Table 1.
Species considered for the ascomycete pan-genome analysis.
Table 2.
Comparison of the number of gene models predicted in the present study and previous reports.
Fig 1.
Clustering analysis of ascomycete pan-genome.
Clustering based on gene content of the orthogroups inferred. The Fusarium complex is highlighted in purple among the number of exclusive genes.
Fig 2.
Number of exclusive specie-specific orthologue groups for each member of the Fusarium complex.
Bar plot including the number of exclusive orthogroups for each Fusarium spp.
Fig 3.
Core phylogeny of ascomycetes.
Phylogenetic reconstruction from the 65 core orthogroups.
Table 3.
Primers designed to be used as specific gene markers in the diagnostic system.
Fig 4.
PCR-based assays using FuSp02 and PC01 [87] as diagnostic markers of fusariosis.
Agarose (0.8%) gels electrophoresis which shows: (A) The amplified ITS region to validate the quality of the template DNA used and isolated from different species of ascomycetes fungi (F. kuroshium [Fk], F. graminearum [Fg], F. verticillioides [Fv], F. oxysporum [Fo], F. tricinctum [Ft], F. solani [Fs], A. alternata [Aa], B. cinereal [Bc], and Neofusicoccum parvum [Np]; lines 1–9, respectively), and (B) The PCR fragments (amplicons) obtained for FuSp02 (odd lanes) and PC01 (even lanes) markers, both of them highly-specific to identify species from Fusarium genus. In (B), lanes from F. tricinctum were not shown only for lack of space reasons in the electrophoresis gel.
Fig 5.
PCR analysis of the markers designed for Fusarium kuroshium diagnosis.
From left to right, every three lanes correspond to each of the different markers (FuKu01, FuKu02, and FuKu03, respectively). These markers were tested using genomic DNA from different species of the Fusarium genus (A) and another phytopathogenic ascomycete fungi (B). Tested species were: On (A), F. kuroshium ([Fk]; lanes 1–3), F. graminearum ([Fg]; lanes 4–6), F. verticillioides ([Fv]; lanes 7–9), F. oxysporum ([Fo]; lanes 10–12), F. tricinctum ([Ft]; lanes 13–15), and F. solani ([Fs]; lanes 16–18); while on (B), Fusarium kuroshium ([Fk]; lanes 1–3), A. alternata ([Aa]; lanes 4–6), B. cinerea ([Bc]; lanes 7–9), and N. parvum ([Np]; lanes 10–12).
Fig 6.
Evaluation of the diagnosis system using as template DNA obtained from a plant tissue artificially infected tissue with F. kuroshium.
This test included the FuSp02 genus-specific marker and FuKu01 and FuKu02 species-specific markers. Lanes 1, 2 and 3 which showed a total absence of any amplicons correspond to negative controls, that is, DNA used as template was isolated from non-infected plant tissue. For the rest of the lines, DNA used as template comes from plant tissue artificially infected tissue with F. kuroshium. Lane 4 represents FuSp02, while lanes 5 and 6 show the expected products for markers FuKu01 and FuKu02. As additional controls, markers designed for F. graminearum were also tested. Two of them designed in the present study (FuGr01 and FuGr02; lanes 7 and 8), and the other one (PC02; lane 9), previously reported [88]. In the same way, the FuOx01 and FuOx02 markers designed on this study for F. oxysporum, were also tested (lanes 10 and 11). Additional results regarding markers designed on this study to F. graminearum and F. oxysporum are shown in sections downstream described.
Fig 7.
Evaluation of the diagnosis system using as template the DNA isolated from plant tissue of E. corallodendron trees, which were collected in the field and it was infested by KSHB and infected with F. kuroshium.
Lanes 1–3 show the amplicons corresponding to ITS, one lane per independently collected tree. Lanes 4–6, FuSp02 marker (genus-specific). Lanes 7–9, and 10–12, species-specific markers designed for F. kuroshium (FuKu01 and Fuku02).
Fig 8.
PCR analysis of primers designed for Fusarium graminearum.
From left to right, every third lane corresponds to the markers (FuGr01, FuGr02, and PC02) tested using genomic DNA from different species of the Fusarium genus and other phytopathogenic ascomycete fungi. (A) Lanes 1–3 correspond to Fusarium kuroshium [Fk], 4–6 to F. graminearum [Fg], 7–9 to F. verticillioides [Fv], 10–12 to F. oxysporum [Fo], 13–15 to F. tricinctum [Ft], and 16–18 to F. solani [Fs]; while on (B), lanes 1–3 correspond to F. graminearum [Fg], 4–6 to Alternaria alternata [Aa], 7–9 to Botrytis cinereal [Bc], and 10–12 to N. parvum [Np].
Fig 9.
Evaluation of primers designed for the diagnosis of F. oxysporum.
From left to right, every two lanes correspond to the markers FuOx01, FuOx02. In (A), lanes 1 and 2 correspond to Fusarium kuroshium [Fk], 3 and 4 to F. graminearum [Fg], 5 and 6 to F. verticillioides [Fv], 7 and 8 to F. oxysporum [Fo], 9 and 10 to F. tricinctum [Ft], and 11 and 12 to F. solani [Fs]. In (B), lanes 1 and 2 showed the PCR products of F. oxysporum [Fo], 3 and 4 of Alternaria alternata [Aa], 5 and 6 from Botrytis cinereal [Bc], and 7 and 8 of N. parvum [Np].
Fig 10.
PCR analysis of the PC03 marker previously reported for the diagnosis of F. oxysporum.
From left to right are shown the PCR products obtained for Fusarium kuroshium [Fk], F. graminearum [Fg], F. verticillioides [Fv], F. oxysporum [Fo], F. tricinctum [Ft], F. solani [Fs], A. alternata [Aa], B. cinereal [Bc], and N.parvum [Np], lanes 1 to 9, respectively.