Veronica Massena Reis

Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems, and leaves, predominantly of Gramineae

Since the first description of Herbaspirillum seropedicae in 1986, few data have been published on this diazotroph, possibly due to difficulties in isolating it from soil. In the present study we found that this bacterium seems to be an obligate endophyte which has been isolated from roots, stems, and leaves of a large number of samples of more than 10 different species of the Gramineae family, but only exceptionally from other plants. H. rubrisubalbicans, previously misnamed as “Pseudomonas” rubrisubalbicans, and known as a mild pathogen of sugarcane causing mottled stripe disease, confirms the endophytie habitat of this genus. This species occurs in roots, stems, and leaves of sugarcane and seems to be restricted to this crop. Inoculation of strains from both species into soil in high numbers resulted in a rapid decline in their numbers. In only 30 days the population of Herbaspirillum spp. in soil decreased below detection limits (<100 cells g-1). When sorghum was planted in this soil, the bacteria reappeared and multiplied within the plant tissues.

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).

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 Potential of C4 Perennial Grasses for Developing a Global BIOHEAT Industry

Unprecedented opportunities for biofuel development are occurring as a result of rising fossil fuel prices, the need to reduce greenhouse gases, and growing energy security concerns. An estimated 250 million hectares (ha) of farmland could be utilized globally to develop a bioenergy industry if efficient and economical perennial biomass crops and bioenergy conversion systems are employed. In temperate zones, C4 or warm-season grass research and development efforts have found switchgrass (Panicum virgatum) and Miscanthus capable of producing biomass yields of 10 to 20 oven dried tonnes (ODT)/ha/yr, while in tropical areas Erianthus and napier grass (Pennisetum purpureum) are producing 25 to 35 ODT/ha/yr. The potential to annually produce 100 barrels of oil energy equivalent/ha with a 25:1 energy output to input ratio appears achievable with high-yielding, N-fixing warm-season grasses grown on marginal lands in the tropics. Commercialization of densified herbaceous plant species has been slow because of the relatively high alkali and chlorine contents of the feedstocks, which leads to clinker formation and the fouling of boilers. This challenge can be overcome by improving biomass quality through advances in plant breeding and cultural management to reduce the chlorine, alkali, and silica content and through the use of new combustion technologies. Warm-season grasses can be readily densified provided suitable grinding and densification equipment and pressure are utilized. The major advantages of producing densified warm-season grasses for BIOHEAT include: it is the most efficient strategy to use marginal farmlands in most temperate and tropical climates to collect solar radiation; it has an excellent energy balance; the feedstocks can be used conveniently in a variety of energy applications; and it is relatively environmentally friendly. Densified warm-season grass biofuels are poised to become a major global fuel source because they can meet some heating requirements at less cost than all other alternatives available today.

Herbaspirillum frisingense sp. nov., a new nitrogen-fixing bacterial species that occurs in C4-fibre plants

The enrichment of nitrogen-fixing bacteria from the C4-fibre plants, Spartina pectinata, Miscanthus sinensis, Miscanthus sacchariflorus and Pennisetum purpureum, with nitrogen-free semi-solid media led to the isolation of Herbaspirillum-like strains among other diazotrophic bacteria. On the basis of physiological properties, phylogenetic analysis comparing 16S rDNA sequences and DNA-DNA hybridization experiments of chromosomal DNA the new isolates could be grouped together in a new species with the proposed name Herbaspirillum frisingense sp. nov. Morphological characteristics, such as cell size and shape, colony appearance, motility and flagellation are largely identical to the known species Herbaspirillum rubrisubalbicans and Herbaspirillum seropedicae. On the basis of utilization of adipate (-), N-acetyl-D-glucosamine (+), meso-erythritol (-), L-rhamnose (-) and meso-inositol (-) Herbaspirillum frisingense sp. nov. can be distinguished from other known Herbaspirillum spp. Nitrogen-fixing capability was examined by PCR amplification of the nifD gene and an acetylene reduction assay, and was found with all isolates tested. 16S rDNA sequence similarity to the other Herbaspirillum spp. is 98.5-99.1%. In genomic DNA-DNA hybridization experiments Herbaspirillum frisingense sp. nov. forms a homogeneous group with 70-100+/-10% similarity, clearly distinct from Herbaspirillum seropedicae and Herbaspirillum rubrisubalbicans with 1-34% similarity. 16S rRNA-targeted oligonucleotide probes, specific for the whole genus Herbaspirillum and for three Herbaspirillum species were designed and are suitable for fluorescence in situ hybridization. The DNA G+C content of Herbaspirillum frisingense sp. nov. is 63+/-2 mol%, in agreement with the values of 61-65% for the genus. PCR fingerprinting exhibits a consistent pattern for groups of strains isolated from the same plant, suggesting a low genomic diversity among bacteria inhabiting C4-gramineous plant tissues. Low genetic DNA diversity seems to be common between probable endophytic bacterial isolates of the same taxon. The type strain of Herbaspirillum frisingense sp. nov. is GSF30T (= DSM 13128T).

Occurrence, physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants

Endophytic diazotrophic bacteria could be isolated from the energy plants Pennisetum purpureum, Miscanthus sinensis, Miscanthus sacchariflorus and Spartina pectinata using semisolid nitrogen free media. Higher levels of diazotrophic bacteria were found if no nitrogen fertilizer was applied. The bacteria were characterized on the basis of typical morphology, physiological tests, and the use of phylogenetic oligonucleotide probes. They belong partially to the species Azospirillum lipoferum and Herbaspirillum seropedicae while others supposedly represent a new species of Herbaspirillum. Using PCR-fingerprinting techniques a limited genetic diversity of these isolates was found which may indicate an adaptation to the specific conditions of the interior of these plants.

Biological Dinitrogen Fixation in Gramineae and Palm Trees

Biological nitrogen fixation (BNF) in the Gramineae family has been well documented, but a complete understanding of this issue is needed to turn the research into a practical approach. The literature has a long and diverse list of diazotrophic bacteria found colonizing several plant tissues, such as roots, stems, leaves, and trash as well as the rhizosphere. However, only a limited amount of research has focussed on existing associations of N2-fixing microorganisms with grasses or cereal, especially for BNF inputs and ecological studies under field conditions. The recent discovery of the endophytic diazotroph bacteria such as Acetobacter diazotrophicus, Herbaspirillum spp. and Azoarcus spp. colonizing the interior of sugarcane, rice, Kallar grass (Leptochloa fusca (L.) Kunth), respectively, and other species of grasses as well as cereals has led to a considerable interest in exploring these novel associations. There is a general consensus that plant genotype is a key factor to higher contributions of BNF...

Diazotrophic Burkholderia Species Associated with Field-Grown Maize and Sugarcane

ABSTRACT Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N2-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N2-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N2-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed.

Yield of micropropagated sugarcane varieties in different soil types following inoculation with diazotrophic bacteria

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF.

A brief story of nitrogen fixation in sugarcane — reasons for success in Brazil

Sugarcane was first introduced into Brazil in 1532, in São Vicente (São Paulo State) by the Portuguese. Since the first cane selection and breeding programs started in Brazil, both local and introduced material were used. In none of the breeding programs were large amounts of nitrogen fertilizer utilized, and this may be the reason why today the best materials have little demand for nitrogen fertilizer, and an effective association has developed between endophytic nitrogen-fixing bacteria and the plant. In some cases high inputs of associated biological nitrogen fixation have been observed. The oil crisis also played a role in the sugarcane story, since the alcohol-from-cane-juice (PRO-ÁLCOOL) program installed to find a substitute for gasoline in cars, stimulated the selection of highly efficient varieties with low nitrogen fertilizer input. The recent promising results involving the inoculation of micropropagated sugarcane plants with endophytic diazotrophic bacteria, along with the ongoing Brazilian sugarcane plant and bacterial genome programs, suggest that the success of the Brazilian sugarcane business may continue for many years to come, considering the potential to be exploited.