DataSheet_1_Rapid molecular assay for the evaluation of clove essential oil antifungal activity against wheat common bunt.pdf

Common bunt of durum wheat (DW), Triticum turgidum L. ssp. durum (Desf.) Husn., is caused by the two closely related fungal species belonging to Tilletia genus (Tilletiales, Exobasidiomycetes, Ustilaginomycotina): Tilletia laevis Kühn (syn. T. foetida (Wallr.) Liro.) and T. caries (DC) Tul. (syn. T. tritici (Bjerk.) G. Winter). This is one of the most devastating diseases in wheat growing areas worldwide, causing considerable yield loss and reduction of wheat grains and flour quality. For these reasons, a fast, specific, sensitive, and cost-effective method for an early diagnosis of common bunt in wheat seedlings is urgent. Several molecular and serological methods were developed for diagnosis of common bunt in wheat seedlings but at late phenological stages (inflorescence) or based on conventional PCR amplification, with low sensitivity. In this study, a TaqMan Real Time PCR-based assay was developed for rapid diagnosis and quantification of T. laevis in young wheat seedlings, before tillering stage. This method, along with phenotypic analysis, was used to study conditions favoring pathogen infection and to evaluate the effectiveness of clove oil-based seed dressing in controlling the disease. The overall results showed that: i) the Real Time PCR assay was able to quantify T. laevis in young wheat seedlings after seed dressing by clove oil in different formulations, greatly reducing times of analysis. It showed high sensitivity, detecting up to 10 fg of pathogen DNA, specificity and robustness, allowing to directly analyze crude plant extracts and representing a useful tool to speed up the tests of genetic breeding for disease resistance; ii) temperature was a critical point for disease development when using wheat seeds contaminated by T. laevis spores; iii) at least one of the clove oil-based formulations tested was able to efficiently control wheat common bunt, suggesting that clove oil dressing could represent a promising tool for managing the disease, especially in sustainable farming.

普通裸麦（DW），即二粒小麦（Triticum turgidum L. ssp. durum (Desf.) Husn.），其病害主要由隶属于黑粉菌属（Tilletiales, Exobasidiomycetes, Ustilaginomycotina）的两个密切相关真菌物种引起：Tilletia laevis Kühn（异名 T. foetida (Wallr.) Liro.）和 T. caries (DC) Tul.（异名 T. tritici (Bjerk.) G. Winter）。该病害是全球小麦种植区最为毁灭性的病害之一，导致产量显著下降以及小麦籽粒和面粉品质的降低。鉴于此，迫切需要一种快速、特定、灵敏且经济的早期诊断小麦种子普通裸麦的方法。虽然已开发了多种分子和血清学方法用于诊断小麦种子的普通裸麦，但这些方法多在后期形态学阶段（花序）或基于传统 PCR 扩增，灵敏度较低。在本研究中，开发了一种基于 TaqMan 实时 PCR 的检测方法，用于快速诊断和量化年轻小麦幼苗中的 T. laevis，在分蘖阶段之前。该方法结合表型分析，用于研究有利于病原体感染的条件，并评估基于丁香油种子处理的疾病控制效果。总体结果如下：i）实时 PCR 检测方法能够量化不同配方中丁香油处理的年轻小麦幼苗中的 T. laevis，显著减少了分析时间。该方法表现出高灵敏度，能够检测到高达 10 fg 的病原体 DNA，特异性强，稳定性好，能够直接分析植物提取物，成为加速抗病性遗传育种测试的有用工具；ii）温度在使用受 T. laevis 菌孢子污染的小麦种子时是病害发展的关键因素；iii）至少有一种测试的丁香油配方能够有效控制小麦普通裸麦，表明丁香油处理可能成为管理该病害的有前景的工具，尤其是在可持续农业中。