Youssef G. Yanni

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.

Ascending Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice Growth Physiology

ABSTRACT Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 1010 rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the worlds most important cereal crops.

The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots

his paper summarizes a multinational collaborative project to search for natural, intimate associations between rhizobia and rice (Oryza sativa L.), assess their impact on plant growth, and exploit those combinations that can enhance grain yield with less dependence on inputs of nitrogen (N) fertilizer. Diverse, indigenous populations of Rhizobium leguminosarum bv. trifolii (the clover root-nodule endosymbiont) intimately colonize rice roots in the Egyptian Nile delta where this cereal has been rotated successfully with berseem clover (Trifolium alexandrinum L.) since antiquity. Laboratory and greenhouse studies have shown with certain rhizobial strain-rice variety combinations that the association promotes root and shoot growth thereby significantly improving seedling vigour that carries over to significant increases in grain yield at maturity. Three field inoculation trials in the Nile delta indicated that a few strain-variety combinations significantly increased rice grain yield, agronomic fertilizer N-use efficiency and harvest index. The benefits of this association leading to greater production of vegetative and reproductive biomass more likely involve rhizobial modulation of the plants root architecture for more efficient acquisition of certain soil nutrients [e.g. N, phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), zinc (Zn), sodium (Na) and molybdenum (Mo)] rather than biological N 2 fixation.

Enhancement of rice production using endophytic strains of Rhizobium leguminosarum bv. trifolii in extensive field inoculation trials within the Egypt Nile delta

This study assessed the ability of biofertilizer inoculants containing Rhizobium leguminosarum bv. trifolii to enhance production of rice (Oryza sativa L.) under actual agricultural conditions in the Nile delta. Large-scale field experiments evaluated 5 rice varieties inoculated with 7 endophytic rhizobial strains during 5 growing seasons, including at sites ranked as the world’s highest in rice production. Inoculation with single strains or multi-strain consortia significantly increased grain yield in 19 of the 24 trials. By combining superior rhizobial inoculants with agricultural extension training, grain yield increased up to 47% in farmers’ fields, with an average increase of 19.5%. Data on rice straw production, harvest index and the agronomic fertilizer N-use efficiency also indicated positive agronomic benefits of rhizobial inoculation. These results establish the merit of deploying our biofertilization strategy using selected rhizobial strains to promote rice production capacity while reducing the need for additional chemical N-fertilizer inputs to maintain agricultural sustainability and acceptable production economy. Technology transfer of this important translational research can significantly help to alleviate hunger and meet the nutritional needs of many people in developing countries.

Ecology and Plant Growth-Promoting Activities of the Natural Association between Rhizobium Leguminosarum Bv. Trifolii and Rice Roots

Recent studies indicate that rhizobia can associate intimately with various Gramineae. We describe a natural, beneficial association of R. leguminosarum by. trifolii and rice grown in Egyptian fields. A diversity of rice-adapted rhizobia was isolated from nodules of berseem clover inoculated with macerates of field-grown, surface-sterilized rice roots. Selected isolates exhibited plant growth-promoting activities on some rice varieties in gnotobiotic culture, and significant grain yield of rice in two cycles of field inoculation trials. These findings indicate their potential ability to enhance rice production in sustainable agriculture, and current studies should help to define how beneficial rhizobia can adapt to cereals and improve productivity in agronomic cropping systems.

Natural Beneficial Association of Rhizobium leguminosarum bv. trifolii and Rice Roots and its Relevance to Sustainable Agriculture

Rhizobia can associate with various Gramineae. However, in order to provide a real-world benefit to agronomic cereal production, rhizobia must be able to successfully colonize grass roots amidst competition with other rhizosphere microorganisms under field conditions, and reduce fertilizer requirements while promoting plant growth without imposing pathological stress on the graminaceous host plant.

Spatial Ecology of Microbial Colonization Intensity and Behavior in Rice Rhizoplane Biofilms Analyzed By CMEIAS Bioimage Informatics Software

CMEIAS (Center for Microbial Ecology Image Analysis System) Bioimage Informatics software is designed to strengthen microscopy-based approaches for understanding microbial ecology at spatial scales directly relevant to ecological functions performed by individual cells and microcolonies. Copyrighted software components are thoroughly documented and provided as free downloads at . The software tools already released include CMEIAS-Image Tool v. 1.28, CMEIAS Color Segmentation, CMEIAS Quadrat Maker and CMEIAS JFrad Fractal Dimension analysis. The spatial ecology module of the next CMEIAS upgrade currently being developed (version 4.0) is designed to extract data from images for analysis of plotless point-patterns, quadrat-lattice patterns, geostatistical autocorrelation and fractal geometry of cells within biofilms. Examples presented here illustrate how selected CMEIAS attributes can be used to analyze the in situ spatial intensity, pattern of distribution, and colonization behavior of an indigenous population of a rhizobial strain on a sampled image of the rhizoplane landscape of a rice plant grown in field soil. The spatial ecology information gained can provide useful insights that help to predict the most likely performance of the biofertilizer test strain in relation to the growth response of the crop under field conditions.