Identity of the downy mildew pathogens of basil, coleus, and sage with implications for quarantine measures

The downy mildew pathogen of basil (Ocimum spp.) has caused considerable damage throughout the past five years, and an end to the epidemics is not in sight. The downy mildew of coleus (Solenostemon spp.) is just emerging and here we report that it was very recently introduced into Germany. Although it has been recognised that these pathogens are a major threat, the identity of the pathogens is still unresolved, and so it is difficult to devise quarantine measures against them. Using morphological comparison and molecular phylogenetic reconstructions we confirmed in this study that the downy mildews of basil and coleus are unrelated to Peronospora lamii, which is a common pathogen of the weed Lamium purpureum. In addition, we conclude by the investigation of the type specimen of P. swingleii and downy mildew specimens on Salvia officinalis that the newly occurring pathogens are not identical to P. swingleii on Salvia reflexa. The taxonomy of the downy mildew pathogens of hosts from the Lamiaceae and, in particular, from the tribes Mentheae and Elsholtzieae, is discussed, and a new species is described to accommodate the downy mildew pathogen of basil and coleus, which is the first downy mildew pathogen known to be parasitic to hosts of the tribe Ocimeae.     

A revision of Bremia graminicola

Bremia graminicola (Chromista, Peronosporales) is a common downy mildew pathogen of Arthraxon spp. (Poaceae) in Central to East Asia and the only species of Bremia parasitic on grasses. Despite its widespread occurrence and apparent differences in host range and morphology compared with other species of the genus, its placement in Bremia has not been challenged for the past 90 y. Its current taxonomic position is revised based on sporangiophore morphology and ultrastructure, haustorium morphology, and nu-rDNA sequence analysis. Haustorium morphology and sporangiophore ultrastructure indicate that B. graminicola is not a member of the genus Bremia, which shows affinities to Plasmopara and Paraperonospora. Based on haustorium morphology, B. graminicola appears to be more closely related to Viennotia oplismeni, although the sporangiophore morphology is strikingly different between these two taxa. This is supported by molecular analyses based on a near-representative sample of nuLSU rDNA sequences of downy mildew genera, whereby B. graminicola is revealed as the sister taxon of V. oplismeni with 100 % BS support under all phylogenetic optimality criteria applied. Relationships of this clade to other groups are less clear. However, network and reduced-consensus analyses show that this lack of resolution is mainly due to the ambiguous molecular affinities of Sclerospora graminicola. Omitting this highly divergent taxon results in considerable support for a clade comprised of taxa with globose to pyriform haustoria, including B. lactucae, and for the sister-group relationship of B. graminicola and V. oplismeni with Hyaloperonospora. Consequently, a new genus, Graminivora, is described to accommodate B. graminicola.     

The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.     

Evolutionary history of the genus Trisopterus

The group of small poor cods and pouts from the genus Trisopterus, belonging to the Gadidae family, comprises four described benthopelagic species that occur across the North-eastern Atlantic, from the Baltic Sea to the coast of Morocco, and the Mediterranean. Here, we combined molecular data from mitochondrial (cytochrome b) and nuclear (rhodopsin) genes to confirm the taxonomic status of the described species and to disentangle the evolutionary history of the genus. Our analyses supported the monophyly of the genus Trisopterus and confirmed the recently described species Trisopterus capelanus. A relaxed molecular clock analysis estimated an Oligocene origin for the group (∼30 million years ago; mya) indicating this genus as one of the most ancestral within the Gadidae family. The closure and re-opening of the Strait of Gibraltar after the Messinian Salinity Crisis (MSC) probably triggered the speciation process that resulted in the recently described T. capelanus.     

Four powdery mildew species with catenate conidia infect Galium: molecular and morphological evidence

The Erysiphaceae are a group of obligately biotrophic fungi that cause powdery mildew disease of angiosperms. Due to their inability to be cultured on artificial media, the taxonomy of the Erysiphaceae has generally been based on the morphological characteristics of fresh and herbarium specimens. Thus, several morphological species with wide host ranges have long been maintained in this family, even though they clearly consist of several biological species. Erysiphe galii has been known as a powdery mildew of Galium spp. Recently, the former E. galii var. galii has been reassessed as Neoerysiphe galii and E. galii var. riedliana as Golovinomyces riedlianus, along with a taxonomic revision of the generic concept of the Erysiphaceae. The present study was conducted to evaluate the validity of the taxonomic revision of the two varieties of E. galii. During the course of this study, we found that the Galium powdery mildews consist of at least four different species, viz. Neoerysiphe galii, Golovinomyces orontii, G. riedlianus, and an unknown species collected in Argentina. The latter species is described as a new species, Golovinomyces calceolariae. The three species belonging to Golovinomyces are morphologically very similar to each other, i.e. the discrimination between them is rather difficult. The morphological differences of the three Golovinomyces species of Galium are discussed.     

Characterization of a Plasmopara species on Ambrosia artemisiifolia, and notes on P. halstedii, based on morphology and multiple gene phylogenies

Common ragweed (Ambrosia artemisiifolia) is an invasive and highly allergenic plant species, on which two species, Plasmopara halstedii and Plasmopara angustiterminalis, have been recognized to cause downy mildew disease. In this study, morphological and molecular patterns of seven Plasmopara specimens collected from A. artemisiifolia in Canada, Hungary, and USA were compared with those of P. halstedii and P. angustiterminalis from Helianthus and Xanthium, respectively. Analyses of partial sequences of three genes, namely those for the large subunit (28S) of rDNA, cytochrome c oxidase subunit II (COX2), and NADH dehydrogenase subunit I (ND1) of mtDNA, were carried out to examine the phylogenetic relationships among these specimens using both Bayesian and maximum parsimony methods. All the phylogenetic analyses revealed that the downy mildew pathogens infecting A. artemisiifolia in Hungary and North America clearly represent a lineage distinct from other Plasmopara taxa investigated. The shape of sporangia and the width of trunks and branches also allowed the separation of the specimens parasitic to A. artemisiifolia from P. halstedii on Helianthus annuus and P. angustiterminalis on Xanthium strumarium. Surprisingly, the Hungarian and the Canadian specimens were more closely related to each other than to those from the USA based on COX2 and ND1 mtDNA data, although the D1/D2/D3 sequences of 28S rDNA were identical in all these Plasmopara specimens. The regional distribution of the mtDNA haplotypes seen in this study suggests a transatlantic migration has occurred and would be interesting to follow up with a more detailed sampling. To investigate the diversity within P. halstedii sensu lato, infecting different host plant species, specimens from six asteraceous genera, Ambrosia, Flaveria, Helianthus, Siegesbeckia, Solidago, and Xanthium, were also included in molecular analyses. These represented six distinct lineages according to the host plant genera. These findings might serve as a basis for a taxonomical reassessment of the P. halstedii complex and also for the delimitation of several well-defined species within this complex.     

Phylogeny of Peronospora, parasitic on Fabaceae, based on ITS sequences

Species concepts are a notoriously difficult taxonomic problem in plant–parasitic fungal-like organisms such as downy mildews (Peronosporomycetes, Peronosporales). This is particularly evident in the largest downy mildew genus, Peronospora, which contains a number of economically important pathogens. Here, we investigate relationships of Peronospora species infecting Fabaceae (angiosperms, Rosidae) originating from various collections from different species of host plants and from different European locations by molecular phylogenetic analysis of ITS sequences. Molecular trees were inferred with ML, MP and Bayesian methods and rooted with Pseudoperonospora. As in other downy mildew groups, molecular data mainly support the use of narrow species delimitations and host range as a taxonomic marker. Fabaceae parasites appear to be subdivided into a number of lineages displaying a considerable degree of host specialization with respect to host genera, as well as host subgenera or species. The number of repeats of a repetitive part of the ITS1 is, within limits, characteristic of subgroups within the cluster of Trifolium parasites. We reveal new hosts for Peronospora found on the Iberian Peninsula.     

Are the microcyclic rust species Puccinia melampodii and Puccinia xanthii conspecific?

The taxonomic relationship between two microcyclic rust species, Puccinia melampodii and Puccinia xanthii, recorded from a number of Asteraceae hosts, was explored by comparing teliospore morphology, host specificity and sequence data. Teliospore morphology varied between and within individual rust accessions but, in general, teliospores of P. xanthii were larger than those of P. melampodii. Field observations and cross-inoculation greenhouse studies showed that P. melampodii accessions were highly specific to their original host species and unable to establish compatible interactions with related Asteraceae species. Though small amounts of genetic variation were detected between P. melampodii and P. xanthii accessions, the ITS and TEF phylogenetic analyses comprising several other rust species grouped these accessions within a well supported clade. Our data indicate that despite differences between P. xanthii and P. melampodii accessions these rusts cannot readily be separated at the species level, supporting earlier taxonomic conclusions and the recent proposal to group them into a morphospecies. Based on host-specificity data, the name Puccinia xanthii var. parthenii-hysterophorae is proposed to accommodate all Mesoamerican records of P. melampodii associated with the host Parthenium hysterophorus.     

Is the mega-diverse genus Ocyptamus (Diptera, Syrphidae) monophyletic? Evidence from molecular characters including the secondary structure of 28S rRNA

Phylogenetic relationships between two New World Syrphinae taxa (Diptera, Syrphidae), i.e. the highly diverse genus Ocyptamus and the large genus Toxomerus, were analysed based on molecular characters. The monophyly of both taxa was tested and the taxonomic status of included subgenera and species groups was examined. Toxomerus constitutes the monogeneric tribe Toxomerini with more than 140 described species, while Ocyptamus (tribe Syrphini) is a very diverse genus (over 300 spp.) with multiple recognised subgenera and species groups. Sequence data from three gene regions were used: the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI) and the nuclear 28S and 18S ribosomal RNA genes. The secondary structure of two expansion segments (D2, D3) of the ribosomal 28S RNA gene is presented for the family Syrphidae and used for the first time in a multiple sequence alignment. Molecular data were analysed using parsimony, maximum likelihood and Bayesian inference. Toxomerus was always recovered as monophyletic within Ocyptamus, and relationships to other New World taxa such as Salpingogaster (Eosalpingogaster) were well-supported. Only the subgenera and species groups of Ocyptamus were consistently recovered as monophyletic lineages, thus the apparent non-monophyly of Ocyptamus demands reclassification of this clade.     

Phylogeny and phylogeography of the genus Patella based on mitochondrial DNA sequence data

Although some molecular phylogenies of Patella species have been published in recent years, unresolved questions concerning the phylogeny and taxonomy of this genus still remain. We sequenced the mitochondrial genes cytochrome c oxidase subunit I, 12S rRNA and 16S rRNA for all Patella species (Patella vulgata, Patella depressa, Patella candei, Patella caerulea, Patella lugubris, Patella ferruginea, Patella pellucida and the continental and Macaronesian forms of Patella ulyssiponensis and Patella rustica) and Cymbula safiana. Our results revealed the occurrence of five strongly supported clades, although relationships between these are not well supported. According to our data P. vulgata is a genetically distinct lineage and the close phylogenetic relationship between this species and P. depressa, found in previous mitochondrial DNA studies, is not supported. The Mediterranean Sea and the Macaronesian Islands seem to have played an important role in the speciation and diversification of this genus, although different clades show different phylogeographic patterns. Our dataset point to the necessity of a taxonomic revision, as P. candei is paraphyletic relative to P. lugubris.     

Tree-based delimitation of morphologically ambiguous taxa: a study of the lizard malaria parasites on the Caribbean island of Hispaniola

Malaria parasites in the genus Plasmodium have been classified primarily on the basis of differences in morphology. These single-celled organisms often lack distinguishing morphological features, and this can encumber both species delimitation and identification. Six saurian malaria parasites have been described from the Caribbean island of Hispaniola. All six infect lizards in the genus Anolis, but only two of these parasites can be distinguished using morphology. The remaining four species overlap in morphology and geography, and cannot be consistently identified using traditional methods. We compared a morphological approach with a molecular phylogenetic approach for assessing the taxonomy of these parasites. We surveyed for blood parasites from 677 Anolis lizards, representing 26 Anolis spp. from a total of 52 sites across Hispaniola. Fifty-five of these lizards were infected with Plasmodium spp., representing several new host records, but only 24 of these infections could be matched to previously described species using traditional morphological criteria. We then estimated the phylogeny of these parasites using both mitochondrial (cytb and coxI) and nuclear (EF2) genes, and included carefully selected GenBank sequences to confirm identities for certain species. Our molecular results unambiguously corroborated our morphology-based species identifications for only the two species previously judged to be morphologically distinctive. The remaining infections fell into two well-supported and reciprocally monophyletic clades, which contained the morphological variation previously reported for all four of the morphologically ambiguous species. One of these clades was identified as Plasmodium floridense and the other as Plasmodium fairchildi hispaniolae. We elevate the latter to Plasmodium hispaniolae comb. nov. because it is polyphyletic with the mainland species Plasmodium fairchildifairchildi and we contribute additional morphological and molecular characters for future species delimitation. Our phylogenetic hypotheses indicate that two currently recognised taxa, Plasmodium minasense anolisi and Plasmodium tropiduri caribbense, are not valid on Hispaniola. These results illustrate that molecular data can improve taxonomic hypotheses in Plasmodium when reliable morphological characters are lacking.     

Phylogeny of <Emphasis Type="Italic">Hyaloperonospora</Emphasis> based on nuclear ribosomal internal transcribed spacer sequences

Phylogenetic relationships in Hyaloperonospora (Oomycetes) were investigated by molecular analyses using internal transcribed spacer (ITS) sequences and collections from different host plants. Trees were inferred with Bayesian Markov chain Monte Carlo, neighbor-joining and maximum parsimony methods and rooted with Perofascia. The results are discussed with respect to host taxonomy and species concepts of downy mildews from the literature. Molecular data mainly support the use of narrow species delimitations and host range as a taxonomic marker. Hyaloperonospora brassicae turns out to be a non-monophyletic assemblage of different species. New combinations are proposed in accordance with the phylogenetic trees.     

Pollen morphology of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae): Taxonomic implications

In order to find new non-molecular evidence to support the phylogenetic and taxonomic position, pollen grains of 20 populations of 16 species of Chinese Curcuma L. and Boesenbergia Kuntz (Zingiberaceae) were investigated under SEM and TEM. The pollen grains are spherical and ovoid, nonaperturate. The pollen wall is composed of a very thin exine and a thick intine. The exine is psilate or echinate. The intine consists of two layers, i.e., a thick, channeled layer (exintine) and an inner homogenous layer (endintine). The results reveal morphological congruence between the pollen grains of species of Curcuma, which according to DNA sequence data appears to be a polyphyletic genus. However the uniform pollen morphology in Curcuma provides no evidence to divide this genus into separate taxonomic entities. Our results on pollen morphology also do not provide any additional evidence to either unite or segregate Boesenbergia albomaculata and Curcumorpha longiflora in the same genus and demonstrate that more taxonomic data on the genus Boesenbergia and its relatives are needed before a final decision can be made.     

Molecular insight into systematics,host associations,life cycles and geographic distribution of the nematode family Rhabdiasidae

Rhabdiasidae Railliet, 1915 is a globally distributed group of up to 100 known species of nematodes parasitic in amphibians and reptiles. This work presents the results of a molecular phylogenetic analysis of 36 species of Rhabdiasidae from reptiles and amphibians from six continents. New DNA sequences encompassing partial 18S rDNA, ITS1, 5.8S rDNA, ITS2 and partial 28S rDNA regions of nuclear ribosomal DNA were obtained from 27 species and pre-existing sequences for nine species were incorporated. The broad taxonomic, host and geographical coverage of the specimens allowed us to address long-standing questions in rhabdiasid systematics, evolution, geographic distribution, and patterns of host association. Our analysis demonstrated that rhabdiasids parasitic in snakes are an independent genus sister to the rest of the Rhabdiasidae, a status supported by life cycle data. Based on the combined evidence of molecular phylogeny, morphology and life cycle characteristics, a new genus Serpentirhabdias gen. nov. with the type species Serpentirhabdias elaphe (Sharpilo, 1976) comb. nov. is established. The phylogeny supports the monophyly of Entomelas Travassos, 1930, Pneumonema Johnston, 1916 and the largest genus of the family, Rhabdias Stiles and Hassall, 1905. DNA sequence comparisons demonstrate the presence of more than one species in the previously monotypic Pneumonema from Australian scincid lizards. The distribution of some morphological characters in the genus Rhabdias shows little consistency within the phylogenetic tree topology, in particular the apical structures widely used in rhabdiasid systematics. Our data suggest that some of the characters, while valuable for species differentiation, are not appropriate for differentiation among higher taxa and are of limited phylogenetic utility. Rhabdias is the only genus with a cosmopolitan distribution, but some of the lineages within Rhabdias are distributed on a single continent or a group of adjacent zoogeographical regions. Serpentirhabdias, Entomelas and Pneumonema show rather strict specificity to their host groups. The evolution of the Rhabdiasidae clearly included multiple host switching events among different orders and families of amphibians as well as switching between amphibians and squamatan reptiles. Only a few smaller lineages of Rhabdias demonstrate relatively strict associations with a certain group of hosts.     

Hebeloma species associated with Cistus

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.     

<Emphasis Type="Italic">Perofascia</Emphasis> is not monotypic: the description of the second taxon affecting the South American crop maca (<Emphasis Type="Italic">Lepidium meyenii</Emphasis>)

Maca (Lepidium meyenii) is an Andean crop of narrow distribution, but because of the nutritional and health value, its cultivation area is rapidly expanding. By a broad-spectrum resistance mechanism against various pathogens, just a few diseases have been reported on maca, among which downy mildew is a potential threat to its cultivation. The occurrence of this disease was, so far, restricted to the native area of maca. However, here we report that it was recently introduced into South Korea. As the causal pathogen has initially been attributed to Hyaloperonospora parasitica (syn. Peronospora parasitica), which was thought to affect various Brassicaceae, but is, in fact, restricted to Capsella bursa-pastoris, the identity of this pathogen remains uncertain. In this study, morphological and phylogenetic data revealed that maca downy mildew is unrelated to any species of Hyaloperonospora and instead belongs to the previously monotypic genus Perofascia. It differs markedly from the type species, Perofascia lepidii, and consequently Perofascia macaicola sp. nov. is described and illustrated here. Considering the quick expansion of cultivated land with maca, quarantine measures for this pathogen might be appropriate for hindering the spread of the disease through the international trade of maca.     

Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa

Downy mildew is probably the most widespread and potentially destructive global disease of spinach (Spinacia oleracea). The causal agent of downy mildew disease on various plants of Chenopodiaceae, including spinach, is regarded as a single species, Peronospora farinosa. In the present study, the ITS rDNA sequence and morphological data demonstrated that P. farinosa from S. oleracea is distinct from downy mildew of other chenopodiaceous hosts. Fifty-eight spinach specimens were collected or loaned from 17 countries of Asia, Europe, Oceania, North and South America, which all formed a distinct monophyletic group. No intercontinental genetic variation of the ITS rDNA within Peronospora accessions causing spinach downy mildew disease was found. Phylogenetic trees supported recognition of Peronospora from spinach as a separate species. Microscopic examination also revealed morphological differences between Peronospora specimens from Spinacia and P. farinosa s. lat. specimens from Atriplex, Bassia, Beta, and Chenopodium. Consequently, the name Peronospora effusa should be reinstated for the downy mildew fungus found on spinach. Here, a specimen of the original collections of Peronospora effusa is designated as lectotype.