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.

Genome-wide variation in the pinewood nematode Bursaphelenchus xylophilus and its relationship with pathogenic traits.

BACKGROUND: Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease across Asia and Europe. In this study, we report the first genome-wide variation analysis of the nematode with an aim to obtain a full picture of its diversity. METHODS: We sequenced six key B. xylophilus strains using Illumina HiSeq sequencer. All the strains were isolated in Japan and have been widely used in previous studies. Detection of genomic variations were done by mapping the reads to the reference genome. RESULTS: Over 3 Mb of genetic variations, accounting for 4.1 % of the total genome, were detected as single nucleotide polymorphisms or small indels, suggesting multiple introductions of this invaded species from its native area into the country. The high level of genetic diversity of the pine wood nematode was related to its pathogenicity and ecological trait differences. Moreover, we identified a gene set affected by genomic variation, and functional annotation of those genes indicated that some of them had potential roles in pathogenesis. CONCLUSIONS: This study provides an important resource for understanding the population structure, pathogenicity and evolutionary ecology of the nematode, and further analysis based on this study with geographically diverse B. xylophilus populations will greatly accelerate our understanding of the complex evolutionary/epidemic history of this emerging pathogen.

Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan.

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of ; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F2 progeny in hybridization tests.

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.

Diversity, migration routes, and worldwide population genetic structure of Lecanosticta acicola, the causal agent of brown spot needle blight.

Lecanosticta acicola is a pine needle pathogen causing brown spot needle blight that results in premature needle shedding with considerable damage described in North America, Europe, and Asia. Microsatellite and mating type markers were used to study the population genetics, migration history, and reproduction mode of the pathogen, based on a collection of 650 isolates from 27 countries and 26 hosts across the range of L. acicola. The presence of L. acicola in Georgia was confirmed in this study. Migration analyses indicate there have been several introduction events from North America into Europe. However, some of the source populations still appear to remain unknown. The populations in Croatia and western Asia appear to originate from genetically similar populations in North America. Intercontinental movement of the pathogen was reflected in an identical haplotype occurring on two continents, in North America (Canada) and Europe (Germany). Several shared haplotypes between European populations further suggests more local pathogen movement between countries. Moreover, migration analyses indicate that the populations in northern Europe originate from more established populations in central Europe. Overall, the highest genetic diversity was observed in south-eastern USA. In Europe, the highest diversity was observed in France, where the presence of both known pathogen lineages was recorded. Less than half of the observed populations contained mating types in equal proportions. Although there is evidence of some sexual reproduction taking place, the pathogen spreads predominantly asexually and through anthropogenic activity.

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.

Transcriptional and morphological changes in the transition from mycetophagous to phytophagous phase in the plant-parasitic nematode Bursaphelenchus xylophilus.

Drastic physiological and morphological changes in parasites are crucial for the establishment of a successful infection. The nematode Bursaphelenchus xylophilus is the pathogenic agent of pine wilt disease, and little is known about the physiology and morphology in this nematode at the initial stage of infection. In this study, we devised an infection system using pine stem cuttings that allowed us to observe transcriptional and morphological changes in the host-infecting phytophagous phase. We found that 60 genes enriched in xenobiotic detoxification were up-regulated in two independent post-inoculation events, whereas down-regulation was observed in multiple members of collagen gene families. After 48 h of inoculation, the tails in some of the adult females exposed to the host changed in morphology. These results suggest that B. xylophilus may change its physiology and morphology to protect itself and to adapt to the host pine wood environment.

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.

Nematodes associated with Dryocoetes uniseriatus (Coleoptera: Scolytidae).

We examined the nematode fauna associated with a species of bark beetle, Dryocoetes uniseriatus Eggers, as part of a biodiversity survey of forest beetle-associated nematodes. Collections were made in a pine stand at an experimental forest station in Ibaraki, Japan, from April to July of 2011; we examined the nematode association in 273 insects collected during this time. In total, 68% of the insects were associated with at least one species of nematode. Six species of nematodes, including two phoretic microbe feeders (Bursaphelenchus rainulfi Braasch & Burgermeister and Micoletzkya sp.), one insect parasite and nematode predator (Devibursaphelenchus cf. eproctatus), one insect parasite (Contortylenchus sp.), one insect parasite and potential microbe feeder (unidentified rhabditid parasite), and one potential insect parasite and fungal feeder (B. sinensis) were recovered from the beetles. D. cf. eproctatus was enclosed in nematangia on the backsides of the elytra, B. rainulfi was isolated from the backsides of the elytra or enclosed in nematangia, Micoletzkya sp. was isolated from under the elytra, Contortylenchus sp. and a rhabditid parasite parasitized the body cavity, and B. sinensis was found in the digestive tract of the insect. The association patterns of the nematode species varied seasonally, although definitive interactions among species (e.g., segregation, competition) were not observed.

A novel approach of preventing Japanese cedar pollen dispersal that is the cause of Japanese cedar pollinosis (JCP) using pollen-specific fungal infection.

In Japan, Japanese cedar pollen dispersal is one of the major causes of pollinosis. Sydowia japonica is an ascomycetous fungus that grows exclusively on the male strobili of Japanese cedar, suggesting a possible mechanism for controlling pollen dispersal. To evaluate this possibility, eleven isolates of S. japonica were collected from around Japan and used as an inoculum to male strobili of Japanese cedar. The treatment demonstrated that the fungus infected only the pollen and prevented pollen dispersal. The fungus did not cause any additional symptoms to other parts of Japanese cedar, such as needles, stems, and buds. All S. japonica isolates collected around Japan could serve to control pollen dispersal. Periodic observation of the fungal pathogenesis with stereomicroscope and scanning electron microscope showed that hyphal fragments and conidia of S. japonica germinated on the surface of male strobili, and the germ tube entered pollen sacs through opening microsporophylls. Within the pollen sacs, the hyphae penetrated pollen gradually, such that all pollen was infected by the fungus by approximately one month before the pollen dispersal season. The infected pollen was destroyed due to the fungal infection and was never released. Our data suggests a novel approach of preventing pollen dispersal using pollen-specific fungal infection.