PCR primers for identification of opine types of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in Japan

The polymerase chain reaction (PCR) is a rapid, precise method for detecting and identifying pathogenic bacteria. In addition to the published primers for identification of Agrobacterium tumefaciens up to species level, two sets of primers were designed to identify the nopaline and octopine types of Agrobacterium tumefaciens. The RBF-RBR primer set designed based on the nopaline type T-DNA right border detected the nopaline type A208 and R225f strains, and the ocsF-ocsR primer set derived from the ocs gene of the octopine type A. tumefaciens detected the octopine type A348 strain. After polymerase Chain reaction (PCR) amplification by the RBF-RBR primers, the A208 and R225f strains could be differentiated from each other by restriction fragment length polymorphism digestion using the restriction enzymes DraI and XbaI. Multiple colonies can be screened at one time in a single PCR tube with satisfactory efficiency, thereby allowing rapid detection of pathogenic A. tumefaciens. Following a rough screening by classical biovar medium and -methyl-d-glucoside medium, the developed PCR system was introduced to identify isolates collected from soil and crown gall samples. Of 42 isolates determined to be A. tumefaciens, 7 were found to be octopine type; all the rest were R225f type.     

Reduced intake of dietary lysine promotes accumulation of intramuscular fat in the Longissimus dorsi muscles of finishing gilts

The present study was conducted to elucidate the effect of dietary lysine levels on the intramuscular fat (IMF) content in the Longissimus dorsi (L. dorsi) muscles of finishing gilts. Eleven gilts in total from two litters of pigs aged 110 days were used. The average initial bodyweight of the pigs was 61.7 kg. Six pigs were assigned to the low lysine (LL) diet group (lysine content: 0.43 or 0.40%) and five pigs were assigned to the control group (lysine content: 0.65 or 0.68%). The diets were iso‐energetic and iso‐protein, and contained all essential amino acids (apart from lysine) in the recommended amounts. The pigs were fed these diets until their live weights reached 110 kg. Live weight gain and feed efficiency tended to be lower in the LL group (P = 0.118 and P = 0.052, respectively). Pigs from the LL group took 5 days longer to reach 110 kg (P < 0.01). The IMF content in the L. dorsi of the LL group was twice as high as that of the control group (6.7 vs 3.5%; P < 0.01). The percentage of oleic acid in the L. dorsi of the LL group tended to be higher than that of the control group (P = 0.052), whereas the percentage of linoleic acid and the total percentage of polyunsaturated fatty acids in the L. dorsi were lower (P < 0.05) in the LL group. Free L‐carnitine content in the L. dorsi was lower (P < 0.05) in the LL group. The average abundance of peroxisome proliferator‐activated receptor gamma mRNA in the L. dorsi of the LL group was threefold higher than that of the control group. The leptin mRNA abundance in the L. dorsi of the LL group was 3.3‐fold higher than that of the control group (P < 0.01). These results suggest that a higher activity of adipogenesis may have been involved in the promoted accumulation of IMF in the L. dorsi muscles of pigs, induced by a dietary LL level.     

Seasonal variations in and effect of incubation water temperature on vertebral number in naturally spawning chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>

Seasonal variations and reaction norms for vertebral number (V _N) in response to incubation water temperature were estimated in adult and juvenile naturally spawning chum salmon Oncorhynchus keta. The mean V _N of adults varied according to spawning time; the early-spawning population had higher V _N values than the late-spawning population. Moreover, the mean V _N values in the early-spawning population decreased with seasonal changes, whereas V _N values in the late-spawning population remained stable. Chum salmon embryos in three full-sib families were incubated at five different temperatures until hatching, and the V _N values of the resulting juveniles were analyzed. The V _N reaction norm to incubation water temperature showed a V-shaped curve that was lowest at an intermediate temperature. The mean V _N at the same incubation temperature varied among the three families. These results suggest that V _N values in chum salmon are influenced by genetic components and incubation water temperatures. V _N may be a useful parameter for estimating the environmental conditions during ontogenesis and the genetic background by detecting population changes.     

Seedling blight of Glycyrrhiza uralensis caused by Pythium myriotylum,P. aphanidermatum and P. spinosum and identifying primary inoculum sources using multiplex PCR detection

Pythium species, isolated from seedlings of Glycyrrhiza uralensis with blight, were identified as P. myriotylum, P. aphanidermatum, and P. spinosum on the basis of morphological characteristics and sequences of the internal transcribed spacer regions of rDNA. In pathogenicity tests, the isolates of the three Pythium species caused blight, producing the original disease symptoms. The primary inoculum source was determined using a multiplex PCR to detect the pathogen. All the Pythium species were detected in the soils of fields with the diseased plants and in soils of adjacent field soils.     

Differences in physiological responses to NaCl between salt-tolerant Sesbania rostrata Brem. & Oberm. and non-tolerant Phaseolus vulgaris L.

Sesbania rostrata ( S. rostrata) Brem. & Oberm., a member of the Fabaceae family, has been used as a promising halophytic plant to ameliorate soil salinity in north-east Thailand. To obtain information regarding the mechanism of salt tolerance, the physiological responses of S. rostrata to NaCl was compared with those of the salt-susceptible species, kidney bean ( Phaseolus vulgaris L. cv. Meal). Seedlings were grown hydroponically with 0, 50, 100 and 150 m m NaCl for 10 days and their effects on growth, chlorophyll content, fluorescence yield ( F _v/ F _m), inorganic elements and amino acid content were determined. The results showed that tolerance to NaCl was clearly different between the two plants. At the highest concentration (150 m m ), the dry weight of S. rostrata was more than 50% greater than the control, whereas the kidney bean could not survive. Chlorophyll a content drastically reduced only in the kidney bean. The F _v/ F _m of S. rostrata did not change with increasing concentrations of NaCl, but that of kidney bean decreased. Greater percentages (≥80%) of absorbed Na⁺ and Cl^– were translocated and accumulated in the shoots of S. rostrata , but remained largely in the roots of kidney bean. The enhancement of contents of amino acids, including proline, with increasing NaCl was observed in both species. These results strongly suggest that the salt tolerance of S. rostrata is associated with the ability of the plant to translocate and sequester Na⁺ and Cl^– in the shoot cells.     

Herbicide pyrazolate causes cessation of carotenoids synthesis in early watergrass by inhibiting 4-hydroxyphenylpyruvate dioxygenase

In aqueous solution, the herbicide pyrazolate [4‐(2,4‐dichlorobenzoyl)‐1,3‐dimethyl‐5‐pyrazolyl p‐toluenesulfonate] is rapidly hydrolyzed to destosyl pyrazolate (DTP), 4‐(2,4‐dichlorobenzoyl)‐1,3‐dimethyl‐5‐hydroxypyrazole, which is an active form of the herbicide. The objective of this study was to examine the effect of pyrazolate and DTP on carotenoids synthesis in susceptible weed, early watergrass (Echinochloa oryzicola Vasing.). Furthermore, their in vitro effect on 4‐hydroxyphenylpyruvate dioxygenase (HPPD) was determined. Roots of the plants at the two‐leaf stage were soaked for 24 h into pyrazolate (5 × 10^–5 mol L^?1) or norflurazon (10^–6 mol L^?1) solution containing 0.5% volume of acetone. At the first sampling time (3 days after treatment: 3 DAT), the chlorophyll content in the third leaves of pyrazolate‐treated plants were not different compared with the untreated control, but it was decreased between 3 and 6 DAT. The declining pattern of β‐carotene in the third leaf of early watergrass was very similar to that of chlorophyll. Both herbicides induced greater accumulation of phytoene in the third leaves of early watergrass 3 DAT, and the levels were kept until 9 DAT. However, feeding of homogentisate reduced the phytoene accumulation only in pyrazolate‐treated plants, suggesting the site of action of the herbicide located in the pathway of plastoquinone synthesis. In a HPPD assay, DTP revealed to inhibit the enzyme with an IC₅₀ value of 13 nmol L^?1 and that of pyrazolate was 52 nmol L^?1. In the pyrazolate solution used in the assay, some of the herbicide possibly has been hydrolyzed to DTP. From the all results obtained, it is strongly suggested that pyrazolate inhibits carotenoids synthesis and causes bleaching on the developing leaves by the similar mechanism with norflurazon, but its action site is not phytoene desaturase and is HPPD.     

Study of the role of the carbohydrate and protein moieties of soy soluble polysaccharides in their emulsifying properties

The objective of this work was to investigate the role played by the protein fraction of soy soluble polysaccharide (SSPS) during its adsorption at oil/water interfaces. SSPS was separated in a high (HMF; 310 kDa) and low (LMF; 20 kDa) molecular weight fraction by gel filtration. SSPS/HMF consisted of 91.6% carbohydrate and 2.2% protein and showed better emulsifying properties than those of the whole SSPS, whereas SSPS/LMF seemed to affect negatively the adsorption behavior of SSPS. SDS-PAGE of the protein fraction obtained from SSPS/HMF showed a molecular mass of 50 kDa, was composed predominantly of proline (23.1%) and glutamic acid (15.2%), and still contained 8.8% of neutral sugar and 5.3% of uronic acid. Results indicated that not all of the protein material present in SSPS contributes to SSPS functionality but that only the material associated with HMF aids in the adsorption of SSPS onto oil/water interfaces.     

Adsorption of OP<Subscript>1</Subscript>-related bacteriophages requires the gene encoding a TonB-dependent receptor-like protein in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Xanthomonas oryzae pv. oryzae strain T7174R is lysed by bacteriophage OP_1h and OP_1h2. Three mutants tolerant to both OP_1h and OP_1h2 were isolated by transposon mutagenesis. The mutants had an insertion of the transposon in XOO1687, which is predicted to encode a TonB-dependent receptor gene. Plasmid pHMIroNB that contained XOO1687 of T7174R was constructed, and the mutant was transformed with the plasmid. The transformant recovered sensitivity to OP_1h and OP_1h2. Electron microscopic analysis demonstrated that OP_1h and OP_1h2 can adsorb to the wild type and the transformant, but they could not adsorb to the phage-tolerant mutant. These results suggest that the TonB-dependent receptor gene relates to adsorption and infection of T7174R by OP_1h2 and OP_1h. Y. Inoue and S. Tsuge have contributed equally to this work.