Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov

The taxonomic affiliation was determined for four Xenorhabdus strains isolated from four Steinernema hosts from different countries. As compared to the five validly described Xenorhabdus species, i.e., X. nematophila, X. japonica, X. beddingii, X. bovienii and X. poinarii, these isolates represented novel species on the basis of 16S rRNA gene sequences and riboprint patterns, as well as by physiological and metabolic properties. They were named Xenorhabdus budapestensis sp. nov., type strain DSM 16342^T, isolated from Steinernema bicornutum; Xenorhabdus ehlersii sp. nov., type strain DSM 16337^T, isolated from Steinernema serratum; Xenorhabdus innexi sp. nov., type strain DSM 16336^T isolated from Steinernema scapterisci; and Xenorhabdus szentirmaii sp. nov., type strain DSM 16338^T, isolated from Steinernema rarum.     

Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae. Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect. The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph. OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria. To study the role of ompR in the lifecyle of Xenorhabdus, an ompR -minus strain was constructed. The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype. Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC. The ompR mutant strain was virulent towards insect hosts. However, when nematodes were grown on a mixture of the wild-type and the ompR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode. The role of ompR in the symbiosis between the bacterium and the nematode is under investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and identification of a symbiotic bacterium fromSteinernema carpocapsae

Xenorhabdus nematophilus sp., an insect-pathogenic bacterium, was newly isolated from Korean entomopathogenic nematode ofSteinernema carpocapsae, which can be used as a useful bioinsecticide. Primary and secondary form variants ofXenorhabdus nematophilus were observed when culturedin vitro. Primary form variants adsorbed bromothymol blue, while secondary form did not. However, many other characters of two variants were very similar. The variants were all rod-shaped and cell size was highly variable ranging from 0.5 by 2.0 μm to 1.0 by 5.0 μm. Both produced highly toxic substances and killed the insect larva within 20–38 hr, indicating that insect pathogenicity ofXenorhabdus is not directly associated with its phase variation. In addition, cell-free culture supernatant ofXenorhabdus was sufficient to kill the insect larva by injecting it into insect hemolymph; however, cell-harboring culture broth was more effective for killing the insect. The use ofXenorhabdus nematophilus may provide a potential alternative toBacillus thuringiensis (Bt) toxins.     

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10¹⁰ bacterial cells ml⁻¹, a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10⁹ and 10⁸ cells ml⁻¹. Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT₅₀ = 17.1 h) than for Steinernema carpocapsae (RT₅₀ = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect’s food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.     

Purification and some properties of endopolygalacturonase from Rhizopus sp. LKN

An endopolygalacturonase of Rhizopus sp. strain LKN, one of several isolates from tempe starter (ragi), was purified 235-fold by CM-Sephadex C-50, DEAE-Sephadex A-50 ion exchange chromatographies and Sephadex G-75 gel filtration. The purified enzyme was homogeneous by SDS-PAGE with a M _r of 38.5 kDa. Its K _m value for pectic acid was 2 mg/ml. It was stable at pH 4.5 to 11 and up to 50°C, with optimum activity at pH 4.5 to 4.75 and 55 to 60°C. Some ionic compounds enhanced the enzyme activity, whereas tannic acid at 0.5 mm caused about 90% inhibition.The authors are with the Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812, Japan.     

<Emphasis Type="Italic">Sulfolobus tengchongensis</Emphasis> sp. nov., a novel thermoacidophilic archaeon isolated from a hot spring in Tengchong,China

A novel thermoacidophilic strain, designated RT8-4, was isolated from an acidic hot spring in Tengchong, Yunnan, China, and characterized phenotypically and phylogenetically. Cells of strain RT8-4 are irregular cocci with peritrechous flagella. The strain grows aerobically in either a lithotrophic or a heterotrophic mode. No anaerobic growth is apparent. Growth on elemental sulfur occurs through the oxidation of sulfur. Strain RT8-4 is capable of utilizing tryptone, d-xylose, d-arabinose, d-galactose, maltose, sucrose, d-fructose, or l-glutamic acid as the sole source of carbon. d-Glucose and d-mannose are not utilized. RT8-4 grows optimally at 85 °C and pH 3.5. The G+C content of the genome of RT8-4 is 34.4 mol%. Phylogenetic analysis based on 16S rDNA sequence as well as DNA–DNA hybridization and phenotypic characterization identifies strain RT8-4 as a novel species in the genus Sulfolobus. It is proposed that strain RT8-4 be designated as Sulfolobus tengchongensis sp. nov. The type strain is RT8-4^T.Communicated by K. Horikoshi     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

Entomopathogenic symbiotic bacteria,Xenorhabdus and Photorhabdus of nematodes

Xenorhabdus and Photorhabdus species are entomopathogenic bacteria with a wide insect host range, that belong to the family Enterobacteriaceae. Xenorhabdus and Photorhabdus species symbiotically associate with nematodes of the families Steinernematidae and Heterorhabditidae respectively. The factor(s) determining the symbiotic interaction between nematodes and bacteria are yet to be identified. Xenorhabdus and Photorhabdus species exist in two main phenotypic forms, a phenomenon known as phase variation. The phase I (or primary form) varies from phase II (or secondary form) in certain physiological and morphological characteristics. There is no variation in the DNA integrity of phase I and phase II and this supports epigenetic regulatory mechanism in phase variation. Certain pathogenic determinants such as pili, lipopolysaccharides and toxins contribute to the pathogenicity of Xenorhabdus and Photorhabdus species, and both appear to be equally pathogenic to insects. The observed similarity in their virulence to insect hosts may reflect possible in vivo conversion of phase II to phase I, however the host cellular invasion and virulence is yet to be properly understood. The virulence of Xenorhabdus variants varies among insects apparently due to factors which include the feeding habits of the insects. The molecular mechanism and biological significance of phase variation are presently unknown.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

Thermalkalibacillus uzonensis gen. nov. sp. nov, a novel aerobic alkali-tolerant thermophilic bacterium isolated from a hot spring in Uzon Caldera, Kamchatka

A novel thermophilic, alkali-tolerant, and CO-tolerant strain JW/WZ-YB58^T was isolated from green mat samples obtained from the Zarvarzin II hot spring in the Uzon Caldera, Kamchatka (Far East Russia). Cells were Gram-type and Gram stain-positive, strictly aerobic, 0.7–0.8 μm in width and 5.5–12 μm in length and produced terminal spherical spores of 1.2–1.6 μm in diameter with the mother cell swelling around 2 μm in diameter (drumstick-type morphology). Cells grew optimally at pH^25°C 8.2–8.4 and temperature 50–52°C and tolerated maximally 6% (w/v) NaCl. They were strict heterotrophs and could not use either CO or CO₂ (both with or without H₂) as sole carbon source, but tolerated up to 90% (v/v) CO in the headspace. The isolate grew on various complex substrates such as yeast extract, on carbohydrates, and organic acids, which included starch, d-galactose, d-mannose, glutamate, fumarate and acetate. Catalase reaction was negative. The membrane polar lipids were dominated by branched saturated fatty acids, which included iso-15:0 (24.5%), anteiso-15:0 (18.3%), iso-16:0 (9.9%), iso-17:0 (17.5%) and anteiso-17:0 (9.7%) as major constituents. The DNA G+C content of the strain is 45 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain JW/WZ-YB58^T is distantly (<93% similarity) related to members of Bacillaceae. On the basis of 16S rRNA gene sequence, physiological and phenotypic characteristics, the isolate JW/WZ-YB58^T (ATCC BAA-1258; DSM 17740) is proposed to be the type strain for the type species of the new taxa within the family Bacillaceae, Thermalkalibacillus uzoniensis gen. nov. sp. nov. The Genbank accession number for the 16S rRNA gene sequence is DQ221694.The Genbank accession number for the 16S rRNA gene sequence of strain JW/WZ-YB58^T is DQ221694.     

<Emphasis Type="Italic">Arthrobacter halodurans</Emphasis> sp. nov., a new halotolerant bacterium isolated from sea water

A novel Gram-positive, halotolerant, non-sporulating, non-motile, catalase-positive, oxidase-negative and aerobic bacterium, designated strain JSM 078085^T, was isolated from sea water collected from the South China Sea. Strain JSM 078085^T exhibited a rod-coccus growth cycle and produced a yellow pigment. The strain was able to grow in the presence of 0–12% (w/v) NaCl and at pH 6.0–9.5 and 4–35°C; optimum growth was observed at pH 7.0 and 25–30°C in the absence of NaCl. The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu). Cell-wall sugars contained galactose and glucose. Strain JSM 078085^T contained menaquinone MK-9(H₂) as the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The major cellular fatty acids were anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0 and the DNA G + C content was 63.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 078085^T should be assigned to the genus Arthrobacter, being most closely related to the type strain of Arthrobacter rhombi (sequence similarity 97.1%), and the two strains formed a distinct lineage in the phylogenetic tree. The level of DNA–DNA relatedness between strain JSM 078085^T and the type strain of Arthrobacter rhombi was 10.6%. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078085^T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter halodurans sp. nov. is proposed. The type strain is JSM 078085^T (=DSM 21081^T=KCTC 19430^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078085^T is EU583729.     

Type studies of <Emphasis Type="Italic">Pleurotus</Emphasis> reported from Japan

Eight type specimens of Pleurotus reported from Japan were examined. Four new combinations, Marasmius alopecius, Omphalotus guepiniformis, Marasmiellus leiophyllus, and Hohenbuehelia squamula, are proposed. Pleurotus cyatheae is accepted in the original genus. The following species are synonyms: Pleurotus harmandii, a synonym of Omphalotus guepiniformis; P. minutoniger, a synonym of Resupinatus striatulus; and P. pulchellus, a synonym of Hohenbuehelia tremula. Omphalotus japonicus (= Lampteromyces japonicus) is a synonum of O. guepiniformis.     

Salinicoccus salitudinis sp. nov., a new moderately halophilic bacterium isolated from a saline soil sample

A novel pale-yellow-pigmented, moderately halophilic, facultatively alkaliphilic, non-motile, non-spore-forming, catalase- and oxidase-positive, obligately aerobic Gram-positive coccus, strain YIM-C678^T was isolated from a saline soil sample collected from a hypersaline habitat in the Qaidam basin, northwest China. The organism grew at 4–37°C and pH 6.0–11.0, with optimum growth at 25°C and pH 8.0. Strain YIM-C678^T grew optimally in the presence of 10–12% (w/v) NaCl and growth was observed in 1–25% (w/v) NaCl. The cell wall murein type was l-Lys-Gly₅. Major cellular fatty acids were anteiso-C_15:0, iso-C_15:0, iso-C_16:0 and C_16:0. Menaquinone 6 (MK-6) was the major respiratory quinone. The DNA G + C content was 46.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain YIM-C678^T belonged to the family Staphylococcaceae and was most closely related to the eight described species of the genus Salinicoccus with sequence similarities from 92.2 (S. luteus YIM 70202^T) to 97.5% (S. kunmingensis YIM Y15^T). The DNA–DNA relatedness between strain YIM-C678^T and S. kunmingensis YIM Y15^T was 35.4%. Chemotaxonomic data and 16S rRNA gene sequence analysis supported the affiliation of strain YIM-C678^T with the genus Salinicoccus. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic differences and DNA–DNA hybridization data supported the view that the bacterium represents a novel species of the genus Salinicoccus, for which the name Salinicoccus salitudinis sp. nov. is proposed, with YIM-C678^T (=DSM 17846 = CGMCC 1.6299) as the type strain.     

The teleomorph of Leptographium yunnanense, discovered in crosses among isolates from Thailand, China, and Japan

A teleomorph was discovered in crosses among isolates of Leptographium yunnanense isolated from Pinus spp. originating in Thailand, China, and Japan. The ascocarps are black, globose to subglobose, and lacking necks. Ascospores are hyaline, 1-celled, surrounded by hyaline sheaths, and appear cucullate in side view, quadrangular in face view, and triangular in end view. Three species were known to have teleomorphs morphologically similar to the present fungus. However, their anamorphs were distinguishable from L. yunnanense. Thus, this teleomorph is described as Grosmannia yunnanensis. Contribution No. 214, Laboratory of Plant Parasitic Mycology, Graduate School of Life & Environmental Sciences, University of Tsukuba.     

Caeoma tsukubaense n. sp., a rhododendron rust fungus of Japan and southern Asia, and its relationship to Chrysomyxa rhododendri

A rust fungus found in Japan on Rhododendron kaempferi, R. kiusianum, and R. dauricum has previously been identified as Chrysomyxa rhododendri. Light and scanning electron microscopy of fresh and herbarium materials of the rust fungus, however, show that the spore surface morphology differs from the urediniospores of C. rhododendri, and the spores are slightly smaller. Furthermore, the DNA sequence of the 5′-end of the large subunit of ribosomal DNA differs from that of C. rhododendri by 3%. Telia have not been found; therefore, it is redescribed as a new anamorphic species, Caeoma tsukubaense. Several specimens from North Korea, Tibet, and Nepal bearing a similar rust fungus are also included in the species.Contribution no.193 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Vesicular-arbuscular mycorrhizal fungi in coastal dune plant communities II. Spore formation ofGlomus spp. predominates under geographically separated patches ofElymus mollis

Five study sites in Japan with pure patches ofElymus mollis near to the shoreline were chosen. Abundance of spore formation ofGlomus spp. underE. mollis patches were studied at four sites to understand the factors determining the development of VA mycorrhizal fungal communities at the edge of coastal sand dune vegetation. At each study site, eight soil samples from two soil depths were collected four times (three times for Niigata) during a year. Spores ofGlomus spp. predominated at every site, but species composition ofGlomus spp. differed from site to site. In a further experiment to measure the soil depth of the highest spore densities underE. mollis, the highest density was found at depths where rhizomes ofE. mollis were present.Contribution No. 119, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Buzz-pollination in three nectariferousBoraginaceae and possible evolution of buzz-pollinated flowers

Buzz-pollination was observed in three nectariferousBoraginaceae spp.:Onosma gigantea Lam.,Trichodesma africana (L.)R. Br. andT. boissieri Post. An evolutionary pathway from usual nectariferous flowers to typical buzz-pollinated flowers is suggested.     

<Emphasis Type="Italic">Exobasidium symploci-japonicae</Emphasis> var. <Emphasis Type="Italic">carpogenum</Emphasis> var. nov. causing Exobasidium fruit deformation on <Emphasis Type="Italic">Symplocos lucida</Emphasis> in Japan

Exobasidium symploci-japonicae var. carpogenum, causing Exobasidium fruit deformation on Symplocos lucida collected in Fukuoka Prefecture, Japan, is newly described based on morphological observations of hymenial structure and mode of basidiospore germination. This new variety differs morphologically from the type variety, particularly in the septal number of basidiospores and in the shapes and sizes of conidia formed on the medium. Colonies of this new variety are also distinguishable from those of the type variety by yeast-like growth, morphology, and color of colonies.Contribution no.178, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.