Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences

Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.     

Parasites, emerging disease and wildlife conservation

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.     

SNS: Adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts

The body wall muscles in the Drosophila larva arise from interactions between Duf/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and sticks-and-stones (SNS)-expressing fusion competent myoblasts in the embryo. Herein, we demonstrate that SNS mediates heterotypic adhesion of S2 cells with Duf/Kirre and IrreC-rst-expressing S2 cells, and colocalizes with these proteins at points of cell contact. These properties are independent of their transmembrane and cytoplasmic domains, and are observed quite readily with GPI-anchored forms of the ectodomains. Heterotypic interactions between Duf/Kirre and SNS-expressing S2 cells occur more rapidly and to a greater extent than homotypic interactions with other Duf/Kirre-expressing cells. In addition, Duf/Kirre and SNS are present in an immunoprecipitable complex from S2 cells. In the embryo, Duf/Kirre and SNS are present at points of contact between founder and fusion competent cells. Moreover, SNS clustering on the cell surface is dependent on Duf/Kirre and/or IrreC-rst. Finally, although the cytoplasmic and transmembrane domains of SNS are expendable for interactions in culture, they are essential for fusion of embryonic myoblasts.     

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.     

Bacterial flora in the alimentary tract of chickens infected with Eimeria brunetti and in chickens immunized with Eimeria maxima and cross-infected with Eimeria brunetti

Differential bacterial counts were made on the intestinal and caecal contents of chickens after inoculation with a standard dose of 320 000 freshly sporulated oocysts of Eimeria brunetti.     

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.     

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective ‘first generation’ cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these ‘second generation’ cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.     

Influence of Nematodes and Light Sources on Growth and Nodulation of Soybean

The influence of nematodes on nodulation of soybean varied according to their modes of parasitism. In the greenhouse, nodule formation was stimulated by the endoparasites, Meloidogyne hapla and Pratylenchus penetrans, but was inhibited slightly by the ectoparasite, Belonolaimus longicaudatus. In an experiment under controlled conditions in a phytotron, Heterodera glycines severely inhibited nodule formation, whereas plants inoculated with B. longicaudatus and P. penetrans had more nodules per g root than nematode-free plants. Nitrogen-fixing capacity, however, was inhibited by all three nematode species. Different light sources used in the phytotron experiment also influenced growth and nodulation of soybean. A fluorescent plus incandescent light regime resulted in plants with the greatest shoot weight, pod number, and nodules per g root. Plants grown under Lucalox lamps had excessive stem elongation.     

Two endemic foci of heterophyids and other intestinal fluke infections in southern and western coastal areas in Korea

Two endemic foci of heterophyid infections were discovered in coastal villages of Puan-gun, Chollabuk-do, and Sachon-gun. Kyongsangnam-do, Korea. Fecal examinations were performed on 153 inhabitants of Puan-gun and 138 of Sachon-gun, using cellophane thick smear and formalin-ether sedimentation technique. The helminth egg and/or protozoan cyst positive rate was 21.5% (33/153) in Puan-gun and 39.1% (54/138) in Sachon-gun. In Puan-gun, the egg positive rate of heterophyids was the highest, 17.6%, and that of other parasites was 0.7-2.6% by parasite species. In Sachon-gun, that of heterophyids was 18.8%, followed by Clonorchis sinensis (12.3%), and other parasites (0.7-5.0%). Twenth-two (Puan-gun) and six (Sachon-gun) heterophyid egg positive cases were treated with praziquantel, and adult flukes were collected from their diarrheic stools. A total of 3,284 adult flukes of Heterophyes nocens was collected from all of the 22 patients treated in Puan-gun (3-778 individually), and other trematodes were also collected from 2-15 patients: Pygidiopsis summa, Stellantchasmus falcalus, Metagonimus yokogawai, M. miyatai, Stictodora fuscata, Heterophyopsis continua, Acanthoparyphium kurogamo, and Gymnophalloides seoi. In Sachon-gun, M. yokogawai (3,007 specimens), H. nocens (120), and S. falcatus (46) were collected from 5 of 6 treated patients, and H. continua and S. lari each from one patient. The present study revealed that heterophyid flukes, especially H. nocens and M. yokogawai, are prevalent in the southern and western coastal areas of Korea where fresh and/or brackish water fishes are popularly eaten raw.     

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren’t. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ² test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ² test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.     

Ent-3,4-seco-labdane and ent-labdane diterpenoids from Croton stipuliformis (Euphorbiaceae)

From a methanolic extract of the leaves of Croton stipuliformis, three ent-3,4-seco-labdanes (1-3) and an ent-labdane (4) together with the known compounds 6-hydroxynidorellol (5), maravuic acid, and sitosterol were isolated and identified from their spectroscopic data. The absolute stereochemistry of compound 4 was determined by application of Mosher's method in the NMR tube.     

Phylogeny of Celastraceae tribe Euonymeae inferred from morphological characters and nuclear and plastid genes

The phylogeny of Celastraceae tribe Euonymeae (∼230 species in eight genera in both the Old and New Worlds) was inferred using morphological characters together with plastid (matK, trnL-F) and nuclear (ITS and 26S rDNA) genes. Tribe Euonymeae has been defined as those genera of Celastraceae with generally opposite leaves, isomerous carpels, loculicidally dehiscent capsules, and arillate seeds (except Microtropis). Euonymus is the most diverse (129 species) and widely cultivated genus in the tribe. We infer that tribe Euonymeae consists of at least six separate lineages within Celastraceae and that a revised natural classification of the family is needed. Microtropis and Quetzalia are inferred to be distinct sister groups that together are sister to Zinowiewia. The endangered Monimopetalum chinense is an isolated and early derived lineage of Celastraceae that represents an important component of phylogenetic diversity within the family. Hedraianthera is sister to Brassiantha, and we describe a second species (Brassiantha hedraiantheroides A.J. Ford) that represents the first reported occurrence of this genus in Australia. Euonymus globularis, from eastern Australia, is sister to Menepetalum, which is endemic to New Caledonia, and we erect a new genus (Dinghoua R.H. Archer) for it. The Madagascan species of Euonymus are sister to Pleurostylia and recognized as a distinct genus (Astrocassine ined.). Glyptopetalum, Torralbasia, and Xylonymus are all closely related to Euonymus sensu stricto and are questionably distinct from it. Current intrageneric classifications of Euonymus are not completely natural and require revision.     

Patterns of plastid and nuclear variation among apomictic polyploids of Hieracium: evolutionary processes and taxonomic implications

Background and Aims

Apomictic species (with asexual seed production) make up for 20–50 % of all taxonomically recognized species in northern Europe, but the phylogenetic relationships of apomictic species and the mode of evolution and speciation remain largely unknown and their taxonomy is consequently disputed.

Methods

In the present study, plastid psbD-trnT sequences (349 accessions) and 12 nuclear microsatellite loci (478 accessions) were used to create an overview of the molecular variation in (mainly) northern European members of the most species-rich of all plant genera, Hieracium s.s. The results are discussed and interpreted in the context of morphological and cytological data on the same species.

Key Results and Conclusions

The complete psbD-trnT alignment was 1243 bp and 50 polymorphisms defined 40 haplotypes. All haplotypes found in the sections of the genus distributed in the northern European lowlands fell into one of two main groups, group H and group V, mutually separated by seven or eight polymorphisms. All accessions belonging to H. sects. Foliosa, Hieracioides (viz. H. umbellatum) and Tridentata and all but one accession of triploid species of H. sects. Oradea and Vulgata showed haplotypes of group V. Haplotypes of group H were found in all accessions of H. sects. Bifida and Hieracium and in all tetraploid representatives of H. sects. Oreadea and Vulgata. Additional haplotypes were found in accessions of the genus Pilosella and in southern European and Alpine sections of Hieracium. In contrast, the distribution of individual haplotypes in the two major groups appeared uncorrelated with morphology and current taxonomy, but polymorphisms within species were only rarely encountered. In total, 160 microsatellite alleles were identified. Levels of variation were generally high with only nine pairs of accessions being identical at all loci (in all cases representing accessions of the same species). In the neighbor-joining analysis based on the microsatellite data, accessions of the same species generally clustered together and some smaller groups of species congruent with morphology and/or current taxonomy were recovered but, except for H. sect. Oreadea, most larger groups were not correlated with morphology. Although the plastid DNA sequences show too little variation and the nuclear microsatellites are too variable to resolve relationships successfully among species or to fully understand processes of evolution, it is concluded that both species and sections as defined by morphology are largely congruent with the molecular data, that gene flow between the sections is rare or non-existent and that the tetraploid species may constitute the key to understanding evolution and speciation in this genus.     

Algicidal and antifungal compounds from the roots of Ruta graveolens and synthesis of their analogs

Bioassay-guided fractionation of the ethyl acetate extract of Ruta graveolens roots yielded rutacridone epoxide with potent selective algicidal activity towards the 2-methyl-isoborneol (MIB)-producing blue-green alga Oscillatoria perornata, with relatively little effect on the green alga Selenastrum capricornutum. The diol-analog of rutacridone epoxide, gravacridondiol, which was also present in the same extract, had significantly less activity towards O. perornata. Rutacridone epoxide also showed significantly higher activity than commercial fungicides captan and benomyl in our micro-bioassay against the agriculturally important pathogenic fungi Colletotrichum fragariae, C. gloeosporioides, C. acutatum, and Botrytis cineara and Fusarium oxysporium. Rutacridone epoxide is reported as a direct-acting mutagen, precluding its use as an agrochemical. In order to understand the structure-activity relationships and to develop new potential biocides without toxicity and mutagenicity, some analogs containing the (2-methyloxiranyl)-dihydrobenzofuran moiety with an epoxide were synthesized and tested. None of the synthetic analogs showed comparable activities to rutacridone epoxide. The absolute stereochemistry of rutacridone was determined to be 2'(R) and that of rutacridone epoxide to be 2'(R), 3'(R) by CD and NMR analysis.     

Annotated checklist of the recent and extinct pythons (Serpentes, Pythonidae), with notes on nomenclature, taxonomy, and distribution

McDiarmid et al. (1999) published the first part of their planned taxonomic catalog of the snakes of the world. Since then, several new python taxa have been described in both the scientific literature and non-peer-reviewed publications. This checklist evaluates the nomenclatural status of the names and discusses the taxonomic status of the new taxa, and aims to continue the work of McDiarmid et al. (1999) for the family Pythonidae, covering the period 1999 to 2010. Numerous new taxa are listed, and where appropriate recent synonymies are included and annotations are made. A checklist and a taxonomic identification key of valid taxa are provided.     

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.