Aya Nishiwaki

The ecology and agronomy of Miscanthus sinensis, a species important to bioenergy crop development, in its native range in Japan: a review.

Among several candidate perennial taxa, Miscanthus×giganteus has been evaluated and promoted as a promising bioenergy crop. Owing to several limitations, however, of the sterile hybrid, both at the taxon and agronomic production levels, other options need to be explored to not only improve M. ×giganteus, which was originally collected in Japan, but to also consider the development of other members of its genus, including Miscanthus sinensis, as bioenergy crops. Indeed, there is likely much to be learned and applied to Miscanthus as a bioenergy crop from the long history of intensive interaction between humans and M. sinensis in Japan, which in some regions of the country spans several thousand years. Combined with its high amount of genetic variation, stress tolerance, biotic interactions with fauna, and function as a keystone species in diverse grasslands and other ecosystems within its native range, the unique and extensive management of M. sinensis in Japan as a forage grass and building material provides agronomists, agroecologists, and plant breeders with the capability of better understanding this species in terms of potential contribution to bioenergy crop development. Moreover, the studies described in this review may serve as a platform for future research of Miscanthus as a bioenergy crop in other parts of the world.

Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan

PREMISE OF THE STUDY Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus ×giganteus, a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus sacchariflorus and Miscanthus sinensis, for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. sacchariflorus and diploid M. sinensis in Japan. METHODS Seeds were counted and DNA content was estimated by flow cytometry for plants of M. sacchariflorus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants. KEY RESULTS Based on seed-set data, M. sacchariflorus has significantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. sacchariflorus plants in southern Japan. CONCLUSIONS This is the first report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. ×giganteus, these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Genetic variation in Miscanthus × giganteus and the importance of estimating genetic distance thresholds for differentiating clones.

Miscanthus × giganteus (Mxg) is an important bioenergy feedstock crop, however, genetic diversity among legacy cultivars may be severely constrained. Only one introduction from Japan to Denmark of this sterile, triploid, vegetatively propagated crop was recorded in the 1930s. We sought to determine if the Mxg cultivars in North America were all synonyms, and if they were derived from the European introduction. We used 64 nuclear and five chloroplast simple sequence repeat (SSR) markers to estimate genetic similarity for 27 Mxg accessions from North America, and compared them with six accessions from Europe, including the species’ type‐specimen. A subset of accessions was also evaluated by restriction‐site associated DNA sequencing (RAD‐seq). In addition, we assessed the potential of new crosses to increase Mxg genetic diversity by comparing eight new triploid Mxg progeny grown from seed, along with samples of the parental species M. sacchariflorus and M. sinensis. Estimates of genotyping error rates were essential for distinguishing between experimental error and true genotypic differences among accessions. Given differences in estimated error rates and costs per marker for SSRs and RAD‐seq, the former is currently more cost‐effective for determining if two accessions are genetically identical. We concluded that all of the Mxg legacy cultivars were derived via vegetative propagation from a single genet. In contrast with the Mxg legacy cultivars, genetic similarity to the type‐specimen of eight new triploid Mxg progeny ranged from 0.46 to 0.56. Though genetic diversity among the Mxg legacy cultivars is critically low, new crosses can provide much‐needed variation to growers.

Genetic structure of Miscanthus sinensis and Miscanthus sacchariflorus in Japan indicates a gradient of bidirectional but asymmetric introgression

Summary Using high-density genetic markers, gene flow is identified from diploid Miscanthus sinensis to tetraploid M. sacchariflorus in Japan, in contrast to genetic isolation between these species in China.

Carbon budget and methane and nitrous oxide emissions over the growing season in a Miscanthus sinensis grassland in Tomakomai, Hokkaido, Japan

Species in the Miscanthus genus have been proposed as biofuel crops that have potential to mitigate elevated atmospheric carbon dioxide (CO2) levels and nitrous oxide (N2O) and methane (CH4) emissions. Miscanthus sinensis is widespread throughout Japan and has been used for biomass production for centuries. We assessed the carbon (C) budget and N2O and CH4 emissions over the growing season for 2 years in a M. sinensis‐dominated grassland that was naturally established around 1972 in Tomakomai, Hokkaido, Japan, which is near the northern limit for M. sinensis grassland establishment on Andisols. Average C budget was −0.31 Mg C ha−1, which indicates C was released from the grassland ecosystem to the atmosphere. Dominant components in the C budget appeared to be aboveground net primary production of plants (1.94–2.80 Mg C ha−1) and heterotrophic respiration (2.27–3.11 Mg C ha−1). The measurement of belowground net primary production (BNPP) of plants in the M. sinensis grassland was extremely variable, thus only an approximate value could be calculated. Mean C budget calculated with the approximated BNPP value was 1.47 and −0.23 Mg C ha−1 for 2008 and 2009, respectively. Given belowground biomass (9.46–9.86 Mg C ha−1) was 3.1–6.5 times higher than that of aboveground biomass may provide additional evidence suggesting this grassland represents a C sink. Average CH4 emissions across years of −1.34 kg C ha−1 would indicate this grassland acts as an atmospheric CH4 sink. Furthermore, average N2O emissions across years were 0.22 kg N ha−1. While the site may contribute N2O to the atmosphere, this value is lower compared with other grassland types. Global warming potential calculated with the approximated BNPP value was −5.40 and 0.95 Mg CO2 Eq ha−1 for 2008 and 2009, respectively, and indicates this grassland could contribute to mitigation of global warming.

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus  ×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.

Effect of Rj genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia

ABSTRACT The nodulation tendency and community structure of indigenous bradyrhizobia on Rj genotype soybean cultivars at cultivation temperatures of 33/28°C, 28/23°C, and 23/18°C for 16/8 h (day/night degrees, hours) were investigated using 780 bradyrhizobial DNA samples from an Andosol with 13 soybean cultivars of four Rj genotypes (non-Rj, Rj 2 Rj 3, Rj 4, and Rj 2 Rj 3 Rj 4). A dendrogram was constructed based on restriction fragment length polymorphism of the PCR products (PCR-RFLP) of the 16S-23S rRNA gene internal transcribed spacer region. Eleven Bradyrhizobium U.S. Department of Agriculture strains were used as a reference. The dendrogram indicated seven clusters based on similarities among the reference strains. The occupancy rate of the Bj123 cluster decreased with increasing cultivation temperature, whereas the occupancy rates of the Bj110 cluster, Be76 cluster, and Be94 cluster increased with increasing cultivation temperature. In particular, the Rj 2 Rj 3 Rj 4 genotype soybeans were infected with a number of Bj110 clusters, regardless of the increasing cultivation temperature, compared to other Rj genotype soybean cultivars. The ratio of beta diversity to gamma diversity (H′ β/H′ γ), which represents differences in the bradyrhizobial communities by pairwise comparison among cultivation temperature sets within the same soybean cultivar, indicated that the bradyrhizobial communities tended to be different among cultivation temperatures. Multidimensional scaling analysis indicated that the infection of the Bj110 cluster and the Bj123 cluster by host soybean genotype and the cultivation temperature affected the bradyrhizobial communities. These results suggested that the Rj genotypes and cultivation temperatures affected the nodulation tendency and community structures of soybean-nodulating bradyrhizobia.

Aboveground plant biomass, carbon, and nitrogen dynamics before and after burning in a seminatural grassland of Miscanthus sinensis in Kumamoto, Japan.

Although fire has been used for several thousand years to maintain Miscanthus sinensis grasslands in Japan, there is little information about the nutrient dynamics in these ecosystems immediately after burning. We investigated the loss of aboveground biomass; carbon (C) and nitrogen (N) dynamics; surface soil C change before and after burning; and carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) fluxes 2 h after burning in a M. sinensis grassland in Kumamoto, Japan. We calculated average C and N accumulation rates within the soil profile over the past 7300 years, which were 58.0 kg C ha−1 yr−1 and 2.60 kg N ha−1 yr−1, respectively. After burning, 98% of aboveground biomass and litter were consumed. Carbon remaining on the field, however, was 102 kg C ha−1. We found at least 43% of C was possibly lost due to decomposition. However, remaining C, which contained ash and charcoal, appeared to contribute to C accumulation in soil. There was no difference in the amount of 0–5 cm surface soil C before and after burning. The amount of remaining litter on the soil surface indicated burning appeared not to have caused a reduction in soil C nor did it negatively impact the sub‐surface vegetative crown of M. sinensis. Also, nearly 50 kg N ha−1 of total aboveground biomass and litter N was lost due to burning. Compared with before the burning event, postburning CO2 and CH4 fluxes from soil appeared not to be directly affected by burning. However, it appears the short time span of measurements of N2O flux after burning sufficiently characterized the pattern of increasing N2O fluxes immediately after burning. These findings indicate burning did not cause significant reductions in soil C nor did it result in elevated CO2 and CH4 emissions from the soil relative to before the burning event.

Carbon sequestration in soil in a semi‐natural Miscanthus sinensis grassland and Cryptomeria japonica forest plantation in Aso, Kumamoto, Japan

Although Miscanthus sinensis grasslands (Misc‐GL) and Cryptomeria japonica forest plantations (Cryp‐FP) are proposed bioenergy feedstock systems, their relative capacity to sequester C may be an important factor in determining their potential for sustainable bioenergy production. Therefore, our objective was to quantify changes in soil C sequestration 47 years after a Misc‐GL was converted to a Cryp‐FP. The study was conducted on adjacent Misc‐GL and Cryp‐FP located on Mt. Aso, Kumamoto, Japan. After Cryp‐FP establishment, only the Misc‐GL continued to be managed by annual burning every March. Mass C and N, δ13C, and δ15N at 0–30 cm depth were measured in 5 cm increments. Carbon and N concentrations, C:N ratio, δ13C, and δ15N were measured in litter and/or ash, and rhizomes or roots. Although C input in Misc‐GL by M. sinensis was approximately 36% of that in Cryp‐FP by C. japonica, mean annual soil C sequestration in Misc‐GL (503 kg C ha−1 yr−1) was higher than that in Cryp‐FP (284 kg C ha−1 yr−1). This was likely the result of larger C input from aboveground litter to soil, C‐quality (C:N ratio and lignin concentration in aboveground litter) and possibly more recalcitrant C (charcoal) inputs by annual burning. The difference in soil δ15N between sites indicated that organic C with N had greater cycling between heterotrophic microbes and soil and produces more recalcitrant humus in Misc‐GL than in Cryp‐FP. Our data indicate that in terms of soil C sequestration, maintenance of Misc‐GL may be more advantageous than conversion to Cryp‐FP in Aso, Japan.

Evaluation of greenhouse gas emissions in a Miscanthus sinensis Andersson-dominated semi-natural grassland in Kumamoto, Japan

ABSTRACT Increasing greenhouse gas emissions from anthropogenic activities continue to be a mounting problem worldwide. In the semi-natural Miscanthus sinensis Andersson; grasslands of Aso, Kumamoto, Japan, which have been managed for thousands of years, we measured soil methane (CH4) and nitrous oxide (N2O) emissions before and after annual controlled burns. We estimated annual soil carbon (C) accumulation, and CH4 and N2O emissions induced by biomass burning in 2009 and 2010, to determine the impacts of this ecosystem and its management on global warming. Environmental factors affecting soil CH4 and N2O fluxes were unknown, with no effect of annual burning observed on short-term soil CH4 and N2O emissions. However, deposition of charcoal during burning may have enhanced CH4 oxidation and N2O consumption at the study site, given that emissions (CH4: −4.33 kg C ha−1 yr−1, N2O: 0.17 kg N ha−1 yr−1) were relatively lower than those measured in other land-use types. Despite significant emission of CH4 and N2O during yearly burning events in early spring, the M. sinensis semi-natural grassland had a large annual soil C accumulation, which resulted in a global warming potential of −4.86 Mg CO2eq ha−1 yr−1. Consequently, our results indicate that long-term maintenance of semi-natural M. sinensis grasslands by annual burning can contribute to the mitigation of global warming.