Biol Fertil Soils (1999) 29:10-23 G. Holguin ⋅ C. L. Patten ⋅ B. R. Glick Genetics and molecular biology of Azospirillum © Springer-Verlag 1999 Abstract Genetic manipulation of Azospirillum spp. has facilitated a better understanding of the mode of action of this plant-growth promoting bacterium and should help to improve its ability to stimulate plant growth and development. This review considers and discusses Agospirillum plasmids, promoter sequences, the isolation of Azospirillum mutants, the genetic trans- formation of Azospirillum, the transfer of foreign genes into Azospirillum by conjugation and the Azospirillum genes that have been isolated and characterized. The Azospirillum genes that are discussed include genes in- volved in nitrogen fixation, plant root attachment, phy- tohormone biosynthesis, tryptophan biosynthesis, car- bon metabolism and a few other less well characterized processes. Key words Azospirillum · Plant interaction genes· Plasmid p90 · Plant-growth-promoting rhizobacteria· Promoter sequences Introduction Azospirillum was first isolated from nitrogen-poor san- dy soil in the Netherlands (Beijerinck 1925). The signif- icance of this discovery was not realized, however, for more than 50 years when Döbereiner and Day (1976) reported that grasses associated with Azospirillum did not exhibit symptoms of nitrogen deficiency seen in surrounding Azospirillum-free grasses. It has since been found that members of this bacterial genera are capable of fixing atmospheric nitrogen and of promoting G. Holguin - C. L. Patten - B. R. Glick (*) Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 e-mail: glick@sciborg.uwaterloo.ca , Tel.: +519-888-4567 ext. 2058, Fax: +519-746-0614 G. Holguin Department of Microbiology, Centro de Investigaciones Biologicas del Noroeste, (CIB), P. O. Box 128, La Paz, B.C.S. 23000, Mexico plant growth. At one time it was thought that the counterpart of Rhizobium, which is found in legumes, had been found for cereals, and by exploiting the capabilities of Azospirillum spp. it would be possible to supply nitrogen to crops of economic importance (Klingmüller 1982). Inoculation with Azospirillum spp. has been known to increase the yield of many cereals in the field by up to 30%, with often even greater increases under greenhouse conditions. However, these results have been difficult to repeat. The factors responsible for these irregularities have not been identified, primarily because the basic features of plant-Azospirillum interactions are not well understood. Unlike rhizobia-produced nodules, inoculation with Azospirillum does not induce a characteristic morphology in the root system. Therefore, the search for a mechanism by which Azospirillum promotes plant growth is somewhat more complicated (Bashan and Levanony 1990; Vande Broek and Vanderleyden 1995). Among the suggested modes of action for Azospirillum are: secretion of phytohormones, nitrogen fixation, production of undefined signal molecules that can interfere with plant metabolism, nitrite production, and the enhancement of mineral uptake by plants (Okon and Itzigsohn 1995). As there is not sufficient evidence to support the notion that one of these mechanisms is solely responsible for plant growth promotion, an additive hypothesis has been proposed (Bashan and Levanony 1990). It suggests that the net beneficial effect to the plant upon Azospirillum inoculation is the result of all the above-mentioned mechanisms operating either simultaneously or sequentially. Moreover, soil parameters, bacterial community interactions, plant growth phase, and growth phase of the bacterial inoculum may influence the participation of one or several of these mechanisms (Bashan and Holguin 1997; Okon and Labandera-Gonzalez 1994). Although the mechanisms involved in the Azospirillum-plant interaction ave not clear, it has been repeatedly shown that Azospirillum has the potential for Received: 11 December 1997