Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection.

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/µl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

Proteome Profiling by 2D-Liquid Chromatography Method for Wheat-Rust Interaction.

Wheat-rust interactions are extremely complex biological processes which are accompanied with the defense/attack responses to survive and overcome pathogen attack or plant defense. Understanding of molecular mechanism of these interactions is a promising way to develop sustainable combat. Therefore, many studies have been performed to reveal the active host and pathogen-derived genes and their products during the infection or defense using different approaches for many decades. Particularly proteomics technology and proteome profiling which is a large scale analysis of a protein mixture to reveal differently expressed proteins under a certain conditions has become a very important tool for providing real insights into the extremely complex interactions. Moreover, this type of research has the potential to explore target proteins/genes such as effectors that can be used in disease management strategies. Hence, in this chapter we describe the proteome profiling protocols by using 2D-LC system.