Characteristics of Ralstonia solanacearum Biovar N2 Strains in Asia

Ralstonia solanacearum biovar N2 strains isolated in Asia were compared by biochemical tests with biovar N2 strains from South America and biovar 2 (race 3) strains from Africa, America, Asia and Europe. Distinct differences were found between Asian and South American strains of biovar N2, and between Asian biovar N2 and biovar 2 strains with respect to their ability to utilize several carbon sources. Using cluster analysis based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) genomic fingerprints, the Asian biovar N2 strains were divided into two groups, group 1 containing Japanese strains and group 2 containing Indonesian and Philippine strains. The fingerprints showed the genetic diversity of biovar N2 strains in Asia.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY,TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES1

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.     

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models

Among ciliates, Paramecium has become a privileged model for the study of “species problem” particularly in the case of the “Paramecium aurelia complex” that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5′LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.     

A multigene phylogeny of theGibberella fujikuroi species complex: Detection of additional phylogenetically distinct species

Phylogenetic relationships within theGibberella fujikuroi species complex were extended to newly discovered strains using nucleotide characters obtained by sequencing polymerase chain reaction (PCR) amplified DNA from 4 loci used in a previous study [nuclear large subunit 28S rDNA, nuclear ribosomal internal transcribed spacer (ITS) region, mitochondriaal small subunit (mtSSU) ribosomal DNA, and β-tubulin] together with two newly sampled protein-encoding nuclear genes, translation elongation factor EF-1α and calmodulin. Sequences from the ribosomal ITS region were analyzed separately and found to contain of two highly divergent, nonorthologous ITS2 types. Phylogenetic analysis of the individual and combined datasets identified 10 new phylogenetically distinct species distributed among the following three areas: 2 within Asia and 4 within both Africa and South America. Hypotheses of the monophyly ofFusarium subglutinans and its two formae speciales, f. sp.pini and f. sp.ananas, were strongly rejected by a likelihood analysis. Maximum parsimony results further indicate that the protein-encoding nuclear genes provide considerably more phylogenetic signal that the ribosomal genes sequenced. Relative apparent synapomorphy analysis was used to detect long-branch attraction taxa and to obtain a statistical measure of phylogenetic signal in the individual and combined datasets.     

Molecular diversity of oligotrophic and neurotropic members of the black yeast genus Exophiala,with accent on E. dermatitidis

Analysis of ITS rDNA of the black yeast Exophiala dermatitidis revealed a close phylogenetic relationship to the meristematic fungus Sarcinomyces phaeomuriformis. As most strains ofS. phaeomuriformis have a yeast-like phenotype corresponding to the anamorph genus Exophiala, a new combination in Exophiala is proposed. On the basis of ITS sequence, M-13 fingerprint and SSU intron data, two main entities could be distinguished within E. dermatitidis. One of these (B) contained prevalently strains from environmental sources, while the other (A) mainly comprised strains from clinical sources. This may be due to a difference in virulence. All strains from severe brain and disseminated infections in East Asia clustered in group A. However, strains of group A caused a relatively mild fungemia in patients outside East Asia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic study of Geitlerinema and Microcystis (Cyanobacteria) using PC‐IGS and 16S–23S ITS as markers: investigation of horizontal gene transfer

Selection of genes that have not been horizontally transferred for prokaryote phylogenetic inferences is regarded as a challenging task. The markers internal transcribed spacer of ribosomal genes (16S–23S ITS) and phycocyanin intergenic spacer (PC‐IGS), based on the operons of ribosomal and phycocyanin genes respectively, are among the most used markers in cyanobacteria. The region of the ribosomal genes has been considered stable, whereas the phycocyanin operon may have undergone horizontal transfer. To investigate the occurrence of horizontal transfer of PC‐IGS, phylogenetic trees of Geitlerinema and Microcystis strains were generated using PC‐IGS and 16S–23S ITS and compared. Phylogenetic trees based on the two markers were mostly congruent for Geitlerinema and Microcystis, indicating a common evolutionary history among ribosomal and phycocyanin genes with no evidence for horizontal transfer of PC‐IGS. Thus, PC‐IGS is a suitable marker, along with 16S–23S ITS for phylogenetic studies of cyanobacteria.     

西南地区6种魔芋属植物基于cpDNA序列的遗传多样性研究

研究野生作物资源的遗传变异及分化机制对种质资源的收集与改良具有重要意义。魔芋是我国西南地区的特色经济作物,但由于受到人为活动干扰,野生种群不断衰退。为评估西南地区魔芋属(Amorphophallus)野生群体的遗传多样性,探究代表性物种的系统发育地位,该研究利用3个叶绿体DNA(cpDNA)片段,分析了魔芋6个物种的遗传多样性,重建了种间系统发育关系。结果表明:(1)西南地区野生魔芋群体的遗传多样性普遍较低,虽然单倍型多样性(H_d)均值为0.428,但近一半群体只有1个单倍型,6个物种整体水平上的单倍型多样性在0.704到0.983之间。(2)在6个物种间检测到高水平的遗传分化,遗传分化系数(F_ST)值在0.481到0.967之间。(3)系统发育分析表明,选取的27个魔芋种主要聚成3个分支:非洲分支、东南亚分支和东亚大陆分支。疣柄魔芋(A. paeoniifolius)隶属于东南亚分支,而东亚大陆分支A包含花魔芋(A. konjac)和西盟魔芋(A. krausei),东亚大陆分支B由东亚魔芋(A. kiusianus)、滇魔芋(A. yunnanensis)和东京魔芋(A. tonkinensis)构成。生境隔离与人为干扰造成了西南地区野生魔芋群体较低的遗传多样性,魔芋属东亚大陆分支的分化可能与早期的快速扩张和生态适应有关。该研究为西南地区魔芋资源的合理保护、可持续利用和杂交育种提供了参考资料。     

Molecular markers for tracking the origin and worldwide distribution of invasive strains of Puccinia striiformis

Investigating the origin and dispersal pathways is instrumental to mitigate threats and economic and environmental consequences of invasive crop pathogens. In the case of Puccinia striiformis causing yellow rust on wheat, a number of economically important invasions have been reported, e.g., the spreading of two aggressive and high temperature adapted strains to three continents since 2000. The combination of sequence‐characterized amplified region (SCAR) markers, which were developed from two specific AFLP fragments, differentiated the two invasive strains, PstS1 and PstS2 from all other P. striiformis strains investigated at a worldwide level. The application of the SCAR markers on 566 isolates showed that PstS1 was present in East Africa in the early 1980s and then detected in the Americas in 2000 and in Australia in 2002. PstS2 which evolved from PstS1 became widespread in the Middle East and Central Asia. In 2000, PstS2 was detected in Europe, where it never became prevalent. Additional SSR genotyping and virulence phenotyping revealed 10 and six variants, respectively, within PstS1 and PstS2, demonstrating the evolutionary potential of the pathogen. Overall, the results suggested East Africa as the most plausible origin of the two invasive strains. The SCAR markers developed in the present study provide a rapid, inexpensive, and efficient tool to track the distribution of P. striiformis invasive strains, PstS1 and PstS2.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

     

Tropical Asian species show that the Old World clade of ‘spiny solanums’ (Solanum subgenus Leptostemonum pro parte: Solanaceae) is not monophyletic

The tropical Asian taxa of the species‐rich genus Solanum (Solanaceae) have been less well studied than their highly diverse New World relatives. Most of these tropical Asian species, including the cultivated brinjal eggplant/aubergine and its wild progenitor, are part of the largest monophyletic Solanum lineage, the ‘spiny solanums’ (subgenus Leptostemonum or the Leptostemonum clade). Here we present the first phylogenetic analysis of spiny solanums that includes broad sampling of the tropical Asian species, with 42 of the 56 currently recognized species represented. Two nuclear and three plastid regions [internal transcribed spacer (ITS), waxy, ndhF‐rpL32, trnS‐trnG and trnT‐trnF] were amplified and used to reconstruct phylogenetic relationships using maximum likelihood and Bayesian methods. Our analyses show that Old World spiny solanums do not resolve in a single clade, but are part of three unrelated lineages, suggesting at least three independent introductions from the New World. We identify and describe several monophyletic groups in Old World solanums that have not been previously recognized. Some of these lineages are coherent in terms of morphology and geography, whereas others show considerable morphological variation and enigmatic distribution patterns. Tropical Asia occupies a key position in the biogeography of Old World spiny solanums, with tropical Asian taxa resolved as the closest relatives of diverse groups of species from Australia and Africa.     

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n = 60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene.Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.     

High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia

The genus Micromonospora has been found in nodules of several legumes and some new species of this genus were isolated from these plant organs. In this study we analysed the taxonomic diversity of Micromonospora strains isolated from alfalfa nodules in Spain and Australia on the basis of three phylogenetic markers, the rrs and gyrB genes and 16S-23S intergenic spacer (ITS). The genome analysis of selected strains representative of different clusters or lineages found after rrs, gyrB and ITS analyses confirmed the results obtained with these phylogenetic markers. They showed that the analysed strains belong to at least 18 Micromonospora species including previously described ones, such as Micromonospora noduli, Micromonospora ureilytica, Micromonospora taraxaci, Micromonospora zamorensis, Micromonospora aurantiaca and Micromonospora tulbaghiae. Most of these strains belong to undescribed species of Micromonospora showing the high taxonomic diversity of strains from this genus inhabiting alfalfa nodules. Although Micromonospora strains are not able to induce the formation of these nodules, and it seems that they do not contribute to fix atmospheric nitrogen, they could play a role related with the mechanisms of plant growth promotion and pathogen protection presented by Micromonospora strains isolated from legume nodules.     

Molecular profiling demonstrates limited diversity amongst geographically separate strains of Ustilago scitaminea

Intraspecies diversity within Ustilago scitaminea isolates from South Africa, Reunion Island, Hawaii and Guadeloupe was assessed by RAPDs, bE mating-type gene detection, rDNA sequence analysis, microscopy and germination and morphological studies. Except for sequence data, the other analyses yielded no differences in the isolates that could be used in a phylogenetic separation. Mycelial DNA of the SA isolate shared 100% sequence identity with that of mycelial DNA cultured from in vitro produced teliospores of the parent cultivar. Overall the ITS1 and ITS2 regions were found to have 96.1% and 96.9% sequence identity with a total of 17 and 21 base changes, respectively, amongst the isolates. The Reunion Island isolate was shown to be most distantly related by 3.6% to the other isolates, indicating a single clonal lineage. The lack of germination in teliospores from Guadeloupe may be attributed to changes in temperature and humidity during transportation.     

African origin and global distribution patterns: Evidence inferred from phylogenetic and biogeographical analyses of ectomycorrhizal fungal genus Strobilomyces

Aim

The ectomycorrhizal genus Strobilomyces is widely distributed throughout many parts of the world, but its origin, divergence and distribution patterns remain largely unresolved. In this study, we aim to explore the species diversity, distribution and evolutionary patterns of Strobilomyces on a global scale by establishing a general phylogenetic framework with extensive sampling.

Location

Africa, Australasia, East Asia, Europe, North America, Central America and Southeast Asia.

Methods

The genealogical concordance phylogenetic species recognition method was used to delimit phylogenetic species. Divergence times were estimated using a Bayesian uncorrelated lognormal relaxed molecular clock. The ancestral area and host of Strobilomyces were inferred via the programs rasp and mesquite . The change of diversification rate over time was estimated using Ape, Laser and Bammtools software packages.

Results

We recognize a novel African clade and 49 phylogenetic species with morphological evidence, including 18 new phylogenetic species and 23 previously described ones. Strobilomyces probably originated in Africa, in association with Detarioideae/Phyllanthaceae/Monotoideae during the early Eocene. The dispersal to Southeast Asia can be explained by Wolfe's “Boreotropical migration” hypothesis. East Asia, Australasia, Europe and North/Central America are primarily the recipients of immigrant taxa during the Oligocene or later. A rapid radiation implied by one diversification shift was inferred within Strobilomyces during the Miocene.

Main conclusions

An unexpected phylogenetic species diversity within Strobilomyces was uncovered. The highest diversity, resulting probably from a rapid radiation, was found in East Asia. Dispersal played an important role in the current distribution pattern of Strobilomyces. The Palaeotropical disjunction is explained by species dispersal from Africa to Southeast Asia through boreotropical forests during the early Eocene. Species from the Northern Hemisphere and Australasia are largely derived from immigrant ancestors from Southeast Asia.     

A phylogenetic study ofPolygonum sect.Tovara (Polygonaceae) based on ITS sequences of nuclear ribosomal DNA

Polygonum sect.Tovara comprises three morphologically very similar species;P. virginianum,P. filiforme, andP. neofiliforme. Sequences of internal transcribed spacers (ITSs) of nuclear ribosomal DNA of these were determined to examine phylogenetic relationships and the levels of differentiation among them. The size of ITS 1 was 241 bp inP. filiforme andP. neofiliforme, and 242 bp inP. virginianum. The size of ITS 2 was 243 bp, and that of the 5.8S rRNA coding region was 163 bp. The ITS sequences clearly separate North AmericanP. virginianum from the eastern Asian species. Nucleotide divergence between them ranges from 3.3% to 3.8% for ITS 1 and from 9.3% to 10.7% for ITS 2. The molecular data also revealed that two eastern Asian species are closely related but should be treated as distinct species.     

Genetic characterization of Mortierella alpina by sequencing the 18S‐28S ribosomal gene internal transcribed spacer region

Aims: The aim of this study was to establish a genetic background of Mortierella alpina, which is a cosmopolitan, soil‐inhabiting Zygomycete that also has important biotechnology potential. Methods and Results: A total of 44 18S‐28S ribosomal gene internal transcribed spacer (ITS1‐5·8S rDNA‐ITS2 DNA) regions of M. alpina from three diverse locations (Far East Asia, North America and West‐Central Europe) were sequenced and investigated. The sequences between M. alpina and the three closely related species (Mortierella macrocystis, Mortierella gamsii and Mortierella humilis) showed 74–84% sequence identity. When a phylogenetic tree was constructed with a neighbour‐joining algorithm, four clades of M. alpina isolates were clearly distinct with high bootstrap values. In addition, isolates from the West‐Central Europe were found to have the highest gene and nucleotide diversities. Conclusions: The ITS region was a suitable tool for distinguishing M. alpina from other closely related species. The region also provided information of the diversity of M. alpina. Significance and Impact of the Study: The study established a genetic background of M. alpina for identification and the diversity of M. alpina provided information for further isolation and screening of the fungus.     

Recent emergence and worldwide spread of the red tomato spider mite, <Emphasis Type="Italic">Tetranychus evansi</Emphasis>: genetic variation and multiple cryptic invasions

Plant biosecurity is increasingly challenged by emerging crop pests. The spider mite Tetranychus evansi has recently emerged as a new threat to solanaceous crops in Africa and the Mediterranean basin, with invasions characterized by a high reproductive output and an ability to withstand a wide range of temperatures. Mitochondrial (868 bp of COI) and nuclear (1,137 bp of ITS) loci were analyzed in T. evansi samples spanning the current geographical distribution to study the earliest stages of the invasive process. The two sets of markers separate the samples into two main clades that are only present together in South America and Southern Europe. The highest COI diversity was found in South America, consistent with the hypothesis of a South American origin of T. evansi. Among the invaded areas, the Mediterranean region displayed a high level of genetic diversity similar to that present in South America, that is likely the result of multiple colonization events. The invasions of Africa and Asia by T. evansi are characterized by a low genetic variation associated with distinct introductions. Genetic data demonstrate two different patterns of invasions: (1) populations in the Mediterranean basin that are a result of multiple cryptic introductions and (2) emerging invasions of Africa and Asia, each likely the result of propagules from one or limited sources. The recent invasions of T. evansi illustrate not only the importance of human activities in the spread of agricultural pests, but also the limits of international quarantine procedures, particularly for cryptic invasions.