A taste of a toxin paradise: Xenorhabdus and Photorhabdus bacterial secondary metabolites against Aedes aegypti larvae and eggs.

Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria are promising sources of effective compounds with important biological activities. This study investigated the effects of cell-free supernatants of X. szentirmaii, X. cabanillasii and P. kayaii against Ae. aegypti eggs and larvae and identified the bioactive larvicidal compound in X. szentirmaii using The EasyPACId method. Among the three tested bacterial species, X. cabanillasii exhibited the highest (96%) egg hatching inhibition and larvicidal activity (100% mortality), whereas P. kayaii was the least effective species in our study. EasyPACId method revealed that bioactive larvicidal compound in the bacterial supernatant was fabclavine. Fabclavines obtained from promoter exchange mutants of different bacterial species such as X. cabanillasii, X. budapestensis, X. indica, X. szentirmaii, X. hominckii and X. stockiae were effective against mosquito larvae. Results show that these bacterial metabolites have potential to be used in integrated pest management (IPM) programmes of mosquitoes.

Natural products from Xenorhabdus and Photorhabdus show promise as biolarvicides against Aedes albopictus.

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.

Potential of 3-octanone as a lure and kill agent for control of the Brown garden snail.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

Arthropod vectors of disease agents: Their role in public and veterinary health in Turkiye and their control measures.

Mosquitoes, sandflies, and ticks are hematophagous arthropods that pose a huge threat to public and veterinary health. They are capable of serving as vectors of disease agents that can and have caused explosive epidemics affecting millions of people and animals. Several factors like climate change, urbanization, and international travel contribute substantially to the persistence and dispersal of these vectors from their established areas to newly invaded areas. Once established in their new home, they can serve as vectors for disease transmission or increase the risk of disease emergence. Turkiye (formerly Turkey) is vulnerable to climate change and has experienced upward trends in annual temperatures and rising sea levels, and greater fluctuations in precipitation rates. It is a potential hotspot for important vector species because the climate in various regions is conducive for several insect and acari species and serves as a conduit for refugees and immigrants fleeing areas troubled with armed conflicts and natural disasters, which have increased substantially in recent years. These people may serve as carriers of the vectors or be infected by disease agents that require arthropod vectors for transmission. Although it cannot be supposed that every arthropod species is a competent vector, this review aims to (1) illustrate the factors that contribute to the persistence and dispersal of arthropod vectors, (2) determine the status of the established arthropod vector species in Turkiye and their capability of serving as vectors of disease agents, and (3) assess the role of newly-introduced arthropod vectors into Turkiye and how they were introduced into the country. We also provide information on important disease incidence (if there's any) and control measures applied by public health officials from different provinces.

Disease caused by Neofusicoccum parvum in pruning wounds of grapevine shoots and its control by Trichoderma spp. and Xenorhabdus szentirmaii.

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.

Antiprotozoal activity of different Xenorhabdus and Photorhabdus bacterial secondary metabolites and identification of bioactive compounds using the easyPACId approach.

Natural products have been proven to be important starting points for the development of new drugs. Bacteria in the genera Photorhabdus and Xenorhabdus produce antimicrobial compounds as secondary metabolites to compete with other organisms. Our study is the first comprehensive study screening the anti-protozoal activity of supernatants containing secondary metabolites produced by 5 Photorhabdus and 22 Xenorhabdus species against human parasitic protozoa, Acanthamoeba castellanii, Entamoeba histolytica, Trichomonas vaginalis, Leishmania tropica and Trypanosoma cruzi, and the identification of novel bioactive antiprotozoal compounds using the easyPACId approach (easy Promoter Activated Compound Identification) method. Though not in all species, both bacterial genera produce antiprotozoal compounds effective on human pathogenic protozoa. The promoter exchange mutants revealed that antiprotozoal bioactive compounds produced by Xenorhabdus bacteria were fabclavines, xenocoumacins, xenorhabdins and PAX peptides. Among the bacteria assessed, only P. namnaoensis appears to have acquired amoebicidal property which is effective on E. histolytica trophozoites. These discovered antiprotozoal compounds might serve as starting points for the development of alternative and novel pharmaceutical agents against human parasitic protozoa in the future.

Natural products from Photorhabdus and Xenorhabdus: mechanisms and impacts.

Insects and fungal pathogens pose constant problems to public health and agriculture, especially in resource-limited parts of the world; and the use of chemical pesticides continues to be the main methods for the control of these organisms. Photorhabdus spp. and Xenorhabdus spp., (Fam; Morganellaceae), enteric symbionts of Steinernema, and Heterorhabditis nematodes are naturally found in soil on all continents, except Antarctic, and on many islands throughout the world. These bacteria produce diverse secondary metabolites that have important biological and ecological functions. Secondary metabolites include non-ribosomal peptides, polyketides, and/or hybrid natural products that are synthesized using polyketide synthetase (PRS), non-ribosomal peptide synthetase (NRPS), or similar enzymes and are sources of new pesticide/drug compounds and/or can serve as lead molecules for the design and synthesize of new alternatives that could replace current ones. This review addresses the effects of these bacterial symbionts on insect pests, fungal phytopathogens, and animal pathogens and discusses the substances, mechanisms, and impacts on agriculture and public health. KEY POINTS: • Insects and fungi are a constant menace to agricultural and public health. • Chemical-based control results in resistance development. • Photorhabdus and Xenorhabdus are compelling sources of biopesticides.

Antifungal activity of different Xenorhabdus and Photorhabdus species against various fungal phytopathogens and identification of the antifungal compounds from X. szentirmaii.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter-activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. KEY POINTS: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified.

The effect of chemical insecticides on the scavenging performance of Steinernema carpocapsae: Direct effects and exposure to insects killed by chemical insecticides.

Entomopathogenic nematodes are used widely in biological insect control. Entomopathogenic nematodes can infect live insects as well as dead insects (i.e., they can act as scavengers). It is important to determine compatibility of entomopathogenic nematodes with other pest management tactics such as chemical insecticides. We hypothesized that chemical insecticides have negative impact on scavenging nematodes. According to our hypothesis, we first investigated the effects of direct exposure of Steinernema carpocapsae infectivity juveniles (IJs) to three chemical insecticides, cypermethrin, spinosad or diflubenzuron in terms of nematode survival and virulence. Subsequently, using the same chemicals, we tested the effects of insecticide-killed insects on scavenger nematode penetration efficiency, time of emergence and the number of nematode progeny. Prior to our study, the impact of pesticides on scavenger nematode fitness had not been studied. Fall webworm, Hyphantria cunea, and greater wax moth, Galleria mellonella, larvae were used as host insects. The survival rate of IJs after direct exposure was 83% for cypermethrin and 93-97% for the other insecticides and control. There were no significant differences in the survival and virulence of the nematodes after 24 h exposure to insecticides. The number of nematodes that invaded the insecticide-killed host was significantly higher in cypermethrin and spinosad treated groups and live H. cunea than in the diflubenzoron treated group and freeze-killed control. However, no significant differences were observed in time of emergence. Significantly more progeny IJs emerged from Spinosad-killed insects than the freeze-killed control. In conclusion, we discovered that the fitness of scavenging IJs is not diminished by insecticides in insect cadavers. In fact, in some cases the exposure to chemical insecticides may enhance virulence.

Relative potency of a novel acaricidal compound from Xenorhabdus, a bacterial genus mutualistically associated with entomopathogenic nematodes.

Our study aimed to identify the novel acaricidal compound in Xenorhabdus szentirmaii and X. nematophila using the easyPACId approach (easy Promoter Activated Compound Identification). We determined the (1) effects of cell-free supernatant (CFS) obtained from mutant strains against T. urticae females, (2) CFS of the acaricidal bioactive strain of X. nematophila (pCEP_kan_XNC1_1711) against different biological stages of T. urticae, and females of predatory mites, Phytoseiulus persimilis and Neoseiulus californicus, (3) effects of the extracted acaricidal compound on different biological stages of T. urticae, and (4) cytotoxicity of the active substance. The results showed that xenocoumacin produced by X. nematophila was the bioactive acaricidal compound, whereas the acaricidal compound in X. szentirmaii was not determined. The CFS of X. nematophila (pCEP_kan_XNC1_1711) caused 100, 100, 97.3, and 98.1% mortality on larvae, protonymph, deutonymph and adult female of T. urticae at 7 dpa in petri dish experiments; and significantly reduced T. urticae population in pot experiments. However, the same CFS caused less than 36% mortality on the predatory mites at 7dpa. The mortality rates of extracted acaricidal compound (xenocoumacin) on the larva, protonymph, deutonymph and adult female of T. urticae were 100, 100, 97, 96% at 7 dpa. Cytotoxicity assay showed that IC50 value of xenocoumacin extract was 17.71 µg/ml after 48 h. The data of this study showed that xenocoumacin could potentially be used as bio-acaricide in the control of T. urticae; however, its efficacy in field experiments and its phytotoxicity need to be assessed in future.

Volatile organic compounds of Metarhizium brunneum influence the efficacy of entomopathogenic nematodes in insect control.

The entomopathogenic fungus (EPF) Metarhizium brunneum occupies the same ecological niche as entomopathogenic nematodes (EPN), with both competing for insects as a food source in the rhizosphere. Interactions between these biocontrol agents can be antagonistic or synergistic. To better understand these interactions, this study focussed on investigating the effect of M. brunneum volatile organic compounds (VOCs), 1-octen-3-ol and 3-octanone, on EPN survival and behaviour. These VOCs proved to be highly toxic to the infective juveniles (IJs) of the EPN Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora with mortality being dose dependent. Chemotaxis studies of H. bacteriophora IJs in Pluronic F127 gel revealed significant preference for the VOCs compared with controls for all tested concentrations. The VOCs also impacted on the test insects in a dose-dependent manner with 3-octanone being more toxic to Galleria mellonella, Cydia splendana and Curculio elephas larvae than 1-octen-3-ol. Mortality of C. splendana and G. mellonella larvae was significantly higher when exposed to relatively high doses (>25%) of 3-octanone. Lower doses of 3-octanone and 1-octen-3-ol immobilised test insects, which recovered after exposure to fresh air for 2 hrs. In depth studies on H. bacteriophora showed that exposure of IJs to > 10% concentration of 3-octanone or 1-octen-3-ol negatively affected infectivity whereas exposure to lower doses (0.1%, 0.01%) had no effect. The VOCs affected IJs, reducing penetration efficacy and the number of generations inside G. mellonella but they failed to inhibit the bacterial symbiont, Photorhabdus kayaii. The ecological significance of VOCs and how they could influence EPF-EPN insect interactions is discussed.

Mode of entry of secondary metabolites of the bacteria Xenorhabdus szentirmaii and X. nematophila into Tetranychus urticae, and their toxicity to the predatory mites Phytoseiulus persimilis and Neoseiulus californicus.

The bacterial metabolites in supernatants of Xenorhabdus species have acaricidal activity, but this mode of entry into mites has not yet been elucidated. Herein, we report on the possible mode of entry of Xenorhabdus szentirmaii and Xenorhabdus nematophila supernatants into Tetranychus urticae (Acari: Tetranychidae) adult females. We also assessed the toxicity of the supernatants against the developmental stages of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus (Acari: Phytoseiidae). Experiments were conducted at 25 ± 1 °C, 70 ± 5% relative humidity, and 16:8h light:dark conditions. Our data showed that the bioactive acaricidal compound is most effective (86.5 to 89% mortality) when the entire integument of T. urticae comes in contact with it compared to contact of the ventral side only (26.5-34%). Against P. persimilis and N. californicus at 6 days post-application (dpa), the eggs were not affected by the X. szentirmaii or X. nematophila supernatant, whereas mortality of the mobile stages (larva, protonymph, deutonymph, adult) was 18.5% to 39.2%. Overall, the predatory mites were less affected by the bacterial metabolites than T. urticae. We hypothesize that the differences in morphology such as longer legs and thicker cuticle, as well as the diet of the predatory mites, reduce the contact of the body parts to the supernatant-treated surfaces. We need to isolate, identify, and characterize the X. szentirmaii and X. nematophila metabolite(s) and demonstrate efficacy to pestiferous mites and safety to plants, non-target organisms and the environment before it can be used as an acaricide.

Evaluation of different sponge types on the survival and infectivity of stored entomopathogenic nematodes.

Sponges are one of the cheapest and most suitable substrates used to formulate and/or store the infective juveniles (IJs) of entomopathogenic nematodes (EPNs). Our study investigated the survival and infectivity of the IJs on five different sponges compared to that in an aqueous suspension (control). The sponges were Oasis® floral, Nanosponge, ScotchbriteTM, or Lysol® and natural sea sponge. EPN species tested were Heterorhabditis bacteriophora, Steinernema carpocapsae and S. feltiae. The recovery efficiency of the IJs from sponges was initially assessed. Subsequently, IJs were stored in the sponges and placed in plastic bags or Falcon tubes and incubated at 10° or 27 °C for 8 months or 11 weeks, respectively. IJ survival and infectivity were monitored monthly for the 10 °C and weekly for 27 °C in these sponge types. The IJs were recovered from the sponges, and their survival was based on observing their movement under a dissecting microscope, and infectivity was based on larval mortality in Galleria mellonella. Recovery efficiency of IJs was best for the Oasis floral sponge for all nematode species ranging between 83 and 91%. The survival and infectivity of stored IJs in all sponge types and control for both 10° and 27 °C gradually decreased over time. IJs stored in Scotchbrite, Lysol, and Nanosponge had the best survival and infectivity, whereas Oasis floral and natural sea sponges showed the poorest results. After 8 months at 10 °C in plastic bags, the survival ratio of all IJs in these three sponges (Scotchbrite, Lysol, and Nanosponge) was approximately 55%. IJs in Scotchbrite and Nanosponge were also able to survive and retain their infectivity at 27 °C for 3 months. IJs stored in Falcon tubes had survival that ranged from 26 to 53% at 27 °C and 55 to 77% at 10 °C. H. bacteriophora IJs lost their infectivity when stored at 27 °C after 10 weeks. However, S. carpocapsae and S. feltiae exhibited 85% infectivity when stored in Scotchbrite and 50% in Nanosponge, respectively. Overall, sponges made from polyurethane (Scotchbrite) followed by melamine (Nanosponge) and cellulose (Lysol) are recommended for long-term nematode storage and transportation of nematode samples. However, Oasis floral sponge may be preferred for short-term IJ formulation for field applications because of easier recovery of IJs.