Inverse correlation between mRNA levels in GFP-silenced transgenic Nicotiana benthamiana and resistance to Potato virus X engineered to contain GFP sequence

Nicotiana benthamiana was transformed with a green fluorescent protein (GFP) gene driven by cauliflower mosaic virus 35S promoter. A GFP-silenced line and a nonsilenced line were selected after ultraviolet irradiation. GFP short-interfering RNAs (siRNAs) were detected in the silenced line but not in the nonsilenced line. T₁ progeny of the silenced line varied in GFP suppression patterns and were grouped into three types (I, II, III) based on the GFP suppression pattern. With Northern blot analysis, different levels of GFP mRNA accumulated, from a very low level in type I and II to an intermediate level in type III, in contrast to a much higher level in the nonsilenced line. Plants were also inoculated with Potato virus X engineered to contain the GFP sequence to evaluate the levels of virus resistance. None to a few GFP spots were observed on inoculated leaves in types I and II, whereas numerous spots and systemic infection appeared in type III. These results showed that virus resistance was inversely correlated with the levels of mRNA, suggesting that the strength of RNA silencing determines the extent of virus resistance.     

Effect of dietary histidine on contents of carnosine and anserine in muscles of broilers

Carnosine (β‐alanyl‐L‐histidine) and anserine (β‐alanyl‐1‐methyl‐L‐histidine) are dipeptides mainly found in skeletal muscle and brain of many vertebrates, and particularly high concentrations are observed in chicken pectoral muscles. It was reported that these peptides have many functions, such as antioxidant activity. In this study, we examined the effect of different levels of dietary histidine on carnosine and anserine contents in broiler muscles. The 14‐days‐old female Chunky strain broilers were given feeds containing three different levels of histidine; 67% (Low‐His), 100% (Control) and 200% (High‐His) of histidine requirement according to the NRC (1994). Chicks were fed experimental diets for 10 days. Both dipeptides in muscle were significantly decreased. In particular, carnosine was not detected at all in the Low‐His group and was significantly increased in the High‐His group. Both dipeptides were not detected in plasma. These results indicated the possibility to produce chicken meat with enhanced amount of these dipeptides by high histidine feeding.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

Regulation of taste-active components of meat by dietary branched-chain amino acids; effects of branched-chain amino acid antagonism

1. The effects of dietary branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) on taste-active components, especially free glutamate (Glu), in meat were investigated. 2. Broiler chickens (28 d old) were given varied dietary BCAA levels for 10 d before marketing. Dietary BCAA content ratios were either 100:100:100 (Low Leu group), 150:100:100 (Control group) or 150:150:150 (High Ile + Val group) for Leu:Ile:Val (% of each BCAA requirement according to NRC, 1994). Taste-related components of meat (free amino acids and ATP metabolites) and sensory scores of meat soup were estimated. 3. Free Glu content, the main taste-active component of meat, was significantly increased by dietary BCAA. Compared to the Control group, free Glu content increased by 30% in the High Ile + Val group. However, the inosine monophosphate (IMP) content in meat did not change among groups. 4. Sensory evaluation of meat soups showed that Control and High Ile + Val groups had different meat flavours. The sensory score of overall taste intensity was significantly higher in the High Ile + Val group. 5. These results suggest that dietary BCAA concentrations regulate free Glu in meat. Increasing dietary Ile + Val induces an increase in free Glu content of meat, improves meat taste and is more effective for increasing free Glu content in meat than decreasing dietary Leu level.     

Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene

Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene ( cytb ) was characterized. All QoI-resistant isolates had the same mutation (GGT to G C T) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b , which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens.     

Potential loss of unique genetic diversity in tomato landraces by genetic colonization of modern cultivars at a non-center of origin

We report the genetic diversity of tomato landraces from Myanmar, with reference to worldwide tomato accessions. Ten simple sequence repeat markers were screened and used to create diversity profiles of tomato germplasm. A total of 133 alleles were amplified from the germplasm investigated; alleles specific to landraces from Myanmar were also observed. Higher genetic diversity for the Myanmar landraces highlighted the broad genetic base of tomato germplasm. Principal component analysis showed that most of the Myanmar landraces were divergent from other accessions. The different approaches used to analyse landrace diversity documented the broader genetic base of germplasm from Myanmar, which is a non‐center of origin for tomato. Genetic diversity in the landraces may be attributable to diverse production systems used by different ethnic groups in Myanmar, ranging from natural hydroponics in eastern areas, to slash‐and‐burn fields in mountain areas and shifting cultivation in many parts of the country. The availability of uniform and high‐yielding cultivars/hybrids threatens the valuable landraces in Myanmar. We emphasize the need to evaluate tomato germplasm from Myanmar for different traits and sustainable use with simultaneous conservation.     

Relationship between the biological activities of methylated derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) and their cell surface binding activities

It was previously reported that (-)-epigallocatechin-3-O-gallate (EGCG) suppresses the expression of the high-affinity IgE receptor FcepsilonRI in human basophilic cells and that this suppressive effect is associated with EGCG binding to the cell surface. This study examined the effects of five methylated derivatives of EGCG, (-)-epigallocatechin-3-O-(3-O-methyl)gallate (EGCG 3' 'Me), (-)-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4' 'Me), (-)-4'-O-methyl-epigallocatechin-3-O-gallate (EGCG 4'Me), (-)-epigallocatechin-3-O-(3,4-O-methyl)gallate (EGCG 3' '4' 'diMe), and (-)-4'-O-methyl-epigallocatechin-3-O-(4-O-methyl)gallate (EGCG 4'4' 'diMe) on FcepsilonRI expression and ERK1/2 phosphorylation, and each of their cell surface binding activities was measured. Of these five methylated derivatives, three that are methylated at the 3' '- and/or 4' '-position, EGCG 3' 'Me, EGCG 4' 'Me, and EGCG 3' '4' 'diMe, suppressed FcepsilonRI expression and ERK1/2 phosphorylation, although the suppressive effects were lower than that of EGCG. EGCG 4'Me and EGCG 4'4' 'diMe, both of which are methylated at the 4'-position, did not demonstrate a suppressive effect. Furthermore, it was found that EGCG 3' 'Me, EGCG 4' 'Me, EGCG 3' '4' 'diMe, and EGCG 4'Me, which are methylated at the 3' '- and/or 4' '-positions or the 4'-position, could bind to the cell surface even though their binding activities were lower than that of EGCG. Only EGCG 4'4' 'diMe, which is methylated at both the 4'- and 4' '-positions, could not bind. These results suggest that the trihydroxyl structure of the B ring is essential for EGCG to exert the suppressive effects and that the hydroxyl groups on both the 4'-position in the B ring and the 4' '-position in the gallate are crucial for the cell surface binding activity of EGCG.     

Association of Verticillium dahliae and Verticillium longisporum with Chinese cabbage yellows and their distribution in the main production areas of Japan

Chinese cabbage yellows caused by Verticillium dahliae and V. longisporum is one of the most economically important diseases in Japan. Verticillium isolates from infected Chinese cabbage in Japan were identified as V. dahliae and V. longisporum based on morphological characteristics and newly developed molecular genotyping methods using group I intron of 18S rDNA, mitochondrial-SSU rDNA and cob gene. Although the two species were equally virulent to Chinese cabbage in the greenhouse, V. longisporum was more virulent in the field compared with V. dahliae. Among the 67 Verticillium isolates from Chinese cabbage, 53 were V. longisporum and considered to be the major pathogen. The isolation frequency of V. longisporum was 98?% in Ibaraki and eastern Gunma where Chinese cabbage was cropped from autumn to winter. In contrast, the frequency was 48?% in Nagano and north to west Gunma where the plant was cropped in cool summer and highland conditions and the rests were V. dahliae. This is the first report to show the different geographical distribution of V. longisporum and V. dahliae in Chinese cabbage production area of Japan.     

Potential of prolonged midseason drainage for reducing methane emission from rice paddies in Japan: a long-term simulation using the DNDC-Rice model

Water management practices, such as midseason drainage (MD) and intermittent irrigation, are effective in reducing methane (CH₄) emission from irrigated rice paddies. In a previous study in which two-year field experiments were conducted at nine sites across Japan, prolonged MD was found to reduce the seasonal total CH₄ emission by 30.5?±?6.7 % (mean?±?95 % confidence interval) compared with conventional MD without compromising rice grain yield. However, the degree of CH₄ reduction by water management is dependent on prevailing weather conditions. To estimate the mean effect of prolonged MD on CH₄ emission at the nine sites with rice straw application, we conducted a long-term (20 years) simulation using a process-based biogeochemistry model, the DNDC-Rice. The model adjusted using site-specific parameters was able to reproduce the measured magnitude of the total CH₄ emission and the suppressive effect of prolonged MD. The number of nonrainy days during MD explained the degree of CH₄ reduction for each site and all sites combined. In the simulation, mean reduction percentage was 20.1?±?5.6 % when acceptable prolonged MD (i.e., having less than 15 % yield loss) was applied compared with conventional MD. The discrepancy of the percentage between measurement and simulation was primarily attributable to longer nonrainy days during prolonged MD at several sites in the measurement than the mean of 20-year simulation. We therefore conclude that the long-term simulation better represents the mean reduction percentage of CH₄ emission by prolonged MD relative to conventional MD at the nine sites across Japan.     

Regulation of free glutamate content in meat by dietary lysine in broilers

Regulation of taste is important for improving meat quality and glutamate (Glu) is one of the important taste‐active components in meat. Here, the effects of dietary lysine (Lys) content on taste‐active components in meat, especially free Glu, were investigated. Fourteen‐day‐old broiler chicks (Gallus gallus) were fed on diets containing 100% or 150% of the recommended Lys content for 10 days. Concentrations of free amino acids in plasma, muscle and liver were measured. The levels of messenger RNAs (mRNAs) for enzymes related to Glu metabolism were determined in muscle and liver. The concentration of muscle metabolites was also determined. The free Glu content in muscle of chicks fed the Lys150% diet was increased by 44.0% compared with that in chicks fed the Lys100% diet (P < 0.01). The mRNA level of lysine α‐ketoglutarate reductase, which is involved in Lys degradation and Glu production, was significantly increased (P < 0.05) in the Lys150% group. Metabolome analysis showed that the Lys degradation products, muscular saccharopine, pipecolic acid and α‐aminoadipic acid, were increased in the Lys150% group. Our results suggest that free Glu content in muscle is regulated by Lys degradation. These results suggest that a short‐term feeding of high‐Lys diet could improve the taste of meat.