We evaluated the effects of fungicides on strawberry powdery mildew (PM) fungus, Podosphaera aphanis, using an electrostatic technique. Thirty-six fungicides were sprayed on single colonies of P. aphanis on leaves of strawberry seedlings (Fragaria × ananassa Duchesne ex Rozier). Colony development varied depending on the tested fungicides. Particularly, pyraziflumid, triflumizole, triforine, polyoxin, sodium hydrogen carbonate + copper wettable powder, and flutianil + mepanipyrim were highly effective for reducing colony development. P. aphanis colonies were histochemically stained to observe the morphological characteristics of fungal cells forming normal and abnormal conidiophores. Abnormal conidiophores were classified into seven types based on their morphological and cytological characteristics. Finally, asexual conidia were collected from single P. aphanis colonies on the leaves spray-treated with fungicides using a dielectrically polarized insulator plate (electrostatic spore collector); conidia attracted to the insulator plates were counted using a high-fidelity digital microscope. Most tested fungicides highly inhibited the production and/or germination of asexual conidia. The germination of asexual conidia was observed only in thiophanate-methyl (methyl benzimidazole carbamates fungicides; MBC fungicides) and azoxystrobin (quinone outside inhibitors; QoI fungicides). Assessing with the electrostatic technique, we clarified that P. aphanis has developed resistance to both thiophanate-methyl and azoxystrobin. Thus, the methodological assessment analyzing the colony development and the number of conidia released from single colonies will be helpful information for screening effective fungicides.

In this study, we evaluated the effectiveness of hyperparasitic fungi in controlling powdery mildew (PM). In a greenhouse, we spray-inoculated single colonies of the melon PM-causing fungus Podosphaera xanthii strain KMP-6N at three different fungal developmental stages (i.e., 5, 10, and 15 days old) with spores of the hyperparasitic fungus Ampelomyces sp. strain Xs-q. After spray inoculation, we collected and counted KMP-6N conidia produced as asexual progeny from PM colonies using an electrostatic rotational spore collector. Collector insulator films were replaced at 24 h intervals until KMP-6N ceased to release additional progeny conidia. Conidial releases from each of the single Xs-q-inoculated KMP-6N colonies gradually reduced, then stopped within ca. 4 and 8 days of the first treatment in 5- and 10-day-old KMP-6N colonies, and within ca. 20 days of the second spray treatment in 15-day-old KMP-6N colonies, respectively. The total numbers of asexual progeny conidia collected from single 5-, 10-, and 15-day-old colonies were ca. 156, 1167, and 44,866, respectively. After electrostatic spore collection, conidiophores in Xs-q-uninoculated KMP-6N colonies appeared normal, whereas almost all conidiophores in 5- and 10-day-old Xs-q-inoculated KMP-6N colonies were completely deformed or collapsed due to the infection of the hyperparasitic fungus. This is the first study to apply electrostatic and digital microscopic techniques to clarify the impact of fungal hyperparasitism on mycohost survival, and, in particular, to assess quantitatively and visually the suppression of conidial release from any PM colonies infected with Ampelomyces.

Powdery mildew fungi produce progeny conidia on conidiophores, and promote the spread of powdery mildew diseases by dispersal of the conidia from conidiophores in the natural environment. To gain insights and devise strategies for preventing the spread of powdery mildew infection, it is important to clarify the ecological mechanism of conidial dispersal from conidiophores. In this study, all of the progeny conidia released from single colonies of strawberry powdery mildew fungus (Podosphaera aphanis (Wallroth) U. Braun and S. Takamatsu var. aphanis KSP-7N) on true leaves of living strawberry plants (Fragaria × ananassa Duchesne ex Rozier cv. Sagahonoka) were consecutively collected over the lifetime of the colony with an electrostatic rotational spore collector (insulator drum) under greenhouse conditions, and counted under a high-fidelity digital microscope. The insulator drum consisted of a round plastic container, copper film, thin and transparent collector film, electrostatic voltage generator, and timer mechanism. When negative charge was supplied from the voltage generator to the copper film, the collector film created an attractive force to trap conidia. The electrostatically activated collector film successfully attracted progeny conidia released from the colony. Experiment was carried out at just one colony on one leaf for each month (in February, May, July, October, November, and December in 2021), respectively. Each collector film was exchanged for a new collector film at 24 h intervals until KSP-7N ceased to release progeny conidia from single colonies. Collection experiments were carried out to estimate the total number of conidia released from a single KSP-7N colony over a 35–45-day period after inoculation. During the fungal lifetime, KSP-7N released an average of 6.7 × 104 conidia from each of the single colonies at approximately 816 h. In addition, conidial release from KSP-7N colonies was largely affected by the light intensity and day length throughout a year; the number of conidia released from single KSP-7N colonies in night-time was clearly smaller than that in daytime, and the time of conidial release from single KSP-7N colonies was shorter by approximately 2 to 4 h in autumn and winter than in spring and summer. The ecological characteristics related to conidial releases from KSP-7N colonies will be helpful information for us to successfully suppress the spread of strawberry powdery mildews onto host plants under greenhouse conditions.

This review examines the progress of electrostatic spore-trapping research and the potential for the practical application of electrostatic apparatuses in powdery mildew control. These apparatuses produce an electric field by charging an insulated conductor wire (ICW). Airborne pathogen spores are subjected to an attractive force in the electric field and are drawn to the charged ICW as a result of dielectrophoretic movement. The strength of the attractive force is commensurate with the field strength (determined by the magnitude of the voltage applied to the ICW). Single-charged monopolar electric field screens (SM screens) are constructed by arraying negatively charged cylindrical ICWs in parallel at a specific interval. The connected electric fields of these ICWs form a gap-free air-shielding barrier. Wind-dispersed spores are precipitated by this barrier to create spore-free air. Oppositely charged SM screens have been combined to develop double-charged dipolar electric field screens, which generate a stronger spore attraction force under lower voltage application. Thus, electric field screens represent a promising physical method for creating spore-free spaces in cropping facilities, where plants can be cultivated without risk of infection by airborne fungal pathogens.

An unattended pest control system was developed to eliminate whiteflies (Bemisia tabaci) that settled on greenhouse tomato plants. The system exploited the whitefly’s habit of flying up from a plant that was mechanically tapped and then heading toward yellow objects. Remote-controlled dollies with arms that tapped plants and yellow-colored double-charged dipolar electric field screens (YDD-EFSs) (oppositely electrified transparent insulator tubes filled with yellow-colored water) attracted and trapped the whiteflies. The whiteflies flew up when the plants were mechanically tapped with the dolly’s arms during reciprocating movements and were subsequently trapped by YDD-EFSs that were automatically translocated to the target plants. The system was applied to rows of whitefly-infested tomato plants. Almost all whiteflies transferred to plants were successfully recovered by two dollies moving on either side of the plants, approaching all plants individually (via programmed movement). In summary, we present an efficient unattended method for controlling whiteflies on tomato plants in greenhouses.

Electrostatic devices generating an electric field (EF) are promising tools for greenhouse tomato cultivation. In these devices, an EF is generated in the space surrounding an insulated conductor (IC) that is charged by a voltage generator. Thus, a physical force is exerted on any insect that enters the EF, as a negatively charged IC (NC-IC) pushes a negative charge (free electrons) out of the insect body. The insect is polarized positively to be attracted to the NC-IC, and a grounded metal net (G-MN) repels the insect. This dual function of the apparatus (insect capture and repulsion) is the core of the electrostatic pest-exclusion strategy. In this study, we applied various innovative EF-based devices to evaluate their efficacy in greenhouse tomato cultivation. Our objective was to determine the optimal apparatus for simple, inexpensive construction by greenhouse workers. The results of this study will contribute to the development of sustainable pest-management protocols in greenhouse horticulture.

In the present study, we analyzed negative electricity released from insects captured by an electric field (EF)-producing apparatus. Adult houseflies (Musca domestica) were used as the model insect. The EF producer consisted of a negatively charged polyvinyl chloride membrane-insulated iron plate (N-PIP) and a non-insulated grounded iron plate (GIP) paralleled with the N-PIP. An EF was formed in the space between the plates. A housefly placed on the GIP was physically attracted to the N-PIP, and electricity released from the fly was detected as a specific transient electric current at the time of attraction and during subsequent confinement of the fly to the N-PIP. The magnitude of the insect-derived electric current became larger as the voltage applied to the N-PIP increased. We determined the total amount of electric current and confinement time within the apparatus necessary to kill all captured flies. These results demonstrate the insecticidal function and insect-capturing ability of the EF-producing apparatus.

Type I trichomes of tomato leaves (Solanum lycopersicum Mill. cv. Moneymaker), as outgrowths of the plant epidermis, are suitable for monitoring infection processes of powdery mildew species using a high-fidelity digital microscope (DM) without fungal staining. On the trichomes, tomato powdery mildew (Pseudoidium neolycopersici L. Kiss) isolate KTP-03 produced a maximum of four vigorously elongated hyphae per conidium, which stopped growth approximately 12 days after inoculation. Single trichome cells, invaded by fungal hyphae at various fungal infection stages during the 12-day period after the inoculation of single conidia, were cut at the bases and directly collected with small precision scissors (i.e., microscissors) held by the manipulator under a DM. Subsequently, suc-polymerase chain reaction (PCR) (reverse transcription (RT)-PCR followed by nested (N)-PCR) was conducted to explore gene expression in the infected trichome. We selected intron-containing genes from tomatoes and powdery mildew fungi for the detection of constitutive gene transcripts, namely plasma membrane H+-ATPase (LHA2) and β-tubulin 2 (TUB2) genes. In suc-PCR, a single band from spliced mRNAs of both LHA2 and TUB2 genes were detected, suggesting that both genes were successfully transcribed in single KTP-03-infected trichomes. With combined primers for both LHA2 and TUB2 (multiplex RT-PCR/N-PCR), two bands were detected through the amplification of intron-spliced mRNAs of both genes. Therefore, our single-trichome cell PCR amplification method is effective for detecting the expression patterns of genes from both tomato and powdery mildew fungus. Combinations of digital microscopy, microscissors, and multiplex RT-PCR/N-PCR amplification techniques will be useful for simultaneously analysing the molecular interactions between plants and powdery mildew fungi at the level of single tomato leaf trichome cells. Also, this employed technique will be of benefit in other plant species and crops, possessing leaf trichome cells, to elucidate the molecular interactions between plants and pathogens.

Powdery mildew fungi infect plant leaves, reducing the yield of infected melon plants. Therefore, an eco-friendly method of controlling powdery mildew in melon plants needs to be developed. A previous study described how the morphological characteristics of the conidiophores of the melon powdery mildew fungus Podosphaera xanthii Pollacci (designated KMP-6N) grown under greenhouse (natural) conditions and red light-emitting diode (LED) irradiation differed from those grown under growth chamber conditions and blue LED irradiation. In the present study, conidiophores with unconstricted conidia under blue light were collected and inoculated onto host leaves through micromanipulation; the unconstricted conidia germinated and infected the leaves, producing vigorously elongated hyphae. The number of conidia collected, the initial times of conidial release from single colonies, and the number of conidia remaining in chains on conidiophores were examined with electrostatic techniques. Under red light, the number of collected conidia gradually increased with the light irradiation period. The initial conidial release occurred between 2 to 4 h; the number of conidia remaining on the conidiophores gradually decreased and, eventually, the conidiophore lengths became shorter. In contrast, under blue light, few conidia were collected at any given time; the number of conidia on the conidiophores gradually increased and, eventually, the conidiophore lengths became longer. Next, the effects of red and blue light on the spread of powdery mildew infection by placing a KMP-6N-infected melon seedling at the centre of a tray containing healthy melon seedlings were examined. Almost all healthy seedlings caused powdery mildew symptoms at ca. 21 days after red light irradiation, whereas only healthy seedlings near the infected seedlings showed symptoms after blue light irradiation. Thus, the spread of melon powdery mildew infection clearly differed between red and blue light irradiation. This is the first report describing the effects of red and blue light on the spread of P. xanthii infection from a single infected seedling to healthy host seedlings; their results provide insight into the ecological mechanisms of powdery mildew conidial scatter from conidiophores.

Our aim was to develop an electrostatic apparatus to lure and capture silverleaf whiteflies (Bemisia tabaci), vegetable leafminers (Liriomyza sativae), and western flower thrips (Frankliniella occidentalis) that invade tomato greenhouses. A double-charged dipolar electric field producer (DD-EFP) was constructed by filling water in two identical transparent soft polyvinyl chloride tubes arrayed in parallel with fixed separation, and then, inserting the probes of grounded negative and positive voltage generators into the water of the two tubes to generate negatively and positively charged waters, respectively. These charged waters electrified the outer surfaces of the opposite tubes via dielectric polarization. An electric field formed between the oppositely charged tubes. To lure these phototactic insects, the water was colored yellow using watercolor paste, then introduced into the transparent insulator tubes to construct the yellow-colored DD-EFP. This apparatus lured insects in a manner similar to commercially available yellow sticky traps. The yellow-colored DD-EFP was easily placed as a movable upright screen along the plants, such that invading pests were preferentially attracted to the trap before reaching the plants. Furthermore, pests settling on the plants were attracted to the apparatus, which used a plant-tapping method to drive them off the plants. Our study provided an experimental basis for developing an electrostatic device to attract and capture insects that enter greenhouses.

This study analysed the mechanism of avoidance behaviour by adult Turkestan cockroaches (Shelfordella lateralis Walker) in response to a static electric field (S-EF) formed in the space between a negatively charged polyvinyl chloride-insulated iron plate (N-PIP) and a grounded metal net (G-MN). The negative surface charge supplied to the iron plate by a voltage generator caused the G-MN to polarise positively via electrostatic induction. In the S-EF, the negative charge of the N-PIP created a repulsive force that pushed free electrons in the field toward the ground via the G-MN. When insects released in the space surrounded by the S-EF inserted their antennae into the S-EF, they pulled them back reflexively and moved backward. The analysis indicated that an electric current flowed transiently toward the ground when an insect inserted its antennae into the S-EF. The insect became positively charged via this discharge and was attracted to the opposite pole (N-PIP). In response to this attractive force, the insect pulled its antennae back quickly. The positive electrification caused by the removal of free electrons from the antenna tip triggered the avoidance behaviour.

A total of 26 Ampelomyces strains were isolated from mycelia of six different powdery mildew species that naturally infected their host plants in Japan. These were characterized based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions and actin gene (ACT) fragments. Collected strains represented six different genotypes and were accommodated in three different clades of the genus Ampelomyces. Morphology of the strains agreed with that of other Ampelomyces strains, but none of the examined characters were associated with any groups identified in the genetic analysis. Five powdery mildew species were inoculated with eight selected Ampelomyces strains to study their mycoparasitic activity. In the inoculation experiments, all Ampelomyces strains successfully infected all tested powdery mildew species, and showed no significant differences in their mycoparasitic activity as determined by the number of Ampelomyces pycnidia developed in powdery mildew colonies. The mycoparasitic interaction between the eight selected Ampelomyces strains and the tomato powdery mildew fungus (Pseudoidium neolycopersici strain KTP-03) was studied experimentally in the laboratory using digital microscopic technologies. It was documented that the spores of the mycoparasites germinated on tomato leaves and their hyphae penetrated the hyphae of Ps. neolycopersici. Ampelomyces hyphae continued their growth internally, which initiated the atrophy of the powdery mildew conidiophores 5 days post inoculation (dpi); caused atrophy 6 dpi; and complete collapse of the parasitized conidiphores 7 dpi. Ampelomyces strains produced new intracellular pycnidia in Ps. neolycopersici conidiophores ca. 8–10 dpi, when Ps. neolycopersici hyphae were successfully destroyed by the mycoparasitic strain. Mature pycnidia released spores ca. 10–14 dpi, which became the sources of subsequent infections of the intact powdery mildew hyphae. Mature pycnidia contained each ca. 200 to 1,500 spores depending on the mycohost species and Ampelomyces strain. This is the first detailed analysis of Ampelomyces strains isolated in Japan, and the first timing and quantification of mycoparasitism of Ps. neolycopersici on tomato by phylogenetically diverse Ampelomyces strains using digital microscopic technologies. The developed model system is useful for future biocontrol and ecological studies on Ampelomyces mycoparasites.

The purpose of this study was to develop a simple electrostatic apparatus to precipitate virus particles spread via droplet transmission, which is especially significant in the context of the recent coronavirus disease 2019 (COVID-19) pandemic. The bacteriophage φ6 of Pseudomonas syringae was used as a model of the COVID-19 virus because of its similar structure and safety in experiments. The apparatus consisted of a spiked, perforated stainless plate (S-PSP) linked to a direct-current voltage generator to supply negative charge to the spike tips and a vessel with water (G-water) linked to a ground line. The S-PSP and G-water surface were paralleled at a definite interval. Negative charge supplied to the spike tips positively polarised the G-water by electrostatic induction to form an electric field between them in which ionic wind and negative ions were generated. Bacteriophage-containing water was atomised with a nebuliser and introduced into the electric field. The mist particles were ionised by the negative ions and attracted to the opposite pole (G-water). This apparatus demonstrated a prominent ability to capture phage-containing mist particles of the same sizes as respiratory droplets and aerosols regardless of the phage concentration of the mist particles. The trapped phages were successfully sterilised using ozone bubbling. Thus, the present study provides an effective system for eliminating droplet transmission of viral pathogens from public spaces.

The purpose of the study was to construct an electrostatic insect-capturing apparatus that could be applied to a drone (quadcopter). For this purpose, a double-charged dipolar electric field screen (DD-screen) was constructed using oppositely charged insulator tubes that was then attached to a drone. For charging, the inner surface of the tubes was coated with a conductive paste and then linked to a negative or positive voltage generator. The opposite charges of the tubes formed an electric field between them and created an attractive force to capture insects that entered the field. The DD-screen constructed here was sufficiently light to enable its attachment to a drone. The screen was hung from the drone perpendicular to the direction of drone movement, so as to receive the longitudinal airflow produced by the movement of the drone. It was positioned 1.8 m below the drone body to avoid the influence of the downward slipstream generated by the rotating propellers. Eventually, the drone was able to conduct a stable flight, with sufficient endurance, and captured airborne insects carried by an airflow of 8 m/s during the flight. This study, therefore, provides an experimental basis for establishing a new method for conducting trap-based monitoring of airborne insects during remote-controlled flight through operation of a DD-screen attached to a drone.

The present study was conducted to establish an electrostatic-based experimental system to enable new investigations of insect behavior. The instrument consists of an insulated conducting copper ring (ICR) linked to a direct current voltage generator to supply a negative charge to an ICR and a grounded aluminum pole (AP) passed vertically through the center of the horizontal ICR. An electric field was formed between the ICR and the AP. Rice weevil (Sitophilus oryzae) was selected as a model insect due to its habit of climbing erect poles. The electric field produced a force that could be imposed on the insect. In fact, the negative electricity (free electrons) was forced out of the insect to polarize its body positively. Eventually, the insect was attracted to the oppositely charged ICR. The force became weaker on the lower regions of the pole; the insects sensed the weaker force with their antennae, quickly stopped climbing, and retraced their steps. These behaviors led to a pole-ascending–descending action by the insect, which was highly reproducible and precisely corresponded to the changed expansion of the electric field. Other pole-climbing insects including the cigarette beetle (Lasioderma serricorne), which was shown to adopt the same behavior.

An electrostatic apparatus was constructed to capture tobacco sidestream smoke. This apparatus consisted of a perforated polypropylene plate with metal spikes and a grounded metal net arrayed in parallel at a defined interval. Spikes were negatively charged to positively polarize the net and an electric field was formed between the opposite charges of the spike tips and the grounded net. Discharge from the spike tips occurred, which depended on the pole distance and the voltage applied to the spikes. At lower voltages (<12.1 kV) that do not cause arc discharge from the tips, a corona discharge occurred with the generation of an ionic wind from the spiked plate to the net. This discharge increased in direct proportion to the applied voltage and relative humidity, while a larger corona discharge generated a stronger ionic wind. The ionic wind involved negative ions and the number of negative ions in the wind increased with increasing applied voltage. The optimal voltage (10 kV) generated sufficient negative ions to ionize smoke particles in the electric field, before the ionized smoke particles were successfully captured by the oppositely charged metal net. Thus, this study provides an experimental basis for the practical application of an electrostatic-based method to prevent the production of tobacco sidestream smoke that leads to passive smoking by non-smokers.

The lengths of conidiophores in fungal colonies of the melon powdery mildew pathogen Podosphaera xanthii Pollacci KMP-6 N cultured under greenhouse (natural) conditions differed markedly from those cultured in a growth chamber. We hypothesized that light wavelength was responsible for the differences in conidiophore length. In this study, we examined the effects of light-emitting diode (LED) irradiation (purple, blue, green, orange, and red light) and white light on colony development and conidiophore formation in KMP-6 N using a stereomicroscope and a high-fidelity digital microscope. Colonies on leaves were flat under greenhouse conditions and under red LED light irradiation but were stacked under growth chamber conditions and under purple, blue, green, and orange LED light irradiation. In addition, KMP-6 N formed catenated conidia comprising six conidia per conidiophore under greenhouse conditions and red light but more than seven conidia per conidiophore under growth chamber conditions and purple, blue, green, and orange light. Furthermore, almost none of the conidia on top of the conidiophores grown under blue light were fully constricted. Therefore, these fungi could not scatter their conidia and spread infection. This is the first report of the effects of LED lights on conidiophore formation in the melon powdery mildew fungus P. xanthii. The results provide insight into the mechanisms underlying the responses of conidiophores to light of specific wavelengths and conidial scatter from conidiophores of melon powdery mildew fungi.

The present study aimed to explore the possibility of using the type I trichomes of tomato (Solanum lycopersicum) to monitor the infection processes of powdery mildews by microscopy. Individual trichome cells of two tomato genotypes were inoculated with pathogenic and non-pathogenic powdery mildew species, Pseudoidium neolycopersici, Erysiphe trifoliorum and Podosphaera xanthii. On the trichome cells of the tomato cultivar Moneymaker, hyphae of the pathogenic Pseudoidium neolycopersici (isolates KTP-03 and KTP-04) grew vigorously whereas hyphal growth of the non-pathogenic Erysiphe trifoliorum and Podosphaera xanthii ceased after appressorium formation, which was associated with papilla formation and hypersensitive cell death, respectively. Similar infection processes of the tested powdery mildews were seen in Moneymaker epidermal cells. Therefore, tomato trichomes are suitable for analysing, at individual cell level, the infection processes of different pathotypes of powdery mildews and for observing the cytological responses of plants by non-pathogenic powdery mildews. On the other hand, it was observed that both isolates KTP-03 and KTP-04 failed to produce conidiophores on the hyphae elongating on Moneymaker trichomes. Similarly, no conidiophores were produced on the hyphae elongating on trichomes of Solanum peruvianum LA2172, which is resistant to KTP-03 and susceptible to KTP-04. Interestingly, delayed cell death occurred in LA2172 epidermal cells, which were attacked by KTP-03 hyphae elongating from trichomes and conidiophores were formed on new hyphae growing from the leaf epidermal cells. Thus, leaf trichomes and epidermal cells of the wild tomato species LA2172 reacted differently to the avirulent isolate KTP-03.

An electrostatic-barrier-forming window (EBW) was devised to capture airborne pollen, which can cause allergic pollinosis. The EBW consisted of three layers of insulated conductor wires (ICWs) and two voltage generators that supplied negative charges to the two outer ICW layers and a positive charge to the middle ICW layer. The ICWs generated an attractive force that captured pollen of the Japanese cedar, Cryptomeria japonica, from air blown through the EBW. The attractive force was directly proportional to the applied voltage. At >= 3.5 kV, the EBW exerted sufficient force to capture all pollen carried at an air flow of 3 m/s, and pollen-free air passed through the EBW. The findings demonstrated that the electrostatic barrier that formed inside the EBW was very effective at capturing airborne pollen; thus, it could allow a home to remain pollen-free and healthy despite continuous pollen exposure.

A microscopy-based needle micromanipulation technique for direct injection of foreign material into target cells was refined as a novel method of aspiration of cellular contents from a single target cell using a micropipette. Subsequently, we performed direct PCR amplification of mRNAs transcribed in target tomato callus cells. Friable calli were induced from leaf explants, and single cells from active small cell aggregates were used as targets for aspiration. The aspirated cellular contents were subjected to RT-PCR and subsequent nested PCR amplification analyses. Five tomato genes (LHA2, GAPDH, CHI3, PI2 and TLC1) were selected from the cDNA database as PCR targets. The GAPDH and LHA2 genes were amplified from all aspirated samples and were used as indicators of successful PCR amplification. Aspiration of only the cytosol (excluding the nucleus), facilitated amplification of mature target gene mRNA that was not contaminated with PCR products derived from the genomic DNA sequences of the target genes. We identified novel stimulus activated genes, such as CHI3 and TLC1, which were constitutively transcribed in tomato callus cells.

Single-cell polymerase chain reaction (PCR) was conducted to detect in situ gene expression in targeted cells of tomato leaf trichomes. The cytoplasm was removed with a micropipette under a light microscope and subsequently used for reverse transcription PCR (RT-PCR), followed by nested PCR. Two intron-containing genes, a glyceraldehyde 3-phosphate dehydrogenase gene and a plasma membrane H+-ATPase gene, were constantly expressed in these cells and therefore used as indicators of successful PCR reactions. In addition, the use of nucleus-free cellular contents for the RT-PCR and subsequent nested PCR analyses was effective for preventing contamination with the products derived from misamplification of corresponding genomic DNA sequences. Using this method, we detected the expression of certain stimuli-activated genes, following the exposure of trichome cells to volatile chemicals. Therefore, the present technique can be used to directly detect gene expression in single trichome cells of tomato leaves in response to external stimulation.

This study examined whether Agrobacterium-mediated T-DNA insertion was useful for obtaining mutant melon plants. For this purpose, we used Agrobacterium rhizogenes because of its frequent hairy root production when inoculated into plants and the easy detection of fruit odours produced by the hairy roots. Of the 6534 hairy root clones obtained, five clones were mutants emitting fruit odours, and the KMH-4196 clone produced the strongest melon fruit odours. These odours were due to the production of at least four compounds that were not detected in non-aromatic hairy root clones: (Z)-3-hexenol, (E)-2-hexenal, 1-nonanoland (Z)-6-nonenol. (Z)-6-nonenol was the most important and was produced stably during successive subculture for 3 years. Molecular analyses indicated that a single copy of T-DNA from the inoculated bacteria was integrated into a chromosome in the clone, suggesting that Agrobacterium-mediated T-DNA insertion is an effective mutagenesis method in higher plants.

Melons (Cucumis melo L.) grown hydroponically in a greenhouse were heavily infested with powdery mildew. We isolated powdery mildew pathogens from the melon leaves and identified the isolate as Podosphaera xanthii KMP-6N, based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions. Host ranges of KMP-6N were determined by estimating the infectivity or pathogenicity after inoculating the conidia onto multiple plant species. The fungi caused severe powdery mildew symptoms on Cucurbitaceae plants, producing scattered conidia on conidiophores. The goal of this study was to observe KMP-6N conidiogenesis on melon leaves. The pathogen formed completely catenated conidiophores approximately 24 h from conidiophore erection to release of mature conidia. Six conidia were produced on the conidiophores and only the conidia at the apex reached maturity. The cycles of conidial release were repeated on melon leaves 14 to 18 times, at approximately 6-h intervals. In the final stage, conidia were released without causing growth and septation of generative cells. Conidiophores produced an average of 36 conidia during a 90-h period. In our study, the modes of conidiogenesis, lifetime of conidiophores and productivity of conidia on a conidiophore were described for powdery mildew fungi.

Our greenhouse tomatoes have suffered from attacks by viruliferous whiteflies Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) over the last 10 years. The fundamental countermeasure was the application of an electric field screen to the greenhouse windows to prevent their entry. However, while the protection was effective, it was incomplete, because of the lack of a guard at the greenhouse entrance area in fact, the pests entered from the entrance door when workers entered and exited. To address this, we developed a portable electrostatic insect sweeper as a supplementary technique to the screen. In this sweeper, eight insulated conductor wires (ICWs) were arranged at constant intervals along a polyvinylchloride (PVC) pipe and covered with a cylindrical stainless net. The ICWs and metal net were linked to a DC voltage generator (operated by 3-V alkaline batteries) inside the grip and oppositely electrified to generate an electric field between them. Whiteflies on the plants were attracted to the sweeper that was gently slid along the leaves. This apparatus was easy to operate on-site in a greenhouse and enabled capture of the whiteflies detected during the routine care of the tomato plants. Using this apparatus, we caught all whiteflies that invaded the non-guarded entrance door and minimized the appearance and spread of the viral disease in tomato plants in the greenhouse.

We examined the defensive responses of leaf surfaces, rhizoids, and protonemata of the moss Aphanoregma patens (Hedw.) Lindb. to inoculation with zoospores and encysted zoospores (cysts) of the oomycete Pythium aphanidermatum OPU849. Aphanoregma patens infected with Pythium exhibited extensive browning of the leaves and stems 4 days after inoculation. The zoospore infection sites were rhizoids and protonemata. Cysts on the rhizoids and protonemata germinated and formed elongated hyphae that invaded the cells of the moss, forming oospores and lobed sporangia. The subsequent production of new zoospores on infected plants indicates that A. patens is a suitable host for manipulating the life cycle of Pythium. However, cysts inoculated onto leaf surfaces did not germinate, and they began to show signs of disruption after only 3 hours. After 2 days, more than 80% of the cysts on leaf surfaces were completely disrupted, possibly due to the secretion of degrading enzymes. Cyst disruption was not observed on rhizoids nor on protonemata.

The apical buds of lateral rose branches (Rosa hybrida 'Carl Red') were asexually propagated by cutting and treatment with chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, 6-azacytidine, and acridine orange), and the growth and differentiation or morphological alterations of the mutagen-treated buds were traced in developed flowers. Variations in size, shape, colour, and the number of petals were detected most frequently in flowers produced from apical buds treated with 100 mu g/ml N-methyl-N'-nitro-N-nitrosoguanidine. The variant petals were cultured on Murashige and Skoog medium supplemented with a-naphthalene acetic acid and 6-benzylaminopurine for in vitro isolation and the reproduction of morphologically altered rose plants. Embryogenic calli were obtained from adventitious roots induced from the petals and were successfully differentiated into intact plants. Consequently, the regenerated plants produced flowers that were different from those originally used for tissue culture. The appearance of the flowers in these rose plants was highly consistent through repeated cycles of cutting, suggesting that the present approach is an easy and rapid procedure for mutation breeding in rose in combination with tissue culture techniques for the in situ isolation and propagation of variant plants. This approach is expected to provide an effective method for easily and rapidly inducing variations in rose flowers and for in vitro morphogenesis through their regeneration.

Meristematic tissues removed from aseptically cultivated tomato seedlings (Solanum lycopersicum Mill.), breeding line KT007, were treated with two chemical mutagens (N-methyl-N'-nitro-N-nitrosoguanidine and 5-azacytosine), and growth and differentiation or morphological changes in the mutagen-treated leaf primordia were traced in developed leaves. Morphological variations were most frequently detected in leaves from meristems treated with 100 mu g/ml N-methyl-N'-nitro-N-nitrosoguanidine. Variegated leaves were cultured to obtain callus tissues for in vitro isolation and to propagate morphologically altered tomato plants using Murashige-Skoog medium supplemented with different concentrations of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid. Eventually, calli were obtained from the leaves and successfully differentiated into plants with variegated leaves using medium supplemented with 2.0 mu g/ml 6-benzylaminopurine. Consequently, the regenerated plants produced variegated leaves similar to those in the plants originally used for tissue culture. This approach using chemical mutagens is an easy and effective way to induce variegated leaves and to rapidly propagate their regenerants in vitro. Our results demonstrate that this approach is an effective method for easily and rapidly inducing variations in tomato leaves and for analysing tomato leaf morphogenesis through regeneration based on an in vitro tissue culture system.

An electric field screen can be used to keep mosquitoes out of houses with open windows. In this study, doubly charged dipolar electric field screens (DD-screens) were used to capture mosquitoes entering through a window. The screen had two components: three layers of insulated conductor iron wires (ICWs) in parallel arrays and two electrostatic direct current (DC) voltage generators that supplied negative or positive voltages to the ICWs. Within each layer, the ICWs were parallel at 5-mm intervals, and connected to each other and to a negative or positive voltage generator. The negatively and positively charged ICWs are represented as ICW(-) and ICW(+), respectively. The screen consisted of one ICW(+) layer with an ICW(-) layer on either side. The Asian tiger mosquito (Aedes albopictus) and house mosquito (Culex pipiens) were used as models of vectors carrying viral pathogens. Adult mosquitoes were blown into the space between the ICWs by sending compressed air through the tip of an insect aspirator to determine the voltage range that captured all of the test insects. Wind speed was measured at the surface of the ICW using a sensitive anemometer. The result showed that at >= 1.2 kV, the force was strong enough that the ICWs captured all of the mosquitoes, despite a wind speed of 7 m/s. Therefore, the DD-screen could serve as a physical barrier to prevent noxious mosquitoes from entering houses with good air penetration.

An electric field screen is a physical device used to exclude pest insects from greenhouses and warehouses to protect crop production and storage. The screen consists of iron insulated conductor wires (ICWs) arrayed in parallel and linked to each other, an electrostatic DC voltage generator used to supply a negative charge to the ICWs, and an earthed stainless net placed on one side of the ICW layer. The ICW was negatively charged to polarize the earthed net to create a positive charge on the ICW side surface, and an electric field formed between the opposite charges of the ICW and earthed net. The current study focused on the ability of the screen to repel insects reaching the screen net. This repulsion was a result of the insect's behaviour, i.e., the insects were deterred from entering the electric field of the screen. In fact, when the screen was negatively charged with the appropriate voltages, the insects placed their antennae inside the screen and then flew away without entering. Obviously, the insects recognized the electric field using their antennae and thereby avoided entering. Using a wide range of insects and spiders belonging to different taxonomic groups, we confirmed that the avoidance response to the electric field was common in these animals.

Old books are highly susceptible to mould infection, and an effective method for avoiding moulding is needed to safely preserve valuable books in library stack rooms. Guarding a bookshelf with an electric field screen is a physical method that prevents airborne spores from entering the space used for book preservation. In this study, insulated conductor wires (ICWs) were used as electrodes to form electric fields. The ICWs were arrayed in parallel and linked to each other and to a direct current voltage generator. The electric field screen consisted of two layers of ICWs, which were negatively and positively charged with equal voltages to make dipoles, ICW(-) and ICW(+). Both ICWs generated an attractive force that captured airborne spores of Penicillium digitatum that were blown inside the screen. The attractive force was directly proportional to the applied voltage. At a parts per thousand 0.9 kV, the screen exerted sufficient force to capture all airflow-carried spores, but a few spores that were once captured were repulsed out of the electric field when subsequent spores were attracted to positions proximal to them. This phenomenon was explained by creeping discharge between spores located close to each other on the ICW surface. This spore-repulsion problem was resolved by adding an additional ICW layer to the electric field screen, namely an electric field screen with an ICW(-) layer on both sides of an ICW(+) layer. The present study demonstrated that the three-layered electric field screen remained mould-free inside a screen-guarded bookshelf, irrespective of continuous spore exposure.

A simple electrostatic apparatus was devised to measure dischargeable electricity and bioelectric potentials produced by flies. The apparatus involved two insulated electrodes, ICW(-) and ICW(+), oppositely charged with equal voltages supplied by two voltage-generators. In the electric field, the flies became net positive by instantaneously discharging their electricity and were attracted to negative surface charges on ICW(-). The tail-lifting movement by the attracted insect was an action creating electric potentials that could cause discharge of ICW(-). The discharge transiently appeared in response to individual movements and was larger when the tail was lifted at higher angles. (C) 2013 Elsevier B.V. All rights reserved.

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet-like leaf tissues) and gemmae (small, flat, sheet-like leaf tissues that propagate asexually via bud-like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP-02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP-4N) and examined their germination and subsequent appressorium formation under a high-fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP-4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP-4N conidia inoculated onto M.polymorpha on both leaf surfaces. No differences in destruction of the KRCP-4N fungal structures between thalli and gemmae were observed. At 4h post-inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6h post-inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP-02 conidia were not destroyed and formed normal, well-lobed appressoria on the surface of M.polymorpha gemmae.

Powdery mildew disease caused by Leveillula taurica is a serious fungal threat to greenhouse tomato and pepper production. In contrast to most powdery mildew species which are epiphytic, L. taurica is an endophytic fungus colonizing the mesophyll tissues of the leaf. In barley, Arabidopsis, tomato and pea, the correct functioning of specific homologues of the plant Mlo gene family has been found to be required for pathogenesis of epiphytic powdery mildew fungi. The aim of this study was to investigate the involvement of the Mlo genes in susceptibility to the endophytic fungus L. taurica. In tomato (Solanum lycopersicum), a loss-of-function mutation in the SlMlo1 gene results in resistance to powdery mildew disease caused by Oidium neolycopersici. When the tomato Slmlo1 mutant was inoculated with L. taurica in this study, it proved to be less susceptible compared to the control, S. lycopersicum cv. Moneymaker. Further, overexpression of SlMlo1 in the tomato Slmlo1 mutant enhanced susceptibility to L. taurica. In pepper, the CaMlo2 gene was isolated by applying a homology-based cloning approach. Compared to the previously identified CaMlo1 gene, the CaMlo2 gene is more similar to SlMlo1 as shown by phylogenetic analysis, and the expression of CaMlo2 is up-regulated at an earlier time point upon L. taurica infection. However, results of virus-induced gene silencing suggest that both CaMlo1 and CaMlo2 may be involved in the susceptibility of pepper to L. taurica. The fact that overexpression of CaMlo2 restored the susceptibility of the tomato Slmlo1 mutant to O. neolycopersici and increased its susceptibility to L. taurica confirmed the role of CaMlo2 acting as a susceptibility factor to different powdery mildews, though the role of CaMlo1 as a co-factor for susceptibility cannot be excluded.

Leveillula taurica is an obligate fungal pathogen that causes powdery mildew disease on a broad range of plants, including important crops such as pepper, tomato, eggplant, onion, cotton, and so on. The early stage of this disease is difficult to diagnose and the disease can easily spread unobserved for example, in pepper and tomato production fields and greenhouses. The objective of this study was to develop a detection and quantification method of L. taurica biomass in pepper leaves with special regard to the early stages of infection. We monitored the development of the disease to time the infection process on the leaf surface as well as inside the pepper leaves. The initial and final steps of the infection taking place on the leaf surface were consecutively observed using a dissecting microscope and a scanning electron microscope. The development of the intercellular mycelium in the mesophyll was followed by light and transmission electron microscopy. A pair of L. taurica-specific primers was designed based on the internal transcribed spacer sequence of L. taurica and used in real-time polymerase chain reaction (PCR) assay to quantify the fungal DNA during infection. The specificity of this assay was confirmed by testing the primer pair with DNA from host plants and also from another powdery mildew species, Oidium neolycopersici, infecting tomato. A standard curve was obtained for absolute quantification of L. taurica biomass. In addition, we tested a relative quantification method by using a plant gene as reference and the obtained results were compared with the visual disease index scoring. The realtime PCR assay for L. taurica provides a valuable tool for detection and quantification of this pathogen in breeding activities as well in plant-microbe interaction studies. © 2013 The American Phytopathological Society.

筆者らは近畿大学農学部(奈良県)においてトマトの一段採り密植栽培を実施しているが，近年，近畿地区においてもトマト黄化葉巻病の発生が問題となってきた。そこで，2007年の栽培から黄化葉巻症状の調査を開始したところ，2009年7月，養液栽培トマトに黄化葉巻の症状が確認され，温室内にタバココナジラミが発生していたことから，トマト黄化葉巻病の可能性が示唆された。本実験では，黄化葉巻の症状を示すトマトからトマト黄化葉巻病ウイルス(TYLCV)の検出を試みるとともに，タバココナジラミのバイオタイプの確認を行ったので報告する。

In our routine surveys for the powdery mildew disease in greenhouse tomatoes, we detected a new pathogen that forms pseudochains consisting of 12 conidia. To identify the original plant that dispersed this pathogen, wild plants infected with powdery mildew were monitored. The pathogen on Japanese mallotus, Mallotus japonicus, produced a similar type of pseudochain, and conidia were infectious to tomatoes. Inversely, the conidia on the tomato leaves infected M. japonicus. Infectivity assays and internal transcribed spacer (ITS)-based phylogenetic analyses indicated that the two pathogens on the tomato and M. japonicus were identical. These results suggest that the conidia on M. japonicus can be transmitted to greenhouse tomatoes. This work documents the ecological transmission of conidia between wild plants and greenhouse tomatoes.

An electric field screen was constructed to examine insect attraction mechanisms in multiple electric fields generated inside the screen. The screen consisted of two parallel insulated conductor wires (ICWs) charged with equal but opposite voltages and two separate grounded nets connected to each other and placed on each side of the ICW layer. Insects released inside the fields were charged either positively or negatively as a result of electricity flow from or to the insect, respectively. The force generated between the charged insects and opposite ICW charges was sufficient to capture all insects. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4767635]

Dual functions (insect repelling and capturing) of a single-charged dipolar electric field screen were evaluated to successfully exclude whiteflies from a window-open greenhouse. The screen consisted of three parts: 1) insulated conductor wires (ICWs) arrayed in parallel at 5 mm intervals, 2) two earthed stainless nets placed within 3 mm of both sides of the ICW layer, and 3) a voltage generator for the negatively charged ICWs. The screen formed two electric fields between the ICW-layer and the ICW-side surface of the earthed net and between the ICWs. At negative charging of 1.5-2.5 kV, all whiteflies reaching the outer surface of the screen net avoided entering the electric field and flew away from the screen. This avoidance was disturbed by 3 m s(-1) wind, as the insects were compulsorily blown inside. However, almost all whiteflies (99.4 %) were captured with the ICW. These results indicate that the insect-capturing function is effective to complement a failure to repel. A greenhouse assay was conducted in the screen-attached and non-screened parts in which a greenhouse was divided with a partition. During the 3-month operation, the screen was durable and functional for excluding pests, and better air ventilation changed the climate conditions in the greenhouse. Thus, the present study demonstrated that our electric field screen can provide an airy condition for tomatoes in a window-open greenhouse and successfully exclude whiteflies using dual screen functions.

An insulated conductor wire (ICW) paralleled with an earthed net was used to observe movements by vinegar flies in relation to their electricity release. ICW was negatively charged to create a positive charge on the net. At particular voltages, flies were attracted to ICW. This attraction was triggered by the deprivation of the insect negative charge with the net. Eventually the insects became net positive and were drawn to the ICW negative charge. The attracted insects generated bioelectricity through skeletal muscular movements. However, the electricity produced was depleted by the net without neutralizing their positive charge in the insect body. (c) 2012 Elsevier B.V. All rights reserved.

Hypersensitive response (HR) of plant cells to the attack of pathogens induces resistance to subsequent attacks by a broad spectrum of pathogens, leading to acquired resistance. In this study, we characterized the localized acquired resistance (LAR) in the epidermal cells of tomato. First, we report the discovery of a new isolate of tomato powdery mildew occurring in Japan, KTP-02, which has a different virulence spectrum compared with the previously-characterized isolate, KTP-01. Using these two isolates, we investigated LAR phenomenon in the epidermal cells of tomato plants carrying the Ol-4 resistance gene. Ol-4 encodes a nucleotide-binding site leucine-rich repeat protein that triggers HR in the epidermal cells in response to KTP-01 but not KTP-02. We mounted a single conidium of KTP-01 on a single tomato epidermal cell and then monitored the progress of HR in that cell by live microscopy. Once HR occurred in that cell, we mounted a single conidium of KTP-02 on cells adjacent to or at one-cell distance from the first challenged cells, in different time points. With a digital microscope, we consecutively tracked the progress of HR (i.e., induction of LAR) in those cells. Results showed that, in tomato plants carrying the Ol-4 gene, HR to KTP-01 results in induction of HR in the adjacent epidermal cells challenged with KTP-02. Our results show that LAR can be triggered only in adjacent cell layer and lasts 24 to 48 h after HR occurred in the first cell. We did not observe the reverse phenomenon, induced susceptibility to KTP-01 by KTP-02. Altogether, we report an advanced technique for investigating LAR phenomena, and provide data on spatiotemporal characteristics of LAR in tomato epidermal cells.

An electric field screen (EF-screen) is a physical device for excluding pest insects from greenhouses and warehouses to protect crops during their production and storage periods. In this study, a simple version of the EF-screen, an insulated conductor iron wire (ICW) paralleled to an earthed net, was constructed to effectively observe the attraction of test insects in relation to their electricity release. The ICW was negatively charged to dielectrically polarise the insulator sleeve of the ICW: negatively on the outer surface and positively on the inner conductor wire surface of the sleeve. The negative surface charge of the ICW caused an electrostatic induction in the earthed net and a resultant positive charge at the ICW-side surface of the net. An electric field formed between the ICW (negative pole) and earthed net (positive pole). Insects were attracted to the ICW when they were placed onto the earthed net. A vital step for the attraction was the creation of a transient bioelectric discharge from an insect. During this discharge, an electric charge of the insect was transferred to the earthed net. Eventually, the insect became net positive and was then attracted to the ICW. The magnitude of the current increased in direct proportion to the increase in voltage applied to the ICW, and the attraction force was directly proportional to the increase in the electric current. Larger voltages were necessary to attract much larger insects because larger insects were stronger and therefore more able to escape from the ICW attraction. Similar results were obtained for a wide range of pest insects belonging to different taxonomic groups (8 orders and 15 families). This study demonstrated that transient bioelectric discharge is common in insects and can be utilised to create an electrostatic force capable of moving insects in a generated electric field.

In an attempt to control insect pests affecting greenhouse tomatoes, we evaluated an electric field screen to create an airy greenhouse condition that successfully excluded insect vectors (whiteflies, green peach aphids, western flower thrips, shore flies) of pathogens. The screen consisted of three parts: 1) insulated conductor wires (ICWs) arrayed in parallel at 5-mm intervals, 2) two stainless-steel nets that were grounded and placed on both sides of the ICWs, and 3) a DC voltage generator to negatively charge the ICWs. An electric field formed between the negative surface charge of the ICWs and the positive charge on the ICW-side surface of the grounded net. The ICWs captured insects that entered the field. Insects that contacted the outer surface of the screen net avoided the electric field and flew away from the screen. During continuous 3-month greenhouse operation, the screen was durable and functional in exerting stable pest exclusion and good air penetration for ventilation under changing greenhouse climate conditions. Thus, our electric field screen provided an airy condition for tomatoes in an open-window greenhouse that successfully excluded flying insect pests.

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector-this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the "two-step germination" of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.

A bifunctional electric field screen was proposed to physically exclude insect pests from warehouses. The screen consists of insulated iron wires (ICW) arranged in parallel and two earthed conductor nets placed on both sides of the ICW. A negative charge (0.1-8.0 kV) was applied to the insulated wires with a voltage generator to polarize an insulator sleeve used to cover the wire, negatively on the outer surface and positively on the inner conductor wire surface of the sleeve. The negative surface charge of the ICW caused an electrostatic induction in the earthed nets and an opposite charge on the net surfaces facing the ICW. An electric field formed in a space between the ICW and the earthed net, and the field strength increased in direct proportion to increasing voltages applied to the ICW. Adults of the test insects (cigarette beetle (Lasioderma serricorne) and vinegar fly (Drosophila melanogaster)) reaching the outer surface of the earthed net were deterred from entering the inside of the charged screen, whereas all insects immediately passed through the screen when the ICW was not charged. This avoidance was directly proportional to the increase in the voltage. In addition, the capability of the screen to capture insects that enter inside the screen was proven by introducing insects into the space between the ICW and the earthed net. Strong capture was observed when the ICW was negatively charged with more than 4.1 kV, under which conditions a short-term electric current (peaking at 0.3-0.6 mu A, for 3 min) occurred transiently. This electric current was due to the release of electricity from the insects, giving a net overall positive charge to the insects, which therefore were attracted more strongly to the negatively charged ICW. A test using an attractant-set chamber showed that the insects were completely prevented from passing through the charged screen, in contrast to a rapid transfer of all insects when the screen was not charged. Thus, the present results show that the described screen is a promising physical tool for controlling insect pests in warehouses. (C) 2010 Elsevier Ltd. All rights reserved.

[要旨] トマトは、栽培種トマトと野生種トマトに大きく分類されている。栽培種トマトは我が国の主要作物であり、その用途は生鮮や加工品など多岐にわたる。また、食品機能性に富み、これまでに、抗酸化作用、抗炎症作用、抗ガン作用、抗アレルギー作用など、様々な機能性が認められている。一方、野生種トマトに関しては、既に著者らのグループにおいて、昆虫に対する忌避作用や抗菌作用などの効果を明らかにしており、さらに多くの機能性物質の存在が示唆されている。そこで本研究では、各種野生種トマトの栽培試験により優良形質のトマトを選抜し、それらの機能性成分の探索、ならびにアンチエイジング素材の開発を目的とした、生物寿命に及ぼす影響についての検討を行った。[Abstract] In order to examine nutraceutical effects of wild tomato species, we screened wild tomato species that were suitable for cultivation and extracted substances useful for longevity prolongment. Namely, twentyone of totally of 41wild tomato species used possibly produced fruits of different shapes, colors（red, green and purple）and sizes. Antioxidative and polyphenolic substances were detected in some fractions of fruit- and leaf-extracts obtained from both common and wild tomato species. All fractions of leafextracts were less effective to prolong life span of the test insect, with no relation to common and wild tomato plants. In point A, there was no significant difference in the longevity prolongment of the test insect among all fractions of both common and wild tomato plants. In point B, two fractions （hexanesoluble and ethyl acetate-soluble fractions）of fruit-extracts from both common and wild tomato plants were positive to the lifespan prolongment of vinegar fly adults. Hexane-soluble fraction of fruit-extracts from wild tomato plants was most effective to prolong lifespan of the test insect. In point C, ethyl acetatesoluble fraction of fruit-extracts from both plants showed the significant effect on the prolongment of lifespan. Antioxidants and polyphenolic substances in ethyl acetate-soluble fractions were effective to prolong lifespan of the test insect. Polyphenols are potential substances to extend lifespan of diverse species including vinegar fly. Hexane-soluble fraction of fruit-extracts from wild tomato plants was positive to the lifespan prolongment, it contained substances effective to prolong lifespan of the test insect. Judging from these results, both common and wild tomatoes are useful sources to search various nutraceutical substances for the longevity prolongment, as assessed by the insect assay system.

The process of host/non-host determination was dissected in interactions of Epilachna vigintioctopunctata, a specialist herbivore of solanaceous plants, with various plant species. On host plants (tomato and egg plant) the ladybird beetle started feeding within 5 min. On red pepper, another solanaceous plant, it also started feeding within 5 min, but did not continue the feeding as vigorously as on tomato or eggplant. This result suggests that the ladybird beetle recognizes red pepper as a host plant but does not overcome its constitutive resistance. On Chinese cabbage, the ladybird beetle did not start feeding as quickly as on the host plants, but once started, it continued feeding as vigorously as on the host plants. This result suggests that the ladybird beetle does not recognize Chinese cabbage as a host plant but overcomes its constitutive resistance. Subsequently, the effect of induced resistance in a host (tomato) and non-hosts (Chinese cabbage and Arabidopsis) was evaluated. The treatment with methyl jasmonate (MeJA) showed no effects in tomato but decreased the damaged area in Chinese cabbage and Arabidopsis. A feeding test with Arabialopsis mutants supported the idea that induced resistance via the jasmonic acid (JA) pathway is effective against the ladybird beetle on the cruciferous plants. We suggest that a specialist herbivore has to overcome not only constitutive resistance but also induced resistance to utilize the non-host plant as a host, and that induced resistance is one of the factors that determine host specificity of the specialist. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Family 19 chitinase from Aeromonas sp. No.10S-24 (72.6 kDa) is composed of two chitin-binding domains (ChBDs), two proline- and threonine-rich (PT-rich) linkers, and a catalytic domain. The purified enzyme was labile in a standard buffer condition and spontaneously degraded into a 46-kDa fragment upon storage at 4 degrees C. The N-terminal sequence of the 46-kDa fragment was found to correspond to the sequence of the G terminal region of the second PT-rich linker, indicating that the 46-kDa fragment is produced by truncation of the two ChBDs and the two PT-rich linkers from the mature protein, and consists only of the catalytic domain. The hydrolytic activities toward insoluble and soluble substrates were significantly reduced by the truncation of two ChBDs. In addition, antifungal activity determined from the digestion rate of haustoria of powdery mildew was reduced by the ChBD truncation. Although the profile of the time-course of N-acetylglucosamine hexasaccharide [ (GlcNAc)(6)] degradation catalyzed by the ChBD-truncated enzyme was similar to that of the mature enzyme protein, the specific activity of the ChBD-truncated enzyme determined from the rate of hexasaccharide degradation was lower than that of the mature enzyme. The two CBDs appear to be responsible for facilitating the hydrolytic reaction. The sugar residue affinities (binding free energy changes) at the individual subsites, (-2) (-1) (+1) (+2) (+3) (+4), were estimated by modeling the hexasaccharide hydrolysis by the mature and ChBD-truncated enzymes. The truncation of ChBDs was found to strongly affect the affinity at the (4) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.

The chitinase secreting strain KPM-012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM-012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM-012A-entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.

The chitinase secreting strain KPM-012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM-012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM-012A-entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for I week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.

The chitinase gene-transformed strain KPM-007E/chi Of Enterobacter cloacae was vitally entrapped in sodium alginate gel beads with its specific virulent bacteriophage EcP-01 to provide a new method for microbially digesting chitinous peritrophic membranes of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, chitinase SH1 from a gram-positive bacterium Kurthia zopfii was overproduced by Escherichia coli cells and purified by affinity column chromatography. The purified enzyme effectively digested peritrophic membranes dissected from the ladybird beetles to expose epithelial tissues beneath the peritrophic membrane, and the beetles that had ingested chitinase after submergence in chitinase solution had considerably reduced their feeding on tomato leaves. KPM-007E/chi, entrapped in the alginate beads, released the chitinase. More chitinase was released when KPM-007E/chi was present with their specific virulent bacteriophage EcP-01 in the beads because of lysis of bacterial cells infected with the bacteriophages. This chitinase release from the microbial beads (containing KPM-007E/chi and EcP-01) was sufficient to digest the peritrophic membrane as well as to suppress feeding of bead-sprayed tomato leaves by the ladybird beetles. A daily supply of tomato leaves treated with the microbial beads considerably suppressed leaf feeding and oviposition by the ladybird beetles, suggesting a possible application of chitinase-secreting bacteria for suppressing herbivorous insect pests.

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.

Volatile oils were obtained from melon hairy roots produced by the infection with Agrobacterium rhizogenes, and their constituents were analyzed by GC and GC-MS spectroscopy. Characteristic major components were 1-nonanal, (Z)-6-nonenol, and (E, Z)-2, 6-nonadienal. The melon hairy roots are thus shown to produce volatile components with the fragrance of ripe melon fruit. © 2003, Japan Oil Chemists' Society. All rights reserved.

Aims: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. Methods and Results: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)(3). To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. Conclusions: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. Significance and Impact of the Study: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.

A chitosan-degrading bacterium, isolated from field soil that had been amended with chitin, was identified as Sphingobacterium multivorum KST-009 on the basis of its bacteriological characteristics. The extracellular chitosanase (SM1) secreted by KST-009 was a 34-kDa protein and could be purified through ammonium sulfate precipitation, gel permeation column chromatography and SDS polyacrylamide gel electrophoresis. A chitosanase gene (csnSM1) was isolated from genomic DNA of the bacteria, and the entire nucleotide sequence of the gene and the partial N-terminal amino acid sequence of the purified SM1 were determined. The csnSM1 gene was found to encode 383 amino acids, 72 N-terminal amino acid residues were processed to produce the mature enzyme during the secretion process. Germinated microconidia of four formae speciales (lycopersici, radicis-lycopersici, melonis, and fragariae) of Fusarium oxysporum were treated with SM1. Chitosanase treatment caused morphological changes, such as swelling of hyphal cells or indistinctness of hyphal cell tips and cessation or reduction of mycelial elongation.

During a year-round survey on the occurrence of powdery mildew on greenhouse-cultivated tomato plants, the disease was most severe in June and July. All tomato plants (45 commercial cultivars and 11 breeding lines) tested were infected with the pathogen but had different degrees of susceptibility. The pathogen was epiphytic and produced white, round pustules mainly on leaves of tomato plants. The pathogen produced conidia singly on conidiophores and forked appressoria on inoculated tomato leaves and seemed to be an Oidium sp. of Erysiphe polygoni type.

[Synopsis] Tissue culture was used for selecting and enhancing the resistant variant cells which indigenously exist in leaf tissues of the susceptible cultivar in order to establish an efficient system for producing crown-rot resistant lines of strawberry. Leaves of the resistant and susceptible cultivars were inoculated with the crown-rot pathogen by needle-prick inoculation method. Two types of lesions (small necrotic spots and extensively expanding lesions) were formed on leaves of both the resistant and susceptible cultivar. The resistant cultivar was characterized by higher percentage of the small necrotic spots. The cells forming the resistant-type lesion were amplified by inducing callus tissues from leaf explants of the susceptible cultivar, and regenerants showing the higher frequencies of the resistant-type lesion were selected. The amplification of these cells to the level of the resistant cultivar was achieved by repeating callus induction and plant regeneration from the selected repenerants. The regenerants finally obtained in the present system showed the resistance in the field inoculation test.

[Synopsis] In this study, we cloned a chitinase gene from Burkholderia cepacia and identified a nucleotide sequence. A genomic library was constructed by the use of λEMBL3, and then phage clones producing chitinase were selected by using 4-MU chitotrioside as a substrate. The inserted DNA was subcloned into a pBluescript vector with restriction enzymes to make pBlueCAK12 (4.4 kb) clone including a chitinase gene. The DNA fragment was deleted by exonuclease III and the chitinase-coding region was determined by chitinase activity detected by the use of 4-MU chitotrioside. The nucleotide sequence of chitinase gene (2.98 kb) was determined by primer walking method, in which ORF of chitinase gene was amplified by specific primers including the expected start and stop codons. The amplified DNA was inserted into a pBluescript vector, and then chitinase activity was detected by the use of 4-MU chitotrioside.

[Synopsis] From the hairy roots induced by an inoculation of Agrobacterium rhizogenes into tobacco (Nicotiana tabacum cv. Bright Yellow) leaf segments, we obtained the hairy root clone which could frequently regenerate shoots from calli produced at the basal sites of lateral roots branched from major hairy roots. The leaflets detached from the regenerated shoots produced secondarily the hairy roots and differentiated into the secondary shoots via calli in a similar manner. This circulatory culture system for the hairy root production and plant regeneration was available for an in vitro assay of multiplication and translocation of TMV. By the inoculation into detached leaves of the regenerated shoots with TMV, the virus-specific mosaic symptom was observed in the leaflets of the newly regenerated shoots, and the localization of TMV in the symptomless tissues such as stems, petioles, and secondary hairy roots of the infected regenerants was easily detected by direct tissue printing immunoblotting assay.Konagaya, Ken-ichi

トマト重要病害である青枯病を効果的に防除するため、 マーカー遺伝子で標識した青枯病菌を作出し、 その感染挙動を明確にするとともに、 体細胞変異を利用して選抜した抵抗性トマト系統におけるその挙動についても検討した。

トマト食葉性昆虫であるニジュウヤホシテントウの生物的防除を行なうため、 トマト葉に生息する細菌を分離・同定し、 その細菌にキチン分解酵素遺伝子を導入して食害抑制効果を検討した。

キチナーゼおよびキトサナーゼ遺伝子を大腸菌発現ベクターに導入し、 それら酵素の大量生産系を確立した。 また、 大量生産したキチナーゼとキトサナーゼをそれぞれ単独および混合してトマトうどんこ病菌に処理した結果、 混合処理区で効率的な分解が確認できた。

本研究の目的は、微生物遺伝子の導入によるメロン毛状根の生産物質を有効成分とする食品香料・香粧品香料を提供することにある。したがって、ここでは一般市販のメロン果実の香気成分とアグロバクテリウムを用い微生物遺伝子導入によって作出したメロン毛状根（選抜網状根系統）KMH.009 およびKMH.003 の香気成分を調べたので報告した。

本実験では、トマトを供試植物としRT.PCR を適用して各種ストレス処理によるgypsy 型レトロトランスポゾンの発現を検出した。その結果、負傷刺激およびDMSO処理を行った場合に、その発現が顕著に誘導され、レトロトランスポゾンが活性化された。

本実験では、KPM.18P の特性を調査し、さらに定着能力を強化することとした。一般に高度葉面定着を示す細菌は生物界面活性剤を生産することが報告されていることから、本細菌の生物界面活性剤生産能について検討した。

本実験では、トマト葉圏から幼虫に対して食害抑制効果を示す細菌の分離を試み、生物防除資材への適用を検討した。トマト葉圏から分離された細菌を幼虫の経口から直接導入し、一定期間トマト葉を摂食させることにより食害抑制効果を示す細菌の選抜を行った。

本実験では、トマトから葉圏に生息するキチナーゼ生産性細菌を分離し、ニジュウヤホシテントウの生物防除を試みた。まず、最も高いキチン分解活性を示す細菌KPM.012A を中腸ペリトロフィック膜に処理したところ、キチナーゼによる分解が確認された。

ガン細胞特異的タンパク質であるCD98 の遺伝子をクローニングし、その遺伝子をアグロバクテリウム法を用いて植物へ導入した。得られた形質転換植物はCD98 遺伝子を発現し、タンパク質の生産も認められた。ガン経口ワクチンとしての有効性について検討した。

植物体の一部に病原体の感染や傷害などのストレスが与えられたとき、直ちにその情報を全身に伝え、そのストレスに対する新たな遺伝子発現が誘導される。本実験では、単一細胞RT.PCR 法をtrichome 細胞に適用し、揮発性物質により誘導される遺伝子の検出を行った。

本実験では、単一細胞から核以外の細胞質をマイクロピペットで吸引する単一細胞RT.PCR 法を確立することとした。実際に単一細胞でRT.PCR を行う場合、その有効性を判定する指標が必要となる。そこでトマトで構成的に発現する遺伝子を検索した。

本実験では、顕微鏡下で標的とした単一細胞からマイクロピペットを用いて細胞内用物を吸引し、そこに存在するmRNA を鋳型とした単一細胞RT.PCR 法を適用することとした。

gypsy 型レトロトランスポゾンの逆転写酵素領域（RT 領域）についてRT.PCR 法による発現解析を行ってきた。そこで今回、gypsy 型レトロトランスポゾンについてゲノムおよびDNA のRT 領域に関して網羅的な解析を行った。

本実験では、マメ科モデル植物であるミヤコグサを用い、根粒形成段階ならびに無機窒素添加条件下で発現するアミノ酸トランスポーター遺伝子について、その遺伝子発現をRT.PCR により解析した。

現在、組織培養を行うにあたり、長い培養期間を要するため、効率的に再生植物体を作出できる培養系の確立が望まれる。本実験では、トマト品種ポンデローザを用い、葉外植片から再分化個体を迅速に得るための再分化条件を明らかにした。

本実験では、シロイヌナズナからアントラニル酸合成酵素遺伝子（asa 遺伝子）をクローニングした後、本遺伝子の一部を塩基置換してフィードバック阻害を受けない酵素を作製するとともに、変異遺伝子を植物体に導入することで5.FI 耐性植物の作出を試みた。

近年、トマトにおいてはうどんこ病が多発する傾向にあるが、うどんこ病菌の感染行動や宿主範囲については必ずしも明確ではない。そこで、本研究では単胞子分離したうどんこ病菌（KTP.01）を使用し、感染挙動の解析と宿主範囲を検討することとした。

本実験では、本菌の単胞子分離株であるKTP.01をトマトに接種し、付着器および吸器の形成に及ぼす湿度の影響を調べるとともに、pseudo-chainの形成について検討した。

筆者らは、トマト葉に感染するうどんこ病菌を分離・同定し、その挙動を解析してきたが、トマトにおける宿主細胞反応については不明な点も多い。そこで親和性および非親和性のオオムギうどんこ病菌分生胞子をトマト葉に接種し、その反応を調べた。

GFP遺伝子を導入したトマト根腐れ萎凋病菌を用い、簡易型水耕装置で育成した水耕トマトでの発病を観察するとともに、本菌のトマト根上での胞子発芽から菌糸伸長の過程をコンピューター画像システムにより解析することとした。

本研究では、トマト根腐れ萎凋病菌の病原性を迅速に検定する方法を考案した。次に高度感受性トマト品種を指標として日本各地で単離されたトマト根腐れ萎凋病菌の病原性検定を行ったところ、各分離菌株間で発病に差異がみられた。

GFP遺伝子を導入したトマト根腐れ萎凋病菌と非病原性フザリウム病菌を用い、トマト根上での同時識別を行うとともに、両菌同時接種による動態解析を検討することとした。その結果、植物根上で両菌を容易に識別および菌叢測定することが可能となった。

本実験では、トマト幼苗を用い、トマト根腐れ萎凋病菌による発病条件を検討するとともに、フザリウム病菌によるトマト根面上での菌糸生育を走査型電子顕微鏡を用いて観察することとした。

5.フルオロインドール（5.FI）耐性植物の作出を目的として、シロイヌナズナ（品種コロンビア）からアントラニル酸合成酵素遺伝子をクローニングした後、フィードバック阻害を受けない変異遺伝子の作製を試みた。

近畿大学農学部キャンパスにおいては、絶滅危惧種に指定された数種の生物種が確認されているが、このような生物種の個体群密度がクズの繁殖によって急速に減少している。そこで、これらの絶滅危惧種の保全を目的とした全学的研究を展開することとした。

オオムギうどんこ病菌の感染を受けた子葉鞘細胞およびその隣接細胞に単一細胞RT.PCR法を適用し、感染特異的に発現する遺伝子の解析を進めている。本研究では、単一細胞から細胞内容物を吸引し、そこに存在するmRNAを鋳型としたRT.PCRを試みた。

トマトのレトロトランスポゾンの発現をRT?PCR法により検出した。Ty1/copia型およびTy3/gypsy型レトロトランスポゾンの逆転写酵素領域は、葉ならびにカルス細胞で発現していた。

本菌は絶対寄生菌であることから、特定の分子ステージにあるうどんこ病菌を大量に得ることは非常に困難であり、それぞれの器官で発現する遺伝子の解析が必ずしも容易ではない。本実験ではPricking RT?PCR法を用い、単一器官を標的として発現遺伝子の解析を行った。

温室栽培トマトにおいてうどんこ病が多発する傾向にあるが、うどんこ病菌の感染行動や宿主範囲については必ずしも明確ではない。そこで、本研究では、単胞子分離したうどんこ病菌を使用し、感染の最適条件を検討することとした。

トマトのレトロトランスポゾンをゲノムからクローニングし、その遺伝子解析を試みた。ゲノム上には多数のレトロトランスポゾンが存在しており、また、培養栽培において発現している新規のTy1/copia型レトロトランスポゾンをクローニングした。

レポーターとして緑色蛍光タンパク質生産遺伝子（GFP）とβ?グルクロニダーゼ生産遺伝子（GUS）を利用し、混合接種した異種分化型菌の検出を試みた。その結果、これらの遺伝子発現がトマト根上で確認できたことから植物根上でも両菌の同時識別が可能となった。

植物根上でのフザリウム病菌の感染挙動解析を行うため、分化型の異なるフザリウム病菌に緑色蛍光タンパク質生産遺伝子（GFP）を導入し、その遺伝子発現検出を利用してメロン根上での追跡を行った。

オオムギに感染しているうどんこ病菌の各分化過程を標的とし、それぞれの器管で発現している遺伝子の分離を行なう為、細胞内RT?PCRを適用し、単一細胞からの遺伝子クローニングを行なった。

Ty1/copia型およびTy3/gypsy型レトロトランスポゾンの逆転写酵素領域で保存されているアミノ酸配列からプライマーを合成し、RT?PCR分析に用いた。トマト葉およびカルスにおいて、これらは発現しており、既知Ty1/copia型とは異なる断片がカルスより増幅した。

経口ガンワクチンの開発を目的とし、ガン細胞特異的タンパク質CD98遺伝子を植物へ導入した。その遺伝子発現をRT?PCRで検出し、さらにタンパク質の翻訳をウエスタンブロット分析で調べた。その結果、組換え植物はCD98タンパク質を生産しており経口ワクチンの可能性が示唆された。

トマトにおける毛状根作出系を確立するため、主要市販トマト43品種と近畿大学交配トマト系統、14品種を用い、Agrobacterium rhizogenesの感染効率を比較検討して、毛状根誘導効率の高いトマトを選抜することとした。

単一細胞RT?PCR法の有効性を正確に評価するため、常時発現する遺伝子を指標に用い、細胞内RT?PCRの成否を判定するシステムを考案した。

pBI121/sGFP を導入した A. rhizogenes を用い、 約 40 品種の市販トマト品種における感染効率を比較検討した。 本菌に対するトマト品種間の差異が明らかになったので、 今後はこれらの品種にアグロインフェクション法を適用すれば効果的に遺伝子導入毛状根が得られる。

既知 Ty1/copia 型および Ty3/gypsy 型レトロトランスポゾンの逆転写酵素領域で保存されているアミノ酸配列をもとにプライマーを合成し、 RT PCR 法によりその発現解析を試みた。 レトロトランスポゾンの RT 領域は、 いずれもトマト葉ならびにカルス細胞で発現していた。

トマトゲノムからレトロトランスポゾンをクローニングし、 その遺伝子解析を試みた。 さらにゲノムライブラリーから Ty3/gyspy 型の RT 領域を含むクローンを選抜して、 遺伝子解析を行ったところ、 レトロトランスポゾンに共通する遺伝子構造が確認された。

E. cloacae 菌株のファージタイピングを目的とし、 17 菌株に特異的に感染するファージの分離を試みた。 単離したファージの宿主範囲を調査したところ、 特定の菌株にのみ感染するタイプと複数の菌株に感染を有するタイプに分けることができた。

キチナーゼ遺伝子をレポーターに用い、 溶菌型ファージの感染系を併用したモニタリング法の開発を試みた。 本法を使用すればトマト葉上における細菌の挙動を迅速に解析できることが示唆され、 効果的な植物病害防除への適用が可能となった。

水耕栽培トマトにおける本菌の防除法を確立するため、 簡易水耕栽培装置の作製とキチン分解性菌を利用したフザリウム病菌の防除について検討した。 その結果、 本システムを利用すればトマト根面上でのフザリウム病菌の生物防除が可能であると考えた。

基礎的アプローチとして、 罹病葉の菌叢から単胞子を分離し、 感染に及ぼす温度や光の影響を調査した。 その結果、 25℃では 2 時間後から発芽管の伸長が認められ、 7 時間後には約 60％の発芽率が得られた。

Sphingobacterium multivorum が生産するキトサナーゼの病原菌細胞壁分解活性を解析するため、 フザリウム病菌の発芽分生胞子に培養濾液から回収したキトサナーゼを処理した。 菌糸細胞の膨張や菌糸先端の部分分解が観察され、 菌糸伸長の抑制も認められた。

既に報告されている構成的発現遺伝子の構造遺伝子内および 3非翻訳領域に対するプライマーを構築し、 細胞内 PCR の成否が判定できる指標遺伝子の検索を試みた。 その結果、 目的遺伝子の領域が増幅されたことから、 GAPDH 遺伝子は細胞内 PCR の指標となった。

REMI 法を用いてメロンつる割病菌を形質転換するとともに、 形質転換株については病原性検定を行った。 さらに GFP 活性を有し、 かつ病原性を保持したメロンつる割病菌と GUS 活性を有するトマト萎ちょう病菌の分生胞子を混合し、 植物根上での同時識別を行った。

GFP を導入したトマト萎ちょう病菌およびメロンつる割病菌を用い、 水耕栽培したトマト根およびメロン根上での本菌の定着と菌糸伸長について検討した。 その結果、 フザリウム病菌は植物種に関係なく、 植物根上に胞子定着した後、 菌糸伸長する過程を示した。

トマト葉面細菌である Enterobacter cloacae にキチナーゼ遺伝子を導入し、 その遺伝子発現をレポーターとするとともに、 本菌に感染するファージを分離し、 両者を組み合せたモニタリングシステムを確立した。

Sphingobacterium multivorum からキトサナーゼ遺伝子をクローニングし、 その塩基配列を決定した後、 大腸菌大量発現システムを適用してキトサナーゼの精製を行なった。 また、 本酵素をフザリウム菌に処理したところ、 病原菌細胞壁分解に有効であると判明した。

南西諸島産のアリモドキゾウムシ個体群について RAPD 法を用いて DNA 増幅パターンの地理的変異の検出法を確立した。

抗青枯病薬剤である 3 （3 インドリル） 酪酸が水耕栽培で問題となる緑藻の増殖を抑制することが明らかとなった。 本実験では 3 （3 インドリル） 酪酸の種々の緑藻に対する効果について検討を行なった。

オオムギ子葉鞘の単一細胞から RT PCR により遺伝子をクローニングするため、 基礎的条件として常時発現している遺伝子のプライマーを構築し、 PCR 時の反応温度および増幅量について検討した。

オオムギうどんこ病菌の感染を受けているオオムギ子葉鞘単一細胞に cDNA 合成溶液と RT PCR 反応液を注入し、 標的細胞内で発現している遺伝子の増幅を試みるとともに、 それら遺伝子のクローニングを行なった。

うどんこ病に対して抵抗性を示すオオムギは、 うどんこ病菌の感染を受けた細胞で抗菌物質としてトリプタミンを生成する。 そこでトリプタミン合成遺伝子であるトリプトファンデカルボキシラーゼ遺伝子を単離し、 その機能を検討した。

土壌病原菌による病害は防除が困難であるため､ 化学的防除法や耕種的防除法に加え､ 拮抗微生物を用いた生物防除が試みられてきた｡ 本研究ではイチゴの萎黄病を対象としてアルギン酸カルシウムビーズに包埋したキチン分解性放線菌の抑制効果を検討した｡

チキン質分解酵素生産性細菌からキチナーゼおよびキトサナーゼ遺伝子をクローニングし､ 大腸菌大量発現システムを利用してそれらを精製した｡ 精製した酵素を用いて植物病原菌の細胞壁分解を検討した結果､ 本酵素の有効性が確認された｡

トマト葉面から安定的に生存している葉面細菌を分離し､ Enterobacter cloacae と同定した｡ 次にこれら細菌にキチン質分解酵素遺伝子を導入し､ それらの酵素による害虫の生物防除の有効性を確認した｡

市販栽培トマト 36 品種を水耕栽培し､ 接種した青枯病菌による品種間の感受性差異を検討した｡ 検定項目としては萎凋初期症状が現われるまでの期間および初期症状から全身萎凋に進行する期間を設け､ その発病状況を 2 週間調査した｡

トマト根腐れ萎ちょう病菌を用い､ 菌糸由来プロトプラストに REMI 法で緑色蛍光タンパク質生産遺伝子 (GFP 遺伝子) を導入し､ 4 菌におけるトマト根での定着および侵入差異について検討した｡

オオムギうどんこ病菌に感染したオオムギ子葉鞘内表皮細胞における感染特異遺伝子の発現機構を解析するため､ マイクロジェクション (MI) 法を適用してきた｡ 本実験では､ このような単一細胞を in situ PCR のチャンバーと考え､ そこで増幅した遺伝子の単離を試みた｡

トマトうどんこ病の生物防除を目的として､ トマト葉面に生息するキチン質分解性細菌を分離し､ 噴霧した分離細菌が葉面上に定着するための生理的特性について検討した｡

トマト葉面で安定的に生息する細菌を分離し､ それらにキチン質分解能を付与することを試みた｡ まず､ 圃場で栽培中のトマト葉から微生物を分離し､ その分解頻度と分離細菌の同定を行った｡

筆者らがクローニングした Kuruthia zopfii のキトサーゼ遺伝子 (chi SH1) および Sphimgobacterlum multivorum のキトサーゼ遺伝子 (cso A) を大腸菌の大量発現系である pET32 ベクターに連結した後､ 大腸菌 BL21 株に導入し､ 生産された酵素をアフィニティーカラムで精製した｡

市販栽培トマト 86 品種を用い､ ガラス温室栽培におけるうどんこ病の発生状況を調査した｡ 調査期間を平成 11 年 12 月から平成 13 年 1 月とし､ ガラス温室内で生育させたトマト苗にうどんこ病菌を自然感染させた｡

マーカー遺伝子挿入法によって fumonisin 生合成遺伝子の特定を試みた｡ Fusarium proliferatum の fumonisin 生産株プロトプラストにマーカー遺伝子 GFP を導入し､ 形質転換体について fumonisin B1 生産能を調べた｡

南西諸島に分布するアリモドキゾウムシには翅鞘色が暗青色と暗褐色の 2 型が認められ､ これらは DNA 増幅パターンの 2 型とよく対応することがわかった｡

本研究では、微生物の葉面処理による植物病害防除を目的とすることから、処理した微生物がどのように葉面で定着し、かつ増殖できるかを解明することとした。申請者は葉面生息細菌の中に高率でトマト葉面に定着性をもつ細菌(高率分離細菌)が生物界面活性物質(バイオサーファクタント)を生産すること、細胞外に多糖類を分泌分解して炭素源に利用することを明らかにしてきた。トマト葉高率分離細菌の変異株を作出するため、エレクトロポーレーションによる本細菌染色体へのトランスポゾン挿入不活化法を行った。すなわち、容易かつ迅速に変異株の選抜と遺伝子解析を行うため、緑色蛍光タンパク質生産遺伝子(GFP)とカナマイシン抵抗性遺伝子を付与したトランスポゾンペクターを使用した。その結果、多くの形質転換株を得ることができた。次に形質転換株の生理・酵素学的機能を検定するため、生物界面活性物質産生能、酵素産生能および色素産生能などの有無を検討したところ、界面活性剤非生産細菌を得ることができた。界面活性剤非生産細菌に関しては、培養後、drop-collapsing法にて容易に分離することができた。本形質転換株をトマト葉上に処理し、走査型電子顕微鏡および蛍光顕微鏡を用いて局在部位を観察したところ、表皮細胞縫合部に沿って生存していることが明らかとなった。また、トマト葉上にあらかじめ本細菌を処理・定着させた後、炭素源(グルコース)を噴霧処理したところ、本細菌の増殖を確認することができた。次にキチナーゼ生産能と界面活性剤生産能をもつ葉面細菌を用いて、トマト葉上に形成されたうどんこ病菌菌叢(Oidium neolycopersici)に処理したところ、本病原菌の菌叢拡大が阻止された。以上のことから、高率分離細菌の定着・増殖メカニズムを明らかにすることで、葉上に発生する病害防除に適用できるものと示唆された。

When leaf segments of a tomato cultivar 'Ponderosa' were inoculated with Agrobacterium rhizogenes MAFF07-20001 carrying the binary vectors pRi and pBI121/sGFP, adventitious roots were developed from calli formed at the edges of the segments. Primordial roots that showed green fluorescence under blue light and elongated vigorously on hormone-free medium without loss of the green fluorescence were obtained. They were easily distinguishable from the non-fluorescing roots on the same segments. Successful integration of the sGFP and rol C genes into the chromosome of tomato roots was confirmed by polymerase chain reaction and Sourhem hybridization. The present method enables us to evaluate the hairy root formation without subculture, isolation and DNA analysis. This method was tested on all of commercial cultivars available in Japan(42 cultivars) and 14 breeding lines of tomato. All but two breeding lines produced the hairy roots. Thus, the present method is useful for hairy root production in tomato. RT-PCR was used to detect gene expression in situ in single selected cells from tomato callus aggregates. The cytoplasm from one cell was removed with a micropipette viewed under a light microscope and used directly for RT-PCR, followed by nested PCR. This method of removing cytosolic contents prevented the introduction of genomic DNA into the RT-PCR, and only intron-spliced products were amplified when intron-containing genes were used as PCR targets. In addition, transcription of the intron-free gene was possibly detected by simultaneously tracing the intron-containing and intron-free genes using mixed primers for the targeted genes. The present study indicated that some stimuli-activated genes, such as CHI3 and TLC1-retrotransposon long terminal repeat, were constitutively transcribed in tomato callus cells. A single-cell RT-PCR was conducted to detect gene expression in situ in pinpointed trichome cells of tomato leaves. The cytoplasm was removed with the micropipette using a light microscope and directly used for RT-PCR, followed by nested PCR. Two intron-containing genes, glyceraldehydes 3-phosphate dehydrogenase gene and plasma membrane H^+-ATPas gene were constantly expressed in this tissues and therefore used as the indicator, because of easy detection of shorter-size PCR-products produced by splicing. In addition, the sucking of nucleus-free cellular contents was effective to prevent contamination of genomic DNA led to miss-amplification of corresponding genomic DNA sequences of the intron-less genes in the process of RT-PCR and subsequent nested PCR. Thus, the present technique could be applicable to single trichome cells of tomato leaves for directly detecting their gene expression in response to chemical and physical stimulation.