Location of diazotrophs in the root interior with special attention to the kallar grass association

There is increasing evidence that nitrogen-fixing bacteria are able to colonize the interior of grass roots. Several techniques have been used to demonstrate the sites of colonization and are discussed. Among the techniques useful for specific labelling, gold-labelled reagents have been frequently successfully applied in other fields. We propose the use of the protein A-gold technique coupled with silver amplification and light microscopy to render diazotrophs visible in semi-thin sections of roots, and we have applied this technique to gnotobiotically grown kallar grass. LR white resin soft grade was a suitable resin for embedding. Diazotrophic rods predominating in the endorhizosphere of naturally growing kallar grass had the potential to colonize the aerenchyma of gnotobiotically-grown plants. Penetration by the bacteria probably occurred at epidermal cell junctions and at points of emergence of lateral roots, as also proposed forAzospirillum. Larger cell aggregates described by us for naturally occurring kallar grass plants were not detected in the gnotobiotic system. Physiological consequences of colonization of the aerenchyma are discussed.