Biological control of rice blast by the epiphytic bacterium <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with a chitinolytic enzyme gene from an antagonistic bacterium, <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

The gene chiA, encoding for the endochitinase ChiA, was cloned from Serratia marcescens strain B2, a tomato epiphytic bacterium, and introduced into the epiphytic bacterium Erwinia ananas NR-1, isolated from rice phylloplane. The gene chiA was introduced under the control of two types of promoter into a broad-host-range plasmid vector. The vector contained various fragments with promoter activity isolated from E. ananas chromosomal DNA. The constructed vectors were designated pchiA-V1pcf9 and pchiA-V1pcf53 for their respective promoters. E. ananas NR-1 transformed with either of these vectors produced and secreted ChiA. The antifungal activity of ChiA produced by transformed E. ananas NR-1 was demonstrated in vitro by the inhibition of Pyricularia oryzae germ tube elongation such as bursting of the hyphal tip. Transformed E. ananas NR-1 suppressed the incidence of rice blast caused by P. oryzae under greenhouse conditions; however, the magnitude of the suppressive effect depended on which promoter was used. Both transformants and the nontransformant E. ananas NR-1 survived on rice phylloplane. It is expected that the rice epiphytic bacterium E. ananas NR-1 carrying a chitinolytic enzyme gene is an efficient biological control agent against rice blast.     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

The use of CPPU for efficient propagation of pineapple

The use of forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea) (CPPU) for efficient propagation of pineapples was investigated. About 85% of axillary buds can be forced to sprout by soaking defoliated stem pieces (12 cm in length) in a 2.5 or 5.0 mg l⁻¹ CPPU solution for more than 3 h. The use of old stems taken from the third or fourth ratoon plants had the advantage of less liability to fungal decay, as compared to young stems from the first crop plants. The CPPU treatment combined with the removal of shoots from stems at monthly intervals significantly increased the number of shoots per stem piece (about 15 shoots per piece at 5.0 mg l⁻¹ CPPU), and resulted in a more uniform shoot size (the percentage of shoots within a range of 5–15 cm in length was about 90% at 5.0 mg l⁻¹ CPPU). The rooting of shoots was easily promoted within 1 month by treating the basal portion of shoots with 20 mg l⁻¹ indole-3-butyric acid (IBA) for 15 min. The CPPU method required about 5 months for plantlet propagation. From these results, we found that pineapple plantlets could be efficiently propagated by the following method: (1) soaking defoliated stems in a 2.5–5.0 mg l⁻¹ CPPU solution for more than 3 h; (2) harvest of developed shoots from the stems at regular intervals; and (3) promotion of rooting on the shoots at 20 mg l⁻¹ IBA for 15 min.     

Cloning of various promoters for foreign gene expression in Erwinia ananas

We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.     

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.     

Relationships between immunoglobulin and fat‐soluble vitamins in colostrum of Japanese Black multiparous cows

Data from 19 Japanese Black multiparous cows were collected to clarify the relationships among immunoglobulin (Ig) G, IgA, β‐carotene, vitamin A and α‐tocopherol contents in colostrum of cows in order to evaluate the role of fat‐soluble vitamins on colostral IgG and IgA production. Mean colostral IgG was 141 mg/mL, ranging from 65 to 208 mg/mL, whereas mean colostral IgA was 8.7 mg/mL, ranging from 1.0 to 34.6 mg/mL. Colostral IgG increased with aging in multiparous cows. There were positive correlations between colostral IgG and colostral vitamin A or colostral α‐tocopherol in cows, and the higher adjusted R² was obtained in the prediction model of colostral IgG from age and colostral vitamin A. Colostral vitamin A was positively correlated with colostral β‐carotene or colostral α‐tocopherol in cows, but there were no relationships between colostral IgA and colostral IgG or colostral fat‐soluble vitamins. These results indicate that fat‐soluble vitamin contents in colostrum of cows may change in similar patterns and high colostral vitamin A is related with high colostral IgG.     

Rice yield and soil carbon dynamics over three years of applying rice husk charcoal to an Andosol paddy field

Rice husk charcoal (RC) produced from the pyrolysis of rice husk (RH) can be one of the cost-effective biochars for use in rice-based farming systems. This study investigated changes in rice yield and soil carbon sequestration over three years of RC application to an Andosol paddy field. The treatments were RC application at 0.02, 0.2, and 2 kg m^?2 (RC0.02, RC0.2, and RC2, respectively), RH application at 0.2 kg m^?2 (RH0.2), and a control with no RC or RH application (CONT). The results showed that RC2 increased culm length by 4% and straw weight by 14% on average over the three years. These increases in plant growth coincided with a higher level of silicon uptake by the rice plants, although they did not significantly affect grain yield. The soil carbon content was progressively increased by RC2 over the three years, whereas it was not significantly affected by RC0.02 or RC0.2. A considerable amount (>72%) of the applied carbon with RC2 remained in the soil by taking account of its downward movement below the 10 cm layer of the paddy field after three consecutive years of RC application. We conclude that rice husk charcoal application to Andosol paddy fields is an effective option for increasing carbon sequestration. Furthermore, the increase in silicon uptake by rice plants suggests that rice husk charcoal can also be functioning as a silicon fertilizer.     

Relationship between watercore and maturity or sorbitol in apples affected by preharvest fruit temperature

The effect of preharvest fruit temperature on the relationships between development of watercore and ethylene evolution, flesh firmness, membrane permeability and sorbitol level in two watercore-susceptible apple (Malus domestica Borkh.) cultivars, ‘Himekami' and ‘Fuji', was investigated at ambient or modified temperatures of 10, 15 or 25°C. Watercore occurrence was completely inhibited at 25°C in both cultivars whereas fruit subjected to 10 or 15°C exhibited significantly higher watercore ratings after two or three weeks of the treatment than those at 25°C and ambient temperatures. Ethylene production of ‘Himekami' apples increased at 25°C and ambient temperature but not at 15°C. Ethylene production was not affected by treatment in ‘Fuji' apples. Flesh firmness of ‘Himekami' tended to maintain a higher level at 15°C than at 25°C, while there was no clear difference among the treatments in ‘Fuji'. The rate of potassium (K) ion elution from the flesh tissues of either cultivar was not affected by fruit temperature treatment. Sorbitol content at 15°C maintained significantly higher level than that at 25°C during the treatment in ‘Himekami' apples although no sorbitol difference was observed among the temperature treatments in ‘Fuji' apples.     

A simple specimen preparation method for histopathological evaluation of vestibular organs

Vestibular organs consist of the maculae staticae, which are located in both the utricle and saccule, as well as the semicircular ducts and their ampullas. There have been no reports on specimen preparation methods for vestibular organs, including maculae staticae or semicircular ducts. In this study, we investigated highly reproducible methods of preparing vestibular organ specimens for histopathological examinations. We established a method that allows researchers to observe the utricle and saccule, including otoliths, the ampulla of a semicircular duct, and parts of semicircular ducts. This highly reproducible method is useful for histopathological analysis of mice with symptoms of abnormal equilibrium caused by medical toxicity and genetic modification.     

The target site of the novel fungicide quinofumelin,Pyricularia oryzae class II dihydroorotate dehydrogenase

The target site of the novel fungicide quinofumelin was investigated in the rice blast fungus Pyricularia oryzae. Quinofumelin-induced mycelial growth inhibition was reversed by orotate but not by dihydroorotate. Recovery tests suggested that the target site of quinofumelin was dihydroorotate dehydrogenase (DHODH), which catalyzes the oxidation of dihydroorotate to orotate. Quinofumelin strongly inhibited P. oryzae class 2 DHODH (DHODH II) (IC₅₀: 2.8 nM). The inhibitory activities of mycelial growth and DHODH II were strongly positively correlated, indicating that DHODH II inhibition by quinofumelin lead to antifungal activity. A P. oryzae DHODH II gene (PoPYR4) disruption mutant (ΔPopyr4), showing the same tendency as the quinofumelin-treated wild strain in recovery tests, was constructed, and disease symptoms were not observed in rice plants infected by ΔPopyr4. Thus, DHODH II, which plays an important role in pathogenicity and mycelial growth, is found to be the target site of quinofumelin.