Camille Eichelberger Granada

Influência da inoculação de rizóbios sobre a germinação e o vigor de plântulas de alface

Os rizobios, conhecidos por sua capacidade de fixar N2 em associacao com leguminosas, tambem se mostram capazes de promover o crescimento de nao-leguminosas, especialmente pela producao de acido indol-acetico (AIA). Neste trabalho, objetivou-se selecionar rizobios produtores de AIA e avaliar o efeito de diferentes concentracoes deste fitormonio sobre a germinacao e o desenvolvimento inicial de plântulas de alface. Foram selecionados quatro isolados de Bradyrhizobium sp. e um isolado de Rhizobium leguminosarum biovar trifolii, os quais foram crescidos por quatro dias em meio levedura-manitol enriquecido com triptofano. Apos esse periodo, avaliou-se a producao de AIA e procedeu-se a inoculacao de sementes de alface com os isolados. O isolado TV-13, de R. leguminosarum biovar trifolii produziu 171,1µg mL-1 de AIA, causando prejuizos para o desenvolvimento das plântulas de alface. Por outro lado, os isolados de Bradyrhizobium sp. produziram entre 1,2 e 3,3µg mL-1 de AIA e aumentaram o vigor das plântulas em relacao ao tratamento sem inoculacao com rizobios. Para verificar se essas diferencas foram decorrentes das concentracoes de AIA, foram realizados mais dois experimentos, nos quais as sementes foram embebidas em culturas de TV-13 com ou sem a presenca de triptofano ou em doses crescentes de AIA sintetico. O isolado TV-13 crescido na presenca de triptofano causou danos progressivos sobre o desenvolvimento das plântulas de alface, o que nao ocorreu na ausencia de triptofano. Tambem foi verificado um retardo na germinacao das sementes quando submetidas a altas concentracoes de AIA sintetico. Os resultados indicam a influencia do AIA sobre os parâmetros de germinacao, de modo que a inoculacao de sementes de alface com rizobios que produzem baixas quantidades de AIA e uma pratica recomendavel.

Occurrence of plant growth-promoting traits in clover-nodulating rhizobia strains isolated from different soils in Rio Grande do Sul state

In the last decades, the use of plant growth-promoting rhizobacteria has become an alternative to improve crop production. Rhizobium leguminosarum biovar trifolii is one of the most promising rhizobacteria and is even used with non-legume plants. This study investigated in vitro the occurrence of plant growth-promoting characteristics in several indigenous R. leguminosarum biovar trifolii isolated from soils in the State of Rio Grande do Sul, Brazil. Isolates were obtained at 11 locations and evaluated for indoleacetic acid and siderophore production and inorganic phosphate solubilization. Ten isolates were also molecularly characterized and tested for antagonism against a phytopathogenic fungus and for plant growth promotion of rice seedlings. Of a total of 252 isolates, 59 produced indoleacetic acid, 20 produced siderophores and 107 solubilized phosphate. Some degree of antagonism against Verticillium sp. was observed in all tested isolates, reducing mycelial growth in culture broth. Isolate AGR-3 stood out for increasing root length of rice seedlings, while isolate ELD-18, besides increasing root length in comparison to the uninoculated control, also increased the germination speed index, shoot length, and seedling dry weight. These results confirm the potential of some strains of R. leguminosarum biovar trifolii as plant growth-promoting rhizobacteria.

Multilocus sequence analysis reveals taxonomic differences among Bradyrhizobium sp. symbionts of Lupinus albescens plants growing in arenized and non-arenized areas.

Lupinus albescens is a leguminous plant that belongs to New World lupine species, which is native to southern Brazil. This Brazilian region is characterized by poor degraded soils with low organic matter and is designated as an arenized area. The symbiosis between Lupinus plants and nitrogen-fixing bacteria belonging to the Bradyrhizobium genus may help the plant establish itself in these areas. To characterize the bradyrhizobial population symbionts of L. albescens plants grown in arenized and non-arenized areas, a multilocus phylogenetic analysis allied to genetic diversity indices were conducted. Seventy-four bradyrhizobial isolates were analyzed, 38 coming from L. albescens plants growing in an arenized area and 36 from a non-arenized area. Isolates were different between arenized and non-arenized areas. Phylogenetic analysis of the 16S rRNA, dnaK, atpD, recA, glnII, rpoB, gyrB, nodA, nodB, and nodZ genes resulted in three supported clades, which were most likely to be three different new Bradyrhizobium species: one species from the arenized area and two from the non-arenized area. Estimates of genetic diversity, which decreased in arenized areas, were positively correlated with habitat variability. These results suggested that a few resistant and efficient Bradyrhizobium sp. strains were capable of forming nodules on L. albescens plants growing in an arenized area. An in vivo inoculation experiment with L. albescens plants showed that Bradyrhizobium ssp. isolated from this extreme environment were more efficient at promoting plant growth than those from the non-arenized area. This result suggested that the environment affected the selection of more efficient plant growth promoters in order to sustain plant growth.

Comparison among bacterial communities present in arenized and adjacent areas subjected to different soil management regimes

AimsThe aims of this work were to characterize the soil bacterial communities in an arenized area in southern Brazil subjected to different management regimes through cultivation-dependent and cultivation-independent methods and to evaluate the potential of selected plant growth-promoting (PGP) bacteria to improve the growth of native Lupinus albescens plants.MethodsBulk soil samples from an arenized site and rhizospheric soil and roots of L. albescens grown in this arenized site as well as samples from soils of the same region outside of the arenized area and rhizospheric soil and roots of L. albescens grown in non-arenized sites were evaluated. Phosphate solubilization, indolic compound and siderophore production abilities of the isolates were screened and compared. Some isolates were selected for in vivo plant growth promotion in greenhouse experiment.ResultsThe samples from the arenized area presented less microbial biomass and less diverse bacterial communities compared with those from non-arenized areas. The PGP characteristics produced by the bacterial isolates showed differences among arenized and non arenized areas. A growth chamber experiment with L. albescens showed that phosphate-insoluble conditions coupled with bacterial inoculation resulted in the best PGP effect.ConclusionsCulture-dependent and culture-independent methods showed converging results regarding diversity indices and the rhizospheric environments increased bacterial diversity and biomass when compared to bulk soils. The PGP traits analyzed in this work were affected by environmental conditions.

Diversidade genética e eficiência simbiótica de rizóbios noduladores de acácia-negra de solos do Rio Grande do Sul

Black wattle is the third most cultivated forest species in Brazil. Besides its economic importance, black wattle is also used to restore degraded areas, where soils are generally acid and Al levels high. This study intended to investigate the genetic diversity of indigenous rhizobia in soils in Rio Grande do Sul and to select isolates that fix N2 efficiently at low pH. Fifty Bradyrhizobium sp. isolates were obtained, which, along with the recommended strains BR 3067 and BR 3068, were molecularly characterized by PCR with primer BOX A 1-R. DNA band patterns of the isolates were used to construct a dendrogram by which the Shannon diversity index was calculated. Ten isolates were further tested for low pH tolerance and Al presence. Eight isolates were selected for a nitrogen-fixing greenhouse trial. High genetic diversity was observed among the isolates. Ten clusters were formed based on a 70 % similarity cut-off level and a diversity index of 4.3. The presence of Al did not affect the tested isolates, while growth decreased at pH 4.5. With respect to the symbiotic efficiency, T6-16 and V-7 were the most effective isolates, similar to the recommended strain BR 3068.

Diversity of native rhizobia isolated in south Brazil and their growth promotion effect on white clover (Trifolium repens) and rice (Oryza sativa) plants

In this study, rhizobia strains isolated from white clover (Trifolium repens) root nodules were evaluated in an effort to identify an efficient nitrogen-fixing rhizobia strain that can also improve the growth of rice plants (Oryza sativa). White clover plants were collected from seven sites in south Brazil, and 78 native rhizobia isolates were obtained. The genetic diversity analysis of those isolates was carried out by BOX-polymerase chain reaction. Overall, the native rhizobia isolated showed a high genetic diversity, but when the bacterial isolates from the same site were compared, the diversity was lower. One native rhizobia, POA3 (isolated from the Porto Alegre locality), was able to promote the growth of both plants and is therefore a good candidate for new inoculant formulation. Finally, we can conclude that the community of native rhizobia symbiont of white clover plants in southern Brazil is highly diverse and the growth promotion effect of rhizobia inoculation on rice plants was more pronounced in a poor nutrient substrate condition than in a rich nutrient substrate condition.

Genome Sequence of the Diazotrophic Gram-Positive Rhizobacterium Paenibacillus riograndensis SBR5 T

Paenibacillus riograndensis SBR5(T), a nitrogen-fixing Gram-positive rhizobacterium isolated from a wheat field in the south of Brazil, has a great potential for agricultural applications due to its plant growth promotion effects. Here we present the draft genome sequence of P. riograndensis SBR5(T). Its 7.37-Mb genome encodes determinants of the diazotrophic lifestyle and plant growth promotion, such as nitrogen fixation, antibiotic resistance, nitrate utilization, and iron uptake.

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants.

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.

Screening of plant growth promoting bacteria associated with barley plants (Hordeum vulgare L.) cultivated in South Brazil

The occurrence of associations between bacteria and plant roots may be beneficial, neutral or detrimental. Plant growth promoting (PGP) bacteria form a heterogeneous group of beneficial microorganisms that can be found in the rhizosphere, the root surfaces or in association with host plant. The aim of this study was to isolate and characterize PGP bacteria associated to barley plants (Hordeum vulgare L.) aiming a future application as agricultural inoculant. One hundred and sixty bacterial strains were isolated from roots or rhizospheric soil of barley based on their growth in nitrogen-free selective media. They were evaluated for their ability to produce indolic compounds (ICs) and siderophores, and to solubilize tricalcium phosphate inin vitro assays. Most of them (74%) were able to synthesize ICs in the presence of the precursor L-tryptophan, while 57% of the isolates produced siderophores in Fe-limited liquid medium, and 17% were able to solubilize tricalcium phosphate. Thirty-two isolates possessing different PGP characteristics were identified by partial sequencing of their 16S rRNA gene. Strains belonging to Cedecea andMicrobacterium genera promoted the growth of barley plants in insoluble phosphate conditions, indicating that these bacteria could be used as bioinoculants contributing to decrease the amount of fertilizers applied in barley crops.

Phylogeny and Phylogeography of Rhizobial Symbionts Nodulating Legumes of the Tribe Genisteae

The legume tribe Genisteae comprises 618, predominantly temperate species, showing an amphi-Atlantic distribution that was caused by several long-distance dispersal events. Seven out of the 16 authenticated rhizobial genera can nodulate particular Genisteae species. Bradyrhizobium predominates among rhizobia nodulating Genisteae legumes. Bradyrhizobium strains that infect Genisteae species belong to both the Bradyrhizobium japonicum and Bradyrhizobium elkanii superclades. In symbiotic gene phylogenies, Genisteae bradyrhizobia are scattered among several distinct clades, comprising strains that originate from phylogenetically distant legumes. This indicates that the capacity for nodulation of Genisteae spp. has evolved independently in various symbiotic gene clades, and that it has not been a long-multi-step process. The exception is Bradyrhizobium Clade II, which unlike other clades comprises strains that are specialized in nodulation of Genisteae, but also Loteae spp. Presumably, Clade II represents an example of long-lasting co-evolution of bradyrhizobial symbionts with their legume hosts.