Antifungal activity of traversianal, a diterpenoid aldehyde from Cercospora sp. ME202, against rice blast fungus Pyricularia oryzae

doi:10.21203/rs.3.rs-4962851/v1

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Antifungal activity of traversianal, a diterpenoid aldehyde from Cercospora sp. ME202, against rice blast fungus Pyricularia oryzae

https://doi.org/10.21203/rs.3.rs-4962851/v1

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Natural products from microorganisms are a rich resource for the identification of effective antifungal agents against plant pathogenic fungi. Traversianal, a diterpenoid produced by Cercospora spp., exhibits antifungal activity against Colletotrichum orbiculare, a pathogen that causes cucumber anthracnose. However, its effects on other fungi have not been investigated. Therefore, this study evaluated the antifungal activity of traversianal, derived from Cercospora sp. ME202, on Pyricularia oryzae, a rice blast pathogen. Traversianal exhibited a concentration-dependent inhibitory effect on conidial germination, with complete inhibition at 30 ppm. In the thin-layer chromatography bioassay, traversianal was detected as an antifungal zone at Rf 0.91, indicating mycelial growth inhibition. In addition, the time course of the fungicidal activity against P. oryzae, evaluated using double-fluorescent staining with fluorescein diacetate and propidium iodide, showed that most of the conidia were killed after 48 h of treatment with 40 ppm traversianal. In experiments with rice plants, blast lesion formation on rice leaves was significantly suppressed by pretreatment with traversianal. These results suggest the potential of traversianal as an inhibitory compound for the control of rice blast disease.

Antifungal compound

Cercospora sp.

Pyricularia oryzae

Rice blast disease

Traversianal

Oryza sativa

Rice blast, caused by Pyricularia oryzae, is one of the most important diseases affecting rice plants. P. oryzae can infect any part of the rice plant, such as the leaves, panicles, and leaf nodes, and is responsible for 10 to 30% of annual rice yield losses worldwide (Talbot 2003; Skamnioti and Gurr 2009; Wilson and Talbot 2009). Furthermore, depending on the cultivar susceptibility and environmental conditions, the disease intensifies and causes yield losses of up to 100% (Chuwa et al 2015; Moktan et al 2021). Chemical control using synthetic chemical fungicides is the major method for efficient and effective management of rice blast (Asibi et al 2019; Rajeswari et al 2024). However, excessive use of fungicides pollutes the environment and leads to the development of pathogen resistance. In 2001, rice blast fungi with reduced susceptibility to melanin-biosynthesis inhibitor fungicides, carpropamid and diclocymet, were isolated in Saga Prefecture, Japan (Yamaguchi et al 2002). Moreover, in 2021, P. oryzae resistant to mitochondrial-respiration inhibitor (quinone outside inhibitors) fungicides was reported in Italy (Tenni et al 2021). Therefore, there is an urgent need to develop alternative disease management strategies that are more effective and environmentally friendly.

Microorganisms produce an extensive range of secondary metabolites such as antibiotics and bioactive compounds, some of which are utilized in pharmaceuticals and pesticides, offering significant benefits to humans (Demain 2009; Keswani et al 2020). Many of these pesticides are easily and quickly degraded in the natural environment and have less impact on other organisms (Kim and Hwang 2007). Thus, pesticides based on natural products derived from microorganisms are an important resource in the agrochemical market (Sparks et al 2023).

We previously reported that traversianal, a diterpenoid aldehyde produced by Cercospora sp. ME202 isolated from Trifolium incarnatum L., showed antifungal activity against the cucumber anthracnose pathogen Colletotrichum orbiculare (Ino et al 2024). Specifically, the compound inhibited conidial germination at concentrations greater than 2 ppm and exhibited fungicidal activity at concentrations of 5 and 10 ppm. It also significantly suppressed the development of anthracnose disease on cucumber leaves (Ino et al 2024). However, the antifungal activity of traversianal against other plant pathogenic fungi has not been investigated. Therefore, the objective of this study was to investigate the antifungal activity of traversianal isolated from Cercospora sp. ME202 against P. oryzae and evaluate its potential as a fungicide against rice blast disease.

Chemical preparation

Traversianal was isolated from Cercospora sp. ME202 as previously described (Ino et al. 2024). The compound was stored at -20°C and used for experiments by dissolving in dimethyl sulfoxide (DMSO) at appropriate concentrations.

Fungal strains and culture conditions

Pyricularia oryzae (strain Naga 69–150, race 007) was used as the rice plant pathogen. The pathogen was grown on rice bran agar (50 g/1 rice bran, 20 g/1 sucrose, 20 g/1 agar, and 150 ml water) at 25 ± 2°C for 14 days. Subsequently, the aerial hyphae were removed and irradiated for 4 days under a black light blue lamp (FL20s BL-B; Panasonic, Osaka, Japan) to induce conidiation. A conidial suspension (1.0 × 10⁵ conidia/ml) was prepared using a hemocytometer (Thoma, Hirschmann, Germany).

Conidial germination assay

P. oryzae conidia (1.0 × 10⁵ conidia/ml) were suspended in traversianal at concentrations adjusted to 1, 2, 5, 10, 20, and 30 ppm in DMSO (0.01–0.3%). The suspensions were placed on glass slides and incubated in a moist chamber (19 × 10 × 2.5 cm) at 25 ± 2°C. DMSO (0.3%) was used as the control. After 24 h, the germination number of 300 conidia in each treatment was counted under a light microscope (Shimadzu BA210E; Shimadzu, Kyoto, Japan). The conidial germination rate was calculated using the following formula: Germinated conidia (%) = (number of germinated conidia/total number of conidia) × 100. All experiments were performed thrice.

Thin-layer-chromatography (TLC) bioassay

Traversianal at 30 ppm (100 µl) was spotted on a silica gel TLC plate (Silica 12 Gel 60; Merck KGaA, Darmstadt, Germany) and developed using a chloroform/ethanol/water (40/1/1, v/v/v) solvent system. After the solvent had completely dried, P. oryzae conidia (> 1.0 × 10⁷ conidia/ml) were suspended in melted potato sucrose agar medium (chloramphenicol, 20 ppm) and sprayed on the plate. The plate was incubated in the moist chamber at 25 ± 2°C for 7 days; thereafter, the inhibition zone of mycelial growth was observed.

Determination of fungicidal activity using double-fluorescence staining

Fluorescein diacetate (FDA; 5 mg/ml in acetone) and propidium iodide (PI; 10 mg/ml in water) were prepared as stock stain solutions and diluted to 200 and 100 µg/ml, respectively, in distilled water before use. Conidia of P. oryzae (1.0 × 10⁵ conidia/ml) were suspended in traversianal (30 and 40 ppm), and the conidial suspensions (24 µl) were placed on glass slides in the moist chamber at 25 ± 2°C. DMSO (0.4%) was used as the control. After 0, 12, 24, 36 and 48 h, the conidial suspensions were mixed with FDA (3 µl) and PI (3 µl), allowing a 10-minute reaction time. Subsequently, the number of conidia exhibiting fluorescence from FDA and PI was counted using a fluorescence microscope (BZ-X700 all-in-one; 17 KEYENCE Corp., Osaka, Japan) with GFP filters (for FDA, living cell, excitation wavelength 470 ± 20 nm/emission wavelength 525 ± 25 nm) and Texas RED filters (for PI, dead cell, excitation wavelength 560 ± 20 nm/emission wavelength 630 ± 37.5 nm) to calculate conidial viability. Image analysis was performed using a BX-Z viewer (KEYENCE Co., Ltd., Osaka, Japan) and BX-Z analyzer (KEYENCE Co., Ltd., Osaka, Japan) built into the device. The conidial viability rate was calculated using the following formula: conidial viability (%) = (number of conidia exhibiting green fluorescence/total number of conidia exhibiting green or red fluorescence) × 100. All experiments were repeated thrice.

Effect of traversianal on rice plants

The rice plants (Oryza sativa L. cv. Koshihikari) used in this study were prepared as described previously (Ashizawa and Zenbayashi 2005). Rice seeds were germinated in tap water, and five germinated seeds were sown in each Petri dish containing Sun soil (Nagata Co., Ltd., Shimane, Japan). The Petri dishes were placed in a plastic case (20 × 10 × 5 cm) and immersed in Uniconazole (Sumiseven; Sumitomo Chemical, Osaka, Japan; 0.25 ppm), a plant growth regulator; and grown to the four-leaf stage in an incubator. The rice plants were treated with traversianal (30 and 40 ppm) and 0.4% DMSO (as a control) by spraying (2 ml per five plants) and kept at 25 ± 2°C for 24 h. The treated rice plants were inoculated by spraying a conidial suspension of P. oryzae (1.0 × 10⁵ conidia/ml; 2 ml per five plants) and maintained in a moist chamber for 24 h in the dark. Seven days after inoculation, the number of blast lesions on the rice leaves was determined. All experiments were repeated thrice, and five rice plants were examined per treatment in each experiment.

Statistical analysis

The data presented in Figs. 1, 3 and 4 are the means and standard deviations and were analyzed for differences using SPSS Statistics ver. 22.0 for Windows (IBM, Armonk, NY, USA). Significant differences in the means between the treatment groups were analyzed using Tukey’s HSD test (P < 0.05).

Antifungal activity of traversianal against P. oryzae determined using conidial germination and TLC bioassays

The inhibitory effect of traversianal on the infection behavior of P. oryzae was evaluated using conidial germination and TLC bioassays. Conidial germination was inhibited by traversianal in a concentration-dependent manner, with over 70% reduction observed at concentrations above 10 ppm compared with the control, and complete inhibition was achieved at 30 ppm (Fig. 1). The percentage of germinated conidia treated with traversianal at 10, 20, and 30 ppm were 25.6 ± 9.8%, 3.7 ± 2.5%, and 0%, respectively, while that treated with 0.3% DMSO (control) was 98.4 ± 2.4% (Fig. 1b). Furthermore, traversianal was detected as a mycelial growth inhibition zone at Rf 0.91 on the TLC plate (Fig. 2).

Fungicidal activity of traversianal against P. oryzae evaluated using double-fluorescence staining

The time course of the fungicidal activity of traversianal against P. oryzae was evaluated using FDA and PI double-fluorescence staining. In the control, treated with 0.4% DMSO, most P. oryzae conidia exhibited green fluorescence (viable conidia) even after 48 h, and no decrease in conidial viability was observed (Fig. 3a). In contrast, the number of conidia exhibiting red fluorescence (non-viable conidia) increased over time in treatments with traversianal at 30 and 40 ppm, indicating a significant reduction in conidial viability (Fig. 3a). The conidial viability of P. oryzae treated with traversianal at 30 and 40 ppm were 60.9 ± 13.7% and 53.7 ± 15.2%, respectively, at 24 h and 14.6 ± 8.8% and 7.9 ± 3.7%, respectively, at 48 h. In contrast, the conidial viability in the control group was 92.4 ± 6.7% and 94.6 ± 2.3% at 24 and 48 h, respectively (Fig. 3b).

Suppressive effect of traversianal on rice blast

Rice leaves were pretreated with traversianal (30 and 40 ppm) to investigate its preventive effect on rice blast disease. A significant decrease was observed in the number of lesions formed on rice leaves treated with 30 and 40 ppm traversianal compared with the control (Fig. 4a). The number of lesions per plant was 109.0 ± 52.6 in the control. In contrast, 69.3 ± 32.8 and 48.7 ± 29.1 lesions were formed on rice leaves treated with traversianal at 30 and 40 ppm, respectively, approximately 36% and 55% fewer than that in the control (Fig. 4b).

The application of chemical pesticides is the main effective method for controlling rice blast and other plant diseases. However, excessive use of chemical fungicides can lead to the development of pathogen resistance. Therefore, there is an urgent need to identify alternative antifungal compounds having novel modes of action.

Terpenoids, the largest class of natural products, possess perhaps the greatest structural diversity and important biological properties (Gershenzon and Dudareva 2007). Natural diterpenoids derived from microorganisms are among the most valuable sources for the identification of novel bioactive compounds (Saha et al. 2022), and some of these compounds possess antifungal activity against plant pathogenic fungi, such as Botrytis cinerea, Colletotrichum coccodes, and Phytophthora infestans (Bi and Yu 2016; Han et al. 2018). Shin et al. (2005) evaluated the antifungal activity of periconicins A and B, which are structurally closely related to traversianal, against various fungal species. Periconicin A showed minimum inhibitory concentration ranging from 3.12 to 200 µg/ml against fungi spanning six different genera. Traversianal inhibits conidial germination and mycelial growth of Colletotrichum orbiculare, a pathogen which causes anthracnose in cucumbers (Ino et al. 2024), and similar results were obtained in P. oryzae in the present study. These findings indicate that traversianal and periconicin A may possess antifungal efficacy against a wide range of fungi.

The fluorescent double staining technique employing FDA and PI is used to assess cell viability, serving as an indicator of compromised cell membrane integrity (Firstencel et al. 1990; Ji et al. 2018). Live conidia exhibit green fluorescence when stained with FDA, and dead conidia exhibit red fluorescence when stained with PI (Palma-Guerrero et al. 2009; Liu et al. 2020). In this study, the proportion of P. oryzae conidia treated with traversianal, which exhibited red fluorescence due to PI, increased over time. This suggests that the membrane integrity of the conidia was progressively compromised by traversianal. In contrast, a small fraction of conidia exhibiting green fluorescence was observed after 48 h of treatment with traversianal at 40 ppm, indicating that higher concentrations or longer treatment times are required to achieve complete fungicidal activity. Further, pretreatment of rice leaves with traversianal 24 h prior to inoculation with P. oryzae significantly suppressed the formation of rice blast lesions. This result indicates that traversianal maintains its antifungal activity on rice leaves for 24 h and is effective in preventing the development of blast disease. However, the mechanism underlying the antifungal activity and the exact duration of suppressive activity on leaves of traversianal are yet to be elucidated and requires further investigation. In conclusion, these results suggest that traversianal, a diterpenoid aldehyde isolated from Cercospora sp. ME202, may contribute to the development of new control agents against rice blasts caused by rice blast fungi.

Acknowledgments

The authors thank the Shimane University for providing financial support for the publication of this report.

Author contributions

Conceptualization: MI, JK, MU; formal analysis and research: MI, MU, AI; original draft preparation: MI, MU, AI; review and editing: MI, MU, AI; funding acquisition: MU; supervision: KJ, MU, AI.

Funding

This study was funded by Shimane University, Japan.

Conflicts of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Antifungal activity of traversianal, a diterpenoid aldehyde from Cercospora sp. ME202, against rice blast fungus Pyricularia oryzae

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Version 1

Abstract

Figures

Introduction

Materials and methods

Chemical preparation

Fungal strains and culture conditions

Conidial germination assay

Thin-layer-chromatography (TLC) bioassay

Determination of fungicidal activity using double-fluorescence staining

Effect of traversianal on rice plants

Statistical analysis

Results

Suppressive effect of traversianal on rice blast

Discussion

Declarations

References

Status:

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