Biosynthesis of selenium nanoparticles using cell-free extract of Xenorhabdus cabanillasii GU480990 and their potential mosquito larvicidal properties against yellow fever mosquito Aedes aegypti.

Nanomaterials are successful due to their numerous applications in various domains such as cancer treatment, environmental applications, drug and gene delivery. Selenium is a metalloid element with broad biological activities and low toxicity especially at the nanoscale. Several studies have shown that nanoparticles synthesized from microbial and plant extracts are effective against important pests and pathogens. This study describes the bio fabrication of selenium nanoparticles using cell free extract of Xenorhabdus cabanillasii (XC-SeNPs) and assessed their mosquito larvicidal properties. Crystallographic structure and size of XC-SeNPs were determined with UV-a spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), Energy-dispersive X-ray spectroscopy (EDAX), Zeta potential and Transmission electron microscopy (TEM). The significant surface plasmon resonance at 275 nm indicated the synthesis of XC-SeNPs from the pure cell-free extract of X. cabanillasii. The XRD result exhibits the crystalline nature of XC-SeNPs. The Zeta potential analysis confirmed that the surface charge of XC-SeNPs was -24.17 mV. TEM analysis revealed that synthesized XC-SeNPs were monodispersed, spherically shaped, and sized about 80-200 nm range. In addition, the larvicidal potentials of the bio-fabricated XC-SeNPs were assessed against the 4th-instar Ae. aegypti. XC-SeNPs displayed a dose-dependent larvicidal effect; the larval mortality was 13.3 % at the minimum evaluated concentration and increased to 72 % at higher dose treatments. The LC50 and LC90 concentration of XC-SeNPs against mosquito larvae were 79.4 and 722.4 ppm, respectively.

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 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.

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.

Environmental tolerance of entomopathogenic nematodes differs among nematodes arising from host cadavers versus aqueous suspension.

Environmental factors such as temperature and desiccation impact the survival and efficacy of entomopathogenic nematodes (EPNs). Most studies on environmental tolerance have focused on EPNs applied in aqueous suspension. Another approach for EPN application is via infected host cadavers. Emergence in host cadavers is also more representative of nematodes in natural populations. In prior studies, certain advantages in fitness have been observed with the cadaver application approach relative to aqueous application, yet the impact of environmental stress on these approaches requires investigation. In this study, we compared the effects of various temperatures (heat and cold) and desiccation intervals (48 and 72 hr) on the survival, virulence and reproductive capacity of Heterorhabditis bacteriophora and Steinernema glaseri when applied via cadaver versus aqueous suspension. In the heat tolerance bioassays, following exposure to 30 °C, 35 °C and 37. 5 °C, nematodes (from both species) in the cadaver treatments exhibited higher survival, and reproductive capacity compared with aqueous application. No survival was observed above 37.5 °C regardless of species or application approach. In cold tolerance, no differences were observed between the cadaver and aqueous treatments after a sequence of exposures from 10 °C to -2 °C. In desiccation assays, following exposure to 85% relative humidity for 2 or 3 days, nematodes (from both species) exhibited higher survival and reproduction in the cadaver treatment than in the aqueous treatment, whereas no differences were observed in virulence. This is the first study to find differential stress tolerance among nematodes emerged from infected host cadavers versus those applied in aqueous suspension. Our findings indicate additional advantages when using the cadaver approach for biocontrol applications, and suggest EPNs existing in natural populations may have broader environmental tolerance than those applied via aqueous suspension.

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.

Fabclavine diversity in Xenorhabdus bacteria.

The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera Xenorhabdus or Photorhabdus. In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine (fcl) biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS2 experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.

Acaricidal effect of cell-free supernatants from Xenorhabdus and Photorhabdus bacteria against Tetranychus urticae (Acari: Tetranychidae).

The effects of secondary metabolites produced by the following symbiotic bacteria, Xenorhabdus szentirmaii, X. nematophila, X. bovienii, X. cabanillasii, Photorhabdus luminescens and P. temperata, associated with entomopathogenic nematodes, were investigated against various developmental stages of Tetranychus urticae (Acari: Tetranychidae) using cell-free bacterial supernatants in Petri dishes. In addition, the effects of the most active bacterial supernatant(s) found in Petri dish experiments were tested on T. urticae in pot experiments. All studies were conducted at 25 ±â€¯1 °C temperature, 70 ±â€¯5% relative humidity and a light cycle of 16 h in a climate room. The result of the Petri dish experiments showed that the supernatants had little or no effect on the egg stage, as less than 4% mortality was recorded. Depending on the bacterial supernatant, mortality in the other stages was 46-97% for larvae, 30-96% for protonymphs, 41-92% for deutonymphs, 92-100% for adult males and 46-93% for adult females. Control mortalities ranged from 1-7% for larvae, 2-9% for protonymphs, 4-10% for deutonymphs, 6-10% for adult males and 4-8% for adult females. Among supernatants tested, X. szentirmaii and X. nematophila were the most efficacious with mortality greater than 90% on the mobile stages of T. urticae. According to the results from pot experiments, the supernatants of X. szentirmaii and X. nematophila, singularly and in combination, significantly reduced the T. urticae population. However, the mixture of X. szentirmaii and X. nematophila supernatants did not increase efficiency to reduce T. urticae population compared to each supernatant alone. Further studies are warranted to find the active compound(s) in the supernatants of X. szentirmaii or and X. nematophila and assess whether the supernatant(s) has the potential of being a practical and economical control agent for T. urticae.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Biological characterization of the entomopathogenic nematode, Steinernema innovationi: a South African isolate.

Entomopathogenic nematode species perform differently under different environmental conditions; therefore, the authors investigated the biological and environmental characteristics that could optimize performance of Steinernema innovationi. The authors studied the effect of temperature on infectivity and reproduction, the foraging behavior and host range. Thermal activity was optimal between 22 and 25°C. Highest infective juvenile (IJ) yields in last instar Galleria mellonella were observed at 22°C (333,014 IJs/g) and 25°C (354,165 IJs/g). An average of 26% of the IJs infected G. mellonella larvae at depths of 15 cm within 24 hr. Steinernema innovationi IJs raised greater than 95% of their body off the substrate and moved in a circular pattern, but did not jump. The hosts, Acheta domesticus, Chilo partellus, and Plutella xylostella showed the least susceptibility. All other hosts, [Eldana saccharina, Sesamia calamistis, Tenebrio molitor, G. mellonella, Cydia pomonella] suffered 100% larval mortality. Pupal mortality ranged from 47 to 68%. An LC50 and LC70 of 3 and 31 IJs/larva, respectively, were calculated for Agrotis ipsilon. The results indicate that S. innovationi is adapted to moderate temperatures. Furthermore, to the author's knowledge this is the first report of a nematode (particularly from 'glaseri group') that shows cruiser-type behavior in movement, but also nictates.Entomopathogenic nematode species perform differently under different environmental conditions; therefore, the authors investigated the biological and environmental characteristics that could optimize performance of Steinernema innovationi. The authors studied the effect of temperature on infectivity and reproduction, the foraging behavior and host range. Thermal activity was optimal between 22 and 25°C. Highest infective juvenile (IJ) yields in last instar Galleria mellonella were observed at 22°C (333,014 IJs/g) and 25°C (354,165 IJs/g). An average of 26% of the IJs infected G. mellonella larvae at depths of 15 cm within 24 hr. Steinernema innovationi IJs raised greater than 95% of their body off the substrate and moved in a circular pattern, but did not jump. The hosts, Acheta domesticus, Chilo partellus, and Plutella xylostella showed the least susceptibility. All other hosts, [Eldana saccharina, Sesamia calamistis, Tenebrio molitor, G. mellonella, Cydia pomonella] suffered 100% larval mortality. Pupal mortality ranged from 47 to 68%. An LC50 and LC70 of 3 and 31 IJs/larva, respectively, were calculated for Agrotis ipsilon. The results indicate that S. innovationi is adapted to moderate temperatures. Furthermore, to the author's knowledge this is the first report of a nematode (particularly from 'glaseri group') that shows cruiser-type behavior in movement, but also nictates.

Trans-cinnamic acid and Xenorhabdus szentirmaii metabolites synergize the potency of some commercial fungicides.

Development of novel approaches for the control of fungal phytopathogens is desirable. In this study we hypothesized that the combination of commercial fungicides with certain enhancing agents could result in synergistic levels of control. Prior research has indicated that trans-cinnamic-acid (TCA), a metabolite of the bacteria Photorhabdus luminescens and metabolites of Xenorhabdus szentirmaii are particularly toxic to various phytpathogenic fungi when compared to metabolites of other Xenorhabdus or Photorhabdus spp. In this study we explored the efficacy of commercial fungicide interactions when combined with either TCA or X. szentirmaii. Fungicides (active ingredient) included Abound® (Azoxystrobin), Serenade® (Bacillus subtilis), Elast® (dodine), Regalia® (extract of Reynoutria sachalinensis), Prophyt® (potassium phosphite) and PropiMax® (propiconazole). In laboratory experiments, singly-applied or combined agents were assessed for fungicidal activity against four plant-pathogenic fungi, Monilinia fructicola, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusarium oxysporum. Fungicidal activity was measured by the phytopathogen's growth on potato dextrose agar with and without fungicide. The interactions between fungicidal agents were determined as antagonistic, additive or synergistic. For suppression of M. fructicola, synergy was observed between TCA when combined with certain concentrations of Elast®, PropiMax®, Regalia®, Prophyte® or Serenade®, and for combinations of X. szentirmaii with Abound®. For suppression of R. solani, synergy was observed between TCA combined with Regalia® or Serenade®. Additionally, when TCA was combined with X. szentirmaii synergistic levels of suppression to M. fructicola were observed. Other combinations of TCA or X. szentirmaii with the fungicides or using alternate concentrations were either additive or occasionally antagonistic in nature. Our results indicate that TCA and X. szentirmaii can each act as strong synergists to enhance fungicidal efficacy. These results may be used to reduce negative environmental impacts of pesticide use while improving control of plant diseases. Additional research is needed to explore the diversity of the synergistic effects and confirm our observations under field conditions.

Response of three cyprinid fish species to the Scavenger Deterrent Factor produced by the mutualistic bacteria associated with entomopathogenic nematodes.

The symbiotic bacteria, Photorhabdus and Xenorhabdus associated with entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema, respectively, produce a compound(s) called the Scavenging Deterrent Factor (SDF). SDF deters a number of terrestrial insect scavengers and predators and one bird species from feeding on host insects killed by the nematode-bacterium complex but has not been tested against aquatic vertebrates. Moreover, the Heterorhabditis-Photorhabdus association is believed to have evolved in an aquatic environment. Accordingly, we hypothesized that SDF will deter fish from feeding on nematode-killed insects and tested the responses of three omnivorous fresh water fish species, Devario aequipinnatus, Alburnoides bipunctatus, and Squalius pursakensis, to SDF in the laboratory. When the fish were exposed to Galleria mellonella larvae killed by the Heterorhabditis- or Steinernema-bacterium complex at 2 or 4days post-infection, all three fish species made several attempts to consume the cadavers but subsequently rejected them. However, all fish species consumed freeze-killed control larvae. In a choice test, when D. aequipinnatus or A. bipunctatus were offered a pair of nematode-killed larvae, both fish species rejected these cadavers; when offered a nematode-killed larva and a freeze-killed larva, both fish species consumed the freeze-killed larva but not the nematode-killed one. In further tests with D. aequipinnatus, there was no significant difference in the number of 2-day-old Bacillus thuringiensis subsp. kurstaki-killed (Btk) larvae consumed compared to freeze-killed larvae, but significantly fewer 4-day-old Btk-killed larvae were consumed compared to freeze-killed larvae. When D. aequipinnatus was fed G. mellonella larvae killed by the symbiotic bacteria, the fish rejected the cadavers. When given freeze-killed or nematode-killed mosquito (Aedes aegypti) larvae, the fish consumed significantly more of the former larvae (99%) compared to the latter (55%). When D. aequipinnatus was placed in a symbiotic cell-free supernatant for 18h, a significant reduction in consumption of freeze-killed larvae compared to cell-free Btk or control broth supernatant was observed. We showed that SDF protects the nematode-killed insects from being consumed by omnivorous fishes and suggests that they will have minimal effects on recycling of EPNs in the aquatic environment.

Multifaceted effects of host plants on entomopathogenic nematodes.

The success of parasites can be impacted by multi-trophic interactions. Tritrophic interactions have been observed in parasite-herbivore-host plant systems. Here we investigate aspects of multi-trophic interactions in a system involving an entomopathogenic nematode (EPN), its insect host, and host plant. Novel issues investigated include the impact of tritrophic interactions on nematode foraging behavior, the ability of EPNs to overcome negative tritrophic effects through genetic selection, and interactions with a fourth trophic level (nematode predators). We tested infectivity of the nematode, Steinernema riobrave, to corn earworm larvae (Helicoverpa zea) in three host plants, tobacco, eggplant and tomato. Tobacco reduced nematode virulence and reproduction relative to tomato and eggplant. However, successive selection (5 passages) overcame the deficiency; selected nematodes no longer exhibited reductions in phenotypic traits. Despite the loss in virulence and reproduction nematodes, first passage S. riobrave was more attracted to frass from insects fed tobacco than insects fed on other host plants. Therefore, we hypothesized the reduced virulence and reproduction in S. riobrave infecting tobacco fed insects would be based on a self-medicating tradeoff, such as deterring predation. We tested this hypothesis by assessing predatory success of the mite Sancassania polyphyllae and the springtail Sinella curviseta on nematodes reared on tobacco-fed larvae versus those fed on greater wax moth, Galleria mellonella, tomato fed larvae, or eggplant fed larvae. No advantage was observed in nematodes derived from tobacco fed larvae. In conclusion, our results indicated that insect-host plant diet has an important effect on nematode foraging, infectivity and reproduction. However, negative host plant effects, might be overcome through directed selection. We propose that host plant species should be considered when designing biocontrol programs using EPNs.

The Effects of Nutrient Concentration, Addition of Thickeners, and Agitation Speed on Liquid Fermentation of Steinernema feltiae.

Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.

Steinernema biddulphi n. sp., a New Entomopathogenic Nematode (Nematoda: Steinernematidae) from South Africa.

A new species of entomopathogenic nematode (EPN), Steinernema biddulphi n. sp., was isolated from a maize field in Senekal, Free State Province of South Africa. Morphological and molecular studies indicated the distinctness of S. biddulphi n. sp. from other Steinernema species. Steinernema biddulphi n. sp. is characterized IJs with average body length of 663 µm (606-778 µm), lateral fields with six ridges in mid-body region forming the formula 2,6,2. Excretory pore located anterior to mid-pharynx (D% = 46). Hyaline layer occupies approximately half of tail length. Male spicules slightly to moderately curved, with a sharp tip and golden brown in color. The first generation of males lacking a mucron on the tail tip while the second generation males with a short filamentous mucron. Genital papillae with 11 pairs and one unpaired preanal papilla. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic data show that S. biddulphi n. sp. belongs to the "bicornutum" clade within the Steinernematidae family.

A novel approach to biocontrol: Release of live insect hosts pre-infected with entomopathogenic nematodes.

As a new application approach, we tested the efficacy of releasing live insect hosts that were pre-infected with entomopathogenic nematodes against insect pests living in cryptic habitats. We hypothesized that the pre-infected hosts could carry the next generation of emerging nematode infective juveniles to hard-to-reach target sites, and thereby facilitate enhanced control in cryptic habitats. Thus, the infected hosts act as "living insect bombs" against the target pest. We tested this approach using two model insect pests: a chestnut tree pest, the goat moth Cossus cossus (Lepidiptera: Cossidae), and a lawn caterpillar, Spodoptera cilium (Lepidoptera: Noctuidae). One pest is considered hard-to-reach via aqueous spray (C. cossus) and the other is more openly exposed in the environment (S. cilium). C. cossus and S. cilium studies were conducted in chestnut logs and Bermudagrass arenas, respectively. The living bomb approach was compared with standard nematode application in aqueous spray and controls (without nematode application); Steinernema carpocapsae (Rize isolate) was used in all experiments. The percentage larval mortality of C. cossus was 86% in the living insect bomb treatment, whereas, all other treatments and controls exhibited less than 4% mortality. The new approach (living bomb) was equally successful as standard aqueous application for the control of S. cilium larvae. Both methods exhibited more than 90% mortality in the turfgrass arena. Our new approach showed an immense potential to control insect pests living in hard-to-reach cryptic habitats.

Viability and Virulence of Entomopathogenic Nematodes Exposed to Ultraviolet Radiation.

Entomopathogenic nematodes (EPNs) can be highly effective biocontrol agents, but their efficacy can be reduced due to exposure to environmental stress such as from ultraviolet (UV) radiation. Our objectives were to 1) compare UV tolerance among a broad array of EPN species, and 2) investigate the relationship between reduced nematode viability (after exposure to UV) and virulence. Nematodes exposed to a UV radiation (254 nm) for 10 or 20 min were assessed separately for viability (survival) and virulence to Galleria mellonella. We compared 9 different EPN species and 15 strains: Heterorhabditis bacteriophora (Baine, fl11, Oswego, and Vs strains), H. floridensis (332), H. georgiana (Kesha), H. indica (HOM1), H. megidis (UK211), Steinernema carpocapsae (All, Cxrd, DD136, and Sal strains), S. feltiae (SN), S. rarum (17C&E), and S. riobrave (355). In viability assessments, steinernematids, particularly strains of S. carpocapsae, generally exhibited superior UV tolerance compared with the heterorhabditids. However, some heterorhabditids tended to be more tolerant than others, e.g., H. megidis and H. bacteriophora (Baine) were most susceptible and H. bacteriophora (Vs) was the only heterorhabditid that did not exhibit a significant effect after 10 min of exposure. All heterorhabditids experienced reduced viability after 20 min exposure though several S. carpocapsae strains did not. In total, after 10 or 20 min exposure, the viability of seven nematode strains did not differ from their non-UV exposed controls. In virulence assays, steinernematids (particularly S. carpocapsae strains) also tended to exhibit higher UV tolerance. However, in contrast to the viability measurements, all nematodes experienced a reduction in virulence relative to their controls. Correlation analysis revealed that viability among nematode strains is not necessarily related to virulence. In conclusion, our results indicate that the impact of UV varies substantially among EPNs, and viability alone is not a sufficient measure for potential impact on biocontrol efficacy as other characters such as virulence may be severely affected even when viability remains high.

Effect of Storage Temperature and Duration on Survival and Infectivity of Steinernema innovationi (Rhabditida: Steinernematidae).

Entomopathogenic nematode species differ in their optimum storage temperature; therefore, we conducted a study on the survival and infectivity of the recently described Steinernema innovationi from South Africa at five storage temperatures (5°C, 10°C, 15°C, 20°C, and 25°C) over 84 d using 20,000 infective juveniles (IJ) in 25 ml aqueous suspension containing 0.1% formalin. Our results showed that survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 d. Infectivity of IJ against Galleria mellonella larvae was >90% for all temperatures except for 5°C at which survival decreased to 10% after 84 d. In addition, we stored 2.5 million IJ on a sponge formulation in 15 ml of 0.1% formalin solution for 84 d at the optimum 15°C followed by 2 wk storage at 25°C. Storage of the IJ on a sponge formulation for 14 d at 25°C post 15°C storage for 84 d did not have a detrimental effect on IJ survival (87%) or infectivity to G. mellonella (95%).