Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA).
Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-α (EF-1α) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated wheat or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals.
Department of Plant Pathology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Mahdi
M
Davari
Anne D
AD
van Diepeningen
Assadollah
A
Babai-Ahari
Mahdi
M
Arzanlou
Mohammed Javad
MJ
Najafzadeh
Theo A J
TA
van der Lee
G Sybren
GS
de Hoog
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004783:Environmental Microbiology; D005456:Fluorescent Dyes / Q000378:metabolism; D005670:Fusarium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D006109:Poaceae / Q000382:microbiology; D011110:Polymorphism, Genetic; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling / Q000379:methods; D014908:Triticum / Q000382:microbiology
D004271:DNA, Fungal; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D020648:Peptide Elongation Factor 1