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Identification of Individual Powdery Mildew Fungi Infecting Leaves and Direct Detection of Gene Expression by Single Conidium Polymerase Chain Reaction

First to fifth and tenth authors: Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan; sixth author: Pharmaceutical Research and Technology Institute, Kinki University, Osaka 577-8502, Japan; seventh author: Research Institute, Kagome Company, Ltd., Tochigi 329-2762, Japan; eighth author: Agricultural, Food and Environmental Sciences Research Center of Osaka Prefecture, Osaka, 583-0862, Japan; and ninth author: Faculty of Bioresources, Mie University, Mie 514-8507, Japan

Greenhouse-grown tomato seedlings were inoculated naturally with two genera of powdery mildew conidia forming appressorial germ tubes that could not be differentiated by length alone. For direct identification, single germinated conidia were removed from leaves by means of a glass pipette linked to the manipulator of a high-fidelity digital microscope. This microscope enabled in vivo observation of the fungi without leaf decoloration or fungal staining. The isolated conidia were subjected to PCR amplification of the 5.8S rDNA and its adjacent internal transcribed spacer sequences followed by nested PCR to attain sensitivity high enough to amplify target nucleotide sequences (PCR/nested PCR). Target sequences from the conidia were completely coincident with those of the pathogen Oidium neolycopersici or Erysiphe trifolii (syn. Microsphaera trifolii), which is nonpathogenic on tomato. Using RT-PCR/nested PCR or multiplex RT-PCR/nested PCR, it was possible to amplify transcripts expressed in single conidia. Conidia at pre- and postgermination stages were removed individually from tomato leaves, and two powdery mildew genes were monitored. The results indicated that the β-tubulin homolog TUB2-ol was expressed at pre- and postgermination stages and the cutinase homolog CUT1-ol was only expressed postgermination. Combining digital microscopic micromanipulation and two-step PCR amplification is thus useful for investigation of individual propagules on the surface of plants.