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 ؋ 10 10 rhizobia per cm 3 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 growthregulating 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 world's most important cereal crops.

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This 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 plant's 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. Inoculation increased total protein quantity per hectare in field-grown grain, thereby increasing its nutritional value without altering the ratios of nutritionally important proteins. Studies using a selected rhizobial strain (E11)

and Leong, 1986), thereby suppressing the diseases they cause. Other mechanisms of GPA include the induction Rice (Oryza sativa L.) is one of the world's most important crops.of host systemic disease resistance (Maurhofer et al.,The present investigation was designed to assess the range of growthpromoting activities of various diazotrophic bacteria on rice seedling 1994), N 2 fixation (Burton, 1976), solubilization of previgor, its carryover effect on straw and grain yield, and the persistence cipitated mineral nutrients (Subba Rao, 1982), and/or of an inoculant strain on rice roots under greenhouse conditions. production of plant growth regulators (Tien et al., 1979; Growth responses to inoculation exhibited bacterial strain-rice variety Bashan et al., 1990) that induce additional root hairs specificity that were either stimulatory or inhibitory. Growth reand/or lateral root formation (Tien et al., 1979), thereby sponses included changes in rates of seedling emergence, radical elonenhancing the plant's ability to take up nutrients from gation, height and dry matter, plumule length, cumulative leaf and root soil and increase yield. areas, and grain and straw yields. Most notable were the inoculation Rhizobial inoculation of legume seed is well studied, responses to Rhizobium leguminosarum bv. trifolii E11 and Rhizoand exploitation of this beneficial N 2 -fixing root-nodule bium sp. IRBG74, which stimulated early rice growth resulting in a symbiosis represents a hallmark of successfully applied carryover effect of significantly (P ϭ 0.05) increased grain and straw yields at maturity, even though their culturable populations on roots agricultural microbiology. However, much less informadiminished to below detectable values at 60 d after planting. The test tion is available regarding the association and GPA of strains were positive for indole-3-acetic acid production in vitro, but rhizobia with nonlegumes. In nature, rhizobia do associonly some reduced acetylene to ethylene in association with rice under ate with roots of nonlegumes without forming true nodlaboratory growth conditions. These studies indicate that certain ules (Ladha et al., 1989; Yanni et al., 1997), but their strains of nonphotosynthetic diazotrophs, including rhizobia, can propopulations decrease in number in the absence of lemote growth and vigor of rice seedlings, and this benefit of early gume-host plants (Ladha et al., 1989; Chabot et al., seedling development can carryover to significantly increased grain 1996b). Direct growth promotion of nonlegumes by rhiyield at maturity.

This paper describes the utility of CMEIAS (Center for Microbial Ecology Image Analysis System) computer-assisted microscopy to extract data from accurately segmented images that provide 63 different insights into the ecophysiology of microbial populations and communities within biofilms and other habitats. Topics include quantitative assessments of: (i) morphological diversity as an indicator of impacts that substratum physicochemistries have on biofilm community structure and dominance-rarity relationships among populations; (ii) morphotype-specific distributions of biovolume body size that relate microbial allometric scaling, metabolic activity and growth physiology; (iii) fractal geometry of optimal cellular positioning for efficient utilization of allocated nutrient resources; (iv) morphotype-specific stress responses to starvation, environmental disturbance and bacteriovory predation; (v) patterns of spatial distribution indicating positive and negative cell-cell interactions affecting their colonization behavior; and (vi) significant methodological improvements to increase the accuracy of color-discriminated ecophysiology, e.g., differentiation of cell viability based on cell membrane integrity, cellular respiratory activity, phylogenetically differentiated substrate utilization, and N-acyl homoserine lactone-mediated cell-cell communication by bacteria while colonizing plant roots. The intensity of these ecophysiological attributes commonly varies at the individual cell level, emphasizing the importance of analyzing them at single-cell resolution and the proper spatial scale at which they occur in situ.