Global nitrogen budgets in cereals: A 50-year assessment for maize, rice, and wheat production systems.

Industrially produced N-fertilizer is essential to the production of cereals that supports current and projected human populations. We constructed a top-down global N budget for maize, rice, and wheat for a 50-year period (1961 to 2010). Cereals harvested a total of 1551 Tg of N, of which 48% was supplied through fertilizer-N and 4% came from net soil depletion. An estimated 48% (737 Tg) of crop N, equal to 29, 38, and 25 kg ha(-1) yr(-1) for maize, rice, and wheat, respectively, is contributed by sources other than fertilizer- or soil-N. Non-symbiotic N2 fixation appears to be the major source of this N, which is 370 Tg or 24% of total N in the crop, corresponding to 13, 22, and 13 kg ha(-1) yr(-1) for maize, rice, and wheat, respectively. Manure (217 Tg or 14%) and atmospheric deposition (96 Tg or 6%) are the other sources of N. Crop residues and seed contribute marginally. Our scaling-down approach to estimate the contribution of non-symbiotic N2 fixation is robust because it focuses on global quantities of N in sources and sinks that are easier to estimate, in contrast to estimating N losses per se, because losses are highly soil-, climate-, and crop-specific.

Nitrate pollution in groundwater and strategies to reduce pollution.

The input-intensive rainfed tropical ecosystem, where wet season (WS) rice (Oriza sativa L.)-dry season (DS) diversified high-value upland crops like vegetables predominate, has resulted in a problem of a large leakage of N into the environment, thereby polluting the water. Excessive use of N fertilizer in high-value crops grown in DS is economically motivated. Out of twenty water sources evaluated in a watershed with a total area of 265 ha located in Magnuang, Ilocos Norte, Philippines, twelve had near or above the World Health Organization's (WHO) NO3-N limit for drinking water of 10 ppm. Soil mineral N (upper 100 cm) observed in seven rice-sweet pepper (Capsicum annuum L.) farmers' fields ranged from 111 to 694 kg ha(-1) which decreased by 10 to 68% in plots with dry-to-wet (DTW) crops like indigo, indigo+mungo and corn. In fallow plots where mineral N was either maintained or increased, it showed movement to lower soil profiles demonstrating NO3 leaching without a crop. On average, maize (Zea mays L.) captured 176 kg N ha(-1) and indigo (Indigofera tinctoria L.) 194 kg N ha(-1). In both fallow and planted plots, mineral N declined to low levels at 100% water-filled pore spaces (WFPS) before rice transplanting. A strategy for including indigo plus maize as a N-catch crop is proposed to decrease NO3 leaching and maximize N use efficiency in a rice-sweet pepper cropping system.

Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice.

A study of the diversity of endophytic bacteria present in seeds of a deepwater rice variety revealed the presence of seven types of BOX-PCR fingerprints. In order to evaluate the plant growth promoting potential the presence of nitrogenase, indole acetic acid production and mineral phosphate solubilization were estimated in the representative BOX-PCR types. The seven representatives of BOX-PCR types produced indole acetic acid, reduced acetylene and showed specific immunological cross-reaction with anti-dinitrogenase reductase antibody. Only four types showed mineral phosphate solubilizing ability. Comparison of cellulase and pectinase activities showed differences among different BOX-PCR types. PCR fingerprinting data showed that one strain isolated from the surface sterilized seeds as well as the aerial parts of the seedlings of rice variety showed low cellulase and pectinase but relatively high ARA. On the basis of 16S rDNA nucleotide sequence and BIOLOG system of bacterial identification, this strain was identified as Pantoea agglomerans. For studying the endophytic colonization this strain was genetically tagged with the reporter gene, gusA. Histochemical analysis of the seedling grown in hydroponics showed that the tagged strain colonized the root surface, root hairs, root cap, points of lateral root emergence, root cortex and the stelar region. Treatment of the roots with 2,4-D produced short thickened lateral roots which showed better colonization by P. agglomerans.

Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens.

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (> or =86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.

Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens.

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.

Synchronizing N release from organic residues: opportunities for integrated management of N.

In intensive cropping systems, mineral nitrogen (N) fertilizers represent the largest component of the N cycle because the indigenous N supply is not adequate. The requirement for mineral fertilizer may be reduced with the use of organic nutrient sources. A more realistic use of organic matter, particularly in sub-Saharan Africa due to limited amounts and availability, is the combined use of organic nutrient sources and mineral fertilizers. The beneficial effects of integrated use of inorganic fertilizers and organic manures on improved nutrient recoveries, soil moisture retention, cation exchange capacity, and erosion control have been reported. However, there are as many reports indicating negligible benefits or even disadvantages of combining nutrient sources on crop production. This is not surprising given the combination of organic residue sources, soils, climatic, crops, and management factors that influence nutrient dynamics. The most widely accepted function of organic materials is improving the nutrient availability to crops by supplying N. The key to both improving efficiency of N use and reducing N losses is synchronization of N supply from soil, biological N2 fixation, organic residues, and inorganic fertilizers with the crop N demand. Organic materials are not magic; N losses also result from their use. Controlling N release from organic sources depends on their nutrient content and quality, soil properties, and the environmental and management factors. This paper will synthesize the information generated from integrated nutrient management trials in sub-Saharan Africa and the Philippines. Management strategies based on an organic resources database and a dynamic soil-crop simulation model are used to identify organic sources as N fertilizers or soil amendments. The decision support tools are also used to attain optimum synchrony between release from organic sources and soils with crop N demand.

Rice ENOD40: isolation and expression analysis in rice and transgenic soybean root nodules.

The early nodulin ENOD40 has been proposed as playing a pivotal role in the organogenesis of legume root nodules. We have isolated the ENOD40 gene homologues ObENOD40 and OsENOD40 from the wild and cultivated rice genotypes Oryza brachyantha and Oryza sativa, respectively. Rice ENOD40s contain a sequence at the 5' end (region I) for encoding an oligopeptide that is highly conserved in all legume ENOD40s. Furthermore, at the 3' end (region II), the nucleotide sequence of rice ENOD40s exhibited a considerable homology to the corresponding region in legume ENOD40s. Among various organs of the rice plant, expression of OsENOD40 was detected only in stems. In situ hybridization studies revealed that, within the stem, transcription of OsENOD40 is confined to parenchyma cells surrounding the protoxylem during the early stages of development of lateral vascular bundles that conjoin an emerging leaf. Expression pattern of OsENOD40 promoter-GUS fusion in nodules developed on transgenic hairy roots of soybean was also found to be restricted to peripheral cells of nodule vascular bundles, thus evidencing that rice ENOD40 promoter activity is essentially the same as that of soybean ENOD40. Taken together, these results strongly suggest that OsENOD40 and legume ENOD40s share common, if not identical, functions in differentiation and/or function of vascular bundles.

Widespread occurrence of the homologues of the early nodulin (ENOD) genes in Oryza species and related grasses.

Eighty accessions representing 23 species from the genus Oryza were examined for the presence of homologues of early nodulin (ENOD) genes. Southern analyses indicated a widespread distribution of homologues of ENOD genes across all the genomes of rice as well as other monocots. The degree of cross-hybridization of the legume ENOD genes with sequences in the genomes of various species, as revealed by hybridization differentials measured in terms of signal intensities, however, suggests that the homologues of ENOD genes are conserved to varied extents in different Oryza species. The presence of homologues of ENOD genes in a wide variety of plant species denotes that the biological functions of early nodulins may be diverse, and not restricted to nodule organogenesis alone. The fact that ENOD gene homologues exist widely both in dicots and monocots provides evidence that these homologues have arisen from a common ancestral plant.

Isolation, analysis and expression of homologues of the soybean early nodulin gene GmENOD93 (GmN93) from rice.

Rice (Oryza sativa var. Nipponbare) possesses two different homologues of the soybean early nodulin gene GmENOD93 (GmN93). Analysis of the cDNA clones of rice homologues showed that OsENOD93a has an open reading frame (ORF) with a coding sequence homology of 58.2% to GmENOD93, whereas the ORF of OsENOD93b has displayed a homology of 42.3%. OsENOD93a and OsENOD93b genes are differentially expressed in different parts of the rice plant, as well as in cultured cells induced or non-induced with chitin oligomer. In intact rice tissues, OsENOD93b was most abundantly expressed in roots and at much lower levels in etiolated and green leaves, whereas the expression of OsENOD93a was very low in roots and etiolated leaves, and was not detected in green leaves. The level of OsENOD93a expression was enhanced markedly in suspension-cultured cells, whereas that of OsENOD93b did not increase. The application of chitin oligomer, an elicitor which induces a defence response in plants, did not significantly alter the expression of both these homologues in suspension cultures.

Non-allelic interaction conditioning spikelet sterility in an F2 population of indica/japonica cross in rice.

Significant segregation of spikelet fertility occurred in an F2 population derived from a spikelet fertility-normal F1 hybrid produced by a cross between 'Palawan', a japonica variety, and 'IR42', an indica variety. To identify factors controlling the fertility segregation, we used 104 RFLP markers covering all 12 rice chromosomes to investigate the association of spikelet fertility and marker segregation. We found that the segregation of two sets of gene pairs was significantly (P < 0.001) associated with fertility segregation. The first pair of genes was linked to RFLP marker RG778 on chromosome 12 and RFLP markers RG690/RG369 on chromosome 1. A significant reduction in fertility was observed when the plants were homozygote at RG778 with the indica allele as well as homozygote at RG690/RG369 with the japonica allele. The second pair of genes was linked to RG218 on chromosome 12 and RG650 on chromosome 7, respectively. The recombinant homozygote at these two loci showed a significant reduction on spikelet fertility. The non-allelic interaction effect was further modified by a gene linked to RG778, resulting in even lower fertility. The results of this study provides the first evidence of chromosomal localization of sporophytic sterility genes whose interaction can result in a reduction of spikelet fertility in the F2 derived from fertility-normal F1.

Molecular-marker-facilitated investigation on the ability to stimulate N2 fixation in the rhizosphere by irrigated rice plants.

An F2 population, consisting of 231 individuals derived from a cross between rice cultivars with a similar growing duration, Palawan and IR42, was utilized to investigate the genetic nature of rice varietal ability to stimulate N2 fixation in the rice rhizosphere. To assess rhizospheric N2 fixation, an isotope-enriched (15)N dilution technique was employed, using (15)N-stabilized soil in pots. IR42, an indica variety, had 23% higher N derived from fixation (Ndfa) than Palawan, a javanica genotype. Normal segregation of atom% (15)N excess was obtained in the F2 population, with an average of 0.218 with 8% of plants below IR42 (0.188) and 10% of plants above Palawan (0.248). One-hundred-and-four RFLP markers mapped on 12 chromosomes were tested for linkage to the putative QTLs. Significant (P<0.01) associations between markers and segregation of atom% (15)N excess were observed for seven marker loci located on chromosomes 1, 3, 6 and 11. Four QTLs defined by the detected marker loci were identified by interval-mapping analysis. Additive gene action was found to be predominant, but for at least one locus, dominance and partial dominance effects were observed. Significant (P<0.01) epistatic effects were also identified. Individual marker loci detected between 8 and 16% of the total phenotypic variation. All four putative QTLs showed recessive gene action, and no phenotypic effects associated with heterozygosity of marker loci were observed. The results of this study suggest that rice genetic factors can be identified which affect levels of atom% (15)N excess in the soil by interacting with diazotrophs in the rice rhizosphere.

Photosynthetic symbionts of Aeschynomene spp. form a cluster with bradyrhizobia on the basis of fatty acid and rRNA analyses.

The relationship between photosynthetic rhizobia that nodulate 10 Aeschynomene species (Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene scabra, Aeschynomene schimperi, and Aeschynomene sensitiva) and reference strains of the genera Bradyrhizobium, Rhizobium, and Azorhizobium was investigated by analyzing cellular fatty acid methyl esters (FAME) and 16S rRNA sequences. The members of each genus produced very distinct FAME patterns, and the photosynthetic rhizobia formed a subcluster in the Bradyrhizobium cluster. The absence of the cyc C19:0 type of fatty acid in all of the photosynthetic rhizobium strains isolated from 10 Aeschynomene species distinguished these microorganisms from other known rhizobia, including strain BTAi 1, a photosynthetic symbiont of A. indica. We sequenced a 264-base segment of the 16S rRNA genes of selected strains after amplification by the PCR and compared the results with previously published sequences for species of rhizobia and related photosynthetic bacteria. Photosynthetic strains IRBG 2 (from A. afraspera), IRBG 230 (from A. nilotica), and ORS 322 (from A. afraspera) had identical sequences but were distinct from strain BTAi (from A. indica) and from strain IRBG 231 (from A. denticulata), which is similar to the type strain (DNA homology group Ia) of Bradyrhizobium japonicum. Nonphotosynthetic strain IRBG 274 (from A. afraspera) was closely related to Bradyrhizobium elkanii (DNA homology group II). All of the photosynthetic rhizobia clearly fell into the Bradyrhizobium cluster. Although the results of the FAME and 16S rRNA analyses were in excellent agreement, our placement of the photosynthetic rhizobia is in apparent conflict with phenotypic data, as determined by numerical taxonomy (Ladha and So, Int. J. Syst. Bacteriol., in press) which placed the photosynthetic rhizobia in a coherent cluster that is as far from the genus Bradyrhizobium as the genera Rhizobium and Azorhizobium are. While the FAME and 16S rRNA data probably provide a more reliable indication of phylogeny, the degree of phenotypic divergence observed raises questions concerning the polyphasic approach to bacterial systematics.

Phylogenetic Analysis of Bradyrhizobium japonicum and Photosynthetic Stem-Nodulating Bacteria from Aeschynomene Species Grown in Separated Geographical Regions.

Nearly complete and short partial 16S rRNA sequences were derived from PCR-amplified ribosomal DNAs of Bradyrhizobium japonicum USDA 136 and USDA 110 and five strains of bacteriochlorophyll-synthesizing bacteria isolated from stem nodules of Aeschynomene indica and other Aeschynomene species growing in different geographic regions, including India, The Philippines and North America. We confirmed that the five stem-nodulating strains examined synthesize bacteriochlorophyll a, and the absorption spectra of methanol-extracted cells contained a major absorbance peak at 770 nm. Strains isolated on different continents and from different Aeschynomene species were found to be phylogenetically homogeneous and exhibited levels of sequence similarity of more than 99%. The bacteriochlorophyll-synthesizing rhizobia, Bradyrhizobium japonicum, Blastobacter denitrificans, Afipia felis, and Rhodopseudomonas palustris exhibited levels of sequence similarity of 97% or greater and belong to a distinct line of descent within the alpha-2 subdivision of the Proteobacteria. Variable regions between positions 995 and 1045 provide potential target sites for design of a probe that is able to distinguish the photosynthetic rhizobia from closely related taxa.

Epiphytic Occurrence of Azorhizobium caulinodans and Other Rhizobia on Host and Nonhost Legumes.

A large population of Azorhizobium caulinodans was present on Sesbania rostrata; up to 5 x 10 cm were found on leaves and fewer were found on flowers. Although A. caulinodans was also present on the leaves of Sesbania aculeata (nonhost), the populations were much smaller than that observed on S. rostrata. The population of S. aculeata rhizobia on host leaves was less than 30 cm, and their presence on host flowers was sporadic. Aeschynomene afraspera and Aeschynomene aspera rhizobia, which are profusely stem nodulating, were found on the leaves of host and nonhost plants and on the flowers of host plants, but, Aeschynomene pratensis and Aeschynomene sensitiva rhizobia were not found on the leaves and flowers of host plants.

Survival of Azorhizobium caulinodans in the Soil and Rhizosphere of Wetland Rice under Sesbania rostrata-Rice Rotation.

The survival of indigenous and introduced strains of Azorhizobium caulinodans in flooded soil and in the rice rhizosphere, where in situ Sesbania rostrata was incorporated before the rice crop, is reported. The azorhizobia studied were both root and stem nodulating. In a pot experiment, two crop cycles each of inoculated and noninoculated Sesbania-rice were compared with two crop cycles of flooded fallow-rice. In a field experiment, the effect of repeated incorporation of in situ S. rostrata in the Sesbania-rice sequence was studied. Soils in which inoculated S. rostrata was incorporated contained about 3,000 times more azorhizobia than did soils in the flooded fallow treatment and about 50 times more azorhizobia than did soils in the noninoculated Sesbania treatment. Azorhizobial numbers in the inoculated Sesbania treatment declined toward rice harvest but remained much higher than in the flooded fallow-rice treatment. Repeated incorporation of S. rostrata increased the population density of indigenous soil azorhizobia, whereas the population of inoculated strain ORS571 (Str Spc) declined to an undetectable level; this finding suggested low competitiveness by the introduced strain. In the incorporated Sesbania treatment, the rice rhizosphere harbored significantly more A. caulinodans and supported higher nitrogenase activity per plant than did the rhizosphere of the flooded fallow-rice treatment. Sterile rice seedlings inoculated with A. caulinodans showed nitrogenase activity comparable to that of seedlings inoculated with Azospirillum lipoferum 34H, a rice root isolate. Rhizobia from Sesbania aculeata, Sesbania sesban, a Trifolium sp., and Vigna unguiculata did not support appreciable nitrogenase activity.

Isolation and identification of N2-fixing Pseudomonas associated with wetland rice.

Semisolid yeast extract medium amended with glucose and tryptic soy agar were used to isolate aerobically N2-fixing (C2H2-reducing) heterotrophic bacteria from the root of wetland rice. The isolates were identified as Pseudomonas by gel immunodiffusion and fluorescent antibody techniques in combination with their morphological, cultural, and biochemical characteristics. The N2-fixing H2-utilizing Pseudomonas described in this paper is a new species.

Immunological techniques to identify Azospirillum associated with wetland rice.

Azospirilla associated with wetland rice were isolated and characterized by employing immunodiffusion and immunofluorescence techniques. Antisera against two strains belonging to Azospirillum lipoferum produced at least one heat-labile precipitation band with most isolates of A. lipoferum and A. brasilense. Antisera against two strains belonging to A. lipoferum and one strain belonging to A. brasilense produced one band only with strains of their respective species. Fluorescent antibody reactions with Azospirillum were species specific. Specificity of these antisera and fluorescent antibodies was further demonstrated with bacteria other than Azospirillum.