Discovery of single-nucleotide mutations in acetolactate synthase genes by Ecotilling

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to discover induced point mutations in genes of interest. The use of the TILLING technique to survey natural variation in genes is called Ecotilling. We report an adaptation of Ecotilling for rapid detection of single-nucleotide mutations in the acetolactate synthase (ALS) genes of sulfonylurea (SU)-resistant (R) Monochoria vaginalis (Pontederiaceae), a paddy weed, in Japan. Genomic DNA of a SU-R plant (target DNA) was mixed with the DNA of a SU-susceptible (S) plant (reference DNA). Ecotilling detected two nucleotide mutations in the ALS gene of SU-R M. vaginalis. These 2 mutations were confirmed by DNA sequencing. A single nucleotide mutation (C to A), in the codon CCT to CAT and another mutation (C to T), in the codon CCT to TCT were identified by sequencing. Both mutations result in the disruption of a Pro codon in the conserved Domain A region with the consequent substitution of a His residue in the first mutation and a Ser residue in the second. Substitution of the Pro residue in Domain A of the ALS gene has been reported to result in insensitivity to SUs in many weed biotypes. This study demonstrates that Ecotilling is a fast, reliable, economical method for detecting single-nucleotide mutations in genes arising from herbicide selection.     

Seasonal characteristics of surface water quality in the wastewater catchment system of an urbanizing basin

Vientiane, Lao PDR, has been subject to extensive and ongoing urbanization plans, including development of natural marshes and residentialization of paddy fields into suburban areas, despite natural marshes playing a vital role in treating wastewater from urban areas. Therefore, it is important to understand the current situation regarding the nutrient balance in these natural wastewater treatment systems to predict future conditions and design appropriate measures against water quality deterioration. However, limited data are available in Vientiane on the hydrological characteristics of water and nutrient runoff that flow into marshes through drainage canals. In this study, we conducted a periodic survey of drainage canals and Mak Hiao River in the wastewater catchment system surrounding Vientiane during the rainy and dry seasons. We monitored the discharge of surface water at 21 observation sites and analyzed water quality of nitrogen, phosphorus, and total organic carbon. These observations revealed that the concentrations of dissolved nitrogen and phosphorus were significantly higher at sites in urban areas, followed by sites in the main river basin and those in agricultural areas. Dissolved nitrogen and phosphorus concentrations varied with runoff discharge, especially in urban and river basin sites, with lower concentrations in the rainy season and higher concentrations in the dry season. On the other hand, we found no significant differences between the rainy and dry seasons in nutrient concentrations in the agricultural basin. Finally, we proposed measures to counteract the deterioration of water quality during dry seasons and simulated the impact of these measures.     

Seasonal and Annual Fluxes of Inorganic Constituents in a Small Catchment of a Japanese Cedar Forest near the Sea of Japan

Fluxes of major ions in rainfall (RF), throughfall plus stemflow (TF + SF), and stream water (SW) were measured for five water years in a small catchment of a Japanese cedar forest near the Sea of Japan. The fluxes of most ions in RF and in TF + SF, including the non-sea-salt constituents, increased from late autumn to midwinter owing to the seasonal westerly wind. The concentrations of most ions in SW showed no obvious seasonal trend during the study period, whereas ${\text{NO}}_3 ^ - $ concentrations were lowest in summer, with a small seasonality. The Ca²⁺ and Mg²⁺ outputs in SW were approximately 3.7 and 1.8 times the TF + SF inputs of these cations, respectively. The large net outputs of base cations in the catchment may indicate a decrease in the soil's acid-neutralizing capacity. Annual dissolved inorganic nitrogen inputs in RF and in TF + SF were 17.7 and 17.9 kg N ha^?1 year^?1, respectively, which exceeded previously published thresholds in Europe and the U.S. (i.e., the values at which these inputs increased ${\text{NO}}_3 ^ - $ levels in SW) and equaled the highest level of nitrogen deposition previously reported in Japan. The ${\text{NO}}_{\text{3}} ^{\text{ - }} $ concentrations in SW were relatively high even in summer. During high-precipitation events, ${\text{NO}}_{\text{3}} ^{\text{ - }} $ concentrations in SW increased with increasing water discharge, and the pH decreased simultaneously during several events. Nitrogen deposition may contribute to the high ${\text{NO}}_{\text{3}} ^{\text{ - }} $ concentrations in SW and the temporary acidification that occurred during the rain events.