Takashi Enomoto

High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing.

Estimation of soil-to-plant transfer factors of radiocesium in 99 wild plant species grown in arable lands 1 year after the Fukushima 1 Nuclear Power Plant accident.

One year after the deposition of radionuclides from the Fukushima 1 Nuclear Power Plant (A formal name is Fukushima Daiichi Nuclear Power Station) in March 2011, radiocesium (134Cs, 137Cs) concentrations ([Cs]) were comprehensively investigated in the wild plants of 99 species most of which were annual or summer green perennial herbs and started to grow from April 2012 at the heavily contaminated fields of paddy (three study sites) and upland (one study site) in Fukushima Prefecture. The survey was conducted three times (April, July and October) in the year. In each site, soils (soil cores of 5-cm depth) and plants (aerial shoots) were collected for determination of [Cs] on a dry weight basis, and then the transfer factor (TF) of radiocesium from soil to plant ([Cs]plant/[Cs]soil) was estimated in each species. The [Cs] values of both soils and plants largely varied. However, some species exhibited relatively high TF values (more than 0.4) (e.g., Athyrium yokoscense, Dryopteris tokyoensis, and Cyperus brevifolius), while others exhibited almost negligible values (less than 0.01) (e.g., Salix miyabeana, Humulus scandens, and Elymus tsukushiensis). In addition, judging from the 11 species grown in both paddy and upland fields, TF values were generally higher in the paddy fields. The estimation of phytoextraction efficiency of soil radiocesium by weed communities in the paddy fields suggests that the weed community is not a practical candidate for phytoremediation technique.

Variations in the foliar δ13C and C3/C4 species richness in the Japanese flora of Poaceae among climates and habitat types under human activity

For 383 Poaceae species harvested over the Japanese islands and stored as herbarium specimens along several decades, we determined C3 and C4 types of photosynthesis from leaf stable carbon isotope ratio (δ13C). Then, we sought the relationships between C4 species richness and climatic factors or habitat types. Except for the two Panicum species (P. lanuginosum and P. scoparium) having the possibility of C3–C4 intermediate, 227 and 154 species were classified into C3 and C4. The C4 species richness increased from northern to southern islands in Japan, positively correlated with mean annual air temperature. Greater C4 species richness in the seashore habitats, and smaller C4 species richness in the shaded, wet and highland habitats would be related to the photosynthetic responses to local environmental factors such as irradiance level and temperature regime. No difference of leaf δ-value of C3 Poaceae was obtained between the habitats with different soil water availability, suggesting the less importance of soil water availability on leaf water-use efficiency in C3 Poaceae species in Japan having humid climate. Additionally, possible effects of human activity around the harvested time or site on leaf δ-value were estimated, because the habitat includes the sites with high human activity. Leaf δ-value was decreased with sampling year, and it was higher in the densely inhabited district for both C3 and C4. They are probably due to a historical decrease in the atmospheric δ-value via increasing human activity, and high gas emission at the districts of high human density.

Population survey of spiders using mt-DNA (COI) sequences in Japanese peach orchards

In this study, we attempted a population survey of spiders in peach orchards with and without pesticide application; however, we had difficulty discriminating them. To identify spiders, we determined the nucleotide sequences of a ca. 640-bp region in the mitochondrial cytochrome oxidase subunit I (COI) gene from 17 species belonging to eight families. To identify five wolf spiders (Araneae: Lycosidae), which are the most abundant spiders in peach orchards in Okayama Prefecture, PCR and restriction fragment length polymorphism were developed on the basis of the COI gene sequences. Population survey results suggest that pesticide applications adversely affect spider populations in peach orchards.