Determination of seven organosulfur compounds in garlic by high-performance liquid chromatography

The properties of garlic (Allium sativum L.) are attributed to organosulfur compounds. Although these compounds change during cultivation and storage, there is no report of their simultaneous analysis. Here, a newly developed analytical method with a rapid and simple sample preparation to determine four sulfoxides and three gamma-glutamyl peptides in garlic is reported. All garlic samples were simply extracted with 90% methanol solution containing 0.01 N hydrochloric acid and prepared for analysis. Alliin, isoalliin, methiin, cycloalliin, and gamma-l-glutamyl-S-methyl-l-cysteine were determined by normal-phase HPLC using an aminopropyl-bonded column. gamma-l-Glutamyl-S-(2-propenyl)-l-cysteine and gamma-l-glutamyl-S-(trans-1-propenyl)-l-cysteine were separated on an octadecylsilane column. The overall recoveries were 97.1-102.3%, and the relative standard deviation values of intra- and interday precision were lower than 2.6 and 4.6%, respectively. This newly developed method offers some advantages over the currently accepted techniques including specificity, speed, and ease of use and would be useful for chemical and biological studies of garlic and its preparations.     

Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts

Cell‐cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB‐GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB‐DsRed. mMB‐GFP and chMB‐DsRed were cocultured and induced to differentiate. After 24 h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP⁺/DsRed⁺ hybrid myotubes were able to survive and grew to hyper‐multinucleated mature form. We also found that undifferentiated mMB‐GFP efficiently fuse to the chMB‐DsRed‐derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast‐based fusogenic technique will open up an alternative direction to create novel hybrid products.     

Development of simple sequence repeat markers for the classification of the clubroot pathogen <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in cruciferous crops. To classify isolates, we developed simple sequence repeat (SSR) markers for P. brassicae. Twenty-four Japanese isolates were used in this study: 12 isolates of an unknown pathotype from the Kyoto Prefecture, as well as 12 isolates of known pathotypes, including three single-spore lines. From the 12 isolates from Kyoto Prefecture, 11 were classified into either pathotype 2 (three isolates) or 4 (eight isolates). We designed 23 SSR markers based on the P. brassicae genome, of which 11 markers from intergenic regions showed polymorphisms in the 24 isolates. Many haploid isolates belonging to pathotypes 2 and 4 were monomorphic, and typical alleles were detected in some isolates not belonging to pathotype 4. Two bands were detected for eight SSR loci in five isolates, indicating that different genotypes were mixed in these isolates. We constructed a phylogram based on the 11 polymorphic SSRs. Pathotypes 2 and 4 formed a cluster, from which pathotypes 3 and 1 were successively placed. These results strongly suggest a close genetic relationship between isolates in pathotypes 2 and 4, consistent with our finding that isolates in these two pathotypes were found at one collection site. In combination with pathotype classification and other marker systems, the SSR markers can be used for more detailed analyses to improve the control of clubroot disease.     

Effects of row deep tillage for the growth base formed by piling up soil in damp lowlands behind coastal sand dunes to construct coastal disaster prevention forest belts on the Kujukuri coastline,Japan

ABSTRACT

To successfully afforest coastal forest belts ensuring high disaster prevention, growth bases for them have been constructed by piling up soil in the low wetlands along the Kujukuri coastline. Ground surfaces in such bases are often covered with water because of soil compaction, leaving them susceptible to stagnant water. Water stagnation in soil is problematic, potentially interfering with afforesting coastal forests. Therefore, row deep tillage was conducted for parts of growth bases to combat the poor physical properties of the existing soil. Here, we surveyed soil profiles and measured vertical soil hardness distribution in two forest stands with piling up soil to evaluate the effectiveness of tillage for man-made soil. Soil hardness measurements indicated that the vertical areas with ‘soft’ and ‘hard/consolidated’ soil alternately appeared in growth base profiles. Generally, soil of the dense and very hard layers was apt to be formed by strong compaction of the filled-up soil because of heavy machinery usage during growth base preparation. Such dense and hard soils in the untilled areas of the profiles were also observed in this study. By contrast, it was confirmed that row deep tillage drastically improved soil physical properties, i.e., decreased hardness and increased water permeability, because the sequential hardened subsoil layers were well-broken-up. Moreover, it was observed many thick and large roots penetrated deeper layers with deep tillage areas. These results suggest that row deep tillage of hardened soil is quite effective at securing the areas and providing the physical conditions for deeply penetrated roots into deeper soils, which increases healthy root development. They show the effects of soil hardness reduction and water permeability improvement have been maintained for two decades, at least, after construction. These findings will be useful for alleviating some problems of soil compaction, water stagnation, and tree-growth hindrance that have been encountered on afforestation sites with man-made soil.