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 brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil

A new entomopathogenic nematode, Steinernema brazilense n. sp., was isolated from a single soil sample collected from a natural forest in Mato Grosso do Sul state, Brazil. S. brazilense n. sp. is characterized morphologically by features of infective juveniles (IJ), males and females. For the IJ, body length averaging 1157 (1023-1284) μm, distance from anterior end to excretory pore 95 (87-102) μm, from anterior end to end of esophagus 148 (139-153) μm, tail length 85 (80-104) μm, D% and E% values 63 (58-70) and 106 (95-118.0), respectively. Lateral field pattern variable; the formula for the arrangement of ridges from head to tail is: 2, 4, 6, 8, 6, 2. For the male, the diagnostic characters include spicule averaging 83 (75-89) μm; D% about 65; the ratio SW% about 192. The length of spicule head is greater than width. Lateral field with one narrow ridge. First generation females are characterized by the presence of a ventral postanal swelling. S. brazilense n. sp. is morphologically close to Steinernema diaprepesi. It can be differentiated from S.diaprepesi by its longer IJ body length (1157 vs 1002 μm), longer distance from anterior end to excretory pore (110 vs 75 μm), a longer tail length (103 vs 83 μm); males of the new species with longer spicule (83 vs 79 μm). The new species can be distinguished further from other members of Steinernema glaseri group by characteristics of rDNA of ITS and D2D3 regions.     

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 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.     

Heterorhabditidoides chongmingensis gen. nov., sp. nov. (Rhabditida: Rhabditidae), a novel member of the entomopathogenic nematodes

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with steinernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP = 90 (80-105) μm, ES = 104 (92-120) μm, tail length = 111 (89-159) μm, and a = 19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

The bacterium associated with the entomopathogenic nematode Steinernema abbasi (Nematoda: Steinernematidae) isolated from Taiwan

A symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, determined to be a species of Xenorhabdus based on its physiological and biochemical characteristics has been determined to be similar to Xenorhabdus indica of S. abbasi Oman isolate as based on sequence analyses of 16S rDNA.     

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.     

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.     

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.     

Susceptibility of Hoplia philanthus (Coleoptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae)

Biological control potential of nine entomopathogenic nematodes, Heterorhabditis bacteriophora CLO51 strain (HbCLO51), H. megidis VBM30 strain (HmVBM30), H. indica, Steinernema scarabaei, S. feltiae, S. arenarium, S. carpocapsae Belgian strain (ScBE), S. glaseri Belgian strain (SgBE) and S. glaseri NC strain (SgNC), was tested against second-, and third-instar larvae and pupae of Hoplia philanthus in laboratory and greenhouse experiments. The susceptibility of the developmental stages of H. philanthus differed greatly among tested nematode species/strains. In the laboratory experiments, SgBE, SgNC, HbCLO51 and HmVBM30 were highly virulent to third-instar larvae and pupae while SgBE was only virulent to second-instar larvae. Pupae were highly susceptible to HbCLO51, HmVBM30, SgBE and SgNC (57–100%) followed by H. indica and S. scarabaei (57–76%). In pot experiments, HbCLO51, SgBE and S. scarabaei were highly virulent to the third-instar larvae compared to the second-instar larvae. Our observations, combined with those of previous studies on other nematode and white grub species, show that nematode virulence against white grub developmental stages varies with white grub and nematode species.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov. and Trigonopsis californica sp. nov

Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain NRRL Y-17858, CBS 7922) was isolated from the ear of an infant in Germany and is closely related to Candida sorbophila. Candida polysorbophila sp. nov. (type strain NRRL Y-27161, CBS 7317) is a member of the Zygoascus clade and was isolated in South Africa as a contaminant from an emulsion of white oil and polysorbate. Candida transvaalensis sp. nov. (type strain NRRL Y-27140, CBS 6663) was obtained from forest litter, the Transvaal, South Africa, and forms an isolated clade with Candida santjacobensis. Trigonopsis californica sp. nov. (type strain NRRL Y-27307, CBS 10351) represents a contaminant from wine in California, and forms a well-supported clade with Trigonopsis cantarellii, Trigonopsis variabilis and Trigonopsis vinaria.     

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.     

Seasonal dynamics of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis as a response to abiotic factors and abundance of insect hosts

The seasonal dynamics of entomopathogenic nematodes (EPNs) of the genus Steinernema and Heterorhabditis were studied during one season in meadow and oak wood habitats, in the vicinity of Ceské Budejovice, Czech Republic. The influences of soil temperature, moisture, and abundance of suitable hosts on EPN dynamics were investigated. The host range of these nematodes, in both habitats was also observed. A total of four EPN species were found in both habitats. Steinernema affine was the dominant species both in oak wood and in meadow. Additionally, the oak wood habitat was inhabited by S. kraussei and S. weiseri; the meadow habitat by Heterorhabditis bacteriophora. The mean abundance of total EPN community was 28,000ind./m(2) in oak wood and 11,000ind./m(2) in meadow. The seasonal dynamics of entomopathogenic nematodes in both habitats were characterized by high nematode densities in the beginning of the season, followed by a rapid decrease, and then stabilization. EPN abundances did not show any apparent correlation with soil temperature and moisture, but they were negatively correlated with the abundance of suitable insect hosts. Inter- and intraspecific competition for limited nutrients (hosts) probably played a major role in EPN seasonal dynamics. Broad host range of entomopathogenic nematodes in both habitats was predominantly represented by dipteran and coleopteran larvae. Most common hosts belonged to the families Asilidae, Bibionidae, and Empididae (Diptera), as well as Carabidae and Curculionidae (Coleoptera).