Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

Complutense University of Madrid, Madrid, Madrid, Spain
International Journal of Food Microbiology (Impact Factor: 3.08). 03/2006; 106(3):297-306. DOI: 10.1016/j.ijfoodmicro.2005.09.005
Source: PubMed


In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.

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Available from: María Teresa González-Jaén
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    • "However, most studies dealt with biocontrol agents, thus fungal inhibition was seeked for. The kind of interactions between two strains of the same species could in part be attributed to the type of secondary metabolites a strain produces and whether such metabolites play any role in the infection process (Llorens et al., 2006). In our case, no longer lag times or reduced growth rates were observed in the mixed inoculum, thus fungal inhibition due to competition was unlikely to occur, and there is no base in our experimental design to support an intraspecies stimulation hypothesis . "
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    • "Different from RAPD method, IGS markers are generated from a single genomic region and molecular changes within the IGS can be followed by varying restriction patterns can be grouped as haplotypes. Even small numbers of isolates can be differentiated by using the IGS variation as shown in Spain originated F. culmorum and F. graminearum isolates [15]. "
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    • "Subtle differences in a single characteristic may delineate species. However, the morphological and physiological characterization of the species is generally time-consuming and only the expert mycologists are able to ensure the correct identification (5). Therefore, in recent years, rapid, sensitive, and reliable methods have received more attention. "
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