Inoculation with Azospirillum argentinense Az19 improves the yield of maize subjected to water déficit at key stages of plant development / La inoculación con Azospirillum argentinense Az19 mejora el rendimiento del maíz expuesto a déficit hídrico durante estadios clave del desarrollo vegetal

Resumen El déficit hídrico constituye una severa limitación a la productividad agrícola. En el marco de la producción sostenible de cultivos, la biotecnología microbiana está cobrando relevancia para aumentar la tolerancia a la sequía y mejorar el rendimiento de los cultivos en condiciones adversas. El propósito de este trabajo fue comparar la acción de la cepa de Azospirillum argentinense Az19, con tolerancia in vitro a estresores abióticos, con la cepa Az39, utilizada ampliamente para la formulación de inoculantes comerciales, al inocularlas en plantas sometidas a déficit hídrico. Se realizaron ensayos de invernadero y de campo. En invernadero, la cepa Az19 evitó el impacto adverso del déficit hídrico en el estadio V2 sobre el crecimiento del maíz. Además, el porcentaje de plantas con espigas y el peso de la espiga disminuyó significativamente con la restricción hídrica aplicada en V2 y en floración en plantas inoculadas con la cepa Az39, pero no en las inoculadas con Az19. En el primer ensayo de campo con el maíz híbrido comercial DOW DS 515 PW las plantas inoculadas con Az19 fueron las que mejor toleraron la deficiencia hídrica. En el segundo ensayo de campo se utilizaron dos genotipos de maíz con sensibilidad diferencial a la sequía. La inoculación con Az19 condujo a una mayor tolerancia al déficit hídrico, con un efecto detectable en algunos componentes del rendimiento en el genotipo sensible. Sobre la base de estos resultados, proponemos el empleo de A. argentinense Az19 para la formulación de inoculantes basados en Azospirillum especialmente indicados para áreas agroecológicas que experimenten períodos de déficit hídrico.

Inoculation with Azospirillum argentinense Az19 improves the yield of maize subjected to water deficit at key stages of plant development.

Water deficit constitutes a severe limitation to agricultural productivity. In the context of sustainable crop production, the potential of microbial biotechnology to increase plant drought tolerance and improve crop yields under adverse conditions is gaining relevance. This work aimed to compare the performance of Azospirillumargentinense strain Az19 to that of strain Az39, the most widely used for commercial inoculants, when inoculated in maize plants exposed to water deficit. For this purpose, greenhouse and field assays were conducted. In the greenhouse experiment, strain Az19 prevented the adverse effect of water deficit at V2 stage on maize growth. Moreover, the percentage of fertile plants and the ear weight decreased significantly under water deficits imposed at V2 and flowering in Az39-inoculated plants but not in Az19-inoculated plants. In the first field trial with the commercial maize hybrid DOW DS 515 PW, Az19-inoculated plants were those which better tolerated the water deficit imposed. In the second field trial, two maize genotypes with differential drought sensitivity (LP 29×LP 2542, sensitive; LP 882 (923)×LP 4703, tolerant) were tested. Higher tolerance to water deficit was detected in plants inoculated with A. argentinense Az19, with a noticeable effect on grain yield components in the sensitive genotype. Based on these results, we propose the use of A. argentinense Az19 for the formulation of more targeted Azospirillum-based inoculants, suitable for agroecological areas subjected to seasonal water deficits.

Beneficial effects of magnetite nanoparticles on soybean-Bradyrhizobium japonicum and alfalfa-Sinorhizobium meliloti associations.

Nanoparticles (NPs)-based growth stimulators have promising usage in agriculture. This research analyzed the impact of citric acid-coated magnetite nanoparticles (Fe3O4-NPs; 50 mg Fe L-1) added once at pre-sowing on soybean and alfalfa seedlings growing in association with their corresponding microsymbiont partners, Bradyrhizobium japonicum and Sinorhizobium meliloti; also on the in vitro growth rate of these microorganisms. Fe-EDTA (50 mg Fe L-1) was used as a comparator. Fe3O4-NPs significantly augmented the growth rate constant (7-17%) and extracellular polysaccharides production of both microsymbionts (B. japonicum: 2-fold; S. meliloti: 43%), which probably favored bacterial adhesion to the root hairs. In both legumes, Fe3O4-NPs increased chlorophyll content (up to 56% in soybean) and improved plant growth, evidenced by a greater root biomass system (80-90% higher than the control), and increased shoot biomass (30-40%). Besides, Fe3O4-NPs addition resulted in earlier nodule formation and enhanced nodule biomass (about 2.5-fold in both species). Nodules were mainly located in the crown of the root in the NP50 treatment, while they were evenly distributed along lateral roots in the control and the comparator. Fe3O4-NPs also augmented significantly nodule leghemoglobin content (∼50-70%) and total N in legumes' shoots (ca. 20%). CAT activity increased only under NP50 treatment and no symptoms of oxidative damage were evidenced. In this work, we found that besides not being toxic neither to soybean and alfalfa plants nor to their microsymbiont partners, Fe3O4-NPs do not exert adverse effects on the symbioses establishment; oppositely, a more efficient nodulation pattern was verified in both plant species.

Azospirillum brasilense Az39 restricts cadmium entrance into wheat plants and mitigates cadmium stress.

In this work, we tested if inoculation with the plant growth-promoting rhizobacteria Azospirillum brasilense strain Az39 alleviates Cd2+ stress in wheat seedlings grown under controlled conditions. Growth, total N, N-related metabolites/enzymes, and oxidative stress parameters were measured. Additionally, the usefulness of a real-time PCR protocol to screen the preferred colonization site of the introduced microorganism was evaluated. Inoculated plants demonstrated mitigation of cadmium-induced adverse effects on plant growth and less reactive oxygen species accumulation in their roots by the end of the experiment, 28 days after sowing. Cd addition resulted in lower NO3- content in the leaves and higher NO3- content in the roots, and a significant rise in NH4+ concentration in both organs in uninoculated plants; in inoculated plants, NH4+ content in the roots did not vary. A. brasilense Az39 enhanced NO levels in wheat root tips, and more adventitious roots and root hairs were observed in inoculated plants. Despite having a more developed root system, inoculated plants showed lower Cd levels in their roots compared to non-inoculated plants. Inoculation with this PGPR favored ion homeostasis in the roots of metal-exposed plants, decreasing Cd/Fe ratio. We corroborated A. brasilense Az39 preference for wheat exorhizosphere using a real-time PCR-based method targeting the nifA gene.

Magnetite nanoparticles coated with citric acid are not phytotoxic and stimulate soybean and alfalfa growth.

In this work, the internalization and distribution of citric acid-coated magnetite nanoparticles (here, Fe3O4-NPs) in soybean and alfalfa tissues and their effects on plant growth were studied. Both legumes were germinated in pots containing an inert growing matrix (vermiculite) to which Hoagland solution without (control, C), with Fe3O4-NPs (50 and 100 mgironL-1, NP50 and NP100), or with the same amount of soluble iron supplied as Fe-EDTA (Fe50, Fe100) was added once before sowing. Then, plants were watered with the standard nutrient solution. The observation of superparamagnetic signals in root tissues at harvest (26 days after emergence) indicated Fe3O4-NPs uptake by both legumes. A weak superparamagnetic signal was also present in the stems and leaves of alfalfa plants. These findings suggest that Fe3O4-NPs are readily absorbed but not translocated (soybean) or scarcely translocated (alfalfa) from the roots to the shoots. The addition of both iron sources resulted in increased root weight; however, only the addition of Fe3O4-NPs resulted in significantly higher root surface; shoot weight also increased significantly. As a general trend, chlorophyll content enhanced in plants grown in vermiculite supplemented with extra iron at pre-sowing; the greatest increase was observed with NP50. The only antioxidant enzyme significantly affected by our treatments was catalase, whose activity increased in the roots and shoots of both species exposed to Fe3O4-NPs. However, no symptoms of oxidative stress, such as increased lipid peroxidation or reactive oxygen species accumulation, were evidenced in any of these legumes. Besides, no evidence of cell membrane damage or cell death was found. Our results suggest that citric acid-coated Fe3O4-NPs are not toxic to soybean and alfalfa; instead, they behave as plant growth stimulators.

Development and testing of a 3D-printable polylactic acid device to optimize a water bioremediation process.

In the present work, a remediation bioprocess based on the use of a native isolate of Chlorella vulgaris immobilized in an alginate matrix inside a polylactic acid (PLA) device is proposed. This microalga immobilized in alginate beads was previously shown to be useful for the reduction of several chemical and microbial contaminants present in the highly polluted water from the Matanza-Riachuelo watershed. However, these beads had a relatively short shelf life in the natural environment. To overcome this limitation, a 3D-printed PLA device was designed. PLA is a biocompatible and biodegradable material suitable for biotechnological applications. We used Erlenmeyers and stirred-tank bioreactors fed batch with Murashige Skoog (MS) culture medium or water from the Cildáñez stream (one of the water bodies of the aforementioned watershed) to estimate the growth kinetics parameters and the bioremediation capacity of immobilized-microalgal cells as an unconfined system (UcS) or a confined system (CfS) inside PLA devices on Cildáñez water. Although alga's growth parameters were maximum in the UcS fed with MS medium as substrate, successful bioremediation of the target water was possible using the CfS: all inorganic nitrogen forms and total phosphorus were reduced at least by 90% after 5 days of bioprocess in an agitated bioreactor, whereas aerobic mesophilic bacteria decreased by about 85%. The number of coliforms also decreased. Standardized cytotoxicity tests using Allium cepa seeds carried out to prove the effectiveness of the bioremediation process, confirmed the high degree of decontamination achieved by the use of immobilized microalga confined in a 3D-printable PLA-device.

Methylobacterium sp. 2A Is a Plant Growth-Promoting Rhizobacteria That Has the Potential to Improve Potato Crop Yield Under Adverse Conditions.

A Gram-negative pink-pigmented bacillus (named 2A) was isolated from Solanum tuberosum L. cv. Desirée plants that were strikingly more developed, presented increased root hair density, and higher biomass than other potato lines of the same age. The 16S ribosomal DNA sequence, used for comparative gene sequence analysis, indicated that strain 2A belongs to the genus Methylobacterium. Nucleotide identity between Methylobacterium sp. 2A sequenced genome and the rest of the species that belong to the genus suggested that this species has not been described so far. In vitro, potato plants inoculated with Methylobacterium sp. 2A had a better performance when grown under 50 mM NaCl or when infected with Phytophthora infestans. We inoculated Methylobacterium sp. 2A in Arabidopsis thaliana roots and exposed these plants to salt stress (75 mM NaCl). Methylobacterium sp. 2A-inoculated plants, grown in control or salt stress conditions, displayed a higher density of lateral roots (p < 0.05) compared to noninoculated plants. Moreover, under salt stress, they presented a higher number of leaves and larger rosette diameter. In dual confrontation assays, Methylobacterium sp. 2A displayed biocontrol activity against P. infestans, Botrytis cinerea, and Fusarium graminearum, but not against Rhizoctonia solani, and Pythium dissotocum. In addition, we observed that Methylobacterium sp. 2A diminished the size of necrotic lesions and reduced chlorosis when greenhouse potato plants were infected with P. infestans. Methylobacterium sp. 2A produces indole acetic acid, solubilizes mineral phosphate and is able to grow in a N2 free medium. Whole-genome sequencing revealed metabolic pathways associated with its plant growth promoter capacity. Our results suggest that Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria (PGPR) that can alleviate salt stress, and restricts P. infestans infection in potato plants, emerging as a potential strategy to improve crop management.

Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum.

The role of endophytic communities of seeds is still poorly characterised. The purpose of this work was to survey the presence of bacterial endophytes in the seeds of a commercial wheat cultivar widely sown in Argentina and to look for plant growth promotion features and biocontrol abilities against Fusarium graminearum among them. Six isolates were obtained from wheat seeds following a culture-dependent protocol. Four isolates were assignated to Paenibacillus genus according to their 16S rRNA sequencing. The only gammaproteobacteria isolated, presumably an Enterobactereaceae of Pantoea genus, was particularly active as IAA and siderophore producer, and also solubilised phosphate and was the only one that grew on N-free medium. Several of these isolates demonstrated ability to restrain F. graminearum growth on dual culture and in a bioassay using barley and wheat kernels. An outstanding ability to form biofilm on an inert surface was corroborated for those Paenibacillus which displayed greater biocontrol of F. graminearum, and the inoculation with one of these isolates in combination with the Pantoea isolate resulted in greater chlorophyll content in barley seedlings. Our results show a significant ecological potential of some components of the wheat seed endophytic community.

Early response of wheat seminal roots growing under copper excess.

Growth reduction caused by copper excess during plant photoautotrophic metabolism has been widely investigated, but information regarding early responses of root apical meristem (RAM) to toxic concentrations of this metal at the initial heterotrophic stage is certainly scarce. We analysed some determinants of seminal root growth in developing wheat seedlings germinated in the presence of 1, 5 and 10 µM CuCl2, focussing on oxidative damage to cell membrane and to proteins, and investigated the expression patterns of some genes relevant to cell cycle progression and cell expansion. The proliferation zone of the RAM was shorter under 5 and 10 µM CuCl2. Cyclin D and CDKA levels remained unchanged in the root apexes of wheat seedlings grown under these Cu(2+) concentrations, but more carbonylated levels of both proteins and less ubiquitinated-cyclin D was detected under 10 µM CuCl2. Increased levels of ROS were revealed by fluorescent probes at this Cu(2+) dose, and severe cell membrane damage took place at 5 and 10 µM CuCl2. Several genes related to retinoblastome phosphorylation and therefore involved in the transition from G1 to S cell cycle stage were found to be downregulated at 10 µM CuCl2, while most expansin genes here analysed were upregulated, even at a non-toxic concentration of 1 µM. These results together with previous findings suggest that a "common" signal which involves oxidative posttranslational modifications of specific cell cycle proteins may be necessary to induce root growth arrest under Cd(2+) and Cu(2+) stress.

Search for endophytic diazotrophs in barley seeds.

Eight endophytic isolates assigned to Pseudomonas, Azospirillum, and Bacillus genera according to pheno-genotypic features were retrieved from barley seeds under selective pressure for nitrogen-fixers. Genetic relationships among related isolates were investigated through RAPD. Six isolates displayed nitrogen-fixing ability, while all could biosynthesize indolacetic acid in vitro and showed no antibiosis effects against Azospirillum brasilense Az39, a recognized PGPR.

Search for endophytic diazotrophs in barley seeds

Eight endophytic isolates assigned to Pseudomonas, Azospirillum, and Bacillus genera according to pheno-genotypic features were retrieved from barley seeds under selective pressure for nitrogen-fixers. Genetic relationships among related isolates were investigated through RAPD. Six isolates displayed nitrogen-fixing ability, while all could biosynthesize indolacetic acid in vitro and showed no antibiosis effects against Azospirillum brasilense Az39, a recognized PGPR.

Response to saline stress and aquaporin expression in Azospirillum-inoculated barley seedlings.

The ability of two strains of Azospirillum brasilense to mitigate NaCl stress in barley plants was evaluated. Barley seedlings were inoculated and subjected to 200 mM NaCl for 18 days. Several days after NaCl treatment, a significant decline in biomass as well as in height was observed in uninoculated plants. However, smaller reductions in biomass and height were detected in plants inoculated with strain Az39. All the stressed plants showed significantly higher Na(+) but lower K(+) contents in their shoots. The growth rate of uninoculated plants was adversely affected by saline treatment, which was associated with higher putrescine content and lower levels of HvPIP2;1 transcripts in the roots. Azospirillum inoculation triggered the transcription of this gene. Our results suggest that barley plants inoculated with A. brasilense may be better prepared to thrive under saline conditions. To our knowledge, this is the first report showing an effect of Azospirillum inoculation on the expression of PIP2;1, a gene involved in the synthesis of root water channels.

Inhibition of root growth and polyamine metabolism in sunflower (Helianthus annuus) seedlings under cadmium and copper stress.

Although sunflower is usually regarded as a highly tolerant crop, impairment of root growth at initial stages of plant development may result in poor crop establishment and higher susceptibility to pathogen attack. In order to evaluate if Cd2+ and Cu2+ may impact on sunflower germination and initial root development, a pot experiment under controlled conditions was carried out. Possible involvement of polyamine metabolism in sunflower response to these stressors was also investigated. Although Cd2+ and Cu2+ treatments affect neither seed germination nor radical emergence, sunflower seedlings grown in the presence of these heavy metals showed significant inhibition of root growth, being this inhibition greater for Cd2+. Both metals caused significant increases in proline contents at the highest concentrations tested (0.5 and 1 mM), and these increments were more pronounced for Cd2+ treatments, especially between days 3 and 10. Metals also increased putrescine (Put) contents at all concentrations assayed from the seventh day onward, causing no variations on this polyamine time-course pattern. Spermine and spermidine contents, however, were increased only by 1 mM Cd2+. Arginine decarboxylase seems to have been the enzyme responsible for Put increases under both metal treatments. This work demonstrates that initial root growth of sunflower seedlings may be significantly impaired in Cd2+ or Cu2+ contaminated soils. It also shows that polyamines are key biological compounds, which are probably involved in signaling pathways triggered under stress environmental conditions.

Heavy metals effects on proteolytic system in sunflower leaves.

Plant proteolytic system includes proteases, mainly localized inside the organelles, and the ubiquitin-proteasome pathway in both, the cytoplasm and the nucleus. It was recently demonstrated that under severe Cd stress sunflower (Helianthus annuus L.) proteasome activity is reduced and this results in accumulation of oxidized proteins. In order to test if under other heavy metal stresses sunflower proteolytic system undergoes similar changes, an hydroponic experiment was carried out. Ten days old sunflower plants were transferred to hydroponic culture solutions devoid (control) or containing 100 microM of AlCl(3), CoCl(2), CuCl(2), CrCl(3), HgCl(2), NiCl(2), PbCl(2) or ZnCl(2) and analyzed for protein oxidative damage and proteolytic activities. After 4 days of metal treatment, only Co(2+), Cu(2+), Hg(2+), and Ni(2+) were found to increase carbonyl groups content. Except for Al(3+) and Zn(2+), all metals tested significantly reduced all proteasome activities (chymotrypsin-like, trypsin-like and PGPH) and acid and neutral proteases activities. The effect on basic proteases was more variable. Abundance of 20S protein after metal treatments was similar to that obtained for control samples. Co(2+), Cu(2+), Hg(2+), Ni(2+), Cr(3+), and Pb(2+) induced accumulation of ubiquitin conjugated proteins. It is concluded that heavy metal effects on proteolytic system cannot be generalized; however, impairment of proteasome functionality and decreased proteases activities seem to be a common feature involved in metal toxicity to plants.

Effect of chlorimuron-ethyl on Bradyrhizobium japonicum and its symbiosis with soybean.

Possible side-effects of the acetolactate synthase (ALS)-inhibiting herbicide chlorimuron-ethyl on Bradyrhizobium japonicum (Kirchner & Jordan) in pure culture and on inoculated soybean plants growing under controlled conditions were investigated. Growth of B japonicum strain E109 was not affected by this herbicide even when exposed to concentrations 150 times higher than recommended field doses. However, nodulation of soybean plants treated 5 days after emergence with chlorimuron-ethyl at standard application rates was impaired: a 38% decrease in the number of nodules per plant was observed four weeks after treatment. Despite nodule number decrease, no changes in shoot nitrogen content could be detected. Total fresh biomass was diminished by 25% in herbicide-treated plants. Leghemoglobin content in nodules did not vary; nevertheless total nodule protein was diminished by 40% in the herbicide-treated group. ALS activity in different soybean tissues and their relative sensitivity to chlorimuron-ethyl were also investigated. Roots and bacteroids had the greatest specific ALS activities. On a fresh weight basis, the bacteroid fraction displayed the highest ALS activity and was also the most tolerant to in vitro chlorimuron addition: 72% of its activity was retained after including 10 microM chlorimuron-ethyl in the reaction mixture. These results indicate that standard application rates of chlorimuron-ethyl will have limited incidence on B japonicum survival, and effects on nodulation may have little long-term consequences on soybean nitrogen fixation potential. The differences found among soybean tissues not only in intrinsic ALS activity but also in their relative sensitivity to this herbicide suggests that, in leguminous plants living in symbiosis with rhizobia, nodules may contribute to an enhanced tolerance to ALS inhibitors.