During surveys of bacteria possibly responsible for N 2 fixation in sugarcane, root and stem samples were collected in four sugarcane-growing regions in Brazil. A new microaerobic N 2-fixing bacterium was isolated from most samples of washed roots and stems from all regions. Isolation procedures were based on semisolid diluted sugarcane juice medium followed by replication to N-free 10% sugar medium acidified with acetic acid to pH 4.5. The new bacterium is an aerobic rod, motile by 1 to 3 lateral flagella, fixes N2 in semisolid media under air but not in liquid media except when a starter dose of N is added. It has no nitrate reductase and N2 fixation proceeds in the presence of 10mM NO~-. Best growth occurs with high sucrose concentrations (10%). Growth occurs up to 30% sucrose but not at 35%. Acid is formed reaching a final pH of below 3.0. Growth and Nz fixation proceed at this acidity. Ethanol is used for growth and is "overoxidised" (oxidized to CO2 and H20). Acetic and lactic acids are also oxidized to CO2 and H20. Acids produced from glucose are consumed with precipitation of CaCO3. Dark brown colonies are formed on potato agar with 10% sugar and dark orange colonies on N poor agar (20mg yeast extract per 1) containing bromothymol blue. In view of the distinct characteristics which do not allow identification within either Frateuria, Gluconobacter, Acetobacter or any known Nz-fixing bacterium a new genus and species are proposed and named Saccharobacter nitrocaptans.

Sixty-one strains of the root-associated nitrogen fixer Spirillum lipoferum exhibited a similar morphology in peptone--succinate salts medium: vibrioid cells having a diameter of 1.0 micrometer. When grown in broth the cells had a single polar flagellum, but when grown on agar at 30 degrees C lateral flagella of shorter wavelength were also formed. The DNA base composition was 69--71 mol% guanine + cytosine when determined by thermal denaturation. DNA homology experiments indicated the occurrence of two distinct but related homology groups: 46 strains were in group I and 15 strains were in group II. Group II strains were distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium. The results indicate that two species exist, and on the basis of their characteristics it is proposed that they be assigned to a new genus, Azospirillum. Strians belonging to group II are named A. lipoferum (Beijerinck) comb. nov., while those belonging to group I are named A. brasilense sp. nov. Strain Sp 59b (ATCC29707) is proposed as the neotype strain for A. lipoferum, and strain Sp 7 (ATCC 29145) is proposed as the type strain for A. brasilense.

During a survey of the occurrence of Azospirillum spp. in cereal roots, we obtained 119 isolates which could not be identified as members of one of the three previously described Azospirillum species. These strains formed a very homogeneous group of N2-fixing, microaerobic, motile, vibrioid, gram-negative rod-shaped organisms which formed a veillike pellicle in semisolid medium similar to that of Azospirillum spp. However, the new isolates differed from Azospirillum spp. by their smaller cell width (0.6 to 0.7 pm), variable flagellation (one to three flagella on one or both poles), moist brownish colonies, and broader pH and oxygen tolerance for nitrogenase activity. Organic acids were the preferred carbon sources, but glucose, galactose, L-arabinose, mannitol, sorbitol, and glycerol were also used. The guanine-plus-cytosine content of the deoxyribonucleic acid was slightly lower than the guanine-plus-cytosine contents of Azospirillum spp. (66 to 67 mol%). Deoxyribonucleic acid hybridization experiments with 17 strains of the group showed 50 to 100% complementarity, while the levels of hybridization with the type strains of Azospirillum brasilense, Azospirillum lipuferum, and Azospirillum amazonense were 23, 15, and 6%, respectively. For these new isolates we propose a new genus, Herbaspirillum (the name refers to the habitat of the orgahisms, the roots of cereals, which are herbaceous seed-bearing plants). The type species is named Herbaspirillum seropedicae after the place where it was first isolated. The type strain is strain 267, which has been deposited in the American Type Culture Collection as strain ATCC 35892.

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Significant nitrogen fixation has recently been demonstrated in Brazilian sugar cane {Saccharum officinarum) cultivars known to form associations with a number of diazotrophs, including Acetobacter diazotrophicus, an acid-tolerant endophytic bacterium which grows best on a sucrose-rich medium. In a series of experiments, aseptically-grown sugar cane plantlets were rooted in a liquid medium and inoculated with A. diazotrophicus originally isolated from field-grown sugar cane. After 4, 7, 9, and 15 d, plants were examined under light, scanning and transmission electron microscopes and the presence of A. diazotrophicus on and within plant tissues was confirmed by immunogold labelling. By 15 d, external bacterial colonization was seen on roots and lower stems, particularly at cavities in lateral root junctions. The loose cells of the root cap at root tips were a site of entry of the bacteria into root tissues. Both at lateral root junctions and root tips, bacteria were also seen in enlarged, apparently intact, epidermal cells. After 15 d, bacteria were present in xylem vessels at the base of the stem, many connected via mucus to spiral secondary thickening. There was no obvious pathogenic reaction to the bacteria within the xylem. From these observations, it is proposed that, under experimental conditions, A. diazotrophicus firstly colonized the root and lower stem epidermal surfaces and then used root tips and lateral root junctions to enter the sugar cane plant where it was distributed around the plant in the transpiration stream. It is further suggested that the xylem vessels in the dense shoots of mature plants are also a possible site of N 2-fixation by diazotrophs as they provide the low pO 2 and energy as sucrose necessary for nitrogenase activity.