Electrochemical Oxo-Fluorosulfonylation of Alkynes under Air: Facile Access to ß-Keto Sulfonyl Fluorides.

Radical fluorosulfonylation is emerging as an appealing approach for the synthesis of sulfonyl fluorides, which have widespread applications in many fields, in particular in the context of chemical biology and drug development. Here, we report the first investigation of FSO2 radical generation under electrochemical conditions, and the establishment of a new and facile approach for the synthesis of ß-keto sulfonyl fluorides via oxo-fluorosulfonylation of alkynes with sulfuryl chlorofluoride as the radical precursor and air as the oxidant. This electrochemical protocol is amenable to access two different products (ß-keto sulfonyl fluorides or α-chloro-ß-keto sulfonyl fluorides) with the same reactants. The ß-keto sulfonyl fluoride products can be utilized as useful building blocks in the synthesis of various derivatives and heterocycles, including the first synthesis of an oxathiazole dioxide compound. Furthermore, some ß-keto sulfonyl fluorides and derivatives exhibited notably potent activities against Bursaphelenchus xylophilus and Colletotrichum gloeosporioides.

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.

Adaptation of pine wood nematode Bursaphelenchus xylophilus to ß-pinene stress.

BACKGROUND: The pine wood nematode (PWN; Bursaphelenchus xylophilus) is the most damaging biological pest in pine forest ecosystems in China. However, the pathogenic mechanism remains unclear. Tracheid cavitation induced by excess metabolism of volatile terpenes is a typical characteristic of pine trees infected by B. xylophilus. ß-pinene, one of the main volatile terpenes, influences PWN colonization and reproduction, stimulating pathogenicity during the early stages of infection. To elucidate the response mechanism of PWN to ß-pinene, pathogenesis, mortality, and reproduction rate were investigated under different concentrations of ß-pinene using a transcriptomics approach. RESULTS: A low concentration of ß-pinene (BL, C < 25.74 mg/ml) inhibited PWN reproduction, whereas a high concentration (BH, C > 128.7 mg/ml) promoted reproduction. Comparison of PWN expression profiles under low (BL, 21.66 mg/ml) and high (BH, 214.5 mg/ml) ß-pinene concentrations at 48 h identified 659 and 418 differentially expressed genes (DEGs), respectively, compared with controls. Some key DEGs are potential regulators of ß-pinene via detoxification metabolism (cytochrome P450, UDP-glucuronosyltransferases and short-chain dehydrogenases), ion channel/transporter activity (unc and ATP-binding cassette families), and nuclear receptor -related genes. Gene Ontology enrichment analysis of DEGs revealed metabolic processes as the most significant biological processes, and catalytic activity as the most significant molecular function for both BL and BH samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) analysis showed that xenobiotics biodegradation and metabolism, carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and transport and catabolism were the dominant terms in metabolism categories. CONCLUSION: In addition to detoxification via reduction/oxidation (redox) activity, PWN responds to ß-pinene through amino acid metabolism, carbohydrate metabolism, and other pathways including growth regulation and epidermal protein changes to overcome ß-pinene stress. This study lays a foundation for further exploring the pathogenic mechanism of PWN.

Pharmacokinetic-pharmacodynamic assessment of the ivermectin and abamectin nematodicidal interaction in cattle.

In a context of nematodicidal resistance, anthelmintic combinations have emerged as a reliable pharmacological strategy to control gastrointestinal nematodes in grazing systems of livestock production. The current work evaluated the potential drug-drug interactions following the coadministration of two macrocyclic lactones (ML) ivermectin (IVM) and abamectin (ABM) to parasitized cattle using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The kinetic behavior of both compounds administered either separately or coadministered was assessed and the therapeutic response of the combination was evaluated under different resistance scenarios. In the pharmacological trial, calves received a single subcutaneous (s.c.) injection of IVM (100 µg/Kg); a single s.c. injection of ABM (100 µg/Kg) or IVM + ABM (50 µg/Kg each) administered in different injection sites to reach a final ML dose of 100 µg/Kg (Farm 1). Plasma samples were taken from those animals up to 20 days post-treatment. IVM and ABM plasma concentrations were quantified by HPLC. A parasitological trial was carried out in three farms with different status of nematodes resistance to IVM. Experimental animals received IVM (200 µg/Kg), ABM (200 µg/Kg) or IVM + ABM (100 µg/Kg each) in Farm 2, and IVM + ABM (200 µg/Kg each) in Farms 3 and 4. The anthelmintic efficacy was determined by fecal egg count reduction test (FECRT). PK analysis showed similar trends for IVM kinetic behavior after coadministration with ABM. Conversely, the ABM elimination half-life was prolonged and the systemic exposure during the elimination phase was increased in the presence of IVM. Although IVM alone failed to control Cooperia spp., the combination IVM + ABM was the only treatment that achieved an efficacy higher than 95% against resistant Cooperia spp. in all farms. In fact, when Cooperia spp. was the main genus within the nematode population and Haemonchus spp. was susceptible or slightly resistant to ML (Farms 2 and 4), the total FECR for the combination IVM + ABM was higher than 90%. Instead, when the predominant nematode genus was a highly resistant Haemonchus spp. (Farm 3), the total FECR after the combined treatment was as low as the single treatments. Therefore, the rational use of these pharmacological tools should be mainly based on the knowledge of the epidemiology and the nematode susceptibility status in each cattle farm.

Morphological adjustment in free-living Steinernema feltiae infective juveniles to increasing concentration of Nemafric-BL phytonematicide.

Third-stage larvae (L3) of Steinernema feltiae exist as free-living infective juveniles (IJ), with suspended development activities. In contrast, parasitic stages (L1, L2, L4, adult) have mutualistic relations with Xenorhabdus species bacteria, along with unique morphological changes and development inside the cadaver of host insects and/or plant-parasitic nematodes. Commercial IJ strains are tolerant to cucurbitacin-containing phytonematicides, but we have scant information on how morphological adjustments in IJ are achieved. In this study, we investigated the nature of morphological adjustments in commercial S. feltiae IJ strains to Nemafric-BL phytonematicide, which contains cucurbitacin B as active ingredient. Post-72 h exposure to phytonematicide concentration, IJ specimens were fixed on mounting slides. Length (body, excretory pore to anterior end, pharynx, rectum, stoma, tail), diameter (head width, neck base, mid-body, anal body), cuticle thickness and De Man ratios were measured with a computer software programme attached to Omax light microscope. Morphometric data against increasing phytonematicide concentration exhibited either density-dependent quadratic, linear or neutral relations. Increase in body length at low phytonematicide concentration was accompanied by decrease in tail length and pharynx length during muscle contraction when IJ were still alive. After death at high phytonematicide concentration, the opposite morphometric effects ensued due to muscle relaxation. The observed changes in morphometric structures were explained on the basis of morphological adjustments that modulated volumes of pseudocoelom cavity in IJ. The modulation is intended to maintain hydrostatic pressure within permissible upper limits in order to avoid structural damage to internal organs embedded in the pseudocoelom fluids.

Pine chemical volatiles promote dauer recovery of a pine parasitic nematode, Bursaphelenchus xylophilus.

Pinewood nematode, Bursaphelenchus xylophilus, a pine parasitic nematode, poses a serious threat to its host pine forests globally. When dispersal-stage larvae 4 (dauer, DL4) of B. xylophilus enters the new pine, it moults into propagative adult (dauer recovery) and reproduces quickly to kill the host pine. Here, we found pine chemical volatiles, rather than the common dauer recovery factors of nematodes (e.g. suitable temperatures, nutrient availability or density), promote B. xylophilus dauer recovery. The results showed that volatilization of chemicals in host pines could attract DL4 and promote DL4 recovery. To identify which chemicals promote this process, we determined the stimulated activity of the main volatiles of pines including six monoterpenes and two sesquiterpenes. Results showed that all the six monoterpenes promoted dauer recovery, especially ß-pinene and ß-myrcene, but the two sesquiterpenes have no effect on the transformation. Furthermore, ß-pinene performed gradient effects on dauer recovery. We hypothesized that when DL4 infect pine trees, the pine volatiles released from the feeding wounds are used as chemical signals for DL4 transformation to adult to reproduce and rapidly kill the pines. Our study identified the B. xylophilus dauer recovery chemical signal and may contribute to preventing pine wilt disease.

Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus / Papel das proteases do látex de Synadenium grantii na atividade nematicida sobre Meloidogyne incognita e Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p <0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Effects of α-pinene on the pinewood nematode (Bursaphelenchus xylophilus) and its symbiotic bacteria.

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is an important plant-parasitic nematode that can cause severe mortality of pine trees. This PWN-induced harm to plants may be closely related to the abundance and diversity of the symbiotic microorganisms of the parasitic nematode. In this study, nematodes were divided into untreated and antibiotic-treated groups. Nematodes were treated by fumigation with different amounts of α-pinene, and the resultant mortality rates were analyzed statistically. Concentrations of symbiotic bacteria were calculated as colony-forming units per nematode. High-throughput sequencing was used to investigate the bacterial community structure. The results showed that the mortality of nematodes increased slightly with an increasing concentration of α-pinene, and nematodes untreated with antibiotics were more sensitive to α-pinene than those treated with antibiotics. The highest abundance of symbiotic bacteria was obtained via medium and low levels of α-pinene, but for which community diversity was the lowest (Shannon and Simpson indexes). The proportion of Pseudomonas spp. in the symbiotic bacteria of nematodes without antibiotics was relatively high (more than 70%), while that of Stenotrophomonas spp. was low (6%-20%). However, the proportion of Stenotrophomonas spp. was larger than that of Pseudomonas spp in the symbiotic bacteria associated with the antibiotic-treated nematodes. Pseudomonas sp. increased after pinene treatment, whereas Stenotrophomonas spp. decreased. These results indicate that although α-pinene has low toxicity to PWNs over a short time period, α-pinene ultimately influences the abundance and community diversity of the symbiotic bacteria of these nematodes; this influence may potentially disturb the development and reproduction of nematodes in the process of infecting pine trees.

Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus.

Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ±â€¯18.38 U g-1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.

Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus.

Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Toxicity of phenolic compounds to entomopathogenic nematodes: A case study with Heterorhabditis bacteriophora exposed to lentisk (Pistacia lentiscus) extracts and their chemical components.

Insects show adaptive plasticity by ingesting plant secondary compounds, such as phenolic compounds, that are noxious to parasites. This work examined whether exposure to phenolic compounds affects the development of insect parasitic nematodes. As a model system for parasitic life cycle, we used Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade) grown with Photorhabdita luminescens supplemented with different concentrations of plant phenolic extracts (0, 600, 1200, 2400 ppm): a crude ethanol extract of lentisk (Pistacia lentiscus) or lentisk extract fractionated along a scale of hydrophobicity with hexane, chloroform and ethyl acetate; and flavonoids (myricetin, catechin), flavanol-glycoside (rutin) or phenolic acids (chlorogenic and gallic acids). Resilience of the nematode to phenolic compounds was stage-dependent, with younger growth stages exhibiting less resilience than older growth stages (i.e., eggs < young juveniles < young hermaphrodites < infective juveniles < mature hermaphrodites). At high concentrations, all of the phenolic compounds studied were lethal to eggs and young juveniles. The nematodes were able to survive in the presence of medium and low concentrations of all studied compounds, but very few of those treatments allowed for reproduction beyond the infective juvenile stage and, at low concentrations, the crude 70% ethanol extract, chloroform and hexane extracts, and myricetin were associated with some impaired reproduction. The ethyl-acetate fraction and gallic acid were extremely lethal to the young stages and allowed almost no development beyond the infective juvenile stage. We conclude that exposure of infective juveniles to phenolics before they infect insects and post-infection exposure of other nematode developmental stages may affect the initiation of the infection, suggesting that the chemistry of dietary phenolics may limit H. bacteriophora's infection of insects.

A species-specific nematocide that results in terminal embryogenesis.

Nematode-insect interactions are ubiquitous, complex and constantly changing as the host and nematode coevolve. The entomophilic nematode Pristionchus pacificus is found on a myriad beetle species worldwide, although the molecular dynamics of this relationship are largely unknown. To better understand how host cues affect P. pacificus embryogenesis, we characterized the threshold of sensitivity to the pheromone (Z)-7-tetradecen-2-one (ZTDO) by determining the minimum exposure duration and developmental window that results in P. pacificus embryonic lethality. We found early-stage embryos exposed to volatile ZTDO for as few as 4 h all display terminal embryogenesis, characterized by punctuated development up to 48 h later, with abnormal morphology and limited cavity formation. To determine if the pheromone arrests pre-hatching development by suffocating or permeabilizing the eggshells, we raised embryos under anoxic conditions and also examined eggshell permeability using the lipophilic dye FM4-64. We found that asphyxiating the embryos arrested embryogenesis in a reversible manner but did not phenocopy the effects of ZTDO exposure, whereas the ZTDO-induced disruption of embryogenesis did correlate with increased eggshell permeability. The effects of ZTDO are also highly specific, as other lipid insect compounds do not produce any detectable embryocidal effect. The high specificity and unusual teratogenic effect of ZTDO may be important in mediating the host-nematode relationship by regulating P. pacificus development.

In vitro larvicidal effect of a hydroalcoholic extract from Acacia cochliacantha leaf against ruminant parasitic nematodes.

The aim of this study was to evaluate the in vitro lethal effect of a hydroalcoholic extract (HAE) from Acacia cochliacantha leaf against three gastrointestinal nematodes species (Haemonchus contortus, H. placei and Cooperia punctata) of domestic ruminants. The HAE was assessed using five concentrations: 100, 125, 175, 150 and 200 mg/ml; 0.5% Ivermectin was used as a positive control and distilled water, as negative control. The data were normalized using the square root and analysed with a completely randomized design through ANOVA analysis using the general lineal model (GLM) of the SAS program. The HAE tannin content was determined through spectrophotometry (UV-visible) and the other major phenols, were identified by chromatographic processes. The results showed an in vitro larvicidal activity of the HAE against the three assessed nematode species with all assessed concentrations. A clear HAE increased concentration dependence effect was observed. The highest activity of the HAE was obtained at the highest concentration (close to 100%, P < 0.05). This result was similar to the one obtained with Ivermectin. On the other hand, the chemical analysis of HAE showed the presence of tannins, caffeoyls and coumaroyl derivates and quercetin as the main compounds. The results suggest that the HAE from this plant species possess in vitro anthelmintic properties. The identified compounds in this study would good candidates for further in vivo researches.

[A new sesquiterpene from Chinese agarwood induced by artificial holing].

In order to study the chemical constituents of n-butanol fraction of ethanol extract from Chinese agarwood induced by artificial holing, various chromatographic techniques were carried out to isolate compounds, and the structures of compounds were determined through a combined analysis of physicochemical properties and spectroscopic evidence. Seven compounds were obtained and identified as selina-3,11-dien-9,15-diol (1), aquilarone D (2), 5α,6ß,7α,8ß-tetrahydroxy-2-[2-(2-hydroxyphenyl)ethyl]-5,6,7,8-tetrahydrochromone (3), 6,7-dimethoxy-2-[2-(4-methoxyphenyl)ethyl]chromone (4), syringin (5), methyl (Z)-p-coumarate (6), and 4'-methoxycinnamic acid (7), among which compound 1 was a new compound and compounds 5-7 were isolated from agarwood for the first time. The bioactivity assay results concluded that compounds 6 and 7 showed certain nematicidal activity against Panagrellus redivivus, and compounds 4, 6 and 7 exhibited cytotoxicity against BEL-7402, SGC-7901 and A549 carcinoma cell lines.

Evaluation of Anthelmintic Activity and Composition of Pumpkin (Cucurbita pepo L.) Seed Extracts-In Vitro and in Vivo Studies.

UNLABELLED: A significant number of studies report growing resistance in nematodes thriving in both humans and livestock. This study was conducted to evaluate the in vitro and in vivo anthelmintic efficiency of Curcubita pepo (C. pepo) L. hot water extract (HWE), cold water extract (CWE) or ethanol extract (ETE) on two model nematodes: Caenorhabditis elegans (C. elegans) and Heligmosoides bakeri (H. bakeri). METHODS: Raman, IR and LC-MS spectroscopy analyses were performed on the studied plant material to deliver qualitative and quantitative data on the composition of the obtained extracts: ETE, HWE and CWE. The in vitro activity evaluation showed an impact of C. pepo extracts on C. elegans and different developmental stages of H. bakeri. The following in vivo experiments on mice infected with H. bakeri confirmed inhibitory properties of the most active pumpkin extract selected by the in vitro study. All of the extracts were found to contain cucurbitine, aminoacids, fatty acids, and-for the first time-berberine and palmatine were identified. All C. pepo seed extracts exhibited a nematidicidal potential in vitro, affecting the survival of L1 and L2 H. bakeri larvae. The ETE was the strongest and demonstrated a positive effect on H. bakeri eggs hatching and marked inhibitory properties against worm motility, compared to a PBS control. No significant effects of pumpkin seed extracts on C. elegans integrity or motility were found. The EtOH extract in the in vivo studies showed anthelmintic properties against both H. bakeri fecal egg counts and adult worm burdens. The highest egg counts reduction was observed for the 8 g/kg dose (IC50 against H. bakeri = 2.43; 95% Cl = 2.01-2.94). A decrease in faecal egg counts (FEC) was accompanied by a significant reduction in worm burden of the treated mice compared to the control group. CONCLUSIONS: Pumpkin seed extracts may be used to control of Gastrointestinal (G.I.) nematode infections. This relatively inexpensive alternative to the currently available chemotherapeutic should be considered as a novel drug candidate in the nearest future.

Stimulation of the Salicylic Acid Pathway Aboveground Recruits Entomopathogenic Nematodes Belowground.

Plant defense pathways play a critical role in mediating tritrophic interactions between plants, herbivores, and natural enemies. While the impact of plant defense pathway stimulation on natural enemies has been extensively explored aboveground, belowground ramifications of plant defense pathway stimulation are equally important in regulating subterranean pests and still require more attention. Here we investigate the effect of aboveground stimulation of the salicylic acid pathway through foliar application of the elicitor methyl salicylate on belowground recruitment of the entomopathogenic nematode, Steinernema diaprepesi. Also, we implicate a specific root-derived volatile that attracts S. diaprepesi belowground following aboveground plant stimulation by an elicitor. In four-choice olfactometer assays, citrus plants treated with foliar applications of methyl salicylate recruited S. diaprepesi in the absence of weevil feeding as compared with negative controls. Additionally, analysis of root volatile profiles of citrus plants receiving foliar application of methyl salicylate revealed production of d-limonene, which was absent in negative controls. The entomopathogenic nematode S. diaprepesi was recruited to d-limonene in two-choice olfactometer trials. These results reinforce the critical role of plant defense pathways in mediating tritrophic interactions, suggest a broad role for plant defense pathway signaling belowground, and hint at sophisticated plant responses to pest complexes.

Attraction Behaviors of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae) to Synthetic Volatiles Emitted by Insect Damaged Potato Tubers.

Entomopathogenic nematodes (EPNs) play a role in indirect defense of plants under attack by root herbivores. Several investigations have shown that EPNs are attracted or repelled by various volatile compounds (VOCs) released from insect damaged plant roots. We hypothesized that the directional responses of EPNs to the VOCs would be affected by foraging strategy and would vary among species, VOC type, and VOC concentrations. We tested the chemotactic responses of four commercial EPN species (Steinernema feltiae, S. carpocapsae, S. kraussei, and Heterorhabditis bacteriophora) to seven compounds released from insect (Melolontha hippocastani)-damaged (decanal, nonanal, octanal, undecane, 6-methyl-5-hepten-2-one, and 1,2,4-trimethylbenzene) and undamaged (2-ethyl-1-hexanol) potato tubers. Our results suggest that EPNs are able to distinguish herbivore-induced VOCs from those that are typical for healthy potato tubers. In our investigation, nonanal, octanal, and decanal had a greater influence on the movement of EPNs than other tested synthetic volatiles. Decanal was an attractant for H. bacteriophora and S. kraussei at both tested concentrations (as a pure compound and at a concentration of 0.03 ppm). The results suggest that the susceptibility to perception of chemical stimuli from the environment is a species-specific characteristic that prevails over the influence of the foraging strategy.

Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4.

A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp.

Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitized animals can self-medicate. As ingested plant phenolics, mainly tannins, reduce strongyle nematode infections in mammalian herbivores. We investigated the effect of plant extracts known to be anthelmintic in vertebrate herbivores on the recovery of the parasitic entomopathogenic nematode Heterorhabditis bacteriophora infecting African cotton leafworm (Spodoptera littoralis). Nematode infective juveniles (IJs) were exposed to 0, 300, 900, 1200, 2400 ppm of Pistacia lentiscus L. (lentisk), Inula viscosa L. (strong-smelling inula), Quercus calliprinos Decne. (common oak) and Ceratonia siliqua L. (carob) extracts on growth medium (in vitro assay). In control treatments, 50-80% of IJs resumed development to J4, young and developed adult hermaphrodites, whereas all extracts, except for C. siliqua at 300 ppm, impaired IJ exsheathment and development. The highest concentration of I. viscosa extract (2400 ppm) had the strongest effect, killing 95% of exposed nematodes. Surviving nematodes did not recover, remaining at the IJ stage. Over the whole cycle, I. viscosa extract inhibited recovery to 25% or less, and did not allow full development to adulthood, whereas 65% of IJs in the control treatment recovered and resumed development, 12% reaching complete maturation within 72 h of incubation. When herbivorous S. littoralis larvae were fed with different plant extracts in vivo, I. viscosa had the strongest effect at concentrations above 300 ppm, with 90% of insect-invading IJs not developing to parasitic stages, whereas in the control treatment, 85% of IJs resumed development. Exposure to C. siliqua extract also inhibited exsheathment and development of 75% of the IJs. Half of those that resumed development reached full maturation. P. lentiscus and Q. calliprinos extracts also inhibited development of 50% IJs. Our results suggest that H. bacteriophora can be used to study herbal medication against parasites in animals.