Phytopythium: molecular phylogeny and systematics.

The genus Phytopythium (Peronosporales) has been described, but a complete circumscription has not yet been presented. In the present paper we provide molecular-based evidence that members of Pythium clade K as described by Lévesque & de Cock (2004) belong to Phytopythium. Maximum likelihood and Bayesian phylogenetic analysis of the nuclear ribosomal DNA (LSU and SSU) and mitochondrial DNA cytochrome oxidase subunit 1 (COI) as well as statistical analyses of pairwise distances strongly support the status of Phytopythium as a separate phylogenetic entity. Phytopythium is morphologically intermediate between the genera Phytophthora and Pythium. It is unique in having papillate, internally proliferating sporangia and cylindrical or lobate antheridia. The formal transfer of clade K species to Phytopythium and a comparison with morphologically similar species of the genera Pythium and Phytophthora is presented. A new species is described, Phytopythium mirpurense.

The expansion of Phytophthora clade 8b: three new species associated with winter grown vegetable crops.

Despite its association with important agricultural crops, Phytophthora clade 8b is a poorly studied group of species. The clade currently consists of three officially described species (Phytophthora porri, P. brassicae and P. primulae) that are host-specific pathogens of leek, cabbages and Primula spp., respectively. However, over the past few decades, several other clade 8b-like Phytophthoras have been found on a variety of different host plants that were all grown at low temperatures in winter seasons. In this study, a collection of 30 of these isolates was subjected to a phylogenetic study using two loci (the rDNA ITS region and the mitochondrial cox1 gene). This analysis revealed a clear clustering of isolates according to their host plants. To verify whether these isolates belong to separate species, a detailed morphological study was conducted. On the basis of genetic and morphological differences and host specificity, we now present the official description of three new species in clade 8b: Phytophthora cichorii sp. nov., P. dauci sp. nov. and P. lactucae sp. nov. Two other groups of isolates (Phytophthora taxon castitis and Phytophthora taxon parsley) might also represent new species but the data available at this time are insufficient for an official description. This brings Phytophthora clade 8b to a group of six species that are all host-specific, slow-growing and specifically infect herbaceous crops at low temperatures.

Taxonomy, DNA barcoding and phylogeny of three new species of Pythium from Canada.

Three new species of Pythium, namely, P. oopapillum, P. emineosum and P. camurandrum are presented in this paper based on morphological descriptions and molecular phylogenetic characterisation. These new species were isolated from various ecological regions in Canada. They have unique morphological features in the genus Pythium, and form distinct clades in maximum parsimony analyses, which are also supported by maximum likelihood phylogeny using general time reversible model (GTR), and Bayesian inference (BI) phylogeny using Markov Chain Monte Carlo (MCMC) analysis methods. A comparative study of the new species with closely related taxa, their clade positions, and morphological features are described in this paper.

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons.

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.

Oligonucleotide array for identification and detection of pythium species.

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.

Taxonomy and phylogeny of the ascomycetous yeast genus Zygoascus, with proposal of Zygoascus meyerae sp. nov. and related anamorphic varieties.

Physiological characters, mating compatibility, PCR-RAPD fingerprints, mol% G + C content, DNA-DNA relatedness, and large-subunit and internal transcribed spacer rRNA gene sequences of strains assigned to the genus Zygoascus were re-examined. On the basis of those data, and after phylogenetic analyses, an emendation of Zygoascus hellenicus (type material is a cross of CBS 6736(T) x CBS 5839(T)) is proposed, comprising two novel anamorphic varieties, Candida steatolytica var. steatolytica (CBS 6736(T)) and C. steatolytica var. inositophila (CBS 5839(T)). A novel teleomorphic species, Zygoascus meyerae sp. nov. (type material is a cross of CBS 4099(T) x CBS 7521(T)) is described, together with two novel anamorphic varieties corresponding to it, Candida hellenica var. hellenica (CBS 4099(T)) and C. hellenica var. acidophila (CBS 7115(T)).

Re-examination of some species of the genus Geotrichum Link: Fr.

The nutritional physiology and the growth rate of thirty-four strains representing species of Geotrichum without known teleomorph states were examined. From twenty-seven strains the mol% G+C were calculated from the DNA melting curves. The first derivatives of the melting curves of seven strains, including the type strain of Geotrichum clavatum, demonstrated the presence of two peaks, 12% away from each other; the remaining strains showed only a single broad peak. DNA homology values among strains of the former group were high, indicating their conspecificity. The strains of the latter group could be subdivided into six DNA homology groups, four of which could be identified with recognized species and two may represent novel taxa. A combined key of Geotrichum and its teleomorph states Galactomyces and Dipodascus is presented.

Natural Hybrids of Phytophthora nicotianae and Phytophthora cactorum Demonstrated by Isozyme Analysis and Random Amplified Polymorphic DNA.

ABSTRACT Three similar isolates of Phytophthora (Phytophthora sp-h) were obtained from diseased Spathiphyllum and Primula plants. Cultural characteristics did not fit any known description of Phytophthora species. The Phytophthora sp-h isolates are papillate, are homothallic, possess 80 to 86% amphigynous antheridia, and have a maximum temperature for growth of 36.5 degrees C. Isozyme analysis of the Phytophthora sp-h isolates revealed a three-banded pattern with malic enzyme and a three-banded pattern with malate dehydrogenase on the second putative locus. The fastest band at both enzyme loci comigrated with the single P. nicotianae band, the slowest band comigrated with the single P. cactorum (and also P. pseudotsugae) band, and one band in between was concluded to represent the heterodimeric isozyme. The random amplified polymorphic DNA patterns of the Phytophthora sp-h isolates almost exclusively consisted of bands that were also present in either P. nicotianae or P. cactorum. Southern hybridization showed that bands specific for P. nicotianae were present as comigrating bands in the Phytophthora sp-h isolates. The same was found for species-specific bands of P. cactorum. It is concluded that the three Phytophthora sp-h isolates represent interspecific hybrids, P. nicotianae being the one parent and P. cactorum the other. Analysis of mito-chondrial DNA with restriction enzymes revealed banding patterns in all the Phytophthora sp-h isolates identical with those of P. nicotianae, confirming that indeed P. nicotianae was one of the parents.

Identification of some oomycetes by reverse dot blot hybridization.

ABSTRACT An assay was developed that can identify unknown isolates of Pythium or Phytophthora species in a single hybridization. This reverse dot blot system is based on arrays of species-specific amplified fragments or oligonucleotides derived from the internal transcribed spacer (ITS) region, which are blotted as dots on a nylon membrane. By using total DNA from a sample as the template, universal primers, and digoxigenin-dUTP, the ITS was amplified and labeled simultaneously by the polymerase chain reaction (PCR). A small aliquot of the resultant labeled and amplified product was used as a probe for hybridization to a dot blot membrane that contained the immobilized species-specific oligonucleotides or amplified PCR fragments. The reverse dot blot system based on arrays of oligonucleotides showed far fewer cross-hybridizations than one based on entire amplified ITS I fragments. Unknown species can be identified simply by visualizing the positive hybridization reaction between the DNA labeled directly from the sample and the immobilized specific oligonucleotide. Currently, the assay can be used to identify Pythium aphanidermatum, P. ultimum, P. acanthicum, and Phytophthora cinnamomi. An oligonucleotide that was originally designed to identify Phytophthora hybridized to 10 of the 14 Phytophthora species tested. Another oligonucleotide designed to identify oomycetes hybridized to the 68 species tested, which represented two of the four orders of this phylum.

Genome comparisons in the yeastlike fungal genus Galactomyces Redhead et Malloch.

The G+C contents of the DNAs of 41 strains belonging to the genus Galactomyces Redhead et Malloch were determined by the thermal denaturation method. Melting profiles revealed that the DNAs of these strains are heterogeneous. Four groups were recognized on the basis of this heterogeneity. However, DNA similarity values, which were calculated by using DNA-DNA reassociation kinetics, revealed that the strains could be divided into six subgroups. Strains belonging to the same subgroup exhibited high levels of DNA similarity (84 to 100%). The members of two subgroups, corresponding to Galactomyces citri-aurantii and Galactomyces reessii, exhibited low levels of DNA similarity with the members of the other subgroups (20 to 27%). The members of the four remaining subgroups, which contained only strains previously identified as Galactomyces geotrichum, exhibited intermediate levels of reassociation (41 to 59%). Some combinations of phenotypic characteristics correlated with the subgroups; a key based on phenotypic characteristics that can be used to distinguish the subgroups is presented.

Polymerase chain reaction-mediated genotyping of Hortaea werneckii, causative agent of tinea nigra.

The black yeast Hortaea werneckii is known to be a causative agent of human tinea nigra but is also found in the environment. Strains from dissimilar sources were studied by polymerase chain reaction fingerprinting of nuclear DNA, using primers annealing to repetitive and random sequences. The seven groups found correspond to those known from restriction fragment length polymorphism (RFLP) studies of the mitochondrial DNA of the same strains. Two main groups contained strains from human as well as from non-human sources. The human strains did not cluster, but were randomly distributed over several populations. It was concluded that these strains are not pathogenic. The factor common to both niches is a relatively high salt concentration.

Pathogenicity of strains of the black yeast Exophiala (Wangiella) dermatitidis: an evaluation based on polymerase chain reaction.

Strains of Exophiala dermatitidis, mainly originating from patients with systemic neurotropic mycosis in Asia and from the lungs of patients with cystic fibrosis (CF) in Europe, were analysed by ribotyping of the small subunit rDNA and by random amplification of polymorphic DNA (RAPD). A characteristic banding pattern for the species was found after restriction analysis of amplified fragments V9 and ITS4. The small subunit rDNA gene of five strains was about 1800 base pairs (bp) long, while in 16 strains its length was about 3000 bp. Using RAPD, seven populations could be distinguished. European CF strains as well as Asian strains from systemic mycoses are mainly distributed over two populations, one of which contained both CF strains and a systemic strain. It is concluded that the two clinical pictures are caused by genetically similar strains. The differences in pathogenicity may be explained by immunological differences in the hosts or by differences in exposure to the fungal propagules.

Population biology of Hortaea werneckii based on restriction patterns of mitochondrial DNA.

Fifteen isolates of Hortaea werneckii, causative agent of tinea nigra in man, were examined with respect to restriction fragment length polymorphisms of mitochondrial DNA. Seven types of mtDNA, interpreted as populations, could be distinguished, with similarities between the restriction patterns ranging from 32 to 79%. Much of the variance originated from length mutations. Of the seven populations four represented isolates from man, two of which also comprised isolates from other sources. This makes adaptation of H. werneckii towards association with man in its evolution unlikely; similarity in the chemical and/or physical characteristics of the different isolation sources, viz. salinity, seems more probable. mtDNA types were not correlated with geographic origin. Isolates with the same mtDNA type are widely geographically distributed.

Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs.

Buoyant density of DNA in CsCl gradients with Hoechst 33258 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (nD) of the isopycnic CsCl/Hoechst 33258 solution over the range of 0-75 %GC was established: %GC = 351762.28 X nD - 123778.66 X nD2 - 249789.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33258 gradients is 1.6 to 2.1 times better than comparative CsCl gradients without the dye. Potential application to %GC determination is discussed.

Pythium insidiosum sp. nov., the etiologic agent of pythiosis.

Pythium insidiosum sp. nov., the etiologic agent of pythiosis, a cosmopolitan disease of horses, cattle, and dogs, is described and illustrated.

Effect of maize (Zea mays L.) endosperm mutants on the surface relief of the kernel pericarp.

Relief studies on the outer surface of the kernel pericarp were conducted using interference microscopy. Comparisons involving pattern, cell relief height and cell width were made between dominant and recessive kernels at the waxy (wx), sugary (su 1), shrunken (sh 2) and opaque (o 2) loci. The pericarp of the dominant and recessive kernels at each locus was genetically identical. The effect of the alleles at the sugary locus was tested in two genetic backgrounds. At some loci, distinct differences in pattern, cell relief height and cell width were found between dominant and recessive kernels. Over all loci and kernel phenotypes, the means ranged from 1.56 to 2.86 µ for cell relief height and from 18.5 to 27.4 µ for cell width. Generally the recessive kernels had a greater relief height and width than the corresponding dominant kernels. The effect of the alleles at the sugary locus was. altered by genetic background. Apparently, the alleles at these loci and genetic background can influence the relief of the outer surface of the kernel pericarp.