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

ABSTRACT 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, Sep 29, 2015
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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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    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.
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    • "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. "
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    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
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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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