Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

Möhlenhoff, Petra; Müller, Lars; Gorbushina, Anna A.; Petersen, Karin

doi:10.1111/j.1574-6968.2001.tb10516.x

Cited by 48 publications

(37 citation statements)

References 14 publications

(16 reference statements)

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“…capabilities may grow or survive [52] [53]. These requirements coincide with the physiological characteristics of some microorganisms detected in this study, as mentioned above.…”

Section: Discussionsupporting

confidence: 87%

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

et al. 2013

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In this study, we investigated the microbial community (bacteria and fungi) colonising an oil painting on canvas, which showed visible signs of biodeterioration. A combined strategy, comprising culture-dependent and -independent techniques, was selected. The results derived from the two techniques were disparate. Most of the isolated bacterial strains belonged to related species of the phylum Firmicutes, as Bacillus sp. and Paenisporosarcina sp., whereas the majority of the non-cultivable members of the bacterial community were shown to be related to species of the phylum Proteobacteria, as Stenotrophomonas sp. Fungal communities also showed discrepancies: the isolated fungal strains belonged to different genera of the order Eurotiales, as Penicillium and Eurotium, and the non-cultivable belonged to species of the order Pleosporales and Saccharomycetales. The cultivable microorganisms, which exhibited enzymatic activities related to the deterioration processes, were selected to evaluate their biodeteriorative potential on canvas paintings; namely Arthrobacter sp. as the representative bacterium and Penicillium sp. as the representative fungus. With this aim, a sample taken from the painting studied in this work was examined to determine the stratigraphic sequence of its cross-section. From this information, “mock paintings,” simulating the structure of the original painting, were prepared, inoculated with the selected bacterial and fungal strains, and subsequently examined by micro-Fourier Transform Infrared spectroscopy, in order to determine their potential susceptibility to microbial degradation. The FTIR-spectra revealed that neither Arthrobacter sp. nor Penicillium sp. alone, were able to induce chemical changes on the various materials used to prepare “mock paintings.” Only when inoculated together, could a synergistic effect on the FTIR-spectra be observed, in the form of a variation in band position on the spectrum.

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“…capabilities may grow or survive [52] [53]. These requirements coincide with the physiological characteristics of some microorganisms detected in this study, as mentioned above.…”

Section: Discussionsupporting

confidence: 87%

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

et al. 2013

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“…The forward primer employed was Fung-GC, and the reverse primer was NS1 (Table 1) [23]. A total of 25 μ L of PCR mixture contained 1 × Ex Taq PCR buffer with MgCl 2 , 100 μ M dNTP, 0.5 μ M of each of the primers, 1U Ex Taq DNA polymerase (Takara), and 50 ng DNA template.…”

Section: Methodsmentioning

confidence: 99%

“…Selected DNA fragments were excised from the DGGE gel and submerged overnight at 4°C in 100 μ L TE buffer. A PCR based on a 1 μ L aliquot of the gel fragment extract as template was performed under the same conditions as described above, with the Fung primer replacing Fung-GC as the forward primer (Table 1) [23]. The resulting amplicons were purified using a Biospin Gel Extraction kit (BioFlux, China) and cloned into the pMD19-T vector (Takara) for sequencing.…”

Section: Methodsmentioning

confidence: 99%

The Abundance and Diversity of Soil Fungi in Continuously Monocropped Chrysanthemum

Song

Zhao

Chen

et al. 2013

The Scientific World Journal

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Chrysanthemum is an important ornamental plant which is increasingly being monocropped. Monocropping is known to affect both fungal abundance and species diversity. Here, quantitative PCR allied with DGGE analysis was used to show that fungi were more abundant in the rhizosphere than in the bulk soil and that the fungal populations changed during the growth cycle of the chrysanthemum. The majority of amplified fragments appeared to derive from Fusarium species, and F. oxysporum and F. solani proved to be the major pathogenic species which are built up by monocropping.

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“…In contrast, ITS sequences vary not only regarding the order of the nucleotides but also their length. For this reason, sequences from the 18S region (Gomes et al 2003;Kowalchuk et al 1997;Pennanen et al 2001;Prakitchaiwattana et al 2004;Smit et al 1999;Vainio and Hantula 2000;Van Elsas et al 2000) or from the 26S region (Cocolin et al 2000;Möhlenhoff et al 2001;Prakitchaiwattana et al 2004) are frequently used. We chose the primer pair published by Cocolin et al (2000) to amplify fragments in the variable D1 region.…”

Section: Comparison Of the Methods Usedmentioning

confidence: 98%

“…Some years later, this method has been used for the direct characterization of fungi (Gomes et al 2003;Kowalchuk et al 1997;Möhlenhoff et al 2001;Pennanen et al 2001;SchabereiterGurtner et al 2001;Smit et al 1999;Vainio and Hantula 2000;Van Elsas et al 2000) and yeast populations (Cocolin et al 2000(Cocolin et al , 2002Prakitchaiwattana et al 2004). Recently, several authors have suggested combining cloning and DGGE to overcome the disadvantages of these methods (Gomes et al 2003;Möhlenhoff et al 2001;SchabereiterGurtner et al 2001). Gomes et al (2003) also recommended the combination of both cultivation-independent and cultivation-dependent approaches, to gain better insights in fungal biodiversity.…”

Section: Introductionmentioning

confidence: 94%

Yeast biodiversity in the guts of several pests on maize; comparison of three methods: classical isolation, cloning and DGGE

2008

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The yeast biodiversity in the guts of several pests (Diabrotica virgifera, Helicoverpa armigera, Ostrinia nubilalis) on maize from two isolation sources was assessed by cultivation-dependent and cultivation-independent methods. These yeasts are considered to bear a potentially high biotechnological relevance due to their potential ability to degrade several mycotoxins incorporated by their hosts. The 97 isolated yeast strains showed 21 different partial sequence types of the 26S rRNA gene which could be assigned to 10 different genera. The determined genera and species are discussed in terms of the meaning of their taxonomic status or their occurrence in nature. Two cultivation-independent methods, cloning and DGGE, were compared. We propose the combination of these methods as well as the combination of both cultivation-independent and cultivation-dependent approaches, for gaining better insights into fungal biodiversity.Abbreviations DGGE denaturing gradient gel electrophoresis TGGE temperature gradient gel electrophoresis SSCP single strand conformation polymorphism

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Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

Cited by 48 publications

References 14 publications

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

Contribution of the Microbial Communities Detected on an Oil Painting on Canvas to Its Biodeterioration

The Abundance and Diversity of Soil Fungi in Continuously Monocropped Chrysanthemum

Yeast biodiversity in the guts of several pests on maize; comparison of three methods: classical isolation, cloning and DGGE

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