Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.     

基于rDNA ITS序列探讨部分腐霉种的系统发育与其形态特征

基于对73株计58种腐霉和6种疫霉的核糖体DNA的ITS序列分析,以海生疫霉为外围群,按邻接法构建系统发育树,对腐霉的系统发育关系进行了研究。结果表明:在58种腐霉中,Pythium ostracodes,P.chamaehyphon,P.carbonicum,P.montanum和P.vexans归为同一组,介于其它腐霉和疫霉之间,这5种腐霉的孢子囊均为球形;现已归为疫霉属的P.undulatum 单独为一组,它与腐霉的亲缘关系比疫霉更近;其余52种腐霉聚成一大组,这52种腐霉基本上按孢子囊或菌丝膨大体形态分成Ⅰ、Ⅱ两组:第1组31种腐霉, 其中30种腐霉的孢子囊或菌丝膨大体为球形;第Ⅱ组21种腐霉,其中19种腐霉的孢子囊为丝状、瓣状或裂片状。基于ITS序列分析,腐霉属的其它性状如藏卵器壁是否光滑、卵孢子是否满器、雄器的着生方式和数量、异宗配合等呈多元演化。     

How do obligate parasites evolve? A multi-gene phylogenetic analysis of downy mildews

Plant parasitism has independently evolved as a nutrition strategy in both true fungi and Oomycetes (stramenopiles). A large number of species within phytopathogenic Oomycetes, the so-called downy mildews, are defined as obligate biotrophs since they have not, to date, been cultured on any artificial medium. Other genera like Phytophthora and Pythium can in general be cultured on standard or non-standard agar media. Within all three groups there are many important plant pathogens responsible for severe economic losses as well as damage to natural ecosystems. Although they are important model systems to elucidate the evolution of obligate parasites, the phylogenetic relationships between these genera have not been clearly resolved. Based on the most comprehensive sampling of downy mildew genera to date and a representative sample of Phytophthora subgroups, we inferred the phylogenetic relationships from a multi-gene dataset containing both coding and non-coding nuclear and mitochondrial loci. Phylogenetic analyses were conducted under several optimality criteria and the results were largely consistent between all the methods applied. Strong support is achieved for monophyly of a clade comprising both the genus Phytophthora and the obligate biotrophic species. The facultatively parasitic genus Phytophthora is shown to be at least partly paraphyletic. Monophyly of a cluster nested within Phytophthora containing all obligate parasites is strongly supported. Within the obligate biotrophic downy mildews, four morphologically or ecologically well-defined subgroups receive statistical support: (1) A cluster containing all species with brownish-violet conidiosporangia, i.e., the genera Peronospora and Pseudoperonospora; (2) a clade comprising the genera with vesicular to pyriform haustoria (Basidiophora, Benua, Bremia, Paraperonospora, Plasmopara, Plasmoverna, Protobremia); (3) a group containing species included in Hyaloperonospora and Perofascia which almost exclusively infect Brassicaceae; (4) a clade including the grass parasites Viennotia oplismeni and Graminivora graminicola. Phylogenetic relationships between these four clades are not clearly resolved, and neither is the position of Sclerospora graminicola within the downy mildews. Character analysis indicates an evolutionary scenario of gradually increasing adaptation to plant parasitism in Peronosporales and that at least the most important of these adaptive steps occurred only once, including major host shifts within downy mildews.     

Two novel species representing a new clade and cluster of Phytophthora

Phytophthora stricta sp. nov. and Phytophthora macilentosa sp. nov. are described based on morphological, physiological and molecular characters in this study. Phytophthora stricta represents a previously unknown clade in the rRNA internal transcribed spacer (ITS)-based phylogeny. Phytophthora macilentosa, along with nine other species, consistently forms a high temperature-tolerant cluster within ITS clade 9. These observations are supported by the sequence analysis of the mitochondrial cytochrome c oxidase 1 gene. Both species are heterothallic and all examined isolates are A1 mating type. Phytophthora stricta produces nonpapillate and slightly caducous sporangia. This species is named after its characteristic constrictions on sporangiophores. Phytophthora macilentosa produces nonpapillate and noncaducous sporangia, which are mostly elongated obpyriform with a high length to breadth ratio. Both species were recovered from irrigation water of an ornamental plant nursery in Mississippi, USA and P. stricta was also recovered from stream water in Virginia, USA.     

Specificity of cytoplasmic and cell-wall antigens from four species of Phytophthora.

Cytoplasmic and cell-wall antigens and antisera were prepared from four Phytophthora species, and cell-wall antigens were prepared from two Pythium species. Immunodiffusion of the Pythium and Phytophthora cell-wall antigens showed that the two Pythium species did not cross-react with the Phytophthora cell-wall antisera. Immunodiffusion analysis of both cell-wall and cytoplasmic antigens of Phytophthora revealed some degree of specificity between species but not between A1 and A2 mating types in Phytophthora cinnamomi. Species specificity was improved by using indirect fluorescent antibody techniques and by the use of cross-absorbed sera. Agglutination and quantitative precipitation techniques did not significantly improve specificity. It was possible to distinguish serologically between Phytophthora cinnamomi and Phytophthora cambivora and the Phytophthora cryptogea-Phytophthora drechsleri group. The absence of consistent serological variation between P. cryptogea and P dreschsleri is consistent with the suggestion (Bumbieris, 1974) that P. cryptogea and P. drechsleri should be considered as one species.     

Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America

A new species of Phytophthora, previously referred to as the 'Dre I' taxon, is named Phytophthora irrigata. Isolates of P. irrigata morphologically and physiologically resemble Phytophthora drechsleri. They are heterothallic, produce nonpapillate sporangia, and grow well at 35 degrees C. The above two species differ significantly in uniformity of mycelium, presence of chlamydospores, DNA fingerprint, and sequences of multiple nuclear and mtDNA regions. Phytophthora irrigata produces smaller sporangia and a distinct DNA fingerprint. Sequence alignments in the ribosomal DNA internal transcribed spacer regions place Phytophthora fallax and Phytophthora captisoa as its closest relatives. The optimal temperature for culture growth is above 30 degrees C and the maximum temperature is 40 degrees C. This new species is abundant in irrigation reservoirs and natural waterways in Virginia and was also isolated in Pennsylvania.     

Co-occurrence and genotypic distribution of Phytophthora species recovered from watersheds and plant nurseries of eastern Tennessee

In 2008 statewide surveys of symptomatic foliage of nursery plants from Tennessee resulted in isolation of 43 isolates of Phytophthora spp. This sample set includes four described species (P. citrophthora, P. citricola, P. nicotianae, P. syringae), and a provisional species of Phytophthora ('P. hydropathica'). At the same time a stream-baiting survey was initiated to recover Phytophthora from eight watersheds in eastern Tennessee, some of which are near plant nurseries. Baiting was accomplished by submerging healthy Rhododendron leaves approximately 1 wk and isolation onto selective media. Six baiting periods were completed, and in total 98 Phytophthora isolates and 45 isolates of Pythium spp. were recovered. Three described species (P. citrophthora, P. citricola and P. irrigata) and the provisional species 'P. hydropathica' were obtained as well as three undescribed Phytophthora taxa and Pythium litorale. Isolates from both surveys were identified to species with morphology and the internal transcribed spacer (ITS) sequence. Isolates from species co-occurring in streams and nurseries (P. citricola, P. citrophthora and 'P. hydropathica') were characterized further with amplified fragment length polymorphism (AFLP) analyses and mefenoxam tolerance assays. Isolates representing a putative clonal genotype of P. citricola were obtained from both environmental and nursery sample sets.     

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.     

Phytophthora polonica, a new species isolated from declining Alnus glutinosa stands in Poland

In a survey of Phytophthora associated with alder decline in Poland, several isolates of a homothallic Phytophthora sp., which could not be assigned to other taxa including Phytophthora alni subspecies, were consistently recovered from rhizosphere soil samples. Their morphology and pathogenicity, as well as sequence data for three nuclear regions (internal transcribed spacer rDNA, elongation factor-1alpha and beta-tubulin) and a coding mitochondrial DNA region (nadh1), were examined. The new Phytophthora species is characterized by the moderate to slow growth rate of its colony in carrot agar at 20 degrees C, high optimal (c. 30 degrees C) and maximum (c. 38 degrees C) growth temperatures, formation of catenulate, often lateral, hyphal swellings, large chlamydospores in agar media and in soil extract, persistent, ovoid to ellipsoid nonpapillate sporangia and large oogonia with paragynous and sometimes amphigynous antheridia. Phytophthora polonica was slightly pathogenic to alder twigs and not pathogenic to trunks of several tree species. In a phylogenetic analysis using either Bayesian inference or maximum likelihood methods, P. polonica falls in clade 8 'sensu Kroon et al. (2004)' of Phytophthora.     

Ancient origin of elicitin gene clusters in Phytophthora genomes

The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs), a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression patterns.     

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.     

Phylogenetic relationships of the downy mildews (Peronosporales) and related groups based on nuclear large subunit ribosomal DNA sequences

In order to investigate phylogenetic relationships of the Peronosporomycetes (Oomycetes), nuclear large subunit ribosomal DNA sequences containing the D1 and D2 region were analyzed of 92 species belonging to the orders Peronosporales, Pythiales, Leptomitales, Rhipidiales, Saprolegniales and Sclerosporales. The data were analyzed applying methods of neighbor-joining as well as maximum parsimony, both statistically supported using the bootstrap method. The results confirm the major division between the Pythiales and Peronosporales on the one hand and the Saprolegniales, Leptomitales, and Rhipidiales on the other. The Sclerosporales were shown to be polyphyletic; while Sclerosporaceae are nested within the Peronosporaceae, the Verrucalvaceae are merged within the Saprolegniales. Within the Peronosporomycetidae, Pythiales as well as Peronosporales as currently defined are polyphyletic. The well supported Albugo clade appears to be the most basal lineage, followed by a Pythium-Lagenidium clade. The third, highly supported clade comprises the Peronosporaceae together with Sclerospora, Phytophthora, and Peronophythora. Peronophythora is placed within Phytophthora, indicating that both genera should be merged. Bremiella seems to be polyphyletic within the genus Plasmopara, suggesting a transfer to Plasmopara. The species of Peronospora do not appear as a monophyletic group. Peronospora species growing on Brassicaceae form a highly supported clade.     

Pythium cryptoirregulare, a new species within the P. irregulare complex

Pythium identification is based on several characteristics with considerable variation, particularly in Pythium irregulare Buis. as currently recognized. Thirty-one isolates of Pythium irregulare Buis. from various hosts and geographic regions were compared by genetic analysis of multiloci DNA fingerprints, sequence analysis of nuclear and mitochondrial genes and morphological and growth rate studies. Previous research indicated two distinct groupings within the species, P. irregulare sensu stricto and a clade referred to here as Pythium sp. Parsimony analyses of 338 AFLP markers divided P. irregulare s.l. into two clades. Comparison of the allele frequencies of 236 polymorphic AFLP loci revealed significant differences between them. The two clades differed in the frequencies of 182 (77%) alleles. P. irregulare s.s. had 122 (52%) polymorphic loci while Pythium sp. had 205 (87%). Pythium sp. had one fixed allele and 79 polymorphic loci absent in P. irregulare s.s. P. irregulare s.s. displayed 16 polymorphic loci absent in Pythium sp. Parsimony and distance analyses of the ribosomal intergenic transcribed spacers (ITS) and the cox II gene sequences support the separation of P. irregulare s.s. and Pythium sp. Amplicon length in P. irregulare s.s. ITS sequences were 936-938 bp and 936-949 bp in Pythium sp. The two clades were separated by two fixed insertion/deletion mutations, nine fixed nucleotide substitutions in the ITS region and three fixed single nucleotide substitutions in the cox II sequences. Average growth rates of the groups differed at 10, 30 and 36 C but not at 15, 21 or 25 C. Statistically significant differences were found in oogonium, oospore and ooplast diameters, antheridial cell length and in ooplast index. We propose that a new species, Pythium cryptoirregulare, be delineated from Pythium irregulare sensu stricto.     

Differential uptake and metabolism of sitosterol and cholesterol by Achlya, Pythium, and Phytophthora species.

The relative ability of isolates of Achlya bisexualis and A. ambisexualis and isolates of Pythium and Phytophthora to take up and metabolize sitosterol and cholesterol was studied. Species of Pythium and Phytophthora took up cholesterol and sitosterol efficiently, whereas Achlya species took up booth sterols inefficiently. Species of Pythium and Phytophthora produced a polar metabolite and esters from sitosterol as they did from cholesterol. Achlya species did not produce the polar metabolite from either sterol. In these experiments Achlya species produced esters only from cholesterol; however, their failure to produce esters from sitosterol may have been due to the higher sitosterol than cholesterol concentration.     

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.     

Potassium homeostasis influences the locomotion and encystment of zoospores of plant pathogenic oomycetes

Zoospores of plant pathogenic oomycetes exhibit distinct swimming speeds and patterns under natural conditions. Zoospore swimming is influenced by ion homeostasis and changes in the ionic composition of media. Therefore, we used video microscopy to investigate swimming patterns of five oomycete species in response to changes in potassium homeostasis. In general, zoospore speed tended to be negatively correlated with zoospore size. Three Phytophthora species (Phytophthora palmivora, Phytophthora megakarya, and Phytophthora infestans) swam in straight patterns with speeds ranging from 50 to 250 microm/s whereas two Pythium species (Pythium aphanidermatum and Pythium dissotocum) swam at similar speeds ranging from 180 to 225 microm/s with a pronounced helical trajectory and varying amplitudes. High external concentrations of potassium salts reduced the swimming speed of Ph. palmivora and induced encystment. This was not observed for Py. aphanidermatum. Application of the potassium ionophores gramicidin, nigericin and valinomycin resulted in reduced swimming speeds and changes in the swimming patterns of the Phytophthora species. Therefore, potassium ions play a key role in regulating zoospore behavior.     

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences

Phytophthora species are devastating plant pathogens in both agricultural and natural environments. Due to their significant economic and environmental impact, there has been increasing interest in Phytophthora genetics and genomics, culminating in the recent release of three complete genome sequences (P. ramorum, P. sojae, and P. infestans). In this study, genome and other large sequence databases were used to identify over 225 potential genetic markers for phylogenetic analyses. Here, we present a genus-wide phylogeny for 82 Phytophthora species using seven of the most informative loci (approximately 8700 nucleotide sites). Our results support the division of the genus into 10 well-supported clades. The relationships among these clades were rigorously evaluated using a number of phylogenetic methods. This is the most comprehensive study of Phytophthora relationships to date, and many newly discovered species have been included. A more resolved phylogeny of Phytophthora species will allow for better interpretations of the overall evolutionary history of the genus.