RNA fingerprinting of microbial community in the rhizosphere soil of grain legumes

Sharma, Shilpi; Aneja, Manish Kumar; Mayer, Jochen; Schloter, Michael; Munch, Jean Charles

doi:10.1016/j.femsle.2004.09.026

Cited by 36 publications

(17 citation statements)

References 20 publications

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“…A 10-mer primer, 5Ј-TCACGATGCA-3Ј, described by Williams et al (37) was used to generate fingerprints. Amplification reactions were performed as described by Sharma et al (31). Controls to rule out the possibility of DNA contamination in the RNA preparation were performed by using DNase-treated but not reverse-transcribed (DNase ϩ RT Ϫ ) nucleic acids in the assays.…”

Section: Methodsmentioning

confidence: 99%

Influence of Freeze-Thaw Stress on the Structure and Function of Microbial Communities and Denitrifying Populations in Soil

Sharma

Szele

Schilling

et al. 2006

Appl Environ Microbiol

253

143

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Microbial N 2 O release during the course of thawing of soil was investigated in model experiment focusing on denitrification, since freeze-thaw has been shown to cause significant physical and biological changes in soil, including a surge of N 2 O and CO 2 . The origin of these is still controversially discussed. The increase in denitrification after thawing may be attributed to the diffusion of organic substrates newly available to denitrifiers from disrupted soil aggregates, leading to an increase in microbial activity. Laboratory experiments with upper soil layer of a grassland were conducted in microcosms for real-time gas measurements during the entire phase of freeze and thaw. Shifts in microbial communities were evident on resolution of 16S and 18S rRNA genes and transcripts by denaturing gradient gel electrophoresis (DGGE). Microbial expression profiles were compared by RNA-arbitrarily primed PCR technique and subsequent resolution of amplified products on acrylamide gels. Differences in expression levels of periplasmic nitrate reductase gene (napA) and cytochrome cd 1 nitrite reductase (nirS) were observed by most-probable-number-reverse transcription-PCR, with higher levels of expression occurring just after thawing began, followed by a decrease. napA and nirS DGGE profiles showed no change in banding patterns with fingerprints derived from DNA, whereas those derived from cDNA showed a clear succession of denitrifying bacteria, with the most complex pattern being observed at the end of the N 2 O surge. This study provides insight into the structural community changes and expression dynamics of denitrifiers as a result of freeze-thaw stress. Also, the results presented here support the belief that the gas fluxes observed during thawing is a result of freezing initiated high microbial activity.The main process that forms the trace gas nitrous oxide (N 2 O) in agricultural soils is denitrification. It contributes significantly to the greenhouse effect. The concentration of N 2 O in the troposphere has increased from 270 ppb in 1750 to concentration of 316 ppb in the year 2000 and continues to rise (15). Moreover, it is involved in the destruction of stratospheric ozone. Therefore, identifying sources of N 2 O has been a subject of research over the last two decades. Several field studies in the temperate regions have indicated that N 2 O emissions in winter and spring, due to freezing and thawing of agricultural soils, can reach between 20 and 70% of the annual budget (28,35).

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Section: Methodsmentioning

confidence: 99%

Influence of Freeze-Thaw Stress on the Structure and Function of Microbial Communities and Denitrifying Populations in Soil

Sharma

Szele

Schilling

et al. 2006

Appl Environ Microbiol

253

143

View full text Add to dashboard Cite

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“…Absence of genomic DNA was confirmed by quantitative PCR without reverse transcriptase. Successful extraction of total RNA from micro-organisms in soil has been restricted to bacterial species (Dineen et al, 2010;Fang et al, 2014;Griffiths et al, 2000;Gunnigle et al, 2014;Hahn et al, 1990;Lillis et al, 2009;Mikkonen et al, 2014;Novinscak & Filion, 2011;Selenska & Klingmüller, 1992;Sessitsch et al, 2002;Sharma et al, 2004;Wang et al, 2009Wang et al, , 2012. To our knowledge, this represents the first report of the isolation of quality fungal RNA from soil.…”

Section: Stage-specific Genes Trichodermamentioning

confidence: 94%

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

et al. 2015

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Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. 'atroviride type B' morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (L-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. 'atroviride type B' growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.

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“…Hence, a RNA-based community analysis is more suitable for elucidating the metabolically active members of a bacterial population, since the amount of rRNA can generally be correlated with the growth activity of bacteria 114) . To date, several reports have described the results of RNAbased community analyses, and distinct differences have been observed between DNA-and RNA-based analyses 18, 57,90,91,100) . In these studies, the RNA-derived community profiles were found to be less complex than their DNAderived counterparts.…”

Section: Rna-based Community Analysismentioning

confidence: 99%

Microbial Community Analysis of the Phytosphere Using Culture-Independent Methodologies

et al. 2007

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The phytosphere is an attractive habitat for microorganisms due to an abundance of nutrients and relative environmental stability. The microorganisms that occupy this habitat assist in the uptake of nutrients from soils and can exert considerable influence upon the overall health of the plant. Recent technical advances in environmental microbiology have enabled the tracing and assessment of these microorganisms using rapid and simple molecular techniques without any culture-dependent bias. We herein review the current status of these modern molecular techniques in the study of plant-associated microbes, and summarize the issues relevant to the phytosphere from the aspect of both basic and applied science.

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RNA fingerprinting of microbial community in the rhizosphere soil of grain legumes

Cited by 36 publications

References 20 publications

Influence of Freeze-Thaw Stress on the Structure and Function of Microbial Communities and Denitrifying Populations in Soil

Influence of Freeze-Thaw Stress on the Structure and Function of Microbial Communities and Denitrifying Populations in Soil

Identification of growth stage molecular markers in Trichoderma sp. ‘atroviride type B’ and their potential application in monitoring fungal growth and development in soil

Microbial Community Analysis of the Phytosphere Using Culture-Independent Methodologies

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