The nematode-trapping fungus Arthrobotrys oligospora detects prey pheromones via G protein-coupled receptors.

The ability to sense prey-derived cues is essential for predatory lifestyles. Under low-nutrient conditions, Arthrobotrys oligospora and other nematode-trapping fungi develop dedicated structures for nematode capture when exposed to nematode-derived cues, including a conserved family of pheromones, the ascarosides. A. oligospora senses ascarosides via conserved MAPK and cAMP-PKA pathways; however, the upstream receptors remain unknown. Here, using genomic, transcriptomic and functional analyses, we identified two families of G protein-coupled receptors (GPCRs) involved in sensing distinct nematode-derived cues. GPCRs homologous to yeast glucose receptors are required for ascaroside sensing, whereas Pth11-like GPCRs contribute to ascaroside-independent nematode sensing. Both GPCR classes activate conserved cAMP-PKA signalling to trigger trap development. This work demonstrates that predatory fungi use multiple GPCRs to sense several distinct nematode-derived cues for prey recognition and to enable a switch to a predatory lifestyle. Identification of these receptors reveals the molecular mechanisms of cross-kingdom communication via conserved pheromones also sensed by plants and animals.

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Discovery of an antitumor compound from xenorhabdus stockiae HN_xs01.

Xenorhabdus, known for its symbiotic relationship with Entomopathogenic nematodes (EPNs), belongs to the Enterobacteriaceae family. This dual-host symbiotic nematode exhibits pathogenic traits, rendering it a promising biocontrol agent against insects. Our prior investigations revealed that Xenorhabdus stockiae HN_xs01, isolated in our laboratory, demonstrates exceptional potential in halting bacterial growth and displaying anti-tumor activity. Subsequently, we separated and purified the supernatant of the HN_xs01 strain and obtained a new compound with significant inhibitory activity on tumor cells, which we named XNAE. Through LC-MS analysis, the mass-to-nucleus ratio of XNAE was determined to be 254.24. Our findings indicated that XNAE exerts a time- and dose-dependent inhibition on B16 and HeLa cells. After 24 h, its IC50 for B16 and HeLa cells was 30.178 µg/mL and 33.015 µg/mL, respectively. Electron microscopy revealed conspicuous damage to subcellular structures, notably mitochondria and the cytoskeleton, resulting in a notable reduction in cell numbers among treated tumor cells. Interestingly, while XNAE exerted a more pronounced inhibitory effect on B16 cells compared to HeLa cells, it showed no discernible impact on HUVEC cells. Treatment of B16 cells with XNAE induced early apoptosis and led to cell cycle arrest in the G2 phase, as evidenced by flow cytometry analysis. The impressive capability of X. stockiae HN_xs01 in synthesizing bioactive secondary metabolites promises to significantly expand the reservoir of natural products. Further exploration to identify the bioactivity of these compounds holds the potential to shed light on their roles in bacteria-host interaction. Overall, these outcomes underscore the promising potential of XNAE as a bioactive compound for tumor treatment.

Mercury concentration in larvae of Eustrongylides sp (Nematoda: Dioctophymatoidea) from fish of the Brazilian Amazon / Concentración de mercurio en larvas de Eustrongylides sp (Nematoda: Dioctophymatoidea) en peces de la Amazonía brasileña

Abstract Introduction: Chemical pollution represents a great concern to aquatic organisms, especially fish. Metals enter the aquatic environment from a variety of sources, including natural biogeochemical cycles and anthropogenic sources such as industrial and residential effluents, mining and atmospheric sources. Objective: To describe the Eustrongylides sp. larvae and the interaction with their fish hosts as indicators of mercury (Hg) contamination in the Brazilian Amazon, and the distribution of Hg in the internal organs of fish species Hoplias malabaricus and Pygocentrus nattereri collected in oxbow lakes on the Tapajós River, in the municipality of Santarém, in the state of Pará. Methods: Total Hg was analyzed using the Direct Hg Analyzer - DMA-80. Concentrations of Hg in Eustrongylides sp. were compared with those found in the tissues/organs of the hosts H. malabaricus and P. nattereri. Hg concentrations in the host/parasite system were statistically compared using Principal Component Analysis. The bioconcentration factor (BCF) was calculated to assess the bioaccumulation capacity of metals in Eustrongylides sp. larvae, comparing the concentration of Hg in the parasite with that accumulated in the musculature of infected hosts. Results: Hg concentrations in all tissues/organs analyzed were higher in the parasitic species Eustrongylides sp. larvae when compared with those found in tissues/organs of H. malabaricus and P. nattereri. There was an inversely proportional relationship, showing that when Eustrongylides sp. larvae are present, the concentration in the parasite is higher than in the musculature of host fish H. malabaricus and P. nattereri. The BCF of Hg was found by comparing Eustrongylides sp. larvae/H. malabaricus muscle and was observed during a flood (BCF Hg = 15 364). Conclusions: The results confirm the greater bioaccumulative capacity of Eustrongylides sp. compared to its host. The data indicated the viability of using Eustrongylides sp. larvae in biomonitoring programs. It is worth mentioning that fish samples for Hg analysis must be free of parasites since their presence can alter the results.

Resumen Introducción: La contaminación química del hábitat acuático representa un gran peligro para organismos acuáticos, especialmente para peces. Los metales ingresan al ambiente acuático desde una variedad de fuentes, incluidos los ciclos biogeoquímicos naturales y fuentes antropogénicas, como efluentes industriales y residenciales, minería y fuentes atmosféricas. Objetivo: Describir las especies de Eustrongylides sp. y la interacción con sus peces hospederos como indicadores de contaminación por mercurio en la Amazonía brasileña, y la distribución en los órganos internos de las especies de peces Hoplias malabaricus y Pygocentrus nattereri recolectadas en cochas del Río Tapajós, en el municipio de Santarém, del estado de Pará. Métodos: El Hg total se analizó utilizando el Direct Hg Analyzer - DMA-80. Las concentraciones de Eustrongylides sp. se compararon con las encontrados en los tejidos/órganos de los hospederos H. malabaricus y P. nattereri. Las concentraciones en el sistema hospedero/parásito se compararon estadísticamente utilizando el análisis de componentes principales. Se calculó el factor de bioconcentración (BCF) para evaluar la capacidad de bioacumulación de metales en larvas de Eustrongylides sp., comparando la concentración en el parásito con la acumulada en la musculatura de los hospederos infectados. Resultados: Las concentraciones de Hg en todos los tejidos/órganos analizados fueron mayores en las larvas de la especie parasitaria Eustrongylides sp. en comparación con las encontradas en los tejidos/órganos de H. malabaricus y P. nattereri. Hubo una relación inversamente proporcional, mostrando que cuando las larvas de Eustrongylides sp. están presentes, la concentración en el parásito es mayor que en la musculatura de los peces hospederos H. malabaricus y P. nattereri. El BCF de Hg se encontró comparando Eustrongylides sp. larvas/ músculo H. malabaricus y se observó durante una inundación (BCF Hg = 15 364). Conclusiones: Los resultados confirman la mayor capacidad bioacumulativa de Eustrongylides sp. en comparación con su hospedero. Los datos indicaron la viabilidad de utilizar larvas de Eustrongylides sp. en programas de biomonitoreo. Cabe mencionar que las muestras de pescado para análisis de Hg deben estar libres de parásitos ya que su presencia puede alterar los resultados.

Photorhabdus aballayi sp. nov. and Photorhabdus luminescens subsp. venezuelensis subsp. nov., isolated from Heterorhabditis amazonensis entomopathogenic nematodes.

Six Gram-negative, rod-shaped bacterial strains isolated from Heterorhabditis amazonensis entomopathogenic nematodes were characterized to determine their taxonomic position. 16S rRNA and gyrB gene sequences indicate that they belong to the class Gammaproteobacteria, family Morganellaceae and genus Photorhabdus, and that some of them are conspecifics. Two of them, APURET and JART, were selected for further molecular characterization using whole genome- and whole-proteome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions using whole genome and whole proteome sequences show that strains APURET and JART are closely related to Photorhabdus luminescens subsp. luminescens ATCC 29999T and to P. luminescens subsp. mexicana MEX47-22T. Moreover, digital DNA-DNA hybridization (dDDH) values between APURET and P. luminescens subsp. luminescens ATCC 29999T, APURET and P. luminescens subsp. mexicana MEX47-22T, and APURET and JART are 61.6, 61.2 and 64.1 %, respectively. These values are below the 70 % divergence threshold that delimits prokaryotic species. dDDH scores between JART and P. luminescens subsp. luminescens ATCC 29999T and between JART and P. luminescens subsp. mexicana MEX47-22T are 71.9 and 74.8 %, respectively. These values are within the 70 and 79 % divergence thresholds that delimit prokaryotic subspecies. Based on these genomic divergence values, APURET and JART represent two different taxa, for which we propose the names: Photorhabdus aballayi sp. nov. with APURET (=CCM 9236T =CCOS 2019T) as type strain and Photorhabdus luminescens subsp. venezuelensis subsp. nov. with JART (=CCM 9235T =CCOS 2021T) as type strain. Our study contributes to a better understanding of the biodiversity of an important bacterial group with enormous biotechnological and agricultural potential.

The cAMP-PKA signalling pathway regulates hyphal growth, conidiation, trap morphogenesis, stress tolerance, and autophagy in Arthrobotrys oligospora.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signalling pathway is evolutionarily conserved in eukaryotes and plays a crucial role in defending against external environmental challenges, which can modulate the cellular response to external stimuli. Arthrobotrys oligospora is a typical nematode-trapping fungus that specializes in adhesive networks to kill nematodes. To elucidate the biological roles of the cAMP-PKA signalling pathway, we characterized the orthologous adenylate cyclase AoAcy, a regulatory subunit (AoPkaR), and two catalytic subunits (AoPkaC1 and AoPkaC2) of PKA in A. oligospora by gene disruption, transcriptome, and metabolome analyses. Deletion of Aoacy significantly reduced the levels of cAMP and arthrobotrisins. Results revealed that Aoacy, AopkaR, and AopkaC1 were involved in hyphal growth, trap morphogenesis, sporulation, stress resistance, and autophagy. In addition, Aoacy and AopkaC1 were involved in the regulation of mitochondrial morphology, thereby affecting energy metabolism, whereas AopkaC2 affected sporulation, nuclei, and autophagy. Multi-omics results showed that the cAMP-PKA signalling pathway regulated multiple metabolic and cellular processes. Collectively, these data highlight the indispensable role of cAMP-PKA signalling pathway in the growth, development, and pathogenicity of A. oligospora, and provide insights into the regulatory mechanisms of signalling pathways in sporulation, trap formation, and lifestyle transition.

A new strain of Volutella citrinella with nematode predation and nematicidal activity, isolated from the cysts of potato cyst nematodes in China.

BACKGROUND: Plant parasitic nematodes (PPNs) are responsible for causing many plant diseases and are extremely difficult to control at present. Currently, due to the negative effects of chemical agents on the environment and human health, the development of new biological pesticides has become an important part of plant nematode control. Nematophagous fungi refers to a class of fungi that kill plant nematodes. Notably, a large number of nematophagous fungi resources remain to be studied. The objective of our study was to use in vitro screening to identify nematophagous fungi and select strains that were highly active against nematodes, providing a primary research for the development and utilization of new nematophagous fungi. RESULTS: A new nematophagous fungal strain (GUCC2219) was isolated from cysts of possibly Globodera spp. and Heterodera spp., identified as Volutella citrinella. The hyphae of V. citrinella produced ring structures of variable size and exhibited predatory and nematicidal activity. The hyphal predation rates (in vitro) against three species of nematodes, Aphelenchoides besseyi, Bursaphelenchus xylophilus, and Ditylenchus destructor, averaged 59.45, 33.35, and 50.95%, respectively, while the fermentation broth produced by the fungus exhibited mortality rates of 100, 100, and 55.63%, respectively, after 72 h. CONCLUSION: V. citrinella is a new strain with nematophagous properties, which are a novel discovery. At the same time, this is the first report of nematicidal and nematode predation activity in the genus Volutella.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria, BA1T, Q614T and PB68.1T, isolated from the digestive system of Heterorhabditis entomopathogenic nematodes, were biochemically and molecularly characterized to clarify their taxonomic affiliations. The 16S rRNA gene sequences of these strains suggest that they belong to the Gammaproteobacteria, to the family Morganellacea, and to the genus Photorhabdus. Deeper analyses using whole genome-based phylogenetic reconstructions suggest that BA1T is closely related to Photorhabdus akhursti, that Q614T is closely related to Photorhabdus heterorhabditis, and that PB68.1T is closely related to Photorhabdus australis. In silico genomic comparisons confirm these observations: BA1T and P. akhursti 15138T share 68.8 % digital DNA-DNA hybridization (dDDH), Q614T and P. heterorhabditis SF41T share 75.4 % dDDH, and PB68.1T and P. australis DSM 17609T share 76.6  % dDDH. Physiological and biochemical characterizations reveal that these three strains also differ from all validly described Photorhabdus species and from their more closely related taxa, contrary to what was previously suggested. We therefore propose to classify BA1T as a new species within the genus Photorhabdus, Q614T as a new subspecies within P. heterorhabditis, and PB68.1T as a new subspecies within P. australis. Hence, the following names are proposed for these strains: Photorhabdus aegyptia sp. nov. with the type strain BA1T(=DSM 111180T=CCOS 1943T=LMG 31957T), Photorhabdus heterorhabditis subsp. aluminescens subsp. nov. with the type strain Q614T (=DSM 111144T=CCOS 1944T=LMG 31959T) and Photorhabdus australis subsp. thailandensis subsp. nov. with the type strain PB68.1T (=DSM 111145T=CCOS 1942T). These propositions automatically create Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov. with SF41T as the type strain (currently classified as P. heterorhabditis) and Photorhabdus australis subsp. australis subsp. nov. with DSM17609T as the type strain (currently classified as P. australis).

Effect of culture conditions on conidia production and enhancement of environmental stress resistance of Esteya vermicola in solid-state fermentation.

AIMS: Esteya vermicola is an endoparasitic fungus producing lunate conidia, which kill pine wood nematode (PWN), and PWN could cause pine wilt disease (PWD). The aims of this study were to increase production and confirm the resistance (temperature and UV irradiation) of lunate conidia, and further determine the effective concentrations of conidia infecting PWN. METHODS AND RESULTS: In this study, rice was used as a carrier to absorb conidial suspension to propagate conidia. The optimal conditions for lunate conidia production were 25°C temperature, 9 days of culture time, 2 : 1 rice/distilled water ratio and 10% inoculum size. The germination rate of E. vermicola cultured on potato dextrose agar was influenced by UV irradiation, similar to growth on rice. Esteya vermicola cultured on rice under heat stress might be more suitable for application in the field. The concentration (1 × 108 conidia per ml) to kill PWN had the highest infectivity among the four conidia concentrations tested after 3 days of inoculation. CONCLUSIONS: This study showed a rice substrate-supported high-quality conidia production and the optimal infectivity concentration of E. vermicola. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide the necessary process of an economical and efficient biological control strategy against PWD.

Peniterpenoids A-C, new sesquiterpenoid metabolites from a wheat cyst nematode Penicillium janthinellum.

Three new sesquiterpenoids, peniterpenoids A - C (1-3), together with six known metabolites (4-9) were isolated from an entomogenous fungus Penicillium janthinellum (LB1.20090001) collected from a wheat cyst nematode. The structures of the new compounds were elucidated based on NMR and HRESIMS spectroscopic analyses. The absolute configuration of the C-8 secondary alcohol of peniterpenoid B (2) was determined by [Rh2(OCOCF3)4]-induced ECD experiment. Subsequently, the antimicrobial and DPPH scavenging activities were determined. Compounds 6-8 exhibited moderate antibacterial activities against Staphylococcus aureus (CGMCC1.2465) with MIC values of 25.0, 50.0 and 12.5 µg/mL, respectively.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Nematode endosymbiont competition: Fortune favors the fittest.

Endosymbiotic bacteria that obligately associate with entomopathogenic nematodes as a complex are a unique model system to study competition. These nematodes seek an insect host and provide entry for their endosymbionts. Through their natural products, the endosymbionts nurture their nematodes by eliminating secondary infection, providing nutrients through bioconversion of the insect cadaver, and facilitating reproduction. On one hand, they cooperatively colonize the insect host and neutralize other opportunistic biotic threats. On the other hand, inside the insect cadaver as a fighting pit, they fiercely compete for the fittest partnership that will grant them the reproductive dominance. Here, we review the protective and nurturing nature of endosymbiotic bacteria for their nematodes and how their selective preference shapes the superior nematode-endosymbiont pairs as we know today.

ngrA-dependent natural products are required for interspecies competition and virulence in the insect pathogenic bacterium Xenorhabdus szentirmaii.

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Bacterial community assemblages in the rhizosphere soil, root endosphere and cyst of soybean cyst nematode-suppressive soil challenged with nematodes.

In disease-suppressive soil, plants rely upon mutualistic associations between roots and specific microbes for nutrient acquisition and disease suppression. Notably, the transmission of suppressiveness by the cysts of sugar beet cyst nematode from suppressive to conducive soils has been previously observed in greenhouse trials. However, our current understanding of the bacterial assemblages in the cyst, root endosphere and rhizosphere soil is still limited. To obtain insights into these bacterial microbiota assemblages, the bacterial communities inhabiting the plant-associated microhabitats and cysts in soybean cyst nematode (SCN)-suppressive soil were characterized by deep sequencing, using soybean grown under growth room conditions with additional SCN challenge. Clustering analysis revealed that the cyst bacterial community was closer to the root endosphere community than to the rhizosphere and bulk soil communities. Interestingly, the cyst bacterial community was initially established by the consecutive selection of bacterial taxa from the soybean root endosphere. We found a set of potential microbial consortia, such as Pasteuria, Pseudomonas, Rhizobium, and other taxa, that were consistently enriched in the rhizocompartments under SCN challenge, and more abundant in the cysts than in the bulk soil. Our results suggest that the soybean root-associated and cyst microbiota may cause the suppressiveness of SCN in suppressive soil.

Characterization of the pixB gene in Xenorhabdus nematophila and discovery of a new gene family.

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Grace's medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Grace's medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Grace's medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.

In vitro efficacy of Duddingtonia flagrans against nematodes of sheep based on in vivo calculations / Eficácia in vitro de Duddingtonia flagrans contra nematoides de ovinos com base em cálculo in vivo

Abstract Duddingtonia flagrans has been tested as an alternative parasite control, but data from in vitro experiments based on in vivo calculations describing nematophagous fungi predation in nematodes are restricted. The objective of this work was to determine the efficacy of D. flagrans against sheep nematode larvae in vitro using in vivo calculations. Fecal samples were introduced to fungi in different concentrations: 0.0/control; 0.05; 0.1; 0.2; 0.4; 0.8; 1.6; 3.2; and 6.4 g corresponding, respectively, to 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 and 74.624.000 chlamydospores/kg of body weight. The material was incubated for 14 days, before the larvae recovery (Assay 1). Assay 2 was carried out with the doses of 0.00625; 0.0125; and 0.025 g. The results showed a negative correlation between fungal concentrations and larval numbers for both assays. The fungus demonstrated an efficacy above 89% in both assays. Thus, we consider that the data from in vitro studies based on in vivo calculations may optimize the fungi quantities for field experiments.

Resumo Duddingtonia flagrans tem sido testado como uma alternativa no controle de parasitos, entretanto, trabalhos in vitro da predação de nematoides por fungos nematófagos correlacionados com cálculos baseados para testes in vivo são restritos. O objetivo deste trabalho foi determinar a eficácia in vitro de D. flagrans contra larvas de nematoides de ovinos tendo como base cálculos in vivo. Amostras fecais receberam a adição do fungo em diferentes concentrações: 0.0/controle; 0,05; 0,1; 0,2; 0,4; 0,8; 1,6; 3,2 e 6,4 gramas correspondendo, respectivamente, às seguintes dosagens: 583.000; 1.166.000; 2.332.000; 4.664.000; 9.328.000; 18.656.000; 37.312.000 e 74.624.000 clamidósporos/Kg de peso vivo animal. O material foi incubado por 14 dias, para recuperação das larvas (Ensaio 1). O Ensaio 2 foi realizado com concentrações de 0,00625; 0,0125 e 0,025 g. Foi observada correlação negativa entre a concentração fúngica e o número de larvas, nos dois ensaios. O fungo demonstrou eficácia acima de 89% em ambos os ensaios. A partir destes dados, acreditamos que ensaios in vitro baseados em cálculos in vivo podem aprimorar as dosagens para a realização de experimentos a campo.

Identification and characterization of a novel ß-glucosidase via metagenomic analysis of Bursaphelenchus xylophilus and its microbial flora.

ß-glucosidases catalyze the final step of cellulose hydrolysis and are essential in cellulose degradation. A ß-glucosidase gene, cen502, was identified and isolated from a metagenomic library from Bursaphelenchus xylophilus via functional screening. Analyses indicated that cen502 encodes a 465 amino acid polypeptide that contains a catalytic domain belonging to the glycoside hydrolase family 1 (GH1). Cen502 was heterologously expressed, purified, and biochemically characterized. Recombinant Cen502 displayed optimum enzymatic activity at pH 8.0 and 38 °C. The enzyme had highest specific activity to p-nitrophenyl-ß-D-glucopyranoside (pNPG; 180.3 U/mg) and had K m and V max values of 2.334 mol/ml and 9.017 µmol/min/mg, respectively. The addition of Fe2+ and Mn2+ significantly increased Cen502 ß-glucosidase activity by 60% and 50%, respectively, while 10% and 25% loss of ß-glucosidase activity was induced by addition of Pb2+ and K+, respectively. Cen502 exhibited activity against a broad array of substrates, including cellobiose, lactose, salicin, lichenan, laminarin, and sophorose. However, Cen502 displayed a preference for the hydrolysis of ß-1,4 glycosidic bonds rather than ß-1,3, ß-1,6, or ß-1,2 bonds. Our results indicate that Cen502 is a novel ß-glucosidase derived from bacteria associated with B. xylophilus and may represent a promising target to enhance the efficiency of cellulose bio-degradation in industrial applications.

Horizontal Gene Transfer from Bacteria Has Enabled the Plant-Parasitic Nematode Globodera pallida to Feed on Host-Derived Sucrose.

The evolution of plant-parasitic nematodes (PPN) is unusual in that these organisms have acquired a range of genes from bacteria via horizontal gene transfer (HGT). The proteins encoded by most of these genes are involved in metabolism of various components of the plant cell wall during invasion of the host. Recent genome sequencing projects for PPN have shown that Glycosyl Hydrolase Family 32 (GH32) sequences are present in several PPN species. These sequences are absent from almost all other animals. Here, we show that the GH32 sequences from an economically important cyst nematode species, Globodera pallida are functional invertases, are expressed during feeding and are restricted in expression to the nematode digestive system. These data are consistent with a role in metabolizing host-derived sucrose. In addition, a detailed phylogenetic analysis shows that the GH32 sequences from PPN and those present in some insect species have distinct bacterial origins and do not therefore derive from a gene present in the last common ancestor of ecdysozoan species. HGT has therefore played at least two critical roles in the evolution of PPN, enabling both invasion of the host and feeding on the main translocation carbohydrate of the plant.

Microbial Communities in Globodera pallida Females Raised in Potato Monoculture Soil.

Globodera spp. are under strict quarantine in many countries. Suppressiveness to cyst nematodes can evolve under monoculture of susceptible hosts. Females developing in potato monoculture soil infested with G. pallida populations Chavornay or Delmsen were examined for inherent microbial communities. In the greenhouse, nonheated and heat-treated (134°C for 10 min) portions of this soil were placed in root observation chambers, planted with Solanum tuberosum 'Selma', and inoculated with G. pallida Pa3 Chavornay. At harvest in Delmsen soil, cysts had fewer eggs in nonheated than heat-treated soil. In denaturing gradient gel electrophoresis analysis, bacterial and fungal fingerprints were characterized by a high variability between replicates; nonheated soils displayed more dominant bands than heated soils, indicating more bacterial and fungal populations. In amplicon pyrosequencing, females from nonheated portions frequently contained internal transcribed spacer sequences of the fungus Malassezia. Specific for the Chavornay and Delmsen population, ribosomal sequences of the bacteria Burkolderia and Ralstonia were abundant on eggs. In this first report of microbial communities in G. pallida raised in potato monoculture, candidate microorganisms perhaps associated with the health status of the eggs of G. pallida were identified. If pathologies on cyst nematodes can be ascertained, these organisms could improve the sustainability of production systems.