The effect of Xenorhabdus bacteria metabolites on Colorado potato beetle (Leptinotarsa decemlineata) adult feeding and larval survival.

Colorado Potato Beetle (CPB) is one of the most destructive potato pests that can quickly develop resistance to insecticides. Therefore, new safe and effective control strategies that are less susceptible to the development of resistance by CPB are urgently needed. Due to their complex mode of action, the likelihood of resistance development by target pests is generally low with antifeedants. In the present study, we assessed the effect of secondary metabolites of various Xenorhabdus bacteria species and strains on CPB adult feeding and on larval development. The metabolites were applied in the form of cell free supernatants (CFSs) from Xenorhabdus cultures. In bioassay 1, leaves treated with ten Xenorhabdus cultures were fed to CPB adults, and their feeding was assessed daily for one week. In bioassay 2, CPB egg masses were placed on the leaves treated with five bacterial cultures, and larval development to pupae was monitored. Out of the ten Xenorhabdus cultures tested, two strains exhibited a significant reduction in the feeding behavior of Colorado Potato Beetle adults, with reductions of up to 70% compared to the control. The effect of CFSs on larval development was variable, and when treated with X. khoisanae SGI 197, over 90% of larvae died in the first few days before reaching the 2nd instar, and complete mortality was achieved on the 8th day of the experiment. Our study is the first study to demonstrate the antifeedant effect of Xenorhabdus cultures towards herbivorous beetles, and the metabolites of these bacteria may have potential for CPB control. Clearly, the metabolites produced by X. khoisanae SGI-197 may be a promising tool for CPB larvae control with the potential to significantly decrease damage to potato plants.

The use of Phasmarhabditis nematodes and metabolites of Xenorhabdus bacteria in slug control.

Many species of slugs are considered serious pests in agriculture and horticulture around the world. In Europe, slugs of the genera Arion and Deroceras are the most harmful pests in agriculture. Therefore, the main goal of this study was to evaluate the effect of the whole-cell metabolites of 10 strains of five Xenorhabdus and three slug-parasitic nematodes (Phasmarhabditis hermaphrodita, Phasmarhabditis bohemica, and Phasmarhabditis apuliae) on the feeding behaviour and repellent effect on target slugs and evaluate a new possible means of biocontrol of these pests. The repellent and anti-feedant effects of nematode-killed insects, metabolites, slug-parasitic nematodes and a combination of metabolites and nematodes were studied through experimental designs: sand-filled plastic boxes divided into two parts in several modifications: with dead Galleria mellonella killed by nematodes, lettuce treated with bacterial metabolites and lettuce placed on the treated sand. We found that slugs avoid eating G. mellonella killed by nematodes, while they eat freeze-killed G. mellonella. Similarly, they avoid the consumption of lettuce in areas treated with bacterial metabolites (the most effective strains being Xenorhabus bovienii NFUST, Xenorhabdus kozodoii SLOV and JEGOR) with zero feeding in the treated side. All three Phasmarhabditis species also provided a significant anti-feedant/repellent effect. Our study is the first to show the repellent and anti-feedant effects of metabolites of Xenorhabdus bacteria against Arion vulgaris, and the results suggest that these substances have great potential for biocontrol. Our study is also the first to demonstrate the repellent effect of P. apuliae and P. bohemica. KEY POINTS: • Slugs avoid eating G. mellonella killed by entomopathogenic nematodes. • Bacterial metabolites have a strong repellent and antifeedant effect on slugs. • Presence of slug parasitic nematodes increases the repellent effect of metabolites.

Efficacy of the Applied Natural Enemies on the Survival of Colorado Potato Beetle Adults.

Colorado potato beetle Leptinotarsa decemlineata is among the most destructive pests of potatoes quickly developing resistance to traditional insecticides. In the present study, we tested the effect of various species and strains of entomopathogenic nematodes on CPB adults, and subsequently, the most effective nematodes were applied alone and in combination with entomopathogenic fungus B. bassiana in pots with potato plants and in the field and their effect on the number of emerging adults was evaluated. In the experimental infections, both the nematode invasion and pathogenicity were variable, and, in several strains, the mortality reached 100%. In pot experiments, soil application of nematodes S. carpocapsae 1343 and S. feltiae Jakub and fungus significantly decreased numbers of emerging CPB adults, while, after the application on leaves, only fungal treatment was effective. The field application of fungus B. bassiana significantly decreased the number of emerging CPB adults in comparison to control sites by ca. 30% while the effect of nematodes and the nematodes-fungus combination was not significant. In conclusion, we demonstrate the necessity of thorough bioassays to select the most effective nematode strains. Entomopathogenic nematodes have the potential to effectively decrease the emergence of CPB adults, but further research is needed to improve the effectiveness in the field.

Dissemination of Isaria fumosorosea Spores by Steinernema feltiae and Heterorhabditis bacteriophora.

Entomopathogenic nematodes and fungi are globally distributed soil organisms that are frequently used as bioagents in biological control and integrated pest management. Many studies have demonstrated that the combination of biocontrol agents can increase their efficacy against target hosts. In our study, we focused on another potential benefit of the synergy of two species of nematodes, Steinernema feltiae and Heterorhabditis bacteriophora, and the fungus Isaria fumosorosea. According to our hypothesis, these nematodes may be able to disseminate this fungus into the environment. To test this hypothesis, we studied fungal dispersal by the nematodes in different arenas, including potato dextrose agar (PDA) plates, sand heaps, sand barriers, and glass tubes filled with soil. The results of our study showed, for the first time, that the spreading of both conidia and blastospores of I. fumosorosea is significantly enhanced by the presence of entomopathogenic nematodes, but the efficacy of dissemination is negatively influenced by the heterogeneity of the testing arena. We also found that H. bacteriophora spread fungi more effectively than S. feltiae. This phenomenon could be explained by the differences in the presence and persistence of second-stage cuticles or by different foraging behavior. Finally, we observed that blastospores are disseminated more effectively than conidia, which might be due to the different adherence of these spores (conidia are hydrophobic, while blastospores are hydrophilic). The obtained results showed that entomopathogenic nematodes (EPNs) can enhance the efficiency of fungal dispersal.

Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand.

Biological control (biocontrol) as a component of pest management strategies reduces reliance on synthetic chemicals, and seemingly offers a natural approach that minimizes environmental impact. However, introducing a new organism to new environments as a classical biocontrol agent can have broad and unanticipated biodiversity effects and conservation consequences. Nematodes are currently used in a variety of commercial biocontrol applications, including the use of Phasmarhabditis hermaphrodita as an agent targeting pest slug and snail species. This species was originally discovered in Germany, and is generally thought to have European origins. P. hermaphrodita is sold under the trade name Nemaslug®, and is available only in European markets. However, this nematode species was discovered in New Zealand and the western United States, though its specific origins remained unclear. In this study, we analyzed 45 nematode strains representing eight different Phasmarhabditis species, collected from nine countries around the world. A segment of nematode mitochondrial DNA (mtDNA) was sequenced and subjected to phylogenetic analyses. Our mtDNA phylogenies were overall consistent with previous analyses based on nuclear ribosomal RNA (rRNA) loci. The recently discovered P. hermaphrodita strains in New Zealand and the United States had mtDNA haplotypes nearly identical to that of Nemaslug®, and these were placed together in an intraspecific monophyletic clade with high support in maximum likelihood and Bayesian analyses. We also examined bacteria that co-cultured with the nematode strains isolated in Oregon, USA, by analyzing 16S rRNA sequences. Eight different bacterial genera were found to associate with these nematodes, though Moraxella osloensis, the bacteria species used in the Nemaslug® formulation, was not detected. This study provided evidence that nematodes deriving from the Nemaslug® biocontrol product have invaded countries where its use is prohibited by regulatory agencies and not commercially available.

Occurrence of Rhabditid Nematodes in the Pet Giant African Land Snails (Achatina fulica).

Gastropods comprise nearly 60,000 species of aquatic as well as terrestrial mollusks, primarily snails and slugs. The giant African land snail (Achatina fulica) is one of the most popular pet snails worldwide. This gastropod mollusk is known as the intermediate host of several parasites that cause severe diseases in pets, free-ranging vertebrates, and humans. The aim of this survey was to investigate the occurrence of parasites in giant African land snails kept as pets in southern Italy. Fresh fecal samples were collected from a total of 60 giant African land snails kept in three private collections in Campania region (Italy). In addition, microscopic analysis of mucus and histological examination of biopsy samples from the foot muscle of 30 individual snails were performed. Coprological examination revealed the presence of rhabditid nematodes identified by both morphological and molecular assessment as Rhabditella axei in two out of three examined samples, and Rhabditis terricola, Cruznema sp., and Pristionchus entomophagus in one coprological sample. No parasites were detected in the muscle biopsy samples, or in the mucus. Due to the potential harm of rhabditid nematodes, their presence in giant African land snails potentially in contact with both pets and humans should not be disregarded.

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.

New record of Steinernema arenarium (Artyukhovsky) (Rhabditida: Steinernematidae) from Ukraine and a note on its distribution.

During a survey of the biodiversity of entomopathogenic nematodes in Ukraine, a population of Steinernema arenarium, strain Ch, was recovered in the sensitive Chornobyl Exclusion Zone. In the present work, this strain was morphologically and molecularly characterised using light microscopy and the sequences of the ITS and D2-D3 region of the 28S rDNA. In addition, we sequenced the ITS and D2-D3 regions of four populations of S. arenarium from a laboratory collection. Phylogenetic analyses were performed and the phylogenetic structure and geographic distribution of S. arenarium are discussed.

Alloionema californicum n. sp. (Nematoda: Alloionematidae): a new alloionematid from USA.

A new species of the family Alloionematidae was isolated from a rotten winged gourd at White Crane Garden, San Francisco, USA, sampled by Christopher Nelson in November 2010, and a live culture is deposited in Félix Lab Strain Database (http://www.justbio.com/worms/index.php), IBENS, Paris, France. Specimens from the culture have been examined. Both morphologically and molecularly, the nematode described herein as Alloionema californicum n. sp. differs from the other alloionematid species, A. appendiculatum and Neoalloionema tricaudatum. It is characterised by having a narrow stoma, 2.5-3.5 or 4 times longer than broad in adults or dauer juveniles respectively. Lateral fields are not present in adults but occur as one prominent ridge in dauers. Males have no bursa, six pairs of genital papillae and one single papilla. Dauers have large apparent phasmids in the middle of the tail. The ecology of the newly described species is unknown but probably it is a saprobic bacteriophagous nematode preferring rotting organic material.

Phasmarhabditis bonaquaense n. sp. (Nematoda: Rhabditidae), a new slug-parasitic nematode from the Czech Republic.

Phasmarhabditis bonaquaense n. sp. is described and illustrated from the body of Malacolimax tenellus, from the locality of Ceské Svýcary near the village of Dobrá Voda, the Czech Republic. Females are characterized by a body length of 2349 (1878-2626) µm and a cupola shaped tail with a long hyaline hair-like tail tip. Extremely prominent papilla-like phasmids present. Males 1829 (1414-2121) µm long. Peloderan bursa with nine pairs of rays (papillae), 1/1/1/2/1/3. One non-paired apparent papilla-like structure located near the ventral appendage anterior to the cloaca. Prominent papilla-like phasmids located close to the tail tip. Small subunit (18S), ITS, and D2-D3 expansion segments of the large subunit of ribosomal DNA were used to analyze the phylogenetic relationships of sequenced species in the genus Phasmarhabditis and other closely related species. Phasmarhabditis bonaquaense n. sp. varied from other related nematodes both in morphological characterizations and phylogenetic analysis. The life cycle of the newly described species is not well known but it is probably a facultative, mollusc-parasitic nematode able to survive permanently in the saprobic phase on decaying organic matter.

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

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

Steinernema goweni n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Zulia State, Venezuela.

A nematode from the genus Steinernema was isolated in Zulia state (North-western, Venezuela). Morphological, morphometric and molecular studies indicated that this nematode belongs to the "bicornutum" group and is described herein as Steinernema goweni n. sp. Steinernema goweni n. sp. is characterised by morphometrics of its infective juveniles, with body length 640 (531-719) µm, pharynx length of 119 (109-126) µm, tail of 67 (59-89) µm, c ratio = 9 (6-11) and E% = 77 (48-94). First generation male specimens can be recognised by the shape and size of spicules (55 (50-57) µm long) and gubernaculum (35 (30-40) µm long), and %D value 42 (28-59), which is at the lower limit within the "bicornutum" group. The number of genital papillae in males is also distinctive for S. goweni n. sp. presenting up to 27 (13 pairs + 1) papillae in 15% of specimens. The phasmids can be seen in scanning electron microscopy in all stages, characters not previously reported within the "bicornutum" group. Phylogenetic analyses of the "bicornutum" group based on both ITS and D2D3 regions showed a clear separation of S. goweni n. sp. from the other species. In both analyses S. goweni n. sp. formed a strongly supported group of American species.

Steinernema poinari sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from the Czech Republic.

A new steinernematid nematode, herein described as Steinernema poinari sp. n., was recovered by baiting soil samples with the last instar wax moth larvae Galleria mellonella in three localities of southwest Bohemia, Czech Republic. Morphological and molecular data confirmed this nematode as a new species which belongs to the affine/intermedium group. S. poinari sp. n. was characterized by male, female and infective juvenile morphological observations. Male spicules are robust with a pointed tip, SW% = 109 (98-123) and GS% = 70 (58-87). The tail terminus of the first generation have a papillated mucron, whereas in the second generation a short filamentous mucron sometimes develops. Females of the first generation have a moderately protruding vulva, postanal swelling absent or slightly developed. Second generation females have a long filamentous mucron on the tail tip. Infective juvenile have a body length of 768 µm, lateral field formula 2, 5, 6, 5, 2, 1, ratio D% of 46 (40-55), E% of 84 (76-95) and H% of 50 (43-56). The new species was characterized by sequences of ITS and D2D3 regions of ribosomal DNA. S. poinari sp. n. can also be separated from S. affine and S. intermedium by cross-breeding tests.