A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast)

Abstract

The intercellular spaces of sugarcane (Saccharum officinarum 1.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation. It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 10' bacterialml extracted fluid) on N-poor medium containing 10% Suc (pH 5.5). This bacterium was identical with the type culture of Acetobacfer diazotrophicus, a recently discovered Nz-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air. Similar bacteria were observed in situ in the intercellular spaces. This demonstrates the presence of an Nz-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium. The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop. This endogenous culture broth consisting of substrate and Nz-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers. It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.In some areas in Brazil, sugarcane has been grown continuously for more than 100 years without any nitrogenous fertilizer. It has long been suspected that substantial N fixation occurs in such systems (Neyra and Dobereiner, 1977). In Hawaii, experiments with "N showed that only a small proportion of fertilizer N given to sugarcane land was recovered in the crops; 70% of the N came from other sources (Takahashi, 1970). Recently, Boddey et al. (1991) and Urquiaga et al. (1992) concluded from I5N and N balance studies that some sugarcane varieties in Brazil are particularly effective in obtaining their N from associative biological fixation. N fixation associated with the roots of Brazilian sugarcane in the soil has been demonstrated by acetylene reduction (Dobereiner et al., 1972) and by 15N2 fixation (Ruschel et al., 1975(Ruschel et al., , 1978Lima et al., 1987). Although at least 11 genera of NZ- fixing bacteria have been isolated from the rhizosphere of sugarcane, none occur in large enough numbers to account for the high rates of N fixation found in these crops.Recently, a new microaerobic N2-fixing bacterium was isolated in large numbers from sugarcane stems, roots, leaves, and rhizosphere soil from sites in Brazil, Australia, and Mexico (Cavalcante and Dobereiner, 1988; MacRae, 1991, 1992;Fuentes-Ramirez et al., 1993). This bacterium, Acetobacter diazotrophicus (Gillis et al., 1989), has most unusual growth requirements. It shows optimal growth with 10% Suc and pH 5.5. It will grow in medium with Suc concentrations up to 30% and rapidly acidifies its surroundings by the formation of acetic acid. It has no nitrate reductase: its N2 fixation continued in...

Infection of mottled stripe disease‐susceptible and resistant sugar cane varieties by the endophytic diazotroph Herbaspirilium

“…diazotrophicus is regarded as the putative organism responsible for Ng fixation reported in sugar cane (Boddey et al, 1991;Dong et al, 1994;James et ah, 1994;Reis et al, 1994). However, other endophytic diazotrophs might be just as important (Dobereiner et al, 1993Boddey, 1995;Boddey et al, 1995).…”

“…Frommel, Nowak & Lazarovits, 1991;Chanway, 1995;Mclnroy & Kloepper, 1995). In addition, non-pathogenic endophytic (symptomless) colonization by diazotrophic bacteria has been reported in specific associations between Acetobacter diazotrophicus and sugar cane (Li & McRae, 1991Dobereiner, 1992;Dong et al, 1994;James et al, 1994), Azoarcus and kaliar grass (Leptochloa fusca) (Hurek et al, 1994), Azospirillum brasilense and wheat (Schloter et al, 1994), and Alcaligenes faecalis and rice (You & Zhou, 1989).…”

Identification and Sterilization of Epiphytic Bacterial Flora near Hydrangea Shoot Apical Meristems

“…Many studies have document plants with endophytes that exist in the xylem, phloem, and other plant cells (Dong et al, 1994;QuadtHallmann and Kloepper, 1996). In some plant species, bacterial contamination occurs in shoot-tip culture even when surface sterilization has been conducted (Bergmann et al, 1997;Mondal et al, 1990).…”

Ocorrência de micorrizas arbusculares e da bactéria diazotrófica Acetobacter diazotrophicus em cana-de-açúcar

“…Quando s e avalia o benefício das bactérias diazotróficas para a fixação biológica de nitrogênio (FBN), a cana-de-açúcar se destaca (Boddey, 1995). Dentre as várias bactérias diazotróficas citadas, Acetobacter diazotrophicus (Gillis et al, 1989) tem sido considerada uma das mais importantes pelo seu caráter endofítico obrigatório (Baldani et al, 1997), isto é, coloniza a planta de cana-de-açúcar desde as raízes até as folhas verdes ou secas (Reis et al, 1994), os vasos do xilema (James & Olivares, 1998) ou mesmo o apoplasto (Dong et al, 1994), mas não sobrevive no solo.…”