Strain SEMIA Research Articles

Effects of growth‐promoting bacteria on soybean root activity, plant development, and yield

AbstractRhizobia and other plant growth‐promoting rhizobacteria (PGPR) have been broadly used as inoculants in agriculture, resulting in morphofunctional improvements in roots and grain yield. This study was carried out during two cropping seasons under field and greenhouse conditions in Brazil to verify the effects of inoculation of two soybean cultivars with PGPR and secondary microbial metabolites (SMMs) on root activity and nodulation, plant development, and grain yield. Inoculation and co‐inoculation treatments consisted of Bradyrhizobium japonicum strain SEMIA 5079 and B. diazoefficiens strain SEMIA 5080 inoculated together, in combination with Bacillus subtilis strain QST 713, Azospirillum brasilense strains Ab‐V5 and Ab‐V6, and SMMs extracted from B. diazoefficiens strain USDA 110 and Rhizobium tropici strain CIAT 889. Root systems were evaluated by direct (optical reading) and indirect (rubidium nitrate application, 85RbNO3) methods. Increases of up to 1.6% in root diameter (0.01‐ to 0.5‐mm class), 28.5% in length, 19.7% in root volume, 17.8% in root surface area, 29% in the number of nodules, 27.2% in nodule dry weight, 13.5% in root dry weight, and 3.8% in shoot dry weight. Greater exploration and activity within and between rows following inoculation at up to 40 and 10 cm in depth, respectively, were observed in plants co‐inoculated with the standard inoculation (only Bradyrhizobium spp.) + SMMs + A. brasilense, resulting in a yield increase of 485 kg ha−1. The results emphasize the biotechnological potential of using secondary metabolites of rhizobia with inoculants containing rhizobia and PGPR to improve the growth and soybean yield in tropical conditions.

Draft Genome Sequence of Bradyrhizobium elkanii Strain SEMIA 938, Used in Commercial Inoculants for Lupinus spp. in Brazil

Due to its high capacity for nitrogen fixation, strain SEMIA 938 is used in commercial inoculants for lupins in Brazil. Its genome was estimated at 8,780,064 bp and indicates that it belongs to the Bradyrhizobium elkanii species, while the analysis of nodulation genes classifies the strain in the symbiovar sojae.

Analysis of the denitrification pathway and greenhouse gases emissions in Bradyrhizobium sp. strains used as biofertilizers in South America.

Greenhouse gases are considered as potential atmospheric pollutants, with agriculture being one of the main emission sources. The practice of inoculating soybean seeds with Bradyrhizobium sp. might contribute to nitrous oxide (N2 O) emissions. We analysed this capacity in five of the most used strains of Bradyrhizobium sp. in South America. We analysed the denitrification pathway and N2 O production by Bradyrhizobium japonicum E109 and CPAC15, Bradyrhizobium diazoefficiens CPAC7 and B. elkanii SEMIA 587 and SEMIA 5019, both in free-living conditions and in symbiosis with soybean. The in silico analysis indicated the absence of nosZ genes in B. japonicum and the presence of all denitrification genes in B. diazoefficiens strains, as well as the absence of nirK, norC and nosZ genes in B. elkanii. The in planta analysis confirmed N2 O production under saprophytic conditions or symbiosis with soybean root nodules. In the case of symbiosis, up to 26.1 and 18.4 times higher in plants inoculated with SEMIA5019 and E109, respectively, than in those inoculated with USDA110. The strains E109, SEMIA 5019, CPAC15 and SEMIA 587 showed the highest N2 O production both as free-living cells and in symbiotic conditions in comparison with USDA110 and CPAC7, which do have the nosZ gene. Although norC and nosZ could not be identified in silico or invitro in SEMIA 587 and SEMIA 5019, these strains showed the capacity to produce N2 O in our experimental conditions. This is the first report to analyse and confirm the incomplete denitrification capacity and N2 O production in four of the five most used strains of Bradyrhizobium sp. for soybean inoculation in South America.

Soybean inoculants in Brazil: an overview of quality control.

The bacterial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens) are recommended for soybean inoculants in Brazil. In several countries, the current regulations are insufficient to induce companies for improving the quality of their products, leading to low performance and subsequent abandonment of inoculant use. From 2010 to 2014, 1086 samples coming mainly from Argentina and the southern region of Brazil were analyzed for viable cells counting, strains identification, and purity analysis according to the SDA/MAPA no. 30/2010 Normative Instruction. Most products were imported and formulated in liquid carriers with 5.0 × 109 colony-forming units (CFU)/mL. The strains most frequently used were SEMIA 5079/5080. Only 2.21% of samples had contaminants. The guaranteed concentration of viable cells in inoculants mostly ranged from 4.1 × 109 to 5.0 × 109CFU/mL or CFU/g. The most frequently found concentration was above 1.1 × 1010CFU/mL or CFU/g, which was higher than the product guarantee. The inoculants used for soybean crop in Brazil have excellent quality, leading the country to the leadership in taking advantage of the biological nitrogen fixation benefits for a productive and sustainable agriculture.

GROWTH AND YIELD OF COMMON BEAN AS AFFECTED BY SEED INOCULATION WITH RHIZOBIUM AND NITROGEN FERTILIZATION

SUMMARYTwo field experiments were conducted during the rainy seasons of 2013 and 2014 to evaluate the effects of biological nitrogen fixation (BNF) and/or sowing N fertilization on the growth and yield of Phaseolus vulgaris L. In addition, leaf soluble protein content (LSPC) was measured at four growth stages in both seasons. Plants were subjected to the following treatments: only fertilized with 20 kg N ha−1 at sowing and with 40 kg N ha−1 at 25 days after emergence (DAE) (F); only inoculated with Rhizobium tropici strain SEMIA 4080 at sowing and fertilized with 40 kg N ha−1 at 25 DAE (I+N); and inoculated and fertilized with 20 kg N ha−1 at sowing and with 40 kg N ha−1 at 25 DAE (IF). An additional treatment was done only in the second season and plants were inoculated with no N fertilization at any phase (I−N). Crop growth rate and leaf area index were enhanced by IF and F treatments as compared to I+N treatment, however, net assimilation rate did not differ among the treatments. At the pollination and flowering stages for seasons, LSPC was significantly increased by IF treatment, which also caused the highest final yield compared with the remaining treatments in both seasons. Therefore, the fertilization with 20 kg N ha−1 at sowing together with inoculation and 40 kg N ha−1 at 25 DAE is recommended for obtaining high yield in bean cultivation with high technology. As the inoculation with Rhizobium at sowing and fertilization with 40 kg N ha−1 at 25 DAE (I+N) had the same crop yield as plants fertilized at sowing and at 25 DAE (F), it can be recommended for a low-cost technology cultivation employed by small farmers.

Effects of single and co-inoculation with native phosphate solubilising strain Pantoea sp J49 and the symbiotic nitrogen fixing bacterium Bradyrhizobium sp SEMIA 6144 on peanut (Arachis hypogaea L.) growth

In the present study, attempts were made to analyze the effect of co-inoculation with an efficient phosphate solubilising native isolate Pantoea sp J49 and the symbiotic nitrogen fixing Bradyrhizobium sp SEMIA 6144 strain on Arachis hypogaea L. plants growth. Single and co-inoculation of peanut plants growing in plastic pots containing soil with low P content were developed. Plants were harvested at R1 and R4 growth stages and were analyzed in different growth parameters. Survival of strain Pantoea sp J49 was analyzed in soil samples and in root tissues. Plants inoculated only with Pantoea sp J49 showed the highest shoot and root weight in both reproductive growth stages evaluated. Plants co-inoculated with this strain and Bradyrhizobium sp SEMIA 6144 showed increase in aerial dry weight at R1 stage. Survival assays demonstrated that Pantoea sp J49 survives not only in the peanut rhizosphere but also inside plant tissues, including nodules formed when it was co-inoculated with Bradyrhizobium sp SEMIA 6144. Results obtained in this study confirm the great potential of the native Pantoea sp J49 isolate in the promotion of peanut plant growth, probably related with its capacity to solubilise phosphate.

Seleção a campo de rizóbios nativos para cornichão

O cornichão (Lotus corniculatus L.) tem sido apontado como uma das forrageiras mais promissoras para o uso no Rio Grande do Sul. No entanto, pouco se conhece a respeito da interação dessas plantas com os rizóbios nativos em condições de campo. Este estudo visou a avaliar a eficiência das interações entre rizóbios e plantas de Lotus corniculatus cv. 'São Gabriel' em condições de campo. O experimento foi conduzido na Estação Experimental Agronômica da UFRGS com delineamento de blocos ao acaso com quatro repetições. Os tratamentos estudados foram: dois controles sem inoculação (um com a adição de 80kg ha-1 de nitrogênio na forma de uréia e outro sem a adição de nitrogênio); inoculação com rizóbios: estirpes SEMIA 816, recomendada no Brasil, U510, recomendada no Uruguai, U512, em estudo no Uruguai, e quatro isolados de rizóbios nativos (UFRGS Lc322, UFRGS Lc349, UFRGS Lc269 e Iso 7). O isolado UFRGS Lc322 e a estirpe U510 são mais eficientes do que a estirpe SEMIA 816, recomendada para cornichão no país. Além disso, a produção de forragem das plantas inoculadas com o isolado UFRGS Lc322 e a estirpe U510 superou a obtida com a adição de 80kg ha-1 de nitrogênio.

Resposta do feijão-caupi à inoculação com estirpes de Bradyrhizobium recomendadas para a soja

O uso de inoculantes contendo bactérias fixadoras de N2 aumenta anualmente na cultura do feijão-caupi nas regiões Norte, Nordeste e Centro-Oeste do Brasil. Entretanto, em muitas ocasiões vêm sendo utilizadas estirpes recomendadas para a cultura da soja, mesmo existindo estirpes próprias para o feijão-caupi. Este trabalho foi conduzido com o objetivo de avaliar a resposta das estirpes de Bradyrhizobium recomendadas para a soja na nodulação e no crescimento e desenvolvimento das plantas de feijão-caupi. Conduziu-se um experimento em condições controladas em casa de vegetação com a cultivar BRS Guariba inoculada com as estirpes recomendadas para a cultura da soja: SEMIA 5079 (= CPAC 15), SEMIA 5080 (= CPAC 7), SEMIA 587 e SEMIA 5019 (= BR 29); duas estirpes recomendadas para o feijão-caupi: SEMIA 6462 (= BR 3267) e SEMIA 6464 (= BR 3262); um tratamento com adubação nitrogenada; e um controle absoluto. Os resultados mostraram que a estirpe SEMIA 587 apresentou eficiência na FBN na cultura do feijão-caupi semelhante à das estirpes BR 3262 e BR 3267. Por outro lado, apesar de as estirpes CPAC 15 e CPAC 7 proporcionarem a formação de elevado número de nódulos no feijão-caupi, ocasionaram crescimento de plantas, eficiência nodular e acúmulo de N inferiores aos das estirpes BR 3262 e BR 3267, não devendo ser utilizadas como inoculante para a cultura.

Rizóbios nativos do Rio Grande do Sul simbioticamente eficientes em Lotus glaber

As leguminosas forrageiras, além de contribuírem para o aumento da oferta de forragem com alto teor de proteína, também são importantes como cobertura vegetal de solos. Entre as leguminosas com grande potencial forrageiro, espécies do gênero Lotus têm se destacado. Este trabalho visou à seleção de rizóbios nativos eficientes em Lotus glaber a partir de amostras de solo de cinco localidades do Rio Grande do Sul. Obtiveram-se 259 isolados de rizóbios, que foram avaliados quanto à morfologia colonial e produção de melanina. Destes, 15 isolados foram selecionados para avaliação da eficiência na fixação simbiótica de nitrogênio em experimento em casa de vegetação e caracterizados geneticamente por comparação do perfil eletroforético dos produtos de amplificação do DNA genômico, por PCR com os oligonucleotídeos iniciadores BOX e ERIC. Apenas cinco isolados produziram melanina. Nove isolados foram mais eficientes do que a estirpe SEMIA 830, que é autorizada para a produção de inoculante para L. glaber no país. Na caracterização genética, observou-se que nenhum isolado apresentou identidade com as estirpes recomendadas, o que demonstra que, nos solos do Rio Grande do Sul, existem rizóbios autóctones eficientes na fixação simbiótica de nitrogênio com L. glaber, podendo ser recomendados para estudos a campo, visando a uma futura produção de inoculantes para estas leguminosas no Brasil.

Symbiotic efficiency and genetic characteristics of Bradyrhizobium sp. strain UFSM LA 1.3 isolated from Lupinus albescens (H. et Arn)

Legume species belonging to the genus Lupinus are annual herb plants. The majority of them are indigenous to the Americas. They are known for nitrogen-fixing symbioses with soil bacteria collectively called rhizobia. The aim of this study was to characterize a rhizobium strain isolated from Lupinus albescens using phenotypic, symbiotic and molecular approaches. Strain UFSM LA 1.3 was tested in vitro according to several parameters: colony size, color and growing rate; acid or alkaline reaction in yeast mannitol media supplemented with bromothymol blue; gum production. Molecular characterization was evaluated by PCR technique using primers BOX A1-R and sequence analysis of the 16S-23S rDNA intergenic region (ITS). ITS sequencing fragments showed genetic similarity with Bradyrhizobium sp. The polymorphism observed by BOX-PCR have shown that strain differs from the reference strain SEMIA 928 and SEMIA 938. The symbiotic efficiency under axenic conditions of UFSM LA 1.3 was 94.6%, without statistical differences compared to the mineral nitrogen fertilized control, to which was applied solution of 400 mg of ammonium nitrate.

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.

Efeito do pH do solo rizosférico e não rizosférico de plantas de soja inoculadas com Bradyrhizobium japonicum na absorção de boro, cobre, ferro, manganês e zinco

Alterações no pH da rizosfera de plantas fixadoras de N2 parecem exercer papel fundamental na absorção de micronutrientes que têm sua disponibilidade dependente de alterações da acidez do solo. Estudaram-se variações na absorção de B, Cu, Fe, Mn e Zn durante o ciclo de crescimento e desenvolvimento da soja, induzidas pela fixação biológica de N2 e pelo pH inicial de amostras de dois solos (um LV argiloso e outro arenoso), em um ensaio conduzido em casa de vegetação. Essas amostras foram incubadas com doses de CaCO3 + MgCO3 (4:1) para elevar o pH (H2O) a valores de 5,2, 5,6, 6,2 e 6,6 no solo argiloso e 5,3, 5,6, 5,9 e 6,3 no solo arenoso. Após 60 dias de incubação, essas amostras receberam 450 mg dm-3 de P e 120 mg dm-3 de K no solo. Sementes de soja (Glycine max (L) Merrill), variedade Paranaíba, inoculadas com Bradyrhizobium japonicum, estirpes SEMIA 587 e SEMIA 5019, foram colocadas para germinar. Foram cultivadas quatro plantas por vaso (2,2 dm³) e colhidas aos 16, 20, 24, 28, 32, 36, 40, 46 e 54 dias após a emergência. Determinaram-se o pH da rizosfera (pHr), o pH do solo entre raízes - não rizosférico (pHnr), os teores de B, Cu, Fe, Mn e de Zn na parte aérea e raiz, o N apenas na parte aérea, o número de nódulos e o peso da matéria seca de parte aérea, raiz e nódulos. Observou-se que as mudanças ocorridas no pHr e pHnr foram dependentes do pH inicial dos solos (pHs) e da fixação biológica de N2. O acúmulo de B e de Fe na parte aérea não foi alterado pelos valores de pHr, modificados em função do pHs, exceto para o Fe no solo argiloso. Todavia, aumentos significativos no acúmulo destes nutrientes na parte aérea ocorreram com o aparecimento dos nódulos, a partir de 24 dias após a emergência. Para Cu, Mn e Zn, as diferenças apareceram sobretudo quanto ao pHs. O conteúdo de micronutrientes na planta revelou-se sensível a mudanças no pH rizosférico, principalmente após a nodulação.

Influence of fungicide seed treatment on soybean nodulation and grain yield

Biological N2 fixation is a major factor contributing to the increased competitiveness of Brazilian soybeans on the international market. However, the contribution of this process may be limited by adverse conditions to symbiotic bacteria, such as fungicide seed treatments. This study aimed to evaluate the effects of the fungicides carbendazim + thiram and carboxin + thiram on soybean nodulation, plant growth and grain yield. Two field experiments were carried out in the Cerrado region of the State of Roraima, in a soil with a low organic matter content and no soybean bradyrhizobia. In 2005, seeds were treated with fungicide carbendazim + thiram and commercial inoculants containing the Bradyrhizobium elkanii strains SEMIA 5019 and SEMIA 587 and B. japonicum strains SEMIA 5079 and SEMIA 5080. In 2006, soybean seeds were treated with the fungicides carbendazim + thiram or carboxin + thiram and inoculated separately with each one of the four strains. The plants were evaluated for number of nodules and dry weight, shoot dry weight and total N accumulated in shoots 35 days after plant emergence, while grain yield and N grain content were determined at harvest. Both fungicides reduced soybean nodulation, especially in the presence of B. elkanii strains. The fungicide carbendazim + thiram reduced nodulation by about 50 % and grain yield by more than 20 % (about 700 kg ha-1), in the treatment inoculated with of strain SEMIA 587.

Characterization of variants of Bradyrhizobium elkanii and B. japonicum and symbiotic behaviour in soybeans

Variation in rhizobia strains isn’t a desirable fact based mainly on the possibility of unexpected results on legume inoculation. In this work, we studied the variability on phenotypic characteristics and genetic stability of rhizobia strains recommended for soybean inoculation. Variants with stable colony morphology were obtained from Bradyrhizobium japonicum strain SEMIA 5080 and from B. elkanii SEMIA 5019. Variants from SEMIA 587 obtained by another author were also used. The variants differed on colony characteristics, nodulation capacity, nitrogen fixation efficiency and competitive ability for nodule formation in two soybean varieties (Jacui 7 and IAS 5). Symbiotic behavior varied according to plant variety. Only the variants 5019 G and 5019 P differed on the isoenzymatic profile. There were differences in antibiotic resistance between variants from two strains. Correlation between symbiotic characteristics and colony morphology or antibiotic resistance wasn’t conclusive. The results indicate that the variability in rhizobia strains might be an important factor to be considered in strain selection and preservation of cultures for inoculant production.

Diversity of a soybean rhizobial population adapted to a Cerrados soil

One hundred isolates were trapped by soybean (Glycine max) plants inoculated with a soil from the Cerrados, the main producing area in Brazil. The soil was originally void of rhizobia able to nodulate soybean, and 15 years before received inoculant containing Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019; the area has been annually cropped with soybean since then, but with no further inoculation for the past 7 years. Enormous diversity was observed among the isolates, with thirteen serologically distinct groups, twelve protein and seven lipopolysaccharide profiles; no more than five isolates shared similar characteristics. An unexpected feature was that 48% of the isolates showed multiple reactions with the antisera to the serogroups established in the soils. Also 40% of the isolates reacted with the antiserum to B. japonicum strain SEMIA 566, that has never been introduced into the soil, probably due to dispersion from other cropping areas, associated with its high saprophytic competence; 13% of the isolates did not react with any of the antisera. Nodulation and N2 fixation capacity also varied considerably among the isolates. Although one third of the isolates were fast growers with an acid reaction in vitro, and many formed pseudo-nodules on common bean (Phaseolus vulgaris), they shared several properties with the Bradyrhizobium inoculant strains. A high level of genetic diversity was confirmed when the DNAs were amplified with BOX and RPO1 primers, and several isolates were positioned in far different clusters in the analysis of interspersed repetitive or nif-directed sequences. Moreover, serological properties showed higher correlation with BOX than with RPO1 products. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Cerrados, and may play an important role as a biological buffer, avoiding the dominance of a particular strain.

Classificação taxonômica das estirpes de rizóbio recomendadas para as culturas da soja e do feijoeiro baseada no seqüenciamento do gene 16S rRNA

As culturas da soja [Glycine max (L.) Merrill] e do feijoeiro (Phaseolus vulgaris L.) são de grande importância econômica e social para o Brasil e ambas podem ter seu requerimento de nitrogênio suprido pela simbiose com bactérias da ordem Rhizobiales. Para garantir a maximização do processo biológico, deve-se proceder à inoculação de estirpes de rizóbio eficientes e competitivas, recomendadas pela pesquisa. No Brasil, foram comercializados, na safra 2001/2002, 14 milhões de doses de inoculantes, dos quais 99 % para as culturas da soja e do feijoeiro. Neste trabalho, determinou-se a posição taxonômica das estirpes utilizadas em inoculantes comerciais para as duas culturas, pelo seqüenciamento da região do DNA que codifica o gene 16S rRNA, que é suficientemente variável, mas carrega as informações necessárias para permitir a análise filogenética de bactérias. O seqüenciamento permitiu definir que duas das estirpes recomendadas para a cultura da soja, SEMIA 587 e SEMIA 5019 (= 29 w), pertencem à espécie Bradyrhizobium elkanii e as duas outras, SEMIA 5079 (=CPAC 15) e SEMIA 5080 (= CPAC 7), à espécie B. japonicum. Determinou-se, ainda, que a estirpe SEMIA 4080 (=PRF 81), recomendada para o cultura do feijoeiro, pertence à espécie Rhizobium tropici. As seqüências obtidas foram depositadas no banco mundial de genes do National Center for Biotechnology Information.

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots.

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.

Soybean response to starter nitrogen and Bradyrhizobium inoculation on a Cerrado oxisol under no-tillage and conventional tillage systems

In Brazil, Bradyrhizobium inoculation has successfully replaced the use of N fertilizer on soybean [Glycine max (L) Merr.] crops. However, with the expansion of no-tillage cropping systems in the Cerrados region, the idea that it is necessary to use small N rates at the sowing to overcome problems related with N immobilization has become widespread, mainly when soybean is cultivated after a non-legume crop. In this study we examined soybean response to small rates of N fertilizer under no-tillage (NT) and conventional tillage (CT) systems. Four experiments (a completely randomized block with five replicates) were carried out in a red yellow oxisol, during the periods of 1998/1999 and 1999/ 2000, under NT and CT. The treatments consisted of four urea rates (0, 20, 30 and 40 kg ha-1 N). All treatments were inoculated with Bradyrhizobium japonicum strains SEMIA 5080 and SEMIA 5079, in the proportion 1 kg of peat inoculant (1,5 x 10(9) cells g-1) per 50 kg of seeds. In both experiments, soybean was cultivated after corn and the N fertilizer was band applied at sowing. In all experiments, N rates promoted reductions of up to 50 % in the nodule number at 15 days after the emergence. Regardless of the management system, these reductions disappeared at the flowering stage and there was no effect of N rates on either the number and dry weight of nodules or on soybean yields. Therefore, in the Brazilian Cerrados, when an efficient symbiosis is established, it is not necessary to apply starter N rates on soybean, even when cultivated under notillage systems.

Phenolic acids: Possible agents of modifying N2-fixing symbiosis through rhizobial alteration?

Phenolic acids, low molecular weight phenolics, are precursors of a variety of antimicrobial compounds, root signalling molecules, and phytoalexins that play an important role in plant defence responses. In agro ecosystem, a large amount of litter is turned over during the cropping season, fallow period and land preparation. This releases a flush of phenolic acids, amounts of which exceed very much the quantities released in root exudation. In rhizobial inoculation of legumes, these phenolic acids, depending on the concentration, may affect the persistence of rhizobia in the soil and their symbiotic efficiency, in terms of N2 fixation. The present study evaluates the effects of different concentrations of four phenolic acids (protocatechuic, p-coumaric, ferulic and vanillic) on population size of four rhizobial strains (Bradyrhizobium elkanii SEMIA 5019, B. japonicum TAL 102 and TAL 620, and Azorhizobium caulinodans ORS 571). Culture media with different concentrations of phenolic acids in the presence or absence of manitol were used to evaluate rhizobial population size on day 6. Rhizobial total proteins were extracted and electrophoresed on polyacrylamide gels. Further, the effects of phenolic acid-affected rhizobia on N2 fixing capacity were also investigated by inoculating two of those strains to soybean. Phenolic acid-treated B. elkanii SEMIA 5019 and B. japonicum TAL 102 were inoculated to soybean, and plant growth, N accumulation and nodule dry weight were assessed in a pot experiment. The population size of TAL 102 was induced when the culture medium was supplied with different phenolic acids as the sole carbon source. In many cases, the presence of manitol in the medium masked the differential effects of phenolic acids on the rhizobial population size. All four phenolic acids used in our study suppressed the population size of TAL 620. Strain ORS 571 showed low population size at low concentrations followed by a growth recovery at high phenolic acid concentrations. Strain SEMIA 5019 treated with 0.03 mM ferulic acid produced the highest increase in shoot growth of soybean, (ca. 65%). Treating strain SEMIA 5019 with 9 mM protocatechuic acid produced the largest decrease in nodule dry weight (ca. 50%) without any significant changes in shoot N accumulation. P-coumaric acid, even at 0.12 mM, could stimulate the N2 fixing activity of SEMIA 5019, whereas the same concentration reduced the effectiveness of TAL102 in a soybean-rhizobium symbiosis. Phenolic acid interactions with rhizobia led to biochemical, and hence physiological changes, resulting in an alteration in their symbiotic ability. Different leguminous plants secrete different phenolic compounds other than phenolic acids during root exudation. Further studies should therefore be conducted to evaluate the effects of those compounds on the symbiosis. It is concluded from this study that the effect of phenolic acids is concentration and structure dependant, and strain-specific. The effect will also be pH dependant. Thus, phenolic acids are possible agents for modifying the legume-rhizobial symbiosis.

Acidity and calcium interaction affect the growth of Bradyrhizobium sp. and the attachment to peanut roots

The present work was designed to study the effect of acidity and calcium concentration on the Bradyrhizobium sp. strain SEMIA 6144 (peanut symbiont). The bacteria showed a growth and viability diminution at low pH (5.0) and at a calcium concentration of 0.05 mM. Increasing concentrations of calcium significantly improved the rhizobial growth under acid stress conditions. Molecules related to the plant-bacteria recognition, such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) showed changes at different pH values and calcium concentrations. Variations in the cellular permeability were also observed under stress conditions. Moreover, the attachment of the bacteria to excised peanut roots was examined. Optimal bacteria root attachment to excised peanut roots was dependent on pH and calcium limitation in the medium.