Further Evidence that the N(inf2)-Fixing Endophytic Bacterium from the Intercellular Spaces of Sugarcane Stems Is Acetobacter diazotrophicus

Z Dong, M Heydrich, K Bernard, M E McCully
1995 Applied and Environmental Microbiology  
Nitrogen-fixing bacteria, isolated from the sugar solution in intercellular spaces of sugarcane stems, were compared with the type strain of Acetobacter diazotrophicus (PAL-5) and found to be congruent with it in all characters studied. These characters were 37 morphological and biochemical tests, cellular fatty acid composition, and nitrogenase activity. The nitrogenase activity was measured by acetylene reduction and H 2 evolution and found to be unusual in that the H 2 evolution was
more » ... d much less than expected by high concentrations of acetylene. Nitrogen-fixing, acid-producing, and acid-tolerant gramnegative bacteria have been isolated from the fluid in intercellular spaces of sugarcane stems (9). These isolates grow best on a sucrose-rich medium and can continue to grow in up to 30% sucrose. Previously, we had found, by use of a small number of biochemical tests, that these bacteria were culturally, metabolically, and phenotypically similar to the N 2 -fixing species Acetobacter diazotrophicus, which was first described in 1989 (9, 13). In the present report, we provide a more complete description of the phenotypic and cultural characteristics of this bacterium by use of a highly comprehensive panel of conventional biochemical tests and determination of cellular fatty acid (CFA) composition by a modern automated system. The production of H 2 by the isolates has been compared with that of the type strain because this gas is an obligatory product of nitrogenase activity, resulting in N 2 fixation. The level of H 2 production can be used to monitor the activity of the N 2 -fixing process (16, 22) . This H 2 production is suppressed to different degrees in different N 2 -fixing bacteria by the presence of acetylene (8). The extent of the suppression has been used to further characterize the isolates from the sugarcane stems. MATERIALS AND METHODS Plants. Two varieties of sugarcane (Saccharum officinarum L.), cv. Media Luna and JA60-5, which had been cultivated in the field in Havana, Cuba, or were first-, second-, or third-generation subcultures of Cuban stocks propagated vegetatively in a greenhouse in Ottawa, Ontario, Canada, were used (9). Bacterial strains. Isolates were obtained by inoculating the fluid removed by aseptic centrifugation from intercellular spaces of the sugarcane stems into semisolid LGI medium (9). Only those eight isolates obtained from different plants which were capable of reducing acetylene were compared. The type strain of A. diazotrophicus, with which all isolates were compared, was PAL-5 (ATCC-49037 ex Döbereiner, from the culture collection of the Departamento de Microbiologia, Universidad de La Habana). The strains designated MC-1, -2, -3, -4, and -7 came from cv. Media Luna (field material from Havana, Cuba), and those designated JO-1, -2, and -4 came from cv. JA60-5 (Carleton University greenhouse). All bacteria were stored in 10% glycerol in a liquid N 2 storage apparatus. Bacteria newly subcultured from these frozen cultures on slopes of modified LGI
doi:10.1128/aem.61.5.1843-1846.1995 fatcat:fm7i6b6bwzdylitlb2o53x5n4i