Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Investigation of the internal bacterial flora of Eurygaster integriceps (Hemiptera: Scutelleridae) and pathogenicity of the flora members

Eurygaster integriceps (Hemiptera: Scutelleridae) is one of the most serious pest of wheat and other cereal crops throughout the Near and Middle East including Turkey. To contribute biological control of this pest, we investigated the culturable bacterial flora of E. integriceps and their virulence against it. We also tested different entomopathogenic fungi (Isaria fumosorosea ARSEF8356, Beauveria bassiana ARSEF8356, Metarhizium brunneum ARSEF8671 and Nomurae rileyi ARSEF1670) against this pest under laboratory conditions. Bacterial isolates were characterized based on morphological, biochemical, physiological and molecular characteristics (16S rRNA sequencing). The isolates were identified as Pantoea sp. (S1, S5, S7, S8, S10 and S11), P. agglomerans (S2, S3 and S4), Pseudomonas sp. S6 and Micrococcus luteus S9. The highest mortality within bacterial isolates was observed from Pantoea sp. S1, P. agglomerans S4 and Pantoea sp. S7 with 100%. Mortality of other bacterial species ranged from 33% to 88%. The highest mortality among the tested entomopathogenic fungi was obtained from Isaria fumosorosea ARSEF 8333 with 100%. Mortality of other fungi ranged from 33% to 50%. Consequently, Pantoea sp. S1, P. agglomerans S4, Pantoea sp. S7, Beauveria bassiana ARSEF 8356 and Isaria fumosorosea ARSEF 8356 seem to be promising candidates in the control of E. integriceps.     

Reinstatement of the genus Hymenaea (Leguminosae: Caesalpinioideae) in Africa

The monotypic genusTrachylobium [T. verrucosum (Gaertn.) Oliv.] from East Africa and adjacent islands is returned toHymenaea verrucosa Gaertn. on the basis of reevaluation of inflorescence, flower, and fruit characters together with new evidence from seedlings, pollen, leaf venation, chromosome number, and chemistry of the resins.Hymenaea verrucosa appears more similar to an Amazonian and Cuban group of species, which may have been derived from a common African ancestral stock in the Tertiary period, than to another group of species that probably evolved from the evergreen forest species and then radiated into an expanding area of drier habitats during the later Tertiary and dry intervals of the Pleistocene. Originally the genusTrachylobium was distinguished fromHymenaea by a morphological comparison with the probably more recently evolved species. A possible explanation is presented for the disjunct amphi-Atlantic distribution ofH. verrucosa and the neotropical species ofHymenaea.     

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae)

Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of root-colonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including putative endophytic and mycorrhizal fungi in addition to AM fungi.     

Identification of a microspordium isolated from Megacopta cribraria (Hemiptera: Plataspidae) and characterization of its pathogenicity in silkworms

A new microsporidium isolated from Megacopta cribraria was characterized by both biological characteristics and phylogenetic analysis. Moreover, its pathogenicity to silkworms was also studied. The spores are oval in shape and measured 3.64 ± 0.2 × 2.20 ± 0.2 μm in size. Its ultrastructure is characteristic of the genus Nosema: a diplokaryon, 13–14 polar filament coils and posterior vacuole. Its life cycle includes meronts, sporonts, sporoblasts and mature spores, with a typical diplokaryon in each stage and propagation in a binary fission. A phylogenetic tree based on SSU rRNA and rRNA ITS gene sequence analysis further indicated that the parasite is closely related to Nosema bombycis and should be placed in the genus Nosema and sub-group ‘true’ Nosema. Furthermore, the microsporidium heavily infects lepidopteran silkworm insect and can be transmitted per os (horizontally) and transovarially (vertically). Our findings showed that the microsporidium belongs to the ‘true’ Nosema group within the genus Nosema and heavily infects silkworms. Based on the information obtained during this study, we named this new microsporidium isolated from M. cribraria as Nosema sp. MC.     

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN?+?I?+?G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN?+?I?+?G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN?+?I?+?G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN?+?I?+?G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN?+?I?+?G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN?+?I?+?G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN?+?I?+?G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN?+?I?+?G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN?+?I?+?G 0.022 to 0.025) shared the same lineage with T. putitora (TrN?+?I?+?G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.     

Leaf flavonoids ofGalium sect.Aparinoides (Rubiaceae)

13 taxa belonging to 4 “species groups” ofGalium L. sect.Aparinoides (Jord.)Gren. produce 15 leaf flavonoids: Apigenin-7-diglucoside, Luteolin-7-monoglucoside and 7-diglucoside, Diosmetin, Diosmetin-7-monoglucoside and 7-diglucoside; Kaempferol-3-rutinoside, Kaempferol-3,7-diglucoside, Quercetin, two Quercetin-3-monoglycosides, Rutin, Quercetin-3-rutinoside-7-glucoside, Quercetin-7-glycoside and an unidentified aglycone. TheG. trifidum, G. obtusum andG. palustre groups (with the exception of theG. tinctorium subspecies andG. elongatum) have similar flavone-flavonole patterns, while theG. antarcticum group produces a specific pattern. Leaf flavonoids of theG. trifidum andG. antarcticum group are inhomogenous, becauseG. tinctorium subsp.tinctorium andG. antarcticum lack flavones. For all taxa (with the exception of those of theG. antarcticum group) intraspecific variation is demonstrated, and 4 populations ofG. trifidum subsp.trifidum, G. tinctorium subsp.tinctorium,G. obtusum subsp.obtusum andG. labradoricum even exhibit intrapopulation variation. The implications of flavonoid data on the systematics and the astonishing intrapopulation and intraspecific variation are discussed.     

A micro-culture technique for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus)

A micro-culture technique was developed to determine the optimum culture conditions for the mitogen stimulation of lymphocytes fromMacaca mulatta (rhesus) andMacaca fascicularis (cynomolgus). The optimum concentrations of PHA and Con A ranged from 10–50 µg/culture, those of lymphocytes from 1?2×10⁵ cells/culture, and those of serum from 10–20%. Tritiated thymidine was used at a concentration of .04µCi/culture.     

Rhodocybe pulchrisperma (Entolomataceae): A new species from North America

Rhodocybe pulchrisperma is described as a new species of Agaricales from North America. It belongs in section Rhodocybe due to the brightly colored pseudocystidia and lack of clamp connections. The very large, handsomely ornamented basidiospores distinguish this species from others in the section. A key to taxa from Florida, Papua New Guinea, and India that exhibit similar features is presented.     

Vibrio madracius sp. nov. Isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago,Mid-Atlantic Ridge,Brazil

Three novel isolates (A-354^T, A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V. mediterranei species, sharing upto 98.1 % 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354^T from V. mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92 %, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V. mediterranei based on the four following features. They do not grow at 8 % NaCl; use d-gluconic acid but not l-galactonic acid lactone as carbon source; and do not have the fatty acid C_18:0. Differentiation from both the other Mediterranei clade species (V. maritimus and V. variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-d-lactose, N-acetyl-d-galactosamine, myo-Inositol, d-gluconic acid, and β-hydroxy-d,l-butyric acid; and present the fatty acids C_14:0 iso, C_15:0 anteiso, C_16:0 iso, C_17:0 anteiso, and C_17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354^T (=LMG 28124^T=CBAS 482^T) is 44.5 mol%.     

Middle Ottnangian (late Burdigalian) mollusks from the Rott Valley (SE Germany): the ultimate marine fauna of the Western Paratethys

The Bivalvia and Gastropoda from the Upper Marine Molasse deposits of the lower Rott Valley (North Alpine Foreland Basin, Lower Bavaria, SE Germany) comprise the first mollusk fauna of middle Ottnangian age (ca 17.5 Ma) to be documented in detail. Altogether, the assemblages from Brombach, Kainerding and Anzenkirchen yielded 27 species of Bivalvia and 9 species of Gastropoda. The bivalve species Spisula brombachensis n. sp. is described as new to science, and Aequipecten camaretensis is first reported from the Ottnangian of the North Alpine Foreland Basin. Furthermore, the well-preserved material allowed for the taxonomic revision of the Ottnangian index fossil Pecten herrmannseni. The bivalve community is dominated by suspension feeders, mostly shallow, soft bottom dwellers (Glycymeris, Anadara) and cementing epifauna (Ostrea). Gastropods almost exclusively comprise carnivorous browsers and predators. The parautochthonous assemblages are indicative of normal marine, well-oxygenized sandy-silty shoals at a water depth of presumably less than 20 m. Except for the presence of a single estuarine Crassostrea, the middle Ottnangian fauna is not indicative of reduced salinities. Consequently, the paleoenvironmental turnover to the brackish-water fauna of the directly overlying upper Ottnangian deposits of the Rzehakia Lake was abrupt. Glycymeris–Anadara–Ostrea communities similar to the one recorded from the Rott Valley are widespread in European seas and beyond during the Neogene and Quaternary.     

Feral pigs as hosts for Amblyomma sculptum (Acari: Ixodidae) populations in the Pantanal,Mato Grosso do Sul,Brazil

The Pantanal in Brazil is the largest floodplain of the world. This ecosystem, rich in wildlife, has a large feral pig population. Such a large host biomass must have a strong influence on the parasite fauna. In this work, we evaluated the role of feral pigs in the maintenance of Amblyomma sculptum (formerly Amblyomma cajennense), the most prevalent tick species in the Pantanal. Tick infestations were evaluated on 243 feral pigs and their environment. The suitability of domestic pigs, representing their feral relatives, to A. sculptum adults and nymphs was assessed experimentally. Tick infestation of feral pigs was strongly associated with that of the environment: 96 and 97 % of the ticks, respectively, were A. sculptum. The infestation prevalence on this host species was close to 90 % in the dry season and 100 % in the wet season and mean infestation intensity was above 30 ticks in both seasons. Suitability of pigs as hosts for A. sculptum was shown by the high proportion of nymphs and female ticks found engorging on captured feral pigs and adequate biological parameters displayed by ticks from experimental infestations of domestic pigs. Other tick species on feral pigs, albeit in much lower numbers, were Amblyomma parvum and Ornithodorus rostratus. Results show that feral pigs feed a high proportion of the A. sculptum adults and nymphs in their territories and should be a target for tick-borne diseases studies. This is particularly relevant to public health because all the main tick species found on feral pigs are aggressive to humans as well.     

Phylogenetic relationships among Phytopythium species,and re-evaluation of Phytopythium fagopyri comb. nov., recovered from damped-off buckwheat seedlings in Japan

We examined the phylogenetic relationships among Phytopythium species using the rDNA ITS region, the LSU rDNA region, and the mitochondrial coxI and coxII genes. The genus was resolved into three monophyletic clades (1–3). Clade 1 was the largest clade, composed of 12 known species. Clades 2 contained two known and one new species candidate and clade 3 contained two known species. Three isolates in clade 2 (FP1, HonMa, and a strain designated as P. helicoides CBS293.35) formed a monophyletic group with high bootstrap support. This monophyletic group was distinct from P. helicoides sensu stricto. All three isolates came from damped-off buckwheat seedlings. The isolates were morphologically identical with one another and were characterized by globose, sub-globose, or pyriform sporangia with apical papillae; internally or internally nested proliferating sporangia; simple sympodia; coiling antheridial stalks; and wavy, sessile, or clavate antheridial cells. The isolates grew at temperatures between 15 °C and 40 °C, and the optimum temperature was 30 °C, with a radial growth rate of 20 mm/24 h. The phylogenetic and morphological analyses indicated that these isolates belong to a distinct species, which was previously under the genus Pythium, named here Phytopythium fagopyri comb. nov.     

Cytogenetics of ticks (Acari: Ixodoidea)

Chromosomes and sex determination of 9 species of Haemaphysalis assigned to 4 subgenera are described. H. (tAlloceraea) kitaokai possesses an XX∶XO sex chromosome system with 18 autosomes plus XX in females; 18 plus X in males. H. (Kaiseriana) hystricis has 18 +XX and 18 + XY in females and males, respectively, in most specimens, but a supernumerary chromosome is present in some individuals. A supernumerary chromosome was also observed in 1 male H. (Aborphysalis) formosensis. These two species are the second and third species of ticks reported to have supernumerary chromosomes. H. formosensis, H. (Kaiseriana) bispinosa, H. (Haemaphysalis) campanulata, H. (H.) flava, H. (H.) megaspinosa, H. (H.) japonica, and H. (H.) pentalagi possess 20 autosomes plus 2 sex chromosomes in females and 20+1 sex chromosomes in males. Phylogenetic relationships within the genus Haemaphysalis are briefly discussed.