In Vitro and Ex Vivo Antifungal Activities of Metconazole against the Rice Blast Fungus Pyricularia oryzae

Abstract

Rice blast, caused by the filamentous fungus Pyricularia oryzae, has long been one of the major threats to almost all rice-growing areas worldwide. Metconazole, 5-(4-chlorobenzyl)-2, 2-dimethyl-1-(1H-1, 2, 4-triazol-1-ylmethyl) cyclopentanol, is a lipophilic, highly active triazole fungicide that has been applied in the control of various fungal pathogens of crops (cereals, barley, wheat), such as the Fusarium and Alternaria species. However, the antifungal activity of metconazole against P. oryzae is unknown. In this study, metconazole exhibited broad spectrum antifungal activities against seven P. oryzae strains collected from rice paddy fields and the wild type strain P131. Scanning electron microscopic analysis and fluorescein diacetate staining assays revealed that metconazole treatment damaged the cell wall integrity, cell membrane permeability and even cell viability of P. oryzae, resulting in deformed and shrunken hyphae. The supplementation of metconazole in vitro increased fungal sensitivity to different stresses, such as sodium dodecyl sulfate, congo red, sodium chloride, sorbitol and oxidative stress (H₂O₂). Metconazole could inhibit key virulence processes of P. oryzae, including conidial germination, germ tube elongation and appressorium formation. Furthermore, this chemical prevented P. oryzae from infecting barley epidermal cells by disturbing appressorium penetration and subsequent invasive hyphae development. Pathogenicity assays indicated a reduction of over 75% in the length of blast lesions in both barley and rice leaves when 10 μg/mL of metconazole was applied. This study provides evidence to understand the antifungal effects of metconazole against P. oryzae and demonstrates its potential in rice blast management.

Keywords: Pyricularia oryzae; antifungal activity; fungicide; metconazole; rice blast.

Figure 1

Inhibitory effects of metconazole on the mycelial growth of the P. oryzae strain P131. (a) Chemical structure of metconazole. (b) Colony morphology of P. oryzae strain P131 grown on CM plate for 5 d (Bar: 1 cm). The statistics of (c) colony diameter and (d) inhibition rates of metconazole on P131 growth. MET: methanol as solvent control, **: p < 0.01.

Figure 2

Microscopic observation of the antifungal effects of metconazole on the P. oryzae strain P131 (a) Bright field (Bar: 200 μm) and (b) SEM images (Bar: 2 μm) of hyphal structures in P. oryzae strain P131 on CM plates. (c) FDA staining of P. oryzae conidia incubated on hydrophobic glasses at 2 hpi (Bar: 100 μm). MET: methanol as solvent control.

Figure 3

Increased sensitivity of P. oryzae in response to various stress upon 0.5 μg/mL metconazole treatment. (a) Colony morphology of indicated strains grown on CM plates (Bar: 1 cm). The statistics of (b) colony diameter and (c) inhibition rates of indicated strains grown on CM plates supplemented with different stresses. CM: as a medium control, HO: 10 mM H₂O₂, CR: 0.2 mg/mL Congo red, SDS: 0.005% Sodium dodecyl sulfate, So: 1 M Sorbitol, Na: 0.7 M NaCl.

Figure 4

Inhibitory effects of metconazole treatment on conidial germination, germ tube elongation and appressorium formation of P. oryzae. (a) Bright field images of P. oryzae strain P131 conidia treated with different concentrations of metconazole at different time points. White arrows indicate germ tubes, red arrows indicate appressoria (Bar: 20 μm). (b) Percentages of conidial germination at 2, 8 and 24 hpi, (c) Average length of germ tube at 2 hpi. (d) The appressorium formation rates at 8 and 24 hpi. MET: methanol as solvent control, ns: p > 0.05, **: p < 0.01.

Figure 5

Metconazole treatment prevented P. oryzae from infecting barley epidermal cells. (a) Representative images of different types of invasive hyphae chosen from different metconazole treatments (Bar: 20 μm). (b) Quantitative analysis of invasive growth in barley leaf. Mean percentage of invasive hyphal growth was calculated based on an analysis of 100 appressorium penetration sites. Hyphal growth was categorized from level I to level IV (I, no penetration; II, with primary invasive hyphae; III, secondary invasive hyphae does not expand to the neighboring plant cells; IV, invasive hyphae expanding into neighboring plant cells). MET: methanol as solvent control.

Figure 6

Metconazole treatment damaged the full virulence of P. oryzae in barley leaves and rice leaves. (a) The lesion image of lesion in barley leaves (Bar: 1 cm). (b) The statistics of the length of lesion in barley leaves. (c) The lesion image of lesion in rice leaves (Bar: 1 cm). (d) The statistics of the length of lesion in rice leaves. MET: methanol as solvent control, M: mock, incubated with distilled water amended with 0.025% tween 20, as negative control, **: p < 0.01.