Eiji Tanaka

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Comparative genomics of Taphrina fungi causing varying degrees of tumorous deformity in plants.

Taphrina fungi are biotrophic plant pathogens that cause plant deformity diseases. We sequenced the genomes of four Taphrina species—Taphrina wiesneri, T. deformans, T. flavorubra, and T. populina—which parasitize Prunus, Cerasus, and Populus hosts with varying severity of disease symptoms. High levels of gene synteny within Taphrina species were observed, and our comparative analysis further revealed that these fungi may utilize multiple strategies in coping with the host environment that are also found in some specialized dimorphic species. These include species-specific aneuploidy and clusters of highly diverged secreted proteins located at subtelomeres. We also identified species differences in plant hormone biosynthesis pathways, which may contribute to varying degree of disease symptoms. The genomes provide a rich resource for investigation into Taphrina biology and evolutionary studies across the basal ascomycetes clade.

Multiple locus genealogies and phenotypic characters reappraise the causal agents of apple ring rot in China

Apple ring rot inflicts severe economic losses in the main apple producing areas of East Asia. The causal agent of the disease has been variously identified as Macrophoma kuwatsukai, Physalospora piricola and Botryosphaeria berengeriana f. sp. piricola, although B. dothidea is currently the most widely accepted pathogen name. The taxonomic uncertainty has delayed research that is needed to manage effectively this destructive disease. In the present study, genealogical concordance phylogenetic species recognition (GCPSR) was applied to pathogenic fungal isolates from apple and pear from several locations in China, along with several reference isolates. Phylogenetic results based on sequences of four nuclear loci (ITS, EF-1α, HIS and HSP) revealed the existence of two species within the examined isolates. One includes an ex-epitype isolate of B. dothidea and the other includes an isolate that was previously designated as B. berengeriana f. sp. piricola. Morphologically, the latter taxon presented an appressed mycelial mat on PDA whereas B. dothidea displayed columns of aerial mycelia reaching the lids, and conidia of the latter species were longer than B. dothidea. Botryosphaeria dothidea had a faster growth rate than the latter taxon under relatively high temperatures. Pathogenicity tests showed that on pear stems the latter taxon caused large-scale cankers along with blisters whereas B. dothidea was non-pathogenic, but on apple shoots the two fungi induced large and small wart-like prominences, respectively. Overall, this cryptic species demonstrated sufficient genetic variations and biological differences from B. dothidea. As a result of taxonomic study, we described here the latter taxon in a new combination, Botryosphaeria kuwatsukai and designate an epitype. Both B. kuwatsukai and B. dothidea are considered to be the main causal agents for apple ring rot in China and Japan.

Mechanisms of bamboo witches' broom symptom development caused by endophytic/epiphytic fungi

Aciculosporium take causes continuous shoot growth but maintains normal leaf-arrangement and branching patterns in the host plant, which eventually resulting in witches’ broom disease of bamboo. An in situ hybridization technique with a species-specific oligonucleotide probe was recently used to demonstrate that endophytic mycelia of A. take is predominantly distributed in the intercellular spaces of the shoot apical meristem of the host. Endophytic hyphae in meristematic tissues, which may produce auxin, are responsible for continuous primordium initiation within the shoot apex. Here I examine another bamboo witches’ broom causal fungus, Heteroepichloë sasae. Both species are biotrophic and belong to family Clavicipitaceae: however, H. sasae does not cause continuous shoot growth. Histological study showed that H. sasae mycelia were distributed superficially, even on shoot apical meristems. These observations suggest that when their stromata develop, endophytic A. take destroys shoot apical meristem and epiphytic H. sasae chokes the shoot apex of the host. Stromata formation consequently causes lateral bud outgrowth because of release from apical dominance. This process repeats and eventually results in the witches’ broom symptoms.

A Brief Chronicle of the Genus Cordyceps Fr., the Oldest Valid Genus in Cordycipitaceae (Hypocreales, Ascomycota)

Abstract The earliest pre-Linnaean fungal genera are briefly discussed here with special emphasis on the nomenclatural connection with the genus Cordyceps Fr. Since its valid publication under the basidiomycetous genus Clavaria Vaill. ex L. (Clavaria militaris L. Sp. Pl. 2:1182, 1753), the genus Cordyceps has undergone nomenclatural changes in the post-Linnaean era, but has stood firmly for approximately 200 years. Synonyms of Cordyceps were collected from different literature sources and analyzed based on the species they represent. True synonyms of Cordyceps Fr. were defined as genera that represented species of Cordyceps Fr. emend. G. H. Sung, J. M. Sung, Hywel-Jones & Spatafora. The most common synonyms of Cordyceps observed were Clavaria and Sphaeria Hall, reported in the 18th and in the first half of the 19th century, respectively. Cordyceps, the oldest genus in the Cordyceps s. s. clade of Cordycipitaceae, is the most preferred name under the “One Fungus = One Name” principle on priority bases.

Localization of helotialean fungi on ectomycorrhizae of Castanopsis cuspidata visualized by in situ hybridization

Non-ectomycorrhizal fungi that associate with typical ectomycorrhizae often remain hidden, and their localization inside ectomycorrhizal (ECM) roots has remained uncharacterized. In this study, the fungal community associated with the ectomycorrhizae of Castanopsis cuspidata was investigated using a culture-dependent isolation technique. Additionally, the species composition and localization were determined using molecular techniques. The results of the isolation and identification of fungal species revealed the predominance of a few species belonging to the order Helotiales. Furthermore, the fungal community structures were significantly different depending on the taxa of the ectomycorrhiza-forming fungi. A taxon-specific probe was developed to analyze the localization of one dominant Hyaloscyphaceae (Helotiales) species in ECM tissues by in situ hybridization. Hybridization signals were detected on the surface of the fungal mantle and around the ECM fungal cells within the mantle. Hyphal penetration into ECM hyphal cells of fungal mantles was also observed. Signals were not detected in the Hartig net or plant tissues inside the mantle in healthy ectomycorrhizae. These findings suggest that the analyzed species interact not only with host plant as root endophyte but also directly with the ECM fungi.

Appressorium-mediated penetration of Magnaporthe oryzae and Colletotrichum orbiculare into surface-cross-linked agar media

Many phytopathogenic fungi form appressoria on some artificial substances. However, it is difficult to induce appressorium-mediated penetration into artificial substances. In the present study, novel artificial agar media were developed to investigate the in vitro penetration process of phytopathogenic fungi. The media contained sodium carboxymethyl cellulose or sodium alginate, and the surfaces were subjected to ionic cross-linking using trivalent metal ions. The hemibiotrophic phytopathogenic fungi, rice blast fungus and cucurbit anthracnose fungus, formed appressoria and penetrated into the surface cross-linked artificial agar media from the base of appressoria. These artificial media appeared to induce fungal infection behaviour that occurred on host plants.

Ophiocordyceps sessilis sp. nov., a new species of Ophiocordyceps on Camponotus ants in Japan

Ophiocordyceps sessilis, a new species of Ophiocordycipitaceae, Hypocreales, was found on Camponotus obscuripes ants, where the ants were primarily infected by another close relative, O. pulvinata. Morphological observation clearly indicated that O. sessilis and O. pulvinata are distinct species. Ophiocordyceps sessilis exhibited superficial development of perithecia and ascospore disarticulation into part-spores, not known in O. pulvinata. Nucleotide sequence data suggested that O. sessilis belongs in Ophiocordyceps, Ophiocordycipitaceae. Molecular data also indicated that O. sessilis is a close relative of O. cuboidea, O. prolifica, O. paracuboidea, and O. ryogamiensis, which are all known to produce part-spores. Since O. sessilis is always associated with ants infected by O. pulvinata, O. sessilis may be a hyperparasite of O. pulvinata.