Susumu Takamatsu

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

First comprehensive phylogenetic analysis of the genus Erysiphe (Erysiphales, Erysiphaceae) I. The Microsphaera lineage

The genus Erysiphe (including powdery mildew fungi only known as anamorph, Pseudoidium) is the largest genus in the Erysiphaceae and contains more than 50% of all species in this family. Little is known about the phylogenetic structure of this genus. We conducted a comprehensive phylogenetic analysis of the Microsphaera-lineage, a monophyletic group including species of sects. Microsphaera and Erysiphe, using 401 sequences of nuc ITS1-5.8S-ITS2 and the 28S rDNA regions. This analysis gave many small clades delimited by the host plant genus or family. We identified two deep branches, albeit with moderate bootstrap supports, that divided the 401 sequences into three large groups. In addition, we identified four large clades consisting of homogeneous sequences of powdery mildews from a wide range of host plants beyond family level, namely, the E. aquilegiae clade, the E. alphitoides clade, the E. quercicola clade, and the E. trifoliorum s. lat. clade. Isolates from herbaceous plants were mostly situated in the E. aquilegiae clade and in Group III that was located at the most derived position of the Microsphaera-lineage. On the other hand, the basal part of the Microsphaera-lineage was occupied by isolates from woody plants except for E. glycines that was used as an outgroup taxon. This supports our previous hypothesis that tree-parasitic powdery mildews are phylogenetically primitive in the Erysiphaceae in general, and host-shift from trees to herbs occurred many times independently during the evolution of powdery mildews. Molecular clock analyses suggested that the divergence of the Microsphaera-lineage began ca. 20 million years ago in the Miocene Epoch of the Neogene Period.

Comprehensive phylogenetic analysis of the genus Golovinomyces (Ascomycota: Erysiphales) reveals close evolutionary relationships with its host plants

The Erysiphaceae were originally parasitic to trees, and host shift from trees to herbs might have occurred many times independently in the tribes and genera. To investigate the evolutionary relationships between Golovinomyces species and their host plants, we conducted a comprehensive molecular phylogenetic analysis of this genus with 183 nucleotide sequences of ITS and 28S rDNA regions from samples collected worldwide. These sequences were divided into 11 distinct lineages. Ten of these lineages consist in each case of sequences from a single plant family or tribe, which suggests close evolutionary relationships of Golovinomyces species and their host plants. The basal five clades were composed of sequences each from a single tribe of the Asteraceae. This result supports speculation that co-speciation occurred between asteraceous hosts and Golovinomyces in the early evolution stage of this genus. Lineage XI at the most derived position of the tree includes sequences from a wide range of host families and is divided into many species with close genetic affinity. Sequences from the putative G. orontii group were separated into three groups, suggesting that G. orontii is a species complex.

Additional rDNA ITS sequences and its phylogenetic consequences for the genus Leveillula with emphasis on conidium morphology

Taxonomy of the genus Leveillula has long been considered as a challenge in powdery mildew systematics. The rDNA diversity has recently been used for phylogenetic analysis of several specimens of the genus Leveillula. In the present study, additional rDNA ITS sequences are provided and a new phylogenetic analysis is carried out aiming at a better understanding of the genetic diversity in the genus Leveillula. New analyses confirmed that L. taurica is unique in the genus, as it exhibits an intraspecific gene sequence diversity considerably higher than in other species. In several cases L. taurica s. lat. on a certain host plant species has a sequence different from L. taurica on other host plants. Moreover, DNA data indicated different lineages among L. taurica specimens which were hardly distinguishable by morphology. More than one genotype occurring on a single host is sometimes possible. According to these results, several races such as Leveillula on Artemisia, Acroptilon, Onobrychis, are molecularly well characterized. While there is enough molecular evidence to delimit such races as independent taxa, clear morphological delimitations between these new and already published taxa are very difficult or even impossible. However, ecological features, and above all, host specificity for biotrophic fungi such as powdery mildew, would be a good criterion to discriminate cryptic taxa along with rDNA sequences. In fact, many collections of Leveillula strains on different hosts show their own type of conidial morphology, which is usually consistent for a strain on a single host species. Hence, we have proposed to describe new species for Leveillula on some host plants such as Artemisia, Acroptilon, Echinops and Onobrychis.

Cystotheca tjibodensis (Erysiphaceae, Ascomycota): rediscovery in Java after 90 years and first finding of anamorph

Cystotheca tjibodensis, formerly known as Lanomyces tjibodensis (Perisporiales), is a fungus found in 1920 in Indonesia. This species, hitherto only known from its type collection, is now regarded as belonging to the Erysiphales. However, molecular data are still required to verify the taxonomic affinity. In March 2011, we rediscovered this fungus at Cibodas Botanical Garden, Java. Detailed characterizations of this tropical powdery mildew are reported in this study based on morphological and molecular examinations. The anamorph of this species that was not found in the type specimen is also reported in this study.

First report of Pseudoidium aff. neolycopersici in Indonesia

Tamarillo was found to be heavily infested with powdery mildew in Bali, February 2012. The cause of the disease, Pseudoidium aff. neolycopersici, was characterised based on the morphology and molecular phylogenetic analysis of the internal transcribed spacer sequence. This is the first report of P. aff. neolycopersici on tamarillo in Indonesia.

Molecular phylogeny reveals phenotypic evolution of powdery mildews (Erysiphales, Ascomycota)

Powdery mildew fungi, comprising 16 genera and ca. 900 species, are exclusively obligate biotrophs of plants. Although several publications have addressed the phylogeny and evolution of powdery mildews based on morphology and host relationships, this review focuses on the evolution of several phenotypic characters based on molecular phylogeny: (1) morphology of appendages; (2) mycelium, ectoparasitic or endoparasitic; (3) number of asci per chasmothecium (fruiting body of powdery mildews), e.g., one to several; (4) conidiogenesis, conidia catenescent (i.e., maturing in chains: Euoidium-type) or maturing one at a time (Pseudoidium-type). Putative ancestral features of the powdery mildews are described as follows. In teleomorphs, large-sized chasmothecia, containing many 8-spored asci, had many appendages with uncinate-circinate apices, arising around the supraequatorial part of chasmothecia. In anamorphs, conidia were produced in chains (Euoidium-type) without distinct fibrosin bodies. Parasitism was ectoparasitic.

Molecular and morphological characterization of Phyllactinia cassiae-fistulae (Erysiphaceae; Ascomycota) from Thailand

Phyllactinia cassiae-fistulae and its Ovulariopsis anamorph, a causal agent of powdery mildew on Cassia fistula, have been found in Thailand for the first time. Phylogenetic analysis using the 28S ribosomal DNA sequences clearly demonstrated that P. cassiae-fistulae distinctly formed a unique clade at the basal part of Phyllactinia with 100% bootstrap support. This phylogenetic analysis supports the unique morphology of P. cassiae-fistulae anamorph having cylindrical-ellipsoil conidia and short conidiophores similar to Oidium species.

Erysiphe havrylenkoana and E. prunastri var. japonica: a new species and a new variety of Erysiphe sect. Uncinula (Erysiphaceae, Ascomycota)

A new species and a new variety of Erysiphe sect. Uncinula are described and illustrated. Erysiphe havrylenkoana is a new species found on Nothofagus alpina and N. obliqua in Argentina, and E. prunastri var. japonica is a new variety collected on Prunus maximowiczii in Japan. Erysiphe havrylenkoana differs from E. nothofagi, E. patagoniaca and E. magellanica in having unique helicoid appendages, brownish from the base to the beginning of helicoid part, spirally twisted at the center and straight or not spiral at the upper part with uncinate to circinate tip. The phylogenetic analyses of rDNA sequences clearly showed that the spiral pattern of helicoid appendages is an important morphological character to delimit species among Nothofagus powdery mildews. Erysiphe prunastri var. japonica is distinct from E. prunastri var. prunastri parasitizing hitherto Prunus species from East-Central Asia to Europe by having mainly eight asci per chasmothecium and smooth, rarely septate appendages.