A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 10³ cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.     

In vitro antimicrobial activity of aminoreductone against the pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA)

In this study, antimicrobial activity of aminoreductone (AR), a product formed during the initial stage of the Maillard reaction, against methicillin-resistant Staphylococcus aureus (MRSA) was evaluated. The significant growth inhibition of all 51 MRSA isolates irrespective of drug susceptibility by AR was observed. The minimum inhibitory concentration (MIC) of AR ranged from 13 to 26 mM. The bactericidal activity of AR was evaluated by a killing assay with multiples of MIC, and it was recognized to depend on its dose. The combined effects of AR and antibiotics frequently used for the treatment of infections caused by Gram-positive and Gram-negative bacteria, such as amikacin (AN), ciprofloxacin, imipenem and levofloxacin, were examined. As a result, AR did not interfere with these antibiotic activities against 12 MRSA isolates selected and showed the advanced effect of growth inhibition in combination with antibiotics. Moreover, the inhibitory effects of AR were similar to those of AN, an antibiotic with known adverse effects, some serious. These findings show that AR is a naturally formed antimicrobial agent present in thermally processed foods with potential health benefits in medical practice.     

Distribution of photoperiod-insensitive alleles Ppd-B1a and Ppd-D1a and their effect on heading time in Japanese wheat cultivars

The genotypes of photoperiod response genes Ppd-B1 and Ppd-D1 in Japanese wheat cultivars were determined by a PCR-based method, and heading times were compared among genotypes. Most of the Japanese wheat cultivars, except those from the Hokkaido region, carried the photoperiod-insensitive allele Ppd-D1a, and heading was accelerated 10.3 days compared with the Ppd-D1b genotype. Early cultivars with Ppd-D1a may have been selected to avoid damage from preharvest rain. In the Hokkaido region, Ppd-D1a frequency was lower and heading date was late regardless of Ppd-D1 genotype, suggesting another genetic mechanism for late heading in Hokkaido cultivars. In this study, only 11 cultivars proved to carry Ppd-B1a, and all of them carried another photoperiod-insensitive allele, Ppd-D1a. The Ppd-B1a/Ppd-D1a genotype headed 6.7 days earlier than the Ppd-B1b/Ppd-D1a genotype, indicating a significant effect of Ppd-B1a in the genetic background with Ppd-D1a. Early-maturity breeding in Japan is believed to be accelerated by the introduction of the Ppd-B1a allele into medium-heading cultivars carrying Ppd-D1a. Pedigree analysis showed that Ppd-B1a in three extra-early commercial cultivars was inherited from ‘Shiroboro 21’ by early-heading Chugoku lines bred at the Chugoku Agriculture Experimental Station.     

Impact of clod (aggregate) size on evaporation rate and soil moisture profile during the drying process

Large clods (centimetres in size) can be formed by tillage in clayey paddy fields where upland crops are planted. These clods cause early water depletion near the soil surface, which decreases crop germination and emergence rates. Because of the difficulty in reducing clod size, desiccation damage to seeds can be avoided by adjusting the seeding depth based on the clod size-dependent soil moisture profile. This study aimed to clarify the effect of clod size on (1) the evaporation rate (E) and soil moisture profile and (2) the mobility of soil water during the drying process. Evaporation experiments were conducted in an air-conditioned greenhouse with natural light using cylindrical columns filled with artificially made clods 3 (L columns) and 1 cm (S columns) in diameter. We measured E, potential evaporation rate (PE), and total soil moisture content (w_tot) throughout the experiment and the soil moisture profiles at the end of the experiment. The water diffusivity (D_w) and apparent vapour diffusion coefficient (d_vap) were calculated as the mobility of soil water and water vapour, respectively. We found that w_tot was lower in the L column than in the S column, although not at the onset of the experiment. At the end of the experiment, the soil moisture content was lower in the L column than in the S column throughout the soil layer. In contrast, E/PE was higher in the L column than in the S column throughout the experiment and even at the same w_tot. Regarding mobility, D_w was slightly greater in the L column than in the S column in the soil moisture content range, where vapour movement could be greater than liquid water movement. In addition, the ratio of d_vap to the diffusion coefficient of water vapour in soil was higher than unity in both columns and was 2.4–3.2 times higher in the L column than in the S column. In summary, larger clods caused a higher evaporation rate and lower soil moisture content, owing to the increased enhancement of water vapour movement probably induced by wind.     

A novel allele of the Arabidopsis phytochelatin synthase 1 gene conferring high sensitivity to arsenic and antimony

To identify genes involved in arsenite [As(III)] tolerance, we screened for As(III)-sensitive mutants using ethyl methanesulfonate (EMS)-mutagenized seeds of Arabidopsis thaliana. One mutant with high sensitivity to As(III) was isolated from the screening. It had a mutation in Glu⁵² of phytochelatin synthase 1 (AtPCS1), and introduction of the wild-type AtPCS1 gene rescued the phenotype, confirming that the mutant represented a new allele of atpcs1 (termed as cad1-7). The expression of AtPCS1 from cad1-7 and cad1-3, a second allele of atpcs1, complemented As-sensitive yeast, but not plants. Our study suggests that AtPCS1 activity is differentially regulated between plants and yeast.     

Estimation of microbial biomass potassium in paddy field soil

Abstract

Potassium (K) in microbial cells, microbial biomass K, in soil has been recently recognized as a K pool for plant growth. We determined soil microbial biomass K in paddy fields to reveal its importance as a K pool in paddy field soil for the first time. Microbial biomass K ranged from 5 to 21 mg K kg^?1 in the soil samples periodically collected from a paddy field and the value corresponded to 41% of the exchangeable K on average. Both microbial biomass K and exchangeable K increased conspicuously due to the long-term application of livestock manure compost or rice straw compost. Biomass K was higher than exchangeable K under K-deficient conditions in the long-term experimental plots without K application. The present study revealed that the microbial biomass contained considerable amounts of K in paddy field soil, indicating the need for evaluation of the microbial biomass K as a source and a stock of K in soil that has been overlooked.     

Diversification and genetic differentiation of cultivated melon inferred from sequence polymorphism in the chloroplast genome

Molecular analysis encouraged discovery of genetic diversity and relationships of cultivated melon (Cucumis melo L.). We sequenced nine inter- and intra-genic regions of the chloroplast genome, about 5500 bp, using 60 melon accessions and six reference accessions of wild species of Cucumis to show intra-specific variation of the chloroplast genome. Sequence polymorphisms were detected among melon accessions and other Cucumis species, indicating intra-specific diversification of the chloroplast genome. Melon accessions were classified into three subclusters by cytoplasm type and then into 12 subgroups. Geographical origin and seed size also differed between the three subclusters. Subcluster Ia contained small-seed melon from Southern Africa and South and East Asia and subcluster Ib mainly consisted of large-seed melon from northern Africa, Europe and USA. Melon accessions of subcluster Ic were only found in West, Central and Southern Africa. Our results indicated that European melon groups and Asian melon groups diversified independently and shared the same maternal lineage with northern African large-seed melon and Southern African small-seed melon, respectively. Cultivated melon of subcluster Ic may have been domesticated independently in Africa. The presence of 11 cytoplasm types in Africa strongly supported African origin of cultivated melon and indicated the importance of germplasm from Africa.     

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells

The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B₄ isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.