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.

Download full-text


Available from: María Teresa González-Jaén,
  • Source
    • "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 . "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this work was to compare the radial growth rate (μ) and the lag time (λ) for growth of 25 isolates of Penicillium expansum at 1 and 20ºC with those of the mixed inoculum of the 25 isolates. Moreover, the evolution of probability of growth through time was also compared for the single strains and mixed inoculum. Working with a mixed inoculum would require less work, time and consumables than if a range of single strains has to be used in order to represent a given species. Suitable predictive models developed for a given species should represent as much as possible the behavior of all strains belonging to this species. The results suggested, on one hand, that the predictions based on growth parameters calculated on the basis of mixed inocula may not accurately predict the behavior of all possible strains but may represent a percentage of them, and the median/mean values of μ and λ obtained by the 25 strains may be substituted by the value obtained with the mixed inoculum. Moreover, the predictions may be biased, in particular, the predictions of λ which may be underestimated (fail-safe). Moreover, the prediction of time for a given probability of growth through a mixed inoculum may not be accurate for all single inocula, but it may represent 92% and 60% of them at 20 and 1ºC, respectively, and also their overall mean and median values. In conclusion, mixed inoculum could be a good alternative to estimate the mean or median values of high number of isolates, but not to account for those strains with marginal behavior. In particular, estimation of radial growth rate, and time for 0.10 and 0.50 probability of growth using a cocktail inoculum accounted for the estimates of most single isolates tested. For the particular case of probability models, this is an interesting result as for practical applications in the food industry the estimation of t10 or lower probability may be required.
    International Journal of Food Microbiology 06/2014; 186C:120-124. DOI:10.1016/j.ijfoodmicro.2014.06.023 · 3.08 Impact Factor
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fusarium culmorum and F. graminearum are the major pathogens for dryland root/foot-rot and head-blight diseases in economically important grain crops. This study was aimed at the molecular characterization of Fusarium spp. isolates which have been collected from cereal fields in three agro-ecological regions in Turkey. Genetic diversity has been analyzed by generating RFLP markers from the intergenic spacer (IGS) region of ribosomal RNA. The selection of restriction enzymes for IGS-RFLP studies has been found critical to maximize polymorphic markers. Only three of fourteen restriction endonucleases were useful in differentiating Fusarium spp. isolates. Pst I was the most efficient enzyme to produce a maximum of 9 DNA markers in one individual and total 22 polymorphic representative banding patterns. Polymorphism based on IGS-RFLP was high and average 88% in both species. There was no association between IGS diversity and geographic locations from which the samples were taken. Both MAT-1 and MAT-2 sequences were amplified in F. graminearum similarly to previous reports. Most of the F. culmorum isolates carried either MAT-1 or MAT-2 sequences, and differently two isolates carried both sequences. Mating type determination was helpful to distinguish F. pseudograminearum from F. graminearum which cannot be discriminated by SCAR markers or morphological assessment. High genetic diversity by IGS-RFLP markers in F. culmorum was discussed in relation to its fitness as the most common pathogen in dryland root rot complex (DLRRC). (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
    Journal of Basic Microbiology 08/2013; 53(8). DOI:10.1002/jobm.201200066 · 1.82 Impact Factor
  • Source
    • "A single fragment of 2600 bp was amplified from all the 74 isolates of Fusarium spp, which was similar to the results obtained from several studies of Fusarium spp in which the size of the IGS region varied from 2200 to 2600 bp (Konstantinova and Yli-Mattila, 2004; Patino et al., 2006; Llorens et al., 2006a,b). Among the six restriction enzymes used in this study to digest the PCR products, HinfI showed the most variable restriction patterns with 11 patterns while EcoRI produced only three patterns. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fusarium species section Liseola namely F. fujikuroi, F. proliferatum, F. andiyazi, F. verticillioides, and F. sacchari are well-known plant pathogens on rice, sugarcane and maize. In the present study, restriction analysis of the intergenic spacer regions (IGS) was used to characterize the five Fusarium species isolated from rice, sugarcane and maize collected from various locations in Peninsular Malaysia. From the analysis, and based on restriction patterns generated by the six restriction enzymes, Bsu151, BsuRI, EcoRI, Hin6I, HinfI, and MspI, 53 haplotypes were recorded among 74 isolates. HinfI showed the most variable restriction patterns (with 11 patterns), while EcoRI showed only three patterns. Although a high level of variation was observed, it was possible to characterize closely related species and isolates from different species. UPGMA cluster analysis showed that the isolates of Fusarium from the same species were grouped together regardless of the hosts. We conclude that restriction analysis of the IGS regions can be used to characterize Fusarium species section Liseola and to discriminate closely related species as well as to clarify their taxonomic position.
    Genetics and molecular research: GMR 01/2012; 11(1):383-92. DOI:10.4238/2012.February.16.4 · 0.78 Impact Factor
Show more