Rapid molecular assay for the evaluation of clove essential oil antifungal activity against wheat common bunt

Abstract

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.

Basic Substances and Potential Basic Substances: Key Compounds for a Sustainable Management of Seedborne Pathogens

“…infection on maize and wheat seeds at different doses, but the effective rates (5 × 10 3 and 5 × 10 4 ppm, respectively, for maize and wheat) had a high phytotoxicity effect [46]. Clove oil has also been tested in field trials, both as a seed soak and as coating (spray) on wheat and field peas against, respectively, Tilletia laevis [47] and Ascochyta blight complex [22], artificially inoculated on seeds, with good effectiveness, which varied from year to year. Submersion application has demonstrated a more reliable effectiveness over the years, compared to coating application.…”

Steps to achieve carvone-rich spearmint (Mentha spicata L.) essential oil: a case study on the use of different distillation methods

“…EO constituents in the proper amount and way of application can numb and ease human disorders (Jamshidi-Kia et al, 2018). EOs have diverse properties such as antifungal (Valente et al, 2023), immune boosting (Sandner et al, 2020), cytotoxicity (Pereira et al, 2017), antioxidant (Nadeem et al, 2022), and antibacterial activities (Wu et al, 2019). Researchers have recently become interested in using Spearmint essential oil (SEO) to preserve foodstuffs, including meat (Shahbazi et al, 2018) and fruit (Yan et al, 2021), rather than occasionally environmentally unfriendly materials.…”

Molecular diagnostic assay for pre-harvest detection of Tilletia indica infection in wheat plants

“…Additionally, many previously developed assays are low-throughput, and international protocols involve time-consuming processes like morphological observations and spore isolation (Valente et al, 2019). A series of DNAbased methods have attempted to distinguish teliospores of Tilletia species from seeds or soil samples, which serve as the primary source of pathogen spread (Eibel et al, 2005;Chen et al, 2016;Valente et al, 2023). In earlier studies, PCR based techniques such as RAPD primer-mediated asymmetric polymerase chain reaction (Yuan et al, 2009), sequencecharacterized amplified region (Liu et al, 2009;Gao et al, 2010), repetitive element polymerase chain reaction (Vesna et al, 2011), inter-simple sequence repeat (Gao et al, 2011), amplified fragment length polymorphism (Zhang et al, 2012), and sequence characterized amplified region (Ren et al, 2022a;Xu et al, 2022) have been successfully demonstrated for the identification of Tilletia species.…”