Diversity of rhizobia, symbiotic effectiveness, and potential of inoculation in Acacia mearnsii seedling production.

Black wattle (Acacia mearnsii) is a forest species of significant economic importance in southern Brazil; as a legume, it forms symbiotic associations with rhizobia, fixing atmospheric nitrogen. Nonetheless, little is known about native rhizobia in soils where the species is cultivated. Therefore, this study aimed to evaluate the diversity and symbiotic efficiency of rhizobia nodulating A. mearnsii in commercial planting areas and validate the efficiency of a potential strain in promoting seedling development. To this end, nodules were collected from four A. mearnsii commercial plantations located in Rio Grande do Sul State, southern Brazil. A total of 80 rhizobia isolates were obtained from black wattle nodules, and thirteen clusters were obtained by rep-PCR. Higher genetic diversity was found within the rhizobial populations from the Duas Figueiras (H' = 2.224) and Seival (H' = 2.112) plantations. Twelve isolates were evaluated belonging to the genus Bradyrhizobium, especially to the species Bradyrhizobium guangdongense. The principal component analysis indicated an association between rhizobia diversity and the content of clay, Ca, Mg, and K. Isolates and reference strains (SEMIA 6163 and 6164) induced nodulation and fixed N via symbiosis with black wattle plants after 60 days of germination. The isolates DF2.4, DF2.3, DF3.3, SEMIA 6164, SEMIA 6163, CA4.3, OV3.4, and OV1.4 showed shoot nitrogen accumulation values similar to the N + control treatment. In the second experiment (under nursery conditions), inoculation with the reference strain SEMIA 6164 generally improved the growth of A. mearnsii seedlings, reinforcing its efficiency even under production conditions.

Crop rotation reduces the frequency of anaerobic soil bacteria in Red Latosol of Brazil.

Crop diversity affects the processes of soil physical structuring and most likely provokes changes in the frequencies of soil microbial communities. The study was conducted for soil prokaryotic diversity sequencing 16S rDNA genes from a 25-year no-tillage experiment comprised of two crop systems: crop succession (Triticum aestivum-Glycine max) and rotation (Vicia sativa-Zea mays-Avena sativa-Glycine max-Triticum aestivum-Glycine max). The hypothesis was that a crop system with higher crop diversification (rotation) would affect the frequencies of prokaryotic taxa against a less diverse crop system (succession) altering the major soil functions guided by bacterial diversity. Soils in both crop systems were dominated by Proteobacteria (31%), Acidobacteria (23%), Actinobacteria (10%), and Gemmatimonadetes (7.2%), among other common copiotrophic soil bacteria. Crop systems did not affect the richness and diversity indexes of soil bacteria and soil archaea. However, the crop rotation system reduced only the frequencies of anaerobic metabolism bacteria Chloroacidobacteria, Holophagae, Spirochaetes, Euryarchaeota, and Crenarchaeota. It can be concluded that crop succession, a system that is poorer in root diversity over time, may have conditioned the soil to lower oxygen diffusion and built up ecological niches that suitable for anaerobic bacteria tolerating lower levels of oxygen. On the other hand, it appeared that crop rotation has restructured the soil over the years while enabling copiotrophic aerobic bacteria to dominate the soil ecosystem. The changes prompted by crop succession have implications for efficient soil organic matter decomposition, reduced greenhouse gas emissions, higher root activity, and overall soil productivity, which compromise to agriculture sustainability.

Genomic Metrics Applied to Rhizobiales (Hyphomicrobiales): Species Reclassification, Identification of Unauthentic Genomes and False Type Strains.

Taxonomic decisions within the order Rhizobiales have relied heavily on the interpretations of highly conserved 16S rRNA sequences and DNA-DNA hybridizations (DDH). Currently, bacterial species are defined as including strains that present 95-96% of average nucleotide identity (ANI) and 70% of digital DDH (dDDH). Thus, ANI values from 520 genome sequences of type strains from species of Rhizobiales order were computed. From the resulting 270,400 comparisons, a ≥95% cut-off was used to extract high identity genome clusters through enumerating maximal cliques. Coupling this graph-based approach with dDDH from clusters of interest, it was found that: (i) there are synonymy between Aminobacter lissarensis and Aminobacter carboxidus, Aurantimonas manganoxydans and Aurantimonas coralicida, "Bartonella mastomydis," and Bartonella elizabethae, Chelativorans oligotrophicus, and Chelativorans multitrophicus, Rhizobium azibense, and Rhizobium gallicum, Rhizobium fabae, and Rhizobium pisi, and Rhodoplanes piscinae and Rhodoplanes serenus; (ii) Chelatobacter heintzii is not a synonym of Aminobacter aminovorans; (iii) "Bartonella vinsonii" subsp. arupensis and "B. vinsonii" subsp. berkhoffii represent members of different species; (iv) the genome accessions GCF_003024615.1 ("Mesorhizobium loti LMG 6,125T"), GCF_003024595.1 ("Mesorhizobium plurifarium LMG 11,892T"), GCF_003096615.1 ("Methylobacterium organophilum DSM 760T"), and GCF_000373025.1 ("R. gallicum R-602 spT") are not from the genuine type strains used for the respective species descriptions; and v) "Xanthobacter autotrophicus" Py2 and "Aminobacter aminovorans" KCTC 2,477T represent cases of misuse of the term "type strain". Aminobacter heintzii comb. nov. and the reclassification of Aminobacter ciceronei as A. heintzii is also proposed. To facilitate the downstream analysis of large ANI matrices, we introduce here ProKlust ("Prokaryotic Clusters"), an R package that uses a graph-based approach to obtain, filter, and visualize clusters on identity/similarity matrices, with settable cut-off points and the possibility of multiple matrices entries.

Use of Mineral Weathering Bacteria to Enhance Nutrient Availability in Crops: A Review.

Rock powders are low-cost potential sources of most of the nutrients required by higher plants for growth and development. However, slow dissolution rates of minerals represent an obstacle to the widespread use of rock powders in agriculture. Rhizosphere processes and biological weathering may further enhance mineral dissolution since the interaction between minerals, plants, and bacteria results in the release of macro- and micronutrients into the soil solution. Plants are important agents in this process acting directly in the mineral dissolution or sustaining a wide diversity of weathering microorganisms in the root environment. Meanwhile, root microorganisms promote mineral dissolution by producing complexing ligands (siderophores and organic acids), affecting the pH (via organic or inorganic acid production), or performing redox reactions. Besides that, a wide variety of rhizosphere bacteria and fungi could also promote plant development directly, synergistically contributing to the weathering activity performed by plants. The inoculation of weathering bacteria in soil or plants, especially combined with the use of crushed rocks, can increase soil fertility and improve crop production. This approach is more sustainable than conventional fertilization practices, which may contribute to reducing climate change linked to agricultural activity. Besides, it could decrease the dependency of developing countries on imported fertilizers, thus improving local development.

Bacterial and Archaeal Communities Change With Intensity of Vegetation Coverage in Arenized Soils From the Pampa Biome.

Arenization occurs in regions that present sandy soils with normal rainfall levels. Predatory use of environmental sources, the dissolution of arenitic rocks and reworking of non-consolidated surface sands intensify this degradation scenario. Thus, this work aimed to evaluate the impact of the arenization process in the Brazilian Pampa Biome and how this phenomenon affects the soil microbial and plant communities. For this purpose, three arenized areas in Southern Brazil (Pampa Biome) were selected and, in each one, three sampling points were studied: arenized (ARA), arenized to grassland transition (AGT), and grassland (GRA) areas. In the three sampling points, soils presented low levels of nutrients, organic matter, mud and pH acidic in all regions but, the presence of vegetation coverage in AGT and GRA areas preserved the topsoil structure. Our study related ARA with bacterial families Alcaligenaceae, Pseudomonadaceae, and Xanthomonadaceae. AGT with bacterial families Bacillaceae and Burkholderiaceae, and plant species Melinis repens (Willd.) Zizka and Paspalum stellatum Humb. and Bonpl. ex Flüggé, and GRA with bacterial families Koribacteraceae, Hyphomicrobiaceae, and Chthoniobacteraceae, and plant species Croton subpannosus Müll.Arg. ex Griseb., Piptochaetium montevidense (Spreng.) Parodi and Elyonurus sp. The three studied areas (as well as sampling points) present soils extremely poor in nutrients with sandy texture, and the bacterial and plant composition well known to be resistant to environmental stresses were dominant. The vulnerability of these areas causes a degradation scenario, which is worsened by agricultural activities. However, in general, this phenomenon is a natural process that occurs mainly due to soil characteristics (poor soils) and climatic variations.

Pindo Palm fruit yield and its relationship with edaphic factors in natural populations in Rio Grande do Sul / Produção de frutos de butiazeiro e sua relação com fatores edáficos em populações naturais do Rio Grande do Sul

ABSTRACT: The objective of this study was to evaluate the fruit yield of naturally occurring populations of Pindo palms ( Butia odorata ) in different habitats and agricultural ecosystems. Three areas were selected in the municipalities of Barra do Ribeiro, Tapes and Arambaré, all of which are located on the Litoral Médio in the state of Rio Grande do Sul, Brazil. Eleven 25x100m² plots were selected in the aforementioned areas, and fruit yield was evaluated in the 2008 and 2009 seasons. Chemical conditions of the soil in each plot, elevation of terrain, height, and density of the reproductive Pindo palms were also assessed. Data analysis revealed a strong correlation between the edaphic conditions and the yield from the Pindo palms. Tapes Pindo palms exhibited higher fruit yield as a result of an increase in the number of bunches produced per plant, along with highly fertile soils and greater availability of calcium and magnesium. The lowest fruit yields were linked to nutrient-poor soils in Brachiaria pastures, as well as sandy plains containing high levels of sodium. Changes in the characteristics of the soils associated with habitat and management influenced the fruit yield of Pindo palms.

RESUMO: O objetivo deste trabalho foi avaliar a produtividade de butiazeiros ( Butia odorata ) em áreas de ocorrência natural sob a influência de diferentes habitats e agroecossistemas. Três áreas correspondentes aos Municípios de Barra do Ribeiro, Tapes e Arambaré, situados no Litoral Médio do Estado do Rio Grande do Sul foram selecionadas. Nessas áreas, foram distribuídas 11 parcelas de 25x100m² e avaliada a produção nas frutificações de 2008 e 2009. Em cada parcela, também foram avaliadas as condições químicas do solo, a elevação do terreno, a altura e a densidade de butiazeiros reprodutivos. As análises dos dados revelaram uma forte relação entre as condições edáficas e a produtividade dos butiazeiros. O butiazal de Tapes apresentou maior produção de frutos, resultante do aumento de cachos produzidos por planta, estando associada aos solos mais férteis e com maior disponibilidade de cálcio e magnésio. A menor produção de frutos foi relacionada com solos pobres em nutrientes, cobertos com pastagens de braquiária, assim como em planícies arenosas, com elevados teores de sódio. Mudanças nas características do solo, associadas com o habitat e o manejo, influenciam na produção de frutos de butiazeiro.

Characterization of plant growth-promoting bacteria inhabiting Vriesea gigantea Gaud. and Tillandsia aeranthos (Loiseleur) L.B. Smith (Bromeliaceae) / Caracterização de bactérias promotoras de crescimento vegetal em Vriesea gigantea Gaud. e Tillandsia aeranthos (Loiseleur) L.B. Smith (Bromeliaceae)

Microorganisms that live inside and around a plant can supply it with essential substances, such as phytohormones and essential nutrients. The present investigation aimed to isolate and characterize the phyllosphere, the endophytic, and the water tank bacteria associated with Vriesea gigantea and Tillandsia aeranthos. The bacteria were tested for siderophore and indole-3-acetic acid (IAA) production, phosphate solubilization, and presence of the nif H gene. Genetic diversity of the bacterial isolates was evaluated by rep-PCR. Sixty-eight bacterial strains were isolated from 3 different microhabitats of V. gigantea and from 2 microhabitats of T. aeranthos bromeliad plants. Gram-positive, spore-forming bacilli comprised most bacterial isolates. All isolates produced IAA in vitro in presence of very low amounts of tryptophan. More than 70% of the evaluated bacteria presented the ability of siderophore production and phosphate solubilization, and possessed the nif H gene. It was not possible to distinguish well-defined groups of isolates based on the bromeliad species and microhabitat they inhabit using genetic characterization by rep-PCR. Water tanks presented the most abundant diversity compared with phyllosphere and endophytes, probably due to the high nutrient concentration, which promotes an ideal environment for complex microbial communities.

Microrganismos que habitam o interior e a superfície podem fornecer substancias essenciais ao crescimento das plantas, como fitormônios e nutrientes essenciais. O presente trabalho teve como objetivo isolar e caracterizar as bactérias da filosfera, do ambiente endofítico e a água de tanque associadas à Vriesea gigantea e Tillandsia aeranthos. As bactérias foram submetidas a testes de verificação de produção de sideróforos e de ácido indol acético (AIA), solubilização de fosfatos, e a presença do gene nif H. A diversidade genética dos isolados bacterianos foi analisada por rep-PCR. Sessenta e oito microrganismos foram isolados de 3 microambientes distintos de V. gigantea e de 2 microambientes de T. aeranthos. A maioria das bactérias isoladas foram bacilos formadores de esporos, gram-positivos. Todos os isolados produziram AIA in vitro na presença de quantidades pequenas de triptofano. Mais de 70% das bactérias analisadas produziram sideróforos, solubilizaram fosfatos e possuíam o gene nif H. Não foi possível distinguir grupos definidos de microrganismos baseados no microhabitat e na espécie de bromélia de onde foram isolados usando rep-PCR. A água do tanque apresentou maior diversidade microbiana quando comparada com a filosfera e o ambiente endofítico, provavelmente devido à alta concentração de nutrientes, que promove um ambiente favorável para o desenvolvimento de comunidades microbianas complexas.

Caracterização da região espaçadora 16-23S rDNA para diferenciação de estirpes de rizóbios utilizadas na produção de inoculantes comerciais no Brasil / Characterization of the spacer region 16-23S rDNA for differentiation of strains of rhizobia used in the production of commercial inoculants in Brazil

A identificação de estirpes de rizóbio tem sido feita pela especificidade por hospedeiros e ensaios microbiológicos tradicionais. Por constituírem um grupo filogeneticamente heterogêneo, diferentes técnicas moleculares têm sido empregadas para auxiliar na caracterização genética e na identificação de estirpes eficientes e competitivas para a produção de inoculantes. Este trabalho teve por objetivos caracterizar a região espaçadora 16S-23S rDNA das estirpes de rizóbios utilizadas nos inoculantes comercializados no Brasil para espécies leguminosas, utilizando a técnica da PCR em combinação com a de RFLP, e avaliar a possibilidade do uso desse marcador molecular como método auxiliar para identificação das estipes. A amplificação da região espaçadora 16-23 S rDNA das estirpes de rizóbios gerou fragmentos com tamanhos que variaram entre 700pb e 1350pb. Os produtos resultantes da amplificação foram submetidos à digestão com as endonucleases. Mps I, Dde I e Hae III. Os resultados obtidos neste estudo indicam a possibilidade do uso da técnica de PCR-RFLP da região espaçadora 16S-23S rDNA como marcador molecular para a diferenciar as estirpes de rizóbios, em complemento às técnicas microbiológicas tradicionais. Contudo, este marcador não é suficientemente discriminatório para ser usado na identificação das estirpes recomendadas para a produção de inoculantes comerciais.

The identification of strains of rhizobia has been made by host specificity and regular microbiological tests. By forming a phylogenetically heterogeneous group, different molecular techniques have been employed to assist in the genetic characterization and identification of efficient and competitive strains for production of inoculants. This study aimed to characterize the spacer region 16S-23S rDNA of the strains of rhizobia used in commercial inoculants in Brazil for legume species, using PCR combined with RFLP, and assess the possibility of using this molecular marker as an auxiliary method for identification of strains. The amplification of the 16-23 S rDNA spacer region of rhizobium strains generated fragments with sizes ranging between 700 and 1350bp. Products from the amplification were subjected to digestion with Mps I, Dde I and Hae III endonucleases. The results indicated the possibility of using the technique of PCR-RFLP of 16S-23S spacer region rDNA as molecular marker to differentiate most strains tested and recommended for production of inoculants, in addition to the traditional microbiological techniques. However, this marker is not sufficiently discriminatory to be used in the identification of the strains recommended for the production of commercial inoculants.

Influência da inoculação de rizóbios sobre a germinação e o vigor de plântulas de alface / Influence of rhizobial inoculation on seedling vigor and germination of lettuce

Os rizóbios, conhecidos por sua capacidade de fixar N2 em associação com leguminosas, também se mostram capazes de promover o crescimento de não-leguminosas, especialmente pela produção de ácido indol-acético (AIA). Neste trabalho, objetivou-se selecionar rizóbios produtores de AIA e avaliar o efeito de diferentes concentrações deste fitormônio sobre a germinação 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. Após esse período, avaliou-se a produção de AIA e procedeu-se à inoculação de sementes de alface com os isolados. O isolado TV-13, de R. leguminosarum biovar trifolii produziu 171,1µg mL-1 de AIA, causando prejuízos 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 relação ao tratamento sem inoculação com rizóbios. Para verificar se essas diferenças foram decorrentes das concentrações de AIA, foram realizados mais dois experimentos, nos quais as sementes foram embebidas em culturas de TV-13 com ou sem a presença de triptofano ou em doses crescentes de AIA sintético. O isolado TV-13 crescido na presença de triptofano causou danos progressivos sobre o desenvolvimento das plântulas de alface, o que não ocorreu na ausência de triptofano. Também foi verificado um retardo na germinação das sementes quando submetidas a altas concentrações de AIA sintético. Os resultados indicam a influência do AIA sobre os parâmetros de germinação, de modo que a inoculação de sementes de alface com rizóbios que produzem baixas quantidades de AIA é uma prática recomendável.

Rhizobia are known by their ability to fix nitrogen in symbiosis with legumes, but they are also capable of promote the growth of non-legume, mainly due to indoleacetic acid production (IAA). In this research, it was aimed to select rhizobia producers of IAA and evaluate the effect of different levels of this hormone over the germination and initial development of lettuce seedlings. Four isolates of Bradyrhizobiumsp. and one isolate of Rhizobium leguminosarum biovar trifolii were grown during four days in yeast manitol medium enriched with tryptophan. After that period, the production of IAA was evaluated and the isolates were inoculated in lettuce seeds. The isolate TV-13, of R. leguminosarum biovar trifolii, produced 171.1µg mL-1 of IAA, causing damages to lettuce seedlings. On other hand, Bradyrhizobium sp. isolates produced between 1.2 and 3.3µg mL-1 of IAA and improved seedlings vigor. In order to verify if these results were due to IAA concentrations, other two assays were carried out, in which lettuce seeds were imbibed in TV-13 cultures with or without tryptophan or in increasing dosages of synthetic IAA. The isolate TV-13 grown in the presence of tryptophan caused progressive damages to lettuce seedlings development, fact that did not occur in the absence of tryptophan. It was also verified a delay in germination of seeds exposed to high levels of synthetic IAA. The results show the influence of IAA on germination parameters, so that the inoculation of lettuce seeds with rhizobia that produce low amounts of IAA is a recommended practice.

Eficiência de Trichoderma harzianum e Gliocladium viride na redução da incidência de Botrytis cinerea em tomateiro cultivado sob ambiente protegido / Efficiency of Trichoderma harzianum and Gliocladium viride in decreasing the incidence of Botrytis cinerea in tomato cultivated in protected environment

A produção de tomates no Estado do RS ocupa um importante papel sócio-econômico, que pode ser constatado pelo crescimento do cultivo dessa hortaliça em ambiente protegido. Essa técnica permite a produção de tomates em período de entressafra; no entanto, ocorrem também condições favoráveis para o desenvolvimento de doenças fúngicas como o mofo cinzento provocado por Botrytis cinerea. O surgimento de raças de patógenos resistentes a fungicidas químicos vem fazendo com que o controle biológico torne-se uma alternativa necessária. Neste trabalho foi realizada seleção in vitro de 24 isolados do fungo Trichoderma harzianum e 12 de Gliocladium viride que inibiram o desenvolvimento do patógeno B. cinerea. Foram selecionados dois isolados (TRIC-30 e GLIO-10) para serem testados em experimentos em condições de campo com tomates cultivados sob ambiente protegido, nos quais a pulverização foliar semanal com uma suspensão com 2x10(7) conídios mL-1 reduziu significativamente a incidência do mofo cinzento, enquanto a aplicação dos antagonistas nas sementes, no substrato e na cova, no momento do plantio, não reduziu a incidência do patógeno.

The production of tomato in the State of Rio Grande do Sul performs an important economical and social role that can be evidenced by the increase in cultivation of this vegetable in protected environment. This practice allows the production of tomato during the off-season periods. However, it can also promote favorable conditions to the development of gray mold caused by Botrytis cinerea, and the arising of pathogen races resistant to fungicides is turning biological control into a necessary alternative. In the present work, an in vitro selection among 24 isolates of the fungus Trichoderma harzianum and 12 of Gliocladium viride that inhibited the development of the pathogen B. cinerea was carried out. Two isolates (TRIC-30 e GLIO-10) were selected to be tested in an experiment in field conditions with tomatoes cultivated under protected environment. In this experiment, the weekly foliar pulverization of a suspension with 2x10(7) conidia mL-1 significantly decreased the incidence of gray mold, while the application of the antagonists on the seeds, substrate and seed hole during seeding did not reduced the incidence of the pathogen.

Imobilização de nitrogênio em solo cultivado com milho em sucessão à aveia preta nos sistemas plantio direto e convencional / Nitrogen immobilization in soil cropped with maize in sequence to black oats in conventional and in no-till systems

O presente trabalho teve como objetivos avaliar a quantidade de nitrogênio imobilizado e a sua remineralização, nos sistemas plantio direto (SPD) e convencional (SC), ao longo do ciclo do milho. Para tal, foram coletadas amostras da camada de 0-5cm de solo semeado com milho em sucessão à aveia preta, sob os sistemas convencional e plantio direto, no dia da semeadura do milho e 46, 62, 88 e 112 dias após. Amostras da parte aérea das plantas de milho foram coletadas nas mesmas épocas, com exceção da primeira, e avaliadas quanto à quantidade de nitrogênio acumulado. As amostras de solo foram avaliadas quanto à quantidade de nitrogênio mineral no solo, de nitrogênio e carbono potencialmente mineralizáveis, atividade de urease e de nitrogênio imobilizado na biomassa microbiana. A imobilização microbiana do nitrogênio foi maior no sistema plantio direto, levando a uma menor quantidade de nitrogênio mineral no solo e resultando em menor acúmulo de nitrogênio na parte aérea do milho ao final do seu ciclo neste sistema, em comparação com o convencional. Não foi observada remineralização do nitrogênio imobilizado, indicando que a biomassa microbiana atuou mais como agente da mineralização de nitrogênio orgânico do que como fonte de nitrogênio potencialmente mineralizável.