Molecular cloning and expression analysis of genes related to phosphatidic acid synthesis in Japanese pear leaves inoculated with <Emphasis Type="Italic">Venturia nashicola</Emphasis>

Phosphatidic acid, which can be generated by the action of phospholipase D (PLD) and by the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK), is a plant defense signal. To identify the role of the PLD, PLC, and DGK genes encoding these enzymes in pear resistance to the scab fungus Venturia nashicola, we report the cloning of these genes and analysis of their expression in inoculated pear leaves. The results showed that PLD mRNA increased in the leaves of the susceptible pear cv. Kousui 1 day after inoculation (dai), then decreased to the basal level. In resistant pear cv. Kinchaku, PLD mRNA did not change significantly except for a small increase at 2 and 3 dai. However, the level of PLD mRNA in Kinchaku was higher than in Kousui 2 dai. The level of PLC mRNA significantly increased in cvs. Kousui and Kinchaku by 1 dai, peaked at day 2, then decreased to the basal level. Thus, the gene expression pattern did not differ between the two cultivars. PLC gene expression was also induced by environmental stress. The DGK gene seemed to be constitutively expressed and was not induced by inoculation in either the susceptible or resistant cultivars. Together, this data showed that both PLD and PLC are possibly involved in a defense response to V. nashicola in Japanese pear leaves, and the PLD gene seems to play a more important role than does the PLC gene.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

Polyclonal and monoclonal antibodies specific to the chrysanthemic acid moiety of pyrethroid insecticides

Two polyclonal and two monoclonal antibodies raised against chrysanthemic acid (CAA) were prepared for ELISA of pyrethroid insecticides with the common CAA moiety. The monoclonal antibody KCA226 showed the highest reactivity towards allethrin among the antibodies in C-ELISA, although KCA226 was more sensitive to higher concentrations of methanol than the polyclonal antibodies in ELISA. KCA226 was reacted linearly with allethrin at concentrations ranging from 1 to 10 μg litre^-1 in 0·5% methanol and from 300 to 10000 μg litre^-1 in 30% methanol. KCA226 reacted specifically with the pyrethroid insecticides with the CAA moiety but was much less reactive with CAA itself. These results suggest that C-ELISA based on KCA226 is useful for the assay of pyrethroid residues with the common CAA moiety. © 1998 Society of Chemical Industry     

Temporal fluctuation in the abundance of alginate‐degrading bacteria in the gut of abalone Haliotis gigantea over 1 year

In this study, we identified and enumerated alginate‐degrading bacteria in the gut of abalone over 1‐year period. From a total of 360 colonies growing on agar medium enriched with alginate, 251 isolates (70%) had the ability to degrade alginate. In addition, a high number of viable alginate‐degrading bacteria were detected throughout the survey period. Alginate‐degrading bacteria were more abundant in the cold season relative to the summer season (10⁷ vs. 10⁴ CFU g^?1, respectively). Strong positive correlation was also observed between the number of alginate‐degrading bacteria and feed intake (R = 0.854; P < 0.01). The identified alginate‐degrading bacteria comprised of 35 species grouped into 11 genera including Algibacter, Formosa, Polarybacter, Tamlana, Tenacibaculum (CFB group), Roseobacter, Ruegeria, Silicibacter (α‐proteobacteria), Agarivorans, Shewanella and Vibrio (γ‐proteobacteria) respectively. More than 80% of the isolated alginate‐degrading bacteria belonged to the genus Vibrio, showing high homology to Vibrio cyclotorophicus, Vibrio splendidus, Vibrio halioticoli and Vibrio neonatus. Based on the results, it was suggested that algal‐polysaccharide (alginate) degrading bacteria (mainly Vibrio) commonly exist in the gut of abalone and may play an important role in the degradation and digestion of the host's feed.     

Chlorella pyrenoidosa Supplementation Reduces the Risk of Anemia, Proteinuria and Edema in Pregnant Women

Pregnancy anemia and pregnancy-induced hypertension (PIH) are common and potentially dangerous disorder in human pregnancy, and nutritional status of pregnant women is one of the leading causes. Chlorella contains large quantities of folate, vitamin B-12 and iron, and can help improve anemia and hypertensive disorder. Our objective was to investigate the preventive effects of Chlorella supplement on pregnancy anemia and PIH in Japanese pregnant women. A total of 70 pregnant women were placed into the control group (n = 38) or the Chlorella group (n = 32). The subjects in the Chlorella group were supplemented daily from 12th–18th wk of gestation until delivery with 6 g of Chlorella supplement. The proportion of anemic (hemoglobin level < 11 g/dL) subjects in the Chlorella group were significantly lower compared with the control group at the second and third trimesters. Additionally, in the Chlorella group, the incidences of proteinuria and edema, signs of PIH, were significantly lower during the third trimester. These results suggest that Chlorella supplementation significantly reduces the risk of pregnancy associated anemia, proteinuria and edema. Chlorella supplement may be useful as a resource of natural folate, vitamin B-12 and iron for pregnant women.     

Impacts of pellet pH on nitrous oxide emission rates from cattle manure compost pellets

Previous reports indicated that the emission of nitrous oxide (N₂O) when manure compost pellets (MCP) were applied to soil was greater than when ordinary manure compost or inorganic fertilizer was applied, but that applying pellets of nitrogen-enriched manure compost, a by-product of deodorizing manure during composting, resulted in N₂O emission rates less than those from MCPs. To investigate the mechanism by which N₂O emission rates and cumulative emissions were reduced in nitrogen-enriched manure composts pellets (N+MCP), we studied the impact of pellet pH on N₂O emission, because pH is different between MCP (pH 8.6) and N+MCP (pH 5.3). In an incubation experiment, the pH of pellets was adjusted to five levels (5.3, 6.0, 7.0, 8.0 and 8.6) with acid or alkaline solutions, and the pellets were incubated without soil in a beaker at 30°C for 90 d (MCP) or 42 d (N+MCP). A large peak in N₂O emission rate was observed soon after beginning the incubation (within 1–3 d) in the neutral and alkaline treatments for both MCP and N+MCP, and these peaks corresponded to a rise in the pellet nitrite contents. Thus, this N₂O emission peak might have been generated by the denitrification of nitrite in the pellets. In the acid treatments of MCP, the N₂O emission was distributed more in the later incubation period (14–90 d), when the reduction of nitrate in MCP occurred. This led to a significant increase in cumulative N₂O emission as compared with the alkaline treatments for MCP. Regarding the mechanism by which N₂O emission was reduced in N+MCP, although larger cumulative N₂O emission rates in the earlier stage (0–14 d for MCP and 0–7 d for N+MCP) were observed when the pellet pH was adjusted close to 7.0, lowering the pH of MCP to 5.3 (the pH of N+MCP) did not demonstrate a significant decrease in cumulative N₂O emission as compared with the original pH treatment (pH 8.6). These results indicate that pellet pH might not relate directly to the mechanism by which N₂O emission was reduced in N+MCP.     

Population dynamics of portunid crab <Emphasis Type="Italic">Charybdis bimaculata</Emphasis> in Ise Bay,central Japan

ABSTRACT:   The present study dealt with seasonal and interannual variations in the abundance and biomass, and spatio-temporal distributions of the portunid crab Charybdis bimaculata dominant in Ise Bay, central Japan. The abundance and biomass of the crab decreased in summer when the oxygen-poor water developed in central or inner parts of the bay, and then increased through new recruits from autumn (October–November) to the following spring (March–May) when the oxygen-poor water disappeared. Berried females were collected mainly from spring to autumn. Recruits were collected in any season. Particularly in winter, most recruits were located in the innermost part of the bay. According to the cohort separation based on size frequency distribution in carapace width of the crab specimens, the cohorts that were derived from spawning in spring to summer largely contributed to establishing and maintaining the benthic populations for the following year in the bay, whereas those from other seasons failed to recruit because of serious damage caused by the oxygen-poor water. Most crab individuals one year post hatch contributed to spawning and then died by the winter of the same year.     

Effects of cryopreservation on sperm structure in Japanese pearl osyter <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

To clarify factors reducing the motility and fertility of cryopreserved spermatozoa of the Japanese pearl oyster Pinctada fucata martensii, the structure of spermatozoa before and after cryopreservation was observed by scanning electron microscopy. Testicular spermatozoa were diluted with cryopreservation diluent (10% methanol+18% fetal bovine serum+72% sea water), and dispensed into 0.25-mL straws. The straws were cooled at a rate of approximately −20 °C/min to −50°C, and subsequently immersed in liquid nitrogen. Percentage motility of spermatozoa before cryopreservation was 69.9±4.2%, and that of cryopreserved spermatozoa was 24.0±1.8%, respectively. In cryopreserved spermatozoa, the percentage that lacked or had a deformed flagellum was 56.6±3.9%, while in fresh spermatozoa this was 8.7±2.0%. In cryopreserved spermatozoa, the percentage of deformed acrosomes was 76.6±5.2%, while in fresh spermatozoa this was only 0.9±0.3%. Cryopreserved spermatozoa with a normal acrosome and flagellum were only 15.4±3.5% of those in fresh spermatozoa. These results indicate that lesion of the flagellum and deformation of the acrosome occurred through the cryopreservation procedure, and both types of damage lead to loss of the motility and fertility in thawed spermatozoa.     

Water (H2 15O) flow in rice is regulated by the concentration of nutrients as monitored by positron multi-probe system (PMPS)

Using H₂ ¹⁵O as a tracer, the effect of the nutrient concentration on the water flow at the discrimination center (DC) of rice was monitored using a positron multi-probe system (PMPS). The maximum velocity of the water flow was achieved by the use of 20× Kasugai's culture solution. The addition of 0.01 to 0.1% NaCl gradually decreased the velocity. In intact plants, the ionic strength of essential elements in the xylem sap may be effective in decreasing the xylem resistance, resulting in the increase of the water flow in the xylem.     

Phytoremediation of the herbicides atrazine and metolachlor by transgenic rice plants expressing human CYP1A1, CYP2B6, and CYP2C19

This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.