Bacteria living in the rhizosphere and favorably affecting plant growth are denominated Plant Growth Promoting Rhizobacteria (PGPR). PGPR of the genus Azospirillum were initially studied because of their association with Gramineae and because of their ability to fix atmospheic nitrogen.38 Field trials, however, indicated that bacterial nitrogen is not responsible for the enhanced plant growth observed upon Azospirillum inoculation.39 The observation that inoculation of plant roots with Azospirillum results in a clear plant root proliferation intensified the study of bacterial phytohormone biosynthesis. For the azospirilla to exert their beneficial effect, it is well recognized that rhizosphere colonization is a prerequisite for enhancing plant growth. Aspects of bacterial rhizosphere colonization include chemotactic motility, adherence to the plant root surface and utilization of rhizo-sphere nutrients.
The study of an associative plant-bacterium interaction is hampered by the lack of an easily detectable plant phenotype following inoculation. To visualize the Azospirillum-plant root association, A. brasilense strains carrying a constitutive gusA fusion (pFAJ31.13) were constructed.31 pFAJ31.13 was constructed by inserting an A. brasilense gene library upstream of a promoterless gusA gene and selecting a fusion for high p-glucuronidase activity under different physiological conditions. This fusion wis then considered useful to localize Azospirillum on wheat roots in colonization assays. During the first days of the association, the bacterial cells were mainly found in the root hair zones (see Fig. 6.3) and at the sites of lateral root emergence. Microscopic analysis of sections through colonized zones o casion-
ally revealed the presence of azospirilla in the epidermis and cortex layers. No bacterial penetration through the endodermis or vascular tissue was observed.
In a second study, the colonization of wheat roots by a selection of A. brasilense mutants impaired in motility, chemotaxis and plant root attachment, carrying pFAJ31.13, was evaluated. Two nonmotile and a generally nonchemotactic mutant were found to be impaired in the primary colonization of the wheat roots. It can be hypothesized, that key compounds are specifically secreted by the root hair zone of the wheat roots to which Azospirillum is attracted.40,41
The expression of a translational A. brasilense nifH-gusA fusion was studied in free-living conditions and during wheat root association.31 Free-living nifH expression was shown to occur only under nitrogen-limiting microaerobic conditions, suggesting the presence of nitrogen and oxygen-dependent control mechanisms for nif gene expression in Azospirillum. Analysis of nifH gene expression and nitrogenase activity during the Azospirillum-wheat root association indicated that both oxygen and the availability of carbon sources are limiting factors for associative nitrogen-fixation.
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