Molecular evidence for cosmopolitan distribution of platyhelminth parasites of tunas (Thunnus spp.)

Global distribution of platyhelminth parasites and their host specificities are not well known. Our hypothesis was that platyhelminth parasites of large pelagic fishes are common around the world. We analysed molecular variation in three different taxa of platyhelminth parasites infecting four species of tunas: yellowfin tuna (Thunnus albacares, Scombridae) from Western Australia, southern bluefin tuna (Thunnus maccoyii, Scombridae) from South Australia, Pacific bluefin tuna (Thunnus orientalis, Scombridae) from Pacific Mexico and northern bluefin tuna (T. thynnus, Scombridae) from two localities in the Mediterranean (Spain and Croatia). Comparisons of ITS2 and partial 28S rDNA demonstrated two congeneric species of blood flukes (Digenea: Sanguinicolidae) from multiple hosts and localities: Cardicola forsteri from southern bluefin and northern bluefin tunas, and Cardicola sp. from Pacific bluefin and northern bluefin tunas; and a gill fluke, Hexostoma thynni (Polyopisthocotylea: Hexostomatidae), from yellowfin, southern bluefin and northern bluefin tunas. Partial 28S rDNA indicates that a second type of fluke on the gills, Capsala sp. (Monopisthocotylea: Capsalidae), occurs on both southern bluefin and Pacific bluefin tunas. This appears to be the first report of conspecific platyhelminth parasites of teleosts with a wide‐ranging geographical distribution that has been confirmed through molecular approaches. Given the brevity of the free‐living larval stage of both taxa of flukes on the gills (H. thynni and Capsala sp.), we conclude that the only feasible hypothesis for the cosmopolitan distribution of these flatworms is migrations of host tunas. Host migration also seems likely to be responsible for the widespread occurrence of the two species of blood flukes (Cardicola spp.), although it is also possible that these were translocated recently by the spread of infected intermediate hosts.     

Phylogenetic Analysis of Sugarcane Rusts Based on Sequences of ITS, 5.8 S rDNA and D1/D2 Regions of LSU rDNA

Phylogenetic analysis of sugarcane rusts based on sequences of ITS and the 5.8 S rDNA revealed two highly divergent ITS groups among isolates of Puccinia sp. sensu Muta, 1987 and P. kuehnii specimens. Although there is sufficient divergence (exceeding normal intraspecific variation) between the ITS regions of the two groups to support separation into different species, unusually high homology of the ITS group I sequences with those of members of Cronartium and identical sequences of the D1/D2 regions of the LSU rDNA for all the isolates of “Puccinia sp.” and P. kuehnii that otherwise exhibited different ITS sequences, suggest that the two highly divergent sequences may have resulted from abnormal genetic events leading to non-orthologous, intraspeciflc polymorphisms. The other sugarcane rust, P. melanocephala and the grass rusts, P. miscanthi and P. rufipes, were separated from “Puccinia sp.” and P. kuehnii and from each other in D1/D2 region analyses, indicating that D1/D2 region sequences may more correctly reflect phylogenetic relationships in these rusts than do the ITS regions. Further studies to examine differences in patho-genicity or finer morphological features within P. kuehnii that may be correlated with the high divergence in ITS sequences and experiments to determine if these two sequence types represent intraspeciflc polymorphism are necessary. Received 11 October 2000/ Accepted in revised form 24 November 2000     

<Emphasis Type="Italic">Platanus</Emphasis> × <Emphasis Type="Italic">acerifolia</Emphasis> genotypes surviving to inoculation with <Emphasis Type="Italic">Ceratocystis platani</Emphasis> (the agent of canker stain): first screening and molecular characterization

Canker stain, caused by the fungus Ceratocystis platani, is a destructive disease in Platanus spp. It has been recently proved that resistant accessions can be produced and grown in Europe. However, additional resistant genotypes are still needed in order to avoid the onset of virulent pathogen strains favoured by the selection pressures exerted by genetically homogeneous resistant plane tree plantings. In this study we present the results of two parallel experiments performed on 975 accessions of P. × acerifolia seedlings and P. × acerifolia clones derived by cutting propagation from mature trees grown in the urban environments. The selection process was based on inoculation with C. platani and yielded 13 accessions that showed different types of resistant reactions and survived in a stable manner thoughout the period of observation. Selected accessions were characterised by sequencing the rDNA-ITS region and by developing PCR procedures capable of detecting P. orientalis and P. occidentalis LEAFY homoeologues. These molecular analyses enabled us to confirm the identification of the species, its hybrid origin and to assess an evident genetic variability among the accessions, which therefore have to be considered as different genotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Note: Molecular identification of three entomopathogenic nematodes from turkey by PCR-RFLP of the ITS regions

Molecular identification methods are widely used for the classification of organisms worldwide. Entomopathogenic nematodes are the most often isolated insect parasitic nematodes in the tropical and subtropical regions. In our investigation, PCR-RFLP (Polymerase Chain Reaction — Restriction Fragment Length Polymorphism) of the ITS region (Internal Transcribed Spacer) on the ribosomal (r) DNA of three entomopathogenic nematodes isolated from Ankara, Turkey, was analyzed for identification. The ITS region of rDNA was amplified by PCR and then digested with the following nine restriction enzymes: Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Hpa II, Rsa I and Sau 3AI. The amplified and restricted sequences of the ITS regions were separated by agarose gel electrophoresis and the RFLP patterns of these three species were shown in this study. According to our results, these species were identified asSteinernema feltiae, Steinernema carpocapsae andHeterorhabditis bacteriophora. http://www.phytoparasitica.org posting Nov. 4, 2005.     

Molecular and Pathogenicity Characteristics of Phytophthora nicotianae Responsible for Root Necrosis and Wilting of Pepper (Capsicum annuum L.) in Tunisia

Isolates of Phytophthora from pepper, produced in Tunisia, were characterised according to molecular and pathogenicity criteria. Polymerase chain reaction amplification of the ITS1 region in the ribosomal DNA resulted in different sized fragments. The pepper isolates and P. nicotianae yielded a fragment of 310bp that distinguished it from P. capsici with a fragment of 270bp. The ribosomal RNA gene amplicons of both internal transcribed spacers and the 5.8 S of the pepper Phytophthora and P. nicotianae were digested with 8 endonucleases. The patterns generated, with the 2 enzymes that cut, were identical for both taxa. This molecular analysis corroborated the morphological and biological characteristics and suggests strongly that the isolates of Phytophthora from pepper belong to the species P. nicotianae. Inoculation of pepper, tomato, eggplant and tobacco plants with the isolates of P. nicotianae from pepper showed they were highly pathogenic on pepper but not on tobacco, while their pathogenicity was weak on tomato and eggplant and was associated with atypical symptoms not observed in the field. These pathogenicity tests suggest that pepper isolates of P. nicotianae are particularly adapted to their host and may thus constitute a forma specialis of P. nicotianae.     

European species of Hypocrea: Part I. The green-spored species

At present 75 species of Hypocrea have been identified in temperate Europe. Nineteen green-spored species and their Trichoderma asexual states are here described in detail. Extensive searches for Hypocrea teleomorphs in 14 European countries, with emphasis on Central Europe, yielded more than 620 specimens within five years. The morphology of fresh and dry stromata was studied. In addition, available types of species described from Europe were examined. Cultures were prepared from ascospores and used to study the morphology of cultures and anamorphs, to determine growth rates, and to extract DNA that was used for amplification and sequencing of three genetic markers. ITS was used for identification, while RNA polymerase II subunit b (rpb2) and translation elongation factor 1 alpha (tef1) were analyzed for phylogenetic reconstruction of the genus.Several unexpected findings resulted from this project: 1) The previous view that only a small number of Trichoderma species form a teleomorph is erroneous. 2) All expectations concerning the number of species in Europe are by far exceeded. Seventy-five species of Hypocrea, two species of Protocrea, and Arachnocrea stipata, are herein identified in temperate Europe, based on the ITS identification routine using fresh material, on species described earlier without molecular data and on species recently described but not collected during this project. 3) Current data suggest that the biodiversity of Hypocrea / Trichoderma above soil exceeds the number of species isolated from soil. 4) The number of Trichoderma species forming hyaline conidia has been considered a small fraction. In Europe, 26 species of those forming teleomorphs produce hyaline conidia, while 42 green-conidial species are known. Three of the detected Hypocrea species do not form an anamorph in culture, while the anamorph is unknown in four species, because they have never been cultured.This work is a preliminary account of Hypocrea and their Trichoderma anamorphs in Europe. Of the hyaline-spored species, H. minutispora is by far the most common species in Europe, while of the green-spored species this is H. strictipilosa.General ecology of Hypocrea is discussed. Specific associations, either with host fungi or trees have been found, but the majority of species seems to be necrotrophic on diverse fungi on wood and bark.The taxonomy of the genus will be treated in two parts. In this first part 19 species of Hypocrea with green ascospores, including six new teleomorph and five new anamorph species, are described in detail. All green-spored species belong to previously recognised clades, except H. spinulosa, which forms the new Spinulosa Clade with two additional new species, and H. fomiticola, which belongs to the Semiorbis Clade and forms effuse to large subpulvinate stromata on Fomes fomentarius, a trait new for species with green ascospores. Anamorph names are established prospectively in order to provide a basis for possible policy alterations towards their use for holomorphs.Taxonomic novelties: Hypocrea aeruginea Jaklitsch, Trichoderma aerugineum Jaklitsch, T. dacrymycellum Jaklitsch, H. danica Jaklitsch, H./T. fomiticola Jaklitsch, H. longipilosa Jaklitsch, H./T. parepimyces Jaklitsch, H. parestonica Jaklitsch, T. parestonicum Jaklitsch.     

Overcoming hybridization barriers between pea and some of its wild relatives

Pea would benefit from the plasticity and adaptability of its cross-incompatible relatives Pisum fulvum and Lathyrus sativus L., and we have tested reciprocal sexual crossings by manually cross-pollinating plants of genotypes of these three species. Studies of in situ germination of pollen grains on stigmata showed that pollen tubes were generally unable to germinate or could not reach the ovary. A few putative hybrid pods were nevertheless harvested, with one grain per pod germinated in vitro, then micropropagated for flow cytometry, isoenzyme, molecular (ribosomal ITS PCR-RFLP) and genomic in situ hybridization (GISH) studies. One such grain was recovered from an inter-generic cross of P. sativum x L. sativus and four from an inter-specific P. sativum x P. fulvumcross. A strong cross-incompatibility was shown between pea and grass pea, where the putative hybrid turned out to be pea. Conversely, with the interspecific, P. sativum x P. fulvum cross, flow cytometry and isoenzymes with leaf tissues strongly suggested hybridity, while molecular approaches and GISH confirmed the production of inter-specific hybrids, and without the need for a wild type P. sativum accession as a bridging cross.     

Phylogenetic relationships in the North African genus <Emphasis Type="Italic">Hedysarum</Emphasis> as inferred from ITS sequences of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were analysed to precise their length (637–643 bp) and resolve phylogenetic relationships among eight Mediterranean species of the genus Hedysarum (Fabaceae). The infra-specific variability levels of the ITS sequences of spontaneous population of H. coronarium proved a lack of polymorphism both in the length and in the sequences examined in this species. Hence, a consensus ITS sequence characterising each Hedysarum species has been investigated for analysis of inter-specific polymorphisms. The level of variation of ITS sequence was high enough to make the ITS1 and ITS2 a useful tool for phylogenetic reconstruction. However, ITS2 seems to be relatively more polymorphic and more informative than ITS1 regarding length or GC percent. The phylogenic relationships in the genus Hedysarum based on ITS1 and ITS2 sequences taken independently or together, are discussed in the context of current work in molecular biosystematics. Results exhibited the distinctiveness of the two H. spinosissimum subspecies (i.e. H. spinosissimum ssp. capitatum and H. spinosissimum ssp. spinosissimum). In addition, the great similarity of the ITS sequences between H. coronarium (the only cultivated species of the genus) and H. carnosum suggests the usefulness of the latter in selection programmes to improve pastoral production in semi-arid areas.