Fig. 1. Epifluorescent microscopy examination of Magnaporthe oryzae invading rice leaves, and determination of shikimate and spireoside contents. A, Epifluorescent microscopy examination of M. oryzae invading rice leaves of susceptible variety Lijiangxintuanheigu (LTH) and resistant variety Digu. The blast isolate Guy11 was tagged with eGFP. Scale bars, 45 μm. hpi, Hours post-inoculation. B, Shikimate content in LTH and Digu post-inoculation with M. oryzae. C, Spireoside content in LTH and Digu post-inoculation with M. oryzae. Data are Mean ± SD (n = 3). Statistical analysis was performed using a two-sided Student’s t-test. **, P < 0.01.

Fig. 2. Effect of spireoside on blast resistance. A, Rice leaves were punched and concurrently inoculated with 50, 100, and 200 μmol/L spireoside (Spi), respectively, along with Guy11 spores. The concentration of DMSO in the negative control was equivalent to that in the 50, 100, and 200 μmol/L spireoside treatments. B‒D, Rice leaves were treated with three different procedures in a field trial: punched and concurrently inoculated with 200 μmol/L spireoside and Guy11 spores (B), sprayed with 200 μmol/L spireoside 12 h before inoculation with Guy11 spores (C), and sprayed with 200 μmol/L spireoside 12 h after inoculation with Guy11 spores (D). The concentration of DMSO in the negative control was matched to that in the 200 μmol/L spireoside treatment. Lesion density was calculated from 5-cm segments of infected rice leaves. DMSO, Dimethyl sulfoxide. Data are Mean ± SD (n = 21‒30 for A, 16 for B, 13‒14 for C, and 15 for D). Statistical analysis was performed using a two-sided Student’s t-test. *, P < 0.05; **, P < 0.01; and ‘ns’ indicates no statistically significant difference at P > 0.05.

Fig. 3. Effect of spireoside on Magnaporthe oryzae. A, Representative microscopy images showing the effects of 200 μmol/L spireoside on conidial germination and appressorium formation in M. oryzae. The blast isolate Guy11 tagged with eGFP was cultured on hydrophobic coverslips at 26 ºC for 0, 4, 12, 24, and 36 h. Scale bars, 50 μm. B, Quantification of conidial germination and appressorium formation from the images in panel A. C, Representative laser scanning microscopy images of sheath cells from Lijiangxintuanheigu (LTH) infected by the eGFP-tagged blast isolate Guy11. LTH sheaths were concurrently inoculated with Guy11 spores and 200 μmol/L spireoside. Scale bars, 30 μm. D, Distribution of fungal infection progression in panel C. DMSO, Dimethyl sulfoxide; hpi, Hours post-inoculation. Data are Mean ± SD (n = 80 for B and 20 for D). Statistical analysis was performed using a two-sided Student’s t-test. **, P < 0.01; ‘ns’ indicates no statistical significance at P > 0.05.

Fig. 4. Transcriptome analysis of Magnaporthe oryzae at 6 h under 200 μmol/L spireoside treatment. A, Differentially expressed genes (DEGs) under 200 μmol/L spireoside treatment. padj, Adjusted P-value. B, Gene Ontology (GO) analysis for all DEGs under 200 μmol/L spireoside treatment. C, Relative expression levels of random DEGs by qRT-PCR. The concentration of DMSO in the negative control was matched with that in the 200 μmol/L spireoside treatment. DMSO, Dimethyl sulfoxide. Values are Mean ± SD (n = 3). Statistical analysis was performed using a two-sided Student’s t-test. **, P < 0.01.

Fig. 5. Effect of spireoside on membrane integrity of Magnaporthe oryzae. A, Guy11 spores were inoculated on hydrophobic slides treated with H2O, 0.92% DMSO, and 200 μmol/L spireoside, respectively. Conidial membrane integrity was stained by fluorescein diacetate (FDA) and propidium iodide (PI). Red fluorescence indicates disrupted membranes, whereas green fluorescence indicates intact membranes. Scale bars, 20 μm. B, Quantification of conidia with green fluorescence in panel A. DMSO, Dimethyl sulfoxide. Values are Mean ± SD (n = 80). The data represent the means from three biologically independent replicates. **, P < 0.01; ‘ns’ indicates no statistical significance at P > 0.05, as determined by a two-sided Student’s t-test.

Fig. 6. Effect of spireoside on rice sheath blight resistance. A and B, Rice sheaths were treated with 200 μmol/L spireoside 12 h before inoculation (A) and 12 h after inoculation (B) with Rhizoctonia solani isolate AG-1-IA in a field trial. Representative leaf sheaths (left panel) and lesion length data (right panel) are shown. Scale bars, 2 cm. The dimethyl sulfoxide (DMSO) concentration in the negative control was equivalent to that in the 200 μmol/L spireoside treatment. Values are Mean ± SD (n = 40‒59 for A and 31‒46 for B). **, P < 0.01; ‘ns’ indicates no statistical significance at P > 0.05, as determined by a two-sided Student’s t-test.