Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum ofBotrytis cinerea

Abstract

A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould.

<p>Distribution of Class B and Class A β-Lactamases in Clinical Strains of <em>Pseudomonas aeruginosa</em>: Comparison of Phenotypic Methods and High-Resolution Melting Analysis (HRMA) Assay</p>

“…While phenotypebased detection methods have many advantages, molecular real-time amplification techniques have gained significant acceptance because they may provide more rapid detection, increased sensitivity, and specificity, and lack the risk of carry-over contamination associated with earlier methods. 17,19 However, normalized and difference graphs were generated to assess the ability of the HRMA method to differentiate between bacterial strains. 18 This study aimed to investigate different methods in diagnosing different strains of P. aeruginosa.…”

Copper stress induces antioxidant responses and accumulation of sugars and phytochelatins in Antarctic Colobanthus quitensis (Kunth) Bartl.

“…ROS causes different changes at cellular level, the most described in the literature is the interaction with unsaturated bonds in membrane lipids generating toxic aldehydes such as MDA [ 13 ], in the analysis of response of C. quitensis we demonstrate a direct correlation between ROS content and MDA content, but not sufficient to generates a lethal outcome, in the case of intracellular ROS in presence of metallic ions, has been described the generation of superoxide, and leads an increase in SOD activity [ 40 ], our results suggest that the excess of copper generates an increase of superoxide [ 41 ], increase the SOD activity generating H 2 O 2 as a product, the H 2 O 2 would be detoxified by CAT, POD and soluble phenolics [ 42 – 45 ].…”

High-Resolution Melting (HRM) Analysis Reveals Genotypic Differentiation of Venturia inaequalis Populations in Greece

“…The HRM is mainly used in species identification like Alternaria and Aspergillus (Garganese et al, 2018;Xanthopoulou et al, 2019) or SNP detection for resistance like in Botrytis cinerea and Colletotrichum spp. (Chatzidimopoulos et al, 2014b;Forcelini et al, 2018). The data of the present assay suggest that HRM is most suitable for SSR genetic analysis in the haploid fungus V. inaequalis.…”