Koji Kakutani

Antioxidant constituents in the dayflower (Commelina communis L.) and their α-glucosidase-inhibitory activity

The dayflower, Commelina communis L., contains 1-deoxynojirimycin (DNJ) and (2R,3R,4R,5R)2,5-bis(hydroxymethyl)-3,4-dihydroxypyrrolidine (DMDP), potent α-glucosidase inhibitors. The extracts and powder of this herb are important food materials for prophylaxis against type 2 diabetes. Eleven flavonoid glycosides as antioxidants, isoquercitrin, isorhamnetin-3-O-rutinoside, isorhamnetin-3-O-β-d-glucoside, glucoluteolin, chrysoriol-7-O-β-d-glucoside, orientin, vitexin, isoorientin, isovitexin, swertisin, and flavocommelin, were identified from the aerial parts of C. communis. Their antioxidant activities were measured using in vitro assays employing the 1,1-diphenyl-2-picrylhydrazyl radical- and superoxide radical-scavenging assays. The results showed that glucoluteolin, orientin, isoorientin, and isoquercitrin are the predominant antioxidants in this herb. Moreover, isoquercitrin, isorhamnetine-3-O-rutinoside, vitexin, and swertisin inhibited the activity of α-glucosidase from rat intestine.

Infectivity of a Japanese isolate of Oidium neolycopersici KTP-01 to a European tomato cultivar resistant to O. lycopersici.

The infectivity of a Japanese isolate of tomato powdery mildew, Oidium neolycopersici KTP-01, to tomato cultivars was examined using a resistant cultivar Grace bred in The Netherlands to O. lycopersici, which was recently proposed to be renamed O. neolycopersici. Grace was severely infected with KTP-01, and its susceptibility was similar to that on susceptible tomato cultivars Moneymaker and Ponderosa, suggesting that KTP-01 differs in pathogenicity on tomatoes from those of European and American isolates.

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

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

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.

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.

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.

In Vitro Suppression of Mycelial Growth of Fusarium oxysporum by Extracellular Chitosanase of Sphingobacterium multivorum and Cloning of the Chitosanase Gene csnSM1

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.

Cytological events in tomato leaves inoculated with conidia of Blumeria graminis f. sp. hordei and Oidium neolycopersici KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.

Consecutive monitoring for conidiogenesis by Oidium neolycopersici on tomato leaves with a high-fidelity digital microscope

Conidiogenesis by Oidium neolycopersici KTP-01 on tomato leaves was vitally monitored with a high-fidelity digital microscope. Conidiophores were initially formed 3 days after inoculation and then elongated to a maximum length within at least 12 h. The apical part was split into two cells after two successive septations, accompanied by apical expansion. These cells subsequently developed into primary and secondary conidia. An additional septation at the stem portion of the conidiophores produced a generative and a foot cell. Subsequent conidiation occurred during repeated cycles of splitting of the generative cell, maturation of the apical cell into a conidium, and abstriction of the conidium. To our knowledge, this report is the first on the developmental process of conidiogenesis by powdery mildew on host leaves as revealed with the digital microscope.

Stable phylloplane colonization by entomopathogenic bacterium Pseudomonas fluorescens KPM-018P and biological control of Phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae)

An entomopathogenic bacterium was isolated from tomato leaves and used as a microbial agent to control larvae of phytophagous ladybird beetles Epilachna vigintioctopunctata. The isolate was identified as Pseudomonas fluorescens KPM-018P on the basis of its bacteriological characteristics. KPM-018P produced extracellular chitinase to form a transparent zone around their colonies by hydrolyzing chitin in a minimal medium. Pale-yellow colonies turned red after a change of incubation temperature. These characteristics were availed as markers for tracking KPM-018P. The bacteria produced biosurfactants that enabled the bacteria to stably colonize the hydrophobic leaf surface; they were recovered without any considerable decrease even after a suspension of KPM-018P was sprayed onto leaves. KPM-018P, transformed with the gfp gene and observed with fluorescence microscopy, stably dwelled in the junctions of epidermal cells of bacteria-sprayed leaves. Ingestion of KPM-018P-sprayed leaves by the larvae caused prompt death of these insects to eventually suppress their pupation. This method is thus effective for decreasing the population of larvae and adult insect pests in the subsequent generation. The study provides an experimental basis for the biocontrol of herbivorous insect pests using a leaf-inhabiting, entomopathogenic strain of P. fluorescens.