Protein secretome of moss plants (Physcomitrella patens) with emphasis on changes induced by a fungal elicitor.

Studies on extracellular proteins (ECPs) contribute to understanding of the multifunctional nature of apoplast. Unlike vascular plants (tracheophytes), little information about ECPs is available from nonvascular plants, such as mosses (bryophytes). In this study, moss plants (Physcomitrella patens) were grown in liquid culture and treated with chitosan, a water-soluble form of chitin that occurs in cell walls of fungi and insects and elicits pathogen defense in plants. ECPs released to the culture medium were compared between chitosan-treated and nontreated control cultures using quantitative mass spectrometry (Orbitrap) and 2-DE-LC-MS/MS. Over 400 secreted proteins were detected, of which 70% were homologous to ECPs reported in tracheophyte secretomes. Bioinformatics analyses using SignalP and SecretomeP predicted classical signal peptides for secretion (37%) or leaderless secretion (27%) for most ECPs of P. patens, but secretion of the remaining proteins (36%) could not be predicted using bioinformatics. Cultures treated with chitosan contained 72 proteins not found in untreated controls, whereas 27 proteins found in controls were not detected in chitosan-treated cultures. Pathogen defense-related proteins dominated in the secretome of P. patens, as reported in tracheophytes. These results advance knowledge on protein secretomes of plants by providing a comprehensive account of ECPs of a bryophyte.

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.

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.

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.

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.

Body Water-Mediated Conductivity Actualizes the Insect-Control Functions of Electric Fields in Houseflies.

In the present study, the relationship between body water loss and conductivity was examined in adult houseflies (Musca domestica). The events an insect experiences in an electric field are caused by the conductive nature of the insect body (i.e., movement of electricity within or its release from the insect). After houseflies were dehydrated, rehydrated, refrigerated, and frozen and thawed, they were placed in static and dynamic electric fields. Untreated houseflies were deprived of their free electrons to become positively charged and then attracted to the insulated negative pole in the static electric field and were exposed to corona and arc discharge from non-insulated negative pole in the dynamic electric field. There was no current in the bodies of dehydrated and frozen flies; hence, there was no attractive force or discharge exposure. In the remaining insects, the results were identical to those in the untreated control insects. These results indicated that the reduction of body water conductivity inhibited the release of electricity from the body in the static electric field and the discharge-mediated current flow through the body in the dynamic electric field. The insect was affected by the electric fields because of its conductivity mediated by body water.

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.

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.

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.

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.

Collection of highly germinative pseudochain conidia of Oidium neolycopersici from conidiophores by electrostatic attraction.

A population of simultaneously germinating conidia is an ideal inoculum of the powdery mildew pathogen, Oidium neolycopersici. In conditions of no or low wind velocity, O. neolycopersici successively stacks mature conidia on conidiophores in a chain formation (pseudochain), without releasing the precedent mature conidia. These pseudochain conidia represent a perfect inoculum, in which all conidia used for inoculation germinate simultaneously. However, we found that conidia must be collected before they fall to the leaf surface, because the germination rate was lower among conidia deposited on the leaf surface. We used an electrostatic spore collector to collect the pseudochain conidia, and their high germination rate was not affected by this treatment. The spore collector consisted of an electrified insulator probe, which created an electrostatic field around its pointed tip, and attracted conidia within its electric field. The attractive force created by the probe tip was directly proportional to voltage, and was inversely proportional to the distance between the tip and a target colony on a leaf. Pseudochain conidia were successfully collected by bringing the electrified probe tip close to target colonies on leaves. In this way, conidia were collected from colonies at 3-d intervals. This effectively collected all conidia from conidiophores before they dropped to the leaf surface. A high germination rate was observed among conidia attracted to the probe tip (95.5+/-0.6%). Conidia were easily suspended in water with added surfactant, and retained their germination ability. These conidia were infective and produced conidia in pseudochains on conidiophores after inoculation. The electrostatic spore collection method can be used to collect conidia as they form on conidiophores, thus obtaining an inoculum population in which all of the conidia germinate simultaneously.