In an attempt to physically protect greenhouse tomato plants from the powdery mildew fungus Oidium neolycopersici, we developed a new electrostatic spore precipitator in which a copper wire conductor is linked to an electrostatic generator and covered with a transparent acrylic cylinder (insulator). The conductor was negatively charged by the generator, and the electrostatic field created by the conductor was used to dielectrically polarize the insulator cylinder. The dielectrically polarized cylinder also produced an electrostatic force without a spark discharge. This force was directly proportional to the potential applied to the conductor and was used to attract conidia of the pathogen. The efficacy of this spore precipitator in protecting hydroponically cultured tomato plants from powdery mildew was evaluated in the greenhouse. The hydroponic culture troughs were covered with a cubic frame installed with the spore precipitator, and the disease progress on precipitator-guarded and unguarded seedlings was traced after the conidia were disseminated mechanically from inoculum on tomato plants. Seedlings in the guarded troughs remained uninfected during the entire experiment, in spite of rapid spread of the disease to all leaves of the unguarded seedlings.

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 beta-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.

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Conidia from living conidiophores of barley powdery mildew ( Blumeria graminis f.sp. hordei ) on host leaves were collected consecutively using an electrostatic spore collector. The collector consisted of an electrical conductor plate linked to an electrostatic voltage generator and insulator plates placed abreast on a timed conveyer. The conductor plate was negatively charged by the potential supplied from the voltage generator. The negatively charged conductor plate caused dielectric polarization of the insulator plate, and the surface charge on the insulator plate attracted mature conidia abstricted from conidiophores on colonies growing on leaves placed 2 cm from the insulator plate. The surface charge on the insulator plate was proportional to the voltage applied to the conductor plate. Under optimized conditions, abstricted conidia were attracted to the electrostatically activated insulator plates without any detriment to their survival. During a colony's life span of c . 460 h, conidia were released throughout the day and c . 12 × 10 4 conidia were collected during the lifetime of the colony. This is the first report on the direct quantification of progeny conidia produced by powdery mildew infecting host leaves.

Two-step PCR (RT-PCR and nested PCR) was used to detect gene expression in powdery mildew pathogen-infected cells of detached inner epidermis of barley coleoptiles. Cellular contents of infected cells were microscopically suctioned with a micropipette and subjected to PCR. Triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase genes involved in the glycolytic pathway and a stimulus-induced endochitinase gene were targeted, and their expression was determined by detecting cDNAs derived from spliced transcripts. The two gycolysis-related genes were constantly expressed in the tissue irrespective of pathogen inoculation. In contrast, chitinase gene expression was induced in non-infected inner epidermis after detachment. After inoculation, this expression was selectively suppressed in pathogen-invaded cells, in spite of continuous expression in non-invaded cells of the same epidermis. Thus, the present method enabled us to directly analyze transcripts in individual cells at the infection site and assess the capability of the pathogen to regulate host gene expression.