Indentification of Acetobacter diazotrophicus,Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans using biochemical and genetic criteria

Nitrogen-fixing Acetobacter diazotrophicus, Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans colonize sugar cane, and are thought to be capable of supplying high levels of fixed nitrogen to this plant. Eight A. diazotrophicus, two H. seropedicae and four H. rubrisubalbicans isolates were identified and compared by complementary biochemical and genetic methods. Utilization of carbon sources and antibiotic resistance patterns allowed differentiation of A. diazotrophicus from Herbaspirillum species. In order to distinguish strains within A. diazotrophicus species, the polymerase chain reaction was employed, using a Rhizobium meliloti dctA primer under low stringency hybridization conditions.     

Herbaspirillum, an endophytic diazotroph colonizing vascular tissue 3Sorghum bicolor L. Moench

Leaves of Sorghum bicolor were examined at 5 d and 14 d afterinoculation with the N₂-fixing endophytic bacteria Herbaspirillumseropedicae and Herbaspirillum rubrisubalbicans. Plants inoculatedwith H. rubrisubalbicans expressed symptoms of ‘red stripedisease’ i.e. red stripes along the secondary veins ofthe leaf blade close to the inoculation point and spreadingup the leaves. Infected leaves showed dense colonization byH. rubrlsubalbicans in regions showing red stripe symptoms at5 d after inoculation. The infection was confined within thevascular system, in particular, the protoxylem and associatedlacunae, which were often completely filled with bacteria, withsome of the latter expressing nitrogenase. The bacteria wererecognized using H. rubrisubalbicans-speciflc antibodies andimmunogold labelling, which also showed that the antibody reactedwith material on the surface of the bacteria, and that thismucus was released into the lumen of the xylem. At 14 d afterinoculation, disease symptoms were slightly more severe, withboth meta- and protoxylem being even more heavily colonizedin parts of the leaf showing red stripes. However, a stronghost defence response was also apparent at this stage, withgums lining the walls of the vessels and enclosing the bacteria,although the latter were still actively dividing. At the edgesof visible disease symptoms, plant gums filled the xylem; bacteriahad formed distinct colonies within these gums, with some ofthe colonies associated with the xylem walls. Plants inoculatedwith H. seropedicae either did not express the disease or showedvery mild symptoms close to the inoculation point. In the lattercase, H. seropedicae were localized within protoxylem vesselsand the metaxylem was partly occluded with plant-derived gums.By contrast with H. rubrisubalbicans, H. seropedicae was alsolocalized in leaves at 14 d without disease symptoms and didnot always appear to elicit a host response, i.e. they colonizedthe walls of metaxylem, with the xylem vessels themselves remainingunoccluded and largely free of gums. The fine line separatingplant pathogens, endophytes and symbioses is discussed in lightof these results. Key words: Herbaspirillum, Sorghum bicolor, nitrogen fixation, endophyte, xylem     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Biological Nitrogen Fixation in Sugar Cane: A Key to Energetically Viable Biofuel Production

The advantages of producing biofuels to replace fossil energy sources are derived from the fact that the energy accumulated in the biomass is captured directly from photosynthesis and is thus renewable, and that the cycle of carbon dioxide fixation by the crop, followed by burning of the fuel makes no overall contribution to atmospheric CO₂ or, consequently, to global warming. However, these advantages are negated if large quantities of fossil fuels need to be used to grow or process the biofuel crop. In this regard, the Brazilian bioethanol program, based on the fermentation/distillation of sugar cane juice, is particularly favorable, not only because the crop is principally hand harvested, but also because of the low nitrogen fertilizer use on sugar cane in Brazil. Recent ¹⁵N and N balance studies have shown that in some Brazilian cane varieties, high yields are possible without N fertilization because the plants are able to obtain large contributions of nitrogen from plant-associated biological N₂ fixation (BNF). The N₂-fixing acid-tolerant bacterium Acetobacter diazotrophicus was first found to occur within roots, stems, and leaves of sugar cane. Subsequently, two species of Herbaspirillum also have been found to occur within the interior of all sugar cane tissues. The discovery of these, and other N₂-fixing bacteria that survive poorly in soil but thrive within plant tissue (endophytic bacteria), may account for the high BNF contributions observed in sugar cane. Further study of this system should allow the gradual elimination of N fertilizer use on sugar cane, at least in Brazil, and opens up the possibility of the extension of this efficient N₂-fixing system to cereal and other crops with consequent immense potential benefits to tropical agriculture.     

Influence of nitrogen fertilisation on the population of diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicus in sugar cane (Saccharum spp.)

We report studies on the possible effects of fertilisation with high level of N (300 kg of N ha^-1) on the occurrence and numbers of the diazotrophic bacteria Herbaspirillum spp. and Acetobacter diazotrophicusin sugar cane plants. In the sugar cane genotype SP79-2312, the N fertilised plants generally showed higher concentrations of this element. These same plants also had lower numbers of A. diazotrophicus, while the population of Herbaspirillum spp. was not affected by N application. These differences in the concentration of N and the numbers of A. diazotrophicus due to N application were not shown in the variety SP70-1143. The numbers of A. diazotrophicus were also shown to be influenced by the harvest time, becoming reduced in the harvests that coincided with dry periods of the year.     

Field inoculation of sorghum and rice withAzospirillum spp. andHerbaspirillum seropedicae

Two field experiments were carried out at the UAPNPBS experimental station, Seropédica, with two sorghum and one rice cultivars. The establishment, and inoculation effects, ofAzospirillum spp. andHerbaspirillum strains marked with antibiotic resistance were investigated. One grain sorghum (BR 300) and one sugar sorghum (Br 505) cultivar were used.Azospirillum lipoferum strain S82 (isolated from surface sterilized roots of sorghum) established in both cultivars and comprised 40 to 80% of theAzospirillum spp. population in roots and stems 60 days after plant emergence (DAE).Azospirillum amazonense strain AmS91 (isolated from surface-sterilized roots of sorghum) reached only 50%. At 90 DAE, S82 almost disappeared (less than 30% of establishment) while the establishment of AmS91 remained constant in roots and stems. No establishment ofH. seropedicae strain H25 (isolated from surface-sterilized roots of sorghum) orA. lipoferum strain S65 (isolated from the root surface of sorghum) could be observed on inoculated roots. Inoculation with S82, AmS91 or S65 but not withH. seropedicae H25, increased plant dry weight of both cultivars and total N in grain of the grain sorghum. In rice,A. lipoferum Al 121 andA. brasilense Sp 245 (isolated from surface sterilized rice and wheat roots respectively) established in the roots but there was no increase inAzospirillum spp. numbers due to inoculation. None of the strains affected plant growth or rice grain yield.Azospirillum amazonense, A82 andH. seropedicae Z95, which did not establish in roots, significantly enhanced seed germination.     

New yeast strains for alcoholic fermentation at higher sugar concentration

Summary New yeast strains for alcoholic fermentation were isolated from samples collected from Brazilian alcohol factories at the end of the sugar cane crop season. They were selected by their capacity of fermenting concentrated sugar cane syrup as well as high sucrose concentrations in synthetic medium with a conversion efficiency of 89–92%. The strains were identified asSaccharomyces cerevisiae.     

Bacterial antagonists of Sunflower (Helianthus annuus L.) fungal pathogens

Bacteria isolated on nutrient agar and King's medium B from sunflower leaves, crown and roots inhibited in vitro growth of the leaf spot and wilt pathogens Alternaria helianthi, and Sclerotium rolfsii, respectively, and also the root rot pathogensRhizoctonia solani and Macrophomina phaseolina. Antagonistic bacteria from leaves were mainly actinomycetes and pigmented Gram-positive bacteria, while those from roots and crowns were identified asPseudomonas fluorescens-putida, P. maltophilia, P. cepacia, Flavobacterium odoratum andBacillus sp. In soil bioassays, when used as seed inoculum in the presence ofS. rolfsii, P. cepacia strain N24 increased significantly the percentage of seedling emergence. Bacterial strains which exhibited broad spectrum in vitro antagonistic activity were tested for colonisation of sunflower roots, when used as a seed inoculum. Good colonisers (10⁴ to 10⁶ bacteria/g root) were consistent in their ability to reduce disease and fungal wilt. A seedling having a primary root length < 5 cm with fewer lateral roots, necrosed cotyledons or crown and a wilted shoot indicated its diseased status. On an average, only 30% of seedlings were diseased when treated with the antagonistic strains, in the presence of the pathogen, while 60% of the seedlings were diseased in the presence of the pathogen alone. In microplots treated with strain N24, only 1 to 3% of the seedlings were wilted, while 14% of the seedlings were wilted in the presence of the pathogen alone. The results obtained show that bacterial antagonists of sclerotial fungi can be used as seed inocula to improve plant growth through disease suppression     

Thermophilic amylase-producing bacteria from Vietnamese soils

Of 25 bacterial isolates from Vietnamese soils, two were identified asBacillus stearothermophilus and one asThermoactinomyces thalpophilus, both thermophilic, amylase-producing bacteria. Amylase activity was highest in the presence of cassava starch as carbon source and (NH₄)₂HPO₄ as nitrogen source. The strains exhibit a high amylase productivity within the first 5 to 7 h of cultivation at 55°C. The crude enzyme had optima of pH 6.5 and 70°C.     

Fermentation pattern ofZymomonas mobilis strains on different substrates—a comparative study

The optimum conditions (pH and initial sugar concentration) of fermentation for the production of ethanol by 4 strains ofZymomonas mobilis (ATCC 10988, ATCC 12526, NRRL B 4286 and IFO 13756) were studied. An initial sugar concentration of 15 % (w/v) at pH 7.0 was found to be optimal for the first two strains and 20 % (w/v) initial sugar at pH 7.0 was found to be optimal for the last two strains. The fermentation pattern of these strains on synthetic medium, cane juice and molasses were compared. Strain NRRL B 4286 showed maximum ethanol production on synthetic medium while on cane juice ATCC 10988 and ATCC 12526 performed well. However, all the strains fermented molasses poorly.     

16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Production of Pleurotus ostreatus mushrooms on sugar cane agrowastes

The growth of different strains of Pleurotus spp. on sugar cane agrowastes was evaluated. Three hybrid strains with good production outcomes and yields exceeding 17% were selected. Strain 184 (P. ostreatus x P. pulmonarius) showed the best results. Three spawn materials (wheat grain, millet grain and milled corn cob) at different spawning levels were tested and a significant influence was found. The obtained results were best explained in terms of total nitrogen content of the initial mixture (spawn + substrate), suggesting a probable nitrogen limited growth of the mushroom on sugar cane residues. A 10% millet grain spawn was found to be a reasonable compromise. Productive responses decreased with an increase in bag's capacity (8–10–12 kg), even though the same diameter was maintained in order to avoid pronounced temperature profiles. Smaller bag's capacities (8–10 kg) were recommended. It was also shown that the utilization of water hyacinth (Eichhornia crassipes) mixed 50/50 with sugar cane residues as substrate caused a twofold increase of crop responses, confirming the advantages of this substrate supplementation. The obtained results identified sugar cane agrowastes as a feasible substrate for Pleurotus spp. production with yields and biological efficiencies comparable and to some extent better than others reported with conventional lignocellulosic residues such as cereal straw.     

Radiation and fermentation treatment of cellulosic wastes

The effect of radiation pasteurization of sugar cane bagasse and rice straw and fermentation using various strains of fungi were studied for upgrading of cellulosic wastes. The initial contamination by fungi and aerobic bacteria both in bagasse and straw was high. The doses of 30 kGy for sterilization and 8 kGy for elimination of fungi were required. Irradiation effect showed that rice straw contained comparatively radioresistant microorganisms. It was observed that all the fungi (Hericium erinacium, Pleurotus djamor, Ganoderma lucidum, Auricularia auricula, Lentinus sajor-caju, Coriolus versicolor, Polyporus arcularius, Coprinus cinereus) grow extending over the entire substrates during one month after inoculation in irradiated bagasse and rice straw with 3% rice bran and 65% moisture content incubated at 30°C. Initially, sugar cane bagasse and rice straw substrates contained 39.4% and 25.9% of cellulose, 22.9% and 26.9% of hemicellulose, and 19.6% and 13.9% of lignin + cutin, respectively. Neutral detergent fibre (NDF) values decreased significantly in sugar cane bagasse fermented byG. lucidum, A. auricula andP. arcularius, and in rice straw fermented by all the 8 strains of fungi. Acid detergent fibre (ADF) values also decreased in bagasse and rice straw fermented by all the fungi.P. arcularius, H. erinacium, G. lucidum andC. cinereus were found to be the most effective strains for delignification of sugar cane bagasse.     

Dynamics,Diversity and Function of Endophytic Siderophore-Producing Bacteria in Rice

Siderophore production confers to bacteria competitive advantages to colonize plant tissues and to exclude other microorganisms from the same ecological niche. This work shows that the community of endophytic siderophore-producing bacteria (SPB) associated to Oryza sativa cultivated in Uruguayan soils is dynamic and diverse. These bacteria were present in grains, roots, and leaves, and their density fluctuated between log₁₀ 3.44 and log₁₀ 5.52 cfu g⁻¹ fresh weight (fw) during the plant growth. Less than 10% of the heterotrophic bacteria produced siderophores in roots and leaves of young plants, but most of the heterotrophic bacteria were siderophore-producers in mature plants. According to their amplified restriction DNA ribosomal analysis (ARDRA) pattern, 54 of the 109 endophytic SPB isolated from different plant tissues or growth stages from replicate plots, were unique. Bacteria belonging to the genera Sphingomonas, Pseudomonas, Burkholderia, and Enterobacter alternated during plant growth, but the genus Pantoea was predominant in roots at tillering and in leaves at subsequent stages. Pantoea ananatis was the SPB permanently associated to any of the plant tissues, but the genetic diversity within this species—revealed by BOX-PCR fingerprinting- showed that different strains were randomly distributed along time and plant tissue, suggesting that a common trait of the species P. ananatis determined the interaction with the rice plant. Several isolates were stronger IAA producers than Azospirillum brasilense or Herbaspirillum seropedicae. In vitro inhibition assays showed that SPB of the genus Burkholderia were good antagonists of pathogenic fungi and that only one SPB isolate of the genus Pseudomonas was able to inhibit A. brasilense and H. seropedicae. These results denoted that SPB were selected into the rice plant. P. ananatis was the permanent and dominant associated species which was unable to inhibit two of the relevant plant growth-promoting bacteria.     

籽用美洲南瓜叶片对白粉病菌的抗逆生理响应

以籽用美洲南瓜(Cucurbita pepo L.)白粉病抗病品系F2和感病品系M3为试材,在人工气候箱内接种白粉病生理小种2US孢子悬浮液,考察在接种白粉病菌后南瓜幼苗植株与白粉病菌的互作、叶片活性氧代谢及保护酶活性的变化,探讨南瓜抵御白粉病的生理机制。结果表明：(1)与感病品系M3相比,接种白粉病菌后,抗病品系F2叶片上病原菌发育缓慢,较难侵染叶片。(2)抗病品系F2在感病初期叶片H₂O₂、O₂^-·含量迅速升高后逐渐下降,而感病品系在感病初期H₂O₂、O₂^-·含量上升缓慢,在达最大值后始终保持较高水平,且感病品系叶片MDA含量始终高于抗病品系;组织化学染色分析发现,抗病品系叶片着色比感病品系快,之后着色面积有所减少并趋于较低水平。(3)抗病品系F2和感病品系M3叶片抗氧化酶CAT、SOD、POD活性及PAL、PPO活性在接种白粉病菌后均显著增加,但抗病品系的活性及其增幅均高于感病品系。研究发现,籽用美洲南瓜抗病品系叶片上白粉病菌发育缓慢,较难受到侵染,生成菌丝体后叶片上粉状斑点较小;抗病品系在被白粉病菌侵染初期依靠活性氧的增加抵御病原菌的入侵,随着活性氧含量增加抗病品系通过迅速增加自身抗氧化酶活性来防止氧化胁迫;与感病品系相比,抗病品系在受病原菌侵染后能迅速增加PAL、PPO活性以抵御病原菌侵染。     

Characterization and identification of bacteria isolated from micropropagated mint plants

Summary Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step in determining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests and subjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned to genera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by a commercial laboratory. Six were classified asAgrobacterium radiobacter; eight asXanthomonas; one each asPseudomonas fluorescens, Micrococcus spp.,Corynebacterium spp., andCurtobacterium spp.; four could not be assigned to genera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration and minimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Cadmium stress in sugar cane callus cultures: Effect on antioxidant enzymes

Catalase (CAT) and superoxide dismutase (SOD) are antioxidant enzymes which are important in the metabolism of reactive oxygen species (ROS), and can be induced by environmental stresses including cadmium (Cd), a heavy metal toxic to living organisms. Sugar cane (Saccharum officinarumL.) in vitro callus cultures were exposed to CdCl₂ and the activities of CAT and SOD were analysed. Lower concentrations of CdCl₂, such as 0.01 and 0.1 mM caused a significant increase in callus growth, whereas 0.5 and 1 mM CdCl₂ strongly inhibited growth of the callus cultures, but only after 9 days of CdCl₂ treatment. Red-brown patches were also observed in calluses exposed to 0.5 and 1 mM CdCl₂. Calluses grown in 0.01 and 0.1 mM CdCl₂ did not exhibit any changes in CAT activity even after 15 days of growth in the presence of CdCl₂. However, for calluses grown in higher concentrations of CdCl₂ (0.5 and 1 mM), a rapid increase in CAT activity was detected, which was 14-fold after 15 days. Furthermore, up to five CAT isoforms were observed in callus tissue. Total SOD activity did not exhibit any major variation. One Mn-SOD and two Cu/Zn-SOD isoenzymes were observed in callus cultures and none exhibited any variation in response to the CdCl₂ treatments. The results suggested that in sugar cane callus cultures, CAT may be the main antioxidant enzyme metabolizing H₂O₂.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.     

Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.