Regulation of elicitin-induced ethylene production in suspension-cultured tobacco BY-2 cells

INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO₂. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.     

Morphological and molecular characterization for a Japanese isolate of tomato powdery mildew Oidium neolycopersici and its host range

 A single conidium of tomato powdery mildew was isolated from heavily infected leaves of tomato (cv. Moneymaker) grown in the greenhouse of Kinki University, Nara Prefecture, Japan. It was successively multiplied so the morphological and taxonomic characteristics of the pathogen and its host range under high humidity conditions could be analyzed. The isolate KTP-01 of the tomato powdery mildew optimally developed infection structures at 25°C under continuous illumination of 3500 lx. More than 90% of the conidia germinated and developed moderately lobed appressoria. After forming haustoria, the pathogen elongated secondary hyphae from both appressoria and conidia. The hyphae attached to leaf surfaces by several pairs of appressoria and produced conidiophores with noncatenated conidia. In addition to its morphological similarity to Oidium neolycopersici, the phylogenetic analysis (based on the sequence of internal transcribed spacer regions of rDNA) revealed that KTP-01 could be classified into the same cluster group as O. neolycopersici. In host range studies, KTP-01 produced abundant conidia on the foliage of all tomato cultivars tested and tobacco (Nicotiana tabacum), and it developed faint colonies accompanied by necrosis on leaves of potato (Solanum tuberosum), red pepper (Capsicum annuum), petunia (Petunia × hybrida), and eggplant (S. melongena). The pathogen did not infect other plant species including Cucurubitaceae plants, which have been reported to be susceptible to some foreign isolates. Thus, the present isolate of the tomato powdery mildew was assigned as O. neolycopersici, a pathotype different from foreign isolates of the pathogen. Received: December 5, 2002 / Accepted: December 26, 2002 Acknowledgments This work was supported in part by a Grant-in-Aid (12660050) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We express our deepest thanks to professor Dr. Y. Sato, Toyama Prefectural University, for his kind and valuable suggestion on taxonomic analysis of the powdery mildew pathogen described in the present study.     

Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages

 In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates (EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains. Received: August 2, 2002 / Accepted: October 31, 2002 Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto, Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations.     

A bolus infusion of xylitol solution in the treatment of cow ketosis does not cause a surge in insulin secretion

When a solution of xylitol was rapidly administered intravenously (bolus infusion) to healthy cattle or those with ketosis, different results were obtained. In healthy cattle, a temporary surge in insulin secretion was observed, whereas in ketotic cattle no such surge was found, but instead a moderate level of secretion continued for a lengthy period. No significant difference in the areas under the insulin curve (AUC) was found between healthy cattle and ketotic cattle up to 120 min after xylitol infusion. These results clearly demonstrated that a bolus infusion of xylitol solution in ketotic cattle does not cause a temporary surge in insulin secretion unlike in healthy animals, but rather results in a continuous, gradual rise in secretion.     

A pea NTPase, PsAPY1, recognizes signal molecules from microorganisms

Apyrases (E.C.3.6.1.5; NTP-NDPases) are distributed in the cytosol, nuclei, cytoskeleton, and on the surface of plant cells. Some may play an important role in signal transduction from exogenous stimuli. We previously found a protein of ca. 55-kDa (CWP-55) in an ATPase-rich fraction from the pea cell wall bound to the elicitor and supprescins (suppressors of defense) from pea pathogen Mycosphaerella pinodes. We cloned the cDNA of CWP-55 that coincided with PsAPY1, one of two NTPase clones in a pea cDNA library. An analysis with a green fluorescent protein fusion protein indicated that PsAPY1 was distributed in the cell wall, nucleus, and cytoplasm. The recombinant PsAPY1 expressed in Escherichia coli had ATP-hydrolyzing activity responsive not only to the elicitor and supprescins from the pea pathogen but also to other elicitors such as a bacterial harpin, a yeast extract, and a synthetic glycopeptide. Biotinylated fungal signal molecules were bound to the recombinant PsAPY1 specifically. Resonant mirror detection confirmed such binding characteristics of PsAPY1. Based on these results, we discuss the role of cell-wall-bound NTPases in recognizing and responding to microorganisms on the cell wall surface.     

An apparatus for collecting total conidia of Blumeria graminis f.sp. hordei from leaf colonies using electrostatic attraction

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 × 10⁴ 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.     

Near infrared spectroscopy for biomonitoring: cow milk composition measurement in a spectral region from 1,100 to 2,400 nanometers

The potential of near infrared spectroscopy (NIRS; 1,100 to 2,400 nm) to measure fat, total protein, and lactose content of nonhomogenized milk during milking and the influence of individual characteristics of each cow's milk on the accuracy of determination were studied. Milk fractions were taken during milking, twice per month, for 6 mo. Samples were taken every 2nd and 4th wk at the morning and the evening milkings. Teatcups were removed at each 3 L of milk yield as determined with a fractional sampling milk meter. A total of 260 milk samples were collected and analyzed with an NIRSystem 6500 spectrophotometer with 1-mm sample thickness. Partial least squares (PLS) regression was used to develop calibration models for the examined milk components. The comparison with the reference method was based on standard error of cross validation (SECV). The obtained SECV varied from .107 to .138% for fat content, from .092 to .125% for total protein, and from .066 to .096% for lactose content, and the accuracy of the reference method (AOAC, 1990, method No 972.16) was .05% for all measured milk components. The obtained models had lower SECV when an individual cow's spectral data were used for calibration. The reduction of SECV for each cow's individual calibration, when compared with SECV for the set of all samples, differed with the different constituents. For fat content determination, the reduction reached 22.46%, for protein 26.40%, and for lactose 31.25%. This phenomena was investigated and explained by principle component analysis (PCA) and by comparing loading of PLS factors that account for the most spectral variations for each cow and the measured milk components, respectively. The results of this study indicated that NIRS (1,100 to 2,500 nm, 1-mm sample thickness) was satisfactory for nonhomogenized milk compositional analysis of milk fractions taken in the process of milking.     

Characterization of the suppressive effects of the biological control strain VAR03-1 of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> on the virulence of tumorigenic <Emphasis Type="Italic">R. vitis</Emphasis>

Rhizobium vitis: strain VAR03-1 is a biological control agent that suppresses grapevine crown gall disease caused by a tumorigenic strain of R. vitis (Ti). Both acetosyringone-induced expression of a virulence gene and the growth of Ti were suppressed in vitro when it was cultivated in the VAR03-1 culture filtrate. These inhibitory effects were reduced by high-temperature treatment or incubation for 72 h. Both activities were detected in the high molecular weight fraction (>?100 kDa) of the filtrate. Our results suggest that the antagonistic effects of VAR03-1 on Ti are mediated by large particle(s) released in the culture media.     

Antibody reactivity in mice and cats to feline enteroepithelial stages of Toxoplasma gondii.

The reactivity of antibodies in mice and cats to feline enteroepithelial stages of Toxoplasma gondii was examined by means of an indirect immunofluorescent antibody test. Mice immunized with feline enteroepithelial stage (FES) parasites produced antibodies not only against FES, but also against tachyzoites, sporozoites/oocysts, tissue cysts and one part of the infected feline enterocytes. After absorption with tachyzoites, the titer of antibodies reactive to enterocytes was significantly reduced. In contrast, the titer of antibodies reactive to FES remained unchanged. The antibodies from cats immunized with FES, reacted specifically to FES, but not to tachyzoites, tissue cysts or enterocytes. These results suggest that FES parasites may have stage-specific antigen(s).     

Gas-liquid chromatographic determination of thiourea in citrus peels

A gas-liquid chromatographic (GLC) method was developed for the detection and determination of thiourea in citrus peels. After the peel is extracted with ethyl ether, the ether extract is adsorbed on sodium sulfate together with water. Thiourea is recoverd from both the sodium sulfate and the peel residue with ethyl acetate-acetone(2+1). The extracted mixture is cleaned on an alumina column, the eluate is concentrated under vacuum, and thiourea is extracted from the concentrate with sodium carbonate solution. GLC was carried out on the prepared benzoyl derivative of thiourea. The average recoveries of thiourea from lemon peel were 85.3, 93.1, and 97.6% at the fortification levels of 1, 10, and 100 ppm, respectively. The detection limit was low as 0.08 ppm.