Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi

Leite, Gonçalo M.; Magan, Naresh; Medina, Ángel

doi:10.1016/j.mimet.2012.01.011

Cited by 65 publications

(57 citation statements)

References 32 publications

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“…Control cultures were performed using the same solvent concentration. Before inoculation, media were covered with sterile cellophane layers (Hutchinson, Chalette-sur-Loing, France) as described by Leite et al (2012) and centrally inoculated with 10 3 spores using a calibrated spore suspension prepared from a seven-day culture.…”

Section: Fungal Strain and Culture Conditionsmentioning

confidence: 99%

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

Caceres

Khoury

Bailly

et al. 2017

Fungal Genetics and Biology

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Aspergillus flavus, a soil-borne pathogen, represents a danger for humans and animals since it produces the carcinogenic mycotoxin Aflatoxin B1 (AFB1). Approaches aiming the reduction of this fungal contaminant mainly involve chemicals that may also be toxic. Therefore, identification and characterization of natural antiaflatoxigenic products represents a sustainable alternative strategy. Piperine, a major component of black and long peppers, has been previously demonstrated as an AFB1-inhibitor; nevertheless its mechanism of action was yet to be elucidated. The aim of the present study was to evaluate piperine's molecular mechanism of action in A. flavus with a special focus on oxidative stress response. For that, the entire AFB1 gene cluster as well as a targeted gene-network coding for fungal stress response factors and cellular receptors were analyzed. In addition to this, fungal enzymatic activities were also characterized. We demonstrated that piperine inhibits aflatoxin production and fungal growth in a dose-dependent manner. Analysis of the gene cluster demonstrated that almost all genes participating in aflatoxin's biosynthetic pathway were down regulated. Exposure to piperine also resulted in decreased transcript levels of the global regulator veA together with an over-expression of genes coding for several basic leucine zipper (bZIP) transcription factors such as atfA, atfB and ap-1 and genes belonging to superoxide dismutase and catalase's families.Furthermore, this gene response was accompanied by a significant enhancement of Version postprintComment citer ce document :

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Section: Fungal Strain and Culture Conditionsmentioning

confidence: 99%

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

Caceres

Khoury

Bailly

et al. 2017

Fungal Genetics and Biology

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show abstract

“…The sensitivity of qPCR is similar to or better than that of other PCR methods; however, it should be underlined that quantification of trace amounts of fungal DNA is often challenging. This is mainly caused by the relatively low fungal load in environmental samples and the structure of the fungal cell wall, which makes its disruption for nucleic acid extraction difficult [Leite et al, 2012]. For example, the detection limit for DNA extracted from M. graminicola cultures was 100 pg/μL using conventional PCR, whereas it was only 50 fg/μL by qPCR; thus qPCR assay was 20-fold more sensitive than conventional PCR [Guo et al, 2006].…”

Section: Future Perspectivesmentioning

confidence: 99%

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat

Kuzdraliński

Kot²,

Szczerba³

et al. 2017

Microb Physiol

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Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient.

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“…Total RNA was extracted from submerged cultures using a modified mini-bead beater method (Leite et al, 2012). Total RNA from the mycelia inside the wafers at different stages of degradation was extracted as described previously (Wei et al, 2010).…”

Section: Rna Preparation and Reverse Transcriptionmentioning

confidence: 99%

Transcriptional profiles of laccase genes in the brown rot fungus Postia placenta MAD-R-698

Wei

Xiao

2015

J Microbiol.

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One of the laccase isoforms in the brown rot fungus Postia placenta is thought to contribute to the production of hydroxyl radicals, which play an important role in lignocellulose degradation. However, the presence of at least two laccase isoforms in this fungus makes it difficult to understand the details of this mechanism. In this study, we systematically investigated the transcriptional patterns of two laccase genes, Pplcc1 and Pplcc2, by quantitative PCR (qPCR) to better understand the mechanism. The qPCR results showed that neither of the two genes was expressed constitutively throughout growth in liquid culture or during the degradation of a woody substrate. Transcription of Pplcc1 was upregulated under nitrogen depletion and in response to a high concentration of copper in liquid culture, and during the initial colonization of intact aspen wafer. However, it was subject to catabolite repression by a high concentration of glucose. Transcription of Pplcc2 was upregulated by stresses caused by ferulic acid, 2, 6-dimethylbenzoic acid, and ethanol, and under osmotic stress in liquid culture. However, the transcription of Pplcc2 was downregulated upon contact with the woody substrate in solid culture. These results indicate that Pplcc1 and Pplcc2 are differentially regulated in liquid and solid cultures. Pplcc1 seems to play the major role in producing hydroxyl radicals and Pplcc2 in the stress response during the degradation of a woody substrate.

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Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi

Cited by 65 publications

References 32 publications

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

Piperine inhibits aflatoxin B1 production in Aspergillus flavus by modulating fungal oxidative stress response

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat

Transcriptional profiles of laccase genes in the brown rot fungus Postia placenta MAD-R-698

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