Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach.

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Dauer juvenile recovery transcriptome of two contrasting EMS mutants of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent against many insect pests. Dauer Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m3 in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL-1) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Entomopathogenic nematodes for biological control of Psylliodes chrysocephala (Coleoptera: Chrysomelidae) in oilseed rape.

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

Stress tolerance in entomopathogenic nematodes: Engineering superior nematodes for precision agriculture.

Entomopathogenic nematodes (EPNs) are soil-dwelling parasitic roundworms commonly used as biocontrol agents of insect pests in agriculture. EPN dauer juveniles locate and infect a host in which they will grow and multiply until resource depletion. During their free-living stage, EPNs face a series of internal and environmental stresses. Their ability to overcome these challenges is crucial to determine their infection success and survival. In this review, we provide a comprehensive overview of EPN response to stresses associated with starvation, low/elevated temperatures, desiccation, osmotic stress, hypoxia, and ultra-violet light. We further report EPN defense strategies to cope with biotic stressors such as viruses, bacteria, fungi, and predatory insects. By comparing the genetic and biochemical basis of these strategies to the nematode model Caenorhabditis elegans, we provide new avenues and targets to select and engineer precision nematodes adapted to specific field conditions.

Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Applying inbreeding, hybridization and mutagenesis to improve oxidative stress tolerance and longevity of the entomopathogenic nematode Heterorhabditis bacteriophora.

Poor shelf-life and sensitivity to environmental stress of entomopathogenic nematodes (EPNs) are traits, which deserve attention for improvement. Recently, a strong positive correlation between oxidative stress tolerance and longevity of Heterorhabditis bacteriophora dauer juveniles (DJs) has been reported. In this study, the improvement of H. bacteriophora DJ longevity was achieved by hybridization and mutagenesis. A hybrid pool deriving from two oxidative stress tolerant and long-living parental strains was generated. This hybrid AU1 × HU2 survived 2.6 days and 18 days longer than its best parent under oxidative stress and control conditions, respectively. In addition to the natural genetic variability, an EMS-mutant pool (M-OXI) with high longevity was generated and one of the derived mutagenized inbred lines (MOX-IL6) survived 5.8 days and 28.4 days longer than its donor line (IL3) under oxidative stress and control conditions, respectively. A genetic cross between the mutagenized inbred line and its donor line (MOX-IL × IL3) still survived 2.5 days and 18.5 days longer than the donor line under oxidative stress and control conditions, respectively. Concerning virulence and reproductive potential, trade-off effects were not observed as a result of hybridization and mutagenesis. These results underline the potential of classical genetic approaches for trait improvement in the nematode H. bacteriophora.

Life history trait analysis of the entomopathogenic nematode Steinernema feltiae provides the basis for prediction of dauer juvenile yields in monoxenic liquid culture.

Entomopathogenic nematodes (Steinernema spp.) are used in integrated pest management to control insect pests in cryptic environments. The nematodes are mass produced in monoxenic liquid culture with their symbiotic bacteria Xenorhabdus spp. For a better understanding of nematode population dynamics, the life history traits (LHTs) of the entomopathogenic nematode Steinernema feltiae were assessed at 25 °C by observing single pairs of male and female nematodes using a hanging drop technique. To investigate the influence of different food supplies on nematode reproduction, the LHTs were assessed with a daily supply of 5 ×, 10 × and 20 × 10(9) cells ml(-1) of the nematode's bacterial symbiont Xenorhabdus bovienii in semi-solid nematode growth gelrite (NGG) medium. Increasing bacterial density had a significant positive influence on the average number of offspring produced, which ranged from 359 to 813 per female. The intrinsic rate of natural increase r m, which ranges from 1.10 to 1.19 day(-1), was neither influenced by the bacterial density, nor was the mean generation time T (5.12-5.25 days) and population doubling time (PDT) (0.64-0.59 days). The average lifespan of reproductive females, which ranged from 6.7 to 7.3 days, was positively correlated with bacterial density. A positive correlation between female body volume and bacterial density was recorded (R = 0.67) as well as a significant positive correlation between female body size and offspring production (R = 0.89) in hanging drops. Whether these data can be used to predict nematode yields in liquid culture was tested. The total female body volume calculated as the average female body volume × total number of parental females per millilitre 3 days after nematode inoculation was positively correlated (R = 0.72) with nematode yields. The total female body volume on process day 3 is thus a good indicator for the estimation of nematode yield at the end of the process (12-15 days post dauer juvenile (DJ) inoculation) in both Erlenmeyer flasks and bioreactors. With a mean deviation of 9467 DJs ml(-1), the error resembles approximately 5 % of the final DJ yields.

Monoxenic liquid culture with Escherichia coli of the free-living nematode Panagrolaimus sp. (strain NFS 24-5), a potential live food candidate for marine fish and shrimp larvae.

The free-living, bacterial-feeding nematode Panagrolaimus sp. (strain NFS 24-5) has potential for use as live food for marine shrimp and fish larvae. Mass production in liquid culture is a prerequisite for its commercial exploitation. Panagrolaimus sp. was propagated in monoxenic liquid culture on Escherichia coli and parameters, like nematode density, population dynamics and biomass were recorded and compared with life history table data. A mean maximum nematode density of 174,278 mL(-1) and a maximum of 251,000 mL(-1) were recorded on day 17 after inoculation. Highest average biomass was 40 g L(-1) at day 13. The comparison with life history table data indicated that the hypothetical potential of liquid culture is much higher than documented during this investigation. Nematode development is delayed in liquid culture and egg production per female is more than five times lower than reported from life history trait analysis. The latter assessed a nematode generation time of 7.1 days, whereas the process time at maximum nematode density in liquid culture was 16 days indicating that a reduction of the process time can be achieved by further investigating the influence of nematode inoculum density on population development. The results challenge future research to reduce process time and variability and improve population dynamics also during scale-up of the liquid culture process.

Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.

A realistic appraisal of methods to enhance desiccation tolerance of entomopathogenic nematodes.

Understanding the desiccation survival attributes of infective juveniles of entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis, is central to evaluating the reality of enhancing the shelf-life and field persistence of commercial formulations. Early work on the structural and physiological aspects of desiccation survival focused on the role of the molted cuticle in controlling the rate of water loss and the importance of energy reserves, particularly neutral lipids. The accumulation of trehalose was also found to enhance desiccation survival. Isolation of natural populations that can survive harsh environments, such as deserts, indicated that some populations have enhanced abilities to survive desiccation. However, survival abilities of EPN are limited compared with those of some species of plant-parasitic nematodes inhabiting aerial parts of plants. Research on EPN stress tolerance has expanded on two main lines: i) to select strains of species, currently in use commercially, which have increased tolerance to environmental extremes; and ii) to utilize molecular information, including expressed sequence tags and genome sequence data, to determine the underlying genetic factors that control longevity and stress tolerance of EPN. However, given the inherent limitations of EPN survival ability, it is likely that improved formulation will be the major factor to enhance EPN longevity and, perhaps, increase the range of applications.

Gluconacetobacter diazotrophicus Pal5 Enhances Plant Robustness Status under the Combination of Moderate Drought and Low Nitrogen Stress in Zea mays L.

Plant growth promoting endophytic bacteria, which can fix nitrogen, plays a vital role in plant growth promotion. Previous authors have evaluated the effect of Gluconacetobacter diazotrophicus Pal5 inoculation on plants subjected to different sources of abiotic stress on an individual basis. The present study aimed to appraise the effect of G. diazotrophicus inoculation on the amelioration of the individual and combined effects of drought and nitrogen stress in maize plants (Zea mays L.). A pot experiment was conducted whereby treatments consisted of maize plants cultivated under drought stress, in soil with a low nitrogen concentration and these two stress sources combined, with and without G. diazotrophicus seed inoculation. The inoculated plants showed increased plant biomass, chlorophyll content, plant nitrogen uptake, and water use efficiency. A general increase in copy numbers of G. diazotrophicus, based on 16S rRNA gene quantification, was detected under combined moderate stress, in addition to an increase in the abundance of genes involved in N fixation (nifH). Endophytic colonization of bacteria was negatively affected by severe stress treatments. Overall, G. diazotrophicus Pal5 can be considered as an effective tool to increase maize crop production under drought conditions with low application of nitrogen fertilizer.

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 x 10(3) DJ per milliliter for S. carpocapsae and 1 to 8 x 10(3) DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 x 10(3) DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3-6 x 10(3) DJs per milliliter in order to obtain >10(3) parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 x 10(3) DJs per milliliter to reach >2 x 10(3) parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 x 10(3) and 105 x 10(3) per mililiter for S. feltiae.

Verticillium Wilt in Oilseed Rape-the Microbiome is Crucial for Disease Outbreaks as Well as for Efficient Suppression.

Microbiome management is a promising way to suppress verticillium wilt, a severe disease in Brassica caused by Verticillium longisporum. In order to improve current biocontrol strategies, we compared bacterial Verticillium antagonists in different assays using a hierarchical selection and evaluation scheme, and we integrated outcomes of our previous studies. The result was strongly dependent on the assessment method chosen (in vitro, in vivo, in situ), on the growth conditions of the plants and their genotype. The most promising biocontrol candidate identified was a Brassica endophyte Serratia plymuthica F20. Positive results were confirmed in field trials and by microscopically visualizing the three-way interaction. Applying antagonists in seed treatment contributes to an exceptionally low ecological footprint, supporting efficient economic and ecological solutions to controlling verticillium wilt. Indigenous microbiome, especially soil and seed microbiome, has been identified as key to understanding disease outbreaks and suppression. We suggest that verticillium wilt is a microbiome-driven disease caused by a reduction in microbial diversity within seeds and in the soil surrounding them. We strongly recommend integrating microbiome data in the development of new biocontrol and breeding strategies and combining both strategies with the aim of designing healthy microbiomes, thus making plants more resilient toward soil-borne pathogens.

Characterization of the phenotypic and genotypic tolerance to abiotic stresses of natural populations of Heterorhabditis bacteriophora.

Entomopathogenic nematodes are effective biocontrol agents against arthropod pests. However, their efficacy is limited due to sensitivity to environmental extremes. The objective of the present study was to establish a foundation of genetic-based selection tools for beneficial traits of heat and desiccation tolerance in entomopathogenic nematodes. Screening of natural populations enabled us to create a diverse genetic and phenotypic pool. Gene expression patterns and genomic variation were studied in natural isolates. Heterorhabditis isolates were phenotyped by heat- and desiccation-stress bioassays to determine their survival rates compared to a commercial line. Transcriptomic study was carried out for the commercial line, a high heat-tolerant strain, and for the natural, low heat-tolerant isolate. The results revealed a higher number of upregulated vs. downregulated transcripts in both isolates vs. their respective controls. Functional annotation of the differentially expressed transcripts revealed several known stress-related genes and pathways uniquely expressed. Genome sequencing of isolates with varied degrees of stress tolerance indicated variation among the isolates regardless of their phenotypic characterization. The obtained data lays the groundwork for future studies aimed at identifying genes and molecular markers as genetic selection tools for enhancement of entomopathogenic nematodes ability to withstand environmental stress conditions.

The structure of the Brassica napus seed microbiome is cultivar-dependent and affects the interactions of symbionts and pathogens.

BACKGROUND: Although the plant microbiome is crucial for plant health, little is known about the significance of the seed microbiome. Here, we studied indigenous bacterial communities associated with the seeds in different cultivars of oilseed rape and their interactions with symbiotic and pathogenic microorganisms. RESULTS: We found a high bacterial diversity expressed by tight bacterial co-occurrence networks within the rape seed microbiome, as identified by llumina MiSeq amplicon sequencing. In total, 8362 operational taxonomic units (OTUs) of 40 bacterial phyla with a predominance of Proteobacteria (56%) were found. The three cultivars that were analyzed shared only one third of the OTUs. The shared core of OTUs consisted mainly of Alphaproteobacteria (33%). Each cultivar was characterized by having its own unique bacterial structure, diversity, and proportion of unique microorganisms (25%). The cultivar with the lowest bacterial abundance, diversity, and the highest predicted bacterial metabolic activity rate contained the highest abundance of potential pathogens within the seed. This data corresponded with the observation that seedlings belonging to this cultivar responded more strongly to the seed treatments with bacterial inoculants than other cultivars. Cultivars containing higher indigenous diversity were characterized as having a higher colonization resistance against beneficial and pathogenic microorganisms. Our results were confirmed by microscopic images of the seed microbiota. CONCLUSIONS: The structure of the seed microbiome is an important factor in the development of colonization resistance against pathogens. It also has a strong influence on the response of seedlings to biological seed treatments. These novel insights into seed microbiome structure will enable the development of next generation strategies combining both biocontrol and breeding approaches to address world agricultural challenges.

Photorhabdus luminescens LN2 requires rpoS for nematicidal activity and nematode development.

Photorhabdus (Enterobacteriaceae) bacteria are pathogenic to insects and mutualistic with entomopathogenic Heterorhabditis nematodes. Photorhabdus luminescens subsp. akhurstii LN2, associated with Heterorhabditis indica LN2, shows nematicidal activity against H. bacteriophora H06 infective juveniles (IJs). In the present study, an rpoS mutant of P. luminescens LN2 was generated through allelic exchange to examine the effects of rpoS deletion on the nematicidal activity and nematode development. The results showed that P. luminescens LN2 required rpoS for nematicidal activity against H06 nematodes, normal IJ recovery and development of H. indica LN2, however, not for the bacterial colonization in LN2 and H06 IJs. This provides cues for further understanding the role of rpoS in the mutualistic association between entomopathogenic nematodes and their symbionts.

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.

Bacterial isolates from nematodes from Turkish soil samples were initially characterized by molecular methods and seven members of the genus Photorhabdus identified to the species level, using riboprint analyses and metabolic properties. Strain 07-5 (DSM 15195) was highly related to the type strain of Photorhabdus luminescens subsp. laumondii DSM 15139T, and was regarded a strain of this subspecies. Strains 1121T (DSM 15194T), 68-3 (DSM 15198) and 47-10 (DSM 15197) formed one, strain 39-8T (DSM 15199T), 39-7 (DSM 15196) and 01-12 (DSM 15193) formed a second cluster that branched intermediate the three subspecies of Photorhabdus luminescens. Based upon moderate 16S rRNA gene sequence similarities and differences in metabolic properties among themselves and with type strains of the three subspecies we consider the two clusters to represent two new subspecies of Photorhabdus luminescens for which the names Photorhabdus luminescens subsp. kayaii, type strain 1121T (DSM 15194T, NCIMB 13951T), and Photorhabdus luminescens subsp. thracensis subsp. nov., type strain 39-8T (DSM 15199T, NCIMB 13952T) are proposed.

Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation.

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.