No abstract

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

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.

The sequences of ITS regions in 30 species and two groups of the genus Pythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/Iobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection of Pythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista. Key WordsChromista; internal transcribed spacer; phylogeny; Pythium; 5.8S rDNA.Pythium is a large genus of the class Oomycetes including more than 80 species (van der Plaats-Niterink, 1981 ), some of which are important plant pathogens with worldwide distribution. The taxonomy of this genus is mainly based on the morphology of reproductive structures such as the oogonium, oospore, antheridium, and sporangium. Since Matthews (1931) and Sideris (1932) proposed the first keys for Pythium spp., several keys have been published as new species have been described. Researchers providing systematic taxonomy in the genus are Middleton (1943), Waterhouse (1967), and van der Plaats-Niterink (1981). Hendrix and Papa (1974) introduced the concept of 'species complexes' in relation to taxonomy of the genus Pythium. This is an arrangement in which the species of Pythium are lumped into 15 species groups based on their morphological characteristics. In the taxonomic keys for Pythium, the size of each structure and common morphological characteristics are taken as criteria, including: (1) the presence of sexual reproductive structures -homothallic or heterothallic; (2) the sporangial morphology-spherical, filamentous or Iobulate; (3) the oogonial wall charactersmooth or ornamental; (4) the oospore character-plerotic or aplerotic; and (5) the antheridial charactermonoclinous or diclinous, However, each author regarded different characteristics as more important. For example, Middleton (1943) used the sporangial morphology at the first branch, whereas Waterhouse (1967) frequently utilized the reproductive structure size in the separaCorresponding author. E-mail: kageyama(~cc.gifu-u.ac.jp tion of species. Van der Plaats-Niterink (1981) regarded the presence of reproductive structures and the oogonial wall character as more important criteria. These differences in the interpretation of the taxonomic value of each character have resulted in a confusing taxonomic system for the Pythium species. It is therefore necessary to determine which morphological characteristic is the ...

No abstract