J. K. Ladha

Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production?

A fundamental shift has taken place in agricultural research and world food production. In the past, the principal driving force was to increase the yield potential of food crops and to maximize productivity. Today, the drive for productivity is increasingly combined with a desire for sustainability. For farming systems to remain productive, and to be sustainable in the long-term, it will be necessary to replenish the reserves of nutrients which are removed or lost from the soil. In the case of nitrogen (N), inputs into agricultural systems may be in the form of N-fertilizer, or be derived from atmospheric N2 via biological N2 fixation (BNF).

Efficiency of Fertilizer Nitrogen in Cereal Production: Retrospects and Prospects

Presently, 50% of the human population relies on nitrogen (N) fertilizer for food production. The world today uses around 83 million metric tons of N, which is about a 100‐fold increase over the last 100 years. About 60% of global N fertilizer is used for producing the worlds three major cereals: rice, wheat, and maize. Projections estimate that 50 to 70% more cereal grain will be required by 2050 to feed 9.3 billion people. This will require increased use of N of similar magnitude if the efficiency with which N is used by the crop is not improved. Fertilizer N‐recovery efficiency by the first crop is 30 to 50%. The remaining N either remains in the soil, the recovery of which in the following crops is very limited (

Opportunities for increased nitrogen-use efficiency from improved resource management in irrigated rice systems

Abstract Research and extension work to improve nitrogen (N) management of irrigated rice has received considerable investment because yield levels presently achieved by Asian farmers depend on large amounts of N fertilizer. Most work has focused on placement, form, and timing of applied N to reduce losses from volatilization and denitrification. In contrast, less emphasis has been given to development of methods to adjust N rates in relation to the amount of N supplied by indigenous soil resources. As a result, N fertilizer recommendations are typically made for districts or regions with the implicit assumption that soil N supply is relatively uniform within these domains. Recent studies, however, document tremendous variation in soil N supply among lowland rice fields with similar soil types or in the same field over time. Despite these differences, rice farmers do not adjust applied N rates to account for the wide range in soil N supply, and the resulting imbalance contributes to low N-use efficiency. A model for calculating N-use efficiency is proposed that explicitly accounts for contributions from both indigenous and applied N to plant uptake and yield. We argue that increased N-use efficiency will depend on field-specific N management tactics that are responsive to soil N supply and plant N status. N fertilizer losses are thus considered a symptom of incongruence between N supply and crop demand rather than a driving force of N efficiency. Recent knowledge of process controls on N cycling, microbial populations, and soil organic matter (SOM) formation and decomposition in flooded soils are discussed in relation to N-use efficiency. We conclude that the intrinsic capacity of wetland rice systems to conserve N and the rapid N uptake potential of the rice plant provide opportunities for significant increases in N efficiency by improved management and monitoring of indigenous N resources, straw residues, plant N status, and N fertilizer.

Weed Management in Direct‐Seeded Rice

Rice ( Oryza sativa L.) is a principal source of food for more than half of the world population, especially in South and Southeast Asia and Latin America. Elsewhere, it represents a high‐value commodity crop. Change in the method of crop establishment from traditional manual transplanting of seedlings to direct‐seeding has occurred in many Asian countries in the last two decades in response to rising production costs, especially for labor and water. Direct‐seeding of rice (DSR) may involve sowing pregerminated seed onto a puddled soil surface (wet‐seeding) or into shallow standing water (water‐seeding), or dry seed into a prepared seedbed (dry‐seeding). In Europe, Australia, and the United States, direct‐seeding is highly mechanized. The risk of crop yield loss due to competition from weeds by all seeding methods is higher than for transplanted rice because of the absence of the size differential between the crop and weeds and the suppressive effect of standing water on weed growth at crop establishment. Of 1800 species reported as weeds of rice, those of the Cyperaceae and Poaceae are predominant. The adoption of direct‐seeding has resulted in a change in the relative abundance of weed species in rice crops. In particular, Echinochloa spp., Ischaemum rugosum, Cyperus difformis , and Fimbristylis miliacea are widely adapted to conditions of DSR. Species exhibit variability in germination and establishment response to the water regime postsowing, which is a major factor in interspecifically selecting constituents of the weed flora. The relatively rapid emergence of “weedy” (red) rice, rice phenotypically similar to cultivars but exhibiting undesirable agronomic traits, has been observed in several Asian countries practicing DSR, and this poses a severe threat to the sustainability of the production system. Stale seedbeds, tillage practices for land leveling, choice of competitive rice cultivars, mechanical weeders, herbicides, and associated water management are component technologies essential to the control of weeds in DSR. Herbicides in particular are an important tool of weed management, but hand weeding is either partially or extensively practiced in countries of Asia, Africa, and Latin America. Though yet to be globally commercialized, transgenic rice varieties engineered for herbicide resistance are a potential means of weed control. The release of herbicide‐resistant rice for red rice control in the United States has indicated the need to critically examine mitigation methods for the control of gene flow. Integrating preventive and interventional methods of weed control remains essential in managing weed communities in DSR, both to prohibit the evolution of herbicide resistance and to maximize the relative contributions of individual components where herbicides are not widely used. There remains a need to further develop understanding of the mechanisms and dynamics of rice weed competition and of the community dynamics of weed populations in DSR to underpin sustainable weed management practices.

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.

How extensive are yield declines in long-term rice–wheat experiments in Asia?

The rice–wheat cropping system, occupying 24 million hectares of the productive area in South Asia and China, is important for food security. Monitoring long-term changes in crop yields and identifying the factors associated with such changes are essential to maintain and/or improve crop productivity. Long-term experiments (LTE) provide these opportunities. We analyzed 33 rice–wheat LTE in the Indo-Gangetic Plains (IGP) of South Asia, non-IGP in India, and China to investigate the extent of yield stagnation or decline and identify possible causes of yield decline. In treatments where recommended rates of N, P and K were applied, yields of rice and wheat stagnated in 72 and 85% of the LTE, respectively, while 22 and 6% of the LTE showed a significant (P<0.05) declining trend for rice and wheat yields, respectively. In the rice–wheat system, particularly in the IGP, rice yields are declining more rapidly than wheat. The causes of yield decline are mostly location-specific but depletion of soil K seems to be a general cause. In over 90% of the LTE, the fertilizer K rates used were not sufficient to sustain a neutral K input–output balance. Depletion of soil C, N and Zn and reduced availability of P, delays in planting, decreases in solar radiation and increases in minimum temperatures are the other potential causes of yield decline. A more efficient, integrated strategy with detailed data collection is required to identify the specific causes of yield decline. Constant monitoring of LTEs and analysis of the data using improved statistical and simulation tools should be done to unravel the cause–effect relationships of productivity and sustainability of rice–wheat systems.

Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth

For over 7 centuries, production of rice (Oryza sativa L.) in Egypt has benefited from rotation with Egyptian berseem clover (Trifolium alexandrinum). The nitrogen supplied by this rotation replaces 25- 33% of the recommended rate of fertilizer-N application for rice production. This benefit to the rice cannot be explained solely by an increased availability of fixed N through mineralization of N- rich clover crop residues. Since rice normally supports a diverse microbial community of internal root colonists, we have examined the possibility that the clover symbiont, Rhizobium leguminosarum bv. trifolii colonizes rice roots endophytically in fields where these crops are rotated, and if so, whether this novel plant-microbe association benefits rice growth. MPN plant infection studies were performed on macerates of surface-sterilized rice roots inoculated on T. alexandrinum as the legume trap host. The results indicated that the root interior of rice grown in fields rotated with clover in the Nile Delta contained ∼106 clover-nodulating rhizobial endophytes g fresh weight of root. Plant tests plus microscopical, cultural, biochemical, and molecular structure studies indicated that the numerically dominant isolates of clover-nodulating rice endophytes represent 3 – 4 authentic strains of R. leguminosarum bv. trifolii that were Nod Fix on berseem clover. Pure cultures of selected strains were able to colonize the interior of rice roots grown under gnotobiotic conditions. These rice endophytes were reisolated from surface-sterilized roots and shown by molecular methods to be the same as the original inoculant strains, thus verifying Kochs postulates. Two endophytic strains of R. leguminosarum bv. trifolii significantly increased shoot and root growth of rice in growth chamber experiments, and grain yield plus agronomic fertilizer N-use efficiency of Giza-175 hybrid rice in a field inoculation experiment conducted in the Nile Delta. Thus, fields where rice has been grown in rotation with clover since antiquity contain Fix strains of R. leguminosarum bv. trifolii that naturally colonize the rice root interior, and these true rhizobial endophytes have the potential to promote rice growth and productivity under laboratory and field conditions.

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.

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.

Biological nitrogen fixation for sustainable agriculture: A perspective

The economic and environmental costs of the heavy use of chemical N fertilizers in agriculture are a global concern. Sustainability considerations mandate that alternatives to N fertilizers must be urgently sought. Biological nitrogen fixation (BNF), a microbiological process which converts atmospheric nitrogen into a plant-usable form, offers this alternative. Nitrogen-fixing systems offer an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. Symbiotic systems such as that of legumes and Rhizobium can be a major source of N in most cropping systems and that of Azolla and Anabaena can be of particular value to flooded rice crop. Nitrogen fixation by associative and free-living microorganisms can also be important. However, scientific and socio-cultural constraints limit the utilization of BNF systems in agriculture. While several environmental factors that affect BNF have been studied, uncertainties still remain on how organisms respond to a given situation. In the case of legumes, ecological models that predict the likelihood and the magnitude of response to rhizobial inoculation are now becoming available. Molecular biology has made it possible to introduce choice attributes into nitrogen-fixing organisms but limited knowledge on how they interact with the environment makes it difficult to tailor organisms to order. The difficulty in detecting introduced organisms in the field is still a major obstacle to assessing the success or failure of inoculation. Production-level problems and socio-cultural factors also limit the integration of BNF systems into actual farming situations. Maximum benefit can be realized only through analysis and resolution of major constraints to BNF performance in the field and adoption and use of the technology by farmers.