Culture-independent analysis of endophytic bacterial communities associated with Brazilian sugarcane

Magnani, Galileo; Cruz, Leonardo M.; Weber, Heroldo; Bespalhok, João Carlos; Daros, Edelclaiton; Baura, Valter A.; Yates, M. G.; Monteiro, Rose A.; Faoro, Helisson; Pedrosa, Fábio O.; Souza, Emanuel Maltempi de

doi:10.4238/2013.october.15.3

Cited by 32 publications

(22 citation statements)

References 33 publications

(42 reference statements)

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“…Studies of endophytic bacterial communities associated with grass species (Poaceae family) represent a well-established and continuously growing field (Seghers et al, 2004;Sun et al, 2008;Pereira et al, 2011;Sessitsch et al, 2012;Magnani et al, 2013); however, in-depth analyses in grasses such as sorghum are not available. Sorghum (and other grasses) is an important staple food in most developing countries (Taylor, 2004;Babalola and Glick, 2012) and, therefore, it is continuously being studied to increase yield of crop and its resistance to environmental and biological stress (Kapanigowda et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Maropola

Ramond

Trindade

2015

Journal of Microbiological Methods

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Keywords:Metagenomic DNA extraction Endophytic bacteria Sorghum root and stem t-RFLP Pyrosequencing Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB-and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDSbased DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.

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Section: Introductionmentioning

confidence: 99%

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Maropola

Ramond

Trindade

2015

Journal of Microbiological Methods

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“…To assess the diversity and taxonomy of crop plant-associated bacteria, many cultivation-dependent and -independent methods are currently in use (14)(15)(16). However, most of these methods are not quantitative and are based on the evaluation of the 16S rRNA gene coding sequences.…”

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confidence: 99%

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Stets

Alquéres

Souza

et al. 2015

Appl Environ Microbiol

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bAzospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N 2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (ϳ10 7 CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.A zospirillum is one of the most important genera of plant growth-promoting rhizobacteria found worldwide under a variety of environmental and soil conditions (1). The diazotroph Azospirillum brasilense is the best-studied species of the genus, is found in close association with many agriculturally important crops, and exerts beneficial effects on plant growth and productivity (2-4). Nitrogen fixation (5, 6) and the production of the auxin 3-indoleacetic acid (IAA) by many representatives of the genus Azospirillum are related to the growth promotion effects observed in inoculated plants, such as increases in root length and the numbers of root hairs and lateral roots (3).The biotechnological use of A. brasilense inoculants in Latin American and in Brazil, in particular, has increased in recent years (7). Strain FP2 is a spontaneous mutant of A. brasilense Sp7 (8). Strain Sp7 has been shown to be capable of stimulating the growth of several members of the family Poaceae and increasing the productivities of wheat and maize crops (2). Strain FP2 can also promote the growth of wheat (9) and enhance maize and wheat productivity under field conditions (unpublished data). Most of the A. brasilense inoculants in B...

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“…Based on this data we can assume that a site specificity of bacterial species distribution may be influencing sugarcane bacterial diversity independent of the plant genotype. In addition, representatives of other genera, such as Kosakonia, Klebsiella, Stenotrophomonas, and some Gram-positive or Gram-variable isolates were also found as previously reported by others, using culture dependent or independent methods (Magnani et al, 2010;Taulé et al, 2012;Fischer et al, 2012;Magnani et al, 2013). The occurrence of representatives of Gluconacetobacter as the most abundant and present in both roots and shoots of sugarcane varieties cropped in Alagoas could be explained by the use of LGI-P medium with addition of sugarcane juice in which the initial pH is 5.7 during isolation procedure.…”

Section: Resultsmentioning

confidence: 66%

“…This variety was used during other surveys of diazotrophic bacteria counts and isolates identification in Brazil (Magnani et al, 2013) and for inoculation studies using five different diazotrophs known to contribute for plant growth promotion Schultz et al, 2014). It is known to be adapted to soils of low fertility throughout the Brazilian territory.…”

Section: Resultsmentioning

confidence: 99%

“…Recent studies using methods independent of cultivation also demonstrated a great diversity of sugarcane natural population of diazotrophs, indication that the contribution of biological nitrogen fixation (BNF) plays an important role in this plant. It was found that Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Burkholderia tropica, B. silvatlantica, Stenotrophomonas, Pantoea and many others are part of the diazotrophic bacteria diversity found distributed in the roots, stems and leaves of sugarcane (Fischer et al, 2012;Taulé et al, 2012;Magnani et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Diazotrophic bacteria associated to sugarcane varieties cropped at Northeast Region of Brazil

Antonio

Rouws

Teixeira

et al. 2016

Agraria

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The objective of this work was to study the diversity of diazotrophic bacteria associated to 10 varieties of sugarcane cultivated in two states of the Northeast of Brazil and to identify the isolates by morphological and molecular methods. Diversity was studied by the application of traditional growth-depending microbiological methods: Gram-staining, comparison of colony morphology and growth on several culture media. Phylogenetic taxonomy was performed using 16S rRNA gene sequencing data. Almost all isolates obtained in semi-solid nitrogen free media were classified as Gram-negative and seven colony morphological groups were distinguished after growth on Potato medium. Among the 10 varieties evaluated, RB72454 resulted in higher number of isolates and diversity. The great majority of isolates from most of the genotypes tested showed similar characteristic to the genus Gluconacetobacter based on its peculiar colony morphology and growth characteristics in semi-solid media. Comparison of the 16S rRNA sequence of each isolate to the Genbank showed that they are closely related to Gluconacetobacter, Burkholderia, Kosakonia, Klebsiella and Stenotrophomonas.Key words: biological nitrogen fixation, ecology of diazotrophic bacteria, sugarcane Bactérias diazotróficas associadas a variedades de cana-de-açúcar cultivadas na região Nordeste do Brasil RESUMOO objetivo deste trabalho foi estudar a diversidade de bactérias diazotróficas associadas a 10 variedades de cana cultivadas em dois estados do Nordeste do Brasil e identificar os isolados por métodos morfológicos e moleculares. A diversidade foi estudada através da aplicação de métodos microbiológicos tradicionais dependentes de crescimento: coloração de Gram, comparação da morfologia de colónias e crescimento em vários meios de cultura. Taxonomia filogenética foi realizada utilizando dados de sequenciamento de gene 16S rRNA. Quase todos os isolados obtidos em meios semissólidos livres de nitrogênio foram classificados como Gram-negativos e sete grupos baseados na morfologia de colônias foram distinguidos após crescimento em meio batata. Entre as 10 variedades avaliadas, RB72454 resultou em maior diversidade e número de isolados. A grande maioria dos isolados dos genótipos testados apresentaram característica similar ao gênero Gluconacetobacter com base em seu peculiar crescimento em meios semissólidos e morfologia da colônia. A comparação da sequência do 16S rRNA de cada isolado com sequencias do GenBank mostrou que eles estão intimamente relacionados com Gluconacetobacter,

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Culture-independent analysis of endophytic bacterial communities associated with Brazilian sugarcane

Cited by 32 publications

References 33 publications

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Diazotrophic bacteria associated to sugarcane varieties cropped at Northeast Region of Brazil

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