Rhizobial Inoculation, Alone or Coinoculated with Azospirillum brasilense, Promotes Growth of Wetland Rice

ABSTRACT Rhizobia and associative bacteria promote growth in rice plants (Oryza sativa L.) through a series of mechanisms, but most studies on inoculation have been performed based on inoculation with these bacteria in a separate or singular manner. The objective of this study was to assess the efficiency of single/isolated inoculation and inoculation combined with symbiotic rhizobia from forage legume and with Azospirillum brasilense on promoting growth and the root colonization process in wetland rice. Two rhizobia among four isolates from a greenhouse and a laboratory experiment were selected that efficiently promoted seed germination and rice plant growth in a sterilized substrate and in soil. The two most efficient isolates (UFRGS Vp16 and UFRGS Lc348) were inoculated alone or in combination with a commercial product containing A. brasilense in two field experiments using two wetland rice cultivars over two growing seasons. In the field experiments, these isolates coinoculated with A. brasilense promoted larger increases in the agronomic variables of wetland rice compared to the control without inoculation. Confocal laser microscopy confirmed the presence of inoculated bacteria tagged with gfp (UFRGS Vp16, UFRGS Lc348, and A. brasilense) colonizing the root surface of the rice seedlings, mainly in the root hairs and lateral roots

In Situ Localization and Strain-Specific Quantification of Azospirillum and Other Diazotrophic Plant Growth-Promoting Rhizobacteria Using Antibodies and Molecular Probes

A central issue in the understanding of the interaction and symbiotic function of diazotrophic bacteria with non-leguminous crop plants is detailed knowledge about the localization of the associated diazotrophic bacteria within the plant, their in situ activities in the plant-associated niches, and strain-specific quantification of inoculated bacteria. In addition to the colonization of rhizosphere soil and the rhizoplane, it has become apparent that an endophytic location of a diazotroph would provide it with a higher potential to interact more closely with the plant, particularly with respect to increasing the availability of carbon and energy nutrients derived from the plant, as well as the possibility, in return, of improving the transfer of bacterial-derived metabolites to the plant. Detailed localization of bacteria was successfully performed using fluorescence labeled ribosome-directed oligonucleotide probes in the fluorescence in situ hybridization (FISH) approach coupled to the use of confocal laser scanning microscopy (CLSM), and via immunolocalization with specific antibodies using transmission electron microscopy (TEM). Furthermore, the fate of inoculated bacteria could be traced by using specifically marked strains by applying the genes for the green or red fluorescent protein (GFP, RFP) and β-glucuronidase (GUS). Strain-specific quantification approaches for inoculants based on quantitative PCR using sequence characterized amplified regions (SCARs) and other genomic marker sequences have been developed and successfully applied. In this chapter major achievements and existing obstacles using these high resolution approaches to analyze bacteria in situ are presented together with some basic protocols