Development and Validation of Specific PCR Assays for Phytopathogens Botrytis cinerea and Xylella fastidiosa, Detection in Vineyard Soil Samples

The cultivation of grapevine (Vitis vinifera) is widely spread in Mexico and globally, the plant’s lifecycle comprises annual fruit production with a potential lifetime of up to 40 years. However, the productivity and longevity of plants are threatened by various diseases. Current control strategies heavily rely on agrochemicals, which implies long-term environmental and soil health risks. Grape producers in Queretaro, Mexico, have observed an increase in diseases that decrements yield and causes crop losses. Experts carry on visual inspections for diagnosis, which are susceptible to failure due to human errors. Accurate and specific disease detection is crucial for establishing effective control measures. In this study, we propose a diagnostic strategy based on PCR tests for the gray mold Botrytis cinerea and Pierce’s disease caused by the bacteria Xylella fastidiosa. Oligonucleotides were designed based on the genes encoding the following products: 16S ribosome, 28S ribosome, cutinase A, pectin methylesterase, and endopolygalacturonase. Through in silico and in vitro analyses, confirmed the specificity of the markers to discriminate between related species. Then we validated their functionality in DNA samples extracted from the soil of the Tierra de Peña vineyard. We analyzed 12 sampling points across 7 sample events from March to October, covering the complete grapevine lifecycle. Our results indicate the absence of these phytopathogens during the 2020 cycle in the study area. The proposed PCR-based tests provide viticulturists with a robust and functional tool for early detection, enabling informed decision-making for disease management in vineyards.