基于FDA-PI双荧光复染法的茄病镰刀菌孢子活性检测

Determination of Spore Viability of Fusarium solani Based on Dual Fluorescence Assay

【目的】茄病镰刀菌(Fusarium solani)孢子活性的检测是病害有效防控的基础。传统的孢子萌发法操作复杂,耗时费力,需要建立一种简便、快速的孢子活性检测方法。研究旨在建立基于荧光素二乙酸酯(fluorescein diacetate,FDA)-碘化丙啶(propidium iodide,PI)双荧光复染法和流式细胞术(flow cytometry,FCM)的茄病镰刀菌孢子活性检测技术。【方法】通过测定FDA和PI的最佳染色时间和最佳工作浓度,建立茄病镰刀菌孢子活性检测的FDA-PI复染法。为评价该方法的准确性,一方面用FDA-PI法检测已知死孢子比例(0、25%、50%、75%、100%)的茄病镰刀菌样品,分析实测死亡率和理论死亡率的相关性;另一方面,经物理、化学和杀菌剂处理后,比较FDA-PI复染法和孢子萌发法的检测结果。【结果】确定了FDA和PI的最佳染色参数,其中PI的最佳工作浓度为3μg·m L-1,最佳染色时间为4℃处理10 min;FDA的最佳工作浓度为100μg·m L-1,最佳染色时间为25℃处理20 min。用该技术检测已知死孢子比例样品,各样品实测孢子死亡率和理论死亡率之间呈极显著正相关(R2=0.99,P<0.05)。经物理和化学处理后,FDA-PI复染法测得的孢子死亡率与孢子萌发法测得的孢子萌发率之间也呈显著负相关(R2=0.99,P<0.05)。经杀菌剂处理后,随着药剂浓度增高,对茄病镰刀菌杀灭效果增强。其中,氰胺化钙处理后,FDA-PI复染法和孢子萌发法检测孢子死亡率结果一致;而咯菌腈和多菌灵处理后,FDA-PI复染法检测孢子死亡率略低于孢子萌发法检测结果。【结论】建立了基于FDA-PI复染法和流式细胞术的茄病镰刀菌孢子活性检测技术,该技术可以替代传统的孢子萌发方法,大幅度缩减病原菌活性检测时间,提高检测效率,对植物病原真菌活性检测平台的建立具有重要的参考价值,将其应用于杀菌剂的筛选还需要进一步的深入研究。

【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·m L-1, incubated for 20 min at 25℃. The optimal working concentration of PI was 3 μg·m L-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.