Shotaro Ando

Two seasons’ study on nifH gene expression and nitrogen fixation by diazotrophic endophytes in sugarcane (Saccharum spp. hybrids): expression of nifH genes similar to those of rhizobia

To explore the presence and expression of the nifH gene of diazotrophic endophytes in sugarcane (Saccharum spp. hybrids) cv. NiF8, we conducted pot experiments for two seasons in a glasshouse from 10 June to 17 September (high temperature and long days) in 2005 and 1 September to 9 December (low temperature and short days) in 2006. The expression of nifH genes in the stems and roots of sugarcane plants at 50 (or 59) and 100 days after transplanting was investigated by reverse transcription (RT)–PCR and by examining the nifH nucleotide sequence diversity. N2 fixation was assessed by the 15N-dilution method. The nifH RNA sequences in the stems and roots of the first experiment were similar to those of Bradyrhizobium sp. and Azorhizobium caulinodans. In the second experiment, nifH expression from these bacteria was not detected in the stems. nifH gene expression in the stems was in accordance with 18–24% N derived from air in the high-temperature season and negligible in the low-temperature season. Both the proliferation of N2 fixers and expression of the nifH gene indicated that bacteria similar to rhizobia may contribute to N2 fixation in sugarcane under our experimental conditions.

Detection of nifH Sequences in Sugarcane (Saccharum officinarum L.) and Pineapple (Ananas comosus [L.] Merr.)

To detect N2-fixing bacteria in a plant without using culture methods, nifH gene segments were amplified with degenerate primers from DNA extracted from stems of sugarcane (Saccharum officinarum L.) and leaves of pineapple (Ananas comosus [L.] Merr.). Sequences of the nifH clones were homologous to those of bacteria in the genera Bradyrhizobium, Serratia, and Klebsiella. The bacteria with nifH sequences homologous to those of the three genera may be promising candidates for predominant endophytic diazotrophs in sugarcane and pineapple, because these sequences were commonly detected in samples from different host plants, which were collected at different locations. On the other hand, no nifH sequence related to Gluconacetobacter diazotrophicus, which is an endophytic N2-fixing bacterium and had been previously isolated from sugarcane, was detected in sugarcane. This indicates the absence or the presence of few G. diazotrophicus in the stems of the sugarcane plants used in the current study.

Effects of high nitrogen application on the dry matter yield, nitrogen content and nitrate-N concentration of sugarcane

Abstract In Japan a high dependency on imported food, particularly livestock feed, has resulted in the accumulation of large amounts of nutrients, especially nitrogen, in the form of livestock manure. Cultivating feed crops that can take up a large amount of N would not only mitigate the water pollution caused by the nitrate leached from the livestock sector, but would also improve food self sufficiency. Sugarcane (Saccharum spp.) is considered to be a promising candidate in this respect. Two field experiments were conducted in 2006 and 2007 in Kagawa to evaluate the dry matter (DM) yield, N content and nitrate-N concentration of sugarcane under high N input. The first experiment was designed as a factorial experiment with two factors, varieties/lines/clones and N rates set at two levels, 30 g m−2 year−1 and 60 g m−1 year−1, with three replicates. In the second experiment, 30 varieties/lines/clones of sugarcane and related genera as well as control crops were planted (n = 2) and a single N rate of 45 g m−2 year−1 was applied. Some varieties/lines of sugarcane produced an above-ground DM yield greater than 4.0 kg m−2 and contained more than 40 g m−2 of N in the above-ground part. Erianthus clones, IK76-126 and IJ76-349 tended to accumulate nitrate-N when high rates of N were applied. No sugarcane varieties out of the 28 varieties/lines tested in the present study accumulated nitrate-N beyond the allowed limit for feed use, irrespective of the N rates tested in the experiments. The ability of sugarcane not to accumulate nitrate-N under conditions of high N input was considered to be an advantage in double-purpose cultivation; that is, sugarcane can be used as a feed and cleaning crop.

Effects of vegetation and cultivation on δ13C values of soil organic carbon and estimation of its turnover in Asian tropics: A case study in Thailand

Abstract The carbon (C) content and natural 13C abundance (δ13C) of soil organic matter were analyzed in soils collected from forests and agricultural fields in Thailand to determine the origin and turnover of soil organic carbon in the Asian tropics. The δ13C values of the soil horizons in two forests were in the range of C3 plant values. Soil δ13C values in fields with continuous cultivation of lowland rice (C3 plant) and sugarcane (C4 plant) were less negative than those in the forests. The organic carbon contents and δ13C values in 226 top-soils from agricultural fields varied in four regions of Thailand and were affected by the cultivation of preceding crops and weeds. The δ13C values of two series of top-soils from forests and sugarcane fields were investigated in the Northeastern and Central Plain Regions. Conversion of forest land to fields for sugarcane cultivation led to a rapid decrease in the content of soil organic carbon and an increase in the soil δ13C values. The calculated forest-derived carbon rapidly decreased with a half-life of 0.35 years (in the Northeastern Region) after conversion to fields for sugarcane cultivation, and sugarcane-derived carbon steadily accumulated. Rapid changes in the δ13C values of soil organic carbon after land conversion to fields with vegetation with different photosynthetic types may result from low organic carbon content and rapid turnover in the tropics.

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from thailand

Abstract A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known ...

Seasonal variations in natural 13C abundances in C3 and C4 plants collected in Thailand and the Philippines

Abstract The natural 13C abundance (δ 13C) of plant leaves collected from fields in Thailand and the Philippines (Asian Monsoon tropics) was analyzed, and changes in the δ 13C values of C3 and C4 plants in wet and dry seasons were characterized. In Thailand, the δ 13C values of C3 plants were −29.2 ± 1.04 (mean ± standard deviation) ‰ in July and August (wet season) and −28.6 ± 1.05‰ in February and March (dry season): these values are not significantly different, whereas the values of C4 plants were −12.7 ± 0.56‰ in the wet season and −14.5 ± 0.68‰ in the dry season (P < 0.01, t-test). In the Philippines, where plants were collected only in October (late wet season), the δ 13C values of C3 plants were −29.5 ± 1.28‰, whereas those of C4 plants were −12.6 ± 1.11‰. These results suggest that under an Asian Monsoon climate, C4 plants exhibit more negative δ 13C values in the dry season than in the wet season, whereas C3 plants as a whole show no clear seasonal changes in δ 13C values.

Overwintering Ability and Dry Matter Production of Sugarcane Hybrids and Relatives in the Kanto Region of Japan

Many kinds of sugarcane hybrids and relatives were planted in two sites, Tsukuba and Nasushiobara, in the northern Kanto region of Japan to evaluate their overwintering ability and biomass production. JW49 (Saccharum spontaneum L.), US56-15-2 (S. spontaneum L.) and Ponape Hurukimura (Saccharum barberi Jeswiet) were able to overwinter in the field experiment conducted in Tsukuba, but not in Nasushiobara. In the field experiment conducted in Nasushiobara, only two lines, JW599 (Saccharum spontaneum L.) and JW630 (Erianthus spp.), overwintered. JW630 also showed good ratooning ability in Tsukuba, even though the results were obtained from the experiment without replication. The newly planted JW599 and JW630 were small in the first year, but from the second year the biomass production of the ratoon plants became huge. The dry matter yields of JW599 and JW630 in the first year were 0.07 and 0.13 kg per hill, respectively, and the average dry matter yields of JW599 and JW630 during three years of ratoon cultivation were 1.46 and 2.63 kg per hill per year, respectively. Assuming that they are planted at 1.5-m inter-row intervals and 0.3 m inter-hill intervals, and each hill can accumulate the same dry matter yield, the dry matter production of the ratoon plant of JW599 and JW630 is estimated to be 32.5 and 58.4 t/ha, respectively.

Phylogenetic and Physiological Characterization of Indigenous Azospirillum Isolates in Thailand(Soil Biology)

Abstract We isolated indigenous Azospirillum bacteria in Thailand and analyzed the physiological characteristics and phylogenetic properties of the 16S rDNA sequences. Azospirillum bacteria were isolated from the rhizosphere soil and surface-sterilized root samples of nonleguminous plants, such as sugarcane, vetiver grass, wild rice, sweet potato, maize, and cultivated rice, grown in Thailand. Nitrogenase activity evaluated by the acetylene reduction activity (ARA) and indole-3-acetic acid (IAA) production enabled to classify the Thai isolates into two groups: Group I, which showed a high lAA production, and Group II, which showed a low ARA and lAA production. Phylogenetic analysis of the 16S rDNA sequences revealed that the 12 isolates in Group I were closest to Azospirillum largimobile, with 97 to 99% homology values, and they were also close to Azospirillum brasilense. The 16S rDNA sequence of TS18 in Group II was distant from all seven known species in the genus Azospirillum, indicating that TS18 may belong to a new species of Azospirillum. Analyses of other physiological characteristics, such as colony morphology, biotin requirement, carbon utilization, and acid formation, also revealed the existence of specificity and diversity among the Thai Azospirillum isolates.

Effects of planting sugarcane and napier grass on n leaching from lysimeters under high application of cattle manure.

Aims: Excess manure accumulated in livestock areas has been a concern in Japan where a great amount of nutrients such as nitrogen (N) has been imported from abroad contained in various agricultural commodities especially feeds for livestock animals. Aims of the present study is to look into the effects of planting sugarcane and napier grass on nitrate leaching from fields overdosed with excessive amount of manure application. Study Design: A lysimeter experiment of 3 treatments with 2 replicates. Place and Duration of Study: NARO-Shikoku Research Center (Kagawa, Japan), three years. Methodology: (1) Monitoring of water samples drained from lysimeters during study period, (2) Sampling of planted crops and soils. Results: The plots planted with either sugarcane or napier grass showed less drainage of water as well as less leaching of mineral N compared to control plots after summer when canopy was fully established. However, leaching of mineral N could not be completely prevented at the events of severe rainfalls except for the plots planted with napier grass. Original Research Article American Journal of Experimental Agriculture, 4(5): 497-514, 2014 498 The reduced amount of drainage water for the plots planted with either sugarcane/napier grass was considered to be attributable to evapotranspiration of these crops. Both sugarcane and napier grass recovered a great amount of N in the above-ground part by the time of harvest. Yet, total N concentration of soil layers did not appear to have been affected by the presence of these crops compared to control plots. Conclusion: It was concluded that planting sugarcane/napier grass was effective in the studied area in reducing leaching of mineral N especially by reducing drainage water.