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. Received 2 May 2001/ Accepted in revised form 21 June 2001     

Occurrence of New Powdery Mildew on Greenhouse Tomato Cultivars

During a year-round survey on the occurrence of powdery mildew on greenhouse-cultivated tomato plants, the disease was most severe in June and July. All tomato plants (45 commercial cultivars and 11 breeding lines) tested were infected with the pathogen but had different degrees of susceptibility. The pathogen was epiphytic and produced white, round pustules mainly on leaves of tomato plants. The pathogen produced conidia singly on conidiophores and forked appressoria on inoculated tomato leaves and seemed to be an Oidium sp. of Erysiphe polygoni type. Received 18 December 2000/ Accepted in revised form 22 July 2001     

A Monitoring System for Green Fluorescence Protein Gene-transformed Fusarium oxysporum in Melon Seedlings

Using melon seedlings at the cotyledon stage and genetically marked fungi, a system for monitoring pathogenic and nonpathogenic Fusarium oxysporum was devised in the present study. Protoplasts were prepared from three formae speciales (melonis, radicis-lycopersici and fragariae )of F. oxysporum and transformed with a synthetic gene for green fluorescence protein. Transformants were primarily isolated in the presence of hygromycin B and then screened by the emission of bright green fluorescence. Roots of melon seedlings were inoculated with fluorescing microconidia of these fungi, and fungal infection behavior was traced. Using fluorescence microscopy, we directly observed not only the fungus at the root surface, but also the mycelia elongating in the trachea of roots. Both pathogenic and nonpathogenic fungi germinated and hyphae elongated superficially on the surface of root. Only pathogenic fungi caused root necrosis at the inoculation site. Hyphae grew within the stem to induce constriction or cracking of lower hypocotyls, then causing wilting of the seedlings. Infection behavior of genetically marked pathogenic and nonpathogenic F. oxysporum could be successfully monitored after inoculation of cotyledons of seedlings. Received 6 June 2001/ Accepted in revised form 3 August 2001     

Detection of intra- and interspecific variation of the dry rot fungus <Emphasis Type="Italic">Serpula lacrymans</Emphasis> by PCR-RFLP and RAPD analysis

We investigated a genotype-based assay to discriminate the dry rot fungi Serpula lacrymans. DNAs were extracted from 74 isolates from the northern half of Japan, and internal transcribed spacers (ITS) were amplified by polymerase chain reaction. Genotypes of isolates were checked by restriction fragment length polymorphism (RFLP) using two enzymes, Taq I and Hha I. Among the 74 isolates identified as S. lacrymans in terms of morphologic features, 5 isolates were shown to have been misidentified. Random amplified polymorphic DNA (RAPD) analysis was conducted in order to detect the intraspecific diversity of S. lacrymans isolated in Japan. Because no relation between geographical origin and genetic distances was observed, the intraspecific diversity of S. lacrymans is suggested to be small.Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002     

Changes in surface properties of tropical wood species exposed to the Indonesian climate in relation to mold colonies

Changes in mold populations and genera on the exposed surfaces of tropical hardwoods — albizia (Paraserianthes falcata), kapur (Dryobalanop lanceolata), mahoni (Switenia macrophylla), nangka (Artocarpus heterophyllus), puspa (Schima wallchii) — were investigated. The wood specimens were exposed to the Indonesian climate for 32 weeks. Properties including mass loss, wettability, mold growth (colony-forming units), and mold genera were evaluated. The change in properties after exposure was significantly affected by the wood species, but there was no clear relation between mass loss and the initial chemical components or between wettability and wood density. The number of mold populations was different by exposure period and wood species, but there was no significant effect of climate conditions, such as rainfall and ultraviolet radiation. Of the genera identified,Aureobasidium, Cladosporium, andPenicillium were dominant molds on the exposed wood surfaces.     

Raman spectroscopic study on the behavior of boric acid in wood

 Raman spectra of Japanese cedar (Cryptomeria japonica D. Don) treated by vacuum impregnation with aqueous boric acid solutions (8.1 × 10⁻² to 7.29 × 10⁻¹ mol dm⁻³) were recorded using a near-infrared laser as an excitation source. Raman spectroscopic measurements were carried out on treated wood blocks of two sizes: 20(T) × 20(R) × 5(L) mm (A-type) and 15(T) × 15(R) × 50(L) mm (B-type). Our attention was focused on a prominent band (ν ₁) assigned to a symmetrical stretching vibration of the BO₃ group because no Raman band due to boron species was observed except bands of B(OH)₃. We observed a change in ν ₁ band intensity with increasing boric acid concentration in the aqueous solution used to treat the A-type wood blocks and investigated the correlation between the intensity and the peak-top wavenumber. Raman line maps in the longitudinal direction of the treated B-type wood blocks revealed that B(OH)₃ is concentrated near the cut ends. These results suggested that two groups of B(OH)₃ exist in wood in terms of the chemical species in the nearest neighbor sphere. Received: March 11, 2002 / Accepted: June 26, 2002     

Adsorption capacities and related characteristics of wood charcoals carbonized using a one-step or two-step process

Sugi (Cryptomeria japonlca D. Don) wood powder was carbonized at varying temperatures by a onestep process up to 1000C and a two-step process using wood charcoal as the raw material up to 1600C. This study was conducted to evaluate the adsorptive properties of wood charcoal and discuss the mechanism of its adsorptive function in relation to the physical and anatomical characteristics of wood after carbonization. Anatomical characteristics of carbonized wood materials were directly observed under heating using an environmental scanning electron microscope (ESEM); the cell wall structures were analyzed by high-resolution transmission electron microscope (HRTEM). The largest weight losses were observed at the highest temperatures, in both the one-step and twostep processes but leveled off above 800C. Shrinkages in the tangential, radial, and longitudinal directions increased with carbonization temperature, peaking at 1000C. Direct observations by ESEM showed distinct shrinkage at around 400C. The first trial observations by HRTEM on the changes in the ultrastructure of cell walls of wood charcoals were done, and it was assumed to affect the formation of micropores. Adsorption was found to follow the Langmuir isotherm model. With the one-step carbonization process, the iodine adsorption capacities of the carbonized wood powders increased with increasing carbonization temperature, peaking at 800C, but decreased at higher temperatures. The wood powder carbonized at 1000C with the two-step process showed the highest capacity, but further heating up to 1400C drastically decreased the adsorption. The shrinkage of cells was related to the increases and decreases in its specific surface area. Specific surface area and total pore volume were evidently related to the adsorptive properties.Part of this paper was presented at the Second International Wood Science Seminar, Indonesia, November 6–7, 1998     

Adsorption of mercury by sugi wood carbonized at 1000°C

The ability of sugi wood carbonized at 1000°C to adsorb mercury was examined using aqueous solutions of mercuric chloride. Parameters studied include contact time, pH, adsorption temperature, and initial concentration of mercury in solution. Results showed that sugi wood carbonized at 1000°C could effectively remove mercury from aqueous solutions. The carbonized wood showed high adsorption ability for mercury at a wide pH range (pH 3–9), but its ability drastically decreased at pH 11. Adsorption decreased with increases in adsorption temperatures, indicating that the processes were exothermic in nature. Adsorption was found to follow the Freundlich isotherm model. The adsorption capacity of carbonized sugi wood was comparable to that of commercial activated carbon.     

Characteristic Raman bands for <Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis> heartwood

The Raman spectrum of Artocarpus heterophyllus heartwood, which proved to be a rich source of flavonoids, exhibited two characteristic bands, at 1247cm^–1 and 745cm^–1. The bands also appeared in the Raman spectrum of the yellow-brown needles extracted from the heartwood with methanol. Based on the Raman measurements of flavones and related compounds, it was predicted that the Raman band at 1247cm^–1 may be attributed to flavonoid-type compounds. No vibrational band corresponding to the characteristic Raman bands was observed by diffuse reflectance infrared spectroscopy. Thus, it was suggested that observation of the characteristic bands is an advantage of Fourier transform-Raman spectroscopy for nondestructive analysis of wood.     

Occurrence of orally administered mulberry 1-deoxynojirimycin in rat plasma

1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, is a characteristic constituent of the mulberry leaf. Dietary mulberry DNJ may be beneficial for the suppression of abnormally high blood glucose levels, thereby preventing diabetes mellitus. Although there is considerable interest in the effects of mulberry DNJ, the intestinal absorption and pharmacokinetic profile of orally administered mulberry DNJ have never been characterized. In this study, we developed a method for determining the level of plasma DNJ by hydrophilic interaction chromatography coupled to a mass spectrometric detector (HILIC-MS) to investigate the absorption and metabolism of orally administered mulberry DNJ in rats. DNJ was separated from plasma extract on a TSK gel Amide-80 column, a representative column for HILIC. At postcolumn, DNJ was concurrently detected and identified by MS. The plasma DNJ concentration in fasted rats was below the detection limit [<1 microg (6 nmol)/mL]; however, the concentration reached a maximum [15 microg (92 nmol)/mL] 30 min after the administration of mulberry DNJ (110 mg/kg of body weight), and the DNJ concentration decreased rapidly thereafter. When the rats received different amounts of mulberry DNJ (1.1, 11, and 110 mg/kg of body weight), dose-dependent incorporation of DNJ into the plasma was confirmed. We did not detect any DNJ metabolites in the plasma. These findings indicate that orally administered mulberry DNJ is absorbed as an intact form from the alimentary tract and then is quickly excreted from the body. The developed HILIC-MS method could be applied in determining levels of DNJ in urine and tissues, and therefore, the method would be a powerful tool for studying the metabolic fate of mulberry DNJ as well as its bioavailability.