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. 2023 Aug 14;9(8):847.
doi: 10.3390/jof9080847.

Increased Conidia Production and Germination In Vitro Correlate with Virulence Enhancement in Fusarium oxysporum f. sp. cucumerinum

Md Jamal Uddin  1   2 , Xiaoqing Huang  1 , Xiaohong Lu  1 , Shidong Li  1
Affiliations

Increased Conidia Production and Germination In Vitro Correlate with Virulence Enhancement in Fusarium oxysporum f. sp. cucumerinum

Md Jamal Uddin et al. J Fungi (Basel). .

Abstract

Cucumber plants commonly suffer from Fusarium wilt disease, which is caused by Fusarium oxysporum f. sp. cucumerinum (Foc). Although resistant cultivars assist with Fusarium wilt disease control, enhancement of the virulence of Foc has been identified after monoculture of wilt-resistant cultivars. To investigate the biological characteristics that contribute to the virulence evolution of Foc, a wildtype strain foc-3b (WT) and its virulence-enhanced variant Ra-4 (InVir) were compared in terms of their growth, reproduction, stress tolerance, and colonization in cucumber plants. The InVir strain showed similar culture characteristics on PDA media to the WT strain but produced significantly more conidia (>two fold), with a distinctly higher germination rate (>four fold) than the WT strain. The colony diameter of the InVir strain increased faster than the WT strain on PDA plates; however, the mycelia dry weight of the InVir was significantly lower (<70%) than that of the WT harvested from PDB. The InVir strain exhibited a significant increase in tolerance to osmolality (1 M NaCl, 1 M KCl, etc.). The GFP-labeled InVir strain propagated in the cucumber vascular faster than the WT strain. These results suggest that increased conidia production and germination in vitro may correlate with virulence enhancement in Fusarium oxysporum f. sp. cucumerinum. This study will provide an insight into its virulence evolution and help us understand the mechanisms underlying the evolutionary biology of F. oxysporum.

Keywords: Fusarium oxysporum; Fusarium wilt; colonization; conidiation; virulence evolution.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphological characters of the wildtype strain foc-3b (A–C) with mild virulence and its virulence-enhanced variant Ra-4 (D–F) grown on PDA medium at 26 °C. Front (A,D) and reverse (B,E) sides of colonies. mi: microconidia; ma: macroconidia; c: chlamydospore. Scale bar = 20 µm.
Figure 2
Figure 2
Mycelia growth of the wildtype strain foc-3b with mild virulence (WT) and its variant Ra-4 with increased virulence (InVir). (A) Diameters of colonies grown on PDA medium at 26 °C and 28 °C, respectively. (B) Dry weight mycelia harvested from PDB medium cultured at 26 °C on a shaker with 180 runs/min for five days. Significant differences indicated by “*” (p < 0.05), “**” (p < 0.01), and “***” (p < 0.0001).
Figure 3
Figure 3
Conidia production and germination of the wildtype strain foc-3b with mild virulence (WT) and its variant Ra-4 with increased virulence (InVir). (A,B) Cultured in PDB and Armstrong medium, respectively. (C) Conidia germination on PDA medium or in sterile distilled water was calculated after 12 h incubation at 26 °C in the dark. Significant differences occurred between WT and InVir for all paired data (p < 0.01).
Figure 4
Figure 4
Tolerance to different stresses of the wildtype strain foc-3b with mild virulence (WT) and its variant Ra-4 with increased virulence (InVir). Inhibition of mycelia growth was determined for two strains grown for seven days on PDA medium amended with 1 M NaCl, 1 M KCl, 1 M glycerin, 1 M sorbitol, 0.03% sodium dodecyl sulphate (SDS), 30 mM H2O2, 0.05% congo red (CR), 0.3 mg/L calcofluor white (CFW), and 0.2 M CaCl2, respectively. Significant differences indicated by “*” (p < 0.05), “**” (p < 0.01) and “***” (p < 0.0001).
Figure 5
Figure 5
Virulence of the wildtype strain foc-3b with mild virulence (WT) and its variant Ra-4 with increased virulence (InVir). Disease incidence and disease index of susceptible (ZN6) and moderately resistant (ZN106) cucumber cultivars inoculated with WT and InVir strains using seed soaking (A) and root-dipping (B) methods, respectively. Significant differences indicated by “*” (p < 0.05), “**” (p < 0.01), and “***” (p < 0.0001).
Figure 6
Figure 6
Colonization of the wildtype strain foc-3b with mild virulence (WT) and its variant Ra-4 with increased virulence (InVir) in roots and stems of susceptible (ZN6) and moderately resistant (ZN106) cucumber cultivars. Observations were made seven days after inoculation with wildtype (WT) and InVir strains, which were marked with GFP and showed no changes in virulence. The inoculation process was performed using the root-dipping method. Scale bar = 100 µm (tap root, hypocotyl, and epicotyl) and 20 µm (vascular in hypocotyl and epicotyl).
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