A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus

Yergeau, Étienne; Filion, Martin; Vujanovic, Vladimir; St‐Arnaud, Marc

doi:10.1016/j.mimet.2004.09.006

Cited by 76 publications

(68 citation statements)

References 35 publications

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“…Fusarium spp. are common soil fungi, which can have important roles not only as plant pathogens, but also as saprotrophic competitors of other pathogenic fungi (Duffy et al, 2004;Yergeau et al, 2005). In this study, the density of Fusarium spp., as judged by quantitative real-time PCR targeting the 18S-rRNA gene, was significantly reduced in the rhizosphere of the mycorrhizal plant (F. rubra) exposed to elevated atmospheric CO 2 concentration.…”

Section: Effects Of Elevated Co 2 On Fungal Communitiesmentioning

confidence: 87%

Specific rhizosphere bacterial and fungal groups respond differently to elevated atmospheric CO2

2009

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Soil community responses to increased atmospheric CO 2 concentrations are expected to occur mostly through interactions with changing vegetation patterns and plant physiology. To gain insight into the effects of elevated atmospheric CO 2 on the composition and functioning of microbial communities in the rhizosphere, Carex arenaria (a non-mycorrhizal plant species) and Festuca rubra (a mycorrhizal plant species) were grown under defined atmospheric conditions with either ambient (350 p.p.m.) or elevated (700 p.p.m.) CO 2 concentrations. PCR-DGGE (PCR-denaturing gradient gel electrophoresis) and quantitative-PCR were carried out to analyze, respectively, the structure and abundance of the communities of actinomycetes, Fusarium spp., Trichoderma spp., Pseudomonas spp., Burkholderia spp. and Bacillus spp. Responses of specific functional groups, such as phloroglucinol, phenazine and pyrrolnitrin producers, were also examined by quantitative-PCR, and HPLC (high performance liquid chromatography) was employed to assess changes in exuded sugars in the rhizosphere. Multivariate analysis of group-specific community profiles showed disparate responses to elevated CO 2 for the different bacterial and fungal groups examined, and these responses were dependent on plant type and soil nutrient availability. Within the bacterial community, the genera Burkholderia and Pseudomonas, typically known as successful rhizosphere colonizers, were significantly influenced by elevated CO 2 , whereas the genus Bacillus and actinomycetes, typically more dominant in bulk soil, were not. Total sugar concentrations in the rhizosphere also increased in both plants in response to elevated CO 2 . The abundances of phloroglucinol-, phenazine-and pyrrolnitrin-producing bacterial communities were also influenced by elevated CO 2 , as was the abundance of the fungal genera Fusarium and Trichoderma.

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Section: Effects Of Elevated Co 2 On Fungal Communitiesmentioning

confidence: 87%

Specific rhizosphere bacterial and fungal groups respond differently to elevated atmospheric CO2

2009

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“…These three species belong to the Gibberella fujikuroi complex, and they are phylogenetically very close, making their differentiation very difficult based on the ITS sequence (26). In this case, we have been able to reach species level based on the ITS sequence with the aid of classical microbiology to ensure the result, but other authors have previously suggested sequencing other loci such as elongation factor alpha to reach species level (40).…”

mentioning

confidence: 91%

Endophthalmitis Caused by Fusarium proliferatum

Ferrer¹,

Alió

Rodrı́guez³

et al. 2005

J Clin Microbiol

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Fusarium proliferatum caused endophthalmitis after cataract surgery. Diagnosis was established by classical microbiology and molecular biology methods (PCR and DNA typing). The treatment with local amphotericin B, oral ketoconazole, and topical natamycin was successful. CASE REPORTA 66-year-old man underwent cataract extraction and intraocular lens implantation in his right eye. Four months after surgery, he complained of eye discomfort and was prescribed a topical steroid (dexamethasone) and tobramicin. Fifteen days later, his symptoms persisted, and the eye began to show severe palpebral edema. Slit lamp examination showed conjunctival hyperemia, hypopyon, capsular fibrosis, and corneal edema, and the intraocular lens had moved to the nasal site. Intraocular pressure was 28 mm Hg, and fundus examination showed vitreous haze. An aqueous humor sample was taken using a 30-gauge needle following examination of the eye. Diagnostic techniques included standard microbiological tests (culture and stains) and PCR. The aqueous humor sample was cultured on several media including Columbia agar plates supplemented with 5% sheep blood chocolate agar, MacConkey agar, and thioglycolate broth and brain heart infusion broth (all media were from Biomérieux, biomérieux Sa, Marcy L'Etoile, France) at 37°C in ambient air. The sample was also inoculated into Sabouraud dextrose agar with chloramphenicol and incubated at 30°C. Two different PCRs were carried out: the first focused on bacterial 16S rRNA gene (16) amplification, and the second focused on specific detection of the fungal internal transcribed spacer (ITS)/5.8S DNA region (5). All the tests on the aqueous humor sample (stains, cultures, and PCRs) were negative. Next, a vitreous sample was taken, and microbiological and molecular tests were repeated. Direct smear of the vitreous sample showed septate hyphae of a filamentous fungus with neutrophils ( Fig. 1). PCR with specific fungal primers was positive, and PCR with bacterial primers was negative. This preliminary result was obtained 6 h after the sample was taken. Antifungal treatment was given as soon as the fungal hyphae were seen in the vitreous sample by direct smear.Intravitreal amphotericin B (5 g/0.1 ml) and oral fluconazole (200 mg/day) were administered the same day the sample was taken.Amplified DNA from fungal PCR was submitted for sequence analysis (PE Applied Biosystems, Foster City, Calif.) and compared to DNA sequences in the BLAST alignment program of the GenBank database (National Institutes of Health) and the EMBL fungal DNA database using Fasta3 sequence similarity searches, which allowed the species identification 48 h after the sample was obtained. DNA database comparison of the sequence showed 100% identity with Fusarium proliferatum (NRRL 31071; USDA Agricultural Research Service Collection, Peoria, Ill.), 99.8% identity with Fusarium fujikuroi, and 99.8 to 99.6% identity with Fusarium annulatum, all of which belong to the Gibberella fujikuroi complex (10, 26).When the molecular identification show...

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“…Between the extreme values, the concentration of denaturing agents creates an increasing gradient. Throughout the run electrophoresis is supplied a constant voltage, typically about 60V for 16 h [52]. In some cases, it can be applied a higher voltage of 130-150 V for 3-6 h, while the bands are then more blurred [53,54].…”

Section: The Denaturing Gradient Gel Electrophoresis Methods To Studymentioning

confidence: 99%

The Molecular‐Based Methods Used for Studying Bacterial Diversity in Soils Contaminated with PAHs (The Review)

Gałązka¹,

Grządziel²

2016

Soil Contamination - Current Consequences and Further Solutions

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Soil contamination could adversely affect microbial diversity, and perhaps also aboveand below-ground ecosystem functioning. It is important to study microbial diversity not only for basic scientific research, but also to understand the link between diversity and community structure and function in the pollution site. The study of microbial diversity and their function in contaminated soil creates a serious problem because they observed significant limitations in methodology and taxonomy of this group. Methodology for the determination of bacterial diversity does not include their function in the soil and other environment areas. Microbes are known for their catabolic activity in bioremediation, but changes in microbial communities are still unpredictable. The bioremediation of a pollutant and its rate depend on the environmental conditions, number and type of the microorganisms, nature and chemical structure of the chemical compound being degraded. However, molecular methods have been used to study soil bacterial communities. While many anthropogenic activities, such as city development, agriculture, and use of pollution, can potentially affect soil microbial diversity, it is unknown how changes in microbial diversity can influence below-ground and aboveground ecosystems. There are problems associated with studying bacterial diversity in soil. These arise not only from methodological limitations, but also from a lack of taxonomic knowledge. Methods to measure microbial diversity in soil can be categorized into two groups: biochemical-based techniques and molecular-based techniques. But more common for studying microbial diversity in soil contaminated with polycyclic aromatic hydrocarbons are the molecular methods.

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A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus

Cited by 76 publications

References 35 publications

Specific rhizosphere bacterial and fungal groups respond differently to elevated atmospheric CO2

Specific rhizosphere bacterial and fungal groups respond differently to elevated atmospheric CO2

Endophthalmitis Caused by Fusarium proliferatum

The Molecular‐Based Methods Used for Studying Bacterial Diversity in Soils Contaminated with PAHs (The Review)

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