Establishment of Arthrobotrys flagrans as biocontrol agent against the root pathogenic nematode Xiphinema index

Plant-parasitic nematodes cause devastating agricultural damage worldwide. Only a few synthetic nematicides can be used and their application is limited in fields. Therefore, there is a need for sustainable and environment-friendly alternatives. Nematode-trapping fungi (NTF) are natural predators of nematodes. They capture and digest them with their hyphae and are starting to being used as bio-control agents. In this study, we applied the NTF Arthrobotrys flagrans (Duddingtonia flagrans) against the wine pathogenic nematode Xiphinema index. A. flagrans reduced the number of X. index juveniles in pot cultures of Ficus carica, an alternative host plant for X. index, significantly. Sodium-alginate pellets with A. flagrans spores were produced for vineyard soil inoculation under laboratory conditions. The NTF A. conoides, A. musiformis and A. superba were enriched from several soil samples, showing their natural presence. Trap formation is an energy-consuming process and depends upon various biotic and abiotic stimuli. Here, we show that bacteria of the genus Delftia, Bacillus, Pseudomonas, Enterobacter and Serratia induced trap formation in NTF like A. conoides and A. oligospora but not in A. flagrans in the absence of nematodes. The application of NTF along with such bacteria could be a combinatorial way of efficient biocontrol in nematode-infested soil.     

A new model system for the study of the animal innate immune response to fungal infections

The association of the model organism Caenorhabditis elegans and the fungus Pleurotus ostreatus gives the possibility to study the molecular and genetic mechanisms of the early stages of the spatial and temporal interactions of animals with fungal pathogens. We identified the stages of the infection process of P. ostreatus on the nematode C. elegans. We found that prior to penetration inside a worm a fungal toxin paralyzed and immobilized, but did not kill C. elegans. This finding opens the possibility for the further study of the effect of paralyzing toxins on host organisms. The membrane permeability of paralyzed worms increased dramatically and leakage products initiated the growth of directional hyphae towards the nematodes. The hyphae penetrated into live C. elegans animals either through natural openings or directly by piercing the cuticle. Upon contact with the nematode cuticle, P. ostreatus attached to it, formed appressoria-like structures and infection pegs, piercing the cuticle and penetrating inside the nematode body. The small zones around the penetration loci are of special interest for the evaluation of initial contacts between two organisms and for the study of the C. elegans local defense response against fungal infection.     

Toward Practical Biological Control of Parasitic Nematodes in Domestic Animals

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Caenorhabditis elegans Is a Model Host for Listeria monocytogenes

Here we report that Caenorhabditis elegans nematodes fed Listeria monocytogenes die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in C. elegans. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of C. elegans, indicating that L. monocytogenes remains extracellular in C. elegans.     

Acid phosphatase activity during the interaction of the nematophagous fungus Duddingtonia flagrans with the nematode Panagrellus sp

The use of Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as a biological control method against free living larvae of gastrointestinal nematodes of livestock animals. This fungus captures and infects the nematode by cuticle penetration, immobilization and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. This report characterizes the acid phosphatase activity during the interaction of D. flagrans with the free-living nematode Panagrellus sp. The optimum pH for the hydrolysis of the acid phosphatase substrate p-nitrophenyl phosphate was 2.2, 2.8 and 5.4 from D. flagrans alone and 2.2 and 5.4 for Panagrellus sp alone, fungus-nematode interaction in liquid medium and fungus-nematode interaction in solid medium. Different acid phosphatase activity bands were detected by SDS-PAGE. Maximum acid phosphatase activity of the fungus or nematode alone and of the fungus-nematode interaction occurred within 70 min of incubation in the presence of the substrate 4-methylumbelliferyl phosphate. The activity of this enzyme was significantly higher for the fungus-nematode interaction when compared to the organisms alone, indicating a synergistic response. Furthermore, structures appeared in the hyphae after 30 min, nematodes were observed adhered after 40 min and many were captured by the typical fungus traps after 70 min of interaction. The participation of acid phosphatase activity and its importance during the interaction of the fungus with the nematode were discussed.     

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.     

Live and dead GFP-tagged bacteria showed indistinguishable fluorescence in Caenorhabditis elegans gut

Caenorhabditis elegans has been used for studying host-pathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.     

Factors responsible for transition of the <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> carnivorous fungus from the saprotrophic to the zootrophic nutrition type

Quantitative investigation of the factors responsible for trap formation in the nematophagous fungus Duddingtonia flagrans F-882 in submerged liquid culture was carried out. The data obtained suggest a complex program for the regulation of zootrophic nutrition in D. flagrans. Optimal concentrations of such carbon and nitrogen sources as sucrose (0.4%), ammonium ions (0.2%), and tryptone (0.2%) promote trap formation in the case of contact with the nematodes Panagrellus redivivus. Increased concentrations of these compounds, however, inhibit trap formation. The sensitivity of the mycelium to nematode excreta depends on the state of the culture and is increased under limitation by certain nutrient components or in the course of prolonged starvation. A direct correlation was found between the number of caught nematodes and the number of chlamydospores formed on the mycelium. The nutrients obtained from the nematode biomass are used for formation of additional chlamydospores (on average, about 20 chlamydospores per nematode). Environmental and evolutionary aspects of the role of zootrophic nutrition in carnivorous fungi are discussed.     

Growth of the Fungus Duddingtonia flagrans in Soil Surrounding Feces Deposited by Cattle or Sheep Fed the Fungus to Control Nematode Parasites

The results reported in this paper represent work from two separate experiments, namely a plot trial using cattle feces conducted at Kungsãngen in Uppsala, Sweden and a plot trial using sheep feces undertaken at Tåstrup in Copenhagen, Denmark. In both trials, a technique was used to monitor the level of Duddingtonia flagrans propagules in soil surrounding feces. The feces were from animals fed or not fed D. flagrans fungal chlamydospores. Also presented are the numbers of soil nematodes in soil surrounding sheep feces. The results indicate that D. flagrans has little growth beyond the fecal environment into surrounding soil when chlamydospores are fed to either sheep or cattle. This is substantiated by the soil nematode data. No statistical differences in the number of nematode taxa identified, Shannon Weiner H′, proportion of various feeding groups, and B/B + F (B and F are the proportions of bacterial and fungal-feeding nematodes) were found when soil surrounding sheep feces containing chlamydospores and parasitic nematode eggs was compared to soil surrounding feces containing parasitic nematode eggs alone. It is unlikely that the application of D. flagrans as a biological control agent against the free-living stages of nematode parasites of these livestock will negatively affect populations of nontarget soil nematodes.     

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37–40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200–300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

The multiple faces of calcineurin signaling in Caenorhabditis elegans: Development, behaviour and aging

Calcineurin, a well-conserved protein phosphatase 2B (PP2B), is a Ca²⁺-calmodulin–dependent serine/threonine protein phosphatase that is known to be involved in a myriad of cellular processes and signal transduction pathways. The biological role of calcineurin has been extensively studied in diverse groups of organisms. Homologues of mammalian and Drosophila calcineurin subunits exist in the nematode, Caenorhabditis elegans. The C. elegans counterpart of the catalytic subunit, calcineurin A, cna-1/tax-6, and the regulatory subunit, calcineurin B, cnb-1, are known to express ubiquitously in multiple tissues including neurons. The characterization of C. elegans calcineurin mutants facilitates identification of its physiological functions and signaling pathways. Genetic interactions between cna-1/tax-6 and cnb-1 mutants with a number of mutants involved in several signaling pathways have exemplified the pivotal role of calcineurin in regulating nematode development, behaviour and lifespan (aging). The present review has been aimed to provide a succinct summary of the multiple functions of calcineurin in C. elegans relating to its development, fertility, proliferation, behaviour and lifespan. Analyses of cna-1/tax-6 and cnb-1 interacting proteins and regulators of the phosphatase in this fascinating worm model have an immense scope to identify potential drug targets in various parasitic nematodes, which cause many diseases inflicting huge economic loss; and also for many human diseases, particularly neurodegenerative and myocardial diseases.