Gluconacetobacter spp

The response of a sugarcane crop to nitrogen fertilizer can be rather low, depending on the cultivar used. A sugarcane crop accumulates between 100-200 kg N ha-1 per season and most of the fixed nitrogen is removed from the field at harvest because the trash representing about 25% of the senescent leaves is almost always burned off before cutting and less than 10% of the fixed-N remains in the field (Oliveira et al. 1994). Thus, the continuous cropping of sugarcane should quickly deplete soil N and cane yields should decline. However, such effects are not usually observed even after decades or centuries of cane cropping. Therefore, it had to be postulated that sugarcane must have a significant N-input through biological nitrogen fixation (BNF) and indeed this could be demonstrated by the quantification of the nitrogen budget including 15N-dilution analysis in sugarcane plants (Urquiaga et al. 1992). When grown with irrigation and ample phosphate fertilizer and molybdenum is applied as foliar spray (500 g/ha), some sugarcane varieties obtain more than 150 kg N ha-1 per year from BNF (Boddey et al. 2003). This is remarkable, because molybdenum is the key trace element required as essential cofactors in the nitrogenase enzyme necessary for N2-fixation and for nitrate reductase in the N-assimilation pathway. Using the 15N-natural abundance technique, 11 sites were studied in sugarcane plantations in Sao Paulo, Minas Gerais, and the Pernambuco States of Brazil. In nine of the sites, BNF inputs were significant and ranged from 25% to 60% of the plant nitrogen (Boddey et al. 2001). Therefore, diazotrophic bacteria need to effectively fix nitrogen in an endophytic association in sugarcane to create this amount of nitrogen nutrition.

The attempts in the late 1980s to isolate diazotrophic bacteria from the internal plant tissue of roots and the aerial parts of sugarcane varieties that are known to have high sugar concentrations (especially in the stem) soon resulted in the discovery of a new nitrogen-fixing species (Cavalcante and Döbereiner 1988). These endophytic bacteria were first named Acetobacter diazotrophicus (Gillis et al. 1989) and later renamed to Gluconacetobacter dia-zotrophicus (Yamada et al. 1997). This bacterium is not able to survive in soil (Baldani et al. 1997) and is transmitted from plant to plant mainly via plant cuttings (Reis et al. 1994). G. diazotrophicus was also found to be associated with spores of arbuscular mycorrhizal fungi (AM). AM fungi are regularly forming symbiotic interactions with the roots of vascular plants increasing the uptake of nutrients, mostly phosphate. Inoculation of plants with AM-fungi harboring G. diazotrophicus increased the translocation of diazotrophic endophytes into the plants (Döbereiner et al. 1993).

G. diazotrophicus is a true endophyte, because it was found inside the roots, stems, and leaves and trash of sugarcane. The bacteria have been localized in the xylem vessels (James et al. 1994, 2001; Sevilla et al. 2001) and in the apoplast space (Dong et al. 1994). Interestingly, it could also be isolated from the sugarcane mealy bug (Saccharococcus sacchari) (Ashbolt and Inkerman 1990). G. diazotrophicus was also found in Pennisetum purpureum (Reis et al. 1994), sweet potato (Paula et al. 1991), coffee (Jiménez-Salgado et al. 1997) pineapple (Tapia-Hernandez et al. 2000), a grass called Eleusine coracana (Loganathan et al. 1999) and several other plants such as tea (root), mango (fruit), banana (root) and ragi (root and stem) (Muthukumarasamy et al. 2002). Two other new diazotrophic Gluconacetobacter species, G. johannae and G. azoto-captans, could be isolated from coffee plants and pineapple (Fuentes-Ramirez et al. 2001a), but these could not be found in sugarcane. Specific PCR-primers are available to identify both G. diazotrophicus (Kirchhof et al. 1998) and G. johannae as well as G. azotocaptans (Fuentes-Ramirez et al. 2001b).

G. diazotrophicus is perfectly able to fix nitrogen with 10% added sucrose, and it still grows well in 30% sucrose (Cavalcante and Döbereiner 1988). G. diazotrophicus does not have an assimilatory nitrate reduction and thus continues to fix nitrogen even in the presence of high amounts of nitrate. G. diazotrophi-cus is not able to transport and respire sucrose, but it excretes the saccharolytic enzyme levan sucrase, which provides the bacterium with glucose for growth and fructose for the formation of the exopolysaccharide levan. It is hypothesized that the exopolysaccharide levan is the "glue" that holds microcolonies of G. diazotrophicus together at the colonization sites inside sugarcane (Reis et al. 2007). This could be of great importance for the protection of nitrogen-fixing micro-colonies against the oxygen damage of nitrogenase. During growth, the bacterium strongly acidifies the environment resulting in pH-values of 3 and below. Nevertheless, this bacterium continues to grow and fix nitrogen at this pH-level for several days (Stephan et al. 1991). In addition to BNF, phytohormone production was demonstrated in this bacterium. The auxin indole-3-acetic acid was shown to be produced in a defined culture medium (Fuentes-Ramirez et al. 1993). It was also shown that it produces a bacteriocin which inhibits Xanthomonas albilineans, the causal agent of leaf scald dis ease in sugarcane (Pinon et al. 2002). Furthermore, an antagonistic effect was demonstrated against the pathogenic fungus Collelotrichum falcatum, a causal agent of red-rot in sugarcane (Muthukumarasamy et al. 2002).

Detailed studies about the mechanism of plant growth stimulation and nitrogen fixation were carried out using a Nif- mutant of G. diazotrophicus PAL-5 (Sevilla et al. 2001). When fixed nitrogen was not limiting, growth promotion was present in plants inoculated with the wild type and the Nif-mutant MAd3a, arguing that a phytohormone effect was operative. However, under limiting conditions of fixed-N, plants inoculated with the wild type grew better and had a higher nitrogen content. This indicates a significant transfer of fixed N from G. diazotrophicus cells to sugarcane resulting in plant growth promotion. These results were confirmed using 15N2 gas incorporation (Sevilla et al. 2001). Interestingly, it could be demonstrated that the colonization of sugar cane by Gluconacetobacter diazotrophicus is inhibited under high N-fertilization conditions (Fuentes-Ramirez et al. 1999). Following the inoculation of sugarcane by G. diazotrophicus specific gene expression was observed (Nogueira et al. 2001).

Inoculation experiments of G. diazotrophicus wild type and mutant strains were also performed with rice and resulted in stimulated growth of rice plantlets after inoculation with the wild type but only a little increase after inoculation with the Nif- mutant strain MAd3a (as reported in Reis et al. (2007)). When PAl5 wild type was inoculated to maize, enhanced plant growth independent of nitrogen fixation was shown, although it did not enter maize roots in significant numbers (Riggs et al. 2001).

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