Abstract Erythrina velutina ("mulungu") is a legume tree from Caatinga that associates with rhizobia but the diversity and symbiotic ability of "mulungu" rhizobia are poorly understood. The aim of this study was to characterize "mulungu" rhizobia from Caatinga. Bacteria were obteined from Serra Talhada and Caruaru in Caatinga under natural regeneration. The bacteria were evaluated to the amplification of nifH and nodC and to metabolic characteristics. Ten selected bacteria identified by 16S rRNA sequences. They were tested in vitro to NaCl and temperature tolerance, auxin production and calcium phosphate solubilization. The symbiotic ability were assessed in an greenhouse experiment. A total of 32 bacteria were obtained and 17 amplified both symbiotic genes. The bacteria showed a high variable metabolic profile. Bradyrhizobium (6), Rhizobium (3) and Paraburkholderia (1) were identified, differing from their geographic origin. The isolates grew up to 45 °C to 0.51 mol L-1 of NaCl. Bacteria which produced more auxin in the medium with l-tryptophan and two Rhizobium and one Bradyrhizobium were phosphate solubilizers. All bacteria nodulated and ESA 90 (Rhizobium sp.) plus ESA 96 (Paraburkholderia sp.) were more efficient symbiotically. Diverse and efficient rhizobia inhabit the soils of Caatinga dry forests, with the bacterial differentiation by the sampling sites.

ABSTRACT Legume-rhizobia symbiosis is a cross-kingdom association that results in large amounts of nitrogen incorporated in food webs. For the Brazilian semi-arid region, data on genetic variability and symbiotic efficiency of Papilionoidae rhizobial communities are very scarce. The aim of this study was to evaluate the genetic variability and the symbiotic efficiency of eight rhizobial isolates obtained from “mulungu” (Erythrina velutina Willd.) nodules. For 16S rRNA gene sequencing, the genomic DNA was extracted using a commercial kit, amplified with universal primers, and subjected to sequencing reactions. For the isolate ESA 71, PCR amplifications for nodC and nodA genes were attempted. Rhizobial efficiency was assessed by two greenhouse experiments. The first assay was carried out under gnotobiotic conditions, with sterile sand as a substrate; the second experiment was conducted in a non-sterile soil. For both experiments, the inoculation treatments consisted of a single inoculation of each isolate, in addition to a treatment with Bradyrhizobium elkanii BR 5609 as a reference strain. Furthermore, two non-inoculated control treatments, supplied and not supplied with mineral N, were also evaluated. Bacterial identification indicated that both α and β-rhizobia could be found in “mulungu” root nodules. Three isolates where classified within the Rhizobium genus, four bacteria belonged to Bradyrhizobium and one isolate clustered with Burkholderia. Positive amplification of an intragenic fragment of the nodA gene using a primer set to β-rhizobia could be found for ESA 71 (Burkholderia). All bacterial isolates were effective in colonizing “mulungu” roots. In the first experiment, all inoculated treatments and N fertilization increased the N concentration in “mulungu” shoot tissues. For total N in the shoots, the isolates ESA 70, ESA 72, and ESA 75 stood out. In the non-sterile substrate experiment, the isolates ESA 70, ESA 71, ESA 72, and ESA 75, together with the reference strains, induced increases in the shoot N concentration and total accumulation compared to the absolute control. The results indicate that “mulungu” is able to establish associations with efficient α and β-rhizobia in Brazilian semi-arid soils.

Resumo Durante a germinação, as sementes ortodoxas perdem sua habilidade de tolerar a dessecação assemelhando-se com as sementes recalcitrantes. Este trabalho objetivou investigar as mudanças celulares que ocorrem durante a re-indução da tolerância à dessecação (TD) em sementes germinadas de Sesbania virgata com 1, 3 e 5 mm de comprimento de radícula. Para restabelecer a TD, as sementes germinadas foram incubadas durante 72 h em solução de polietilenoglicol (PEG, -2,04 MPa), posteriormente foram desidratadas em sílica gel (10% de teor de água) e reidratadas. A viabilidade celular foi avaliada pelo teste de TUNEL e através de microscopia eletrônica de transmissão em radículas que foram submetidas à secagem ou não. O TUNEL-positivo confirmou a degradação do DNA por meio da fluorescência dos núcleos nas células da radícula com cinco mm de comprimento e as avaliações ultraestruturais detectaram a perda da integridade do conteúdo celular nas radículas com 3 e 5 mm de comprimento e que não sobreviveram à desidratação a 10% de teor de água.

Abstract During germination, orthodox seeds lose their ability to tolerate desiccation resembling recalcitrant seeds. This research aimed to investigate the cell changes during the re-induction of the desiccation tolerance (DT) in Sesbania virgata germinated seeds with 1, 3 and 5 mm long radicles. To re-establish DT, germinated seeds were incubated for 72 h in polyethylene glycol (PEG, -2.04 MPa) before dehydration in silica gel (at 10% moisture content) followed by rehydration. Cell viability was assessed through TUNEL test in dry radicles and transmission electron microscopy in both fresh and dry radicles. The positive-TUNEL confirmed the DNA degradation, through the green fluorescence of the cell nuclei from 5 mm radicle length and the ultra structural evaluations detected loss of cellular content integrity in 3 and 5 mm cell radicles that did not survive dehydration to 10%.

Esta pesquisa objetivou investigar as alterações celulares durante a perda e o restabelecimento da tolerância à dessecação (TD) em sementes germinadas de Sesbania virgata.A perda da TD foi caracterizada em sementes germinadas com 1, 2, 3, 4 e 5 mm de comprimento de radícula, submetidas à desidratação em sílica gel seguida de reidratação. Para restabelecer a TD, as sementes germinadas foram incubadas por 72h em PEG (-2,04 MPa) com e sem ABA (100 μM) antes da secagem em sílica gel. A viabilidade celular foi avaliada pela sobrevivência de plântulas e a integridade do DNA foi avaliada por meio de eletroforese em gel. Sementes com 1 mm de radícula sobreviveram à secagem até o teor de água original (aproximadamente 10%). O tratamento com PEG foi eficiente para restabelecer a TD, parcialmente, em sementes com 2, 3 e 4 mm, exceto com 5 mm de radícula. Sementes germinadas tratadas com PEG+ABA apresentaram melhor desempenho em relação às sementes sem ABA, sendo que a TD foi restabelecida em sementes com 5 mm de radícula. Dentre as sementes tratadas com PEG e secas até 10% de teor de água, a integridade do DNA foi mantida em sementes com 1 mm de radícula.

This research aimed to investigate the cellular alterations during the loss and re-establishment of desiccation tolerance (DT) in germinated Sesbania virgata seeds. The loss of DT was characterized in germinated seeds with increasing radicle lengths (1, 2, 3, 4 and 5 mm) when subjected to dehydration in silica gel, followed by rehydration. To re-establish DT, the germinated seeds were incubated for 72h in polyethylene glycol (PEG, -2.04 MPa) with or without ABA (100 μM) before dehydration in silica gel. Cell viability was assessed by seedling survival, and DNA integrity was evaluated by gel electrophoresis. Seeds with 1 mm radicle length survived dehydration to the original moisture content (MC) of the dry seed (approximately 10%). PEG treatment was able to re-establish DT, at least partially, with 2, 3 and 4 mm but not in 5 mm radicle lengths. Germinated seeds treated with PEG+ABA performed better than those treated only with PEG, and DT was re-established even in germinated seeds with a 5 mm radicle length. Among the PEG-treated germinated seeds dehydrated to 10% MC, DNA integrity was maintained only in those with a 1 mm radicle length.

Conduziu-se este trabalho, com o objetivo de avaliar o efeito do condicionamento osmótico sobre a germinação e o vigor de sementes de Sesbania virgata. Foram utilizadas sementes escarificadas em ácido sulfúrico concentrado durante 40 minutos e, posteriormente, embebidas em soluções de PEG 8000 com os potenciais osmóticos de 0,0; -0,2; -0,4; -0,6 e -0,8 MPa durante 12, 24 e 48 horas. Após o condicionamento, as sementes foram submetidas à secagem em sílica gel até que fosse atingido o grau de umidade inicial das sementes e, posteriormente, foram envelhecidas artificialmente (40 ºC/48 h/100% UR). Os efeitos do condicionamento e do envelhecimento foram avaliados por meio do teste de germinação, primeira contagem e índice de velocidade de germinação. O osmocondicionamento das sementes beneficiou a germinação e o vigor das sementes de Sesbânia, submetidas ou não ao envelhecimento acelerado. O período de 24 horas de condicionamento favoreceu o desempenho das sementes osmocondicionadas.

This study evaluated the effect of the osmopriming on germination and vigour of Sesbania virgata seeds. Seeds were chemically scarified in concentrated sulphuric acid for 40 minutes and put to germinate either directly or after being submitted to osmopriming, drying and accelerated aging. Osmopriming was carried out with polyethylene glycol solutions (PEG 8000) at the following osmotic potentials-0.2; -0.4; -0.6 and -0.8 MPa for 12, 24 and 48 hours. After osmopriming, seeds were dried in silica gel until the initial moisture content was reached, and then submitted to the accelerated aging (48 h/100% RH). The effects of osmopriming and accelerated aging were evaluated through germination test, first counting germination and germination speed index. The osmopriming, followed or not by accelerated aging, positively influenced germination and vigour of Sesbania virgata seeds.