Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria

Abstract

Identification of Fusarium species has always been difficult due to confusing phenotypic classification systems. We have developed a fluorescent-based polymerase chain reaction assay that allows for rapid and reliable identification of five toxigenic and pathogenic Fusarium species. The species includes Fusarium avenaceum, F. culmorum, F. equiseti, F. oxysporum and F. sambucinum. The method is based on the PCR amplification of species-specific DNA fragments using fluorescent oligonucleotide primers, which were designed based on sequence divergence within the internal transcribed spacer region of nuclear ribosomal DNA. Besides providing an accurate, reliable, and quick diagnosis of these Fusaria, another advantage with this method is that it reduces the potential for exposure to carcinogenic chemicals as it substitutes the use of fluorescent dyes in place of ethidium bromide. Apart from its multidisciplinary importance and usefulness, it also obviates the need for gel electrophoresis.

PCR-based determination of colonization patterns during potato tuber infection by single and multiple pathogens

“…PCR primers based on sequenced internal transcribed space (ITS) regions of rDNA have been proven useful in differentiating and detecting a wide range of fungi in plant tissues including P. infestans (Lee & Taylor, 1992;Tooley et al, 1998), Pythium (Chen, 1992), Verticillium (Nazar et al, 1991), Fusarium (O'Donnell, 1992 and others (Henson and French, 1993;White et al, 1990). In this study, PCR based on specific primers directed against each pathogen (de Boer and Ward, 1995;Kageyama et al, 1997;Liu et al, 1994;Mishra, et al, 2003;Nazar et al, 1991;Robb, 1994;Tooley et al, 1998;White et al, 1990;Zotobowska and Pospieszny, 1998) was successfully used to detect them either alone or in association with P. infestans. Pathogens were detected earlier than visual symptoms, very often a few centimetres ahead of the expanding lesion depending on the pathogen.…”

Genetic analysis and PCR-based identification of major Fusarium species causing head blight on wheat in Japan

“…Recently, molecular technique has been developed to monitor and quantify the Fusarium species causing FHB and producing mycotoxins (Bluhm et al 2002;Edel et al 1997;Jurado et al 2005;Konstantinova and Yli-Mattila 2004;Kulik et al 2004;Láday et al 2004;Mishra et al 2003;Nicholson et al 1998Nicholson et al , 2003Nicholson et al , 2004Parry and Nicholson 1996;Suga et al 2004;Waalwijk et al 2004;Wilson et al 2004;Yli-Mattila et al 2002, 2004aYoder and Christianson 1998). These specific molecular markers were mostly designed from internal transcribed spacer (ITS) (Bluhm et al 2002;Edel et al 1997;Mishra et al 2003;Wilson et al 2004;Yli-Mattila et al 2002, 2004a or intergenic spacer (IGS) regions of rDNA (Jurado et al 2005;Konstantinova and Yli-Mattila 2004;Yli-Mattila et al 2002, 2004a, b-tubulin gene (Yli-Mattila et al 2002, 2004b or mitochondrial DNA (mtDNA) (Láday et al 2004), and are highly specific for identifying Fusarium species.…”

“…These specific molecular markers were mostly designed from internal transcribed spacer (ITS) (Bluhm et al 2002;Edel et al 1997;Mishra et al 2003;Wilson et al 2004;Yli-Mattila et al 2002, 2004a or intergenic spacer (IGS) regions of rDNA (Jurado et al 2005;Konstantinova and Yli-Mattila 2004;Yli-Mattila et al 2002, 2004a, b-tubulin gene (Yli-Mattila et al 2002, 2004b or mitochondrial DNA (mtDNA) (Láday et al 2004), and are highly specific for identifying Fusarium species. However, some of these species-specific markers have a differential sensitivity for isolates of same species of Fusarium from different areas (Yli-Mattila et al 2004b).…”

Morphological and molecular identification of Fusarium species associated with head blight on wheat in East Croatia

“…It has proven to be very useful and more suitable than whole seed plating for the identification of Fusarium species (Demeke et al 2005;Yli-Mattila et al 2004;Rahjoo et al, 2008). Species-specific primers have been developed and used for PCR detection and screening of F. graminearum (Nicholson et al 1998;Waalwijk et al 2003Waalwijk et al , 2004, F. pseudograminearum (Aoki and O'Donnell 1999), F. acuminatum (Williams et al 2002), F. avenaceum (Schilling et al 1996;Waalwijk et al 2003Waalwijk et al , 2004, F. crookwellense (Yoder andChristianson 1998), F. culmorum (Nicholson et al 1998), F. equiseti (Mishra et al 2003) and F. poae (Parry and Nicholson 1996). Some authors state that PCR can be used for the routine detection and identification of Fusarium species without the need for isolation and morphological investigation of this fungus (Koncz et al 2008).…”