Abstract：
BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds.
RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds\' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds\' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application.
CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds\' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds\' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application.
CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
摘要：
背景：真菌植物病原体是对作物和粮食安全的重大威胁，并且一直需要开发安全有效的化合物来拮抗它们。植物内测定是复杂且繁琐的,因此不适合于新的候选抗真菌化合物的初始高通量筛选。我们提出了一种体外筛选管道，该管道集成了五种快速定量和定性方法，以评估前瞻性抗真菌化合物的功效和作用方式。
结果：使用五种有记录的抗真菌化合物（苯菌灵，儿茶酚,环己酰亚胺,2,4-二乙酰间苯三酚，和苯乙酸）具有不同的作用方式和功效，针对模型土壤传播的真菌病原体尖孢镰刀菌f。sp。黄瓜根。我们最初使用绿色荧光蛋白（GFP）标记的尖孢酵母和PrestoBlue染色评估了五种化合物抑制真菌生长和代谢活性的能力，分别,在多孔板测定中。我们测试了化合物对分生孢子萌发和菌丝伸长的抑制作用。然后我们采用FUN-1和SYTO9/碘化丙啶染色，再加上共聚焦显微镜,在细胞水平上区分真菌生长抑制和死亡。最后,使用活性氧（ROS）检测测定法，我们能够量化响应化合物应用的ROS产生。
结论：总的来说，拟议的管道提供了大量的定量和定性数据的测试化合物，可以帮助查明有希望的新化合物;这些可以更积极地评价使用植物筛选试验。©2024作者害虫管理科学由JohnWiley&SonsLtd代表化学工业学会出版。
结果：使用五种有记录的抗真菌化合物（苯菌灵，儿茶酚,环己酰亚胺,2,4-二乙酰间苯三酚，和苯乙酸）具有不同的作用方式和功效，针对模型土壤传播的真菌病原体尖孢镰刀菌f。sp。黄瓜根。我们最初使用绿色荧光蛋白（GFP）标记的尖孢酵母和PrestoBlue染色评估了五种化合物抑制真菌生长和代谢活性的能力，分别,在多孔板测定中。我们测试了化合物对分生孢子萌发和菌丝伸长的抑制作用。然后我们采用FUN-1和SYTO9/碘化丙啶染色，再加上共聚焦显微镜,在细胞水平上区分真菌生长抑制和死亡。最后,使用活性氧（ROS）检测测定法，我们能够量化响应化合物应用的ROS产生。
结论：总的来说，拟议的管道提供了大量的定量和定性数据的测试化合物，可以帮助查明有希望的新化合物;这些可以更积极地评价使用植物筛选试验。©2024作者害虫管理科学由JohnWiley&SonsLtd代表化学工业学会出版。
