Molecular Variability of the Fusarium solani Species Complex Associated with Fusarium Wilt of Melon in Iran

Abstract

Species of the Fusarium solani species complex (FSSC) are responsible for the Fusarium wilt disease of melon (Cucumis melo), a major disease of this crop in Iran. According to a recent taxonomic revision of Fusarium based primarily on multilocus phylogenetic analysis, Neocosmospora, a genus distinct from Fusarium sensu stricto, has been proposed to accommodate the FSSC. This study characterized 25 representative FSSC isolates from melon collected in 2009-2011 during a field survey carried out in five provinces of Iran. Pathogenicity assays showed the isolates were pathogenic on different varieties of melon and other cucurbits, including cucumber, watermelon, zucchini, pumpkin, and bottle gourd. Based on morphological characteristics and phylogenetic analysis of three genetic regions, including nrDNA internal transcribed spacer (ITS), 28S nrDNA large subunit (LSU) and translation elongation factor 1-alpha (tef1), Neocosmospora falciformis (syn. F. falciforme), N. keratoplastica (syn. F. keratoplasticum), N. pisi (syn. F. vanettenii), and Neocosmospora sp. were identified among the Iranian FSSC isolates. The N. falciformis isolates were the most numerous. This is the first report of N. pisi causing wilt and root rot disease in melon. Iranian FSSC isolates from different regions in the country shared the same multilocus haplotypes suggesting a long-distance dispersal of FSSC, probably through seeds.

Keywords: Cucumis melo; FSSC; Neocosmospoa sp.; Neocosmospora falciformis; Neocosmospora keratoplastica; Neocosmospora pisi; haplotypes; pathogenicity; phylogenetic analysis.

Figure 1

Distribution of FSSC isolates recovered from melons in Iran and examined in this study. Each isolate was recovered from a distinct field. In the map, each dot represents an isolate and each color represents a multilocus haplotype (MH). Dark green: MH 4; Light green: MH 12; Dark blue: MH 2; Yellow: MH 10; Pink: MH 22; Brown: MH 16; Light blue: MH 13; Red: MH 11; Gray: MH 15; Purple: MH 3.

Figure 2

Results of pathogenicity assays of four isolates of diverse FSSC species on different cucurbits. The FSSC species tested included F. keratoplasticum (isolate Iv-km-50), F. falciforme (isolate Iv2-r-30), F. vanettenii (isolate Far-317), and FSSC 5 (isolate FS-Spa). Nine plants of each cucurbit species or variety were inoculated per fungal isolate, and the same number of plants was mock-inoculated with water. Symptom severity was evaluated 30 days after inoculation. (a) Symptom severity induced by each Fusarium isolate on 11 diverse cucurbits (means of nine values ± SE). (b) Values of symptoms severity induced by each Fusarium isolate on 11 diverse cucurbits were pooled together to compare the virulence of isolates (means of 99 values ± SE).

Figure 2

Results of pathogenicity assays of four isolates of diverse FSSC species on different cucurbits. The FSSC species tested included F. keratoplasticum (isolate Iv-km-50), F. falciforme (isolate Iv2-r-30), F. vanettenii (isolate Far-317), and FSSC 5 (isolate FS-Spa). Nine plants of each cucurbit species or variety were inoculated per fungal isolate, and the same number of plants was mock-inoculated with water. Symptom severity was evaluated 30 days after inoculation. (a) Symptom severity induced by each Fusarium isolate on 11 diverse cucurbits (means of nine values ± SE). (b) Values of symptoms severity induced by each Fusarium isolate on 11 diverse cucurbits were pooled together to compare the virulence of isolates (means of 99 values ± SE).

Figure 3

Maximum likelihood phylogeny of FSSC isolates from melons based on concatenated sequencing data of three gene regions (ITS, LSU, and tef1). Scale bar indicates number of substitutions per site. Bootstrap values higher than 70 are shown. Fusrium staphyleae (NRRL 22316) was used as the outgroup. Red dots indicate Iranian isolates recovered in this study.

Figure 4

TCS multilocus haplotype network generated using the POPArt program by combining three gene regions (ITS, LSU, and tef1) of FSSC isolates recovered from melons in Iran. The size of the circles indicates the relative frequency of sequences of a given multilocus haplotype (MH). Hatch marks along the branches indicate the number of mutations. Each color represents one of the five provinces where the FSSC isolates were recovered. The isolates retrieved from GenBank and FS-Spa reference isolate received from Spain are assigned to the category ‘Abroad’. The numbers on the left of a slash indicates the MH number (Table 1), while the numbers on the right of the slash indicates the number of isolates in a given MH.