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Simple Summary: Xenorhabdus is a bacterial genus that symbioses with entomopathogenic nematodes, specifically classified within Steinernema. These nematodes transport the bacteria into target insects, where the bacteria suppress the host's immune responses and induce septicemia. Within the cadaver of the insect, the nematodes reproduce and re-establish their association with the symbiotic bacteria. This study identifies significant variability in insecticidal virulence across different Xenorhabdus species or strains. This variability arises from differences in their growth rates and suppressive activities against host immune functions. Analysis of secondary metabolites revealed the presence of several peptides in the virulent strains. Further analysis of non-ribosomal peptide synthetase genes suggested cyclo Pro-Phe (cPF) as a virulence factor, which suppressed insect immune responses and enhanced the bacterial virulence. Entomopathogenic bacteria, classified into the genus Xenorhabdus, exhibit a dual lifestyle as mutualistic symbionts to Steinernema nematodes and as pathogens to a broad range of insects. Bacterial virulence depends on toxin proteins that induce toxemia and various immunosuppressive secondary metabolites that cause septicemia. Particularly, the immunosuppressive properties of Xenorhabdus bacteria determine the variability of their insecticidal activities. This study explored the role of peptide metabolites in virulence and its variation among six bacterial strains across three species: X. nematophila, X. bovienii, and X. hominickii. Initially, their virulence significantly varied against a susceptible lepidopteran host, Maruca vitrata, but showed less variation against a tolerant coleopteran host, Tenebrio molitor, with high median lethal bacterial doses. In M. vitrata, virulence was strongly correlated with bacterial growth rate and inhibitory activity against phospholipase A2. Secondly, the six strains differed in the compositions of their secreted secondary metabolites, analyzed by GC-MS following ethyl acetate extraction. Notably, there was significant variation in the production of di- or tetra-peptides. Highly virulent strains commonly produced the cyclic Pro-Phe (cPF). Thirdly, the expression of non-ribosomal peptide synthetase (NRPS) genes varied greatly among the strains. NRPS genes were minimally expressed in the tolerant T. molitor and highly expressed in the susceptible M. vitrata. In M. vitrata, specific NRPS genes were markedly expressed in the virulent strains. Finally, cPF demonstrated potent immunosuppressive activity against the cellular and humoral responses of M. vitrata. The addition of cPF significantly enhanced the virulence against the tolerant T. molitor. These findings suggest that immunosuppression is necessary for the pathogenicity of Xenorhabdus bacteria, wherein NRPS products play a critical role in suppressing immune-associated factors in target insects. [ABSTRACT FROM AUTHOR]

Viral diseases like yellow fever, dengue, and Zika have an alarming impact on public health. These diseases can be transmitted by Aedes mosquito species, such as Ae. albopictus, which is now found in many countries outside its original range. Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of insect-preying nematodes and are known to produce an array of natural products with various activities including larvicidal activity. In this study, the effects of natural products produced by four Xenorhabdus and one Photorhabdus bacteria on the ovipositional behavior of Ae. albopictus mosquitoes were assessed. Utilizing a binary choice assay in insect cages, gravid female mosquitoes were presented with two oviposition cups containing water supplemented with varying concentrations of bacterial supernatants (50–1% concentrations) versus control medium. After 72 h, the eggs deposited on filter papers were counted. The oviposition attractant index (OAI) feature of the bacterial supernatant was evaluated using the number of eggs laid in the cups. Notably, all tested supernatants exhibited concentration-dependent deterrence of oviposition. Xenorhabdus cabanillasii displayed the strongest deterrent effect, inhibiting egg-laying at 50–5% concentrations (OAI: − 0.87 to − 0.35), followed by X. nematophila (50–10%, OAI: − 0.82 to − 0.52). Xenorhabdus szentirmaii, X. doucetiae, and P. kayaii showed significant deterrence at ≥ 20% concentrations. Using promoter exchange mutants generated by the easyPACId approach, fabclavine from X. szentirmaii was identified as the bioactive compound with evident deterrent effects. Such deterrents targeting egg-laying could be valuable for controlling populations by disrupting their breeding in suitable habitats. [ABSTRACT FROM AUTHOR]

BACKGROUND: In the perpetual struggle to manage mosquito populations, there has been increasing demand for the development of biopesticides to supplant/complement current products. The insecticidal potential of Xenorhabdus and Photorhabdus has long been recognized and is of interest for the control of important mosquitoes like Aedes albopictus which vectors over 20 different arboviruses of global public health concern. RESULTS: The larvicidal effects of cell‐free supernatants, cell growth cultures and cell mass of an extensive list of Xenorhabdus and Photorhabdus spp. was investigated. They were quite effective against Ae. albopictus causing larval mortality ranging between 52–100%. Three Photorhabdus spp. and 13 Xenorhabdus spp. release larvicidal compounds in cell‐free supernatants. Cell growth culture of all tested species exhibited larvicidal activity, except for Xenorhabdus sp. TS4. Twenty‐one Xenorhabdus and Photorhabdus bacterial cells (pellet) exhibited oral toxicity (59–91%) against exposed larvae. The effect of bacterial supernatants on the mosquito eggs were also assessed. Bacterial supernatants inhibited the hatching of mosquito eggs; when unhatched eggs were transferred to clean water, they all hatched. Using the easyPACId approach, the larvicidal compounds in bacterial supernatant were identified as fabclavine from X. szentirmaii and xencoumacin from X. nematophila (causing 98 and 70% mortality, respectively, after 48 h). Xenorhabdus cabanillasii and X. hominickii fabclavines were as effective as commercial Bacillus thuringiensis subsp. israelensis and spinosad products within 5 days post‐application (dpa). CONCLUSION: Fabclavine and xenocoumacin can be developed into novel biolarvicides, can be used as a model to synthesize other compounds or/and can be combined with other commercial biolarvicides. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Xenorhabdus species are bacterial symbionts of entomopathogenic Steinernema nematodes, in which they produce diverse secondary metabolites implicated in pathogenesis. To expand resources for natural product prospecting and exploration of host-symbiont-pathogen relationships, the genomes of Xenorhabdus cabanillasi, Xenorhabdus ehlersii, Xenorhabdus japonica, Xenorhabdus koppenhoeferii, and Xenorhabdus mauleonii were analyzed.

We present the characterization of the entomopathogenic nematode isolate LD_CU, and its associated symbiotic entomopathogenic bacterium. BLASTn analysis of the ITS rRNA sequence of the nematode revealed 100% similarity with Steinernema siamkayai, confirming conspecificity. The pairwise alignment showed no nucleotide differences with the type population of S. siamkayai. Phylogenetic analysis based on ITS rRNA gene sequences supported these findings, placing isolate LD_CU in a monophyletic clade with S. siamkayai, which forms a sister clade with S. huense, S. cumgarense, S. tami, and S. minutum. Biochemical tests identified the associated bacterial symbiont as Xenorhabdus stockiae. The isolate tested negative for oxidase, catalase, nitrate reductase, O-Nitrophenyl-β-D galactopyranoside (ONPG), methyl red, tryptophan deaminase, indole production, ornithine, lysine, citrate, malonate, hydrogen sulfide, acetoin, and phenylalanine deaminase, but positive for urea and esculin hydrolysis, motility, and acid production from glucose fermentation. The bacteria exhibited neutral red adsorption on MacConkey agar and formed brownish pigmented colonies on nutrient agar. Molecular characterization using 16S rRNA gene sequences revealed 100% similarity with X. stockiae and 90% similarity with X. innexi, indicating significant divergence from other Xenorhabdus species. Phylogenetic analysis showed a close relation to X. stockiae VP-2016b and distinct differences from X. innexi DSM 16336T. Pairwise alignment confirmed no nucleotide differences between the present bacterial strain and X. stockiae. This detailed and comprehensive profiling supports the accurate identification of Steinernema siamkayai and its bacterial symbiont, Xenorhabdus stockiae, and contributes valuable information to the taxonomy and phylogeny of these organisms. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, along with their bacterial symbionts from the genera Xenorhabdus and Photorhabdus, respectively, are important biological control agents against agricultural pests. Rapid progress in the development of genomic tools has catalyzed a transformation of the systematics of these organisms, reshaping our understanding of their phylogenetic and cophlylogenetic relationships. In this review, we discuss the major historical events in the taxonomy and systematics of this group of organisms, highlighting the latest advancements in these fields. Additionally, we synthesize information on nematode–bacteria associations and assess the existing evidence regarding their cophylogenetic relationships. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between X. nematophila secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new N-acyl amides, GKA-Arg (1) and GKA-Pro (2) which harbor a γ-keto acyl appendage. A lrhA null mutant produced elevated levels of compound 1 and reduced levels of compound 2 relative to wild type. N-acyl amides 1 and 2 were shown to be selective agonists for the human Gprotein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist 2 deleteriously affected the hatch rate and length of Steinernema nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. [ABSTRACT FROM AUTHOR]

Abstract Background Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. Results In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. Conclusions The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

Background: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. Results: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. Conclusions: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum (Lib.) de Bary, a polyphagous necrotrophic fungal pathogen, has brought about significant losses in agriculture and floriculture. Until now, the most common method for controlling S. sclerotiorum has been the application of fungicides. Xenocoumacin 1 (Xcn1) is a potential biopesticide having versatile antimicrobial activities, generated by Xenorhabdus nematophila. This study was intended to isolate Xcn1 from X. nematophila YL001 and clarify its efficacies for S. sclerotiorum control. Xcn1 demonstrated a wider antifungal spectrum against 10 plant-pathogenic fungi. It also exhibited a strong inhibitory effect on the mycelial growth of S. sclerotiorum with an EC50 value of 3.00 μg/mL. Pot experiments indicated that Xcn1 effectively inhibited disease extension on oilseed rape and broad bean plants caused by S. sclerotiorum. Morphological and ultrastructural observations revealed that the hyphae of S. sclerotiorum became twisted, shriveled, and deformed at the growing points after treatment with Xcn1 at 3.00 μg/mL and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. Moreover, Xcn1 also increased cell membrane permeability and decreased the content of exopolysaccharide as well as suppressing the activities of polygalacturonase and cellulase of S. sclerotiorum, but exerted no effects on oxalic acid production. This study demonstrated that Xcn1 has great potential to be developed as a new biopesticide for the control of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Xenorhabdus, known for its symbiotic relationship with Entomopathogenic nematodes (EPNs), belongs to the Enterobacteriaceae family. This dual-host symbiotic nematode exhibits pathogenic traits, rendering it a promising biocontrol agent against insects. Our prior investigations revealed that Xenorhabdus stockiae HN_xs01, isolated in our laboratory, demonstrates exceptional potential in halting bacterial growth and displaying anti-tumor activity. Subsequently, we separated and purified the supernatant of the HN_xs01 strain and obtained a new compound with significant inhibitory activity on tumor cells, which we named XNAE. Through LC-MS analysis, the mass-to-nucleus ratio of XNAE was determined to be 254.24. Our findings indicated that XNAE exerts a time- and dose-dependent inhibition on B16 and HeLa cells. After 24 h, its IC50 for B16 and HeLa cells was 30.178 µg/mL and 33.015 µg/mL, respectively. Electron microscopy revealed conspicuous damage to subcellular structures, notably mitochondria and the cytoskeleton, resulting in a notable reduction in cell numbers among treated tumor cells. Interestingly, while XNAE exerted a more pronounced inhibitory effect on B16 cells compared to HeLa cells, it showed no discernible impact on HUVEC cells. Treatment of B16 cells with XNAE induced early apoptosis and led to cell cycle arrest in the G2 phase, as evidenced by flow cytometry analysis. The impressive capability of X. stockiae HN_xs01 in synthesizing bioactive secondary metabolites promises to significantly expand the reservoir of natural products. Further exploration to identify the bioactivity of these compounds holds the potential to shed light on their roles in bacteria-host interaction. Overall, these outcomes underscore the promising potential of XNAE as a bioactive compound for tumor treatment. [ABSTRACT FROM AUTHOR]

The, antifungal activities of trans-cinamic acid (TCA) and cell-free supernatant (CFS) of X. szentirmaii were evaluated against Alternaria brassicicola that cause Black spot disease. The results showed that TCA was more suppressive than CFS of X. szentirmaii in the control of A. brassicicola. In petri assays, the highest suppressive effect on spore germination was in TCA treatments compared to CFS of X. szentirmaii. Moreover, no germ tube elongation was observed in any of tested concentrations (0.25%, 0.50%, 1%, 2%) of TCA. The highest level of mycelial growth inhibitions (100% and 92%) were exhibited by TCA (2%) and TCA (1%), respectively. The curative and protective activity of TCA on disease severity of A. brassicicola were also evaluated on broccoli plants. In the curative activity assays, disease severities were 71.00%, 56.33% and 54.03% for control, TCA (1%) and TCA (2%), respectively. Whereas in the protective activity, TCA (2%) suppressed the disease severity significantly greater than TCA (1%). The disease severity of A. brassicicola after TCA application on broccoli plants was 25.21% and 17.37% for TCA (1%) and TCA (2%) respectively. Similar data were obtained in the efficacy of TCA on A. brassicicola. In the curative activity assays, TCA (1%) and TCA (2%) exhibited the efficacy with rates of 20.57% and 23.02%, respectively. However, TCA (2%) showed significantly higher efficacy than TCA (1%) in the protective activity. The current study provides that TCA has a great potential to suppress A. brassicicola and can be a good alternative to synthetic fungicides.

Entomopathogenic bacteria, classified into the genus Xenorhabdus, exhibit a dual lifestyle as mutualistic symbionts to Steinernema nematodes and as pathogens to a broad range of insects. Bacterial virulence depends on toxin proteins that induce toxemia and various immunosuppressive secondary metabolites that cause septicemia. Particularly, the immunosuppressive properties of Xenorhabdus bacteria determine the variability of their insecticidal activities. This study explored the role of peptide metabolites in virulence and its variation among six bacterial strains across three species: X. nematophila, X. bovienii, and X. hominickii. Initially, their virulence significantly varied against a susceptible lepidopteran host, Maruca vitrata, but showed less variation against a tolerant coleopteran host, Tenebrio molitor, with high median lethal bacterial doses. In M. vitrata, virulence was strongly correlated with bacterial growth rate and inhibitory activity against phospholipase A2. Secondly, the six strains differed in the compositions of their secreted secondary metabolites, analyzed by GC-MS following ethyl acetate extraction. Notably, there was significant variation in the production of di- or tetra-peptides. Highly virulent strains commonly produced the cyclic Pro-Phe (cPF). Thirdly, the expression of non-ribosomal peptide synthetase (NRPS) genes varied greatly among the strains. NRPS genes were minimally expressed in the tolerant T. molitor and highly expressed in the susceptible M. vitrata. In M. vitrata, specific NRPS genes were markedly expressed in the virulent strains. Finally, cPF demonstrated potent immunosuppressive activity against the cellular and humoral responses of M. vitrata. The addition of cPF significantly enhanced the virulence against the tolerant T. molitor. These findings suggest that immunosuppression is necessary for the pathogenicity of Xenorhabdus bacteria, wherein NRPS products play a critical role in suppressing immune-associated factors in target insects.

Understanding how parasites evolved is crucial to understand the host and parasite interaction. The evolution of entomopathogenesis in rhabditid nematodes has traditionally been thought to have occurred twice within the phylum Nematoda: in Steinernematidae and Heterorhabditidae families, which are associated with the entomopathogenic bacteria Xenorhabdus and Photorhabdus, respectively. However, nematodes from other families that are associated with entomopathogenic bacteria have not been considered to meet the criteria for "entomopathogenic nematodes." The evolution of parasitism in nematodes suggests that ecological and evolutionary properties shared by families in the order Rhabditida favor the convergent evolution of the entomopathogenic trait in lineages with diverse lifestyles, such as saprotrophs, phoretic, and necromenic nematodes. For this reason, this paper proposes expanding the term "entomopathogenic nematode" considering the diverse modes of this attribute within Rhabditida. Despite studies are required to test the authenticity of the entomopathogenic trait in the reported species, they are valuable links that represent the early stages of specialized lineages to entomopathogenic lifestyle. An ecological and evolutionary exploration of these nematodes has the potential to deepen our comprehension of the evolution of entomopathogenesis as a convergent trait spanning across the Nematoda. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus. [ABSTRACT FROM AUTHOR]

Many species of slugs are considered serious pests in agriculture and horticulture around the world. In Europe, slugs of the genera Arion and Deroceras are the most harmful pests in agriculture. Therefore, the main goal of this study was to evaluate the effect of the whole-cell metabolites of 10 strains of five Xenorhabdus and three slug-parasitic nematodes (Phasmarhabditis hermaphrodita, Phasmarhabditis bohemica, and Phasmarhabditis apuliae) on the feeding behaviour and repellent effect on target slugs and evaluate a new possible means of biocontrol of these pests. The repellent and anti-feedant effects of nematode-killed insects, metabolites, slug-parasitic nematodes and a combination of metabolites and nematodes were studied through experimental designs: sand-filled plastic boxes divided into two parts in several modifications: with dead Galleria mellonella killed by nematodes, lettuce treated with bacterial metabolites and lettuce placed on the treated sand. We found that slugs avoid eating G. mellonella killed by nematodes, while they eat freeze-killed G. mellonella. Similarly, they avoid the consumption of lettuce in areas treated with bacterial metabolites (the most effective strains being Xenorhabus bovienii NFUST, Xenorhabdus kozodoii SLOV and JEGOR) with zero feeding in the treated side. All three Phasmarhabditis species also provided a significant anti-feedant/repellent effect. Our study is the first to show the repellent and anti-feedant effects of metabolites of Xenorhabdus bacteria against Arion vulgaris, and the results suggest that these substances have great potential for biocontrol. Our study is also the first to demonstrate the repellent effect of P. apuliae and P. bohemica. Key points: • Slugs avoid eating G. mellonella killed by entomopathogenic nematodes. • Bacterial metabolites have a strong repellent and antifeedant effect on slugs. • Presence of slug parasitic nematodes increases the repellent effect of metabolites. [ABSTRACT FROM AUTHOR]

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Extensive surveys of entomopathogenic nematodes (EPNs) have been conducted globally to identify native species. In Africa, EPN-related research has gained much attention over the past two decades, with several surveys having been undertaken in different countries, including Benin, Cameroon, Egypt, Ethiopia, Kenya, Morocco, Nigeria, Rwanda, South Africa and Tanzania. Such surveys have led to the description of 37 EPN species and 16 species of symbiotically associated bacteria being documented from Africa. Whereas South Africa and Egypt have made significant progress in the discovery and identification of EPN, in terms of their symbiotic bacteria and pathogenicity testing against pest insects, in many other African countries, the research is still in its early stages, or is else non-existent. Most research conducted so far has focused on laboratory bioassays, while research on the use of nematodes in more natural environments is scant. The present review provides a comprehensive overview of EPNs and their associated symbiotic bacteria from the African continent. The biological control potential of local EPN isolates against various economically important insect pests in Africa is also discussed. [ABSTRACT FROM AUTHOR]

Photorhabdus and Xenorhabdus are the bacterial symbionts of insect pathogenic nematodes, Heterorhabditis and Steinernema, respectively. This study aims to characterize the bacterial symbionts from Mizoram, North-east India and to evaluate their antibacterial potential. The bacterial isolates were characterized using recA and gyrB gene regions. The ethyl acetate extract of bacterial isolates was tested against pathogenic bacterial strains, viz. Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 10145), and Bacillus subtilis (ATCC 11774) using disk diffusion method. Analysis of recA and gyrB genes revealed that the Photorhabdus isolates were P. hindustanensis, and P. namnaonensis. This study constitutes the first documentation of P. namnaonensis from India. The two isolated Xenorhabdus belong to X. vietnamensis and X. stockiae. The ethyl acetate extracts of the studied bacteria suppressed the development of all the microorganisms tested. Based on MIC and MBC values, the highest activity was exhibited by TS (P. hindustanensis) and TD (P. namnaonensis) isolates against P. aeruginosa and K. pneumoniae respectively. The lowest inhibitory activity was observed on both Xenorhabdus isolates (RF and PTS) against B. subtilis. This study focuses on the existence and identification of symbiotic bacteria from Mizoram, an Indo-Burma biodiversity hotspot region, and details their activity against different pathogenic bacteria. Since these metabolites could be potent antibiotics, further research is required to better understand the genetic information, chemical composition, and method of action against other microorganisms.

Three entomopathogenic nematode populations were isolated from agricultural fields in the Anantnag district of Jammu and Kashmir (India). Sequences of multiple gene regions and phenotypic features show that they are conspecific and represent a novel species. Molecular and morphological features provided evidence for placing the new species into the “Kushidai” clade. Within this clade, analysis of sequence data of the internal transcribed spacer (ITS) gene, the D2D3 region of the 28S rRNA gene, the mitochondrial cytochrome oxidase I (mtCOI) gene, and the mitochondrial 12S (mt12S) gene depicted the novel species as a distinctive entity closely related to Steinernema akhursti, S. kushidai, and S. populi. Phylogenetic analyses also show that the new species is a sister species to S. akhursti, and these two species are closely related to S. kushidai and S. populi. Additionally, the new species does not mate or produce fertile progeny with any of the closely related species, reinforcing its uniqueness from a biological species concept standpoint. The new species is further characterized by the third-stage infective juveniles with almost straight bodies (0.7–0.8 mm length), poorly developed stoma and pharynx, and conoid-elongate tail (49–66 µm) with hyaline posterior part. Adult females are characterized by short and conoid tails bearing a short mucron in the first generation and long conoid tails with thin mucron in the second generation. Adult males have ventrally curved spicules in both generations. Moreover, the first-generation male has rounded manubrium, fusiform gubernaculum, conoid and slightly ventrally curved tails with minute mucron, and the second generation has rhomboid manubrium anteriorly ventrad bent, and tails with long and robust mucron. The morphological, morphometrical, molecular, and phylogenetic analyses support the new species status of this nematode, which is hereby described as Steinernema anantnagense n. sp. The bacterial symbiont associated with S. anantnagense n. sp. represents a novel species, closely related to Xenorhabdus japonica. These findings shed light on the diversity of entomopathogenic nematodes and their symbiotic bacteria, providing valuable information for future studies in this field.

ABSTRACT 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.

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests. [ABSTRACT FROM AUTHOR]

Summary: The bacterial strain SF857 was isolated from the entomopathogenic nematode, Steinernema bakwenae SF857, found in soil samples of St Lucie cherry, a species of cherry tree, from the Muriti farm, close to Kroondal (25°44′E, 59°99′S) in the northwest province of South Africa. Strain SF857 is Gram-negative, catalase and oxidase-negative, and lecithinase-positive. Strain SF857 shared the highest 16S rRNA gene sequence similarity (97.7%) with the type strain of Xenorhabdus ishibashii. However, a comparison of housekeeping gene sequences showed that strain SF857 grouped closer to X. griffiniae , X. ehlersii and X. thuongxuanensis than to X. ishibashii. Digital DNA-DNA hybridisation (dDDH) between strain SF857 and all Xenorhabdus spp. is less than the 70% species threshold, confirming that the strain belongs to a separate species. Furthermore, the biochemical characteristics of strain SF857 differ from other Xenorhabdus spp. Based on these findings, strain SF857 represents a novel species within the genus Xenorhabdus , for which the name Xenorhabdus bakwenae sp. n. (type strain SF857T) is proposed. Pathogenicity bioassays with last-instar Cydia pomonella showed S. bakwenae to be highly virulent, with potential use as a biocontrol agent in apple orchards. [ABSTRACT FROM AUTHOR]

Aims Black scurf disease, caused by Rhizoctonia solani , is a severe soil-borne and tuber-borne disease, which occurs and spreads in potato growing areas worldwide and poses a serious threat to potato production. New biofungicide is highly desirable for addressing the issue, and natural products (NPs) from Xenorhabdus spp. provide prolific resources for biofungicide development. In this study, we aim to identify antifungal NPs from Xenorhabdus spp. for the management of this disease. Methods and results Out of the 22 Xenorhabdus strains investigated, Xenorhabdus budapestensis 8 (XBD8) was determined to be the most promising candidate with the measured IC50 value of its cell-free supernatant against R. solani as low as 0.19 ml l−1. The major antifungal compound in XBD8 started to be synthesized in the middle logarithmic phase and reached a stable level at stationary phase. Core gene deletion coupled with high-resolution mass spectrometry analysis determined the major antifungal NPs as fabclavine derivatives, Fcl-7 and 8, which showed broad-spectrum bioactivity against important pathogenic fungi. Impressively, the identified fabclavine derivatives effectively controlled black scurf disease in both greenhouse and field experiments, significantly improving tuber quality and increasing with marketable tuber yield from 29 300 to 35 494 kg ha−1, comparable with chemical fungicide fludioxonil. Conclusions The fabclavine derivatives Fcl-7 and 8 were determined as the major antifungal NPs in XBD8, which demonstrated a bright prospect for the management of black scurf disease. [ABSTRACT FROM AUTHOR]

Photorhabdus and Xenorhabdus are the bacterial symbionts of insect pathogenic nematodes, Heterorhabditis and Steinernema, respectively. This study aims to characterize the bacterial symbionts from Mizoram, North-east India and to evaluate their antibacterial potential. The bacterial isolates were characterized using recA and gyrB gene regions. The ethyl acetate extract of bacterial isolates was tested against pathogenic bacterial strains, viz. Escherichia coli (ATCC 10536), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 10145), and Bacillus subtilis (ATCC 11774) using disk diffusion method. Analysis of recA and gyrB genes revealed that the Photorhabdus isolates were P. hindustanensis, and P. namnaonensis. This study constitutes the first documentation of P. namnaonensis from India. The two isolated Xenorhabdus belong to X. vietnamensis and X. stockiae. The ethyl acetate extracts of the studied bacteria suppressed the development of all the microorganisms tested. Based on MIC and MBC values, the highest activity was exhibited by TS (P. hindustanensis) and TD (P. namnaonensis) isolates against P. aeruginosa and K. pneumoniae respectively. The lowest inhibitory activity was observed on both Xenorhabdus isolates (RF and PTS) against B. subtilis. This study focuses on the existence and identification of symbiotic bacteria from Mizoram, an Indo-Burma biodiversity hotspot region, and details their activity against different pathogenic bacteria. Since these metabolites could be potent antibiotics, further research is required to better understand the genetic information, chemical composition, and method of action against other microorganisms. [ABSTRACT FROM AUTHOR]

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic–nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic–nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential. [ABSTRACT FROM AUTHOR]

Vector-borne diseases pose a severe threat to human and animal health. Culex pipiens L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress Cx. pipiens populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [Xenorhabdus and Photorhabdus spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) (Sterinernema spp. and Heterorhabditis spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 107 cells mL−1) of four different symbiotic bacteria (Xenorhabdus nematophila, X. bovienii, X. budapestensis, and P. luminescens subsp. kayaii) was assessed against different development stages of Cx. pipiens (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the X. bovienii KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides. [ABSTRACT FROM AUTHOR]

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has caused extreme losses in amphibian biodiversity. Finding bacteria that produce metabolites with antifungal properties may turn out to be invaluable in the fight against this devastating disease. The entomopathogenic bacteria, Xenorhabdus szentirmaii and X. budapestensis produce secondary metabolites that are effective against a wide range of fungal plant pathogens. To assess whether they may also be effective against Bd, we extracted cell-free culture media (CFCM) from liquid cultures of X. szentirmaii and X. budapestensis and tested their ability to inhibit Bd growth in vitro. As a second step, using juvenile common toads (Bufo bufo) experimentally infected with Bd we also tested the in vivo antifungal efficacy of X. szentirmaii CFCM diluted to 2 and 10% (v/v), while also assessing possible malign side effects on amphibians. Results of the in vitro experiment documented highly effective growth inhibition by CFCMs of both Xenorhabdus species. The in vivo experiment showed that treatment with CFCM of X. szentirmaii applied at a dilution of 10% resulted in infection intensities reduced by ca. 73% compared to controls and to juvenile toads treated with CFCM applied at a dilution of 2%. At the same time, we detected no negative side effects of treatment with CFCM on toad survival and development. Our results clearly support the idea that metabolites of X. szentirmaii, and perhaps of several other Xenorhabdus species as well, may prove highly useful for the treatment of Bd infected amphibians. Key points: First report of high anti-Bd efficacy of Xenorhabdus metabolites in vitro. Metabolites of X. szentirmaii can be applied effectively on live toads. Application of X. szentirmaii metabolites on live toads was safe. [ABSTRACT FROM AUTHOR]

The cabbage butterfly, Pieris brassicae Linnaeus (Lepidoptera: Pieridae), is an oligophagous and invasive insect pest of various economically important cole crops. Recently, there have been reports about an increase in the incidence and damaging activities of cabbage butterflies, signifying that the existing control methods fail to meet the grower's expectations. Entomopathogenic nematodes (EPNs) and their endosymbiotic bacteria have immense potential for the control of a wide range of insect pests. In this investigation, the EPN species Steinernema carpocapsae and its associated bacterial species, Xenorhabdus nematophila, were isolated and identified through morphological and molecular techniques. The laboratory bioassay experiment was performed using S. carpocapsae and X. nematophila against the 3rd instar larvae of P. brassicae (25 ± 1 °C; RH = 60%). The efficacy of EPN suspension (30, 60, 90, 120, 150 IJs/mL) and bacterial suspension (1 × 104, 2 × 104, 3 × 104, 4 × 104, and 5 × 104 CFU/mL) via contact and oral routes showed significant mortality among the larvae. Surprisingly, 100% insect mortality within 48 h was recorded in the bacterial inoculum 5 × 104 CFU/mL. However, in the case of EPNs (S. carpocapsae), 150 IJs/mL caused the highest, 92%, larval mortality rate after 96 h. The results signify that both indigenous EPNs and their associated bacteria can provide efficient control against P. brassicae larvae and could effectively contribute to IPM programs. However, further analyses are required to authenticate their effectiveness in field conditions. [ABSTRACT FROM AUTHOR]

Steinernema glaseri is a potential biocontrol agent against white grubs of the Phyllophaga spp. complex; however, its suitability for in vitro multiplication has been scarcely investigated. In this study, the effects of the surface culture of NJ-43 strain with its symbiotic bacteria Xenorhabdus poinarii on egg-yolk agar (P2), chickenliver agar and nutrient meat-peptone (MP) agar on infective juvenile (IJ) productivity and their virulence against Galleria mellonella larvae were investigated. The bacteria on the surface of the agar were first incubated in darkness at 30 °C for 54-102 h, and then 100 surface-sterilised IJs were added. After two harvests, the accumulated productivity was higher on chicken-liver agar (536 × 10³ IJs/m² day) and P2 agar (534 × 10³ IJs/m² day) than on nutrient MP agar (58 × 10³ IJs/m2 day). The mean virulence of the in vitro produced IJs was 46-60% and showed no statistically significant difference among the three culture media. In conclusion, the maximum multiplication factor of S. glaseri NJ-43 on solid media was 385, and its original virulence was retained. [ABSTRACT FROM AUTHOR]

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Nematode–microbe symbiosis plays a key role in determining pathogenesis against pests. The modulation of symbiotic bacteria may affect the virulence of entomopathogenic nematodes (EPNs) and the biological management of pests. We tested the influence of asafoetida (ASF) extract on the virulence of Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila, in Pyrrhocoris apterus. A total of 100 mg of ASF killed 30% of EPNs in 48 h, while P. apterus remained unaffected. The EPNs pre-treated with 100 mg of ASF influenced P. apterus's mortality by 24–91.4% during a period of 24 to 72 h. The topical application of ASF acted as a deterrent to S. carpocapsae, lowering host invasion to 70% and delaying infectivity with 30% mortality for 168 h. Interestingly, Steinernema's symbiotic bacterium, Xenorhabdus, remained unaffected by ASF. An in vitro turbidity test containing 100 mg of ASF in a medium increased the growth rate of Xenorhabdus compared to a control. A disc diffusion assay confirmed the non-susceptibility of Xenorhabdus to ASF compared to a positive control, streptomycin. Pro-phenol oxidase (PPO) and phenol oxidase (PO) upregulation showed that ASF influences immunity, while EPN/ASF showed a combined immunomodulatory effect in P. apterus. We report that ASF modulated the virulence of S. carpocapsae but not that of its symbiotic bacterium, X. nematophila, against P. apterus. [ABSTRACT FROM AUTHOR]

Alternatives to hazardous insecticides are urgently needed for an environmentally friendly and effective management of insect pests. One such option is the use of entomopathogenic nematodes (EPN). To increase the availability of EPN with potential for biocontrol, we surveyed agricultural soils in the Republic of Rwanda and collected two Steinernema isolates. Initial molecular characterization showed that they represent a new species, for which we propose the name S. africanum n. sp. To describe this new species, we reconstructed phylogenetic relationships, calculated sequence similarity scores, characterized the nematodes at the morphological level, conducted crossing experiments, and isolated and characterized their symbiotic bacteria. At the molecular level, S. africanum n. sp. is closely related to S. litorale and S. weiseri. At the morphological level, S. africanum n. sp. differs from closely related species by the position of the nerve ring and also because the stoma and pharynx region is longer. The first-generation males have ventrally curved spicules with lanceolate manubrium and fusiform gubernaculum and the second-generation males have rounded manubrium and anteriorly hook-like gubernaculum. Steinernema africanum n. sp. does not mate or produce fertile progeny with any of the closely related species.

Symbiotic bacteria form a mutualistic relationship with nematodes and are pathogenic to many insect pests. They kill insects using various strategies to evade or suppress their humoral and cellular immunity. Here we evaluate the toxic effects of these bacteria and their secondary metabolites on the survival and phenoloxidase (PO) activation of Octodonta nipae larvae using biochemical and molecular methods. The results show P. luminescens H06 and X. nematophila All treatments caused significant reductions in the number of O. nipae larvae in a dose-dependent manner. Secondly, the O. nipae immune system recognizes symbiotic bacteria at early and late stages of infection via the induction of C-type lectin. Live symbiotic bacteria significantly inhibit PO activity in O. nipae whereas heat-treated bacteria strongly increase PO activity. Additionally, expression levels of four O. nipae proPhenoloxidase genes following treatment with P. luminescens H06 and X. nematophila All were compared. We found that the expression levels of all proPhenoloxidase genes were significantly down-regulated at all-time points. Similarly, treatments of O. nipae larvae with metabolites benzylideneacetone and oxindole significantly down-regulated the expression of the PPO gene and inhibited PO activity. However, the addition of arachidonic acid to metabolite-treated larvae restored the expression level of the PPO gene and increased PO activity. Our results provide new insight into the roles of symbiotic bacteria in countering the insect phenoloxidase activation system. [ABSTRACT FROM AUTHOR]

On the basis of biological activities of the ethyl acetate extracts of four Xenorhabdus sp. including Xenorhabdus nematophila FUM 220, Xenorhabdus nematophila FUM 221, Xenorhabdus bovienii FUM 222, and Xenorhabdus bovienii FUM 223, X. nematophila FUM 220 was preferentially selected to track the isolation of responsible compounds. Chemical study on the ethyl acetate extract of X. nematophila isolate FUM220 which is derived from the native nematode Steinernema carpocapsae (Rhabditida: Steinernematidae), was evaluated, and eleven compounds, including xenocoumacin II (1), xenortide-396 (2), xenortide A (3), xenortide-410 (4), xenortide-449 (5), xenematide A 663 (6), rhabdopeptide-574 (7), rhabdopeptide-588 (8), rhabdopeptide-687 (9), rhabdopeptide-701 (10), and nematophin-273 (11) were characterized. In this experimental study, we surveyed the antitumoral potential of bacterial extract and bacterial metabolites to treat human breast cancer (MCF-7), human lung cancer (A549), and murine Tumor (B16) cell lines. We observed that all samples were cytotoxic, but bacterial extracts of X. nematophila FUM 220 and X. bovienii FUM 223 showed higher toxicity on mentioned cell lines. Potent cytotoxic activity was found for compounds 6 and 11 with IC50 of 6.2 μg/ml against human lung cancer A549 cell lines, too. These compounds showed moderated antibacterial activity against Xanthomonas oryzae pv. oryzae strain Xoo-IR42 (Xanthomonadales: Xanthomonadaceae) (MIC of 62.5 μg/ml) and Staphylococcus aureus strain 1112 (Bacillales: Staphylococcaceae) (MIC of 100 μg/ml). The bacterial extracts from X. bovienii FUM 222 showed strong inhibition of the growth of S. aureus strain 1112, by a minimal inhibitory concentration assay (MIC of 53.5 μg/ml). Xenorhabdus genera produce metabolites with potent cytotoxic and antibacterial activity. Single compounds can be isolated, identified, and commercialized, but various species or strains may change their anticancer or antimicrobial potential. The present study brings new clues regarding the qualified of Xenorhabdus as future peptide sources for supplying natural bioactive compounds and challenge multidrug-resistant bacteria, treat cancer, and plant diseases. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes (EPNs) are effective biocontrol agents against different insect pests. However, very little work has been done in Pakistan on the symbiotic bacteria associated with EPNs and their metabolites. In this laboratory study, two bacterial isolates i.e. Xenorhabdus spp. associated with EPN Steinernema glaseri (Steiner) (Steinernematidae) and Photorhabdus spp. associated with EPN Heterorhabditis bacteriophora (Poinar) (Heterorhabditidae), were evaluated against armyworm Spodoptera litura Fabricius (Lepidoptera: Noctuidae) which is a destructive lepidopterous pest. These EPNs associated bacterial isolates were applied @ 4×107 CFUs/mL against different larval instars of S. litura. At day one post-treatment, maximum larval mortality (59.40%) was exhibited by Xenorhabdus spp. against 2nd instar larvae followed by 52.80, 46.20 and 46.20% mortality of 5th, 3rd and 4th instar larvae, respectively. In case of Photorhabdus spp., maximum mortality (26.40%) was observed against 2nd instar larvae. In other bioassay, 40% suspensions of bacterial metabolites were evaluated against different larval instars of S. litura and were found significantly effective against all larval instars. Maximum average mortality (52.80%) was observed in Xenorhabdus spp. derived metabolites against 2nd instar larvae as compared to Photorhabdus spp. In both bioassays, mortality was increased along with the exposure time and reached 100% in all S. litura larval instars at fourth day posttreatment. Findings of this in-vitro study demonstrate that EPNs and their symbiotic bacterial isolates would be effective biorational control tools against S. litura. [ABSTRACT FROM AUTHOR]

Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

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.

This study was carried out to determine the antifungal effects of the supernatant produced by the bacterium Xenorhabdus szentirmaii, which is associated with soil-inhabiting entomopathogenic nematodes, on important plant pathogenic fungi, Fusarium verticilliodes, Fusarium oxysporum f.sp lycopersici, Fusarium oxysporum f.sp radicis lycopersici, Botrytis cinerea, Sclerotinia sclerotiorum and Phytophthora nicotianae. The effects of 1, 3, 5 and 7% concentrations of the supernatant produced by X. szentirmaii bacteria on mycelium growth of the fungal pathogens was determined at 3, 7 and 14 days after application (dap). The effects of the supernatant on the formation of sporangia and the emergence of zoospores from the sporangium of Phytophthora nicotianae was also determined. According to the results of the study, the highest dose of the supernatant (7% concentration) prevented mycelium development of all tested pathogens. The effect of the supernatant decreased at 14 dap against F. verticilliodes, F. oxysporum f.sp lycopersici, F. oxysporum f.sp radicis lycopersici. The antifungal effect on S. sclerotiorum completely disappeared in 14 days whereas the effect continued even in 14 days against B. cinerea and P. nicotianae. Xenorhabdus szentirmaii supernatant at a concentration of 7% was highly efficacious on the formation of sporangia and the emergence of zoospores within the sporangium of P. nicotianae at a concentration of 7%.

Abstract Background The biological diversity on planet earth is declining day by day, due to different factors such as excessive applications of pesticides. The utilization of chemical pesticides affected environment as well as microorganisms. The awareness among the peoples towards the hazards by the residual toxicity of chemical pesticides should be developed for agro-environmental sustainability. Main body Entomopathogenic nematodes (EPNs) are the bacto-helminth parasites which show classical mutualism with the genera Xenorhabdus and Photorhabdus. The nematodes along with its endosymbiotic bacteria have a biocontrol potential which could be used to reduce chemical pesticides. Applications of bioagents have been reported and resulted in considerable reduction in pathogens. Furthermore, these bioagents are biodegradable, eco-friendly and easy to apply for protection of crops against diverse pathogenic organism. The nematode-bacterium complexes are effective against huge range of bacteria, fungi, nematodes and insects that are harmful to the crops. Along with biocontrol potential, the endosymbionts produce diverse secondary metabolic compounds, exoenzymes and toxic compounds that show antibiotic, antimycotic, nematicidal, miticidal and anticancerous properties. Conclusion The present review deals with the diversity of endosymbiotic microbes from EPNs and their role in biocontrol for the agro-environmental sustainability.

Neofusicoccum parvum , is a fungal pathogen and one of the etiological agents of dieback disease in grapevines. The fungus causes deterioration of vines due to vascular colonization and/or production of toxins. We report herein the inhibitory effects of Trichoderma spp. isolates and the antifungal effects of cell-free supernatants (CFS) from Xenorhabdus and Photorhabdus bacteria against N. parvum in agar plates. We also evaluated the effects of the most effective fungi and bacteria against the pathogen in pruning wounds of vine shoots. All isolates of Trichoderma exhibited antifungal activity ranging between 82 and 97.5% at 14 days of post-treatment. All Xenorhabdus and Photorhabdus CFS at 10 and 33% concentrations inhibited mycelial growth with X. szentirmaii PAM 11 and PAM 25 causing the highest inhibition (>74%). In the shoot experiments, T. asperellum IB 01/13 and T. asperellum Quality®, X. szentirmaii PAM 11 (undiluted growth culture and CFS) suppressed the fungus by ≥ 93%. Our study highlights the potential of Trichoderma and X. szentirmaii PAM 11 for use as biofungicides in the management of N. parvum in grapevines. Further studies should be conducted to develop formulations of Trichoderma and Xenorhabdus that enhance stability in shelf-life and increase the efficacy of N. parvum control in grapevines under field conditions. [Display omitted] • Fungal diseases cause serious problems in grape production. • Neofusicoccum parvum is a fungal agent of dieback disease in grapevines. • Trichoderma is antagonistic against N. parvum in vines. • Xenorhabdus spp. produce putative biofungicide/s against dieback in vines. [ABSTRACT FROM AUTHOR]

Xenorhabdus can produce a large number of secondary metabolites with insecticidal, bacteriostatic, and antitumor activities. Efficient gene editing tools will undoubtedly facilitate the functional genomics research and bioprospecting in Xenorhabdus. In this study, BlastP analysis using the amino acid sequences of Redαβ or RecET recombinases as queries resulted in the identification of an operon (XBJ1_operon 0213) containing RecET-like recombinases encoding genes from the genome of Xenorhabdus bovienii strain SS-2004. Three proteins encoded by this operon was indispensable for full activity of recombineering, namely XBJ1-1173 (RecE-like protein), XBJ1-1172 (RecT-like protein), and XBJ1-1171 (single-strand annealing protein). Using this newly developed recombineering system, a gene cluster responsible for biosynthesis of a novel secondary metabolite (Min16) was identified from X. stockiae HN_xs01 strain. Min16 which exhibited antibacterial and cytotoxic activities was determined to be a cyclopeptide composed of Acyl-Phe-Thr-Phe-Pro-Pro-Leu-Val by using high-resolution mass spectrometry and nuclear magnetic resonance analysis, and was designated as changshamycin. This host-specific recombineering system was proven to be effective for gene editing in Xenorhabdus, allowing for efficient discovery of novel natural products with attractive bioactivities. Key points: • Screening and identification of efficient gene editing tools from Xenorhabdus • Optimization of the Xenorhabdus electroporation parameters • Discovery of a novel cyclopeptide compound with multiple biological activities [ABSTRACT FROM AUTHOR]

Two families of entomopathogenic nematodes (EPNs), Steinernematidae and Heterorhabditidae, symbiotically associated with Xenorhabdus and Photorhabdus bacteria, are effective biological control agents of insect pests. Native isolates are likely to be better candidates for insect pest control than exotic specimens due to their adaptation to native environmental conditions. In this study, Steinernema feltiae isolate FUM221, was recovered from soil samples collected from the Ardabil Province, Iran. Morphological and morphometric investigations of the first and second-generation adults, infective juveniles, and molecular characterizations were conducted based on ITS and 18S rDNA genes. Molecular analysis based on the 16S rRNA region and phenetic data revealed Xenorhabdus bovienii as this isolate symbiont bacterium. The scanning electron microscopy (SEM) verified the identification of this isolate. The molecular characterization using two loci and phylogenetic analyses provided more evidence on the classification of this steinernematid and its distinction from same species from other countries. Moreover, molecular and phenetic characterizations of its symbiotic bacterium indicated minor variations compared to other isolates. Herein, the comprehensive taxonomic data of this steinernematid and its symbiont bacterium, is presented. [ABSTRACT FROM AUTHOR]

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic–nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic–nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered “easy-PACId” research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

BACKGROUND:Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS:Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS:The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Txp40 is a ubiquitous toxin from Xenorhabdus and Photorhabdus bacteria, exhibits insecticidal activity against a wide range of insect pests belonging to Lepidoptera and Diptera orders. Initially, Txp40 affects midgut of the target insect and further damages some other tissues like fat bodies but the detailed mode of action is not known. Txp40 shares no significant sequence match to any proteins with known structure or function, suggesting that it is a novel type of insecticidal toxin. Here, we report purification, toxicity and biophysical characterization of the Txp40b toxin from X. nematophila (ATCC, 19061). The recombinant Txp40b was found toxic to Galleria mellonella larvae with LD 50 of 30.42 ng larva−1. Circular dichroism spectroscopy revealed that purified Txp40b is an α-helix rich protein with a relatively lower melting temperature of 45 °C. In-silico model generated suggests two domain structure of Txp40b toxin. Detailed structural analysis of Txp40b will provide new insights about the mode of action and possibly it would illustrate a new domain and/or motif in the area of insecticidal proteins. [Display omitted] • Txp40 is a ubiquitous toxin protein from Xenorhabdus and Photorhabdus bacteria however, its mode of action is unknown. • Purification, toxicity and biophysical characterization of Txp40b toxin from Xenorhabdus nematophila are reported. • Txp40b is a monomeric alpha-helix rich protein having a melting point of 45 °C. • Recombinant Txp40b exhibits insecticidal activity against Galleria mellonella with median lethal dose of 30.42 ng larva−1. [ABSTRACT FROM AUTHOR]

Simple Summary: An entomopathogen, Bacillus thuringiensis (Bt), has been used to control insect pests. On the other hand, insect immune responses defend the bacterial pathogenicity. An idea to enhance the Bt virulence was to inhibit insect immune defense using immunosuppressant. Secondary metabolites from another entomopathogen, Xenorhabdus hominickii, are known to inhibit the insect immune responses, but they alone give little virulence against insects. The addition of the bacterial culture broth containing the secondary metabolites to Bt spores significantly enhanced the Bt virulence in laboratory and field trials. This study demonstrates an integrated biological control by ideally combining two entomopathogenic bacteria. Insect immunity defends against the virulence of various entomopathogens, including Bacillus thuringiensis (Bt). This study tested a hypothesis that any suppression of immune responses enhances Bt virulence. In a previous study, the entomopathogenic bacterium, Xenorhabdus hominickii (Xh), was shown to produce secondary metabolites to suppress insect immune responses. Indeed, the addition of Xh culture broth (XhE) significantly enhanced the insecticidal activity of Bt against S. exigua. To analyze the virulence enhanced by the addition of Xh metabolites, four bacterial secondary metabolites were individually added to the Bt treatment. Each metabolite significantly enhanced the Bt insecticidal activity, along with significant suppression of the induced immune responses. A bacterial mixture was prepared by adding freeze-dried XhE to Bt spores, and the optimal mixture ratio to kill the insects was determined. The formulated bacterial mixture was applied to S. exigua larvae infesting Welsh onions in a greenhouse and showed enhanced control efficacy compared to Bt alone. The bacterial mixture was also effective in controlling other Spodopteran species such as S. litura and S. frugiperda but not other insect genera or orders. This suggests that Bt+XhE can effectively control Spodoptera-associated pests by suppressing the immune defenses. [ABSTRACT FROM AUTHOR]

In bacteria, DNA-methyltransferase are responsible for DNA methylation of specific motifs in the genome. This methylation usually occurs at a very high rate. In the present study, we studied the MTases encoding genes found in the entomopathogenic bacteria Xenorhabdus. Only one persistent MTase was identified in the various species of this genus. This MTase, also broadly conserved in numerous Gram-negative bacteria, is called Dam: DNA-adenine MTase. Methylome analysis confirmed that the GATC motifs recognized by Dam were methylated at a rate of >99% in the studied strains. The observed enrichment of unmethylated motifs in putative promoter regions of the X. nematophila F1 strain suggests the possibility of epigenetic regulations. The overexpression of the Dam MTase responsible for additional motifs to be methylated was associated with impairment of two major phenotypes: motility, caused by a downregulation of flagellar genes, and hemolysis. However, our results suggest that dam overexpression did not modify the virulence properties of X. nematophila. This study increases the knowledge on the diverse roles played by MTases in bacteria. [ABSTRACT FROM AUTHOR]