Iron necessity for chlamydospore germination in Fusarium oxysporum f. sp. cubense TR4

Abstract

Fusarium wilt disease of banana, caused by the notorious soil-borne pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), is extremely difficult to manage. Manipulation of soil pH or application of synthetic iron chelators can suppress the disease through iron starvation, which inhibits the germination of pathogen propagules called chlamydospores. However, the effect of iron starvation on chlamydospore germination is largely unknown. In this study, scanning electron microscopy was used to assemble the developmental sequence of chlamydospore germination and to assess the effect of iron starvation and pH in vitro. Germination occurs in three distinct phenotypic transitions (swelling, polarized growth, outgrowth). Outgrowth, characterized by formation of a single protrusion (germ tube), occurred at 2 to 3 h, and a maximum value of 69.3% to 76.7% outgrowth was observed at 8 to 10 h after germination induction. Germination exhibited plasticity with pH as over 60% of the chlamydospores formed a germ tube between pH 3 and pH 11. Iron-starved chlamydospores exhibited polarized-growth arrest, characterized by the inability to form a germ tube. Gene expression analysis of rnr1 and rnr2, which encode the iron-dependent enzyme ribonucleotide reductase, showed that rnr2 was upregulated (p < 0.0001) in iron-starved chlamydospores compared to the control. Collectively, these findings suggest that iron and extracellular pH are crucial for chlamydospore germination in Foc TR4. Moreover, inhibition of germination by iron starvation may be linked to a different mechanism, rather than repression of the function of ribonucleotide reductase, the enzyme that controls growth by regulation of DNA synthesis.

Keywords: Germ tube; Outgrowth; Polarized; Quiescence; Ribonucleotide reductase.

Fig. 1

Bright-field and scanning electron microscopy micrographs showing the transitions during germination of chlamydospores of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4): rounded (A, D), polarized growth (B, E), and outgrowth  (C, F). The stages of germination are further illustrated in the schematic (G). The site of polarity establishment (indicated by arrow heads) on polarized chlamydospores, is the point where the germ tube (indicated by white asterisk) emerges during outgrowth. Scale bar = 20 µm. Phenotypic changes during the process of germination of chlamydospores in Foc TR4 (H)

Fig. 2

Effect of iron starvation on cellular metabolic activity and germination on chlamydospores of Fusarium oxysporum f. sp. cubense Tropical Race 4 (A, B). Boxplots show the upper and lower quartile, median (bold horizontal bar), mean (white circle), and whiskers (vertical lines)

Fig. 3

Effect of iron starvation on the expression of genes rnr1 and rnr2 that encode the Rnr1 and Rnr2 subunits, respectively, of the ribonucleotide reductase enzyme in chlamydospores of Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) (A). Effect of pH on chlamydospore germination in Foc TR4 (B). The dotted line denotes the 50% germination of chlamydospores