We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 g/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.

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The ecological biochemistry of flavonoids, in which I have been engaged for 25 years, is summarized in this review article. The review covers (1) a survey of rare bio-active flavonoids in higher plants; (2) the fungal metabolism of prenylated flavonoids; (3) flavonoids antidoting against benzimidazole fungicides; (4) dihydroflavonol ampelopsin in Salix sachalinensis as a feeding stimulant towards willow beetles; and (5) flavones as signaling substances in the life-cycle development of the phytopathogenic Peronosporomycete Aphanomyces cochlioides, a cause of spinach root rot and sugar beet damping-off diseases. Finally recent trends in the ecological biochemistry of flavonoids are briefly described.

Two new perylenequinones, named hypocrellin B and C, together with the known hypocrellin A were isolated from the stromatal tissues of Shiraia bambusicola. The structures of these compounds, including the absolute stereochemistry, were determined.

Substantial levels of trehalose accumulate in bacteria, fungi, and invertebrates, where it serves as a storage carbohydrate or as a protectant against environmental stresses. In higher plants, trehalose is detected at fairly low levels; therefore, a regulatory or signaling function has been proposed for this molecule. In many organisms, trehalose‐6‐phosphate phosphatase is the enzyme governing the final step of trehalose biosynthesis. Here we report that OsTPP1 and OsTPP2 are the two major trehalose‐6‐phosphate phosphatase genes expressed in vegetative tissues of rice. Similar to results obtained from our previous OsTPP1 study, complementation analysis of a yeast trehalose‐6‐phosphate phosphatase mutant and activity measurement of the recombinant protein demonstrated that OsTPP2 encodes a functional trehalose‐6‐phosphate phosphatase enzyme. OsTPP2 expression is transiently induced in response to chilling and other abiotic stresses. Enzymatic characterization of recombinant OsTPP1 and OsTPP2 revealed stringent substrate specificity for trehalose 6‐phosphate and about 10 times lower Km values for trehalose 6‐phosphate as compared with trehalose‐6‐phosphate phosphatase enzymes from microorganisms. OsTPP1 and OsTPP2 also clearly contrasted with microbial enzymes, in that they are generally unstable, almost completely losing activity when subjected to heat treatment at 50 °C for 4 min. These characteristics of rice trehalose‐6‐phosphate phosphatase enzymes are consistent with very low cellular substrate concentration and tightly regulated gene expression. These data also support a plant‐specific function of trehalose biosynthesis in response to environmental stresses.

For evaluating N 2 fixation of diazotrophic bacteria, nitrogen-poor liquid media supplemented with at least 0.5% sugar and 0.2% agar are widely used for acetylene reduction assays. In such a soft gel medium, however, many N 2 -fixing soil bacteria generally show only trace acetylene reduction activity. Here, we report that use of a N 2 fixation medium solidified with gellan gum instead of agar promoted growth of some gellan-preferring soil bacteria. In a soft gel medium solidified with 0.3% gellan gum under appropriate culture conditions, bacterial microbiota from boreal forest bed soils and some free-living N 2 -fixing soil bacteria isolated from the microbiota exhibited 10-to 200-fold-higher acetylene reduction than those cultured in 0.2% agar medium. To determine the N 2 fixation-activating mechanism of gellan gum medium, qualitative differences in the colony-forming bacterial components from tested soil microbiota were investigated in plate cultures solidified with either agar or gellan gum for use with modified Winogradsky's medium. On 1.5% agar plates, apparently cryophilic bacterial microbiota showed strictly distinguishable microbiota according to the depth of soil in samples from an eastern Siberian Taiga forest bed. Some pure cultures of proteobacteria, such as Pseudomonas fluorescens and Burkholderia xenovorans, showed remarkable acetylene reduction. On plates solidified with 1.0% gellan gum, some soil bacteria, including Luteibacter sp., Janthinobacterium sp., Paenibacillus sp., and Arthrobacter sp., uniquely grew that had not grown in the presence of the same inoculants on agar plates. In contrast, Pseudomonas spp. and Burkholderia spp. were apparent only as minor colonies on the gellan gum plates. Moreover, only gellan gum plates allowed some bacteria, particularly those isolated from the shallow organic soil layer, to actively swarm. In consequence, gellan gum is a useful gel matrix to bring out growth potential capabilities of many soil diazotrophs and their consortia in communities of soil bacteria.In 1967, Schöllhorn and Burris discovered that nitrogenase from an N 2 -fixing rhizobium of soybean can reduce acetylene to produce ethylene (C 2 H 4 ) (32), a reaction analogous to the conversion of the natural substrate N 2 into ammonia. Shortly afterwards, it was shown that this acetylene reduction activity parallels N 2 reduction by nitrogenase (13), and since then, acetylene reduction assays have been widely used in the evaluation of biological N 2 fixation. An acetylene reduction assay is generally performed under the following conditions: precultured bacterial cells are suspended into N-free or -deficient liquid medium containing a carbon source, usually D-glucose or D-mannitol (35) at 0.5 to 2.0%, and exposed for 24 h or less at a representative room temperature, e.g., 25°C (2). However, this method is not applicable to free-living, microaerobic N 2 -fixing bacteria, which have been regarded as notoriously difficult to culture. To solve this problem, Döbereiner and her group developed a soft g...