Phosphorylation status of a conserved residue in the adenylate cyclase of Botrytis cinerea is involved in regulating photomorphogenesis, circadian rhythm, and pathogenicity.

Adenylate cyclase (AC) regulates growth, reproduction, and pathogenicity in many fungi by synthesizing cyclic adenosine monophosphate (cAMP) and activating downstream protein kinase A (PKA). Botrytis cinerea is a typical necrotrophic plant-pathogenic fungus. It shows a typical photomorphogenic phenotype of conidiation under light and sclerotia formation under dark; both are important reproduction structures for the dispersal and stress resistance of the fungus. The report of B. cinerea adenylate cyclase (BAC) mutation showed it affects the production of conidia and sclerotia. However, the regulatory mechanisms of the cAMP signaling pathways in photomorphogenesis have not been clarified. In this study, the S1407 site was proven to be an important conserved residue in the PP2C domain which poses a remarkable impact on the phosphorylation levels and enzyme activity of the BAC and the overall phosphorylation status of total proteins. The point mutation bacS1407P , complementation bacP1407S , phosphomimetic mutation bacS1407D , and phosphodeficient mutation bacS1407A strains were used for comparison with the light receptor white-collar mutant Δbcwcl1 to elucidate the relationship between the cAMP signaling pathway and the light response. The comparison of photomorphogenesis and pathogenicity phenotype, evaluation of circadian clock components, and expression analysis of light response transcription factor genes Bcltf1, Bcltf2, and Bcltf3 showed that the cAMP signaling pathway could stabilize the circadian rhythm that is associated with pathogenicity, conidiation, and sclerotium production. Collectively, this reveals that the conserved S1407 residue of BAC is a vital phosphorylation site to regulate the cAMP signaling pathway and affects the photomorphogenesis, circadian rhythm, and pathogenicity of B. cinerea.

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.

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.

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.

Soil Surface-Trapping of Tomato Leaf-Miner Flies Emerging from Underground Pupae with a Simple Electrostatic Cover of Seedbeds in a Greenhouse.

In the present study, an electrostatic apparatus for trapping adult tomato leaf miner flies (Liriomyza sativae) emerging from underground pupae at the surface of a seedbed in an organic greenhouse was developed. The apparatus consisted of insulated iron rods arranged in parallel at set intervals and linked to a voltage generator, which supplied a negative charge to the rods, as well as non-insulated grounded iron rods with the same configuration. The two layers of insulated and non-insulated iron rods were arrayed in parallel to form a static electric field between the layers. The electric field created a strong attractive force capable of capturing flies that entered the field. In a greenhouse assay, the apparatus was placed horizontally above a seedbed in a greenhouse and surveyed for its ability to capture adult flies emerging from pupae that were introduced onto the seedbed beneath the apparatus. The results revealed that the apparatus effectively trapped all adult flies that emerged from the pupae and that it functioned stably while continuously operated during the entire period of the experiment. Thus, our novel apparatus is a promising tool for the physical control of adult tomato leaf miners in the insecticide-independent cultivation of greenhouse tomatoes.

Insect Physical Control: Electric Field-Based Pest Management Approach.

The Special Issue 'Insect physical control: electric field-based pest management approach' was launched to showcase valuable new research on pest control using applied electrostatic engineering. Some phenomena generated in static and dynamic electric fields can be used to build new devices to capture or kill target insects using an attractive force or a force striking insects entering an electric field. This research field is new, and there are few researchers currently working within it. Consequently, this editorial introduces the history and general principles of electric field generation. I then discuss future directions for this field.

High Voltage Electric Fields Have Potential to Create New Physical Pest Control Systems.

An electric field is the space surrounding an electric charge, within which it is capable of exerting a perceptible force on another electric charge. Especially under high voltage, electric fields induce various electrostatic phenomena, some of which could be utilized to provide remarkable pest control measures. The main focus of the present study was to introduce an attractive force generated by a surface charge on an insulated electrified conductor, which was successfully used to construct an electric field screen that prevented airborne nuisances (spores, flying insects, pollen, and fine smoke) from entering the interiors of various facilities. Another focus was the disinclination of insects to enter the electric field, thus, giving the electric field screen the ability to repel insects. Charges accumulated on the surfaces of non-insulated conductors are mobile through discharge, based on their potential difference. Such arc discharge was strong enough to destroy insects that were exposed to it. Some precedent illustrative examples are cited to explain the principles of attraction, dielectrophoretic movement of spores, and discharge-mediated positive electrification of insects, and to discuss how electric fields are generated and used in electric field-based pest control strategies.

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.

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.

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.

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.

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.

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation.

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.

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

ABSTRACT In an attempt to physically protect greenhouse tomato plants from the powdery mildew fungus Oidium neolycopersici, we developed a new electrostatic spore precipitator in which a copper wire conductor is linked to an electrostatic generator and covered with a transparent acrylic cylinder (insulator). The conductor was negatively charged by the generator, and the electrostatic field created by the conductor was used to dielectrically polarize the insulator cylinder. The dielectrically polarized cylinder also produced an electrostatic force without a spark discharge. This force was directly proportional to the potential applied to the conductor and was used to attract conidia of the pathogen. The efficacy of this spore precipitator in protecting hydroponically cultured tomato plants from powdery mildew was evaluated in the greenhouse. The hydroponic culture troughs were covered with a cubic frame installed with the spore precipitator, and the disease progress on precipitator-guarded and unguarded seedlings was traced after the conidia were disseminated mechanically from inoculum on tomato plants. Seedlings in the guarded troughs remained uninfected during the entire experiment, in spite of rapid spread of the disease to all leaves of the unguarded seedlings.

Family 19 chitinase from Aeromonas sp. No.10S-24: role of chitin-binding domain in the enzymatic activity.

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 C-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 (-1) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.

Identification of individual powdery mildew fungi infecting leaves and direct detection of gene expression by single conidium polymerase chain reaction.

ABSTRACT Greenhouse-grown tomato seedlings were inoculated naturally with two genera of powdery mildew conidia forming appressorial germ tubes that could not be differentiated by length alone. For direct identification, single germinated conidia were removed from leaves by means of a glass pipette linked to the manipulator of a high-fidelity digital microscope. This microscope enabled in vivo observation of the fungi without leaf decoloration or fungal staining. The isolated conidia were subjected to PCR amplification of the 5.8S rDNA and its adjacent internal transcribed spacer sequences followed by nested PCR to attain sensitivity high enough to amplify target nucleotide sequences (PCR/nested PCR). Target sequences from the conidia were completely coincident with those of the pathogen Oidium neolycopersici or Erysiphe trifolii (syn. Microsphaera trifolii), which is nonpathogenic on tomato. Using RT-PCR/nested PCR or multiplex RT-PCR/nested PCR, it was possible to amplify transcripts expressed in single conidia. Conidia at pre- and postgermination stages were removed individually from tomato leaves, and two powdery mildew genes were monitored. The results indicated that the beta-tubulin homolog TUB2-ol was expressed at pre- and postgermination stages and the cutinase homolog CUT1-ol was only expressed postgermination. Combining digital microscopic micromanipulation and two-step PCR amplification is thus useful for investigation of individual propagules on the surface of plants.