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Detection and Quantification of Leveillula taurica Growth in Pepper Leaves

First, fifth, and eighth authors: Wageningen UR Plant Breeding, Wageningen University & Research Center, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands; second, fourth, and sixth authors: Laboratory of Phytoprotection Science and Technology, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan; third author: Department of Plant Anatomy, Eötvös Loránd University, H-1117 Budapest, Pázmány Péter sétány 1/C, Hungary; and seventh author: Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, H-1525 Budapest, P.O. Box 102, Hungary.

Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved; for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The real-time PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant–microbe interaction studies.