title: Real-time PCR detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.
abstract: Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.
authors.affiliation: Instituto Tecnológico Agrario de Castilla y León Zamadueñas, Valladolid, Spain. marvilte@itacyl.es
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Martín
authors.forename: Rebeca
authors.initials: R
authors.lastname: Cobos
authors.forename: Laura
authors.initials: L
authors.lastname: Martín
authors.forename: Lorena
authors.initials: L
authors.lastname: López-Enríquez
mesh_terms: D001203:Ascomycota / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013172:Spores, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D027843:Vitis / Q000382:microbiology*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal