Hayato Masuya

Diversity of Stag Beetle-Associated Nematodes in Japan

ABSTRACT A survey of stag beetle (Lucanidae)-associated nematodes was conducted in Japan to construct a preliminary species inventory and to obtain their molecular profiles as a first step for elucidating their host fidelity and ecological roles. Eight species of stag beetles (94 individuals in total), Dorcus rubrofemoratus, D. montivagus, D. striatipennis, D. rectus, D. titanus, Prismognathus angularis, Prosopocoilus inclinatus, and Lucanus maculifemoratus were collected from 12 localities in Japan, dissected and examined for nematode associates. Isolated nematodes were used to attempt the establishment of voucher cultures. Successfully cultured nematodes were observed under a light microscope for morphological identification at genus or species level, and were sequenced for their near full length SSU and D2/D3 LSU. All investigated lucanid individuals harbored at least one species of nematode, and eight species (Bursaphelenchus sp., Koerneria luziae, K. lucani, Pristionchus cf. pacificus, Pristionchus sp., Pseudodiplogasteroides composites, Pseudodiplogasteroides sp., and Rhabditidoides sp.: 40 populations of nematodes in total), were successfully cultured from the insects. Within these eight species, four species were considered to predominate and had low carrier specificity and wide distribution, i.e., K. luziae, P. composites, Pristionchus sp. and Rhabditidoides sp. were isolated from various species of beetles and from several different localities, and K. luziae, K. lucani, and P. composites have been previously described from native lucanids in Germany. Considering the recent world-wide trade of living insects, especially lucanids as pets, we suggest the use of caution to prevent the invasion of their associated nematodes and other associated organisms.

Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi

BackgroundHorizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown.ResultsIn order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species.ConclusionsThe close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit.

Blue Stain Fungi Associated with Tomicus piniperda (Coleoptera: Scolytidae) on Japanese Red Pine

A number of various species of blue-stain fungi were isolated fromTomicus piniperda adults at various stages of development, as well as from the galleries, pupal chambers and sapwood underneath galleries on Japanese red pine. This study was an attempt to identify the species, composition of blue-stain fungi associated withT. piniperda, the frequency of occurrence of the fungi, and their role in the sapwood-staining of Japanese red pine in Tsukuba City, central Japan. Among the seven species of blue-stain fungi isolated, an undescribed species ofOphiostoma together withO. minus were the dominant species and closely associated withT. piniperda. These two species occurred on newly emerging adults more frequently than the overwintered adults.Hormonema dematioides was also associated with the beetle, however, its frequency of occurrence from the emerged new adults was very low. Although the two other species,O. ips andGraphium sp. were also isolated from emerged beetles, the frequency of these fungi from gallery systems suggested that they were accidentally carried byT. piniperda. Leptographium wingfieldii, known to be associated with the beetle in Europe, was also isolated at a very low frequency and the fungus seemed not to be closely associated with the beetle.Ophiostoma sp. andO. minus appear to be the most important causes of blue-stain of Japanese red pine sapwood after infestation byT. piniperda.

Comparisons of ophiostomatoid fungi associated with Tomicus piniperda and T. minor in Japanese red pine

Five species ofOphiostoma, twoLeptographium species and aGraphium species were isolated from two morphologically and ecologically similar bark beetle species,Tomicus piniperda andT. minor, and their infested Japanese red pine (Pinus densiflora) in Yamanashi Prefecture, central Honshu, Japan. An underscribedOphiostoma species andO. minus were isolated mainly fromT. piniperda and its galleries.Ophiostoma canum which was found for the first time in Japan was mainly fromT. minor and its galleries. Specific relationships between the beetles and fungal species are suggested.

Ophiostoma species associated with bark beetles infesting three Abies species in Nikko, Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.

Colonization process of the root endophytic fungus Heteroconium chaetospira in roots of Chinese cabbage

Abstract Dark septate endophytic fungi (DSE) may have an important functional relationship with host plants, but these functions and the colonization process remain unknown. We made microscopic observations of the growth of an endophytic hyphomycete in Chinese cabbage roots to understand its colonization process. This hyphomycete was Heteroconium chaetospira, a suspected DSE. Three weeks post inoculation, some hyphae became irregularly lobed and formed microsclerotia within host epidermal cells of healthy plants. In stunted plants, hyphae formed closely packed masses of fungal cells within host epidermal cells, but conidiophores rarely broke through the cell walls to produce conidia.

Bursaphelenchus tadamiensis n. sp. (Nematoda: Aphelenchoididae), isolated from a stag beetle, Dorcus striatipennis (Coleoptera: Lucanidae), from Japan

Bursaphelenchus tadamiensis n. sp. is described. The new species was isolated during a field survey of nematodes associated with stag beetles. It was recovered from the dissected body of a Dorcus striatipennis, from Tadami, Fukushima, Japan. The new species is medium sized for the genus, with males 486-772 μm and females 643-997 μm in body length, and has four lateral lines, six male genital papillae (P1 ventral single papilla is missing or vestigial), mitten-shaped spicule with clear dorsal and ventral limbs, rather large squared bursal flap, vulva lacking flap, female tail long, tapering and ventrally curved. Based upon its diagnostic morphological characters, the new species belongs to the B. fungivorus group and is close to B. thailandae, B. willibaldi and B. braaschae. It is distinguished from these three species by morphology of the male bursal flap and several morphometric values, e.g., the V value of B. tadamiensis n. sp. is clearly larger than that of the above three species. The molecular phylogenetic analyses inferred from near full-length SSU and D2/D3 LSU supported the morphological observations, i.e., the new species is molecularly similar to B. thailandae, B. willibaldi, B. braaschae and B. kiyoharai. The stag beetle association of the new species may be an accidental phoresy, although the nematode could be undergoing carrier (vector) switching, because only two adult (no dauer juveniles) individuals were isolated from only one of 100 beetles examined. The insect organ with which the nematode was associated is unknown.

Symbiotic Fungal Flora in Leaf Galls Induced by Illiciomyia yukawai (Diptera: Cecidomyiidae) and in Its Mycangia

We investigated the association between a gall midge, Illiciomyia yukawai, and its symbiotic fungi on Japanese star anise, Illicium anisatum. The number of fungal species isolated from the galls increased with development of the galls, whereas those from the leaves showed a different trend. Botryosphaeria dothidea was dominant in the galls from June to October, and after that Phomopsis sp. 1, Colletotrichum sp., and Pestalotiopsis sp. became dominant. Although B. dothidea was not isolated from the leaves, it was detected from mycangia (abdominal sternite VII) of egg-laying adults at a high isolation frequency (>90%). However, B. dothidea was not isolated from mycangia of adults emerging from galls that were enclosed by plastic bags. This indicates that I. yukawai is closely associated with B. dothidea and that its newly emerged adults do not take the fungus into mycangia directly from the galls where they had developed. Also, the fungus from the fungal layers of ambrosia galls has less ability to propagate on artificial media despite the presence of its mycelial mass in mature galls.

Root Fungal Endophytes Enhance Heavy-Metal Stress Tolerance of Clethra barbinervis Growing Naturally at Mining Sites via Growth Enhancement, Promotion of Nutrient Uptake and Decrease of Heavy-Metal Concentration.

Clethra barbinervis Sieb. et Zucc. is a tree species that grows naturally at several mine sites and seems to be tolerant of high concentrations of heavy metals, such as Cu, Zn, and Pb. The purpose of this study is to clarify the mechanism(s) underlying this species’ ability to tolerate the sites’ severe heavy-metal pollution by considering C. barbinervis interaction with root fungal endophytes. We measured the heavy metal concentrations of root-zone soil, leaves, branches, and fine roots collected from mature C. barbinervis at Hitachi mine. We isolated fungal endophytes from surface-sterilized root segments, and we examined the growth, and heavy metal and nutrient absorption of C. barbinervis seedlings growing in sterilized mine soil with or without root fungal endophytes. Field analyses showed that C. barbinervis contained considerably high amounts of Cu, Zn, and Pb in fine roots and Zn in leaves. The fungi, Phialocephala fortinii, Rhizodermea veluwensis, and Rhizoscyphus sp. were frequently isolated as dominant fungal endophyte species. Inoculation of these root fungal endophytes to C. barbinervis seedlings growing in sterilized mine soil indicated that these fungi significantly enhanced the growth of C. barbinervis seedlings, increased K uptake in shoots and reduced the concentrations of Cu, Ni, Zn, Cd, and Pb in roots. Without root fungal endophytes, C. barbinervis could hardly grow under the heavy-metal contaminated condition, showing chlorosis, a symptom of heavy-metal toxicity. Our results indicate that the tree C. barbinervis can tolerate high heavy-metal concentrations due to the support of root fungal endophytes including P. fortinii, R. veluwensis, and Rhizoscyphus sp. via growth enhancement, K uptake promotion and decrease of heavy metal concentrations.

Comparative virulence of blue-stain fungi isolated from Japanese red pine

Abstract We made inoculations to determine the comparative virulence of blue-stain fungi isolated from Japanese red pine (Pinus densiflora Sieb. & Zucc.). Based on the symptoms caused by each fungus, the most virulent fungus was Leptographium wingfieldii. Also, Ophiostoma minus was relatively virulent while lesions induced by some species did not differ from those in the control treatment. Based on the results, we concluded that the pathogenicity of the blue-stain fungi varied widely, and thus their impact on the host most likely varies too.