Quantitative nested real-time PCR detection of <Emphasis Type="Italic">Verticillium longisporum</Emphasis> and <Emphasis Type="Italic">V. dahliae</Emphasis> in the soil of cabbage fields

Verticillium longisporum and V. dahliae, causal agents of Verticillium wilt, are spreading through the cabbage fields of Gunma Prefecture. Using the V. longisporum-specific intron within the 18S rDNA and differences between ITS 5.8S rDNA sequences in Japanese isolates of V. longisporum and V. dahliae, we developed three quantitative nested real-time (QNRT) PCR assays. The QNRT-PCR quantification of V. longisporum or V. dahliae in cabbage field soil was consistent with the severity of Verticillium wilt disease in those fields. In field trials of resistant cultivar YR Ranpo grown for three seasons in soil infested with the pathogen, disease severity and pathogen density in the soil were significantly reduced in a field moderately contaminated by V. dahliae, but only slightly reduced in a highly contaminated field. These results suggest that continuous cultivation of a resistant cultivar is an effective way to reduce the pathogen population. QNRT-PCR assays provide a powerful analytical tool to evaluate the soil population dynamics of V. longisporum and V. dahliae for disease management.     

Sexual mating preferences in vitro and in planta among Japanese isolates of Phytophthora infestans

The sexual preferences of Japanese isolates of Phytophthora infestans were determined by mating on agar, in broth, or in plants. The influence of their sexual preference was confirmed in the host tissues. Three wild-type isolates and a -glucuronidase (GUS) transformant were co-cultured to identify the origin of antheridia and oogonia. Japanese A1 isolate had a unique sexual preference compared with foreign isolates. It produced self-fertile oospores with about 40% of total gametangia but tended to form antheridia on V-8 agar medium. In addition, oospores were formed in plants, but their sexual preference could not be reflected in vitro.     

Role of perithecia as an inoculum source for stem rot type of pepper root rot caused by Fusarium solani f. sp. piperis (teleomorph: Nectria haematococca f. sp. piperis)

Pepper (Piper nigrum L.) root rot caused by Fusarium solani f. sp. piperis (FSP; teleomorph: Nectria haematococca f. sp. piperis) includes two symptom types called root rot (RR) type and stem rot (SR) type. In this study, the temporal and spatial associations between perithecial formation of FSP and development of SR were investigated in naturally infested fields to verify the hypothesis that ascospores from the perithecia are the major inoculum source of the SR type on vines in the field. In surveys of all vines in two neighboring pepper fields every month from December 2005 to November 2006, I mapped the locations of all vines with perithecia and all vines the SR type. The frequency of vines with perithecia increased during April and May, the late rainy season. In June, the early dry season, the number of vines with SR type greatly increased. The vertical range of perithecial formation on the vines extended to 200 cm in height, but was restricted to 30 cm in the dry season in both fields. The join-count statistics showed a significant spatial association between vines with perithecia and vines with SR type in one field (P = 0.042), while no significant spatial association was recognized in another field. The results suggested that ascospores from perithecia of FSP on pepper vines are likely to be one of the main inoculum sources of the SR type of the disease on adjacent vines, but they may not be the exclusive source.     

A new spore precipitator with polarized dielectric insulators for physical control of tomato powdery mildew

ABSTRACT 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.     

Sequential observation of 2,6-dimethylaniline-induced nasal lesions in a rat two-stage nasal carcinogenesis model after initiation with N-bis(2-hydroxypropyl) nitrosamine

Male F344 rats received diet containing 3,000 ppm 2,6-dimethylaniline (DMA) after initiation with a single subcutaneous injection of 2,400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN), and histological and electron microscopic examinations of the nasal cavity were performed at 4, 13, 26 and 52 weeks to examine sequential changes induced by DMA. Severe atrophy of Bowman's glands and epithelial disarrangement were apparent from week 4, followed by dilatation and/or proliferation of Bowman's glands, degeneration of epithelial cells, and proliferation of undifferentiated epithelial cells from week 13. Focal glandular hyperplasias, dysplastic foci, and adenomas were observed from week 26, and carcinomas at 52 week. These nasal lesions were mostly evident in the olfactory mucosa in the nasal cavity, and their severity and/or incidences, other than atrophy of Bowman's glands, increased with the treatment period. Electron microscopically, carcinoma cells demonstrated desmosomes, dense secretory granules identical to those in normal Bowman's glands, a basement membrane, and microvilli. These results suggest that Bowman's glands are the target of DMA, giving rise to nasal carcinomas after DHPN-initiation.     

Cytological characteristics of microconidia of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

The inner cellular structure of microconidia of Magnaporthe oryzae was examined using fluorescent probes and electron microscopic techniques. The volume of the nucleus relative to the cell was significantly larger in microconidia than in macroconidia or vegetative hyphae, similar to observations for spermatia of other fungi. Selective fluorescent staining revealed that cytosolic RNA was less abundant in microconidia than in macroconidia and germ tubes, suggesting that general metabolic activity in microconidia is low. Consistently, GFP expression driven by the TrpC promoter was highly active during the formation of phialides and microconidia but gradually decreased as the microconidia matured. Such data suggest that microconidia are in a quiescent or dormant state.     

Use of microsatellite marker Pi26 for rapid discrimination of five Japanese genotypes of Phytophthora infestans

Microsatellite markers were tested to rapidly discriminate five Japanese genotypes (US-1, JP-1, JP-2, JP-3, and JP-4) of Phytophthora infestans. Collected from 1958 to 2007, 111 isolates of Japanese P. infestans were examined using a fluorescent-labeled primer and capillary electrophoresis. Microsatellite marker Pi26 generated specific products for each genotype without any differences in terms of isolation area or year for a particular genotype. The Pi26 marker is a powerful tool for obtaining information on the structure of Japanese populations of P. infestans.