RESUMEN

Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic nematodes associated with symbiotic bacteria (Xenorhabdus stockiae and Photorhabdus luminescens) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against E. coli, S. aureus, B. subtilus, P. mirabilis, E. faecalis, and P. stutzeri. The characterization of these secondary metabolites by Fourier transforms infrared spectroscopy revealed amine groups of proteins, hydroxyl and carboxyl groups of polyphenols, hydroxyl groups of polysaccharides, and carboxyl groups of organic acids. Furthermore, the obtained crude extracts were analyzed by high-performance liquid chromatography for the basic identification of potential bioactive peptides. Gas chromatography-mass spectrometry analysis of ethyl acetate extracts from Xenorhabdus stockiae identified major compounds including nonanoic acid derivatives, proline, paromycin, octodecanal derivatives, trioxa-5-aza-1-silabicyclo, 4-octadecenal, methyl ester, oleic acid, and 1,2-benzenedicarboxylicacid. Additional extraction from Photorhabdus luminescens yielded functional compounds such as indole-3-acetic acid, phthalic acid, 1-tetradecanol, nemorosonol, 1-eicosanol, and unsaturated fatty acids. These findings support the potential development of novel natural antimicrobial agents for future pathogen suppression.

Asunto(s)

Antibacterianos , Cromatografía de Gases y Espectrometría de Masas , Simbiosis , Cromatografía Líquida de Alta Presión/métodos , Antibacterianos/farmacología , Antibacterianos/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolismo Secundario , Photorhabdus/química , Photorhabdus/metabolismo , Xenorhabdus/química , Xenorhabdus/metabolismo , Pruebas de Sensibilidad Microbiana , Animales

RESUMEN

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

Asunto(s)

Mariposas Nocturnas , Nematodos , Xenorhabdus , Animales , Xenorhabdus/genética , Filogenia , Argentina , Nematodos/genética , Mariposas Nocturnas/genética , Análisis de Secuencia , Simbiosis

RESUMEN

Xenorhabdus is a symbiotic group of bacteria associated with entomopathogenic nematodes of the family Steinernematidae. Although the described Steirnernema species list is extensive, not all their symbiotic bacteria have been identified. One single motile, Gram-negative and non-spore-forming rod-shaped symbiotic bacterium, strain VLST, was isolated from the entomopathogenic nematode Steinernema unicornum. Analyses of the 16S rRNA gene determined that the VLST isolate belongs to the genus Xenorhabdus, and its closest related species is Xenorhabdus szentirmaii DSM 16338T (98.2 %). Deeper analyses using the whole genome for phylogenetic reconstruction indicate that VLST exhibits a unique clade in the genus. Genomic comparisons considering digital DNA-DNA hybridization (dDDH) values confirms this result, showing that the VLST values are distant enough from the 70 % threshold suggested for new species, sharing 30.7, 30.5 and 30.3 % dDDH with Xenorhabdus khoisanae MCB, Xenorhabdus koppenhoeferi DSM 18168T and Xenorhabdus miraniensis DSM 18168T, respectively, as the closest species. Detailed physiological, biochemical and chemotaxonomic tests of the VLST isolate reveal consistent differences from previously described Xenorhabdus species. Phylogenetic, physiological, biochemical and chemotaxonomic approaches show that VLST represents a new species of the genus Xenorhabdus, for which the name Xenorhabdus lircayensis sp. nov. (type strain VLST=CCCT 20.04T=DSM 111583T) is proposed.

Asunto(s)

Filogenia , Rabdítidos , Xenorhabdus , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Rabdítidos/microbiología , Análisis de Secuencia de ADN , Xenorhabdus/clasificación , Xenorhabdus/aislamiento & purificación

RESUMEN

BACKGROUND: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches. METHODS: Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction. RESULTS: Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity. CONCLUSIONS: For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.

Asunto(s)

Rabdítidos/genética , Rabdítidos/aislamiento & purificación , Animales , Chile , Femenino , Larva/parasitología , Estadios del Ciclo de Vida , Masculino , Mariposas Nocturnas/parasitología , Control Biológico de Vectores , ARN Ribosómico 16S/genética , Rabdítidos/anatomía & histología , Rabdítidos/clasificación , Infecciones por Rhabditida/parasitología , Simbiosis , Temperatura , Xenorhabdus/genética , Xenorhabdus/fisiología

RESUMEN

The fungus, Sclerotinia sclerotiorum, causes white mold disease and infects a broad spectrum of host plants (> 500), including soybean with yield losses of up to 70%. Biological control is a potential alternative for management of this severe plant pathogen, and relative to chemical fungicides, provides broad benefits to the environment, farmers and consumers. The symbiotic bacteria of entomopathogenic nematodes, Xenorhabdus spp. and Photorhabdus spp., are characterized by the production of antimicrobial compounds, which could serve as potential sources for new bio-fungicides. The objectives of this study were to assess cell-free supernatants (CFS) of 16 strains of these bacteria cultures on S. sclerotiorum mycelium growth; assess the volatiles of X. szentirmaii cultures on the fungus mycelium and sclerotium inhibition; and evaluate the X. szentirmaii cultures as well as their CFS on the protection of soybean seeds against the white mold disease. Among the 16 strains, the CFS of X. szentirmaii showed the highest fungicidal effect on growth of S. sclerotiorum. The CFS of X. szentirmaii inhibited > 98% of fungus growth from mycelium and sclerotia, whereas the volatiles generated by the bacterium culture inhibited to 100% of fungus growth and 100% of sclerotia production. The bacterial culture diluted to 33% in water and coated on soybean seeds inhibited S. sclerotiorum and protected soybean plants, allowing 78.3% of seed germination and 56.6% of plant development. Our findings indicate potential for a safe and novel control method for S. sclerotiorum in soybean. Moreover, this is the first study to indicate that volatile organic compounds from Xenorhabdus spp. can be used in plant disease suppression.

Asunto(s)

Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Glycine max/microbiología , Photorhabdus/fisiología , Xenorhabdus/fisiología , Animales , Germinación/efectos de los fármacos , Micelio/efectos de los fármacos , Nematodos/microbiología , Desarrollo de la Planta/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Semillas/microbiología , Simbiosis/efectos de los fármacos , Compuestos Orgánicos Volátiles/farmacología

RESUMEN

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

Asunto(s)

Aedes/microbiología , Control de Mosquitos/métodos , Photorhabdus , Xenorhabdus , Aedes/virología , Animales , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Fiebre Chikungunya/prevención & control , Fiebre Chikungunya/transmisión , Dengue/prevención & control , Dengue/transmisión , Genes Bacterianos , Insectos/microbiología , Insecticidas , Larva/microbiología , Larva/virología , Mosquitos Vectores/microbiología , Control Biológico de Vectores/métodos , Photorhabdus/genética , Photorhabdus/metabolismo , Photorhabdus/patogenicidad , Enfermedades Transmitidas por Vectores/prevención & control , Enfermedades Transmitidas por Vectores/transmisión , Xenorhabdus/genética , Xenorhabdus/metabolismo , Xenorhabdus/patogenicidad , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/transmisión

RESUMEN

We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa.

Asunto(s)

Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Familia de Multigenes , Spodoptera/enzimología , Xenorhabdus , Animales , Proteínas de Insectos/genética , Monofenol Monooxigenasa/genética , Mutación , Control Biológico de Vectores , Spodoptera/genética , Xenorhabdus/genética , Xenorhabdus/metabolismo

RESUMEN

Leaf-cutter ants (Hymenoptera: Formicidae) have evolved as dominant herbivores on the American continent. These social insects remove the leaves of economically important plant species to maintain their colony's food reserves, the symbiotic fungus Leucocoprinus gongylophorus, a basidiomycete. Such fungus can be used for applications of fungicide molecules from metabolites generated by symbiont bacteria (Xenorhabdus and Photorhabdus) from entomopathogenic nematodes (Steinernema and Heterorhabditis). Through isolation and multiplication in tryptic soy broth (TSB) medium of the bacteria Xenorhabdus szentirmaii isolated PAM 25, we conducted laboratorial tests using treatments with 10, 25, and 50% of the metabolites obtained in the sixth day of cultivation. The treatments were centrifuged and filtered to generate a supernatant, which was diluted in potato + dextrose + agar (PDA), to verify the consequences of exposure to the fungus L. gongylophorus in Petri dishes. To confirm metabolite efficiency, the control treatments in PDA only and mixed (PDA+TSB) media were conducted simultaneously for 14 days. We observed total inhibition of the symbiont fungus in both the 25 and 50% dilutions during the first days of the tests. Our results support that these metabolites have inhibitory effect on the development of symbiont fungus of leaf-cutter ants.(AU)

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As formigas-cortadeiras (Hymenoptera: Formicidae) evoluíram estabelecendo-se no continente americano como herbívoros dominantes. Esses insetos sociais praticam a desfolha de espécies vegetais de interesse econômico, com a finalidade de manter a reserva alimentar da colônia, o fungo simbionte Leucocoprinus gongylophorus, um basidiomiceto. Tal fungo pode ser alvo de aplicações de moléculas fungicidas encontradas em metabólitos gerados por bactérias simbiontes (Xenorhabdus e Photorhabdus) de nematoides entomopatogênicos (Steinernema e Heterorhabditis). Por meio do isolamento e da multiplicação em meio caldo triptona de soja (TSB) da bactéria Xenorhabdus szentirmaii (isolado PAM 25), foram realizados testes laboratoriais com tratamentos contendo 10, 25 e 50% do metabólito obtido no sexto dia de cultivo. Para tanto, o metabólito foi centrifugado e filtrado, gerando um sobrenadante, o qual foi diluído em batata + dextrose + ágar (BDA), para verificar as consequências da exposição do fungo L. gongylophorus em placas de Petri. Os tratamentos de controle apenas com meio BDA e misto (BDA + TSB) também foram conduzidos simultaneamente por 14 dias, a fim de confirmar a eficiência dos metabólitos. Tanto na diluição de 25 e 50% houve total inibição do fungo simbionte já nos primeiros dias da condução dos testes. Diante dos resultados obtidos, pode-se afirmar que os metabólitos são a causa do efeito inibitório do desenvolvimento do fungo simbionte das formigas-cortadeiras.(AU)

Asunto(s)

Control Biológico de Vectores , Xenorhabdus , Photorhabdus/virología , Himenópteros , Nematodos , Hongos/patogenicidad

RESUMEN

Leaf-cutter ants (Hymenoptera: Formicidae) have evolved as dominant herbivores on the American continent. These social insects remove the leaves of economically important plant species to maintain their colony's food reserves, the symbiotic fungus Leucocoprinus gongylophorus, a basidiomycete. Such fungus can be used for applications of fungicide molecules from metabolites generated by symbiont bacteria (Xenorhabdus and Photorhabdus) from entomopathogenic nematodes (Steinernema and Heterorhabditis). Through isolation and multiplication in tryptic soy broth (TSB) medium of the bacteria Xenorhabdus szentirmaii isolated PAM 25, we conducted laboratorial tests using treatments with 10, 25, and 50% of the metabolites obtained in the sixth day of cultivation. The treatments were centrifuged and filtered to generate a supernatant, which was diluted in potato + dextrose + agar (PDA), to verify the consequences of exposure to the fungus L. gongylophorus in Petri dishes. To confirm metabolite efficiency, the control treatments in PDA only and mixed (PDA+TSB) media were conducted simultaneously for 14 days. We observed total inhibition of the symbiont fungus in both the 25 and 50% dilutions during the first days of the tests. Our results support that these metabolites have inhibitory effect on the development of symbiont fungus of leaf-cutter ants.(AU)

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As formigas-cortadeiras (Hymenoptera: Formicidae) evoluíram estabelecendo-se no continente americano como herbívoros dominantes. Esses insetos sociais praticam a desfolha de espécies vegetais de interesse econômico, com a finalidade de manter a reserva alimentar da colônia, o fungo simbionte Leucocoprinus gongylophorus, um basidiomiceto. Tal fungo pode ser alvo de aplicações de moléculas fungicidas encontradas em metabólitos gerados por bactérias simbiontes (Xenorhabdus e Photorhabdus) de nematoides entomopatogênicos (Steinernema e Heterorhabditis). Por meio do isolamento e da multiplicação em meio caldo triptona de soja (TSB) da bactéria Xenorhabdus szentirmaii (isolado PAM 25), foram realizados testes laboratoriais com tratamentos contendo 10, 25 e 50% do metabólito obtido no sexto dia de cultivo. Para tanto, o metabólito foi centrifugado e filtrado, gerando um sobrenadante, o qual foi diluído em batata + dextrose + ágar (BDA), para verificar as consequências da exposição do fungo L. gongylophorus em placas de Petri. Os tratamentos de controle apenas com meio BDA e misto (BDA + TSB) também foram conduzidos simultaneamente por 14 dias, a fim de confirmar a eficiência dos metabólitos. Tanto na diluição de 25 e 50% houve total inibição do fungo simbionte já nos primeiros dias da condução dos testes. Diante dos resultados obtidos, pode-se afirmar que os metabólitos são a causa do efeito inibitório do desenvolvimento do fungo simbionte das formigas-cortadeiras.(AU)

Asunto(s)

Control Biológico de Vectores , Xenorhabdus , Photorhabdus/virología , Himenópteros , Nematodos , Hongos/patogenicidad

RESUMEN

Entomopathogenic nematodes of the families Heterorhabditidae Poinar, 1976 and Steinernematidae Chitwood & Chitwood, 1937 are used for biological control of insect pests. An isolate of Steinernema diaprepesi Nguyen & Duncan, 2002 was recovered from a carrot field in the locality of Santa Rosa de Calchines (Santa Fe Province, Argentina). These nematodes were characterised based on morphological, morphometric and molecular studies. Their symbiotic bacterium was identified as Xenorhabdus doucetiae Tailliez, Pagès, Ginibre & Boemare, 2006 by sequencing the 16S rRNA gene. The isolate of S. diaprepesi studied exhibits some morphometric differences with the original description, especially in the first generation adults. This is the first description of the species in Argentina.

Asunto(s)

Rabdítidos/clasificación , Rabdítidos/microbiología , Xenorhabdus/fisiología , Animales , Argentina , ARN Ribosómico/genética , ARN Ribosómico 16S/genética , Rabdítidos/anatomía & histología , Rabdítidos/genética , Especificidad de la Especie , Xenorhabdus/genética , Xenorhabdus/aislamiento & purificación

RESUMEN

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

Asunto(s)

Aedes/microbiología , Control de Mosquitos , Control Biológico de Vectores , Photorhabdus/fisiología , Xenorhabdus/fisiología , Aedes/crecimiento & desarrollo , Animales , Larva/crecimiento & desarrollo , Larva/microbiología

RESUMEN

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae have a symbiotic association with bacteria which makes them virulent against insects. EPNs have been mass produced using in vivo and in vitro methods, including both solid and liquid fermentation. This study assessed the effect of nematode inoculum age on the production of Steinernema feltiae in liquid, solid and biphasic processes. Several physical parameters were also assessed: the effect of medium viscosity, flask size and aeration speed on the recovery and yield of infective juveniles (IJs). Inoculum age treatments included inoculum liquid cultures that were 7, 14, 21 and 28 days old. Nematodes from the same inoculum were added to one liquid medium (liquid culture), one solid medium with bacteria previously grown in sponge (solid culture) and a variation of the solid medium (a biphasic culture), in which the bacteria were first grown in liquid and, then, soaked into the sponges, with the purpose of providing a more homogeneous bacterial culture before nematode inoculation. Experiments were conducted in Erlenmeyer flasks. Eight treatments were established involving combinations of three variables: two media (with and without 0.2% agar), two flask sizes (250 and 150 ml) and two agitation speeds (180 and 280 rpm). The study showed increases in nematode yield for liquid cultures, but not for solid or biphasic cultures, with the advance of the inoculum age up to 28 days of growth. Furthermore, the addition of 0.2% agar to the liquid medium and increasing the aeration rate by using larger flasks with higher agitation speed may increase nematode recovery and final yield. The experiments were conducted using shake flasks but the results may also be applicable for bioreactors.

Asunto(s)

Insectos/parasitología , Rabdítidos/crecimiento & desarrollo , Animales , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Rabdítidos/metabolismo , Rabdítidos/microbiología , Xenorhabdus/crecimiento & desarrollo , Xenorhabdus/metabolismo , Xenorhabdus/fisiología

RESUMEN

Las bacterias Xenorhabdus y Photorhabdus están asociadas simbióticamente con nematodos de las familias Steinernematidae y Heterorhabditidae respectivamente. Se caracterizan por producir un complejo de sustancias como toxinas, antibióticos y enzimas extracelulares que matan los insectos. Con el objetivo de seleccionar los nematodos más patogénicos y su nivel de producción de antibióticos se propuso evaluar la patogenicidad de 13 aislamientos nativos. Las bacterias se aislaron de juveniles infectivos (JIS) por macerado directo, se cultivaron en medios microbiológicos selectivos (NBTA y MacConkey) y se describieron fenotípica y bioquímicamente. La patogenicidad se evaluó en larvas de último instar de G. mellonella L. (Lepidóptera: Pyralidae), mediante diluciones seriadas de inóculo bacteriano con una concentración de 104cel/ml. Se registró el porcentaje de mortalidad a las 12, 24 y 48 horas y las unidades formadoras de colonia (UFC) en agar NBTA, en los mismos intervalos de tiempo. Los datos se sometieron a un análisis de varianza y a la prueba de comparación de medias de Duncan. Las pruebas bioquímicas y enzimáticas resultaron positivas para el género Xenorhabdus. Los análisis estadísticos mostraron que los aislamientos, UNPX04, UNPX15 provenientes de La Florida-Risaralda y Llano Bajo-Valle del Cauca respectivamente, causaron el 100 % de mortalidad a las 12 y 24 horas en contraste con siete aislamientos que causaron sólo el 70% de mortalidad.

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Xenorhabdus and Photorhabdus bacteria are symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae respectively. They are characterized by producing a complex of substances such as toxins, antibiotics and extracellular enzymes that kill insects. In order to select the most pathogenic nematodes and antibiotic production level, it was propose to evaluate the pathogenicity of 13 native isolates. Bacteria were isolated from infective juveniles (JIS) by direct macerated, cultured on selective media (NBTA and MacConkey) and described phenotypic and biochemically. The pathogenicity was evaluated on the last instar larvae of G. mellonella, using serial dilutions of the bacterial inoculum with a concentration of 104cel/ml. The mortality rate was registered at 12, 24 and 48 hours and the colony forming units (CFU) in NBTA agar in the same intervals of time. The data were analyzed by variance analysis and mean comparison by Duncan test. Biochemical and enzymatic tests were positive for the genus Xenorhabdus. The results showed that the isolates UNPX04, UNPX15 from agricultural soils of Florida -Risaralda and Llano Bajo- Valle del Cauca respectively, caused 100% of mortality at 12 and 24 hours in contrast with seven isolations that caused only 70% of mortality.

Asunto(s)

Virulencia , Xenorhabdus , Antibacterianos , Profilaxis Antibiótica , Factores de Virulencia

RESUMEN

Dengue fever is an important vector-borne disease, mainly transmitted by Aedes aegypti. To date, there are no vaccines or effective drugs available against this arboviral disease. As mosquito control is practically the only method available to control dengue fever, alternative and cost-effective pest control strategies need to be explored. The gram-negative enteric bacteria Xenorhabdus and Photorhabdus are symbiotically associated with nematode parasites, which themselves are highly pathogenic for insect larvae. Here, we evaluate the oral toxicity of these entomopathogenic bacteria in A. aegypti larvae. The susceptibility of larvae (third late or fourth early instars) was assessed by exposing them to suspensions containing Photorhabdus luminescens or Xenorhabdus nematophila, respectively. Two diet treatments were tested with larvae fed on pet food and unfed larvae. After 24 h, larvae began to die when exposed to the bacteria. Exposure to P. luminescens killed 73% of the fed and 83% of the unfed larvae, respectively. In comparison, X. nematophila was less pathogenic, killing 52% of the larvae in the fed and 42% in the unfed treatment. Remarkably, cannibalism was observed in all bioassays after exposing larvae to either of the bacterial species. To our knowledge, this is the first report demonstrating the efficiency of these entomopathogenic bacteria for oral A. aegypti killing. Our results provide a promising basis for using these bacteria as bioinsecticides for mosquito control in the future.

Asunto(s)

Aedes/microbiología , Control de Mosquitos/métodos , Photorhabdus/fisiología , Xenorhabdus/fisiología , Animales , Interacciones Huésped-Patógeno , Larva/microbiología , Control Biológico de Vectores

RESUMEN

The submerged culture of the entomopathogenic nematode Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila, was carried out in orbitally agitated bottles using a culture medium containing whey (in grams per litre: 500 whey, 20 yeast extract, 10 dried egg yolk-food grade, 3 sodium chloride, 37 corn oil-food grade). Maximum total viable nematode concentrations of 198,333ml(-1) were achieved within fermentations of 24 days with 64% of the nematode population within the infective juvenile stage (IJ) (126,666ml(-1)) at the end. The kinetics of the bioprocess was well modelled using the four-parameter Sigmoidal model and the corresponding maximum specific rates of nematode production (0.47 day(-1)), carbohydrates consumption (0.0008g(carbohydrates)g(nematodes)(-1)day(-1)) and nitrogen consumption (4.44g(nitrogen)g(nematodes)(-1)day(-1)) are first proposed. Besides, X. nematophila appears to have the capacity of lactose hydrolysis.

Asunto(s)

Medios de Cultivo/metabolismo , Proteínas de la Leche/metabolismo , Nematodos/crecimiento & desarrollo , Rabdítidos/crecimiento & desarrollo , Xenorhabdus/crecimiento & desarrollo , Animales , Técnicas Bacteriológicas/métodos , Biomasa , Carbohidratos/química , Técnicas de Cocultivo/métodos , Medios de Cultivo/química , Fermentación , Cinética , Lactosa/química , Lactosa/metabolismo , Proteínas de la Leche/química , Nematodos/metabolismo , Nematodos/microbiología , Nitrógeno/química , Rabdítidos/metabolismo , Rabdítidos/microbiología , Simbiosis , Proteína de Suero de Leche , Xenorhabdus/aislamiento & purificación , Xenorhabdus/metabolismo

RESUMEN

The production of infective juvenile stages (IJ) of the entomopathogenic nematode Steinernema carpocapsae in the presence of its symbiotic bacterium Xenorhabdus nematophilus was carried out in orbitally agitated bottles. Four complex culture media (M1-M4) were used, containing from 8% to 28% (by vol.) agave juice (aguamiel) from Mexican maguey-pulquero (Agave spp) as the main carbohydrate source. After 20 days of fermentation, a maximum viable IJ concentration of 249,000 IJ/ml and an initial nematode population multiplication factor of x620 were achieved when medium M4 was used (aguamiel concentration in this medium was 28% by vol.). M4 medium contained (w/v): 0.3% total nitrogen, 3.2% total carbohydrates and 3.0% total fat. According to the results obtained, total carbohydrates concentration appeared to be of great importance in obtaining high IJ concentrations.

Asunto(s)

Agave/química , Metabolismo de los Hidratos de Carbono , Medios de Cultivo/química , Grasas/metabolismo , Nitrógeno/metabolismo , Rabdítidos/crecimiento & desarrollo , Animales , México , Control Biológico de Vectores , Rabdítidos/metabolismo , Rabdítidos/microbiología , Factores de Tiempo , Xenorhabdus

RESUMEN

This article presents the evolution of culture broth rheological properties during monoxenic cultures of Steinernema carpocapsae in cylindrical bottles agitated orbitally. Rheological properties were evaluated in simple-shear flow conditions and were well-modeled by the Ostwald-de Waele model. Rheological properties varied from slightly dilatant, n = 1.2 (-), to moderately pseudoplastic flow behavior, n = 0.6 (-). Nematode concentrations increased from 750 +/- 190 to 130 900 +/- 6900 nematodes/mL, and the apparent viscosity (eta(a)) evolved from 4.5 +/- 0.7 to 46.6 +/- 3.2 mPa.s during the fermentations. Rheological behavior did not appear to be strongly influenced by nematode number and/or its stage of development; however, the release of substances from the decomposition of nematode cadavers appeared to be of great importance. Among the different developmental stages of the nematodes, only juveniles of the first stage (J1) were highly susceptible to the shearing conditions tested (shear stress, tau(r)()(theta), from 0.9 to 3.5 Pa during periods of 80-100 min), resulting in the viability loss of 85% of J1 nematodes.

Asunto(s)

Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Incubadoras , Nematodos/crecimiento & desarrollo , Dinámica Poblacional , Reología/métodos , Xenorhabdus/crecimiento & desarrollo , Animales , Evolución Biológica , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Técnicas de Cocultivo/métodos , Medios de Cultivo/química , Movimiento (Física) , Nematodos/clasificación , Nematodos/metabolismo , Nematodos/microbiología , Control de Calidad , Resistencia al Corte , Especificidad de la Especie , Simbiosis/fisiología , Viscosidad , Xenorhabdus/citología , Xenorhabdus/patogenicidad