V. L. D. Baldani

Recent advances in BNF with non-legume plants

Abstract It is now well accepted that nitrogen-fixing bacteria colonising graminaceous plants can be grouped into three categories: 1, rhizosphere organisms; 2, facultative endophytes and 3, obligate endophytes. In the first category are included all species that colonise the root surface such as Azotobacter paspali, Beijerinkia spp. Facultative endophytes are these nitrogen-fixing bacteria that can colonise the surface and interior of the roots principally the four species of Azospirillum , except Azospirillum halopraeferans . The third category is constituted mainly by diazotrophs isolated more recently such as Acetobacter diazotrophicus, Herbaspirillum spp and Azoarcus spp which are able to colonise the root interior and aerial tissues of the plants. Although most of the studies related to nitrogen fixing bacteria have been concentrated on Azospirillum spp, it is the obligate endophytes, isolated more recently, that have attracted the attention of scientists working in this field. The ability to colonise the entire plant interior and locate themselves within niches protected from oxygen or other factors make them the most promising group of diazotrophs associated with graminaceous and other non-leguminous plants. In this review we compare these three groups of nitrogen-fixing bacteria, their interaction with the host plants and discuss the potential of their use in agriculture.

Host-plant specificity in the infection of cereals with Azospirillum spp

Abstract The specificity of the infection of maize, wheat and rice roots by N2-fixing Azospirillum spp was studied in four greenhouse experiments using pots with unsterilized soil and in two field experiments. In all experiments A. lipoferum was most frequently isolated from externally sterilized roots of maize, and A. brasilense nir− (nitrite reductase negative) from wheat and rice. In pot experiments, A. brasilense nir+ was isolated with moderate frequency from within maize roots but rarely from within wheat or rice roots. Inoculation of the pots with a mixture of representative strains of the three Azospirillum groups had no effect on the proportion of strains recovered from each plant species. In the field experiments, inoculation with spontaneous streptomycin-resistant mutants of two of the representative strains confirmed the apparent specificity of A. lipoferum for maize roots and of A. brasilense for wheat but the results were partially obscured by the unexpectedly high proportion of streptomycin-resistant strains isolated from within the roots of uninoculated plants.

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.

Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

SummaryFour field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience.

This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of which was coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bacteria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants.

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

SummaryTwo experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and15N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the15N and acetylene reduction data it was apparent that these increases were not due to N2 fixation.In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources.The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N2 fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots.

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...

Isolation and characterization of diazotrophic bacteria from banana and pineapple plants

Banana and pineapple fruit crops are widely cultivated in tropical areas where high amounts of fertilizers are applied, principally nitrogen. Over 200 kg N.ha-1.yr-1 is often applied to these crops. Nevertheless, developing countries face the problem of high costs of chemical fertilizers. As already demonstrated for other tropical crops, like sugar cane, the utilization of nitrogen-fixing bacteria may support the growth of these fruit plants. In this work, we demonstrate the association of nitrogen-fixing bacteria with banana and pineapple. Samples from roots, stems, leaves and fruits of different genotypes showed the occurrence of diazotrophic bacteria, when evaluated in semi-specific semi-solid media. These isolates could be separated into seven different groups according to their morphological and physiological characteristics. Additional, phylogenetic assignments were performed with group- and species-specific oligonucleotide probes. Bacteria related to the groups of Azospirillum amazonense, Azospirillum lipoferum, Burkholderia sp. and a group similar to the genus Herbaspirillum could be detected in samples of both crops. However, Azospirillum brasilense and another two groups of Herbaspirillum-like bacteria were detected only in banana plants. Two isolates of the latter group were identified as Herbaspirillum seropedicae, whereas the other isolates may represent a new Herbaspirillum species.

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.

Endophytic Occurrence of Diazotrophic Bacteria in Non-Leguminous Crops

The recent discovery of obligate endophytic diazotrophs, like Acetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans colonizing sugar cane roots, stems and leaves, may finally explain the high contributions of BNF to this crop. A. diazotrophicus so far has only been isolated from sugar cane, sweet potatoe and Cameroon grass, all three with high sugar contents and propagated by stem cuttings. Azoarcus spp., another obligate endophytic genus, isolated from Kallar grass (Leptochloa fusca), has been found to establish within wetland rice roots when inoculated. Herbaspirillum seropedicae has been isolated from many graminaceous plants, forage grasses as well as from maize, sorghum and rice, but it does not seem to occur in other plant families. H. rubrisubalbicans so far has only been isolated from sugar cane roots, stems and leaves. These two species, even though clearly identified as two distinct species by DNA homology, are extremely similar and can only be distinguished by growth on n-acetyl-glucosamine (H. seropedicae) and that on meso-erythritol (H. rubrisubalbicans). Transmission of H. seropedicae may occur within seeds or via graminaceous weed plants because even high numbers of this organism inoculated into soil, do not survive for more than one month. Inoculation of endophytic diazotrophs, especially A. diazotrophicus is better achieved by the use of VA mycorrhizae together with bacterial cultures or with spores containing the bacteria. Spores of various VAM species were found to contain this organism and also A. lipoferum and Klebsiella sp. The infection of the root cap zone in micropropagated sugar cane plants with A.diazotrophicus and its colonization of xylem, has been confirmed by optical and electron microscope observations with immunogold-silver-enhanced techniques. Also Herbaspirillum spp. have been localized in the metaxylem after artificial inoculation of sugar cane and sorghum, using the same technique.