Fábio Bueno dos Reis

Nodulation and nitrogen fixation by Mimosa spp. in the Cerrado and Caatinga biomes of Brazil

*An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus. *Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815. *Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the delta(15)N ((15)N natural abundance) technique, contributions by biological N(2) fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp. *It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N(2) fixation by beta-rhizobial symbioses in the field.

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N2 fixation in natural ecosystems. Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N2 fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their 15N natural abundance. Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their 15N natural abundance was carried out by searching the ISI Web of Science database using relevant key words. Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N2 fixation. N input into ecosystems from N2 fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system. Conclusions: Measurement of 15N natural abundance has given greater understanding of where legume and actinorhizal plant N2 fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.

Biological nitrogen fixation associated with tropical pasture grasses

The semi-humid or humid tropics are ideal for the production of large quantities of biomass from fast-growing C 4 grasses, but high yields normally require large quantities of fertiliser, especially N, which has a very high input from fossil fuels (natural gas). A program has been started recently to use elephant grass (Pennisetum purpureum Schum.) to substitute firewood as a fuel and also to make charcoal for iron production. In this case, any large N fertiliser additions would mean that the yield of bio fuel per unit of fossil fuel invested would be detrimentally affected. In this study, we report on the potential for the selection of genotypes of fast-growing C 4 tropical grasses ofthe genera Pennisetum and Brachiaria for their capacity to obtain N inputs from plant-associated biological nitrogen fixation (BNF). Fourteen genotypes each of Brachiaria and Pennisetum were screened for BNF contributions by growing them in 15 N-labelled soil. In the case of the Pennisetum, after a suitable cutting height for the crop had been selected, there were large differences in dry matter production, N accumulation and 15 N enrichment. The differences in 15 N enrichment between genotypes were statistically significant and BNF inputs were estimated as high as 41% of accumulated N. In the study on Brachiaria genotypes, potential inputs of BNF seemed lower. Only one or two genotypes of B. brizantha and B. ruziziensis obtained more then 20% of their N from BNF. The N 2 -fixing bacteria that were most commonly associated with shoots and roots the Pennisetum genotypes were of the genus Herbaspirillum, but predominantly of a recently described new species. The Brachiaria spp. from three different sites (Rio de Janeiro, Goania, Bahia) were predominately colonised by Azospirillum spp., most of the isolates being of the species Azospirillum amazonense. Very few Herbaspirilla were isolated from these plants.

In vivo proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the host plant Brassica oleracea

The genus Xanthomonas is composed of several species that cause severe crop losses around the world. In Latin America, one of the most relevant species is Xanthomonas campestris pv. campestris, which is responsible for black rot in cruciferous plants. This pathogen causes yield losses in several cultures, including cabbage, cauliflower and broccoli. Although the complete structural genome of X. campestris pv. campestris has been elucidated, little is known about the protein expression of this pathogen in close interaction with the host plant. Recently, a method for in vivo analysis of Xanthomonas axonopodis pv. citri was developed. In the present study, this technique was employed for the characterization of the protein expression of X. campestris pv. campestris in close interaction with the host plant Brassica oleracea. The bacterium was infiltrated into leaves of the susceptible cultivar and later recovered for proteome analysis. Recovered cells were used for protein extraction and separated by two-dimensional electrophoresis. Proteins were analysed by peptide mass fingerprinting or de novo sequencing and identified by searches in public databases. The approach used in this study may be extremely useful in further analyses in order to develop novel strategies to control this important plant pathogen.

Comparative proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the susceptible and the resistant cultivars of Brassica oleracea

Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris, causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea. The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction.

Nitrogen-Fixing Endophytes: Recent Advances in the Association with Graminaceous Plants Grown in the Tropics

Several definitions of endophytic bacteria have been proposed during the last few years (Quispel, 1992; Kloepper, Beuchamp, 1992; Fisher et al. 1992). While they differ among themselves, there is a general agreement that endophytic bacteria are those that live, at least part of their life cycle, in living plant tissues without causing substantial harm and showing no disease symptoms. Symptomless has been considered as the key word to express the nature of the endophytism (Mishagi, Donndelinger, 1990).

N2 Fixation in Sugar Cane: the Role of Acetobacter Diazotrophicus

Almost 4 million cars in Brazil are fueled by hydrated (95%) ethanol produced from sugar cane and the remaining 6 to 8 million light vehicles run on gasohol (gasoline mixed with 15 to 22% ethanol). The bio-ethanol programme has great environmental advantages: Not only does the addition of ethanol to gasoline obviate the need for tetra-ethyl lead, but engines running on hydrated alcohol produce less carbon monoxide, nitrogen oxides and hydrocarbon in the exhaust gases than conventional gasoline engines (Boddey, 1993). Furthermore, unlike gasoline or other fossil fuels, the combustion of this biofuel makes no net contribution to global CO2 emissions as the carbon was fixed from the atmosphere by the sugar cane crop. However, when considering the CO2 emission budget, the amount of fossil fuel used to grow the crop must be taken into account. This includes diesel fuel for agricultural machinery and transport of cane to the mill, as well as the energy used to process the cane and distill the ethanol. As sugar cane in Brazil is still largely harvested manually, and the energy to power the mills and distilleries is all provided from burning bagasse, this ratio of energy value of the ethanol to fossil fuel used in its production (the energy balance) is very favourable (approximately 4.5).

Charaterisation of different polyclonal antisera to quantify Herbaspirillum spp. in Elephant grass (Pennisetum purpureum Schun)

Immunological techniques can be applied to detect micro-organisms in different ecosystems. Polyclonal antisera are produced when the immune system of a mammal is exposed to foreign macromolecules. They consist of a mixture of different antibodies synthesised in response to various antigenic determinants. To use this antisera as a probe for microbiological detection on plant/soil material is necessary to characterise the quality of the antibodies/antisera for “in situ” detection and quantification. To follow the fate of Acetobacter diazotrophicus strain BR 11280 (PAL 3), Herbaspirillum seropedicae strain BR 11175 (Z67) and H. rubrisubalbicans strain BR 11192 (M4) in the rhizosphere of elephant grass (Pennisetum purpureum Schun. var. Capim Cana D’Africa), polyclonal antisera were raised against these strains. All antisera were purified by protein-A, followed by a primary characterisation for cross-reactivity. To reduce cross-reactivity for these sera, affinity purification was used. After that, all sera were species specific. Bacterial colonisation was quantified by the ELISA protocol. The detection limit was 105 cells ml−1 for both the anti A. diazotrophicus and H. rubrisubalbicans serum and 106 cells ml−1 for the anti H. seropedicae serum. The bacterial numbers were high and present in/on the root tissue. No contamination was detected in the control. It was not possible to detect A. diazotrophicus inoculated on these plants. It is possible that its numbers were below the detection limit but this bacterium is not so effective as Herbaspirillum sp. in colonising the root system efficiently. Only strains of Herbaspirillum spp could be detected in the rhizosphere of elephant grass in high densities mostly on the rhizoplane. The numbers by ELISA estimated were 107 cells g−1 fresh weight.