Quantitative analysis of phytopathogenic ascomycota on leaves of pedunculate oaks (Quercus robur L.) by real-time PCR

• Published in: FEMS microbiology letters 2002-04, Vol.209 (2), p.295-299
• Main Authors: Heuser, T, Zimmer, W
• Format: Article
• Language: English
• Subjects: Ascomycota - genetics; Ascomycota - isolation & purification; autumn; Biological and medical sciences; Cladosporium; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA, Fungal - analysis; DNA, Ribosomal - genetics; Erysiphe alphitoides; Freezing; Fundamental and applied biological sciences. Psychology; Fungal plant pathogens; Fungi; Generalities. Techniques; Hyphae; Leaves; Microbiology; Microorganisms; Microsphaera alphitoides; Mycology; Oak; Pathogenicity, host-agent relations, miscellaneous strains, epidemiology; pathogens; Phytopathology. Animal pests. Plant and forest protection; Plant Diseases - microbiology; Plant Leaves - microbiology; Polymerase Chain Reaction; Powdery mildew; Quantitative analysis; quantitative polymerase chain reaction; Quercus - microbiology; Quercus robur; Ramularia; Real time; Real‐time polymerase chain reaction; ribosomal DNA; Species
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2002.tb11147.x

Leaves of oak trees are often infected by various pathogenic fungi. As it is difficult to remove such organisms quantitatively from the leaf surface and as it is often impossible to grow these organisms independently from their host, there are almost no molecular data available from these oak leaf specific pathogens. For the quantitative removal of the microorganisms a procedure was developed combining a wax and microorganism freezing method with a DNA extraction technique. For the development of a species specific detection, DNA of pathogenic filamentous fungi was isolated from hyphae of the upper leaf surface of Quercus robur. Three different species could be identified as (i) Cladosporium sp., (ii) Ramularia sp. and (iii) Microsphaera alphitoides by amplifying and sequencing an 18S-28S segment of their rDNA. For the final quantification a real-time PCR protocol was established allowing the species specific quantification of the three pathogenic filamentous fungi. The whole procedure was successfully applied to quantify the amount of the three species on oak leaves collected in autumn.