Shuhei Takemoto

Combined Analyses of Bacterial, Fungal and Nematode Communities in Andosolic Agricultural Soils in Japan

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.

Population Ecology of Bursaphelenchus xylophilus

Population ecology is the study of biological populations, and it includes diverse subfields: population dynamics concerns the size and structure of a population and examines the effects of environmental factors on it. In the study of population genetics, the changes of allele frequencies within populations and their distribution are especially interesting. The evolutionary change of populations may be studied using some biological characteristics.

Establishment of a set of inbred strains of the pine wood nematode, Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae), and evidence of their varying levels of virulence

Pine wilt disease (PWD) caused by the pine wood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, has become a worldwide problem. The pathogenic mechanism of PWD continues to remain controversial, which in part may be attributed to the lack of universal materials of B. xylophilus with a high genetic purity. The intrinsic high genetic diversity in B. xylophilus isolates/populations must be a fatal obstacle for performing forward genetics and other molecular approaches to controlling them. We conducted a series of successive full-sib mating of conventional isolates of B. xylophilus to establish a set of inbred strains. Using DNA markers, we also determined their genetic diversity and biological characteristics, such as virulence and reproductive ability. Consequently, the newly established strains yielded a higher genetic purity than the conventional isolates and showed varying virulence despite sharing a common ancestor. The significance of this study lies not only in establishing a set of inbred strains of B. xylophilus with the certification of their purity but also in demonstrating that avirulent strain(s) with a genotype similar to the virulent strains can be obtained by simple successive full-sib mating. This technique is one of the most powerful tools for elucidating the pathogenic mechanism(s) of PWD.

Cohabitation of the pine wood nematode, Bursaphelenchus xylophilus , and fungal species in pine trees inoculated with B. xylophilus

The relationship between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and fungi cohabiting in 15-year-old Japanese black pine, Pinus thunbergii, was examined at intervals of 2 months over a year following inoculation with PWN. The population of PWN in the trees was large in August, but decreased slightly in December then increased again in February. Eighteen species of fungi were isolated from wood samples of the pine trees examined. Among them were Phialophora repens, Sphaeropsis sapinea, Pestalotiopsis spp. and Rhizoctonia sp., which were detected most frequently every season. All of these dominant fungi had positive effects on increasing the nematode population except for Rhizoctonia sp., on which PWN propagated less. Under laboratory conditions, 18 species of fungi isolated from pine trees and Botrytis cinerea cultured on potato dextrose agar served as food for PWN, and PWN population build up was compared at 20°C. PWN increased significantly on Pestalotiopsis sp. 1, Pestalotiopsis sp. 2, Sphaeropsis sapinea, Phialophora repens and B. cinerea from 10-15 days after inoculation. From the viewpoint of food quality and their cohabiting ability, we concluded that the species of fungi that were dominant in the pine trees, except for Rhizoctonia sp., had a compatible relationship with PWN, whilst Rhizoctonia sp. and Penicillium spp. were neutral, and Trichoderma spp. had an incompatible relationship with PWN.

Behaviour in constrained spaces reflects habitat adaptations of terrestrial nematodes

Terrestrial nematodes occupy a wide variety of habitats, including soils, plants and animals (McSorley, 2003). Habitat has a major influence on physical adaptation and species diversity, and it seems likely that the variety of habitat characteristics also drives behavioural adaptation, resulting in different physical abilities of nematodes. The inherent obstacle to studying the behaviour of nematodes lies in observing them in natural habitats because their movement is usually constrained by soil or host tissues. The development of micro-moulded substrates may solve this problem by allowing direct observation and can be a promising new tool for nematode research. We test the hypothesis that different environmental structures are a factor that causes behavioural adaptation of nematodes by analysing the movement characteristics of nematodes in three different structures. We devised two micro-moulded substrates (approximately 5 × 5 mm) made of flexible polydimethylsiloxane. One had six long straight pathways 60 μm wide, 50 μm deep and 2 mm long (Fig. 1A). We designed the substrate to reproduce long and narrow pathways in plants, such as resin ducts and tracheids. We measured the locomotion speed of individuals for 30 to 60 s. The other was a micropore network (50 μm deep) designed to investigate the moving pattern in soil (Fig. 1B). We counted the number of 200 μm blocks traversed by the nematodes forwards and backwards and calculated the percentage of distance moved backwards. The speed in the micropore network indicates the total distance moved forward and backward per unit of time. In the network we formed a T-shaped

The Relationship Between the Pinewood Nematode (PWN) and Fungi Cohabiting in aine Trees Inoculated with the PWN

The relationship between the pinewood nematode (PWN) and fungi cohabiting with the nematodes in 15-year-old Japanese black pine (Pinus thunbergii) was examined bimonthly over a year after inoculation with PWN. The population of PWN in the trees was high in August, but slightly decrease in December then increased again in February. From wood samples of the pine trees examined, 18 species of fungi have been isolated. Among the 18 fungi detected, Phialophora repens, Sphaeropsis sapinea, Pestalotiopsis sp., Rhizoctonia sp. were the most frequently isolated in every season. All of these fungi had positive effects on the increase of nematode population, though the population of PWN on Rhizoctonia sp. was less than those on the other three dominant fungi. Under laboratory conditions, 19 species of fungi cultured on potato dextrose agar (PDA) served for PWN as food source, and the PWN’s population built up on each fungus was compared at 20 ˆC. PWN dramatically increased on Pestalotiopsis sp. 1, Pestalotiopsis sp. 2, Sphaeropsis sapinea, Phialophora repens, and Botrytis cinerea (control), from 10 to 15 days after inoculation. From the point of view in terms of the food quality and their cohabitating ability we conclude that the species of fungi that are dominant in pine trees, except Rhizoctonia sp., have a compatible relationship with the PWN, while Rhizoctonia sp. and Penicillium sp. proved to be neutral, and Trichoderma sp. an incompatible relationship with PWN.