Norio Sahashi

Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.

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.

Cross Inoculation Tests with Phellinus noxius Isolates from Nine Different Host Plants in the Ryukyu Islands, Southwestern Japan

Brown root rot, caused by Phellinus noxius, leads to problems in management of shade, ornamental, and windbreak trees in subtropical regions of the world, and it has been causing serious problems in Japan since 1988. To identify the pathogenicity, host specialization, and virulence of the pathogen, cross inoculation tests were carried out using isolates of the fungus obtained from nine different tree species. P. noxius was pathogenic to all of the nine tree species tested, and it killed inoculated seedlings. Among the 810 trees inoculated with the fungus, a total of 141 trees (17.4%), including all nine species, were dead within 110 days after inoculation. The first symptom of infection was rapid wilt with discoloration of leaves 20 to 30 days after inoculation, and then the plants quickly declined. Only one isolate of P. noxius that was tested caused significantly higher mortality in its original host than in other species, suggesting that the pathogen has little to no host specificity. However, the fungus did show variation in virulence, with the isolates originating in Bischofia javanica and Casuarina equisetifolia causing significantly higher mortality than those collected from other host species. More isolates of P. noxius from the same host species and/or from various countries should be studied to understand host specialization and virulence of the pathogen.

Virulence of Bursaphelenchus xylophilus Isolated from Naturally Infested Pine Forests to Five Resistant Families of Pinus thunbergii

Pine wilt disease is one of the most serious epidemic tree diseases in Japan, and resistant pine trees have been developed through a breeding program. To evaluate resistance of resistant families of Japanese black pine, Pinus thunbergii, to the pinewood nematode, Bursaphelenchus xylophilus, isolated from the field, and to determine whether differentiation of pathogenicity to resistant pine families appears in the nematode isolates, seedlings of five resistant pine families were inoculated with 25 nematode isolates. Disease incidence 18 weeks after inoculation was significantly different among nematode isolates and among pine families but there was no interaction effect between nematode isolate and pine family. This indicates that nematode isolates did not have differential host specificity to resistant families of P. thunbergii. Isolate Shimabara, a test isolate of the breeding program, showed the same degree of virulence as the highly virulent isolates frequently used in experiments. However, more virulent isolates than Shimabara were found among the isolates collected from natural pine forest. This indicated that B. xylophilus populations with higher virulence than Shimabara exist in the natural population. These findings are important in development of more efficient breeding procedures for resistant pine trees.

Seasonal Variation of Paulownia Witches'-Broom Phytoplasma in Paulownia Trees and Distribution of the Disease in the Tohoku District of Japan

Seasonal variation of paulownia witches’-broom (PWB) phytoplasma within different organs (leaves, branch and trunk bark and roots) in paulownia trees was investigated by the amplification of a PWB-specific DNA fragment by the polymerase chain reaction (PCR). In leaf samples, PWB phytoplasma was first detected in June and the incidence gradually increased. On the other hand, the PWB was detected at relatively low incidence in branch bark, trunk bark and roots and the incidence did not change among seasons. A survey of PWB in 27 fields in the Tohoku district of Japan showed that malformed flower buds were observed in paulownia trees in almost all of the fields. PWB-phytoplasma was also detected by PCR from paulownia trees in almost all of the fields in Iwate and Fukushima Prefectures. The frequencies of trees in which phytoplasma was detected by PCR were higher than those in which symptoms were observed. These results indicated that PCR amplification of a PWB-specific DNA fragment is an effective tool for practical diagnose and that PWB is widely distributed in the Tohoku district of Japan.

Genetic Differentiation and Spatial Structure of Phellinus noxius, the Causal Agent of Brown Root Rot of Woody Plants in Japan

Phellinus noxius is a pathogenic fungus that causes brown root rot disease in a variety of tree species. This fungus is distributed in tropical and sub-tropical regions of Southeast and East Asia, Oceania, Australia, Central America and Africa. In Japan, it was first discovered on Ishigaki Island in Okinawa Prefecture in 1988; since then, it has been found on several of the Ryukyu Islands. Recently, this fungus was identified from the Ogasawara (Bonin) Islands, where it has killed trees, including rare endemic tree species. For effective control or quarantine methods, it is important to clarify whether the Japanese populations of P. noxius are indigenous to the area or if they have been introduced from other areas. We developed 20 microsatellite markers from genome assembly of P. noxius and genotyped 128 isolates from 12 of the Ryukyu Islands and 3 of the Ogasawara Islands. All isolates had unique genotypes, indicating that basidiospore infection is a primary dissemination method for the formation of new disease foci. Genetic structure analyses strongly supported genetic differentiation between the Ryukyu populations and the Ogasawara populations of P. noxius. High polymorphism of microsatellite loci suggests that Japanese populations are indigenous or were introduced a very long time ago. We discuss differences in invasion patterns between the Ryukyu Islands and the Ogasawara Islands.

Mortality of Shaded Pine Trees Inoculated With Virulent and Less-Virulent Isolates of Pine Wood Nematodes

ABSTRACT The virulence of two species (three isolates) of pine wood nematodes in stressed adult Japanese red and black pines was tested using an inoculation experiment. Three thousand nematodes, Bursaphelenchus xylophilus isolate ‘Ka4’ (virulent), isolate ‘C14-5’ (less virulent) or B. mucronatus isolate ‘M’ (less virulent), were inoculated into 15-yr-old naturally shaded and stressed pine trees planted in two separated experimental stands, in which shelterwood cutting had not been carried out after planting. The inoculation was conducted at the beginning of August 2010, and the trees were then visually examined every month for disease symptoms until February 2011. Trees that died during the experimental period were cut and brought back to the laboratory for reisolation attempts of the pathogenic nematodes. The Ka4 isolate killed all inoculated trees within one to 2-mo, whereas C14-5 and M each killed about half. The inoculated nematodes were reisolated from all the dead trees and some of the surviving ones. These results corroborated those of previous research by using small saplings (i.e., that less virulent nematodes can kill shaded (stressed) trees). Further, a feeding preference experiment using their vector beetles, Monochamus alternatus Hope, showed that both healthy and stressed trees are at equal risk of being used by beetles and hence of getting infected by the nematodes. Therefore, pine wilt disease in shading-stressed trees is assumed to occur in susceptible pine trees in natural pine stands. The dead tree is used by insects as an oviposition resource.

Comparative and population genomic landscape of Phellinus noxius: A hypervariable fungus causing root rot in trees

The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood‐decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin‐degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3‐beta‐glucan synthases in P. noxius, which may account for its fast‐growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole‐genome sequencing showed this multinucleate species contains abundant poly‐allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra‐isolate polymorphism reflect mono‐/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.

Brown root rot caused by phellinus noxius in the Ogasawara (Bonin) islands, southern Japan - current status of the disease and its host plants

An unusual mortality of several woody plant species was recently found in the Ogasawara Islands. Here, we show that brown root rot caused by Phellinus noxius was the primary cause of this unusual tree death. Typical symptoms of the disease were confirmed on 41 plant species in 29 plant families. Among them, 23 were novel hosts of P. noxius, including 15 species endemic to the Ogasawara Islands. Six species were new host records in Japan.

Distribution of genets of Cylindrobasidium argenteum in a river valley forest as determined by somatic incompatibility, and the significance of basidiospores for its dispersal.

Cylindrobasidium argenteum, a causal pathogen of white stem blight of a variety of broadleaf trees and shrubs, is thought to be transmitted to healthy trees through contact with infected branches of neighboring trees. To clarify whether basidiospores of C. argenteum are involved in the dispersal and infection process, we investigated distribution of genets of the fungus in a river valley forest using a somatic incompatibility test. Thirty-eight isolates were collected from diseased trees along two census lines at the bottom of Kikuchi River valley in southern Japan. In addition, 50 isolates were collected from four experimental plots on the slope of the valley. When the isolates from the valley bottom were paired in all combinations, a narrow clear zone line appeared in 701 out of 703 pairings, indicating that the material included 36 different genotypes, and suggesting that airborne basidiospores play an important role for dispersal of C. argenteum. In contrast, vegetative spread proved to be more frequent on the slope of the valley. On lower parts of such slopes, opportunities for healthy trees to meet infectious agents may increase because infected broken branches frequently move downwards on the slope. Based on these results, we suggest that C. argenteum adopts following dissemination strategies: (1) it spreads chiefly by basidiospores and infects trees, and (2) after colonizing stems or branches and producing an extensive mycelial mat, secondary infections of adjacent trees occur by contact with infectious agents such as diseased branches.