Morphological and molecular taxonomy of Pythium longisporangium sp. nov. isolated from the Burgundian region of France

During the course of an investigation on the Pythiaceous oomycetes occurring in the Burgundian vineyards, some species of Pythium possessing mainly hypogynous antheridia were found. These had been classified as oomycetes belonging to the "Pythium rostratum" group for a long time. Three of these isolates, having similar structures and growth, are very closely related to a recently described species, Pythium bifurcatum Paul. A close look at these, however, underlines some fundamental differences with the latter. Not all of them produce zoospores but have very large sporangia. The type specimen is F-1200 (B 76a) which is a medium-slow growing saprophyte. The sequence of the ITS region of the rDNA also shows a very close relationship with P. bifurcatum. On the basis of morphological and molecular analysis, we now describe this species as Pythium longisporangium sp. nov. Morphological features of this new species, the sequences of the ITS region of its nuclear ribosomal DNA, and its comparison with related species are discussed.     

PYTHIUM SYLVATICUM鉴定及其专一性PCR引物

从杭州采集的水稻、棉花和大豆猝倒苗中分离到国内新记录腐霉种Pythium sylvaticum。该腐霉为异宗配合种,菌丝膨大体球形或柠檬形,雄器异丝生,藏卵器光滑,每个藏卵器上1~3个雄器,雄器常在接近藏卵器处形成二叉状分枝,卵孢子不满器。测定了该种4 个菌株的核糖体内转录间隔区(ITS)的序列,根据与59种腐霉ITS序列的比较,设计了P. sylvaticum种专一性引物PSF1和PSR2。实际结果表明:该引物能从11种共14株腐霉DNA中特异性地扩增P. sylvaticum,从而与其它10种腐霉区分。     

Two new species,Pythium agreste and P. wuhanense,based on morphological characteristics and DNA sequence data

In an investigation of Pythium species in China, two new species, P. agreste and P. wuhanense, were identified based on morphological characteristics and DNA sequence data. Pythium agreste has slightly inflated sporangia, oogonia encompassed by antheridia and antheridial stalks forming a very complicated knot, and plerotic oospores. It differs from the morphologically similar P. volutum which has inflated sporangia, bigger oogonia, and aplerotic oospores; from P. kashmirense which has contiguous inflated sporangia, catenulate oogonia and coiled or bent oogonial stalks; and from P. pectinolyticum which has catenulate oogonia and bigger oogonia and oospores. Pythium wuhanense can be differentiated from morphologically similar P. emineosum and P. paroecandrum by its possession of intercalary cylindrical to elongated sporangia and intercalary oogonia catenulate with sporangia and antheridia. Phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species, based on the internal transcribed spacer (ITS) region and cytochrome C oxidase subunit I (COI) gene sequences using maximum parsimony and Bayesian methods. The two new species are described and illustrated in detail.     

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.     

分离自蔬菜地的腐霉一新种及其rDNAITS序列分析(英文)

从广西南宁收集的蔬菜地土壤样本中,分离得到一个新的腐霉种,命名为广西腐霉Pythium guangxiense。不同于其近似种,广西腐霉的孢子囊球形或亚球形;藏卵器球形、亚球形、椭圆形或囊形;雄器同丝生或异丝生,柄常分枝且缠绕藏卵器。对构成广西腐霉rDNAITS区段的772个碱基进行扩增和测序。在根据ITS序列建立的分子系统发育树中,广西腐霉与禾生腐霉Pythium graminicola、周雄腐霉P.periilum和肿囊腐霉P.inflatum等产生瓣状孢子囊的腐霉种关系密切。对广西腐霉进行了分类描述和其与近似种的比较。     

ITS region of the rDNA of Pythium longandrum, a new species; its taxonomy and its comparison with related species

Pythium longandrum (F-73.0) was isolated, from soil samples taken in Lille in northern France. Morphologically the fungus resembles closely Pythium rostratum, however its antheridial characters are unique. The oogonia of this species are provided with hypogynous and monoclinous antheridia. The antheridial cells are inflated and are probably the largest and longest for the genus. The internal transcribed spacer region of its nuclear ribosomal DNA indicates that it is entirely different from all other species of Pythium. This new species is characterized by its spherical to elongated sporangia, smooth-walled oogonia and hypogynous to monoclinous antheridia bearing long antheridial cells closely applied to the oogonia. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and comparison with related species are discussed here.     

Pythium burgundicum sp. nov. isolated from soil samples taken in French vineyards

During the course of investigation on pythiaceous fungi occurring in the Burgundian vineyards, a new species of Pythium has been isolated. This oomycete is characterized by its nonproliferating and nonsporulating, spherical to cylindrical type of sporangia (hyphal bodies) germinating through germ tubes, smooth-walled oogonia that are supplied with hypogynous, monoclinous or rarely diclinous antheridia, and smooth-walled oospores. The antheridial cells are very prominent and are reminiscent of Pythium bifurcatum, Pythium segnitium and Pythium longandrum described previously by the author. The internal transcribed spacer region of the rRNA of this new species is composed of 883 bases, which is quite different from that of the closest relatives. Morphological and molecular features of this new species, named as Pythium burgundicum , are discussed in this article.     

Phytophthora aquimorbida sp. nov. and Phytophthora taxon 'aquatilis' recovered from irrigation reservoirs and a stream in Virginia, USA

Two distinct subgroups (L2 and A(-2)) were recovered from irrigation reservoirs and a stream in Virginia, USA. After molecular, morphological and physiological examinations, the L2 subgroup was named Phytophthora aquimorbida and the A(-2) designated as Phytophthora taxon 'aquatilis'. Both taxa are homothallic. P. aquimorbida is characterized by its noncaducous and nonpapillate sporangia, catenulate and radiating hyphal swellings and thick-walled plerotic oospores formed in globose oogonia mostly in the absence of an antheridium. P. taxon 'aquatilis' produces plerotic oospores in globose oogonia mostly with a paragynous antheridium. It has semi-papillate, caducous sporangia with variable pedicels, but it does not have hyphal swelling. Analyses of ITS, CO1, β-tubulin and NADH1 sequences revealed that P. aquimorbida is closely related to P. hydropathica, P. irrigata and P. parsiana, and P. taxon 'aquatilis' is related to P. multivesiculata. The optimum temperature for culture growth is 30 and 20 C for P. aquimorbida and P. taxon 'aquatilis' respectively. Both taxa were pathogenic to rhododendron plants and caused root discoloration, pale leaves, wilting, tip necrosis and dieback. Their plant biosecurity risk also is discussed.     

Pythium carbonicum,a new species isolated from a spoil heap in northern France,the ITS region,taxonomy and comparison with related species

Pythium carbonicum (F-72) sp. nov. was found in soil samples taken on the top of a spoil heap in northern France. The morphology of this new species resembles that of a recently described species: Pythium megacarpum. However, the antheridial and oogonial characteristics of this new species are unique, and the comparison of its ITS region of the nuclear ribosomal DNA indicates that this species is also related to the genus Phytophthora. The fungus does not sporulate, the sporangia germinate directly into mycelium through germ tubes. The oogonia of P. carbonicum are smooth-walled and also papillated, and are provided with monoclinous and diclinous antheridia that wrap around, forming a complicated knot. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and its comparison with related species are discussed here.     

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.     

ITS1 region of the rDNA of Pythium proliferatum, a new species: its taxonomy and its comparison with related species

A new species, Pythium proliferatum (F-382), was isolated from soil samples taken in Genlis in the burgundian region of France. The fungus is unique because of the character combinations of its large, spherical to elongated, proliferating sporangia, and its smooth walled oogonia supplied with different types of antheridia like hypogynous, monoclinous sessile, monoclinous stalked, diclinous and wrapping around the oogonia. Almost all types of antheridia that are found in the genus are present in this new species. Morphological features of this new species, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are discussed in this article.     

Pythium abappressorium--a new species from eastern Washington

A new species of Pythium isolated from wheat and apple roots in eastern Washington is described. Pythium abappressorium sp. nov. is characterized by abundant appressoria. Plerotic oospores and sporangia are formed from the appressoria and remnants of the appressoria remain attached to the base of sporangia at maturity. Smaller appressorial swellings, reminiscent of hyphal swellings, are also formed within the appressoria. Pythium abappressorium is pathogenic to wheat, causing damping-off and stunting, but is not pathogenic to apples. The fungus can grow in the temperature range 5 to 30 C, with an optimum of 20 C. The sequence of the ITS1 region of the rDNA did not match the sequences from a worldwide collection of over 1200 isolates, including types and neotypes, suggesting that this species has not been previously described.     

<Emphasis Type="Italic">Pythium montanum</Emphasis> sp. nov., a new species from a spruce stand in the Bavarian Alps

During a study on soilborne Pythiaceae in the Bavarian Alps, 3 isolates of a papillate Pythium species with aplerotic oospores were found in rhizosphere soil from a declining spruce stand. The isolates showed a unique combination of sporangial and gametangial characters, and could not be assigned to any known species of the genus. They were characterised by a relatively low growth rate, internally proliferating sporangia and extremely variable, monoclinous, sessile or shortly stalked antheridia. Direct germination of sporangia frequently occurred in water culture. Internal transcribed spacer (ITS) regions of the ribosomal DNA showed very low levels of similarity to those of all known species of Pythium. This paper describes the isolates as Pythium montanum sp. nov., gives details on its morphology and provides preliminary information on its pathogenicity. Additional molecular evidence for its status as a new species is also provided. Phylogenetic analyses with a set of Pythium spp. showed that P. montanum groups in a clade together with P. vexans, a species whose taxonomical status still remains unclear, and that is considered a ‘border species’ between Pythium and Phytophthora.     

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.     

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.