The success of BNF in soybean in Brazil

Approximately forty years after commercial cropping of soybean in Brazil began, the total area under this crop has reached over 13 M ha with a mean productivity of 2400 kg ha^–1. Soybean varieties introduced from the USA and varieties rescued from early introductions in Brazilian territory were part of the Brazilian soybean-breeding programme which spread the crop from high to low latitudes. Disease-resistance, pest-resistance, tolerance to low fertility soils, as well as production of plants with pods sufficiently high above the ground for efficient mechanical harvesting, were all aims of the programme. Although BNF was not explicitly considered as a trait for selection in the breeding/selection programme, maximisation of biological nitrogen fixation (BNF) was favoured by conducting selection and breeding trials on soils low in N, in which the seeds were inoculated with efficient Bradyrhizobium inoculants but without N fertiliser application. Several efficient imported Bradyrhizobium strains were found to be unable to compete with native soil micro-flora and other previously-introduced Bradyrhizobium strains. Surprisingly, after being in the soil for many years one or two of these strains had become more competitive while maintaining their high BNF capacity. Today, these strains are included amongst the recommended Brazilian inoculants and have promoted significant improvements in grain yields. The breeding of soybeans in conditions that made grain yield highly dependent on BNF, and the continuous attention paid to the selection of Bradyrhizobium strains appropriate for the newly released varieties, have been the main contributors to today's high yields and their great benefit to the Brazilian economy. There seems to be no reason why this ongoing research programme should not serve as an appropriate model to improve BNF inputs to grain legumes in other countries of the world.     

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

We investigated the effects of an autumn sowing of contrasting cover crops (oats, rye and a combination of oats and rye) on soil aggregate stability, mycorrhizal colonization, phosphorus uptake and yield of sweet corn planted the following summer. Rye is a common cover crop in the middle Atlantic region of the United States of America. It grows slowly in the autumn, survives the winter, grows rapidly in the spring and flowers in the summer. Thus, herbicide is commonly used to kill rye prior to planting spring crops. Oats, in contrast, grows rapidly in the autumn but is killed by frost during the winter. Thus, with oats, potentially less herbicide is needed to prepare the field for spring planting. When compared to fallow, oats was as effective as rye in increasing mycorrhizal colonization of sweet corn, density of mycorrhizal hyphae, and soil aggregate stability. An oats cover crop may thus be a viable alternative to rye. The combination of cover crops (rye and oats), however, was significantly better than single species of cover crops in terms of sweet corn mycorrhizal colonization, P uptake and yield of sweet corn.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N₂-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.     

Electrical transmission among neurons in the buccal ganglion of a mollusc, Navanax inermis

The opisthobranch mollusc, Navanax, is carnivorous and cannibalistic. Prey are swallowed whole by way of a sudden expansion of the pharynx. The buccal ganglion which controls this sucking action was isolated and bathed in seawater. Attention was focused upon 10 identifiable cells visible on the ganglion''s rostral side. Two cells were observed simultaneously, and each was penetrated with two glass microelectrodes, one for polarizing the membrane and the other for recording membrane potential variations. The coupling coefficients for direct current flow and action potentials of several identified cells were tabulated. Attenuation was essentially independent of the direction of current flow, but depended upon the relative size of the directly and indirectly polarized cells. The attenuation of subthreshold sinusoidally varying voltages increased with frequency above about 1 Hz. The coupling coefficient for spikes was lower than for DC due to greater high frequency attenuation. There is considerable similarity in the spontaneous PSP''s of all cells, which is not due to the electrical coupling but to input from a common source. The 10 cells were not chemically interconnected but some were electrically connected to interneurons which fed back chemically mediated PSP''s. The feedback can be negative or positive depending upon the membrane potential of the postsynaptic cell. We conclude that electrical coupling among the 10 cells plays a minor role in sudden pharyngeal contractions but that the dual electrical-chemical coupling with interneurons may be important in this respect.     

Statistical Signs of Synaptic Interaction in Neurons

The influence of basic open-loop synaptic connections on the firing of simultaneously recorded neurons has been investigated with auto- and cross-correlation histograms, using experimental records and computer simulations. The basic connections examined were direct synaptic excitation, direct synaptic inhibition, and shared synaptic input. Each type of synaptic connection produces certain characteristic features in the cross-correlogram depending on the properties of the synapse and statistical features in the firing pattern of each neuron. Thus, empirically derived cross-correlation measures can be interpreted in terms of the underlying physiological mechanisms. Their potential uses and limitations in the detection and identification of synaptic connections between neurons whose extracellularly recorded spike trains are available are discussed.     

New insights into the signaling pathways controlling defense gene expression in rice roots during the arbuscular mycorrhizal symbiosis

Mycorrhizal fungi form a mutualistic relationship with the roots of most plant species. This association provides the arbuscular mycorrhizal (AM) fungus with sugars while the fungus improves the uptake of water and mineral nutrients in the host plant. Moreover, the induction of defense gene expression in mycorrhizal roots has been described. While salicylic acid (SA)-regulated Pathogenesis-Related (PR) proteins accumulate in rice roots colonized by the AM fungus G. intraradices , the SA content is not significantly altered in the mycorrhizal roots. Sugars, in addition to being a source of carbon for the fungus, might act as signals for the control of defense gene expression. We hypothesize that increased demands for sugars by the fungus might be responsible for the activation of the host defense responses which will then contribute to the stabilization of root colonization by the AM fungus. An excessive root colonization might change a mutualistic association into a parasitic association.Key words: Glomus intraradices, glucose, fructose, Oryza sativa, pathogenesis-related (PR), salicylic acid (SA), sucrose, sugarsThe arbuscular mycorrhizal (AM) fungi are obligate biotrophs that establish mutualistic associations with the roots of over 90% of all plant species. AM fungi improve the uptake of water and mineral nutrients in the host plant, mainly phosphorus and nitrogen, in exchange for sugars generated from photosynthesis. The benefits of the AM symbiosis on plant fitness are largely known, including increased ability to cope with biotic and abiotic stresses.^1,2 In fact, the amount of carbon allocated to mycorrhizal roots might be up 20% of the total photosynthate income.³ During root colonization, the AM fungus penetrates into the root through the epidermal cells and colonizes the cortex. In the root cortical cells, the fungus forms highly branched structures, called arbuscules, which are the site of the major nutrient exchange between the two symbionts.^4,5 The legumes Medicago truncatula and Lotus japonicus have been widely adopted as the reference species for studies of the AM symbiosis. Cereal crops and rice in particular are also able to establish symbiotic associations with AM fungi.^6,7 Arabidopsis thaliana, the model system for functional genomics in plants, has no mycorrhization ability.It is also well known that plants have evolved inducible defense systems to protect themselves from pathogen invasion. Challenge with a pathogen activates a complex variety of defense reactions that includes the rapid generation of reactive oxygen species (ROS), changes in ion fluxes across the plasma membrane, cell wall reinforcement and production of antimicrobial compounds (e.g., phytoalexins).⁸ One of the most frequently observed biochemical events following pathogen infection is the accumulation of pathogenesis-related (PR) proteins.⁹ For some PR proteins antimicrobial activities have been described (e.g., chitinases, β-1,3-glucanases, thionins or defensins). The plant responses to pathogen attack are activated both locally and systemically. The phytohormones salicyclic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) act as defense signaling molecules for the activation of defense responses.¹⁰ Whereas SA-dependent signaling often provides resistance to biotrophic pathogens, JA/ET-dependent signaling is effective against necrotrophic pathogens.¹¹ During plant-pathogen interactions, cross-talk between SA and JA/ET signaling pathways provides the plant with the opportunity to prioritize one pathway over another to efficiently fine-tune its defense response to the invading pathogen. Contrary to biotrophic pathogens which exhibit a high degree of host specificity, the AM fungi manage to colonize a broad range of plant species.Evidence also exists on the existence of common mechanisms and signaling pathways governing responses to AM and pathogenic fungi.^2,12,13 Alterations in the content of hormones acting as defense signals also appear to occur during the AM symbiosis. As an example, JA and its derivatives (jasmonates) are believed to play an important role during the AM symbiosis in M. truncatula or tomato plants.^14,15 However, controversial data exists in the literature concerning the involvement of the various defense-related hormones during AM functioning. In particular, our current understanding of SA signaling during AM symbiosis is not clear.We recently documented the symbiotic proteome of the rice roots during their interaction with the AM fungus Glomus intraradices.⁶ A majority of the proteins identified in the rice symbiotic proteome are proteins with a function in defense responses or sugar metabolism. Among the proteins that accumulated at high levels in mycorrhizal rice roots compared to non mycorrhizal roots were PR proteins belonging to different PR families, such as PR1, chitinases (PR3), PR5 and several PR10 proteins. The PR1 and PBZ1 (a member of the PR10 family of PR proteins) genes are considered markers of the activation of defense responses in rice plants.^16,17 Of interest, the expression of many of the AM-regulated PR genes was previously reported to be induced by SA.^16,18–20 Proteins acting as oxidative stress protectors, such as ascorbate peroxidases, peroxidases and glutathione-S-transferases, also accumulated in mycorrhizal rice roots. Together, these observations support that the plant''s immune system is activated in the mycorrhizal rice root.To gain further insights into the molecular mechanisms governing PR gene expression in mycorrhizal roots, the SA and sugar contents of mycorrhizal roots were determined. Towards this end, rice (Oryza sativa ssp. japonica cv. Senia) plants were inoculated with the AM fungus G. intraradices.⁶ At 42 days post-inoculation (dpi), the overall colonization of the rice roots ranged from 25 to 30% as judged by microscopical observations of trypan blue-stained roots (results not shown; similar results were reported previously in reference ⁶). By this time, all the events related to fungal development, namely intraradical hyphae, arbuscules at different morphological stages of formation and vesicles, were present in G. intraradices-inoculated roots, thus confirming the establishment of the symbiotic association in the rice roots.Knowing that many AM-regulated proteins are also regulated by SA in rice roots, it was of interest to determine whether the level of endogenous SA increases in mycorrhizal roots compared to non mycorrhizal roots. In plants, intracellular SA is found predominantly as free SA and its sugar conjugate SA-glucoside (SAG). Root samples were analyzed for SA content, by measuring the level of both free SA and SAG as previously described in reference ²¹. This analysis revealed no significant differences, neither in free nor in SAG, between mycorrhizal and non mycorrhizal roots (Fig. 1). Then, it appears that although the expression of PR genes (functioning in a SA-dependent manner) is activated during the AM symbiosis, the fungus G. intraradices do not exploit the SA-mediated signaling pathway for induction of PR genes.Open in a separate windowFigure 1SA content, free SA and SA-glucoside (SAG) conjugate, in roots of mock-inoculated (−Gi) and G. intraradices-inoculated (+Gi) rice plants. SA determination was carried out at 42 days post-inoculation with G. intraradices. Three independent biological samples and three replicates per biological sample were used for quantification of SA. Two out of the three samples were the same ones used for the characterization of the symbiotic proteome in which the accumulation of SA-regulated PR genes was observed in reference ⁶. FW, fresh weight. Bars represent the means ± standard error.On the other hand, a direct link between sugar metabolism and the plant defense response has been established, including the phenomenon of high sugarmediated resistance and the finding that various key PR genes are induced by sugars. Transgenic approaches that lead to alterations in photoassimilate partitioning, either sucrose or hexoses, also alter PR gene expression.^22,23 In other studies, a SA-independent induction of PR genes by soluble sugars, sucrose, glucose and fructose, was reported in reference ²⁴.Sucrose, the main form of assimilated carbon during photosynthesis, is transported to the root tissues via the phloem where it becomes available to the root cells. As previously mentioned, characterization of the rice symbiotic proteome revealed alterations in the accumulation of proteins involved in sugar metabolism, such as enzymes involved in glucolysis/gluconeogenesis (e.g., fructose-1,6-bisphophate aldolase, enolase) or in pentose interconversions (e.g., UDP-glucose dehydrogenase).⁶ Because the plant provides sugars to the fungus, it is not surprising to find alterations in enzymes involved in sugar metabolism in the mycorrhizal roots. Evidence also supports that AM fungi acquire hexoses from the host cell and transform it into trehalose and glycogen, the typical sugars in the fungus.²⁵ Utilization of sucrose then requires hydrolysis in the plant cell which can be performed by sucrose synthase, producing UDP-glucose and fructose or invertases, producing glucose and fructose. Along with this, increased activities of invertases and sucrose synthases or increased expression of their corresponding genes, have been described during AM symbiotic interactions.^26,27 Very recently, the MtSucS1 sucrose synthase gene was reported to be essential for the establishment and maintenance of the AM symbiosis in Medicago truncatula.²⁸ In this context, we decided to explore whether colonization by G. intraradices has an effect on the accumulation of soluble sugars in rice roots.Sucrose, glucose and fructose content were measured enzymatically²³ in the rice roots at 42 days post-inoculation with G. intraradices . A tendency to a higher sucrose level was observed in mycorrhizal roots compared to non-mycorrhizal roots (Fig. 2). Concerning the hexose content, the mycorrhizal roots had a significantly lower hexose, both glucose and fructose levels, compared to non-mycorrhizal roots (p ≤ 0.05, Fig. 2). This finding is in agreement with results reported by other authors indicating that the fungal symbiont takes up and uses hexoses within the root.^29,30 The observation that the sucrose content is not significantly affected by mycorrhiza functioning, indicates that the host cell is able to sense sucrose concentration in order to maintain it at sufficient but constant levels to satisfy the demand for sugars by the fungal symbiont.Open in a separate windowFigure 2Sugar content in roots of rice plants inoculated with G. intraradices (+Gi) or mock-inoculated (−Gi). (A) Sucrose content. (B) Glucose content. (C) Fructose content. Measurements were made at 42 days post-inoculation with G. intraradices. Bars represent the means ± standard error.Clearly, the outcome of the AM symbiosis is an overall improvement of the fitness of both partners: the plant supplies the fungus with photosynthates whereas the fungus delivers nutrients from the soil to the host plant. Variations in the extent of colonization of the AM fungi will impose different carbon demands on the plants. However, a high demand of photosynthates by the mycorrhizal root might result in increased mycorrhization which, in turn, might be detrimental for the host plant. The rate of colonization and the amount of fungal biomass must then be tightly controlled by the host plant. We postulate that an increased sink strength by AM colonization might result in transient and/or localized increases in sugar concentrations in the root cell which might be the signal for the activation of defense gene expression. A schematic representation of plant responses associated with increased demands for sugars and deployment of defense responses is shown in Figure 3. According to this model, sugars might play a dual role during the AM symbiosis: (1) sugars are transferred from the plant to the fungus in exchange of mineral nutrients and (2) sugars alter host gene expression, leading to the activation of defense-related genes. This will allow the host plant to avoid an excessive root colonization by the AM fungus that might cause negative effects on the plant''s fitness. A complex exchange and interplay of signals between plant roots and AM fungi must then operate during functioning of the AM symbiosis for coordination of joint nutrient resource explotation strategies and control of the plant''s immune system. During evolution, co-adaptation between the two symbionts, the AM fungi and the host plant, must have occurred for stabilization of mycorrhizal cooperation and optimal functioning of mycorrhizal associations along the mutualism-parasitism continuum.Open in a separate windowFigure 3Proposed model for a sugar mediated-activation of defense-related genes in mycorrhizal roots. In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. Arbuscules are the site of mineral nutrient transfer to the plant and the site of carbon acquisition by the fungus. Although arbuscules form within the root cortical cells, they remain separated from the plant cell cytoplasm by a plant-derived membrane, the periarbuscular membrane. In this way, an interface is created between the plant and fungal cells which appears to be optimal for nutrient transfer. Sucrose is transported through the phloem into the root. In the root cell, sucrose is hydrolyzed by host invertase and sucrose synthase activities before uptake by the AM fungus. Hexose uptake at the plant-fungus interfase might be passive with a concentration gradient maintained by rapid conversion of hexoses taken up by the fungus to trehalose and glycogen. Active mechanisms might also operate for hexose transport processes between the host cell and the symbiont. Under conditions of a high demand for sugars by the AM fungus, transient increases in sugar content will occur in the root cells which would be the signal for the activation of the host defense responses. The host-produced defense compounds would stabilize the level of root colonization by the AM fungus. An excessive root colonization might change the mutualistic association into a parasitic one.     

Influence of the temporal distribution of electric pulses on transcallosal single unit responses

We examined how differently timed stimuli to one auditory cortex affect the spike trains they drive in the controlateral homotopic field of anesthetized rats. Bipolar electrical stimulations consisted of trains of pulses (100 micro s, <500 micro A) at rates of 25, 50 or 125 pulses/s and with different stimulus patterns (i.e., dispersions, sequences), called "pacemaker", "accelerando" or "decelerando". Trains lasted for 342 ms and were separated by 4 s. When trains were evaluated over times comparable to the stimulus duration changes characteristically involved an initial slowing followed by recovery and several discharges both stimulus- and neuron-dependent. When evaluated by cross-correlations between cortical cell pairs, the changes extended far beyond the stimulus end. Results suggest that interhemispheric projections, by way of their averages and patterns, play key, long duration roles in the spike-dependent properties of cortical synapses (e.g., potentiation, depression) and thus of cortical circuit operations.