Fig 1: Transmission electron micrograph of Azospirillum lipoferum 4B grown in liquid culture. (F, polar flagellum; bar, 1 µm). Source: Alexandre et al. 1999, Applied Environmental Microbiology, 65, 7401.

    Azospirillum is a Gram negative motile bacteria belonging to the order Rhodospirillales, associated with roots of monocots, including important crops, such as wheat, corn and rice. Both in greenhouse and in field trials, several strains of Azospirillum were shown to exert beneficial effects on plant growth and crop yields, under various soil and climatic conditions, and are thus qualified as Plant Growth-Promoting Rhizobacteria (PGPR).
Actually, Azospirillum is the primary commercial phytostimulator inoculant for cereals worldwide. For instance, up to one million hectares of maize are successfully bio-fertilized with Azospirillum in Mexico. In the context of sustainable agriculture, plant inoculation with Azospirillum is a good alternative to reduce chemical inputs.

Fig 2: Effect of Azospirillum lipoferum inoculation on rice root morphology. (A) uninoculated control; (B) rice inoculated with a strain of A. lipoferum. Source: UMR-CNRS 5557 Centre d'Ecologie Microbienne.

Azospirillum can establish an associative symbiosis with cereals but unlike mutualistic symbiosis (such as rhizobia with leguminous plants), the association is not accompanied by the formation of new organs. Azospirillum benefits the plant directly, via associative nitrogen fixation, synthesis of phytohormones (notably indole-3-acetic acid, IAA), and modulation of plant hormonal balance by deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). These traits may lead to enhanced root system branching and root elongation, which in turn will favour the uptake of soil water and minerals (Fig. 2). The establishment of the associative symbiosis between Azospirillum PGPR and the plant starts at a very early stage, i.e. during bacterial colonization of germinating seeds.

Two species of Azospirillum were initially described in 1978: A. brasilense and A. lipoferum. Eleven species have now been described. The majority of the studies on Azospirillum, notably on plant-beneficial properties, have been performed on Azospirillum brasilense where implementation of genetics is easier than in Azospirillum lipoferum. Genome sequencing of a strain of A. brasilense isolated from wheat is ongoing at the University of Tennessee and Oak Ridge National Laboratory (USA) by the team of Igor B. Zhulin (supported by the US National Science Foundation grant EF-0412186). In order to perform comparative genomics, the project ongoing in Genoscope was focused on strain 4B of Azospirillum lipoferum species.

Fig 3: Colonization of rice root by A. lipoferum 4B labelled with EGFP, analyzed by confocal laser scanning microscopy. Source: UMR-CNRS 5557 Centre d'Ecologie Microbienne.

Azospirillum lipoferum 4B has been isolated from a rice field of Camargue (South of France) in 1982 and has been studied for its high efficiency in nitrogen fixation and for its successful properties of rice colonization (Fig. 3) and growth promotion. Next to its potential use in agronomy, Azospirillum lipoferum 4B displays interesting features such as the presence of an inducible prophage and occurrence of phase variation, an adaptive process generating intrapopulation diversity and important in niche adaptation or to escape host defenses. Usually, variants spontaneously emerge from the wild-type at a frequency of 10-4 to 10-3 per cell per generation in vitro. Moreover, the strain A. lipoferum 4T, an atypical non-motile laccase-positive isolate of A. lipoferum isolated simultaneously and from the same rice rhizsophere than A. lipoferum 4B, is likely to be the variant form generated within the soil ecosystem. Recently, phase variation was shown to be accompanied by major genome rearrangements, with the loss of a 750-kb plasmid, both in the variant and in A. lipoferum 4T. This genome plasticity raises numerous questions about microevolution of Azospirillum in a fluctuating environment like soil.