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.     

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10³ cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10⁷/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.The authors are with EMBRAPA-CNPAB, Cx Postal 74.505, Seropédica, Rio de Janeiro, 23851-970, Brazil     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

Spittlebug indicators of nitrogen-fixing plants

Abstract.

• 1 Spittlebugs (Homoptera: Cercopoidea) are preferentially associated with nitrogen-fixing angiosperms. Generally, the Aphrophoridae associate with legumes, the Clastopteridae with actinorhizal plants, and the Cercopidae with associative nitrogen-fixing grasses. This is the first reported insect—host association specific to nitrogen-fixing plants.
• 2 It is probable that spittlebugs are attracted to nitrogen-fixing plants because these hosts provide a relatively rich, reliable source of organic nitrogen compounds for xylem-sucking insects.
• 3 Many spittlebugs live on plants that do not fix nitrogen, and ecological, mechanical or chemical factors may bar some spittlebugs from otherwise suitable nitrogen-fixing plants.
• 4 Among nitrogen-fixing hosts, spittlebugs appear to prefer plants that transport fixed nitrogen as amino acids and amides to plants that transport fixed nitrogen as ureides.

     

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.