Use of PCR-RFLP markers of the rDNA IGS region for genetic diversity studies in Fusarium species associated with babaco and naranjilla crops in Ecuador.

Abstract

ABSTRACT

Among the biotic factors, which determine greater economic losses in agricultural crops, are the phytopathogenic fungi. Fusarium oxysporum, constitutes a complex of species with considerable morphological and physiological variation; which makes its identification difficult, this has led to the development of molecular tools for the study of special forms of this pathogen. In particular, F. oxysporum f. sp. vasconcellea (Fov) and F. oxysporum f. sp. quitoense (Foq) infect babaco and naranjilla crops causing babaco vascular wilt (BVW) and naranjilla vascular wilt (NVW). In this research, the polymerase chain reaction technique and polymorphic length restriction fragments (PCR-RFLP) of the intergenic spacer (IGS) of ribosomal DNA (rDNA) were applied to determine the genetic diversity of special forms (f. sp.) of F. oxysporum, the causal agent of MVB and MVN. The IGS sequences were amplified by PCR and sequenced in both directions. The IGS sequences were used for phylogenetic tree construction and identification of specific restriction sites for EcoRI and ApaI enzymes and identification of single nucleotide polymorphisms (SNPs). The size of the PCR products of the IGS region was 1935 and 2603 nucleotides, which were determined based on sequence alignment and molecular weight marker. The phylogenetic analysis and the PCR-RFLPs technique placed the babaco and naranjilla sequences in different groups. In the first group are located the sequences NA4 and NA6; in the second group BA3, BA4, BA5, BA6 and NA2; in the third group BA1, BA2, NA1, NA3 and NA5. These results show that each group evolved from the same common ancestor, therefore, each group would have a polyphyletic origin.

Key words: endonucleases, restriction, polymorphisms, haplotypes, biotrophs, quitoense, vasconcellea.