Analysis of sources of oxolinic acid-resistant field strains of <Emphasis Type="Italic">Burkholderia glumae</Emphasis> based on rep-PCR analysis and nucleotide sequences of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Repetitive sequence-based polymerase chain reaction (rep-PCR) analysis using BOX and ERIC as primers showed a highly divergent phylogeny among field strains of Burkholderia glumae. To elucidate the sources of oxolinic acid (OA) resistance in field strains of B. glumae isolated from rice seedlings cultivated in Mie, Toyama, and Iwate prefectures, Japan, the amino acid at position 83 of GyrA (GyrA83), which is involved in OA resistance, and the DNA patterns from the rep-PCR and the partial nucleotide sequences of gyrB and rpoD from various strains were analyzed. The ten Mie strains, in which GyrA83 was isoleucine (Ile), were divided into two groups based on the band patterns in rep-PCR analysis, although the nucleotide sequences of gyrB and rpoD were identical among the strains. Based on the band patterns in the rep-PCR analysis and the gyrB and rpoD sequences, two highly OA-resistant Toyama strains, Pg-13 and Pg-14, for which GyrA83 was serine (Ser) and Ile, respectively, were in the same lineage. This suggests that the bacteria might acquire OA resistance faster than phylogenic diversity as determined with the repetitive sequences BOX and ERIC and with gyrB and rpoD. Furthermore, three Iwate strains (H95, H101, and H104), isolated from seedlings of different cultivars grown in different years and having Ile at GyrA83, are probably in the same lineage, suggesting that OA-resistant bacteria might be transferred among different cultivars.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

In the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex-PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.     

Molecular characterisation of equine group A rotavirus, Nasuno, isolated in Tochigi Prefecture, Japan

In this study, equine group A rotavirus (RV-A), Nasuno, isolated from foal diarrhoea in Tochigi Prefecture, Japan was characterised genetically by sequence analysis of the genome segments encoding VP4 and VP7. The nucleotide and deduced amino acid sequences revealed high homology with P[12] RV-As (94.0-99.3% and 94.9-99.4%) and G3 RV-As (86.9-99.5% and 91.1-99.4%). Nasuno was also classified into P[12] and G3 in the phylogenetic analysis of the nucleotide sequences of the genome segments encoding VP4 and VP7.     

Development of new self-feeding system for mass rearing of ayu <Emphasis Type="Italic">Plecoglossus altivelis altivelis</Emphasis> under artificial and natural light-dark cycles

Self-feeding of grouped ayu Plecoglossus altivelis altivelis was examined under an artificial light-dark (LD) cycle and natural day length using a trigger with a photosensitive sensor. In Experiment 1, approximately 15 juvenile fish (mean body weight, 0.6 g) were kept in 60-L glass tanks (four tanks) under LD 12∶12 (lighting period 06∶00–18∶00 hours) and self-feeding and locomotor activities were simultaneously recorded. Self-feeding was detected within one day and feeding activities were strongly synchronized to the LD cycle, with almost strictly diurnal feeding (98.2%). Although locomotor activities in the upper part of the tank were also greatest during the light phase (76.8%), the activity profiles were different from those of self-feeding, suggesting the validity of the photosensitive sensor. In Experiment 2,25 fish (mean body weight, 15 g) were kept in outdoor 1000-L tanks (four tanks) under natural day length and water temperatures in May, and self-feeding was recorded from mid-August. Stable self-feeding was observed from early September in all the tanks. Nearly all food demands were diurnal, and usually crepuscular. Somatic growth was seen in all tanks. These results demonstrate that the self-feeding system using a photosensitive sensor is applicable for grouped juvenile ayu as small as 0.6 g and adult fish reared under natural conditions.     

Circadian rhythms and individual variability of self-feeding activity in groups of rainbow trout Oncorhynchus mykiss (Walbaum)

An experiment was conducted to examine whether circadian self‐feeding rhythms exist in groups of rainbow trout Oncorhynchus mykiss (Walbaum) and to investigate individual variability of self‐ feeding and its influence on growth. Ten trout of 144.9 ± 12.0 g (mean ± SD) were transferred to each of eight 290‐L tanks and given free access to feed via a self‐feeder. Fish in four of the eight groups were individually tagged, and the individual activating the trigger was identified using video observations. The self‐feeding activity of each group was recorded under a light:dark cycle of 16.5:7.5 h and under continuous light (LL) conditions. Although diel self‐feeding rhythms in the groups of trout were, in general, diurnal, night‐feeding activity was observed in the initial LD phase, but there was a progressive reduction and eventual cessation in most groups. Endogenous circadian self‐feeding rhythms were detected in the groups of trout under LL conditions. The trout formed dominance hierarchies of self‐feeding activity, with one or two individuals accounting for the majority of trigger actuations. However, the dominants did not occupy the area surrounding the trigger. Growth rates did not differ significantly between the dominants and the subordinates within the groups.     

Relationship between peripartal plasma oxytocin and prostaglandin F(2alpha) metabolite and placental expulsion time in heavy draft mares

The aim of this study was to clarify the relationship between circulating oxytocin (OT) and PGF(2alpha) metabolite (PGFM) in mares at the third stage of labor and placental expulsion time in order to investigate a cause of retained placenta of which the incidence increase in a heavy draft mare. Blood was sampled every 5 min from foaling to expulsion of the placenta in 18 heavy draft mares to evaluate circulating OT and PGFM. The relationships between OT and PGFM concentration and recorded placental expulsion times were investigated. The results were as follows (1) The highest level of OT concentration was observed close to foaling in 15 mares. (2) The OT concentrations close to foaling were variable with a large difference from the lowest concentration, 22.1 pg/ml, to the highest concentration, 209.3 pg/ml. (3) The highest level of PGFM was observed close to foaling in 17 mares. (4) During the 60 min following foaling, the OT concentrations of the mares (n=11) that had a shorter placental expulsion time (i.e., <1 h), were significantly higher than those of the mares (n=7) that had a longer placental expulsion time (i.e., >1 h; P<0.05). Collectively, the OT concentration immediately after foaling is negatively related to the placental expulsion time. Deficiency of OT secretion at foaling have should be considered as one of the causes of retained placenta in heavy draft mares.