Biological nitrogen fixation and prospects for ecological intensification in cereal-based cropping systems.

The demand for nitrogen (N) for crop production increased rapidly from the middle of the twentieth century and is predicted to at least double by 2050 to satisfy the on-going improvements in productivity of major food crops such as wheat, rice and maize that underpin the staple diet of most of the world's population. The increased demand will need to be fulfilled by the two main sources of N supply - biological nitrogen (gas) (N2) fixation (BNF) and fertilizer N supplied through the Haber-Bosch processes. BNF provides many functional benefits for agroecosystems. It is a vital mechanism for replenishing the reservoirs of soil organic N and improving the availability of soil N to support crop growth while also assisting in efforts to lower negative environmental externalities than fertilizer N. In cereal-based cropping systems, legumes in symbiosis with rhizobia contribute the largest BNF input; however, diazotrophs involved in non-symbiotic associations with plants or present as free-living N2-fixers are ubiquitous and also provide an additional source of fixed N. This review presents the current knowledge of BNF by free-living, non-symbiotic and symbiotic diazotrophs in the global N cycle, examines global and regional estimates of contributions of BNF, and discusses possible strategies to enhance BNF for the prospective benefit of cereal N nutrition. We conclude by considering the challenges of introducing in planta BNF into cereals and reflect on the potential for BNF in both conventional and alternative crop management systems to encourage the ecological intensification of cereal and legume production.

Fertilizing benefits of biogenic phosphorous nanonutrients on Solanum lycopersicum in soils with variable pH.

Nanoformulations of Phosphorous (P) have recently been proposed as alternatives to P fertilizers. In this study, the fertilizing efficacies of P-based nanomaterials (NMs), nanohydroxyapatite (nHAP) and nanophosphorus (nP), were examined on Solanum lycopersicum (Pusa Rohini, Indian tomato) in growth room pot experiments. These NMs differed in their mode of synthesis, chemical composition, size and shape. Rock-phosphate (RP), phosphoric acid (PA) and di-ammonium phosphate (DAP) were included as bulk materials for comparison. Three varieties of artificial soils were included in the study, neutral (pH 7.2), acidic (pH 4.3) and basic (pH 9.8). The effects of the NMs on germination, plant growth, and P content were assessed at the 15th and 30th days after treatment. The results showed that P-based NMs enhance the overall germination and plant growth by increasing P levels in all types of soils for the tomato plants in comparison to the bulk P sources. Analysis using X-ray fluorescence revealed enhanced P content in the plants indicating the uptake of P-based NMs. Evaluation of H2O2, total phenolics and total flavonoids contents after NM treatment suggest that there is no stress caused due to the application of NMs to the plant. The results of this study indicate the beneficial role of P-based NMs as fertilizers at the early stages of plant development, which opens a scope for further investigation of underlying metabolic and molecular pathways and field trials.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato.

KEY MESSAGE: Host mediated silencing of COM1 gene of Colletotrichum gloeosporioides disables appressorial differentiation and effectively prevents the development of Anthracnose disease in chilli and tomato. Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target the Colletotrichum gloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed extremely low abundance of CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

Nitrogen Fixation in Cereals.

Cereals such as maize, rice, wheat and sorghum are the most important crops for human nutrition. Like other plants, cereals associate with diverse bacteria (including nitrogen-fixing bacteria called diazotrophs) and fungi. As large amounts of chemical fertilizers are used in cereals, it has always been desirable to promote biological nitrogen fixation in such crops. The quest for nitrogen fixation in cereals started long ago with the isolation of nitrogen-fixing bacteria from different plants. The sources of diazotrophs in cereals may be seeds, soils, and even irrigation water and diazotrophs have been found on roots or as endophytes. Recently, culture-independent molecular approaches have revealed that some rhizobia are found in cereal plants and that bacterial nitrogenase genes are expressed in plants. Since the levels of nitrogen-fixation attained with nitrogen-fixing bacteria in cereals are not high enough to support the plant's needs and never as good as those obtained with chemical fertilizers or with rhizobium in symbiosis with legumes, it has been the aim of different studies to increase nitrogen-fixation in cereals. In many cases, these efforts have not been successful. However, new diazotroph mutants with enhanced capabilities to excrete ammonium are being successfully used to promote plant growth as commensal bacteria. In addition, there are ambitious projects supported by different funding agencies that are trying to genetically modify maize and other cereals to enhance diazotroph colonization or to fix nitrogen or to form nodules with nitrogen-fixing symbiotic rhizobia.

A rhamnose-deficient lipopolysaccharide mutant of Rhizobium sp. IRBG74 is defective in root colonization and beneficial interactions with its flooding-tolerant hosts Sesbania cannabina and wetland rice.

Rhizobium sp. IRBG74 develops a classical nitrogen-fixing symbiosis with the aquatic legume Sesbania cannabina (Retz.). It also promotes the growth of wetland rice (Oryza sativa L.), but little is known about the rhizobial determinants important for these interactions. In this study, we analyzed the colonization of S. cannabina and rice using a strain of Rhizobium sp. IRBG74 dually marked with ß-glucuronidase and the green fluorescent protein. This bacterium colonized S. cannabina by crack entry and through root hair infection under flooded and non-flooded conditions, respectively. Rhizobium sp. IRBG74 colonized the surfaces of wetland rice roots, but also entered them at the base of lateral roots. It became endophytically established within intercellular spaces in the rice cortex, and intracellularly within epidermal and hypodermal cells. A mutant of Rhizobium sp. IRBG74 altered in the synthesis of the rhamnose-containing O-antigen exhibited significant defects, not only in nodulation and symbiotic nitrogen fixation with S. cannabina, but also in rice colonization and plant growth promotion. Supplementation with purified lipopolysaccharides from the wild-type strain, but not from the mutant, restored the beneficial colonization of rice roots, but not fully effective nodulation of S. cannabina Commonalities and differences in the rhizobial colonization of the roots of wetland legume and rice hosts are discussed.

Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules.

A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.

Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules.

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Both these proteins contain approximately 100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I, but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia.

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

Altered kinetic properties of tyrosine-183 to cysteine mutation in glutamine synthetase of anabaena variabilis strain SA1 is responsible for excretion of ammonium ion produced by nitrogenase.

A L-methionine- D, L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N(2) reduction. In order to determine the biochemical basis for the NH(4)(+)-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn(+2) for optimum activity, while SA0-GS was Mg(+2) dependent. SA0-GS had the following apparent K(m) values at pH 7.5: glutamate, 1.7 m M; NH(4)(+), 0.015 m M; ATP, 0.13 m M. The apparent K(m) for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 m M; NH(4)(+), 12.4 m M; ATP, 0.17 m M). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH(4)(+)-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K(m) values for NH(4)(+) and glutamate, and thus limited the assimilation of NH(4)(+) generated by N(2) reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH(4)(+) binding and metal ion coordination.

Infection and colonization of rice seedlings by the plant growth-promoting bacterium Herbaspirillum seropedicae Z67.

A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.