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

Abstract

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.

Natural woody plant, Mallotus japonicus, as an ecological partner to transfer different pathotypic conidia of Oidium neolycopersici to greenhouse tomatoes

“…Excised leaves were placed in a Petri dish and incubated in a growth chamber for 7 days at 25 ± 1°C and 50-70% relative humidity under continuous illumination of 4000 lux. Conidiophores on the leaves were observed using a KH-2700 high-fidelity digital microscope (Hirox, Tokyo, Japan) according to methods described previously (Matsuda et al 2005).…”

“…This approach is highly effective for collecting living conidia after observation, as no use of chemical treatments for fixation, decolorisation or staining of samples is required (Matsuda et al 2005). We targeted conidiophores for microscopic observations, because of the easy and rapid detection of conidial pseudochains on them (Oichi et al 2004(Oichi et al , 2006.…”

Successional Changes in Powdery Mildew Pathogens Prevailing in Common and Wild Tomato Plants Rotation-cultivated in a Greenhouse

“…Conidiophores in the colonies were observed using a high-fidelity digital microscope KH-2700 (Hirox, Tokyo, Japan) according to the method described previously (Matsuda et al, 2005). In all colonies, conidiophores formed pseudochains with a different number of mature conidia according to their developmental stages; a maximum of four conidia were observed on conidiophores (four-cell pseudochains) (Fig.…”

“…The digital microscope was applied to pathogens living on leaves without any fixation or staining (Matsuda et al, 2005), allowing us to collect living conidia that formed on conidiophores after observation. We next examined the infectivity of isolated single conidia.…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…RT-PCR has been used to detect viable populations of Mycosphaerella graminicola in wheat (Guo et al, 2005). A RT-nested-PCR method was applied for detection of Oidium neolycopersici in tomato (Matsuda et al, 2005). Even so, the most frequent application of this technique in phytopathology is the analysis of plant and fungal gene expression during disease development (Yang et al, 2010).…”