Nobuaki Matsuyama

Novel hydrogen peroxide metabolism in suspension cells of Scutellaria baicalensis Georgi

We identified a rapid and novel system to effectively metabolize a large amount of H2O2 in the suspension cells ofScutellaria baicalensis Georgi. In response to an elicitor, the cells immediately initiate the hydrolysis of baicalein 7-O-β-d-glucuronide by β-glucuronidase, and the released baicalein is then quickly oxidized to 6,7-dehydrobaicalein by peroxidases. Hydrogen peroxide is effectively consumed during the peroxidase reaction. The β-glucuronidase inhibitor, saccharic acid 1,4-lactone, significantly reduced the H2O2-metabolizing ability of theScutellaria cells, indicating that β-glucuronidase, which does not catalyze the H2O2 degradation, plays an important role in the H2O2 metabolism. As H2O2-metabolizing enzymes, we purified two peroxidases using ammonium sulfate precipitation followed by sequential chromatography on CM-cellulose and hydroxylapatite. Both peroxidases show high H2O2-metabolizing activity using baicalein, whereas other endogenous flavones are not substrates of the peroxidase reaction. Therefore, baicalein predominantly contributed to H2O2 metabolism. Because β-glucuronidase, cell wall peroxidases, and baicalein pre-exist inScutellaria cells, their constitutive presence enables the cells to rapidly induce the H2O2-metabolizing system.

Burkholderia gladioli associated with symptoms of bacterial grain rot and leaf-sheath browning of rice plants

Rice plants with bacterial leaf-sheath browning and grain rot were observed in Fukuoka Prefecture in Japan during the autumn seasons of 1995 and 1996. Burkholderia spp. were consistently isolated from the infected leaf sheaths and grains. These isolates were pathogenic and induced symptoms of seedling rot, grain rot, and leaf-sheath browning in rice plants, as well as in some orchidaceous plants (cymbidium, dendrobium, and oncidium leaves), gladiolus leaves, and onion bulbs. On the basis of morphological, physiological and pathological tests, and species-specific polymerase chain reaction, the isolates were identified as belonging to either Burkholderia glumae or Burkholderia gladioli. B. gladioli, as well as B. glumae, attacked rice plants after artificial inoculation and reproduced the symptoms similar to those after natural infections. We confirmed that rice is an additional natural host of B. gladioli. It is clarified that bacterial grain rot of rice is caused not only by B. glumae but also by B. gladioli.

Grouping of isolates in AG 2 of Rhizoctonia solani by total cellular fatty acid analysis

Total-cellular fatty acid compositions of 34 isolates ofRhizoctonia solani belonging to intraspecific groups (ISGs) of anastomosis group (AG) 2, i.e., AG 2-1, AG 2-2 IIIB (mat rush), AG 2-2 IV (sugar beet), AG 2-2 LP (turfgrass), and AG 2–3 (soybean), were compared. The major fatty acids identified were palmitic, stearic, and oleic acids. Principal component analysis based on the percentage composition of total cellular fatty acids revealed consistently low variability among isolates of a single ISG of AG 2. Average linkage cluster analysis showed that isolates obtained from turfgrass representing a newly proposed group, AG 2-2 LP, were differentiated from other AG 2 ISGs. Isolates of another newly proposed group AG 2–3, from diseased soybean were also closely related to AG 2-1 and AG 2-2 IIIB but distinguishable from the AG 2-1 and AG 2-2 LP isolates by the average linkage cluster analysis. These results suggested that the percentage composition of total-cellular fatty acids is a distinct characteristic for the five ISGs belonging to AG 2, and fatty acid analysis is useful for the differentiation and characterization of these ISGs of AG 2 inR. solani.

RFLP analysis of the PCR-amplified 28S rDNA in Rhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.

Bacterial Wilt of Russell Prairie Gentian Caused by Burkholderia caryophylli

A new bacterial disease of Russell prairie gentian (Eustoma grandiflorum) was found in Fukuoka Prefecture, Japan, in 1997. This disease was characterized by wilting and yellowing of the foliage. A cross section of the stem of a diseased plant revealed a tan to yellow-brown discoloration of the vascular tissue. A nonfluorescent, aerobic, Gram-negative bacterium was consistently isolated from infected plants. The bacteriological characteristics of 10 isolates of the bacterium coincided with those of the reference strains of Burkholderia caryophylli that were isolated from carnations. The bacterium, as well as the reference strains, attacked Russell prairie gentian and carnation after artificial inoculation and reproduced the symptoms similar to those after natural infections. On the basis of bacteriological characteristics and pathogenicities, the bacterium was identified as B. caryophylli. This is the first report of a disease caused by B. caryophylli on Russell prairie gentian ; therefore, bacterial wilt of Russell prairie gentian is proposed as the name of the disease.

Rapid detection of Rhizoctonia species, causal agents of rice sheath diseases, by PCR-RFLP analysis using an alkaline DNA extraction method

Restriction fragment length polymorphism (RFLP) analysis for DNA products amplified by the polymerase chain reaction (PCR) was used for the direct detection ofRhizoctonia solani AG 1 IA and AG 2-2 IIIB,R. oryzae, R. oryzae-sativae andR. fumigata from the diseased rice sheaths. A rapid DNA extraction method with a solution of sodium hydroxide was conducted to extract parasite DNA from diseased rice sheaths. 28S ribosomal DNA (rDNA) derived from fungal genomic DNA extracted by the alkaline method was specifically PCR-amplified. The results of PCR-RFLP analysis for DNA samples from artificially inoculated rice sheath tissues with eachRhizoctonia spp. and the corresponding culture on the medium using two restriction enzymes.HhaI andMspI, showed identical polymorphisms. PCR-RFLP analysis using DNA samples from naturally infected rice sheath tissues also revealed the possibility of direct diagnosis ofR. solani AG 1 IA,R. oryzae andR. oryzae-sativae.

Crown gall of tobacco caused by Agrobacterium tumefaciens biovar 1 in tobacco fields

Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.