, and for X. miraniensis was S. khoisanae. The Photorhabdus species were identified as P. luminescens (n = 56) and P. asymbiotica (n = 1). Phylogenenic analysis divided P. luminescens into five groups. Groups 1 and 2 consisted of 45 and 8 isolates defined as subspecies hainanensis and akhurstii, respectively. One isolate was related to hainanensis and akhurstii, two isolates were related to laumondii, and one isolate was the pathogenic species P. asymbiotica subsp. australis. H. indica was the major EPN host for Photorhabdus. This study reveals the genetic diversity of Xenorhabdus and Photorhabdus spp. and describes new associations between EPNs and their bacterial symbionts in Thailand.

A multigene approach for assessing evolutionary relationships of Xenorhabdus spp. (γ-Proteobacteria), the bacterial symbionts of entomopathogenic Steinernema nematodes

Xenorhabdus spp., are gram-negative bacterial symbionts of entomopathogenic nematodes in the genus Steinernema. A specialized and intimate relationship exists between nematode and bacteria, affecting many of their life history traits, such as nutrition, dispersal, host-finding, foraging and defense from biotic and abiotic factors. Xenorhabdus currently comprises more than 20 species isolated from Steinernema spp. with diverse host range, host foraging behavior, reproductive modes and environmental tolerance. Xenorhabdus phylogenies have historically been based on 16s rDNA sequence analyses, and only recently has data from housekeeping genes been employed. The prevalence of lateral gene transfer among bacteria calls for a wider perspective when considering their phylogeny. With the increasing number of Xenorhabdus species and strains, various perspectives need to be considered for investigating the evolutionary history of these nematode bacterial symbionts, In this study, we reconstruct the evolutionary histories of 30 species of Xenorhabdus considering the traditional 16s rDNA gene region as well as the housekeeping genes recA and serC. Datasets were analyzed individually and then combined, using a variety of phylogenetic criteria.

Xenorhabdus antibiotics: a comparative analysis and potential utility for controlling mastitis caused by bacteria

The role of antibiotics produced by bacterial symbionts of entomopathogenic nematodes is to suppress growth of microbes in the soil environment. These antibiotics are active against Gram-positive and Gram-negative bacteria, and were tested against mastitis isolates from dairy cows. Two bioassays were adapted for Xenorhabdus antibiotics; an overlay method on agar plates, and serially diluted, cell-free, Xenorhabdus cultures. The antimicrobial activities of the liquid cultures of 13 strains from five Xenorhabdus species were further evaluated. Antimicrobial activities of the type strains of X. nematophila, X. budapestensis and X. szentirmaii were tested on mastitis isolates of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae with both bioassays. A previously reported antibiotic from X. nematophila, nematophin, was synthesized in three steps from tryptamine and 4-methyl-2-oxovaleric acid sodium salt. The antibiotics of all three Xenorhabdus strains were powerful in either bioassay, but the sensitivity of the isolates differed from each other. While Kl. pneumoniae was the least susceptible, Staph. aureus had the highest sensitivity to each Xenorhabdus strain. Xenorhabdus szentirmaii and X. budapestensis were more potent antibiotic producers than X. nematophila, and raceme nematophin was ineffective against all mastitis isolates. These results indicate that Xenorhabdus antibiotics are effective against mastitis isolates and should be further evaluated for their potential in mastitis control or prevention.

Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nema- todes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. bron- gniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial pro- duction; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 lg/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 lg/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 lg/ml).

Comparison of the bioluminescence ofPhotorhabdus species and subspecies type strains

Natural bioluminescence of all recently accepted Photorhabdus species and subspecies type strains (bacterial symbionts of entomopathogenic nematodes) was measured using a commercial luminometer; optimum conditions for the measurement were described. Cultures emitted reliably measurable bioluminescence with characteristic level and kinetics for each strain. Bioluminescence of all strains was significantly higher at 37 than at 25 degrees C at the beginning of the measurement, no effect of bacterial concentration on the intensity of bioluminescence was observed. The technique can provide reliable and quick information for the determination of Photorhabdus taxons.

Hemocyte Apoptosis Induced by Entomopathogenic Bacteria, Xenorhabdus and Photorhabdus, in Bombyx mori

Entomopathogenic bacteria of Xenorhabdus nematophila, Xenorhabdus sp., and Photorhabdus temperata subsp. temperata are symbionts of entomopathogenic nematodes including Steinernema and Heterorhabditis. These bacteria have potent insecticidal pathogenicity causing hemolymph septicemia. To explain a mechanism of the septicemia, this research raises a hypothesis that the hemolymph septicemia is due to the induction of the programmed cell death (=apoptosis) of the hemocytes by the entomopathogenic bacteria. Injection of the bacteria into the hemocoel of the fifth instar larvae of Bombyx mori led to septicemia (few viable hemocytes) at 12h in all three bacterial treatments. During the pathogenicity, the infected hemocytes exhibited the membrane blebbing, nuclear chromatin condensation and DNA fragmentation, which reflected typical morphological changes of cells undergoing apoptosis. The apoptosis began as early as 4h after bacterial injection and increased with post-injection time. Three pathogenic bacteria, however, differed in the degree of apoptosis-inducing effect on B. mori by the effective bacterial dose and time. These results support the hypothesis that hemolymph septicemia caused by the bacteria is due to their apoptosis-inducing effect.

The PhoP-PhoQ two-component regulatory system of Photorhabdus luminescens is essential for virulence in insects.

Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg(2+)-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg(2+)-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.

Characterisation of symbionts of entomopathogenic nematodes by universally primed-PCR (UP-PCR) and UP-PCR product cross-hybridisation.

This work introduces and demonstrates the applicability of universally primed-PCR (UP-PCR) for differentiating bacterial symbionts of entomopathogenic nematodes. Furthermore, typing by UP-PCR product cross-hybridisation was successfully introduced to cluster the bacterial strains. The work was initiated by isolating 10 isolates of Photorhabdus temperata (S172) from the nematode host Heterorhabditis sp. (DK172) and 12 isolates of Xenorhabdus bovienii (S1) from the nematode Steinernema feltiae (DK1). The isolates were compared by UP-PCR using different primers. The two bacterial species (P. temperata and X. bovienii) could be distinguished on the basis of the banding pattern whereas isolates isolated from the same nematode host had identical banding patterns. Three isolates obtained from DK172 and DK1, respectively, were then selected along with a number of reference strains (Hb, HP88, C1, K122, HSH2, HL81, T228, 61, AN6, Q58) and further characterised by UP-PCR product cross-hybridisation. The Xenorhabdus strains (Q58, AN6, 61, T228, S1) represented three species and these species were separated by the hybridisation technique. The Photorhabdus strains (Hb, HP88, C1, K122, HSH2, HL81, S172) represented two species and were also separated according to this in the cross-hybridisation. Within each species of Photorhabdus, two subgroups were formed as a result of intensity of the hybridisation signals. This grouping was in agreement with previous studies in other laboratories.

Characterisation of symbionts of entomopathogenic nematodes by universally primed-PCR (UP-PCR) and UP-PCR product cross-hybridisation

This work introduces and demonstrates the applicability of universally primed-PCR (UP-PCR) for differentiating bacterial symbionts of entomopathogenic nematodes. Furthermore, typing by UP-PCR product cross-hybridisation was successfully introduced to cluster the bacterial strains. The work was initiated by isolating 10 isolates of Photorhabdus temperata (S172) from the nematode host Heterorhabditis sp. (DK172) and 12 isolates of Xenorhabdus bovienii (S1) from the nematode Steinernema feltiae (DK1). The isolates were compared by UP-PCR using different primers. The two bacterial species ( P. temperata and X. bovienii) could be distinguished on the basis of the banding pattern whereas isolates isolated from the same nematode host had identical banding patterns. Three isolates obtained from DK172 and DK1, respectively, were then selected along with a number of reference strains (Hb, HP88, C1, K122, HSH2, HL81, T228, 61, AN6, Q58) and further characterised by UP-PCR product cross-hybridisation. The Xenorhabdus strains (Q58, AN6, 61, T228, S1) represented three species and these species were separated by the hybridisation technique. The Photorhabdus strains (Hb, HP88, C1, K122, HSH2, HL81, S172) represented two species and were also separated according to this in the cross-hybridisation. Within each species of Photorhabdus, two subgroups were formed as a result of intensity of the hybridisation signals. This grouping was in agreement with previous studies in other laboratories.