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Bacterial diseases cause colossal losses in vegetable crops and are considered major limiting factors for the commercial production of vegetables around the globe. Among bacterial diseases, angular leaf spot of cucumber caused by Pseudomonas syringae is one of the most economically important diseases that cause substantial economic loss to the cucumber crop. In this study, the in‐vitro and in‐vivo effects of different strains (X1–X15) of the nematode symbiotic bacterium Xenorhabdus nematophila was determined on the growth of P. syringae in different selected media (TB, SB, SOB, SOC, YT and LB) and at different pH levels (6.5, 7.5 and 8.5) and on the incidence of angular leaf spot disease on potted cucumber plants. According to the results, maximum growth inhibition of P. syringe (25 mm) was observed in the case of strain X12, followed by strains X10 (23 mm), X8 (21 mm) and X6 (20 mm), while maximum colony growth was observed in the control. Moreover, this P. syringae growth inhibition by the X12 strain of X. nematophila was significantly maximum in SB medium and at pH 7.5. Results of the in‐vivo experiment with potted plants showed a maximum incidence (90%) of angular leaf spot disease in control plants, while a minimum disease incidence (15%) was observed for the plants treated with two times dilution of X. nematophila culture, followed by ten times (23%) and fifty times (35%). The overall results of this study demonstrate the inhibitory effect of X. nematophila on the growth and incidence of P. syringae and underscore its putative role in the biological management of plant pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Abstract Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its excellent activity against bacteria, oomycetes and fungi. However, the current low yield of Xcn1 limits its development and utilization. To improve the yield of Xcn1, response surface methodology was used to determine the optimal composition of fermentation medium and one factor at a time approach was utilized to optimize the fermentation process. The optimal medium composed of in g/L: proteose peptone 20.8; maltose 12.74; K2HPO4 3.77. The optimal fermentation conditions were that 25 °C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flask with an agitation rate of 150 rpm for 48 h. Xenorhabdus nematophila YL001 was produced the highest Xcn1 yield (173.99 mg/L) when arginine was added to the broth with 3 mmol/L at the 12th h. Compared with Tryptic Soy Broth medium, the optimized fermentation process resulted in a 243.38% increase in Xcn1 production. The obtained results confirmed that optimizing fermentation technology led to an increase in Xcn1 yield. This work would be helpful for efficient Xcn1 production and lay a foundation for its industrial production.

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]

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]

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.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematodebacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematodebacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes. [ABSTRACT FROM AUTHOR]

Attempts were made to evaluate the purified bioactive compounds of Xenorhabdus nematophila against Meloidogyne incognita. In order to extract the purified compounds, a solid-supported liquid-liquid extraction system with a flow rate (1 mL/min) was used to purify bioactive molecules. Compounds were individually collected concentrated and evaluated against M. incognita. Among 25 fractions the L19 fraction, exhibited 98% inhibition in egg hatching and mortality of juveniles. The biomolecules were identified through Liquid Chromatography- Mass Spectroscopy (LC-MS) technique. To decipher the mode of action of compounds, molecular docking studies were performed with potential protein targets such as acetylcholinesterase, β-1,4-endoglucanase, glutathione S-transferase-1, cytochrome c oxidase, G-protein coupled receptor and Fatty acid and retinol-binding proteins of M. incognita. The results revealed that among eight compounds from the L19 fraction, malonate and pidopidon exhibited greater binding affinity towards the selected protein targets of M. incognita. In vitro studies with malonate and pidopidon against M. incognita showcased a 99% reduction in egg hatching and juvenile mortality. Moreover, greenhouse experiments revealed that malonate compounds not only reduced 94% of the M. incognita population but also enhanced the plant growth parameters in tomato by 60%. Hence the present study stands novel in exploiting the nematicidal compounds from X. nematophila giving limelight to explore pidopidon and malonate as novel nematicidal compounds for the management of M. incognita. Graphical abstract:The nematicidal efficacy of entomopathogenic nematodes (EPNs) associated with their symbiotic bacteria Xenorhabdus nematophila against Meloidogyne incognita. In this study we used different concentration of biomolecules to inhibit egg hatching and juvenile mortality. Molecular docking was carried out using different protein to understand the effect of biomolecules. [Display omitted] • Bioactive molecules from Xenorhabdus nematophilus were extracted and purified using a solid-supported liquid-liquid extraction system at 1 mL/min, resulting in 25 fractions screened for egg hatching and juvenile mortality. • LCMS analysis revealed Malonate, and Pidopidon, as major compounds. • The pure compounds revealed inhibition in egg hatching and juvenile mortality. [ABSTRACT FROM AUTHOR]

Chitinase plays a vital role in plant defense against insect pests and fungal pathogens due to its ability to hydrolyse chitin, which forms the major portion of the lining of gut membrane of insects and the cell wall of fungi. Earlier we reported a ~ 76 kDa chitinase protein isolated from Xenorhabdus nematophila strain (ATCC 19061). Purified chitinase protein was tested against the larvae of Helicoverpa armigera,a major crop pest and when fed orally to the larvae, it proved to be toxic. In the present study, we report the generation of transgenic tobacco plants expressing the chitinase gene of X. nematophila. The stable integration of the chitinase gene was confirmed by Southern blot while the expression of the gene was checked by RT-PCR analysis and quantification of chitinolytic activity. The transgenic lines were phenotypically normal and healthy. The result of bioassays revealed that the tobacco lines expressing increased level of chitinase protein showed 93–100% mortality of larvae of H. armigera when fed on the transgenic leaves expressing chitinase. A considerable reduction in leaf damage (55–76%) was also observed in transgenic lines as compared to the damage in untransformed control plants when the larvae were allowed to feed on the transgenic leaves. The larvae feeding on transgenic leaves showed adverse effect on their growth, development and survival. Thus we propose, the chitinase gene can prove to be a potent candidate for pest control in crop plants in future. Key message: Overexpression of insecticidal chitinase from Xenorhabdus nematophila protects transgenic tobacco plants against Helicoverpa armigera. Our results indicate that the gene encoding chitinase is a promising candidate for crop protection. [ABSTRACT FROM AUTHOR]

Spodoptera litura, one of the polyphagous pests, causes huge economical lose and use of chemical pesticide causes impact to the environmental. The present study deals with the use of cell- free supernatant of bacteria Xenorhabdus nematophila NP-1 strain for synthesizing silver nanoparticles and analyzing its larvicidal ability against Spodoptera litura. Color change from yellow to dark brown specifies the synthesis of AgNPs. UV–Vis spec indicates the presences of AgNPs at 440 nm λmax and functional groups; alcohols, carboxylic acids, aromatics, alkylhalides, ethers and phenols were confirmed by FTIR. SEM revealed the synthesized AgNPs is in spherical shape, EDaX confirms the elemental composition and the crystalline nature were observed using XRD. GC–MS analysis showed presence of Benzencepropanoic acid, 1, 3, 5 Trichloropent-2-ene, 1,1-Dichloro-2,3- dicmethycycloprone and 1,2-benzenedicarboxylic acid bioactive compounds some of which may be responsible for insecticidal and antibacterial activity. The antibacterial activity against S. aureus, B. subtilis and K. pneumoniae showed maximum zone of inhibition at 100 µL/mL. Larvicidal activity of S. litura shows highest mortality at 48 h. In potted plant experiment, AgNPs treated plants showed less damage, with less leaf consumption by S. litura larvae. Further, the synthesis of AgNPs were targeted to zebrafish embryos (non- target organism) and it didn't exhibit any toxic effect even at higher concentration. Our experiment concludes that, AgNPs synthesized using NP-1 strain has highest antimicrobial and insecticidal activity, which can be used in biomedical and biopesticides. [ABSTRACT FROM AUTHOR]

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.

Background: Entomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species. Methods: In the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response (recA and lexA) associated with mitomycin C and UV exposure. Results: We described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions. [ABSTRACT FROM AUTHOR]

Abstract The control of insects of medical importance, such as Aedes aegypti and Aedes albopictus are still the only effective way to prevent the transmission of diseases, such as dengue, chikungunya and Zika. Their control is performed mainly using chemical products; however, they often have low specificity to non-target organisms, including humans. Also, studies have reported resistance to the most commonly used insecticides, such as the organophosphate and pyrethroids. Biological control is an ecological and sustainable method since it has a slow rate of insect resistance development. Bacterial species of the genera Xenorhabdus and Photorhabdus have been the target of several research groups worldwide, aiming at their use in agricultural, pharmaceutical and industrial products. This review highlights articles referring to the use of Xenorhabdus and Photorhabdus for insects and especially for mosquito control proposing future ways for their biotechnological applicability. Approximately 24 species of Xenorhabdus and five species of Photorhabdus have been described to have insecticidal properties. These studies have shown genes that are capable of encoding low molecular weight proteins, secondary toxin complexes and metabolites with insecticide activities, as well as antibiotic, fungicidal and antiparasitic molecules. In addition, several species of Xenorhabdus and Photorhabdus showed insecticidal properties against mosquitoes. Therefore, these biological agents can be used in new control methods, and must be, urgently considered in short term, in studies and applications, especially in mosquito control.

Background Entomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species. Methods In the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response (recA and lexA) associated with mitomycin C and UV exposure. Results We described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions.

Xenorhabdus nematophila strain ATCC 19061 is an insect pathogen that produces various protein toxins which intoxicate and kill its larval host. In the present study, we have described the cloning, expression and characterization of a 76-kDa chitinase protein of X. nematophila. A 1.9 kb DNA sequence encoding the chitinase gene was PCR amplified and cloned. Further, the chitinase protein was expressed in Escherichia coli and purified by using affinity chromatography. Two highly conserved domains were identified GH18 and ChiA. The purified chitinase protein showed chitobiosidase activity, β-N-acetylglucosaminidase and endochitinase activity, when enzyme activity was measured using respective substrates. The purified chitinase protein was found to be orally toxic to the larvae of a major crop pest, Helicoverpa armigera when fed to the larvae mixed with artificial diet. It also had adverse effect on the growth and development of the surviving larvae. Surviving larvae showed 9-fold reduction in weight, as a result the transformation of larvae into pupae was adversely affected. Our results demonstrated that the chitinase protein of X. nematophila has insecticidal property and can prove to be a potent candidate for pest control in plants. [ABSTRACT FROM AUTHOR]

The control of insects of medical importance, such as Aedes aegypti and Aedes albopictus are still the only effective way to prevent the transmission of diseases, such as dengue, chikungunya and Zika. Their control is performed mainly using chemical products; however, they often have low specificity to non-target organisms, including humans. Also, studies have reported resistance to the most commonly used insecticides, such as the organophosphate and pyrethroids. Biological control is an ecological and sustainable method since it has a slow rate of insect resistance development. Bacterial species of the genera Xenorhabdus and Photorhabdus have been the target of several research groups worldwide, aiming at their use in agricultural, pharmaceutical and industrial products. This review highlights articles referring to the use of Xenorhabdus and Photorhabdus for insects and especially for mosquito control proposing future ways for their biotechnological applicability. Approximately 24 species of Xenorhabdus and five species of Photorhabdus have been described to have insecticidal properties. These studies have shown genes that are capable of encoding low molecular weight proteins, secondary toxin complexes and metabolites with insecticide activities, as well as antibiotic, fungicidal and antiparasitic molecules. In addition, several species of Xenorhabdus and Photorhabdus showed insecticidal properties against mosquitoes. Therefore, these biological agents can be used in new control methods, and must be, urgently considered in short term, in studies and applications, especially in mosquito control. [ABSTRACT FROM AUTHOR]

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157 bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. Key Points: •Functional characterization of hipBAXnTA module from Xenorhabdus nematophila. •hipBAXnTA module is a functional type II TA module. •Transcriptional characterization of hipBAXnTA module. •hipBAXnTA module is a well regulated TA module. [ABSTRACT FROM AUTHOR]

PirAB toxin was initially found in the Photorhabdus luminescens TT01 strain and is a demonstrated binary toxin with high insecticidal activity. In this paper, we co-expressed the pirAB gene of Xenorhabdus nematophila HB310 in a prokaryotic expression system, and we found that the PirAB protein showed high hemocoel insecticidal activity against Galleria mellonella, Helicoverpa armigera and Spodoptera exigua. LD50 values were 1.562, 2.003 and 2.17 μg/larvae for G. mellonella, H. armigera, and S. exigua, respectively (p > 0.05). Additionally, PirAB-interaction proteins were identified from G. mellonella by 6 × His Protein Pulldown combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Of which, arylphorin of G. mellonella showed the highest matching rate. A protein domain conservative structure analysis indicated that arylphorin has three domains including Hemocyanin-N, Hemocyanin-M, and Hemocyanin-C. Among these protein domains, Hemocyanin-C has immune and recognition functions. Further, Hemocyanin-C domain of arylphorin was identified to interact with PirA but not PirB by Yeast two-hybrid system. These findings reveal, for the first time, new host protein interacting with PirAB. The identification of interaction protein may serve as the foundation for further study on the function and insecticidal mechanism of this binary toxin from Xenorhabdus. [ABSTRACT FROM AUTHOR]

Drosophila suzukii, Spotted Wing Drosophila (SWD), is a serious economic issue for thin-skinned fruit farmers. The invasion of this dipteran is mainly counteracted by chemical control methods; however, it would be desirable to replace them with biological control. All assays were performed with Bacillus thuringiensis (Bt), Xenorhabdus nematophila (Xn), and Xn secretions, administered orally in single or combination, then larval lethality was assessed at different times. Gut damage caused by Bt and the influence on Xn into the hemocoelic cavity was also evaluated. In addition, the hemolymph cell population was analyzed after treatments. The data obtained show that the combined use of Bt plus Xn secretions on larvae, compared to single administration of bacteria, significantly improved the efficacy and reduced the time of treatments. The results confirm the destructive action of Bt on the gut of SWD larvae, and that Bt-induced alteration promotes the passage of Xn to the hemocoel cavity. Furthermore, hemocytes decrease after bioinsecticides treatments. Our study demonstrates that combining bioinsecticides can improve the efficacy of biocontrol and such combinations should be tested in greenhouse and in field in the near future.

ABSTRACT Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium Xenorhabdus nematophila, a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector Steinernema carpocapsae. We found that phenotypic variants cluster in three groups, one of which is composed of lrp defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of X. nematophila with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted. IMPORTANCE Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila. We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila, we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host.

Toxin-antitoxin (TA) modules, initially discovered on bacterial plasmids and subsequently identified within chromosomal contexts, hold a pivotal role in the realm of bacterial physiology. Among these, the pioneering TA system, ccd (Control of Cell Death), primarily localized on the F-plasmid, is known for its orchestration of plasmid replication with cellular division. Nonetheless, the precise functions of such systems within bacterial chromosomal settings remain a compelling subject that demands deeper investigation. To bridge this knowledge gap, our study focuses on exploring ccdAB Xn2 , a chromosomally encoded TA module originating from the entomopathogenic bacterium Xenorhabdus nematophila . We meticulously delved into the system's genomic assignments, structural attributes, and functional interplay. Our findings uncovered intriguing patterns-CcdB toxin homologs exhibited higher conservation levels compared to their CcdA antitoxin counterparts. Moreover, we constructed secondary as well as tertiary models for both the CcdB toxin and CcdA antitoxin using threading techniques and subsequently validated their structural integrity. Our exploration extended to the identification of key interactions, including the peptide interaction with gyrase for the CcdB homolog and CcdB toxin interactions for the CcdA homolog, highlighting the intricate TA interaction network. Through docking and simulation analyses, we unequivocally demonstrated the inhibition of replication via binding the CcdB toxin to its target, DNA gyrase. These insights provide valuable knowledge about the metabolic and physiological roles of the chromosomally encoded ccdAB Xn 2 TA module within the context of X. nematophila , significantly enhancing our comprehension of its functional significance within the intricate ecosystem of the bacterial host.Communicated by Ramaswamy H. Sarma.

[Display omitted] • Xenorhabus nematophila is a entomopathogenic bacterium. • X. nematophila produces a wide range of broad-spectrum bioactive compounds. • Bioactive compounds includes antibacterial, antifungal, nematocidal and insecticidal. • Toxin-antitoxin modules are essential for its adaptive survival. • Distribution of type-II TAs & their different mechanisms of activation under stress conditions. Mutualistic symbiosis exists in the natural world between the Enterobacteriaceae genus Xenorhabdus and the entomopathogenic nematode genus Steinernema. Despite the fact that the bulk of the relationships are species-specific, a single Xenorhabdus sp. may infect more than one Steinernema sp. During the complex and stressful life cycle inside the gut of insect as well as in soil, Xenorhabdus nematophila produces various bioactive compounds. These bioactive compounds are responsible for killing of infected insect larvae. It also ensures the reproduction and proliferation of the bacteria and nematodes. In this review, we have discussed various bioactive compounds produced by Xenorhabdus nematophila, proving it extremely critical for novel antibiotic discovery in the era of current antibiotic resistance crisis. The wide range of bioactive compounds produced by X. nematophila have antibacterial as well as antifungal properties. However, some of the bioactive compounds are effective against insects, nematodes, protozoa, and has anti-cancer potential too. Further, we have also discussed the increased production and gene regulation of several antimicrobial compounds. In the end, we have also discussed the presence of Toxin-Antitoxin (TA) systems and their involvement in adaptation to multiple adverse environments for the bacteria. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes have been proposed as biological agents for the control of Drosophila suzukii, an invasive pest of small-stone and soft-skinned fruits. Larvae of the fly are susceptible to Steinernema carpocapsae infection but the reaction of immune defenses of the host are unknown. To determine the immune response, larvae were infected with S. carpocapsae and Xenorhabdus nematophila to evaluate the effector mechanisms of both humoral and cellular processes. The symbiont bacteria presented an inhibitory effect on the phenoloxidase cascade with a low level of melanization. Besides, X. nematophila activated the synthesis of putative antimicrobial peptides on the hemolymph of infected larvae. However, those peptides presented a lower antimicrobial activity compared to hemolymph from larvae infected with non-symbiont bacteria. Xenorhabdus nematophila avoided also the phagocytosis response of hemocytes. During in vitro and in vivo assays, S. carpocapsae was not encapsulated by cells, unless the cuticle was damaged with a lipase-treatment. Hemocyte counts confirmed differentiation of lamellocytes in the early phase of infection despite the unrecognition of the nematodes. Both X. nematophila and S. carpocapsae avoided the cellular defenses of D. suzukii larvae and depressed the humoral response. These results confirmed the potential of entomopathogenic nematodes to control D. suzukii.

ABSTRACT Animal-microbe symbioses are ubiquitous in nature and scientifically important in diverse areas, including ecology, medicine, and agriculture. Steinernema nematodes and Xenorhabdus bacteria compose an established, successful model system for investigating microbial pathogenesis and mutualism. The bacterium Xenorhabdus nematophila is a species-specific mutualist of insect-infecting Steinernema carpocapsae nematodes. The bacterium colonizes a specialized intestinal pocket within the infective stage of the nematode, which transports the bacteria between insects that are killed and consumed by the pair for reproduction. Current understanding of the interaction between the infective-stage nematode and its bacterial colonizers is based largely on population-level, snapshot time point studies on these organisms. This limitation arises because investigating temporal dynamics of the bacterium within the nematode is impeded by the difficulty of isolating and maintaining individual living nematodes and tracking colonizing bacterial cells over time. To overcome this challenge, we developed a microfluidic system that enables us to spatially isolate and microscopically observe individual, living Steinernema nematodes and monitor the growth and development of the associated X. nematophila bacterial communities—starting from a single cell or a few cells—over weeks. Our data demonstrate, to our knowledge, the first direct, temporal, in vivo visual analysis of a symbiosis system and the application of this system to reveal continuous dynamics of the symbiont population in the living host animal. IMPORTANCE This paper describes an experimental system for directly investigating population dynamics of a symbiotic bacterium, Xenorhabdus nematophila, in its host—the infective stage of the entomopathogenic nematode Steinernema carpocapsae. Tracking individual and groups of bacteria in individual host nematodes over days and weeks yielded insight into dynamic growth and topology changes of symbiotic bacterial populations within infective juvenile nematodes. Our approach for studying symbioses between bacteria and nematodes provides a system to investigate long-term host-microbe interactions in individual nematodes and extrapolate the lessons learned to other bacterium-animal interactions.

Graphical abstract Highlights • First essentially complete TAome analysis for type II toxin-antitoxin systems. • A comparative TAome study with Photorhabdus luminescens. • Comprehensive evolutionary relationship of toxins from Xenorhabdus nematophila. • Functional and transcriptional charaterization of relBE TA module. • Give a new sight for rid of multi-drug tolerance in bacterial population. Abstract Here we report the first essentially complete TAome analysis for type II toxin-antitoxin (TA) system, a major class of TA modules found in bacterial system, from entomopathogenic bacterium Xenorhabdus nematophila ATCC 19061 (NCBI RefSeq NC_014228). We summarize this analysis in terms of TA locus, accession identifier, hits in conserved domain database, toxin superfamily, antitoxin superfamily and chromosomal/mobile genome/plasmid occurrences. Moreover, for TA context analyses we use six different specifications namely virulence factors, mobile genetic elements (MGE), antibiotic resistance genes, secretion systems, prophage and a classification of mobile genetic elements (ACLAME); among these hits are found for only MGE, ACLAME and prophage. A total 39 sets of TA have been discovered in which numbers of TA encoded for MGE, ACLAME and prophage are 15, 15 and 5 respectively while the remaining four have no context hit. In addition, a comparative analysis of TAome was also done with closely related bacterium Photorhabdus luminescens subsp. laumondii TTO1 (NCBI RefSeq NC_005126) and results shows that a total 8 sets of TA are conserved. Further, a bootstrap Neighbor-Joining phylogenetic tree was also constructed for major toxin protein superfamily found namely RelE, HigB, GNAT, CcdB and MazF explored in the TAome of X. nematophila. We also characterized, the most abundantly found TA module (relBE) in this TAome, functionally and transcriptionally. This first TAome analysis of type II TA modules provides new insights in multi-drug tolerance in bacterial populations. [ABSTRACT FROM AUTHOR]

BACKGROUND: Steinernema carpocapsae is a nematocomplex widely used as an alternative to chemicals for the biological control of insect pests; this nematode is symbiotically associated with the bacterium Xenorhabdus nematophila and both contribute to host death. The architecture and functions of structures and molecular components of the surface of nematodes and their symbiont bacteria are integral to early interactions with their hosts; thus, we assessed the role of protein pools isolated from the surface of S. carpocapsae and from phase I X. nematophila against Galleria mellonella. RESULTS: Using high‐salt treatments, we isolated the surface proteins and assayed them on G. mellonella haemocytes; haemocyte viability and phagocytic activity were investigated in the presence of surface proteins from nematodes or bacteria. Proteins from live S. carpocapsae possessed mild cytotoxicity on the haemocytes, whereas those from live X. nematophila markedly affected the host cells' viability. Bacterial proteins inhibited phagocytic activity, although they strongly triggered the host proPO (prophenoloxidase‐phenoloxidase) system. CONCLUSION: Nematocomplex surface compounds play a key role in immunoevasion/depression of insect hosts, causing a severe physiological disorder. Natural compounds newly identified as active against pests could improve the pest management of species potentially harmful to plants in urban green spaces and agriculture. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

A strain from a soil sample was obtained by screening of chemical and biological activities. This strain was identified as Xenorhabdus nematophila using 16S rDNA method and it was named as SN313. The fermented broth of SN313 was isolated and purified using microorganism fermentation, liquid extraction, column chromatography, high performance preparative liquid chromatography and other techniques. Three compounds were obtained and identified as N-(2-hydroxyphenylacetyl) tryptamine (1), phenazine-1-carboxylic acid (2), cyclo(Pro-Trp) (3) based on the results of MS, NMR and published literatures. Compound 1 is a new derivative of β-indolyl ethylamine. Mycelium growth rate method was performed to determine the antifungal activities of the three compounds. The results showed that compound 1 significantly inhibited the growth of Phytophthoa capsici and Botrytis cinerea (IC50 values were 11.20 μg/mL and 28.94 μg/mL, respectively). Compound 2 had significant inhibitive effect agianst B. cinerea, P. capsici, Thanatephorus cucumeris and Bipolaris sorokiniana (IC50 < 40 μg/mL). And compound 3 could inhibit the growth of B. cinerea (IC50 = 41.58 μg/mL). Of note, isolation of compound 2 from soil microorganism provides a new natural source of phenazine-1- carboxylic acid. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host’s immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host’s hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of pro-phenoloxidase (PO) system phenylthiourea (PTU), even if these effects were not observed in in vitro tests. Furthermore, biochemical modifications of X. nematophila cell wall implement in vivo phagocytosis, suggesting that this bacterium avoid phagocytosis because the ligand of phagocytic receptors is somehow buried or disguised in the cell wall. Finally, dead X. nematophila escapes engulfment even by human phagocytes suggesting that X. nematophila could be a useful model to investigate escape from phagocytosis by mammalian macrophages.

PirAB ( Photorhabdus insect-related proteins, PirAB) toxin was initially found in the Photorhabdus luminescens TT01 strain and has been shown to be a binary toxin with high insecticidal activity. Based on GenBank data, this gene was also found in the Xenorhabdus nematophila genome sequence. The predicted amino acid sequence of pirA and pirB in the genome of X. nematophila showed 51% and 50% identity with those gene sequences from P. luminescens . The purpose of this experiment is to identify the relevant information for this toxin gene in X. nematophila . The pirA , pirB and pirAB genes of X. nematophila HB310 were cloned and expressed in Escherichia coli BL21 (DE3) using the pET-28a vector. A PirAB-fusion protein (PirAB-F) was constructed by linking the pirA and pirB genes with the flexible linker (Gly) 4 DNA encoding sequence and then efficiently expressed in E. coli. The hemocoel and oral insecticidal activities of the recombinant proteins were analyzed against the larvae of Galleria mellonella . The results show that PirA/B alone, PirA/B mixture, co-expressed PirAB protein, and PirAB-F all had no oral insecticidal activity against the second-instar larvae of G. mellonella . Only PirA/B mixture and co-expressed PirAB protein had hemocoel insecticidal activity against G. mellonella fifth-instar larvae, with an LD 50 of 2.718 μg/larva or 1.566 μg/larva, respectively. Therefore, we confirmed that PirAB protein of X. nematophila HB310 is a binary insecticidal toxin. The successful expression and purification of PirAB laid a foundation for further studies on the function, insecticidal mechanism and expression regulation of the binary toxin. [ABSTRACT FROM AUTHOR]

Dengue, Chikungunya, and Zika are important vector-borne diseases, and Aedes aegypti L. is their main transmitter. As the disease management is mainly based on mosquito control strategies, the search for alternative and cost-effective approaches is ongoing. The Gram-negative bacteria Xenorhabdus nematophila and Photorhabdus luminescens are symbiotically associated with entomopathogenic nematodes and are highly pathogenic for insect larvae. After we have recently confirmed the toxicity of these bacteria in Ae. aegypti larvae, we here evaluated the toxic activity of culture fluids on the development of this mosquito species. Larval susceptibility was assessed by exposing larvae to different concentrations of P. luminescens or X. nematophila culture fluids to confirm whether secondary metabolites might cause the mosquitos' death. Xenorhabdus nematophila culture fluid was more effective and stable during the mosquito pathogenicity bioassays compared to that of P. luminescens. Larval mortality started a few hours after exposure of the insects to the fluids. Furthermore, the residual effect of larvicidal activity of X. nematophila fluid persisted at full efficiency for 4 d. Particularly, larval mortality was still higher than 50% for up to 8 d. Exposure of larvae to a sublethal dose of X. nematophila fluid delayed pupation as well as emergence of adult mosquitoes and caused cumulative larval mortality higher than 90% by day 14. Here, we describe for the first time the use of stable culture fluids and therefore secondary metabolites of P. luminescens and X. nematophila as a promising basis for the use as biopesticide for control of Ae. aegypti in the future. [ABSTRACT FROM AUTHOR]

In the present circumstances, toxin-antitoxin (TA) modules have a great consideration due to their elusive role in bacterial physiology. TA modules consist of a toxic part and a counteracting antitoxin part and these are abundant genetic loci harbored on bacterial plasmids and chromosomes. The control of cell death ( ccd ) TA locus was the first identified TA module and its unitary function (such as plasmid maintenance) has been described, however, the function of its chromosomal counterparts is still ambiguous. Here, we are exploring the genomic assortment, structural and functional association of chromosomally encoded ccdAB TA homolog ( ccdAB Xn 1 ) in the genome of an entomopathogenic bacterium Xenorhabdus nematophila . This bacterium is a symbiotic model with the nematode Steinernema carpocapsae that infects and kills the host insect. By genomic assortment analysis, our observations suggested that CcdA antitoxin homologs are not more closely related than CcdB toxin homologs. Further results suggest that the ccdAB Xn 1 TA homolog has sulphonamide (such as 4C6, for CcdA homolog) and peptide (such as gyrase, for CcdB homolog) ligand partners with a typical TA interaction network that may affect essential cellular metabolism of the X. nematophila . Collectively, our results improve the knowledge and conception of the metabolic interactive role of ccdAB TA homologs in X. nematophila physiology.Communicated by Ramaswamy H. Sarma.

The gram-negative bacterium Xenorhabdus is mutualistically associated with entomopathogenic nematode, Steinernema. Three bacterial strains were isolated from the entomopathogenic nematodes, Steinernema glaseri (Steiner) and S. carpocapsae (Weiser), of the white grubs, Polyphylla olivieri (Laporte de Castelnau) in Iran. This study was focused on the characterization of the bacterial strains by using phenetic characters and 16S ribosomal RNA gene sequence. This polyphasic approach led to the identification of all strains as Xenorhabdus spp., separated as X. nematophila and X. poinarii. The phylogenetic relationship between Iranian and global strains of Xenorhabdus was analyzed and discussed. Characterization of symbiotic bacteria associated with the entomopathogenic nematodes is a main step to the study of different aspects of this complex. These results provide new insights for the biodiversity of bacto-helminthic complex in Iran.

Entomopathogenic nematodes (EPNs) are widely used as biological control agents against insect pests, the efficacy of these organisms strongly depends on the balance between the parasitic strategies and the immune response of the host. This review summarizes roles and relationships between insect hosts and two well-known EPN species, Steinernema feltiae and Steinernema carpocapsae and outlines the main mechanisms of immune recognition and defense of insects. Analyzing information and findings about these EPNs, it is clear that these two species use shared immunosuppression strategies, mainly mediated by their symbiotic bacteria, but there are differences in both the mechanism of evasion and interference of the two nematodes with the insect host immune pathways. Based on published data, S. feltiae takes advantage of the cross reaction between its body surface and some host functional proteins, to inhibit defensive processes; otherwise, secretion/excretion products from S. carpocapsae seem to be the main nematode components responsible for the host immunosuppression.

Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici . However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus .

The diamondback moth (DBM)Plutella xylostella(L.) is one of the most destructive pests of crucifer crops in many parts of the world. Inappropriate applications have caused DBM to become resistant to most insecticides. We tested the combined effects of the bacteriaBacillus thuringiensis(Bt) andXenorhabdus nematophila(Xn) against DBM. Populations ofBt-resistant moths (DBM-R), selected repeatedly withB. thuringiensissubsp.kurstakiHD-73 (BtHD-73), showed resistance rates of 105.43 and 0.92 forBtHD-73 andX. nematophilaHB310 (XnHB310), respectively. These results not only indicated that DBM-R is highly resistant toBtHD-73 and only slightly resistant toXnHB310, but also showed that there is no interactive resistance between the two bacteria. For the third-instar larvae of DBM-R and DBM-S, no significant differences were found in the oral insecticidal activities ofBtHD-73 + XnHB310 combination treatments orBtHD-73 alone. However, mortality rates were significantly increased in response toXnHB310 + BtHD-73 when compared with theXnHB310-alone treatment. These results demonstrate a synergism ofBtonXn, but no such effect ofXnonBt. Therefore, our findings highlight the potential for both bacteria to be used in a mixture for field applications. [ABSTRACT FROM PUBLISHER]

Xenorhabdus and Photorhabdus spp. (Enterobacteriaceae) can synthesize and release secondary metabolites that play crucial roles in their pathogenicity by suppressing the immunity of target insects. The insect immunity contributes to defense against the pathogenicity of Bacillus thuringiensis (Bt). This study tested a hypothesis that bacterial immunosuppresants could enhance the susceptibility of mosquitoes (Aedes albopictus and Culex pipiens pallens) to Bt. Three symbiotic bacteria [X. nematophila (Xn), X. hominickii (Xh), and P. temperata temperata (Ptt)] were cultured in nutrient broth to allow them to produce secondary metabolites. Bacillus thuringiensis israelensis (BtI) was highly toxic to both culicid mosquitoes with median lethal concentration (LC50, spores/ml) of 2.9 × 105 and 2.2 × 105 at 16 h after treatment, respectively. Addition of each bacteria-cultured broth enhanced BtI toxicity to these mosquito larvae. The LC50 values of BtI to Ae. albopictus larvae were reduced to 1.5 × 105 in Xn mixture, 1.7 × 105 in Xh mixture, and 1.9 × 105 in Ptt mixture. The LC50 values of BtI t o Cx. pipiens pallens larvae were also reduced to 1.2 × 105 in Xn mixture, 1.3 × 105 in Xh mixture, and1.5 × 105 in Ptt mixture. Adding benzylideneacetone or oxindole produced f r om Xn and Ptt also enhanced BtI toxicities to these mosquito larvae. Based on these results, we developed a new mosquitocidal Bt formulation called "Dip- Kill" consisting of 80% Xn-cultured broth, 10% BtI (1010 spores/ml), and 10% preservative. Dip-Kill at 1,000 ppm was superior to a commercial BtI product at its recommended dose. [ABSTRACT FROM AUTHOR]

Relationships between parasites and hosts can be drastic, depending on the balance between parasite strategies and the efficiency of the host immune response. In the case of entomopathogenic nematodes and their insect hosts, we must also consider the role of bacterial symbionts, as the interaction among them is tripartite and each component plays a critical role in death or survival. We analyzed the effects induced by the nematode–bacteria complex Steinernema carpocapsae , against red palm weevil (RPW) larvae, Rhynchophorus ferrugineus . We examined the antimicrobial response of the insect when in the presence of nematocomplexes or of its symbionts, Xenorhabdus nematophila . In detail, we investigated the potential interference of live and dead S. carpocapsae , their isolated cuticles, live or dead bacterial symbionts and their lipopolysaccharides, on the synthesis and activity of host antimicrobial peptides. Our data indicate that both live nematodes and live bacterial symbionts are able to depress the host antimicrobial response. When nematodes or symbionts were killed, they lacked inhibitory properties, as detected by the presence of antimicrobial peptides (AMPs) in the host hemolymph and by assays of antimicrobial activity. Moreover, we isolated S. carpocapsae cuticles; when cuticles were injected into hosts they revealed evasive properties because they were not immunogenic and were not recognized by the host immune system. We observed that weevil AMPs did not damage X. nematophila , and the lipopolysaccharides purified from symbionts seemed to be non-immunogenic. We believe that our data provide more information on the biology of entomopathogenic nematodes, in particular concerning their role and the activity mediated by symbionts in the relationship with insect hosts. [ABSTRACT FROM AUTHOR]

Key message: Overexpression of insecticidal pilin subunit from Xenorhabdus nematophila protects transgenic tobacco and tomato plants against Helicoverpa armigera. Abstract: Xenorhabdus nematophila is a pathogenic bacterium producing toxins that kill the larval host. Previously, we characterized a pilin subunit of X. nematophila which was found to be a pore-forming toxin and cytotoxic to the larval hemocytes of Helicoverpa armigera by causing agglutination and lysis of the cells. In the present study, we report the efficacy of the insecticidal pilin subunit expressed in transgenic tobacco and tomato plants for control against H. armigera. A 537 bp mrxA gene encoding the 17 kDa insecticidal pilin subunit was transferred into the genome of tobacco and tomato, respectively, via Agrobacterium-mediated transformation. The stable integration of the 537 bp mrxA gene in the transgenic plants was confirmed by Southern blot analysis and expression of mrxA gene was confirmed by RT-PCR and Western blot analyses. The transgenic plants appeared healthy and phenotypically normal but proved toxic to the insects in insect bioassays, showing 100 % insect mortality and reduced damage of the transgenic plants. Based on these observations, it is suggested that pilin subunit can be used as a potential candidate for control of H. armigera and may open new strategies for pest control in agricultural plants. [ABSTRACT FROM AUTHOR]

The GroEL homolog XnGroEL protein of Xenorhabdus nematophila belongs to a highly conserved family of molecular chaperones/heat shock proteins (Hsps). XnGroEL was shown to possess oral insecticidal activity against a major crop pest Helicoverpa armigera. Under normal conditions, the Hsps/chaperones facilitate folding, assembly, and translocation of cellular proteins, while in stress conditions they protect proteins from denaturation. In this study, we describe generation of transgenic tomato plants overexpressing insecticidal XnGroEL protein and their tolerance to biotic and abiotic stresses. Presence of XnGroEL in the transgenic tomato lines conferred resistance against H. armigera showing 100 % ( p ≤ 0.001) mortality of neonates. In addition, XnGroEL provided thermotolerance and protection against high salt concentration to the tomato plants. Expression of XnGroEL minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane system of the plants under heat and salt stress. The enhanced tolerance to abiotic stresses correlated with increase in the anti-oxidative enzyme activity and reduced HO accumulation in transgenic tomato plants. The variety of beneficial properties displayed by XnGroEL protein provides an opportunity for value addition and improvement of crop productivity. [ABSTRACT FROM AUTHOR]

Yellow fever mosquito, Aedes aegypti , is an important medical insect that can transmit vector borne disease to human and animal. Mosquito control is the only way to stop the spread of arboviral diseases since no vaccines and effective drugs are available to vector borne diseases. In this study, Bacillus thuringiensis subsp. israelnsis Cry4Ba has high toxicity to A. aegypti larvae and the calculated 50% lethal concentration of Cry4Ba was 21.28 ng/mL. The gram-negative bacteria Xenorhabdus and Photorhabdus are symbiotically associated with nematode, which is one of biological agents for insect control with high pathogenicity. In here, X. nematophila and P. luminescens showed 52% and 43% of oral toxicity against Aedes aegypti larvae at 72 h after bacterial ingestion. Remarkable, the mortality of larvae was enhanced up to 95% or 87% by X. nematophila or P. luminescens at 48 h, when larvae were exposed to these bacteria with Cry4Ba toxin (5 ng/mL), which has 20% of larval mortality at 48 h after toxin ingestion along. This study reports that entomopthogenic bacteria enhance Cry4Ba's toxicity against A. aegypti larvae. These results may have promising basis for practical application to mosquito control in preventing arboviral diseases transfer and reducing the Bt resistance risk to mosquito. [ABSTRACT FROM AUTHOR]

[Display omitted] • Presence of three hipBA TA paralogs in the genome of X. nematophila. • HipA homologs represents a class of serine/threonine-protein kinase. • HipB homologs represents a class of transcriptional regulator. • hipBA TA paralogs in the genome of X. nematophila are functionally associated. Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila. [ABSTRACT FROM AUTHOR]

Xenorhabdus nematophila is an entomopathogenic bacteria. It secretes a GroEL homolog, XnGroEL protein, toxic to its larval prey. GroEL belongs to the family of molecular chaperones and is required for proper folding of cellular proteins. Oral ingestion of insecticidal XnGroEL protein is toxic to Helicoverpa armigera, leading to cessation of growth and development of the larvae. In the present study, the insecticidal efficacy of XnGroEL against H. armigera has been evaluated in transgenic tobacco plant expressing the protein. A 1.7-kb gene encoding the 58-kDa XnGroEL protein was incorporated into the tobacco genome via Agrobacterium-mediated transformation. The stable integration of the transgene was confirmed by Southern blot analysis and its expression by RT-PCR and western blot analyses in transgenic plants. The transgenic lines showed healthy growth and were phenotypically normal. Insect bioassays revealed significant reduction of 100 % in the survival of larvae ( p < 0.001) and 55-77 % reduction in plant damage ( p < 0.05 and p < 0.001) compared to the untransformed and vector control plants. The results demonstrate that XnGroEL is a novel potential candidate for imparting insect resistance against H. armigera in plants. [ABSTRACT FROM AUTHOR]