Polymorphism of mycotoxin biosynthetic genes among Fusarium equiseti isolates from Italy and Poland

Abstract

Fusarium equiseti (Corda) Saccardo is a soil saprophyte and a weak pathogen, associated with several diseases of fruit and other crops in subtropical and tropical areas, but also in countries with temperate climate. A wide range of secondary metabolites has been identified among natural F. equiseti populations, with zearalenone (ZEA), fusarochromanone and fusarenon-X being the most common. In present study, the genetic diversity of strains from two populations (from Italy and Poland) was evaluated by analysing the translation elongation factor 1α (tef-1α) sequences, two polyketide synthases from the ZEA biosynthetic pathway (PKS13 and PKS4) and the TRI5 gene from the trichothecene biosynthetic pathway. ZEA was produced in rice cultures by 20 of the 27 tested isolates in concentrations ranging from 1.34 ng/g to 34,000 ng/g). The ability to produce enniatins and trichothecenes was evaluated in all strains by identifying esyn1, TRI13 and TRI4 genes. The presence of PKS4 and PKS13 genes was confirmed by polymerase chain reaction (PCR) in only some ZEA-producing isolates. Similarly, the TRI5 gene was found in 14 of the 27 isolates tested. This is likely to have been caused by the divergence of those genes between F. equiseti and F. graminearum (the latter species was used for the primers design) and can be exploited in phylogenetic studies. The analysis of the mycotoxin biosynthetic gene sequences can be used to differentiate the studied genotypes even more precisely than the analysis of the non-coding regions (like tef-1α).

Fig. 1

Most parsimonious tree for the isolates studied, created on the basis of the translation elongation factor 1α (tef-1α) sequences. The tree was obtained using the maximum parsimony approach and tested by bootstrapping (10,000 replicates) with a cut-off value of 50%. Abbreviations used for the origin and host species: IT – Italy, PL – Poland, AR – Argentina, Os – Oryza sativa, Zm – Zea mays, Ea – Equisetum arvense, Ac – Allium cepa, Ch – Chenopodium, Pi – Pinus root, Sl – Solanum lycopersicum, Or – Orobanche ramosa, Tr – Triticum sp., Le – Lycopersicon esculentum, Tc – triticale, Ta – Triticum aestivum

Fig. 2

Colony diameter of the strains used in the study after four days of culturing on potato dextrose agar (PDA) medium at room temperature (mean of two replicates)

Fig. 3

Zearalenone (ZEA) production by Fusarium equiseti isolates used in this study

Fig. 4

Most parsimonious tree for six chosen isolates based on the sequences of the polyketide synthase (PKS13) gene from the ZEA metabolic pathway. The tree was obtained using the maximum parsimony approach and tested by bootstrapping (10,000 replicates) with a cut-off value of 70%. The F. graminearum KF 371 strain was used as the reference. Abbreviations used for the origin and host species: IT – Italy, PL – Poland, Or – Orobanche ramosa, Tc – triticale, Ta – Triticum aestivum

Fig. 5

Most parsimonious tree for the 14 strains tested, based on the trichodiene synthase (TRI5) gene sequences. The maximum parsimony approach was used and tested by bootstrapping (10,000 replicates) with a cut-off value of 50%. Two strains of F. graminearum (DON chemotype, KF 1413, and NIV chemotype, KF 371) were used as references. Abbreviations used for origin and host species: IT – Italy, PL – Poland, Os – Oryza sativa, Zm – Zea mays, Or – Orobanche ramosa, Le – Lycopersicon esculentum, Tc – triticale, Ta – Triticum aestivum