Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Steinernema sichuanense n. sp. (Rhabditida, Steinernematidae), a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China

Steinernema sichuanense n. sp. is characterized by male, female and IJ. For male, the spicules are robust with prominent rostrum; gubernaculum has blunt anterior end; cuneus is arrow-shaped, pointed posteriorly. Second-generation male has a prominent mucron. For female, tail usually has one to four papillae-like projections on tail tip; post anal swelling is absent. For IJ, body length is about 710 microm; lateral field has six ridges; the formula of lateral field is 2, 5, 6, 4, 2 with two prominent submarginal ridges; tail usually has a dorsal depression. In Steinernema affine/intermedium group, the IJ of S. sichuanense n. sp. differs from S. affine by its absence of the internal tail spine; differs from Steinernema beddingi by its six ridges in lateral field compared to 4 for S. beddingi. For male mucron is absent in both generations of S. affine, S. intermedium and S. beddingi, whereas it is present in the second-generation of S. sichuanense sp. n. Morphology and morphometrics of spicules and gubernacula of the four species in S. affine/intermedium group are quite different based on SEM photographs. For female, the postanal swelling is absent in the first-generation of S. sichuanense n. sp. whereas S. affine and S. intermedium have slight swelling and S. beddingi has conspicuous swelling. The new species is further recognized by characterization of sequences of ITS and D2/D3 regions of the ribosomal DNA. The symbiotic bacterium associated to S. sichuanense belongs to the species Xenorhabdus bovienii.     

Steinernema weiseri n. sp. (Rhabditida,Steinernematidae), a new entomopathogenic nematode from Europe

Steinernema weiseri n. sp. is described from a roadside with apple trees near Ceské Budejovice, Czech Republic. The species is also widely distributed in Germany and Slovakia, from where it had previously been reported as Steinernema spec. F. The British Steinernema sp. D1 is considered conspecific with S. weiseri n. sp. Males of the new species are mainly characterised by light brown, slightly curved spicules with a long manubrium and the presence of a short tail mucron in the second generation. Third-stage infective juveniles are characterised by a `medium size' body and tail length, short hyaline tail portion (mostly around 1/3 of tail length), the excretory pore situated in the mid-pharynx region, lip region slightly offset, angular and flattened, and the lateral field having nine equally developed lines separated by eight distinct ridges. S. weiseri n. sp. is most similar to S. feltiae, with which it did not hybridise. RFLP analysis of the ITS region of the rDNA repeat shows S. weiseri n. sp. to be distinct from 50 other Steinernema species and isolates. The new species was found in a wide range of habitats and is readily maintained on Galleria mellonella larvae.     

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy)

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866 ± 61 (767-969) μm, distance from head to excretory pore of 63 ± 2.7 (59-68) μm, tail length of 81 ± 3.2 (76-89) μm, ratio E (%) 77 ± 3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66 ± 1.4 (64-67) μm and gubernaculum length of 44 ± 1.4 (43-46) μm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53 ± 4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.     

Steinernema cholashanense n. sp. (Rhabditida, Steinernematidae) a new species of entomopathogenic nematode from the province of Sichuan, Chola Shan Mountains, China

During a random survey of entomopathogenic nematodes in the provinces of Sichuan and Gansu (eastern Tibet) in 2004, soil samples from several sites were collected and tested for the incidence of entomopathogenic nematodes. A new species was collected in this survey and it is described herein as Steinernema cholashanense n. sp. Steinernema cholashanense n. sp. is characterized by morphology and morphometry of the IJ and male. For the IJ, the new species can be recognized by the average body length 843 microm, esophagus length 125 microm, H%=39% and E%=81%. The lateral field pattern is 2, 5, 7, 4, 2. The male of the first generation is characterized by spicule shape and length and especially with prominent velum and the presence of a mucron on both generations. The average body length of the IJ of S. cholashanense n. sp. (843 microm) is shorter than that of S. oregonense (980 microm), S. kraussei (951 microm) and S. litorale (909 microm), similar to that of S. feltiae (849 microm), but longer than that of S. weiseri (740 microm), S. jollietti (711 microm) and S. hebeiense (658 microm). Esophagus length of the new species (125 microm) is closer to that of S. jollieti (123 microm) but longer than that of S. weiseri (113 microm) and shorter than that of S. oregonense (132 microm), S. kraussei (134 microm) and S. feltiae (136 microm). E% of the new species (81) is similar to that of S. kraussei (80), but smaller than that of S. jollieti (88), S. weiseri (95), S. oregonense (100) and S. feltiae (119). Spicule head length of the new species is almost the same as its width, this character is similar to that of S. kraussei but it is different from this species by its prominent velum. The new species can be recognized further by characteristics of sequences of ITS and D2D3 regions and cross hybridization with closely related species, S. feltiae, S. kraussei and S. oregonense.     

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae), an entomopathogenic nematode from Thailand

Steinernema siamkayai n. sp. (Rhabditida: Steinernematidae) is a new entomopathogenic nematode isolated in Lohmsak, Thailand. Morphology, hybridisation and molecular studies indicated the distinctness of S. siamkayai n. sp. from other Steinernema spp. Diagnostic characters include: the total body length (398–495 m) and tail length (31-41m) of the third-stage infective juvenile and lateral field pattern with 6-8 longitudinal ridges; the presence of a tail mucro in both first and second generation females and males; the size and shape of the spicules and gubernaculum, and the arrangement of the genital papillae of the first and second generation males; and the shape of the vulva and tail of the first generation female and second generation female     

Oscheius basothovii n. sp. (Nematoda: Rhabditidae), a new entomopathogenic nematode isolated from an uncultivated grassland in South Africa

During a recent soil sample survey in Heidelberg at the Suikerbosrand Nature Reserve near Johannesburg in South Africa, a new entomopathogenic nematode species, collected from the grassland was discovered. Oscheius basothovii was isolated from soil samples using the Galleria mellonella bait method. Amplification and Sanger sequencing of the 18S ribosomal DNA placed the nematode in genus Oscheius. Morphological studies with light and scanning electron microscopy confirmed its anatomical affinities with the genus. The new species is characterised by various traits including its original 18S rDNA sequence, six lips, and amphidelphic reproduction. Males were characterised by the presence of spicules towards their posterior end and short blunt tails. Females had a body with irregular ridges, a vulva opening, and long pointed tails. The infective juveniles of this new species are able to invade Galleria mellonela and Tenebrio molitor larvae and cause mortality within 24–72?h after inoculation under laboratory conditions.     

Steinernema ceratophorum n. sp. (Nematoda: Steinernematidae), a new entomopathogenic nematode from north-east China

Steinernema ceratophorum n. sp. was isolated from soil in Liaoning and Jining Provinces of China. Morphological, cross-breeding and DNA studies support the distinctiveness of S. ceratophorum n. sp. in comparison with four morphologically similar Steinernema species: S. affine, S. intermedium, S. riobrave and S. bicornutum. Diagnostic characters include: body length of infective juvenile, juvenile lip region with two horn-like structures and lateral field with six or eight longitudinal ridges; tail tip of first and second generation males lacking mucron; spicules of first generation male curved, dark yellow in colour and 71 µm long; tail tip of first generation female with small mucronate projection; and second generation female with fine mucronate process about 5.5 µm long. In addition, the restriction fragment length pattern (RFLP) of the ITS region of the ribosomal DNA repeat unit is different from other Steinernema spp., and S. ceratophorum n. sp. did not hybridise with S. riobrave, the species with the most similar RFLP.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Metarhizium brachyspermum sp. nov. (Clavicipitaceae), a new species parasitic on Elateridae from Japan

Ascomata of a Metarhizium species were collected from Tochigi Prefecture, Japan. The ascomata were similar to M. kalasinense in its host preference for elaterids, olive-green clavate stromata, and obliquely immersed perithecia, but the asci and ascospores were significantly shorter in length than those of the latter species. Furthermore, phylogenetic analyses based on the internal transcribed spacer region of nuclear ribosomal DNA and the elongation factor 1-alpha gene showed that the Japanese species was phylogenetically distinctive in the M. anisopliae lineage, including M. kalasinense. Accordingly, we describe a new species, M. brachyspermum. Isolates of this species produced olive-green conidial masses on the surface of the colony. Also, hyphal anastomoses between two metulae were often observed on conidiophores.     

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 scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Xylodon subflaviporus sp. nov. (Hymenochaetales,Basidiomycota) from East Asia

Xylodon subflaviporus (Hymenochaetales, Basidiomycota) is described as a new species collected from tropical and subtropical regions of East Asia. This new species is characterized by resupinate basidiocarps, a poroid hymenophore, pseudodimitic hyphal system, nodose-septate hyphae, four types of cystidia (capitate, acicular to cylindrical, subulate to ventricose, and apically-encrusted), and thin-walled, colorless, smooth and ellipsoid basidiospores. Bayesian, Maximum Likelihood and Maximum Parsimony algorithms were used to construct phylogenies inferred from the internal transcribed spacer (ITS) region of rDNA. Morphological and molecular studies, along with intercompatibility tests, confirmed the independent status of this species. Also, based on morphological and molecular evidence, six new combinations of Xylodon are proposed to accommodate species originally classified under Hyphodontia s.l., i.e. X. bubalinus, X. chinensis, X. mollissimus, X. nongravis, X. reticulatus and X. subtropicus. A key to known species of Hyphodontia s.l. with poroid, irpicoid or raduloid hymenophores is provided for the convenience in identification.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae

A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernemacarpocapsae. The purified protease had an apparent molecular mass of 30 kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K_m, V_max and k_cat values of 409 μM/min, 0.389 μM/min and 24.9 s⁻¹, respectively. The protease was most active at pH 8.0 and 35 °C, and its proteolytic activity was almost completely reduced after incubation at 75 °C for 30 min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24 h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.     

A new entomopathogenic nematode, <Emphasis Type="Italic">Steinernema robustispiculum</Emphasis> n. sp. (Rhabditida: Steinernematidae), from Chumomray National Park in Vietnam

Steinernemar robustispiculum n. sp. (Rhabditida: Steinernematidae) was isolated from woodland in Chumomray National Park, Sason, Sathay, Kontum, Vietnam. Its morphology, morphometrics, cross-hybridisation and the ITS-rDNA sequence analysis revealed that S. robustispiculum clearly differs from other known Steinernema spp. As in the cases of S. intermedium (Poinar, 1985), S. robustispiculum has very robust spicules, but it can be distinguished by the longer tail of the infective juvenile, lower E%, shorter spicules, the shape of the spicules, the number of genital papillae in the caudal region and the presence of a mucron on the male tail. S. robustispiculum has a lateral field resembling that of S. sangi Phan, Nguyen & Moens, 2001, but can be distinguished by a higher E%, higher D%, smaller length to width ratio of the spicules and the morphology of both the spicule head (manubrium) and the dorsal lobe of the spicule. The morphometrics of infective juveniles of S. robustispiculum are similar to those of S. monticolum Stock, Choo & Kaya, 1997; these species can be distingusihed by the position of the excretory pore, the smaller length to width ratio of the spicules, and the length and morphology of the spicule head (manubrium). The phylogenetic relationships within Steinernema Travassos, 1927, including the newly sequenced Vietnamese species S. robustispiculum n. sp., S. loci Phan, Nguyen & Moens, 2001, S. thanhi Phan, Nguyen & Moens, 2001 and S. sangi, are presented based on analyses of the ITS-rDNA. The ITS RFLP profiles obtained from 17 different restriction enzymes are also presented.