M. Teresa González-Jaén

PCR detection assay of fumonisin-producing Fusarium verticillioides strains.

Fusarium verticillioides is considered to be the main source of fumonisins, a group of toxins that contaminate commodities and result in chronic and acute diseases affecting humans and animals. The detection and control of this species is crucial to prevent fumonisins from entering the food chain. The objective of the present research was to develop a specific, sensitive, and robust PCR assay to detect F. verticillioides strains using two pairs of specific primers for F. verticillioides, which have been designed on the basis of the intergenic spacer region of the rDNA units. The first pair of primers was F. verticillioides species specific, whereas the second pair of primers detected fumonisin-producing F. verticillioides strains. This second pair of primers allowed for the discrimination between the major group of F. verticillioides strains, fumonisin-producing strains that are mainly associated with crops, and a minor group of strains, non-fumonisin-producing strains that are associated with bananas. Fifty-four strains of F. verticillioides from different geographical regions and hosts were tested using both sets of primers. Sixteen additional Fusarium species were examined. The specificity of the primer sequences provides the basis for a simple, rapid, accurate, and sensitive detection and identification method of this fungal species that represents a risk for human and animal health.

Genetic variability and fumonisin production by Fusarium proliferatum.

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.

Genetic markers for the analysis of variability and for production of specific diagnostic sequences in fumonisin-producing strains of Fusarium verticillioides

Fusarium verticillioides(Gibberella moniliformis, Gibberella fujikuroi mating population A) is the main source of fumonisins, a group of toxins which contaminates commodities, causing chronic and acute diseases in humans and animals. Fumonisins are produced during colonisation and infection of host plants even when disease symptoms are not recognisable. Early detection and control of F. verticillioides is crucial to prevent fumonisins from entering the food chain. DNA-based strategies have been used to search for markers to develop sensitive, robust and specific diagnostic assays, mainly based on PCR. The different approaches used, based either on DNA markers unrelated to fumonisin production or on information about the genes involved in fumonisin production, are described and discussed. The ability of these methods to discriminate between the two populations occurring within F. verticillioides, fumonisin-producing and fumonisin non-producing strains, is also addressed.

Potential effects of environmental conditions on the efficiency of the antifungal tebuconazole controlling Fusarium verticillioides and Fusarium proliferatum growth rate and fumonisin biosynthesis

Fusarium verticillioides and Fusarium proliferatum are important phytopathogens which contaminate cereals in the Mediterranean climatic region with fumonisins. In this study we examined the interaction between the fungicide efficacy of tebuconazole and water potential (Ψw) (-0.7-7.0MPa)×temperature (20-35°C) on growth and FUM1 gene expression by real time RT-PCR (an indicator of fumonisin biosynthesis) in strains of both Fusarium species. Concentrations of tebuconazole required to reduce growth by 50 and 90% (ED50 and ED90 values) were determined. Growth of strains of both species was largely reduced by tebuconazole, with similar efficacy profiles in the interacting water potential×temperature conditions. In contrast, FUM1 expression was not generally reduced by tebuconazole. Moreover, sub-lethal doses in combination with mild water stress and temperatures less than 35°C significantly induced FUM1 expression with slight differences in both species. These results suggest that the efficacy of antifungal compounds to reduce mycotoxin risk would be more effective if consideration is given to both growth rate and toxin biosynthesis in relation to interacting environmental conditions. This is the first study linking fungicide efficacy of tebuconazole with environmental factor effects on control of growth and FUM1 gene expression of F. verticillioides and F. proliferatum.

Comparative analysis of an endopolygalacturonase coding gene in isolates of seven Fusarium species

Polygalacturonase (PG) synthetized by Fusarium spp. is a polymorphic enzyme with complex isoform patterns influenced by culture conditions and which are variable at the intra- and inter-specific levels. The partial sequence of an endopolygalacturonase coding gene (endopg) has been analyzed in isolates of seven species of Fusarium, most of them associated with Pinus pinea. The genomic fragment analyzed, 740 bp long, included two introns and the exon 4, which contained most of the amino acid motifs conserved in PGs from different origins. The high similarity of the amino acid sequences found among the isolates would indicate that differences in amino acid composition are not probably the main reason of the variability of this enzyme. Therefore, differences in the gene expression and regulation patterns of endopg gene among the isolates should be considered. Both types of sequences, the coding and the two intron sequences contained in the endopg fragment, were compared among the isolates to evaluate their potential use for phylogenetic studies. The different levels of variability of these sequences and the similar results obtained from the phylogenetic analyses when the three sequences (coding, intron and amino acid sequences) were used indicate that this endopg region would be very useful for phylogenetic analysis in the genus Fusarium.

Phylogenetic analysis, fumonisin production and pathogenicity of Fusarium fujikuroi strains isolated from rice in the Philippines

BACKGROUND Fusarium fujikuroi Nirenberg is a maize and rice pathogen causing important agricultural losses and produces fumonisins - mycotoxins which pose health risk to humans and farm animals. However, little information is available about the phylogenetics of this species and its ability to produce fumonisins in rice. We studied 32 strains isolated from rice in the Philippines and performed a phylogenetic analysis using the partial sequence of Elongation Factor 1 alpha (EF-1α) including isolates belonging to closely related species. Fumonisin B1 (FB1 ) production was analyzed in 7-day-old cultures grown in fumonisin-inducing medium by an enzyme-linked immunosorbent assay-based method and by real-time reverse transcriptase-polymerase chain reaction using primers for FUM1 gene, a key gene in fumonisin biosynthesis. RESULTS Nucleotide diversities per site (π) were 0.00024 ± 0.00022 (standard deviation) for the 32 F. fujikuroi strains from the Philippines and 0.00189 ± 0.00143 for all 34 F. fujikuroi strains, respectively. F. fujikuroi isolates grouped into one cluster separated from the rest of isolates belonging to the closely related F. proliferatum and showed very low variability, irrespective of their geographic origin. The cluster containing strains of F. proliferatum showed higher intraspecific variability than F. fujikuroi. Thirteen of the 32 strains analyzed were FB1 producers (40.62%), with production ranging from 0.386 to 223.83 ppm. All isolates analyzed showed FUM1 gene expression above 1 and higher than the CT value of the non-template control sample. Both seedling stunting and elongation were induced by the isolates in comparison with the control. CONCLUSION F. fujikuroi are distinct from F. proliferatum isolates based on phytogenetic analysis and are potential fumonisin producers because all are positive for FUM1 gene expression. No relationship between fumonisin production and pathogenicity could be observed.

Comparative analysis of polygalacturonases in isolates of seven species of Fusarium from Pinus pinea

Polygalacturonases (PGs) are important pectolytic enzymes produced by phytopathogenic fungi during the process of infection and colonistation of the host plants. In this work, PGs produced by isolates of seven Fusarium species associated to Pinus pinea have been analysed for: activity, isoform pattern observed by isoelectric focusing endo- or exo-mode of action and their production in culutres growing on pectin and galacturonic acid. Of the seven isolates, those of F. oxysporum and F. moniliforme exhibite high PG activity and the most complex isoform patterns including acidic ones. These isolates were also more efficient in degrading the PG substrate, during which both endo and exo type polygalacturonase activities were detected. It is suggested that these features could be useful to the fungus during infection and colonisation of its host, specially during seed germination and early development.

Growth rate and TRI5 gene expression profiles of Fusarium equiseti strains isolated from Spanish cereals cultivated on wheat and barley media at different environmental conditions.

Fusarium equiseti is a toxigenic species that often contaminates cereal crops from diverse climatic regions such as Northern and Southern Europe. Previous results suggested the existence of two distinct populations within this species with differences in toxin profile which largely corresponded to North and South Europe (Spain). In this work, growth rate profiles of 4 F. equiseti strains isolated from different cereals and distinct Spanish regions were determined on wheat and barley based media at a range of temperatures (15, 20, 25, 30, 35 and 40°C) and water potential regimens (-0.7, -2.8, -7.0, and -9.8MPa, corresponding to 0.99, 0.98, 0.95 and 0.93 aw values). Growth was observed at all temperatures except at 40°C, and at all the solute potential values except at -9.8MPa when combined with 15°C. Optimal growth was observed at 20-30°C and -0.7/-2.8MPa. The effect of these factors on trichothecene biosynthesis was examined on a F. equiseti strain using a newly developed real time RT-PCR protocol to quantify TRI5 gene expression at 15, 25 and 35°C and -0.7, -2.8, -7.0 and -9.8MPa on wheat and barley based media. Induction of TRI5 expression was detected between 25 and 35°C and -0.7 and -2.8MPa, with maximum values at 35°C and -2.8MPa being higher in barley than in wheat medium. These results appeared to be consistent with a population well adapted to the present climatic conditions and predicted scenarios for Southern Europe and suggested some differences depending on the cereal considered. These are also discussed in relation to other Fusarium species co-occurring in cereals grown in this region and to their significance for prediction and control strategies of toxigenic risk in future scenarios of climate change for this region.

Divergence of the IGS rDNA in Fusarium proliferatum and Fusarium globosum reveals two strain specific non-orthologous types

A phylogenic analysis of Fusarium proliferatum and closely related species was performed using the most variable part within the intergenic spacer of the nuclear ribosomal DNA (IGS) and compared with a previously reported phylogeny performed in the same group of samples with a partial region of the nuclear single copy gene encoding the elongation factor 1α (EF-1α). The phylogenies from both genomic sequences were not concordant and revealed the presence of two non-orthologous IGS types, named types I and II, in F. proliferatum and Fusarium globosum.Two specific PCR assays designed to amplify either IGS type I or type II revealed that only one IGS type was present in each individual in these two species. The presence of both IGS types at the species level indicates that homogenization has not been achieved yet. This might be retarded if panmictic sexual reproduction was affected by certain levels of clonal reproduction and/or by the diverse hosts that these species are able to colonize. This study indicates that taxonomic studies carried out with the IGS rDNA, which has been widely used in Fusarium, should be undertaken with caution.

Evaluating aflatoxin gene expression in aspergillus section flavi

The determination of aflatoxin production ability and differentiation of aflatoxigenic strains can be assessed by monitoring the expression of one or several key genes using reverse transcription polymerase chain reaction (RT-PCR). We herein describe the methods for RNA induction, extraction, and quality determination, and the RT-PCR conditions used to evaluate the ability of a given Aspergillus strain to produce aflatoxins.