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: 3.16). 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.

Download full-text


Available from: María Teresa González-Jaén, Jul 01, 2015
1 Follower
  • Source
    [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 · 3.16 Impact Factor
  • Source
    [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 · 0.85 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fusarium virguliforme is the cause of sudden death syndrome in soybean. Physiological variability among isolates of the fungus is unknown. One way to measure physiologic variability is to analyze growth on different carbon sources. The carbon source utilization profiles of 18 F. virguliforme isolates were examined using the Biolog FF 96-well microplate, which contains 95 different carbon sources. The utilization of dextrin,D-mannitol, maltotriose,D-lactic acid methyl ester, N-acetyl-D-galactosamine, salicin, D-trehalose, and L-alanine differed significantly among isolates (P = 0.05). Carbon sources were grouped into 3 clusters based on their ability to promote growth of F. virguliforme, after calculating Euclidean distances among them. About 12% of the carbon sources promoted a high amount of mycelial growth, 39% promoted a medium amount of growth, and 49% promoted a low amount of mycelial growth; the latter was not significantly different from the water blank control. A hierarchical tree diagram was produced for the 18 isolates based on their carbon source utilization profiles using Ward's hierarchical analysis method. Two main clusters of isolates were formed. One cluster represented greater average mycelial growth on all of the carbon sources than the other cluster. In this study, variability in carbon source utilization among F. virguliforme isolates was evident, but the results were not associated with geographic origin of the isolates, year collected, or published data on aggressiveness. Additional research is needed to determine if these carbon utilization profiles are associated with other biological characteristics, like spore germination, propagule formation, and saprophytic competitiveness.
    Canadian Journal of Microbiology 12/2010; 56(12):979-86. DOI:10.1139/W10-085 · 1.18 Impact Factor