Real-Time Collection of Conidia Released from Living Single Colonies of Podosphaera aphanis on Strawberry Leaves under Natural Conditions with Electrostatic Techniques.

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.

Physical Methods for Electrical Trap-and-Kill Fly Traps Using Electrified Insulated Conductors.

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.

Developing a Phototactic Electrostatic Insect Trap Targeting Whiteflies, Leafminers, and Thrips in Greenhouses.

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.

Turkestan Cockroaches Avoid Entering a Static Electric Field upon Perceiving an Attractive Force Applied to Antennae Inserted into the Field.

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.

Selective Arcing Electrostatically Eradicates Rice Weevils in Rice Grains.

We developed an arc discharge exposer (ADE) that kills rice weevils nesting in dried rice. The ADE features multiple identical metal plates, half of these are linked to a voltage generator and the others are grounded. The plates were arrayed in parallel and an electric field formed between them. Any insect entering the field was arced from the negatively charged plate and killed. The ADE was placed on a vessel containing pest-infested rice grains; the insects were lured out of the grains by mechanically vibrating the vessel. When rice grains move, insects tend to climb upward, thus, the weevils were effectively removed. Our electrostatic apparatus is easy to construct and could be used to control pests in stored rice.

Impact of Nerve-Sparing Status on Positive Surgical Margin Location and Biochemical Recurrence in Patients with Prostate Cancer Post Radical Prostatectomy.

PURPOSE: This study was designed to assess the relationship between nerve-sparing (NS) status, positive surgical margin (PSM) location, and biochemical recurrence (BCR) based on a multicenter, radical prostatectomy (RP) database. METHODS: We retrospectively reviewed data from 726 patients who underwent RP without any neoadjuvant or adjuvant treatment between 2010 and 2014. We statistically assessed the impact of NS sides on PSM location and BCR. RESULTS: PSM rates were 21.9% in the 726 patients studied, 13.2% in patients with ≤pT2, and 46.8% in patients with ≥pT3. Regarding PSM locations, the anterior-apex (AA) was the most common site for PSM (43.3%). After adjusting for confounding factors, bilateral nerve sparing (BNS) had a significantly higher odds ratio of PSM than the absence of NS did (odds ratio [OR] 3.04, 95% confidence interval [CI] 1.85-4.99). In the UNS RP in patients with ≤pT2, non-AA PSM on the non-NS side was significantly higher than that on the NS side (92.9% vs. 45.5%, p = 0.009). In all patients, 5.8% experienced BCR during a median follow-up of 43.5 months. PSM was significantly associated with BCR-free survival in patients with ≤pT2 (p = 0.013), but not in patients with ≥pT3 (p = 0.185). Non-AA PSM at the non-NS side was an independent risk factor for BCR (hazard ratio [HR] 2.56, 95% confidence interval [CI] 1.12-5.85), whereas AA PSMs, including NS/non-NS sides and non-AA PSM at the NS side, were not associated with BCR-free survival. CONCLUSIONS: Avoidance of non-AA PSM on the non-NS side may be rather important for maintaining BCR-free survival after RP.

A Simple Electrostatic Precipitator for Trapping Virus Particles Spread via Droplet Transmission.

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.

Ampelomyces strains isolated from diverse powdery mildew hosts in Japan: Their phylogeny and mycoparasitic activity, including timing and quantifying mycoparasitism of Pseudoidium neolycopersici on tomato.

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.

Use of Electric Discharge for Simultaneous Control of Weeds and Houseflies Emerging from Soil.

An electrostatic apparatus was developed to control weeds and houseflies emerging from ground soil in a greenhouse simultaneously. Identical iron plates were placed in parallel at a defined interval and fixed in an iron frame. Two sets of fixed iron plates were used, one for weed control and one for fly control. For weed control, all of the iron plates were negatively charged, and negative charges accumulated on the plates were released to weed shoots through arc discharge. Houseflies were introduced into the space between the negatively charged and grounded plates, then subjected to arc discharge from the charged plates. Both plant shoots and adult houseflies are electrically conductive; thus, they were killed by discharge-exposure in the electric field between the charged iron plate and the ground soil, and between the charged and grounded plates, respectively. In practical use, these two devices were assembled as a two-level apparatus for simultaneous control of both targets. Several apparatuses were linked together, which increased the total electricity charge on the plates and produced a stronger discharge force sufficient to kill all targets. Thus, this study provides an electrostatics-based pest-control method for pesticide-independent greenhouse farming.

Impact of nuclear YAP1 expression in residual cancer after neoadjuvant chemohormonal therapy with docetaxel for high-risk localized prostate cancer.

BACKGROUND: Although docetaxel-based chemohormonal therapy (CHT) is one of the standard treatments for castration-resistant prostate cancer (CRPC), pertinent biomarkers and precise mechanisms involved in the resistance for CHT for CRPC remain unknown. We investigated the relationship between chemohormonal resistance and the expression of steroid receptors and Hippo pathway proteins using a docetaxel-resistant prostate cancer (PCa) cell line and human PCa tissues in patients who underwent surgery with and without neoadjuvant therapy. METHODS: A docetaxel-resistant subline (22Rv1-DR) was generated to assess Hippo pathway protein expression and the effect of YAP1 inhibition on cellular characteristics. A tissue microarray with 203 cores from 70 high-risk localized PCa tissues was performed to assess steroid receptor and Hippo pathway protein expressions. RESULTS: Nuclear YAP (nYAP) expression was higher in 22RV-1-DR than in parental 22Rv-1 and YAP1 knockdown suppressed cell proliferation of 22Rv1-DR. Steroid receptor and Hippo pathway protein expressions varied among three different neoadjuvant groups, and nYAP1 expression was the highest in the CHT group. The patients with high nYAP in residual cancer after neoadjuvant CHT had a significantly higher biochemical recurrence (BCR) rate than those with low nYAP1. On multivariate analysis, the high nYAP1 was an independent prognostic factor for BCR. CONCLUSIONS: nYAP expression is a potential biomarker in high-risk patients treated with docetaxel-based CHT. Steroid receptors and Hippo pathway proteins may play a role in the chemohormonal resistance in advanced PCa.

A patient with synchronous bilateral low-grade upper tract urothelial carcinoma who underwent nephroureterectomy and total ureterectomy with ileal ureteric replacement.

We present the case of a synchronous bilateral low-grade upper-tract urothelial carcinoma patient who underwent left nephroureterectomy and right total ureterectomy with ileal ureteric replacement resulting in a favorable prognosis. A 66-year-old male presented with bilateral hydronephrosis. Computed tomography revealed left pelvic and right upper-middle ureteral tumors with no lymph node swelling or distant metastasis. The patient underwent a left nephroureterectomy with a bladder cuff, and histopathology revealed a low-grade urothelial carcinoma. Considering the contralateral low-grade nature as revealed in histopathology and the right retrograde ureterography being noninvasive, he underwent a right total ureterectomy with ileal ureteric replacement. Histopathology of the right ureter revealed a low-grade urothelial carcinoma. The patient successfully avoided an anephric status without renal function deterioration for 4 years with a transurethral resection for a recurrent small papillary bladder tumor on 18 months after the total ureterectomy.

Analysis of Pole-Ascending-Descending Action by Insects Subjected to High Voltage Electric Fields.

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-Barrier-Forming Window that Captures Airborne Pollen Grains to Prevent Pollinosis.

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.

[Two Cases of Urinary Retention with Prostatic Abscess Drained by Transurethral Resection].

A 58-year-old male with alcoholic liver cirrhosis and a 58-year-old male with diabetes mellitus presented with high fever and urinary retention. Prostatic abscess was diagnosed by a computed tomography scan, which showed fluid collection in the prostate in both patients. Despite systematic antibiotic therapies, urinary retention persisted even after fever diminished. They underwent prostatic abscess drainage by transurethral resection (TUR). Urinary retention was released, and the recurrence of prostatic abscess has not been observed after TUR.

Electrostatic Insect Sweeper for Eliminating Whiteflies Colonizing Host Plants: A Complementary Pest Control Device in An Electric Field Screen-Guarded Greenhouse.

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.

Loss of function in Mlo orthologs reduces susceptibility of pepper and tomato to powdery mildew disease caused by Leveillula taurica.

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.

Detection and quantification of Leveillula taurica growth in pepper leaves.

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 real-time 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.

Characterization of polygenic resistance to powdery mildew in tomato at cytological, biochemical and gene expression level.

Extensive research in the area of plant innate immunity has increased considerably our understanding of the molecular mechanisms associated with resistance controlled by a dominant resistance gene. In contrast, little is known about the molecular basis underlying the resistance conferred by quantitative trait loci (QTLs). In this study, using the interaction of tomato (Solanum lycopersicum) with Oidium neolycopersici, we compared the cytological, biochemical and molecular mechanisms involved in both monogenic and polygenic resistances conferred by a dominant gene (Ol-1) and three QTLs (Ol-qtls), respectively. Our results showed that the three Ol-qtls jointly confer a very high level of broad-spectrum resistance and that the resistance is associated with both the hypersensitive response and papillae formation, with the hypersensitive response being prevalent. Both H(2)O(2) and callose accumulation, which are coupled with Ol-1-mediated resistance, are also associated with the resistance conferred by Ol-qtls. Further, we analysed the pathogen-induced transcript profiles of near-isogenic lines carrying the three Ol-qtls and the Ol-1 gene. Transcript profiles obtained by cDNA-amplified fragment length polymorphism analysis showed that, on fungal challenge, about 70% of the transcript-derived fragments are up-regulated in both susceptible and resistant genotypes. Most of the sequenced transcript-derived fragments showed homology to genes with functions in defence responses, suggesting that defence-responsive genes responsible for basal defence are involved in both monogenic and polygenic resistances conferred by Ol-1 and Ol-qtls, respectively. Although about 18% of the identified transcript-derived fragments are specific for either monogenic or polygenic resistance, their expression patterns need to be further verified by quantitative reverse transcriptase-polymerase chain reaction.

An avirulent tomato powdery mildew isolate induces localized acquired resistance to a virulent isolate in a spatiotemporal manner.

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.

Polymorphic change of appressoria by the tomato powdery mildew Oidium neolycopersici on host tomato leaves reflects multiple unsuccessful penetration attempts.

The appressorial shapes of the powdery mildews are an important clue to the taxonomy of the powdery mildew fungi, but the conidia of the tomato powdery mildew Oidium neolycopersici KTP-01 develop non-lobed, nipple-shaped, and moderately lobed or multilobed appressoria on the same leaves. To remove this ambiguity, we performed consecutive observations of sequential appressorial development of KTP-01 conidia with a high-fidelity digital microscope. Highly germinative conidia of KTP-01, collected from conidial pseudochains formed on the tomato leaves, were inoculated into host tomato and nonhost barley leaves or an artificial hydrophobic membrane (Parafilm). Events from germination initiation to appressorium formation were synchronous in all conidia on all materials used for inoculation, but post-appressorial behaviors varied among the materials. Appressoria on the membrane-stuck glass slide formed several projections at different portions of the appressoria to repeat unsuccessful penetration attempts. Similar unsuccessful penetration behavior by KTP-01 conidia was observed in the inoculations into leaves of barley plants, wild tomato species Lycopersicon peruvianum LA2172 (carrying the Ol-4 gene for powdery mildew resistance), and a susceptible host tomato (Lycopersicon esculentum) that had been inoculated with the barley powdery mildew (Blumeria graminis f. sp. hordei, race 1) conidia. On the barley leaves, all penetrations of KTP-01 were impeded by the papillae formed beneath the sites of the appressorial projections. On both the wild tomato and the race 1-inoculated cultivated tomato plants, KTP-01 conidia were prevented from forming functional haustoria by hypersensitive epidermal cell death; this hypersensitive reaction involved the Ol-4 gene in the wild tomato plants or the 'induced resistance' acquired by the nonpathogenic conidia previously inoculated into the cultivated tomato plants. All these KTP-01 conidia produced several projections on the appressoria during the repeated unsuccessful penetration attempts and eventually exhibited multilobed appressoria. On the host tomato leaves inoculated singly with KTP-01 conidia, fewer than 20% of the conidia located appressoria on the central part of target epidermal cells and succeeded in forming functional haustoria at the first penetration attempt without forming an appressorial projection. These conidia exhibited non-lobed appressoria. The remaining conidia, however, whose appressoria were located on/near the border of the target epidermal cells, were more likely to fail to penetrate at the first penetration, and then to develop additional projections for subsequent penetrations. Most conidia succeeded in forming functional haustoria at the second to fourth penetration attempts, but a few conidia failed to produce haustoria at all attempted penetrations. Eventually, the conidia that succeeded at the second penetration possessed a single appressorial projection (exhibiting the nipple-shaped appressoria), whereas the remaining conidia exhibited moderately lobed appressoria with two to four appressorial projections and multilobed appressoria, with more projections. Thus, the present study revealed that the basic shape of appressoria of KTP-01 was the non-lobed type, and that polymorphic changes of the appressoria occurred as a result of successive production of projections during repeated unsuccessful penetration attempts.