Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

Removal of sperm tail using trypsin and pre-activation of oocyte facilitates intracytoplasmic sperm injection in mice and rats

We examined various methods to enhance the accessibility of intracytoplasmic sperm injection (ICSI) technology to more users by making the technique easier, more efficient, and practical. First, the methods for artificially removing the mouse sperm tail were evaluated. Trypsin treatment was found to efficiently remove the sperm tails. The resultant sperm cells had a lower oocyte activation capacity; however, the use of activated oocytes resulted in the same fecundity as that of fresh, untreated sperm. Pre-activated oocytes were more resistant to physical damage, showed higher survival rates, and required less time per injection. Testing this method in rats yielded similar results, although the oocyte activation method was different. Remarkably, this method resulted in higher birth rates of rat progeny than with conventional methods of rat ICSI. Our method thereby streamlines mouse and rat ICSI, making it more accessible to laboratories across many disciplines.     

Loss-of-function of Nicotiana tabacum L. eukaryotic translation initiation factors eIF4E1-S and eIF(iso)4E-T synergistically confers high-level resistance to both Potato virus Y (PVY) and resistance-breaking PVY

The plant eukaryotic translation-initiation factors eIF4E and eIF(iso)4E play key roles in infection by plant RNA viruses, especially potyviruses. Mutations in the genes that encode these factors reduce susceptibility to the viruses. In the amphidiploid plant tobacco (Nicotiana tabacum L.), eIF4E1-S deletion mutants resist Potato virus Y (PVY), but resistance-breaking strains (RB-PVY) have appeared. In an earlier study, we demonstrated that the loss-of-function of eIF(iso)4E-T reduces susceptibility to RB-PVY. Here, we show that simultaneous inhibition of eIF4E1-S and eIF(iso)4E-T synergistically confers enhanced resistance to both PVY and RB-PVY without host growth or development defects. PVY symptoms and accumulation in a tobacco line lacking eIF4E1-S were detected at 14 days post-inoculation (dpi) and RB-PVY symptoms in lines without functional eIF(iso)4E-T were observed at 24 dpi. RB-PVY emerged in a PVY-infected tobacco line lacking eIF4E1-S. In contrast, lines without functional eIF4E1-S and eIF(iso)4E-T were nearly immune to PVY and RB-PVY, and little accumulation of either virus was detected even at 56 dpi. Thus, the lines will be promising for PVY-resistance breeding. This study provides a novel strategy to develop tobacco highly resistant to PVY and RB-PVY, and insights into the mechanisms responsible for high-level resistance.     

Effects of porcine leptin receptor gene polymorphisms on backfat thickness,fat area ratios by image analysis,and serum leptin concentrations in a Duroc purebred population

The leptin receptor (LEPR) gene is considered a candidate gene for fatness traits. It is located on SSC 6 in a region in which quantitative trait loci (QTLs) for backfat thickness (BF), fat area ratios, and serum leptin concentration (LEPC) have previously been detected in a Duroc purebred population. The objectives of the present study were to identify porcine LEPR polymorphisms and examine the effects of LEPR polymorphisms on fatness traits in this same population. The Duroc pigs (226 to 953 pigs) were evaluated for BF, fat area ratios using image analysis, and LEPC. A total of seven single nucleotide polymorphisms (SNPs) in the full‐length LEPR coding region were identified in pigs from the base population. Four non‐synonymous SNPs of the LEPR gene and 15 microsatellite markers on SSC 6 were then genotyped in all pigs. During candidate gene analysis, we detected significant effects of the non‐synonymous SNP c.2002C>T in exon 14 on all traits. In fine mapping analysis, significant QTLs for BF, fat area ratios, and LEPC were detected near the LEPR gene in the same region. These results indicated that the c.2002C>T SNP of LEPR has a strong effect on BF, fat area ratios and LEPC.     

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars,combinations of permeating cryoprotectants and equilibration regimens

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.     

Molecular identification of methane oxidizing bacteria in a Japanese rice field soil

By using cultivation-independent techniques, community changes of methane-oxidizing bacteria (MOB) in rice bulk soils were investigated under field conditions in a Japanese rice field. The representative soil samples were collected during the typical rice growing season and nonrice growing period all year round. Statistical characterization of denaturing gradient gel electrophoresis (DGGE) community patterns of MOB pmoA/amoA functional gene fragments showed that MOB community structures in the rice bulk soils remained largely unchanged throughout the investigated period. The total intensity of six common DGGE bands that appeared consistently throughout the investigated period accounted for 64% of the total intensity of all 18 different DGGE bands detected. The low squared distance of the Ward cluster analysis of the DGGE pattern and the high Sorensen similarity coefficient (81%) also implied the high similarity of the MOB community structures. The stable MOB community structure did not couple well with the wide variation of soil water contents all year round. Sequencing analysis of the nine characteristic bands including six common bands revealed the presence of Type I, Type II methanotrophs, and β-proteobacterial ammonia oxidizers in rice bulk soils. In comparison with MOB type species, three DGGE bands showed a wide variation of the highly conserved amino acid residues, implying the presence of novel MOB bacteria inhabiting the rice bulk soil. The high diversity of MOB composition suggested that rice bulk soils might serve as an ideal reservoir for the dynamic changes of MOB in a rice field ecosystem in response to environment changes.     

Xenografting of gonadal tissues into mice as a possible method for conservation and utilization of porcine genetic resources

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early‐stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro‐matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice.