Seasonal and geographical differences in the ruminal microbial and chloroplast composition of sika deer (Cervus nippon) in Japan.

To understand the nutritional status of culled wild sika deer (Cervus nippon), we compared the ruminal microbes of deer living in habitats differing in food composition (Nagano winter, Nagano spring, and Hokkaido winter) using next-generation sequencing. Twenty-nine sika deer were sampled. Alpha and beta diversity metrics determined via 16S and 18S rRNA amplicon-seq analysis showed compositional differences. Prevotella, Entodinium, and Piromyces were the dominant genera of bacteria, fungi and protozoa, respectively. Moreover, 66 bacterial taxa, 44 eukaryotic taxa, and 46 chloroplastic taxa were shown to differ significantly among the groups by the linear discriminant analysis effect size (LEfSe) technique. Total RNA-seq analysis yielded 397 significantly differentially expressed transcripts (q < 0.05), of which 48 (q < 0.01) were correlated with the bacterial amplicon-seq results (Pearson correlation coefficient > 0.7). The ruminal microbial composition corresponded with the presence of different plants because the amplicon-seq results indicated that chloroplast from broadleaf trees and Stramenopiles-Alveolates-Rhizaria (SAR) were enriched in Nagano, whereas chloroplast from graminoids, Firmicutes and the dominant phylum of fungi were enriched in Hokkaido. These results could be related to the severe snow conditions in Hokkaido in winter and the richness of plants with leaves and acorns in Nagano in winter and spring. The findings are useful for understanding the nutritional status of wild sika deer.

Genetic diversity and population structure of feral rapeseed (Brassica napus L.) in Japan.

Rapeseed (Brassica napus L.) is one of the most economically important oilseed crops worldwide. In Japan, it has been cultivated for more than a century and has formed many feral populations. The aim of this study was to elucidate the genetic diversity of feral rapeseeds by genotyping 537 individuals (among which 130 were determined to be genetically modified) sampled from various regions in Japan. Analysis of 30 microsatellite markers amplified 334 alleles and indicated moderate genetic diversity and high inbreeding (expected heterozygosity, 0.50; observed heterozygosity, 0.16; inbreeding coefficient within individuals, 0.68) within the feral populations. The Mantel test showed only an insignificant weak positive correlation between geographic distance and genetic distance. Analysis of molecular variance showed a greater genetic diversity among individuals than between populations. These results are in accordance with population structure assessed by using principal coordinate analysis and the program STRUCTURE, which showed that the 537 individuals could be assigned to 8 genetic clusters with very large genetic differences among individuals within the same geographic population, and that among feral individuals, many are closely related to rapeseed accessions in the NARO Genebank but some have unknown origins. These unique feral rapeseeds are likely to be affected by strong selection pressure. The results for genetically modified individuals also suggest that they have two different sources and have a considerable degree of diversity, which might be explained by hybridization with nearby individuals and separation of hybrid cultivars. The information obtained in this study could help improve the management of feral rapeseed plants in Japan.

Mangrove-diazotroph relationships at the root, tree and forest scales: diazotrophic communities create high soil nitrogenase activities in Rhizophora stylosa rhizospheres.

BACKGROUND AND AIMS: The tidal flats on which mangrove plants grow tend to have low soil nitrogen contents because nitrogen-containing litter is repeatedly washed offshore by ebb tides. Under such circumstances, it is unclear how mangrove plants acquire the nitrogen required to support their vigorous growth. In the present work, chemical and biological characteristics of diazotrophy around mangrove plant roots were surveyed under natural conditions to elucidate mangrove-diazotroph relationships. METHODS: Soil nitrogenase activity of a representative mangrove plant, Rhizophora stylosa, which has a broad geographical distribution, was measured using the acetylene reduction assay at forest, tree and prop root scales. In addition, diazotrophic community composition was compared between rhizosphere and bulk soil based on sequencing of nifH genes. KEY RESULTS: Soil nitrogenase activity was high near prop roots, and this pattern was enhanced as soil live root content increased. At the forest scale, we observed high soil nitrogenase activity (acetylene-reducing activity) inside the forest (the highest value was 90.9 µmol C2H2 min-1 cm-3, average 46.8 ± 18.2 µmol C2H2 min-1 cm-3). Rates decreased sharply from the forest to the tidal flat (range 1.2-22.2 µmol C2H2 min-1 cm-3, average 7.9 ± 4.5 µmol C2H2 min-1 cm-3). The nifH operational taxonomic unit composition differed significantly among forest and tree rhizospheres and the bulk soil (P < 0.0001). CONCLUSIONS: Our results suggest that the accumulation of diazotrophs around R. stylosa mangrove trees enhances the supply of biologically fixed nitrogen to the mangrove roots. This supply is especially important when the soil naturally contains little nitrogen. This nitrogen acquisition system may be a key process that explains the high productivity of mangrove ecosystems.

Tracking the route of atmospheric nitrogen to diazotrophs colonizing buried mangrove roots.

Nitrogen-fixing activity has been observed in the rhizosphere of mangrove ecosystems, suggesting a close mangrove-diazotroph relationship. In regularly flooded soil, however, the pathway by which atmospheric nitrogen reaches the diazotrophs in the rhizosphere is unknown. This study provides evidence that mangrove aerial roots serve as pathways that supply nitrogen gas to the diazotrophs colonizing buried roots. A plastic chamber was attached on the exposed part of a Rhizophora stylosa Griff prop root, and 15N2 tracer gas was injected into it. The entire root, including the below-ground part, was collected for analysis of 15N labelling and nitrogenase activity. We detected 15N labelling in buried root materials 2 h after gas injection. Compared with the δ15N contents in root material from an untreated tree, the increment was >10‰ in lateral roots. The nitrogenase activity measured on the other R. stylosa roots was highest in lateral roots, matching well with the results of 15N labelling. Our results indicate that atmospheric nitrogen is taken into aerial mangrove roots through lenticels, diffuses into the buried root system and is fixed by diazotrophs. The unusual appearance of mangrove aerial roots, which has intrigued researchers for many years, could be a key to the high productivity of mangrove ecosystems.

Characterization of Natural and Simulated Herbivory on Wild Soybean (Glycine soja Seib. et Zucc.) for Use in Ecological Risk Assessment of Insect Protected Soybean.

Insect-protected soybean (Glycine max (L.) Merr.) was developed to protect against foliage feeding by certain Lepidopteran insects. The assessment of potential consequences of transgene introgression from soybean to wild soybean (Glycine soja Seib. et Zucc.) is required as one aspect of the environmental risk assessment (ERA) in Japan. A potential hazard of insect-protected soybean may be hypothesized as transfer of a trait by gene flow to wild soybean and subsequent reduction in foliage feeding by Lepidopteran insects that result in increased weediness of wild soybean in Japan. To assess this potential hazard two studies were conducted. A three-year survey of wild soybean populations in Japan was conducted to establish basic information on foliage damage caused by different herbivores. When assessed across all populations and years within each prefecture, the total foliage from different herbivores was ≤ 30%, with the lowest levels of defoliation (< 2%) caused by Lepidopteran insects. A separate experiment using five levels of simulated defoliation (0%, 10%, 25%, 50% and 100%) was conducted to assess the impact on pod and seed production and time to maturity of wild soybean. The results indicated that there was no decrease in wild soybean plants pod or seed number or time to maturity at defoliation rates up to 50%. The results from these experiments indicate that wild soybean is not limited by lepidopteran feeding and has an ability to compensate for defoliation levels observed in nature. Therefore, the potential hazard to wild soybean from the importation of insect-protected soybean for food and feed into Japan is negligible.

Distribution of Petrosavia sakuraii (Petrosaviaceae), a rare mycoheterotrophic plant, may be determined by the abundance of its mycobionts.

Petrosavia sakuraii (Petrosaviaceae) is a rare, mycoheterotrophic plant species that has a specific symbiotic interaction with a narrow clade of arbuscular mycorrhizal (AM) fungi. In the present study, we tested the hypothesis that the distribution and abundance of mycobionts in two P. sakuraii habitats, Nagiso and Sengenyama (central Honshu, Japan), determine the distribution pattern of this rare plant. Nagiso is a thriving habitat with hundreds of P. sakuraii individuals per 100 m(2), whereas Sengenyama is a sparsely populated habitat with fewer than 10 individuals per 100 m(2). AM fungal communities associated with tree roots were compared at 20-cm distances from P. sakuraii shoots between the two habitats by molecular identification of AM fungal partial sequences of the small subunit ribosomal RNA gene. The percentage of AM fungal sequences showing over 99 % identity with those of the dominant P. sakuraii mycobionts was high (54.9 %) in Nagiso, but low (13.2 %) in Sengenyama. Accordingly, the abundance of P. sakuraii seems to reflect the proportion of potential mycobionts. It is likely that P. sakuraii mycobionts are not rare in Japanese warm temperate forests since 11.2 % of AM fungal sequences previously obtained from a deciduous broad-leaved forest devoid of P. sakuraii in Mizuho, central Honshu, Japan, were >99 % identical to those of the dominant P. sakuraii mycobionts. Thus, results suggest that the abundant mycobionts may be required for sufficient propagation of P. sakuraii, and this quantitative trait of AM fungal communities required for P. sakuraii may explain the rarity of this plant.

Transportability of confined field trial data from cultivation to import countries for environmental risk assessment of genetically modified crops.

Requirement of in-country confined field trials for genetically modified (GM) crops prior to unrestricted release is well-established among countries with domestic regulations for the cultivation approval of GM crops. However, the requirement of in-country confined field trials is not common in countries where the scope of the application does not include cultivation. Nonetheless, Japan and China request in-country confined field trials for GM crops which are intended only for use as food, feed and processing. This paper considers the transportability of confined field trial data from cultivation countries (e.g. United States, Canada, and South American countries) to import countries like Japan for the environmental risk assessment of GM crops by reviewing: (1) the purpose of confined field trial assessment, (2) weediness potential, defined as "an ability to establish and persist in an unmanaged area that is frequently disturbed by human activity", of host crops, and (3) reliability of the confined field trial data obtained from cultivation countries. To review the reliability of the confined field data obtained in the US, this paper describes actual examples of three confined field trials of approved GM corn events conducted both in the US and Japan. Based on the above considerations, this paper concludes that confined field data of GM corn and cotton is transportable from cultivation countries to importing countries (e.g. from the US to Japan), regardless of the characteristics of the inserted gene(s). In addition, this paper advocates harmonization of protocols for confined field trials to facilitate more efficient data transportability across different geographies.