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Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
2005 (English)In: BMC Microbiology, ISSN 1471-2180, Vol. 5, 65- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. RESULTS: We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. CONCLUSION: The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.

Place, publisher, year, edition, pages
2005. Vol. 5, 65- p.
URN: urn:nbn:se:umu:diva-4872DOI: 10.1186/1471-2180-5-65PubMedID: 16280082OAI: diva2:144137
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2009-11-25Bibliographically approved
In thesis
1. Development of molecular techniques for fungal diagnostic research
Open this publication in new window or tab >>Development of molecular techniques for fungal diagnostic research
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fungi are present everywhere in indoor and outdoor environments. Many fungi are toxigenic or pathogenic that may cause various public health concerns. Rapid detection, quantification and characterization of fungi in living and working environments are essential for exposure risk assessment to safe guard public health.

Rapid and accurate detection and identification of fungi using molecular method require specific markers. In this thesis, partial mt SSU and LSU rDNA were amplified and sequenced from 31 fungal species of 16 genera. Sequence alignments showed that fungal mt SSU and LSU rDNA contained sufficient amount of variation for the development of markers that can discriminate even among closely related species. Forty-eight probes were designed and were verified as highly specific to 25 fungal species commonly detected in living and working environments. These specific probes would have potential applications in clinical diagnosis and public health-related environmental monitoring.

Nested PCR is a highly sensitive and specific method. Based on the nuclear 18S rDNA sequence variation pattern, three nested PCR systems were developed to detect the conifer tree pathogen Gremmeniella abietina, an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The three nested PCR systems showed high specificity and sensitivity. These methods could have broad applications in forest protection and disease management programs.

Quantitative real-time PCR offers the ability of simultaneous detection and quantification of DNA of a specific microbe in one reaction. Based on the 18S rDNA sequence, two real-time PCR assays were developed to detect and quantify Wallemia sebi, a deuteromycete fungus commonly found in agricultural environments and is suspected to be a causative agent of farmer’s lung disease. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. Application of the real-time PCR methods in the quantification of W. sebi in the aerosols of a farm revealed a high concentration of W. sebi spores (107/m3). The study indicates that W. sebi is a dominant fungus in agriculture environments.

Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. A TaqMan probe and a SYBR Green I based real-time PCR assay were developed to detect and quantify Cladosporium in aerosols. The two real-time PCR systems proved to be highly specific and sensitive for Cladosporium. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. High spore concentration of Cladosporium (107/m3) was observed in a cow barn. Cladosporium spore concentration in paper and pulp factory and countryside house also exceeded threshold value for clinical significance. Prolonged exposure in these environments could impose certain health risk. Thus, monitoring Cladosporium spore concentration in indoor environments is important for indoor air quality control.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2005. 75 p.
Molecular biology, Fungi, DNA markers, Aerosols, Detection and quantification, Environmental monitoring, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-656 (URN)91-7305-994-3 (ISBN)
Public defence
2006-01-18, Stora föreläsningssalen, Arbetslivsinstitutet, Petrus Laestadius väg, Umeå, 09:00 (English)
Available from: 2005-12-12 Created: 2005-12-12 Last updated: 2009-11-24Bibliographically approved

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