TaqMan qPCR Detection and Quantification of Phytophthora cinnamomi in Soil and Plant Tissues for Walnut Disease Management

Abstract

Phytophthora cinnamomi is a devastating soil-borne plant pathogen. The primary source of P. cinnamomi infection is the soil, where the pathogen can persist for long periods. Effective prevention and management of this pathogen in tree crops requires an early and reliable detection method. In this study, we developed a simple, fast, reliable, and sensitive method based on real-time quantitative PCR (qPCR) for P. cinnamomi detection and quantification directly in plant or soil samples. Primers were developed targeting the nuclear single-copy ras-related protein gene Ypt1, suitable for Phytophthora-specific PCR. The specificity of the assay was confirmed by testing it against genomic DNA from 50 isolates across eight different Phytophthora clades, including the very similar P. parvispora. The efficiency and reliability of the qPCR protocol were evaluated in challenging environmental samples, such as plant tissue of different host trees (walnut, chestnut, oak) and naturally infected soils in walnut orchards. The main outcome was the development of a qPCR method for the specific identification and quantification of P. cinnamomi in natural soil samples. Additionally, this study established a systematic and repeatable soil sampling method and developed an efficient soil DNA extraction technique to apply the developed qPCR in naturally infested soils of walnut orchards.