DataSheet_2_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.

普通黑粉病，又称杜氏小麦黑粉病（Triticum turgidum L. ssp. durum Husn.），由隶属于黑粉菌属（Tilletiales, Exobasidiomycetes, Ustilaginomycotina）的两种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）。该病是全球小麦种植区最具破坏性的病害之一，导致产量损失显著，小麦籽粒和面粉品质下降。鉴于这些原因，开发一种快速、特异、灵敏且经济的早期诊断普通黑粉病小麦幼苗的方法迫在眉睫。虽然已开发出多种分子和血清学方法用于诊断小麦幼苗的普通黑粉病，但这些方法多在后期生育阶段（花序）进行或基于传统PCR扩增，敏感性较低。在本研究中，开发了一种基于TaqMan实时PCR的检测方法，用于快速诊断和定量检测小麦幼苗中的T. laevis，在分蘖期之前。该方法结合表型分析，用于研究有利于病原体感染的条件，并评估基于丁香精油种衣剂控制病害的有效性。总体结果如下：i) 实时PCR检测能在不同配方的丁香精油种衣剂处理后的小麦幼苗中定量检测到T. laevis，显著减少了分析时间。该方法表现出高灵敏度，能检测到高达10 fg的病原体DNA，具有特异性和稳健性，可直接分析粗植物提取物，成为加速遗传育种抗病性测试的有用工具；ii) 温度在使用被T. laevis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精油配方能够有效控制小麦普通黑粉病，表明丁香精油种衣剂可能成为管理病害的有前途的工具，特别是在可持续农业中。