Development of a multiplex PCR for the identification of Fusarium solani and F. oxysporum in a single step

Fusarium solani and F. oxysporum are plant pathogenic fungi that cause root rot and wilt, respectively, in many economically important crops. Identification of both species by morphological characteristics is complex and time-consuming, especially when a crop is host to several species, as in the case of strawberry and other many crops. The objective of this research was to develop a molecular method for the precise and sensitive identification of Fusarium solani and F. oxysporum by multiplex PCR. Specific PCR conditions were developed for the amplification of about 170-bp PCR product from the internal transcribed spacer of F. oxysporum and a PCR product of about 650 bp from the elongation factor 1-alpha gene of F. solani. The specificity of the multiplex PCR was tested using gDNA extracted from 232 strains of F. solani and F. oxysporum, 12 strains of other Fusarium spp. and 14 strains of other genera. The sensitivity of each primer set was tested individually and simultaneously with one strain of F. oxysporum and F. solani. This protocol was then used to identify both species in natural isolates from diseased plant and soil samples from strawberry nursery. Each primer set was validated using genomic DNA from non-sterile and sterile soils artificially inoculated with both species. Under these conditions, both Fusarium species were detected and distinguished over a range of concentrations (1 × 107 to 1 × 102 conidia g−1 soil) by successful amplification of corresponding PCR products for each species. This multiplex PCR technique is a fast tool to identify natural isolates of both species, F. solani and F. oxysporum, in a single PCR reaction.