Abstract: 【Objective】Quantitative determination of spore viability of Fusarium solani is the foundation for effective control of the diseases. The traditional spore germination assay is complicated and time consuming, and often with poor sensitivity. Rapid and effective determination methods are needed. The objective of this study is to develop a dual fluorescence assay by combining fluorescein diacetate (FDA) with propidium iodide (PI) for quantitative detection of spore viability of F. solani sensitively and accurately. 【Method】The optimal incubation time, optimal concentrations of FDA and PI were determined, and then the FDA-PI dual fluorescence assay were established. To evaluate the accuracy of FDA-PI assay, pre-defined percentages of dead F. solani spores (0, 25%, 50%, 75% and 100% dead) were detected by FDA-PI assay. The practical mortalities (via FDA-PI assay) were compared with theoretical mortalities. Moreover, spores of F. solani were subjected to physical, chemical and fungicide stress. Relationship between spore mortality (via FDA-PI dual fluorescence assay) and rate of spore germination (via spore germination assay) were analyzed. 【Result】A FDA-PI dual fluorescence assay was developed to assess the activity of F. solani spores. The optimal working concentration of FDA was 100 µg?mL^-1, incubated for 20 min at 25℃. The optimal working concentration of PI was 3 µg?mL^-1, and incubated for 10 min at 4℃. For pre-defined percentages of dead spores, a correlation existed between practical (via FDA-PI assay) and theoretical mortality of spores (R²=0.99, P＜0.05). The result demonstrated that FDA-PI assay allowed for quantification of spores viability of F. solani. Physical treatments (pressurised heat, dry heat and UV light) and chemical treatments (ethanol and hydrogen peroxide) induced gross changes in the activity of the spores of F. solani. There was a negative correlation between spore mortality (via FDA-PI assay) and rate of spore germination (via spore germination assay) (R²=0.99, P＜0.05). For fungicide (fludioxonil, carbendazim and calcium cyanamide) treatment, spore mortalities of F. solani raised as the concentration of fungicide increased. As to spores of F. solani treated by calcium cyanamide, the spore mortality detected by FDA-PI assay was consistent with the result detected by spore germination assay. While the results of FDA-PI assay was below than spore germination assay after fludioxonil and carbendazim treatment. 【Conclusion】A new method was developed for rapid, high-throughput and automatic detection of spore viability of F. solani based on FDA-PI dual fluorescence assay and flow cytometry. This method would replace the traditional spore germination assay, and could shorten the detection time and improve the efficiency greatly.

Key words: fluorescein diacetate (FDA), propidium iodide (PI), fluorescence staining, flow cytometry (FCM); Fusarium solani, viability analysis