Development of a PCR protocol for the identification and detection of Fusarium solani f. sp. piperis from soil and roots of black pepper (Piper nigrum)

Abstract

The main disease of black pepper (Piper nigrum) in Brazil is caused by Fusarium solani f. sp. piperis. The disease diagnosis is problematic because, besides this pathogen, there are other different phylogenetic lineages within the Fusarium solani species complex (FSSC) associated with black pepper that can not be reliably identified based solely on morphological characteristics. Based on differences in the translation elongation factor 1-alpha (EF-1α) gene sequences of species of the FSSC, one pair of species-specific primers, Fpiper1F/Fpiper1R, was synthesized to accurately detect and identify F. solani f. sp. piperis. Primer specificity was tested with DNA from 18 isolates of F. solani f. sp. piperis, 24 representative isolates of other lineages of the FSSC associated with P. nigrum, and isolates representing eight strains of other species. The primers amplified only a single PCR band of 230 bp from F. solani f. sp. piperis. Nested PCR was more sensitive than conventional PCR, using the primer pair EF1/EF2 for the first round and specific primers Fpiper1F/Fpiper1R for the second round. Detection sensitivity was 1 fg of purified F. solani f. sp. piperis DNA template and 101 macroconidia/g soil infected. The specific primers will greatly facilitate the detection and identification of F. solani f. sp. piperis, and have potential as a diagnostic tool for detecting this pathogen in field-grown black pepper soil.