Evaluation of Medicinal Plant Extracts for the Control of Rice Blast Disease

File S1. METHODS

Plant materials and preparation of plant extracts

Therice () cultivar Nipponbare was grown in a greenhouse on soil (Bonsol Number 2; Sumitomo Chemical Corp., Tokyo) at 28 oC/23 oC (day/night) and 50% humidity.

The medicinal plants were collected in the Wuling Mountains. The whole plants of each plant species were extracted with six different solvents (water, ethanol, petroleum ether,-butanol, chloroform, and ethyl acetate), followed by distillation, extraction, and filtration to yield 48 crude extracts, as described previously (Han et al, 2018; Castells-Graells and Lomonossoff, 2021). The solution was dried using a rotary evaporator at 70 oC under reduced pressure and finally completely dissolved in 70% ethanol to a final concentration of ～100 mg/mL.

Ethanol extracts of the roots, stem, leaves, pericarps and seeds ofwere also prepared as described above.

Pathogen culture and inoculation

(race 007.0, MAFF101511, isolated in Aichi prefecture, Japan) was grown on oat agar (30 g oatmeal, 7 g agar, diluted to 1 L with distilled water) at 26 oC in the dark. When the entire plate was covered with a fungal colony (～10 d), mycelia on the surface were gently scraped off with a brush and incubated under continuous black-blue light (FL15BLB; Toshiba, Osaka, Japan) for 3–4 d to generate spores. The spores were washed off with sterile water using a brush, filtered through 2 layers of Kimwipe S-200 (Nippon paper Crecia; Tokyo, Japan). The density of spores was counted using a Fuchs-Rosenthal Haemocytometer under a microscope at 100× magnification, and the spore concentration was adjusted to a density of 1 ×105/mL with sterile water (Akagi et al, 2015; Wang et al, 2019; Li et al, 2021).

For blast inoculation,spores were suspended in 0.02% Tween 20 at a density of 1 × 105/mL and sprayed onto rice seedlings at the four-leaf stage. After incubation in a dew chamber (Ozawa Ltd., Kyoto, Japan) at 24 oC for 24 h, the rice seedlings were moved back to the greenhouse. All disease assays were performed with three biological replicates, and each replicate consisted of 4 plants (= 12). The fourth leaf blades were collected (～100 mg) after 3 or 7 d of inoculation, wrapped in aluminum foil, labeled, snap-frozen in liquid nitrogen, and stored at -80 oC for later use.

Determination of antifungal activity of plant extracts

For spore germination test, the plant extract (50 μL) was mixed with 300 μL of spore suspension (race 007.0), spread on an agar plate (7 g/L agar) and incubated in a standard incubator for 24 h at 26 oC. The final concentration of the plant extract was 14.28 mg/mL (7-fold dilution with distilled water), with the spore density about 0.85 × 105/mL. Isoprothiolane (Fuji-one?, diisopropyl 1, 3-dithiolan-2-ylidene-malonate), a potent fungicide mostly used to control the rice blast fungus, was used as positive control at the final concentration of 20mg/mL, and 10% ethanol was used as negative control. Spore germination was observed under an inverted microscope (Motic BA210; Motic Medical Diagnostic Systems, Co., Ltd., Xiamen, China). A total of 150–350 spores from five microscopic views (replicates), 30–70 spores per view, were counted for each treatment. Spores with a germ tube length longer than the short spore radius were regarded as germinated. The inhibition rate of spore germination caused by each extract was calculated as follows: Inhibition rate (%) = (1 – Germinated spores / Total spores) × 100.

For blast resistance test, the rice seedlings at the four-leaf stage were foliar sprayed withpericarp extract (14.28 mg/mL, average 0.5 mL/plant) prepared in 0.02% Tween 20. After incubation for 24 h, they were inoculated withspores (race 007. 0, 1 ×105/mL). In the control group (Mock), water spray was followed byspore incubation.

Evaluation of blast disease development

The disease development was evaluated by relative fungal growth using qRT-PCR. The fourth leaf blades of blast-inoculated rice seedlings were sampled after 7 d of inoculation, and genomic DNA was extracted using MagExtractor (Toyobo, Osaka, Japan) following the manufacturer’s instructions. qRT-PCR was performed forgenomic 28S rDNA and() on a Thermal Cycler Dice Real Time System TP800 (Takara, Tokyo, Japan) using SYBR premix Ex Taq II (Takara, Tokyo, Japan) with cycles at 95 oC for 5 s (denaturation), 55 oC for 20 s (annealing), and 72 oC for 20 s (extension) (Liu et al, 2020). Primer sequences used for qRT-PCR analysis were showed in Table S3. Relative fungal growth was expressed as the fold ofrDNA amplification relative to the rice() amplification.

Gene expression analysis

The fourth leaf blades of blast-inoculated rice seedlings were sampled after 3 d of inoculation, and Total RNA was extracted using a TRIzol kit(Takara Bio., Tokyo, Japan), cDNA was synthesized using PrimeScript RT reagent kit with gDNA Eraser (Takara Bio., Tokyo, Japan), and gene expression was analyzed by qRT-PCR using a SYBR Premix Ex Taq kit (Takara Bio., Tokyo, Japan).() was used as the internal reference gene (Liu et al, 2020). The gene-specific PCR primers were designed using Primer-Blast 3.0 (National Center for Biotechnology Information, Bethesda, MD) (Table S1), and the specificity of the primers was confirmed by the presence of a single peak in the melting curve obtained after completion of the amplification reaction (data not shown).

Statistical analysis

All experiments were conducted with three replicates. Statistical data analysis was performed using the Statistical Tool for Agriculture Research (STAR) Version 2.0.1 software (International Rice Research Institute, the Philippines). The results were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test with the control (10% ethanol) for the spore germination tests. For the blast inoculation and gene expression analysis experiments, non-parametric-test were performed using Excel software 365 (Microsoft Corporation, Tokyo, Japan). A value of< 0.05 was regarded as statistically significant.

Fig. S1. Inhibition ofspore germination by ethanol extracts from different plant species and parts ofplants.

A–D, Inhibition ofspore germination by extracts from different plant species. A, Isoprothiolane (20 mg/mL) as the positive control; B, Ethanol (10%) as the negative control; C, Ethanol extract ofplants; D,-butanol extract ofplants. E–H, Inhibition ofspore germination by extracts from parts ofplants. E, Isoprothiolane (20 mg/ml) as the positive control; F, Ethanol (10%) as the negative control; G, Root extract (7.14 mg/mL); H, Pericarp extract (7.14 mg/mL).

Table S1.Primer sequences used for qRT-PCR.

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