The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi

Soil-dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN–bacteria–arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non-volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non-volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A. faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens.     

British and European Legislation Regulating Rhabditid Nematodes

Rhabditid nematodes are invertebrates that introduce pathogenic bacteria to the insects and molluscs they parasitise. Marketed as biopesticides, they provide safe and effective alternatives to chemical pesticides in several important sectors of horticulture. In Great Britain, the introduction of non-indigenous species of nematodes is strictly controlled, as is the contained use and deliberate release of genetically modified nematodes and their bacterial symbionts. Currently, indigenous, unmodified nematodes do not have to be registered for use as biopesticides in the UK, but in some other European countries registration is mandatory. The European Commission is seeking to harmonize procedures for the authorization of plant protection products, including rhabditid nematodes, under the provisions of Council Directive 91/414/EEC.     

Pathogenic effect of entomopathogenic nematode-bacterium complexes on terrestrial isopods

In this study, we evaluated the effect of entomopathogenic nematodes (EPNs) Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora, symbiotically associated with bacteria of the genera Xenorhabdus or Photorhabdus, on the survival of eight terrestrial isopod species. The EPN species S. carpocapsae and H. bacteriophora reduced the survival of six isopod species while S. feltiae reduced survival for two species. Two terrestrial isopod species tested (Armadillidium vulgare and Armadillo officinalis) were found not to be affected by treatment with EPNs while the six other isopod species showed survival reduction with at least one EPN species. By using aposymbiotic S. carpocapsae (i.e. without Xenorhabdus symbionts), we showed that nematodes can be isopod pathogens on their own. Nevertheless, symbiotic nematodes were more pathogenic for isopods than aposymbiotic ones showing that bacteria acted synergistically with their nematodes to kill isopods. By direct injection of entomopathogenic bacteria into isopod hemolymph, we showed that bacteria had a pathogenic effect on terrestrial isopods even if they appeared unable to multiply within isopod hemolymphs. A developmental study of EPNs in isopods showed that two of them (S. carpocapsae and H. bacteriophora) were able to develop while S. feltiae could not. No EPN species were able to produce offspring emerging from isopods. We conclude that EPN and their bacteria can be pathogens for terrestrial isopods but that such hosts represent a reproductive dead-end for them. Thus, terrestrial isopods appear not to be alternative hosts for EPN populations maintained in the absence of insects.     

Masters of conquest and pillage: Xenorhabdus nematophila global regulators control transitions from virulence to nutrient acquisition

Invertebrate animal models are experimentally tractable and have immunity and disease symptoms that mirror those of vertebrates. Therefore they are of particular utility in understanding fundamental aspects of pathogenesis. Indeed, artificial models using human pathogens and invertebrate hosts have revealed conserved and novel molecular mechanisms of bacterial infection and host immune responses. Additional insights may be gained from investigating interactions between invertebrates and pathogens they encounter in their natural environments. For example, enteric bacteria in the genera Photorhabdus and Xenorhabdus are pathogens of insects that also mutualistically associate with nematodes in the genera Heterorhabditis and Steinernema respectively. These bacteria serve as models to understand naturally occurring symbiotic associations that result in disease in or benefit for animals. Xenorhabdus nematophila is the best-studied species of its genus with regard to the molecular mechanisms of its symbiotic associations. In this review, we summarize recent advances in understanding X. nematophila –host interactions. We emphasize regulatory cascades involved in coordinating transitions between various stages of the X. nematophila life cycle: infection, reproduction and transmission.     

Identification, Pathogenicity and Comparative Virulence of Fusarium spp. Associated with Diseased Euphorbia spp. in Europe

Fusarium spp. isolated from diseased Euphorbia spp. in Europe were assessed for pathogenicity to North American accessions of leafy spurge ( Euphorbia esula/virgata ). Of the nine strains of Fusarium spp. isolated from diseased E. stepposa or E. virgata in the Caucasus region of Russia and E. esula/virgata in southern France, all were pathogenic to leafy spurge. There were significant differences in virulence among strains. Four strains, including the two that were most virulent, were identified as F. oxysporum . Four of the five other strains were identified as F. solani and one was identified as F. proliferatum . Three of the four most virulent strains to leafy spurge were isolated from E. stepposa . The most virulent strain was associated with root damage caused by insect biological control agents, as found earlier with domestic strains of Fusarium spp. pathogenic to leafy spurge. Two strains identified as F. solani were vegetatively compatible. It was concluded that further screening of a larger set of strains of foreign Fusarium spp. under quarantine conditions in the US or in limited overseas facilities would be justified, and could yield promising biological control agents for leafy spurge.     

Bacteria used in the biological control of plant-parasitic nematodes: populations, mechanisms of action, and future prospects

As a group of important natural enemies of nematode pests, nematophagous bacteria exhibit diverse modes of action: these include parasitizing; producing toxins, antibiotics, or enzymes; competing for nutrients; inducing systemic resistance of plants; and promoting plant health. They act synergistically on nematodes through the direct suppression of nematodes, promoting plant growth, and facilitating the rhizosphere colonization and activity of microbial antagonists. This review details the nematophagous bacteria known to date, including parasitic bacteria, opportunistic parasitic bacteria, rhizobacteria, Cry protein-forming bacteria, endophytic bacteria and symbiotic bacteria. We focus on recent research developments concerning their pathogenic mechanisms at the biochemical and molecular levels. Increased understanding of the molecular basis of the various pathogenic mechanisms of the nematophagous bacteria could potentially enhance their value as effective biological control agents. We also review a number of molecular biological approaches currently used in the study of bacterial pathogenesis in nematodes. We discuss their merits, limitations and potential uses.     

Endosymbionts of arthropods and nematodes: allies to fight infectious diseases?

Arthropods and nematodes are important protagonists in human health because either they act as vectors of pathogens (bacteria, protozoa, viruses or fungus), or are themselves parasites. Fighting infectious diseases is based essentially on vaccination (prevention) or chemotherapeutic (curative) approaches in human, but one can envisage as an alternative to reduce the number of vectors or limit their ability to spread pathogens. Such strategies controlling dissemination will undoubtedly benefit from the knowledge accumulated by recent works on powerful mechanisms developed by symbiotic insect bacteria such as Wolbachia to popagate in arthropods and nematods. This review summarizes these recent data, and indicate how these mechanisms can be manipulated to reduce the dissemination of insect vectors propagating human diseases.     

Susceptibility of cockroaches (Gromphadorhina portentosa,Nauphoeta cinerea and Blaptica dubia) exposed to entomopathogenic nematodes

Cockroaches are major pests, vectors of pathogenic bacteria and induce allergies. Current control methods use chemical pesticides, but they can be ineffective and costly and there are reports of resistance in the field; hence new control methods are needed. There are conflicting reports about the susceptibility of cockroaches to entomopathogenic nematodes (EPNs); so we investigated if EPNs could kill several diverse cockroach species, including the Madagascan hissing roach (Gromphadorhina portentosa), the Lobster roach (Nauphoeta cinerea) and Blaptica dubia. Female adult cockroaches were exposed to either commercial products containing Steinernema kraussei or a combination of Heterorhabditis spp. and Steinernema spp. at 50 and 150 nematodes per cm² for 21 days. We also monitored feeding and the numbers of infective juveniles that were produced from each cockroach corpse. We found that S. kraussei were harmless to all cockroach species (at both doses) but when exposed to a mixture of Heterorhabditis spp. and Steinernema spp. B. dubia died after 6 days and its feeding was strongly inhibited. We also found that the mixture of Heterorhabditis spp. and Steinernema spp. could proliferate in the cadavers of B. dubia whilst S. kraussei could only reproduce in G. portentosa and B. dubia but not N. cinerea. In conclusion, S. kraussei was harmless to all three cockroach species but B. dubia was killed when exposed to Heterorhabditis spp. and Steinernema spp., highlighting the differences in the host range of EPNs.     

Comparison of the methods applicable for the pathogenicity assessment of entomopathogenic nematodes

Single infective juveniles of Heterorhabditis bacteriophora, H. megidis (Nematoda: Heterorhabditidae), Steinernema arenarium, S. carpocapsae and S. feltiae (Nematoda: Steinernematidae) were used to infect single Galleria mellonella (Lepidoptera: Pyralidae) larvae. Four parameters of entomopathogenic nematodes pathogenicity were assessed: the mortality of insects, infectivity of nematodes, number of nematodes established per single G. mellonella, and degree of infective juveniles colonization (percent of infective juveniles which intestine was colonized by symbiotic bacteria). The accuracy, repeatability, and versatility for different species of EPNs in bioassay arenas were compared. Our modifications of the original methods yielded ~ 50% higher efficiency of infective juveniles in cell culture plates and > 20% higher efficiency in centrifuge test tubes. The efficiency of nematodes in cell culture plates (39–77%) was relatively low, especially in the case of Heterorhabditis spp. In the bioassay arena, infective juveniles migrated between cells. The results of our studies indicate that the pathogenicity of EPNs should be assessed in centrifuge test tubes. In these arenas, the infectivity of single IJs was ~ 90% for Heterorhabditis spp. and ~ 95% for Steinernema spp. The degree of colonization of the EPN isolates by symbiotic bacteria was in the range of 96–98%.     

The endosymbiont and the second bacterial circle of entomopathogenic nematodes

Single host–symbiont interactions should be reconsidered from the perspective of the pathobiome. We revisit here the interactions between entomopathogenic nematodes (EPNs) and their microbiota. We first describe the discovery of these EPNs and their bacterial endosymbionts. We also consider EPN-like nematodes and their putative symbionts. Recent high-throughput sequencing studies have shown that EPNs and EPN-like nematodes are also associated with other bacterial communities, referred to here as the second bacterial circle of EPNs. Current findings suggest that some members of this second bacterial circle contribute to the pathogenic success of nematodes. We suggest that the endosymbiont and the second bacterial circle delimit an EPN pathobiome.     

Why bacteria matter in animal development and evolution

While largely studied because of their harmful effects on human health, there is growing appreciation that bacteria are important partners for invertebrates and vertebrates, including man. Epithelia in metazoans do not only select their microbiota; a coevolved consortium of microbes enables both invertebrates and vertebrates to expand the range of diet supply, to shape the complex immune system and to control pathogenic bacteria. Microbes in zebrafish and mice regulate gut epithelial homeostasis. In a squid, microbes control the development of the symbiotic light organ. These discoveries point to a key role for bacteria in any metazoan existence, and imply that beneficial bacteria‐host interactions should be considered an integral part of development and evolution.     

Plant-Associated Symbiotic Burkholderia Species Lack Hallmark Strategies Required in Mammalian Pathogenesis

Burkholderia is a diverse and dynamic genus, containing pathogenic species as well as species that form complex interactions with plants. Pathogenic strains, such as B. pseudomallei and B. mallei, can cause serious disease in mammals, while other Burkholderia strains are opportunistic pathogens, infecting humans or animals with a compromised immune system. Although some of the opportunistic Burkholderia pathogens are known to promote plant growth and even fix nitrogen, the risk of infection to infants, the elderly, and people who are immunocompromised has not only resulted in a restriction on their use, but has also limited the application of non-pathogenic, symbiotic species, several of which nodulate legume roots or have positive effects on plant growth. However, recent phylogenetic analyses have demonstrated that Burkholderia species separate into distinct lineages, suggesting the possibility for safe use of certain symbiotic species in agricultural contexts. A number of environmental strains that promote plant growth or degrade xenobiotics are also included in the symbiotic lineage. Many of these species have the potential to enhance agriculture in areas where fertilizers are not readily available and may serve in the future as inocula for crops growing in soils impacted by climate change. Here we address the pathogenic potential of several of the symbiotic Burkholderia strains using bioinformatics and functional tests. A series of infection experiments using Caenorhabditis elegans and HeLa cells, as well as genomic characterization of pathogenic loci, show that the risk of opportunistic infection by symbiotic strains such as B. tuberum is extremely low.     

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).     

Environmental predators as models for bacterial pathogenesis

Environmental bacteria are constantly threatened by bacterivorous predators such as free-living protozoa and nematodes. In the course of their coevolution with environmental predators, some bacteria developed sophisticated defence mechanisms, including the secretion of toxins, or the capacity to avoid lysosomal killing and to replicate intracellularly within protozoa. To analyse the interactions with bacterial pathogens on a molecular, cellular or organismic level, protozoa and other non-mammalian hosts are increasingly used. These include amoebae, as well as genetically tractable hosts, such as the social amoeba Dictyostelium discoideum, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Using these hosts, the virulence mechanisms of opportunistic pathogenic bacteria such as Legionella, Mycobacterium, Pseudomonas or Vibrio were found to be not only relevant for the interactions of the bacteria with protozoa, nematodes and insect phagocytes, but also with mammalian hosts including humans. Thus, non-mammalian model hosts provide valuable insight into the pathogenesis of environmental bacteria.     

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.     

Loop-mediated isothermal amplification based identification of entomopathogenic nematodes Heterorhabditis spp. and Steinernema spp. from soil DNA

Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema kill insects with the help of their symbiotic bacteria. They are widely used as biocontrol agents to manage insect pests of crops. The infective juveniles (IJ) of EPNs are isolated from soil by insect baiting technique, which is labour-intensive, time-consuming, wasteful, and inefficient. Here, we present loop-mediated isothermal amplification (LAMP) assays for rapid detection of Heterorhabditis spp. (Het-LAMP) and Steinernema spp. (Ste-LAMP) from total soil DNA. The primers for Het-LAMP and Ste-LAMP were designed using ITS and 18S rDNA regions of genomic DNA. The LAMP reactions could be completed in 60 min, at 66 °C and 68 °C, respectively, followed by termination at 85 °C for 5 min. The assays were highly sensitive and could detect up to 0.02 picograms of Heterorhabditis DNA and 96 picograms of Steinernema DNA in a 25 μl reaction. Both the assays were specific for the target nematode species and detected the presence of a single IJ in the total DNA extracted from 250 mg of soil. The assays developed in this study would be of immense utility for the efficient detection and identification of native EPNs in large-scale surveys. These assays are amenable to automation and could be used to develop convenient detection kits for point-of-service diagnosis of EPNs in the field without the need for a trained and experienced personnel.

     

Spread and Transmission of Bacterial Pathogens in Experimental Populations of the Nematode Caenorhabditis elegans

Caenorhabditis elegans is frequently used as a model species for the study of bacterial virulence and innate immunity. In recent years, diverse mechanisms contributing to the nematode''s immune response to bacterial infection have been discovered. Yet despite growing interest in the biochemical and molecular basis of nematode-bacterium associations, many questions remain about their ecology. Although recent studies have demonstrated that free-living nematodes could act as vectors of opportunistic pathogens in soil, the extent to which worms may contribute to the persistence and spread of these bacteria has not been quantified. We conducted a series of experiments to test whether colonization of and transmission between C. elegans nematodes could enable two opportunistic pathogens (Salmonella enterica and Pseudomonas aeruginosa) to spread on agar plates occupied by Escherichia coli. We monitored the transmission of S. enterica and P. aeruginosa from single infected nematodes to their progeny and measured bacterial loads both within worms and on the plates. In particular, we analyzed three factors affecting the dynamics of bacteria: (i) initial source of the bacteria, (ii) bacterial species, and (iii) feeding behavior of the host. Results demonstrate that worms increased the spread of bacteria through shedding and transmission. Furthermore, we found that despite P. aeruginosa''s relatively high transmission rate among worms, its pathogenic effects reduced the overall number of worms colonized. This study opens new avenues to understand the role of nematodes in the epidemiology and evolution of pathogenic bacteria in the environment.     

Plant diversity and identity effects on predatory nematodes and their prey

There is considerable evidence that both plant diversity and plant identity can influence the level of predation and predator abundance aboveground. However, how the level of predation in the soil and the abundance of predatory soil fauna are related to plant diversity and identity remains largely unknown. In a biodiversity field experiment, we examined the effects of plant diversity and identity on the infectivity of entomopathogenic nematodes (EPNs, Heterorhabditis and Steinernema spp.), which prey on soil arthropods, and abundance of carnivorous non‐EPNs, which are predators of other nematode groups. To obtain a comprehensive view of the potential prey/food availability, we also quantified the abundance of soil insects and nonpredatory nematodes and the root biomass in the experimental plots. We used structural equation modeling (SEM) to investigate possible pathways by which plant diversity and identity may affect EPN infectivity and the abundance of carnivorous non‐EPNs. Heterorhabditis spp. infectivity and the abundance of carnivorous non‐EPNs were not directly related to plant diversity or the proportion of legumes, grasses and forbs in the plant community. However, Steinernema spp. infectivity was higher in monocultures of Festuca rubra and Trifolium pratense than in monocultures of the other six plant species. SEM revealed that legumes positively affected Steinernema infectivity, whereas plant diversity indirectly affected the infectivity of Heterorhabditis EPNs via effects on the abundance of soil insects. The abundance of prey (soil insects and root‐feeding, bacterivorous, and fungivorous nematodes) increased with higher plant diversity. The abundance of prey nematodes was also positively affected by legumes. These plant community effects could not be explained by changes in root biomass. Our results show that plant diversity and identity effects on belowground biota (particularly soil nematode community) can differ between organisms that belong to the same feeding guild and that generalizations about plant diversity effects on soil organisms should be made with great caution.     

Compositional properties of nuclear genes from cold-blooded vertebrates

Summary We have investigated the compositional properties of coding sequences from cold-blooded vertebrates and we have compared them with those from warm-blooded vertebrates. Moreover, we have studied the compositional correlations of coding sequences with the genomes in which they are contained, as well as the compositional correlations among the codon positions of the genes analyzed.The distribution of GC levels of the third codon positions of genes from cold-blooded vertebrates are distinctly different from those of warm-blooded vertebrates in that they do not reach the high values attained by the latter. Moreover, coding sequences from cold-blooded vertebrates are either equal, or, in most cases, lower in GC (not only in third, but also in first and second codon positions) than homologous coding sequences from warm-blooded vertebrates; higher values are exceptional. These results at the gene level are in agreement with the compositional differences between cold-blooded and warm-blooded vertebrates previously found at the whole genome (DNA) level (Bernardi and Bernardi 1990a,b).Two linear correlations were found: one between the GC levels of coding sequences (or of their third codon positions) and the GC levels of the genomes of cold-blooded vertebrates containing them; and another between the GC levels of third and first+ second codon positions of genes from cold-blooded vertebrates. The first correlation applies to the genomes (or genome compartments) of all vertebrates and the second to the genes of all living organisms. These correlations are tantamount to a genomic code.