Microsatellite and mitochondrial DNA analyses reveal no genetic difference between two pufferfish species torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and karasu <Emphasis Type="Italic">T. chinensis</Emphasis>

It has been reported that the nucleotide sequences of the mitochondrial and nuclear genes of Takifugu pufferfish torafugu T. rubripes and karasu T. chinensis show 99–100% sequence identity, indicating a very close relationship between the two species. To further investigate this genetic relationship, we compared genetic variation at four microsatellite loci and at the mitochondrial control region (CR) (561 bp) between groups of T. rubripes caught at two locations [TrG, caught in the Genkai Sea off Tsushima Island in 2003 (n = 50); TrS, caught in the Suwo Sea off Kita-Kyushu in 2008 (n = 50)] and T. chinensis caught at one location (TcK, caught off the east coast of Korea in 2004; n = 50). Analyses using microsatellite loci showed that genetic diversity index values of the TrG, TrS and TcK groups were 0.9505, 0.9350 and 0.9335, respectively, while values of genetic distance and genetic differentiation between TrG and TcK (0.0543 and 0.0189, respectively) were smaller than those between TrG and TrS (0.0857 and 0.0194, respectively). Analyses using CR for the same specimens showed that genetic distances were consistent with those obtained using microsatellite loci. These results, together with our previous observations, suggest that T. rubripes and T. chinensis are very closely related and possibly can be regarded as the same species.     

Symbiotic efficiency and compatibility of native rhizobia in northern Thailand with different soybean cultivars

Symbiotic efficiency and compatibility of 81 isolates of native bradyrhizobia from irrigated areas in northern Thailand with four soybean cultivars and one cowpea cultivar were evaluated under laboratory conditions. Effectiveness and / or compatibility of the tested isolates were compared with those of a standard strain (Bradyrhizobium japonicum CB 1809) by using plants grown on plastic seed bags. Effectiveness of the isolates was also estimated using uninoculated control plants grown in a nitrogen-free solution. Nodulation of a wide range of host plants by the majority of the tested isolates was observed, which agreed well with the results of our previous field experiment (Shutsrirung et al. 2002: Soil Sci. Plant Nutr., 48, 491–499). Up to 75% of the tested isolates induced a higher growth efficiency than that of the uninoculated control in association with one of the tested cultivars, Black soybean. Comparision with uninoculated control plants, enable to estimate the proportion of the tested isolates leading to effective growth promotion (E + e) of each cultivar, namely, Black soybean (local Thai cultivar), 75%; Cowpea, 82%; SJ5 (commercial Thai cultivar), 33%; Bragg (US cultivar), 33%; and Improved Pelican (US cultivar), 9%. These results indicated that although isolates with a high infectiveness with both “Asian-type” and “US-type” soybeans could be found, a high frequency of isolates leading to inefficient nodules was observed in the US cultivar, suggesting the presence of genetic differences in the soybean cultivars that express high-preference (efficient nodules) or low-preference (inefficient nodules) for a certain group of tested isolates. Based on the results of this laboratory experiment together with our previous field experiment, native rhizobial populations in the irrigated area of northern Thailand could be separated into three groups; Group 1: rhizobium strains showing a high effectiveness with only Asian cultivars, Group 2: strains showing a high effectiveness with only US origin cultivars, and Group 3: strains showing a high effectiveness with both Asian and US origin cultivars. The majority of the native rhizobial populations belonged to Group 1. The isolates in Group 3 may display a high potential for manipulating useful rhizobial inoculant.     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.     

Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof

Reasons for performing study: Abnormal epidermal stem cell regulation may contribute to the pathogenesis of equine chronic laminitis. Objective: To analyse the involvement of p63, a regulator of epidermal stem cell proliferative potential, in chronic laminitis. Methods: Epidermal tissues from skin, coronet and lamellae of the dorsal foot were harvested from 5 horses with chronic laminitis and 5 control horses. Tissues were analysed using histopathology, immunofluorescence microscopy and quantitative immunoblotting Results: Hoof lamellae of laminitic horses had a lower frequency of p63 positive cells than control lamellae, particularly in the distal region. Quantitative immunoblotting confirmed reduced p63 expression in the laminitic distal lamellar region. The decreased p63 expression in laminitic epidermal lamellae was most apparent in the abaxial region adjacent to the hoof wall and highly associated with the formation of terminally differentiated, dysplastic and hyperkeratotic epidermis in this region, whereas lamellae from control horses maintained high p63 expression throughout the axial‐abaxial axis. Conclusions: Expression of p63 in equine skin resembles that reported in other species, including man and rodents, suggesting that p63 can serve as a marker for the proliferative potential of equine epidermal stem cells. p63 expression was significantly lower in the chronic laminitic hoof than in that of control horses, suggesting laminitic hoof epithelium has more limited proliferative potential with a shift towards differentiation. This may reflect reduced activity of epidermal stem cells in laminitic hoof. It is proposed that p63 contributes to the maintenance of hoof lamellae and that misregulation of p63 expression may lead to epidermal dysplasia during lamellar wedge formation. Potential relevance: This study suggests that loss of epidermal stem cells contributes to the pathogenesis of equine laminitis. Autologous transplantation of p63‐positive epidermal stem cells from unaffected regions may have regenerative therapeutic potential for laminitic horses.     

Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999     

Thermal tolerance of a thermally selected strain of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> and the pedigrees of its F1 and F2 generations indicated by their critical thermal maxima

A thermally selected strain of rainbow trout has been established by selective breeding since 1966 in Miyazaki, Japan. In the present study, we compared the critical thermal maxima (CTMs), the temperatures at which organisms reach a predefined sublethal endpoint and lose their equilibrium, between a thermally selected and two normal (Donaldson) strains of rainbow trout. The CTM of one normal strain from Nikko (Nikko strain) acclimated to 20 °C (29.7 °C) was significantly lower than those of the thermally selected strain (30.0 °C) and the other Donaldson strain from Aomori (29.9 °C) (P?<?0.05). The F1 generations, F1T and F1N, were produced by crossing thermally selected strain females with Nikko strain males and Nikko strain females with thermally selected strain males, respectively. No significant difference was observed in the CTM between F1T [30.1?±?0.15 °C (n?=?30)] and F1N [30.1?±?0.16 °C (n?=?30)] (P?>?0.05) for fish acclimated to 20 °C, suggesting that the F1 offspring inherited the thermal tolerance trait from one thermally selected strain parent irrespective of whether it was the male or female. F2 offspring of F1T or F1N also showed the thermal tolerance trait. The coefficients of variation for CTM were also compared among all the datasets obtained in the present study and their values for F1 hybrids were lower than those of the parental generation of the Nikko strain (P?<?0.05). In contrast, the coefficients of variation of F2s were the same as those of their parental generation. Furthermore, the thermally selected strain and Nikko strain as a reference family provide a F2 generation for segregating phenotypes, which is required for in-depth genetic analysis of the thermally selected rainbow trout strain.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

Bacterial leaf blight of sweet pepper (Capsicum annuum) caused by Pseudomonas cichorii in Japan

Sweet peppers (cv. Tosajishi beauty) with leaf blight symptoms were observed in Kochi Prefecture, 2003. Initially, small spots formed on leaves, and later enlarged to brown to dark brown spots, and eventually blighted leaves fell. Several bacteria that caused the same symptoms were isolated and subsequently reisolated. These bacteria were identified as Pseudomonas cichorii on the basis of bacterial characteristics and the 16S rRNA gene sequence. This is the first report of bacterial leaf blight in the sweet pepper in the world.     

Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation.