Stella Castro

Interaction of the fungicide mancozeb and Rhizobium sp. in pure culture and under field conditions

Abstract Information on the compatibility of Rhizobium sp. with seed-protectant chemicals is controversial because of variations in the methods used and the lack of quantitative data. The present study was conducted to determine the influence of the fungicide mancozeb (ethylenebis-dithiocarbamate), at recommended doses, on the growth, survival and symbiotic properties of Rhizobium sp. infecting peanut plants (Arachis hypogaea) under laboratory and field conditions. The results indicated that mancozeb decreased growth in pure culture by 50% of both Rhizobium sp. USDA 3187 and a strain isolated from peanut nodules. However, no differences were found in peanut seed yields under field conditions. These results suggest that the soil environment could reduce the probability of the direct, harmful effects of mancozeb on bacterial growth.

Nodulation in peanut (Arachis hypogaea L.) roots in the presence of native and inoculated rhizobia strains

There is considerable interest in improving nitrogen fixation in legumes to increase soil fertility, particularly in the developing countries. The effect of inoculation of peanut (Arachis hypogaea L.) seed with Bradyrhizobium sp. USDA 3187 was examined under field conditions. In order to determine nodule occupancy, bacterial cells from surface-sterilized nodules of inoculated plants were isolated and characterized by their intrinsic antibiotic resistance and by agglutination test. Results obtained revealed that peanut roots were nodulated by native strain rather than the introduced one, indicating that strain USDA 3187 was not as competitive as naturalized peanut rhizobia. Data also showed that there were no significant differences in nodule dry weight, plant dry biomass, nitrogenase activity and seed yield between uninoculated and inoculated treatments.

Contribution of phytochelatins to cadmium tolerance in peanut plants

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H₂O₂) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.

Antioxidant enzyme activities and gene expression patterns in peanut nodules during a drought and rehydration cycle

Drought stress is one of the most important environmental factors that affect plant growth and limit biomass production. Most studies focus on drought stress development but the reversibility of the effects receives less attention. Therefore, the present work aims to explore the biological nitrogen fixation (BNF) of the symbiotic association between peanut (Arachis hypogaea L.) and Bradyrhizobium sp. during a drought-recovery cycle with a focus on the response of enzyme activity and gene expression of the antioxidant system. Peanuts exposed to drought stress had impaired BNF, as indicated by lower nitrogenase activity, and decreased leghaemoglobin content; the latter was reversed to control values upon rehydration. Previous results demonstrated that reactive oxygen species (O2·- and H2O2) were accumulated as a consequence of drought stress, suggesting that nodules experience oxidative stress. In addition, marker transcripts responsive to drought, abscisic acid and H2O2 were upregulated. Increased transcript levels of glutathione reductase were associated with an increased enzyme activity but superoxide dismutase and glutathione S-transferase activities were unchanged, despite upregulated gene transcription. In contrast, increased activity of ascorbate peroxidase (APX) was unrelated with changes in cytosolic APX transcript levels suggesting isogene specificity. In conclusion, the work exemplarily demonstrates the efficient and dynamic regulation of antioxidant enzymes and marker compounds during drought cycling, which is likely to be a prerequisite for functional optimisation of nodule metabolism.

Response of Azospirillum brasilense Cd to Sodium Chloride Stress

Growth of Azospirillum brasilense Cd in the presence of different NaCl concentrations showed that it tolerates up to 200 mM NaCl in the medium, without appreciable decline in growth rate. At 300 mM NaCl, a decrease of 66% in growth was observed at 24 h of culture. At 48 h of culture, bacteria in the presence of 300 mM NaCl reached the maximum optical density value that was attained at 12 h by control cultures. This investigation was designed to elucidate the effect of saline stress on Azospirillum brasilense Cd and the physiologic mechanism involved in its possible salinity tolerance. For this reason, studies of other osmolytes, as well as of putrescine metabolism and protein patterns were done with bacteria grown with this NaCl concentration in the medium, at 24 and at 48 hours. A. brasilense responded to saline stress elevating the intracellular concentration of glutamate at 24 h, and of K+at 48 h. Glucan pattern, putrescine metabolism, and total and periplasmic protein patterns of the treated group showed several changes with respect to the control. In spite of the several cellular functions affected by saline stress, the results imply that A. brasilense Cd shows salinity tolerance in these experimental conditions.

Biochemical alterations in Bradyrhizobium sp USDA 3187 induced by the fungicide Mancozeb

We have previously shown that fungicide Mancozeb causes a 50% decrease in Bradyrhizobium sp USDA 3187 growth rate and affects the bacteria-root symbiotic interaction. In order to elucidate the fungicide toxicity mechanism we determined the effects of Mancozeb on cell chemical composition, glutathione (GSH) content (molecule involved in the detoxification process), glutathione S-transferase (GST) activity and on polyamine, exopolysaccharides, capsular polysaccharides and lipopolysaccharides. Mancozeb produced biochemical alterations in membrane composition, polysaccharides and polyamines. In spite of the increment of GSH content and GST activity, they are not enough to prevent the growth diminution.

Importance of glutathione in the nodulation process of peanut (Arachis hypogaea).

GSH appears to be essential for proper development of the root nodules during the symbiotic association of legume-rhizobia in which the entry of rhizobia involves the formation of infection threads. In the particular case of peanut-rhizobia symbiosis, the entry of rhizobia occurs by the mechanism of infection called crack entry, i.e. entry at the point of emergence of lateral roots. We have previously shown the role of GSH content of Bradyrhizobium sp. SEMIA 6144 during the symbiotic association with peanut using a GSH-deficient mutant obtained by disruption of the gshA gene, encoding gamma-glutamylcysteine synthetase (gamma-GCS), which was able to induce nodules in peanut roots without alterations in the symbiotic phenotype. To investigate the role of the peanut GSH content in the symbiosis, the compound L-buthionine-sulfoximine (BSO), a specific inhibitor of gamma-GCS in plants, was used. There were no differences in the plant growth and the typical anatomic structure of the peanut roots when the plants grew in the Fahraeus medium either in presence or in absence of 0.1 mM BSO. However, the GSH content was reduced by 51% after treatment with BSO. The BSO-treated plants inoculated with wild-type or mutant strains of Bradyrhizobium sp. showed a significant reduction in the number and dry weight of nodules, suggesting that GSH content could play an important role in the nodulation process of root peanut with Bradyrhizobium sp.

The interaction of 2,4-dichlorophenoxyacetic acid, ribosomes and polyamines in Azospirillum brasilense

2,4-Dichlorophenoxyacetic acid (2,4-D) is an herbicide used extensively in agriculture. We had previously determined that 1 mM 2,4-D could inhibit cell growth, DNA and protein synthesis of Azospirillum brasilense. The present work was designed to determine if these alterations are a consequence of 2,4-D action on polyamine biosynthesis and if the protein synthesis inhibition is a result of ribosomal impairment. In this paper we demonstrate that 2,4-D alters the metabolism of polyamines and, thus, affects protein synthesis at the ribosomal level.

Influence of cadmium on the symbiotic interaction established between peanut (Arachis hypogaea L.) and sensitive or tolerant bradyrhizobial strains.

Heavy metals in soil are known to affect rhizobia-legume interaction reducing not only rhizobia viability, but also nitrogen fixation. In this work, we have compared the response of the symbiotic interaction established between the peanut (Arachis hypogaea L.) and a sensitive (Bradyrhizobium sp. SEMIA6144) or a tolerant (Bradyrhizobium sp. NLH25) strain to Cd under exposure to this metal. The addition of 10xa0μM Cd reduced nodulation and nitrogen content in both symbiotic associations, being the interaction established with the sensitive strain more affected than that with the tolerant one. Plants inoculated with the sensitive strain accumulated more Cd than those inoculated with the tolerant strain. Nodules showed an increase in reactive oxygen species (ROS) production when exposed to Cd. The histological structure of the nodules exposed to Cd revealed a deposit of unknown material on the cortex and a significant reduction in the infection zone diameter in both strains, and a greater number of uninfected cells in those nodules occupied by the sensitive strain. In conclusion, Cd negatively impacts on peanut-bradyrhizobia interaction, irrespective of the tolerance of the strains to this metal. However, the inoculation of peanut with Bradyrhizobium sp. NLH25 results in a better symbiotic interaction suggesting that the tolerance observed in this strain could limit Cd accumulation by the plant.

2,4-Dichlorophenoxyacetic acid affects the attachment of Azospirillum brasilense Cd to maize roots

2.4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide widely applied to forage, grain and cereals. We previously determined that 1 mM 2,4-D diminished cell growth and cellular activity of Azospirillum brasilense Cd. The present work was designed to determine the possible effect of this herbicide--at concentrations used on crops--on the attachment of the bacteria to maize roots, since this step is of prime importance for the growth stimulation of the plant obtained with Azospirillum brasilense. In this paper we demonstrate that 2,4-D alters the bacterial adhesion to maize roots.