Tatsuya Inamura

Isolation and Expression of Genes for Acetolactate synthase and Acetyl - CoA carboxylase in Echinochloa phyllopogon, a Polyploid Weed species

BACKGROUND Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon

Resistance to two herbicides in Echinochloa phyllopogon is associated with overexpression of two cytochrome P450s that are simultaneously controlled by a putative single genetic element. Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon.

Cytochrome P450 genes induced by bispyribac‐sodium treatment in a multiple‐herbicide‐resistant biotype of Echinochloa phyllopogon

BACKGROUND Incremental herbicide metabolism by cytochrome P450 monooxygenases (P450s) has been proposed as the basis for resistance to bispyribac-sodium (bispyribac) in a multiple-herbicide-resistant biotype of Echinochloa phyllopogon. Upon exposure to bispyribac, strong induction of bispyribac-metabolising P450 activity has been reported in the resistant line, indicating that P450s induced by bispyribac are involved in the bispyribac resistance. RESULTS A polymerase chain reaction (PCR)-based cloning strategy was used to isolate 39 putative P450 genes from the bispyribac-resistant line of E. phyllopogon. Expression analysis by real-time PCR revealed that seven of the isolated genes were upregulated in response to bispyribac treatment of seedlings at the three-leaf stage. The transcript levels and protein sequences of the seven genes were compared between the bispyribac-resistant line and a susceptible line. CYP71AK2 and CYP72A254 were transcribed prominently in the bispyribac-resistant line. Amino acid polymorphisms were found in three genes, including CYP72A254. CONCLUSION Upregulated expression of these genes is consistent with the inducible herbicide-metabolising P450 activity under bispyribac stress that was reported in a previous study. This is the first study to compare P450 genes in arable weed species in order to elucidate the mechanism for P450-mediated herbicide resistance.

Irrigation system and land use effect on surface water quality in river, at Lake Dianchi, Yunnan, China.

The surface water samples were collected in river Dahe and its tributaries, which flow into severely eutrophic lake Dianchi, Yunnan Province, China, in order to elucidate factors controlling water quality fluctuations. The temporal and spatial distribution of water quality tendency was observed. The water quality of each river is dependent on the hydrology effect such water gate and circulating irrigation system. We must consider the hydrology effect to accurately understand water quality variations of river in this study field. In river without highly circulating irrigation system or water gate effect, the downstream nitrate nitrogen (NO3-N) concentration increase occurred in area dominated by open field cultivation, whereas the NO3-N concentration was constant or decreased in area dominated by greenhouse land use. This result suggests that greenhouse covers the soil from precipitation, and nitrate load of greenhouse could be less than that of open field cultivation while the rainfall event. In the upper reaches of river, where is dominated by open field cultivation, there were no sharp increase dissolved molybdate reactive phosphorus and total phosphorus concentration, but P load was accumulated in the lower reaches of river, whose predominant land use is greenhouse. Although the P sources is unclear in this study, greenhouse area may have potential of P loads due to its high P content in greenhouse soil. Considering hydrology effect is necessary to determine what the major factor is influencing the water quality variation, especially in area with highly complicated irrigation system in this studying site.

Geostatistical Analysis of Yield, Soil Properties and Crop Management Practices in Paddy Rice Fields

Abstract We examined the possible benefit of rational site-specific crop management practices in 17 paddy fields located in an area of 2.0 ha for the prevalence of precision agriculture methods on a cooperatively managed large-scale farm in Sakurai, Nara Japan. Data on grain yield, soil physicochemical properties and farmers crop management practices were collected in each paddy field. Unhulled rice yield was estimated at a resolution of 5m ą 5m in an area of 1.2 ha using a yield-monitoring combine. The spatial distribution of the variations for the collected data was characterized using geostatistical procedures. The kriged map of the unhulled rice yield drawn from the results of geostatistical analysis indicated the potential value of rational site-specific crop management using the yield-monitoring combine. The ratio of spatially structured variation to the total variation of brown rice yield, that is, the controllable proportion to total variation, was 75.4%. Each yield component i.e., the number of spikelets per square meter, filled-spikelet percentage and 1000-grain weight contributed 33.7%, 54.7% and 11.6%, respectively, to brown rice yield. These three yield components combined contributed to 96.5% of the brown rice yield variation. The agronomic factors (soil fertility factor, early growth factor, N dressing and uptake factor) contributed 79.7%, 52.1% and 41.8%, respectively, to the variation of these three yield components. Therefore these agronomic factors accounted for 58.1% of the total variation of the brown rice yield and 77.1% (i.e. 58.1% out of 75.4%) of the spatially structured variation of the brown rice yield. This controllable proportion may be a criterion for the prevalence of site-specific crop management in large-scale farm management in general, although only one case study was conducted.

Changes in Soil Physicochemical Properties Following Land Use Change from Paddy Fields to Greenhouse and Upland Fields in the Southeastern Basin of Dianchi Lake, Yunnan Province, China

Paddy fields in the southeastern basin of Dianchi Lake have rapidly changed to greenhouses since 1999. A total of 61 surface soil samples, including 43 greenhouse soils, 12 upland soils, and 6 paddy soils, were collected from a flat lowland area mainly used for agricultural production fields in the southeastern basin of Dianchi Lake. Analyses of the soil samples indicated that the greenhouse soils were characterized by a lower organic matter content, lower pH, and higher soluble nutrients than the paddy soils in the area. The lower organic matter content of the greenhouse soils was ascribed to environmental or management factors rather than the clay content of the soil. Accumulation of soluble nutrients, especially inorganic N, was due to over-application of fertilizers, which also caused soil acidification. The average amount of readily available N, P, and K accumulated in the greenhouse soils was estimated to be equal to or higher than the annual input of these nutrients as a fertilizer, indicating that a reduction in fertilizer application was possible and recommended. In contrast, a very low available Si content was observed in the paddy soils, suggesting the need for Si application for rice production.

Growth and nitrogen uptake of paddy rice as influenced by fermented manure liquid and squeezed manure liquid

Abstract A field experiment was conducted to estimate the effects of fermented manure liquid (FL) and squeezed manure liquid (SL) on the growth and nitrogen (N) uptake of paddy rice and to determine the threshold dose for application to irrigated rice. The rice plants were grown in plots without fertilizer application (NF) or with chemical fertilizer application (60, 60, 60 kg N, P2O5 K2O ha−1, respectively) (CF), FL application (25, 35, and 45 t ha−1) (FL25, FL35, and FL45) or SL application (35, 55, and 75 t ha−1)(SL35, SL55, and SL75). The effects of the manure liquids (FL and SL) on plant length, number of tillers, leaf area index (LAI), grain yield, or biomass of rice were not significantly different from those of CF except for a significant increase in grain yield and biomass at a high levels of application. FL and SL showed a similar quadratic equation of applied N to grain yield, indicating that they played the same role in rice growth. The recovery rate of applied N was 45.3% and the agronomic efficiency for N from manure liquid was more than 20 kg kg−1 when less than 55 t ha−1 manure liquid was applied. The response of grain yield to applied N from both manure liquids and CF showed a significant quadratic equation (r = 0.945**). The maximum yield of rice (6.3 t ha−1) was obtained by applying the threshold dose of 136 kg N ha−1. Neither manure liquids were detrimental to rice growth during the growing season at an application level of about 68 t ha−1. These findings indicated that the manure liquids could be utilized as a substitute for nitrogen fertilizer in rice cultivation under irrigated conditions.

Effects of the Long-Term Application of Anaerobically-Digested Cattle Manure on Growth, Yield and Nitrogen Uptake of Paddy Rice (Oryza sativa L.), and Soil Fertility in Warmer Region of Japan

Abstract The suitability of anaerobically-digested manure (ADM) from a biogas plant, as an alternative to chemical fertilizers for rice cultivation was evaluated by a long-term study. At the standard nitrogen (N) application rate (10 g m−2), the aboveground biomass, N uptake, and grain yield in rice plots with ADM application (MF) were not significantly different from those in the plots treated with chemical fertilizer (CF). Split application of ADM improved the apparent N efficiency. The N application rate corresponding to maximum grain yield was approximately 15 g m−2 by a split application, and more N application by using ADM saturated grain yield due to decrease in ripened grain ratio and individual grain weight. On the whole, the soil total-C, total-N and available N in the MF plot were not significantly different from those in the CF plot. The available phosphate (P) levels was lower in the MF plot than in either the CF plot or plot without N application (NF), mainly due to lower content of P in ADM. However, the P level remained much higher than the fatal threshold level for the growth of paddy rice. These findings suggest that under appropriate fertilization conditions, ADM is a valuable organic resource, and can be used continuously as an alternative to chemical fertilization for rice cultivation, without substantial changes in soil C and N fertility.

Correlation of the Amount of Nitrogen Accumulated in the Aboveground Biomass at Panicle Initiation and Nitrogen Content of Soil with the Nitrogen Uptake by Lowland Rice during the Period from Panicle Initiation to Heading

Abstract To estimate the site-specific optimal amount of nitrogen (N) to be applied as top-dressing (N top-dressing) at panicle initiation, we examined the effects of the amount of N accumulated in the aboveground biomass at the panicle initiation stage (NJ, the total N content of soil and the amount of mineralizable soil N (Nm) on the amount of soil N taken up by the plants during the period from panicle initiation to heading (Ns) and the recovery ratio of applied chemical N during this period (J). We analyzed the data obtained in paddy fields with a similar soil type and weather condition in the growth period from 1999 to 2001. The growth of rice plants was regulated by the amounts of N applied before transplanting and at panicle initiation. The relative N uptake rate during the period from panicle initiation to heading (RNR) in the plots without N top-dressing was explained about 89% by multiple regression of N„ the total N of soil and Nm. Ns in the plots with N top-dressing that was estimated using RNR showed a significantly negative partial correlation with N1; the total N of soil and Nm. J in the plots with N top-dressing showed a significantly negative partial correlation with N1: but a significantly positive partial correlation with Nm. These results suggest that the fertilizer N should be applied considering the contribution of Nl5 the total N content of soil and Nm to Ns and J to fit the site-specific N demand of rice.

Comparison of Root System Development in Two Rice Cultivars During Stress Recovery from Drought and the Plant Traits for Drought Resistance

Summary A greenhouse experiment was conducted to compare root system development of two upland rice cultivars, IRAT 13 and Senshou, during recovery from drought stress and to identify the plant traits that confer drought resistance. From 62 days after sowing (62 DAS), drought stress was given for 6 d followed by rewatering for 14 d. Root length density (RLD) and root diameter (thickness) were measured at the end of the stress and rewatering periods. Control plants were well-watered throughout the study. Gultivar IRAT 13 had thicker roots and higher relative RLD (ratio of RLD in drought-stressed plants to that in control plants) than under drought stress, and significantly higher root growth recovery after rewatering cultivar Senshou. Related plant traits such as evapotranspiration (ET), leaf and stem dry weights and weight of senescent leaves (dead leaves) in IRAT 13 were significantly more favorable for drought resistance compared to Senshou.