Community structure of microeukaryotes in a rice rhizosphere revealed by DNA-based PCR-DGGE

Rice roots provide a specific habitat for microorganisms in the rhizosphere of a submerged field through supply of oxygen and organic matter. Many studies have focused on the microbial community in the rice rhizosphere, but less is still known about the microeukaryotic community structure of rice rhizosphere. This study explored the microeukaryotic community structure of a rice rhizosphere through denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA gene. The rice roots and the rhizosphere soil samples, which were collected from a field under rice-wheat rotation system, were separately analyzed. To characterize the rice rhizosphere-specific community, the bulk soil of rice field and the wheat rhizosphere samples were also examined. DGGE fingerprints showed that the microeukaryotic community of rice roots were distinct from the community of the bulk soil and showed a temporal shift with the growth stage. The rhizosphere soil community was distinct from the root and bulk soil communities, but this could be explained by that the root and bulk soil communities were shared in the rhizosphere. The rice rhizosphere community was also distinct from those in the wheat rhizosphere. Microeukaryotes that characterized the rice rhizosphere (roots and the rhizosphere soil) community could be affiliated to Polymyxa, flagellates, and oomycetes, which suggested that microeukaryotes with various ecological roles, e.g., parasites, bacterial grazers, and decomposers, inhabit the rice rhizosphere. The results showed that the rice root and its growth stages are key factors shaping the microeukaryotic community structure in the rhizosphere.     

Areal variation in the relationship between natural 15N abundances of rice (Oryza sativa L.) and soil

This study was performed to clarify whether areal variation exists in the relationship between natural ¹⁵N abundances (δ¹⁵N values) of rice (Oryza sativa L.) and soil without an applied nitrogen (N) source, and to explore possible reasons for any areal variation. We investigated the relationships between δ¹⁵N values of rice and those of unamended soil with no applied N source in two locations; Daisen and Ogata, in Akita Prefecture, Japan. The δ¹⁵N values of rice in Daisen were higher than those in Ogata from 2007 to 2009, irrespective of the cropping year. Results demonstrated areal variation in the relationship between δ¹⁵N values of rice and those of unamended soil. The variation might be attributed to variation in the δ¹⁵N of natural N input and to ammonia nitrification and subsequent denitrification. When the relationship between δ¹⁵N values of rice and those of unamended soil is used to discriminate between organic and conventional rice, the areal variation of the relationship in the target area should be taken into account, from the point of the δ¹⁵N value of natural N input and N transformation in the soil.     

Grazing of a common species of soil protozoa (Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice (Oryza sativa L.)

We performed a controlled experiment with rice seedlings (Oryza sativa L.) growing in Petri dishes on homogeneous nutrient agar containing a simple rhizosphere food web consisting of a diverse bacterial community and a common soil protozoa, Acanthamoeba castellanii, as bacterial grazer. Presence of amoebae increased bacterial activity and significantly changed the community composition and spatial distribution of bacteria in the rhizosphere. In particular, Betaproteobacteria did benefit from protozoan grazing. We hypothesize that the changes in bacterial community composition affected the root architecture of rice plants. These effects on root architecture affect a fundamental aspect of plant productivity. Root systems in presence of protozoa were characterized by high numbers of elongated (L-type) laterals, those laterals that are a prerequisite for the construction of branched root systems. This was in sharp contrast to root system development in absence of protozoa, where high numbers of lateral root primordia and short (S-type) laterals occurred which did not grow out of the rhizosphere region of the axile root. As a consequence of nutrient release from grazed bacteria and changes in root architecture, the nitrogen content of rice shoots increased by 45% in presence of protozoa. Our study illustrates that interactions over three trophic levels, i.e. between plants, bacteria and protozoa significantly modify root architecture and nutrient uptake by plants.     

Establishment of Azorhizobium caulinodans in the rhizosphere of wetland rice (Oryza sativa L.)

 Azorhizobium caulinodans strongly colonized the rhizosphere of rice plants after incorporation of Sesbania rostrata in a field trial throughout the growing season and during the fallow period until 19 weeks after incorporation of S. rostrata. A. caulinodans became well established in the rhizosphere (7.17 log cfu g^–1 dry rice root) and colonized subsequent S. rostrata test plants. Three traditional and three improved high-yielding rice varieties were inoculated with A. caulinodans under gnotobiotic conditions. In none of the combinations did acetylene reduction activity significantly increase. Ethylene production on colonized rice roots only started after the growth medium had been supplemented with an extra C source (0.1 to 0.25% Na-lactate). This indicates that the bacterial nitrogenase activity is limited by energy supply. Four possible inoculant-carriers (peat, coir dust, bagasse, rice straw) were compared for long-term survival of the bacterial strain. Independent of the storage temperature (26  °C or 4  °C), the survival of A. caulinodans in peat and coir dust was very high during a 12-month period (>8 log cfu g^–1 dry carrier), whereas the bagasse and rice straw carriers showed a serious decline from 3 months onwards. Received: 6 April 1999     

Increased zinc supply does not inhibit cadmium accumulation by rice (Oryza sativa L.)

Rice (Oryza sativa L.) grown on cadmium (Cd)-contaminated soils has caused health problems in Asian subsistence rice farmers. For other crops, normal co-contaminant zinc (Zn) inhibits the increased uptake of Cd. We used a multi-chelator-buffered nutrient solution to characterize the interaction of Zn and Cd in uptake-translocation of Cd in “Lemont” rice. The activity of free Zn²⁺ varied from 10^?7.6 to 10^?5.2 M, while free Cd²⁺ held constant at 10^?10.7 M. Zinc activity 10^?5.6 M and higher was phytotoxic to rice, resulting in severe chlorosis, reduced growth, and increased Cd transport to shoots. In contrast to previous studies with wheat, lettuce, and spinach, free Zn²⁺ maintained at adequate to sub-phytotoxic levels (10^?7.6 to 10^?6.1) did not inhibit Cd uptake by rice. The inability of Zn to inhibit Cd uptake by rice is a key factor in Cd risk from zinc-lead mine waste contaminated soil compared with other crops.     

Spatio-temporal variability of microbial abundance and community structure in the puddled layer of a paddy soil cultivated with wetland rice (Oryza sativa L.)

The puddled layer of paddy soils represents a highly dynamic environment regarding the spatio-temporal variability of biogeochemical conditions. To study these effects on the abundance and community structure of microbial populations, a rhizotron experiment was conducted throughout an entire growing season of wetland rice. Soil samples were taken from selected areas of the puddled layer (bulk soil, oxidized layer, rhizosphere) at main plant developmental stages such as (i) the initial stage, (ii) tillering, (iii) panicle initiation, (iv) flowering, and (v) maturity. Cell numbers of archaea, bacteria, and selected phyla were assessed by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The structure and diversity of microbial communities was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) along with sequencing of selected bands. Following submergence of the paddy soil, shifts of bacterial community structure were observed in the oxidized layer and the rhizosphere. Members of the β-Proteobacteria became predominant in the rhizosphere at tillering stage and were affiliated with aerobic, iron-oxidizing bacteria of the genus Sideroxydans. Seasonal effects were mainly visible in the rhizosphere, as several phylogenetic subgroups including methanotrophic bacteria showed increased cell numbers at flowering stage. Cell numbers of methanogenic archaea were also highest at flowering stage (bulk soil, rhizosphere) and members of the Methanocellales were identified as predominant archaeal populations in areas of oxic and anoxic conditions. In contrast to bacteria, the communities of archaea in the puddled layer of the studied paddy soil were less influenced by spatio-temporal variations of biogeochemical conditions.     

Increased yield of direct seeded rice (Oryza sativa L.) by foliar fertilization through multi-component fertilizers

Foliar application of fertilizers can guarantee the availability of nutrients to rice for obtaining higher yield. Rice responds favorably to macro- and micronutrients and the tolerance to salinity hazards improves by decreasing the N/S ratio. In this study, results showed that nutrient concentrations (g L^?1) for rice are: nitrogen (N) 108.0, phosphorous (P₂O₅) 6, potassium (K₂O) 81.0, calcium (CaO) 15.0, and magnesium (MgO) 6 g L^?1; and for iron (Fe), manganese (Mn), zinc (Zn), cupper (Cu), boron (B), molybdenum (Mo) and silicon (Si) the recommended concentrations are 0.6, 0.45, 0.21, 0.06, 0.09, 0.0002 and 0.004 g L^?1, respectively. A significant increase was recorded in number of panicles m^?2, 1000 grain weight, biological yield and grain yield with foliar application of nutrients. Five foliar applications of nutrients resulted in maximum number of panicles m^?2, grains panicle^?1, 1000 grain weight and biological yield. It is concluded that five foliar applications of balanced amounts of fertilizers at the seedling stage (two sprays), tillering (single spray) and at panicle initiation and panicle differentiation (two sprays) helped in enhancing yield and yield components of rice. In this research, five foliar applications produced the smallest damaging effects of blast (Pyricularia oryzae) in rice.     

Crop growth and nitrogen transformations in wheat (Triticum aestivum L.) planted after wetland rice (Oryza sativa L.)

Summary A field study was undertaken to examine the effects of various management strategies on wheat (Triticum aestivum L.) performance and N cycling in an intensively cropped soil. Microplots receiving 100 kg N ha^–1 as¹⁵NH₄ ^{+ 15}NO₃ ^– at sowing, tillering or stem elongation were compared with unfertilized microplots. Stubble from the previous rice crop was either incorporated, burnt without tillage, burnt then tilled or retained on the surface of untilled soil. Wheat grain yield ranged from 1.5 to 5.1 t ha^– and was closely related to N uptake. Plant accumulation of soil N averaged 36 kg N ha^–1 (LSD 5% = 10) on stubble-incorporation plots and 54 kg N ha^–1 on stubble-retention plots. Fertilizer N accumulation averaged 18 kg N ha^–1 (LSD 51% = 6) on stubble-incorporation plots and 50 kg N ha^–1 on stubble-retention plots. Tillage had little effect on burnt plots. Delaying N application from sowing until stem elongation increased average fertilizer N uptake from 26 to 39 kg N ha^–1 (LSD 5% = 6), but reduced soil N uptake from 50 to 37 kg N ha^– (LSD 5% = 10).Immobilization and leaching did not vary greatly between treatments and approximately one-third of the fertilizer was immobilized. Less than 1% of the fertilizer was found below a depth of 300 mm. Incorporating 9 t ha^–1 of rice stubble 13 days before wheat sowing reduced net apparent mineralization of native soil N from 37 to 3 kg ha^–1 between tillering and maturity. It also increased apparent denitrification of fertilizer N from an average 34 to 53 kg N ha^–1 (LSD 5% = 6). N loss occurred over several months, suggesting that denitrification was maintained by continued release of metabolizable carbohydrate from the decaying rice stubble. The results demonstrate that no-till systems increase crop yield and use of both fertilizer and soil N in intensive rice-based rotations.     

Cytosolic glutamine synthetase is present in the phloem sap of rice (Oryza sativa L.)

The presence of glutamine synthetase (GS) in the rice sieve tube was examined. Proteins in the rice phloem sap from leaf sheaths were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes and immunoblotted with anti-GS1 antibody. A cross-reacting band, thought to be GS1, was detected in the phloem sap. Moreover, the phloem sap contained a significant amount of GS transferase activity. Previous studies have shown that the concentrations of substrates and cofactors in the rice phloem sap are sufficient for cytosolic GS reaction. These data suggest that physiologically active GS1 is present in rice phloem sap, which might convert glutamate to glutamine in vivo .     

Genetic diversity in Japanese aromatic rice (Oryza sativa L.) as revealed by nuclear and organelle DNA markers

Aromatic rice (Oryza sativa L.) cultivated in Japan is regionally differentiated by geographical distribution and characteristics. We aimed to characterize the lineage of Japanese aromatic rice using DNA markers. Based on analyses with nuclear SSR markers, we found that Japanese aromatic rice cultivars belong, with one exception, to japonica but showed some differences from authentic japonica and were divided into two clades that were distributed in western and eastern Japan, respectively. Further analyses with organelle markers showed that most of the cultivars in eastern Japan had cytoplasm characterized by tropical japonica, whereas most of those in western Japan had cytoplasm characterized by temperate japonica. We postulate that the ancestor of the cultivars in eastern Japan differs from those of the cultivars in western Japan, and that the two groups may have been separately introduced from Taiwan into Japan. The cytoplasm of aromatic rice cultivars in western Japan may have originated from tropical japonica and been substituted into the cytoplasm of temperate japonica through hybridization between tropical japonica as a male parent and temperate japonica as a female parent.     

Effect of the number of tillages in fallow season and fertilizer type on greenhouse gas emission from a rice (Oryza sativa L.) paddy field in Ehime,southwestern Japan

Agricultural fields, including rice (Oryza sativa L.) paddy fields, constitute one of the major sources of atmospheric methane (CH₄) and nitrous oxide (N₂O). Organic matter application, such as straw and organic fertilizer, enhances CH₄ emission from paddy fields. In addition, rice straw management after harvest regulates CH₄ emissions in the growing season. The interaction of tillage times and organic fertilizer application on CH₄ and N₂O emissions is largely unknown. Therefore, we studied the effects of fallow-season tillage times and fertilizer types on CH₄ and N₂O emissions in paddy fields in Ehime, southwestern Japan. From November 2011 to October 2013, four treatments, two (autumn and spring) or one (spring) in the first year, or two (autumn and spring) or three (autumn, winter, and spring) in the second year times of tillage with chemical or organic fertilizer application, were established. Gas fluxes were measured by the closed-chamber method. Increasing the number of tillage times from one to two decreased succeeding CH₄ emission and the emission factor for CH₄ (EF_CH4) in the rice-growing season, suggesting that the substrate for CH₄ production was reduced by autumn and spring tillage in the fallow season. Higher EF_CH4 [1.8–2.0 kg carbon (C) ha^?1 d^?1] was observed when more straw was applied (6.9–7.2 Mg ha^?1) in the second year. Organic fertilizer application induced higher CH₄ emission just after the application as basal and supplemental fertilizers, especially at a lower straw application rate. This indicated that EF_CH4 in the organically managed fields should be determined individually. Organic fertilizer application with two tillage times induced N₂O efflux during the rice-growing season in the second year, but N₂O emissions were not affected by winter tillage. Although paddy fields can act as an N₂O sink because of reduced soil conditions when straw application was high, application of organic C and nitrogen as fertilizer can enhance N₂O production by the denitrification process during the growing season, especially in the ripening stage when soil anaerobic conditions became moderate. These results suggest that negative emission factors for N₂O (EF_N2O) can be applied, and EF_N2O of organic fertilizer should be considered during the estimation of N₂O emission in the paddy field.     

Suppression of the fungal pathogen Magnaporthe grisea by Stenotrophomonas maltophilia,a seed-borne rice (Oryza sativa L.) endophytic bacterium

The present work was carried out to study the potential of bacteria isolated from the seeds of rice plant for the biocontrol of five rice pathogenic fungi. Eleven endophytic bacteria isolated from rice seeds were evaluated for their antagonistic potential. Of five pathogens studied, only the growth of Magnaporthe grisea was inhibited by one bacterial isolate in an in vitro dual culture assay. Based on 16S rDNA sequence analysis, and biochemical and morphological characteristics, this strain was closely related to Stenotrophomonas maltophilia. We named this new isolate to be S. maltophilia SEN₁ (seed endophyte). This isolate was further tested for the production of volatile and diffusible antibiotics against M. grisea, for plant growth-promoting (PGP) traits and colonization of some rice cultivars. In addition, S. maltophilia SEN₁ was tested for its ability to promote plant growth and reduce the incidence of rice blast disease under greenhouse conditions. When applied to the soil, this isolate increased seedling growth and suppressed blast disease in plants of three studied cultivars. This study also showed this isolate could colonize the root interior of other rice cultivars. This study indicates that the S. maltophilia isolate studied has an excellent potential to be used as biocontrol agents of M. grisea or biofertilizer under in vitro and in vivo conditions.     

Selenium Speciation and Distribution Characteristics in the Rhizosphere Soil of Rice (Oryza sativa L.) Seedlings

A five-step sequential extraction procedure was used for the fractionation of selenium (Se) in rhizosphere and nonrhizosphere soils with rice (Oryza sativa L.) seedlings. Results showed that in rhizosphere soils without the addition of Se, the soluble Se (Sol-Se), exchangeable Se and Se bound to carbonates (Exc-Se), Se bound to organic-sulfide matter and elemental Se (OM-Se), and total Se contents were significantly greater than those in nonrhizosphere soils, whereas the residual Se (Res-Se) was less than that in the nonrhizosphere soils. After the addition of Se, the Sol-Se and OM-Se contents in the rhizosphere soils were still evidently greater than those in nonrhizosphere soils, but the Exc-Se was significantly less in rhizosphere soils than in nonrhizosphere soils. Our overall results suggest that the Se bioavailability in rice rhizosphere soils is greater than that in nonrhizosphere soils. Selenium bioavailability in the rhizosphere soil is not correlated with Se accumulation in rice seedlings.     

Effect of seed phosphorus and soil phosphorus applications on early growth of rice (Oryza sativa L.) cv. IR66

Abstract

The vigour and size of rice seedlings in the nursery are generally correlated with final grain yield. The present study examined the possibility that increasing seed phosphorus (P) concentration would stimulate early growth of rice seedlings and therefore would have the potential to increase rice yield. Rice seeds with a uniform size and three levels of P concentration (0.115, 0.173, and 0.240% on a dry weight basis) were sown in pots on a P deficient soil with three levels of P supply (0, 7.75, and 38.8 mg P kg^?1 soil) to investigate their effect on root and shoot dry weight and P accumulation at three harvest times, 10, 20, and 30 d after sowing (DAS). The effect of seed P concentration on plant growth was greatest at a low soil P concentration and it was less pronounced with increasing soil P concentration and with time at all levels of soil P. At 10 DAS, shoot dry weight was 15% higher at a high seed P concentration (0.240%) (p < O.O1) than at a low seed P concentration (0.115%) at each level of soil P supply whereas at subsequent harvests (20 and 30 DAS) the effect of seed P concentration was observed only when the soil P supply was deficient. In contrast with its effects on shoot dry weight, high seed P concentration increased root dry weight only at the latest harvest (30 DAS). The fact that high seed P increased P concentrations in shoot tips, and in roots at 10 DAS suggests that improved P nutrition of seedlings in the first 10 DAS may be the mechanism by which high seed P concentration stimulates early growth, especially in soils with low P concentration. Sowing rice seed with high P concentration may be beneficial for increasing farmer's rice yields, in P deficient soil, and requires further field investigations.     

Selection of efficient vesicular-arbuscular mycorrhizal fungi for wetland rice (Oryza sativa L.)

Summary We tested the response of the wetland rice cultivar Prakash to inoculation with ten vescular-arbucular mycorrhizal (VAM) fungi (three selected from the first screening and seven isolated from local paddy fields) in a pot experiment under flooded conditions in order to select the most efficient mycorrhizal fungi to inoculate the rice nursery. A sandy clay loam soil was used as the substrate, fertilized with the recommended N and K levels (100 kg N ha^–1 as ammonium sulphate and 50 kg K ha^–1 as muriate of potash) and half the recommended level of P (25 kg ha^–1 as super phosphate). The inoculation was made into dry nursery beds and the beds were flooded when the seedlings were about 25 cm high, in 15 days. Twenty-eight-day old seedlings were transferred to pots filled with well puddled soil flooded with 5 cm of standing water. Based on the increase in grain yield and total biomass, Glomus intraradices and Acaulospora sp. were considered efficient and suitable for inoculation into rice nurseries.     

水稻苗期不同阶段与低氮耐性相关的QTL分析

以超级杂交稻协优9308(协青早B/中恢9308)的重组自交系(R IL)为材料,通过溶液培养试验检测苗期不同阶段与低氮耐性相关的数量性状基因座(QTL)。结果共检测到14个QTLs,单个QTL可解释的表型变异为7.13%1~3.03%。其中,处理后15 d检测到6个QTLs,分别位于第1、7、1、7、10和11染色体上;处理后30 d检测到8个QTLs,分别位于第3、8、3、10、3、8、10和4染色体上。处理后15 d,在第1染色体RM297-RM212区间检测到同时控制相对冠干重和相对总干重的QTL,与氮循环有关,此染色体区域可能富含关键的氮代谢基因。定位结果表明,两个时间检测出的低氮耐性QTL的差异表达与水稻不同发育阶段基因的时空表达密切相关,从而反映在低氮耐性位点的差异上。     

Effects of inorganic fertilizers and micronutrients on methane production from wetland rice (Oryza sativa L.)

We compared the effects of adding different forms of nitrogenous fertilizers on the production of CH₄ in soil and on CH₄ emission from rice plants, Urea and diammonium phosphate gave the highest rates of CH₄ production from the soil and emission through rice plants, followed by (NH₄)₂SO₄. NaNO₃ was the least effective. The effects of micronutrients like Mo, Ni, or B were more prominent than those of Fe, Mn, Zn, V, or Co. It is concluded that CH₄ emission from rice paddies is influenced by both macro- and micronutrients, through effects on both microbial methanogenesis in soil and elimination through rice plants as a consequence of the effects on plant growth.     

N2 fixation in two Sesbania species and its transfer to rice (Oryza sativa L.) as revealed by 15N technology

Summary We used ¹⁵N technology to investigate N₂ fixation by Sesbania speciosa and Sesbania rostrata and its transfer to a lowland rice crop after incorporation of the Sesbania spp. into soil as green manure. During the first 50 days after establishment in November–December 1989, S. speciosa and S. rostrata produced 1126 and 923 kg dry matter ha^-1 respectively. They gathered 31 and 23 kg N ha^-1 respectively, of which 62%±5% and 55%±3% respectively, came from N₂ fixation. Both these species produced a greater biomass during September–October 1989, with S. rostrata producing more than S. speciosa. These results reflected differential responses by the plants to different day lengths at different times of the year. Furthermore, the dry matter yield and %N of ¹⁵N-labelled S. speciosa were smaller than those of the unlabelled plants, possibly due to inhibition of N₂ fixation in root nodules by the chemical N fertilizers added during labelling. These differences were not so pronounced in the stem-nodulated S. rostrata. The increased grain yield of rice fertilized with N in the form of chemical fertilizer or green manure was a result of an increased number of panicles per hill. The rice crop manured with S. speciosa produced a lower grain yield, with a lower grain weight than that manured with S. rostrata. This was due to a low uptake of soil N by rice manured with S. speciosa. Recovery of N from the green manure in rice straw with S. speciosa was significantly higher than from rice manured with S. rostrata, because of the higher applied N uptake by rice manured with the former.