Comparative effect of human urine and ammonium nitrate application on maize (Zea mays L.) grown under various salt (NaCl) concentrations

The present study investigates the effect of urine and ammonium nitrate on maize (Zea mays L.) vegetative growth, leaf nutrient concentration, soil electrical conductivity, and exchangeable‐cations contents under various concentrations of NaCl in a soil substrate. The experiment was arranged in a completely randomized block design with eight replications under greenhouse conditions. The experimental soil substrate was made from a 1 : 1 : 1 volume‐ratio mixture of compost, quartz sand, and silty‐loam soil. Salinity was induced by adding 0, 15, and 30 mL of 1 M NaCl solution per kg of substrate to achieve an electrical conductivity (EC) of 1.3 (S0), 4.6 (S1), and 7.6 (S2) dS m^–1. Nitrogen sources were urine and ammonium nitrate applied at 180 and 360 mg N (kg soil substrate)^–1. Basal P and K were added as mono potassium phosphate in amounts equivalent to 39 mg P and 47 mg K (kg substrate)^–1, respectively. In the S0 treatment, a 3‐fold increase in EC was measured after urine application compared to an insignificant change in ammonium nitrate–fertilized substrates 62 d after sowing. Under saline conditions, application of 360 mg N (kg soil)^–1 as urine significantly decreased soil pH and maize shoot dry weight. At the highest salt and N dose (S2, N360) 50% of urine‐fertilized plants died. Regardless of salinity there was no significant difference between the two fertilizers for investigated growth factors when N was supplied at 180 mg (kg soil)^–1. Leaf N and Ca contents were higher after urine application than in ammonium nitrate–fertilized plants. At an application rate of 180 mg N (kg soil)^–1, urine was a suitable fertilizer for maize under saline conditions. Higher urine‐N dosages and/or soil salinity exceeding 7.6 dS m^–1 may have a deleterious effect on maize growth.     

缺锌玉米植株的傅立叶变换红外光谱研究

【目的】傅里叶变换红外光谱（fourier transform infrared spectroscopy, FTIR）是一种基于化合物中官能团和极性键振动的结构分析技术。本文利用傅立叶变换红外光谱仪检测缺锌和正常供锌玉米植株不同器官的组分变化,同时比较两个玉米品种植株不同部位的生物量和锌含量,以期为缺锌影响玉米生长与生理代谢的机理研究提供参考。【方法】选取农大108和郑单958两个玉米品种,利用营养液培养方式,设置缺锌和正常处理。1）当玉米出现缺锌症状后,将地上部和根系分开,测量株高和根长,烘干至恒重测干重。2）烘干至恒重的植株样品用HNO3-HClO4（3∶1）消煮,原子吸收分光光度计（型号WFX-120C,北京瑞利分析仪器公司）测定消煮液中锌浓度,计算植株中锌含量和锌积累量。3）收获玉米根系放入FAA固定液（70%酒精∶38%甲醛∶乙酸体积=90∶5∶5）中,利用扫描仪（EsponV700）扫描根系样品获取数字化图像,利用WinRHIZO根系分析软件（Regent Instruments Inc., Canada）对图像进行分析,获得根长、 根面积、 根体积等指标。4）取玉米根、 茎、 叶部分烘干样品,磨碎过0.2 mm筛,采用溴化钾压片法,利用傅立叶变换红外光谱仪（VERTEX 70,Bruker）检测不同部位的光谱特性,OPUS 6.5软件采集数据并进行基线校正。【结果】缺锌胁迫下, 植株地上部锌含量明显下降,低于临界水平（20 g/g）,生物量降低; 缺锌根系面积与体积变小,总根长变小。用缺锌与施锌植株生物量比来表征玉米对缺锌敏感性,品种农大108较郑单958对缺锌更为敏感。缺锌玉米根系和叶片FTIR谱在波数3410、 2920、 1650、 1380、 1055 cm-1附近处透过率较高,茎FTIR谱在这些波数处透过率较低,表明缺锌导致根系和叶片中碳水化合物、 脂类、 蛋白质及核酸含量下降,而在茎中有所积累。农大108植株中各组分变化受缺锌影响较大。【结论】缺锌导致玉米植株生长受抑,利用FTIR技术研究发现缺锌植株中碳水化合物、 脂类、 蛋白质及核酸组分发生变化,农大108植株中各组分变化受缺锌影响较大,品种农大108可能较郑单958对缺锌更为敏感。     

Nitrogen fertilization does not affect micronutrient uptake in grain maize (Zea mays L.)

Abstract

Due to continuous single nitrogen fertilization, we hypothesized a built-up deficiency of micronutrients in crops that would limit plant growth and crop quality. In 2-year field experiments using urea-N fertilized grain maize (Zea mays L.), hybrid KWS 2376 at 0, 120 and 240 kg N ha^?1 crop uptake of Zn, Mn, Cu and Fe was studied at DC 32, DC 61 and in the grain harvested. Micronutrient contents at DC 32 stage – 1st node (aboveground phytomass) and DC 61 – flowering (ear leaf) were all at levels indicative of adequate micronutrient supply to the crop. At both sampling occasions the Fe:Zn and Fe:Mn ratios were adequate implying that Fe did not inhibit the uptake of Zn and Mn. The application of nitrogen increased the Fe content at the 1st sampling in both years; in the second year the same was also the case for the Zn content. Nitrogen nutrition increased the contents of Mn and Fe at the 2nd sampling only in year 2; in the other treatments no changes were observed in the micronutrient contents. Micronutrient correlations in the grain were discovered between Zn and Mn contents and between Fe and Mn contents. In the second year the highest N-rate significantly increased the Fe and Zn content of the grain compared with the lower rates of nitrogen fertilization. Grain yields were not affected by the rate of nitrogen and ranged between 13.65 and 14.34 t ha^?1 (1st year) and between 13.68 and 14.18 t ha^?1 (2nd year). Nitrogen fertilization did not reduce the content of micronutrients in the plant or grain of maize. It is evident that the continuous single use of N fertilization so far has not resulted in a micronutrient deficiency of the plants limiting the nutrient density of the grain or reducing its quality.     

Salinity stress on various physiological and biochemical attributes of two distinct maize (Zea mays L.) genotypes

Present study investigates the effect of salinity stress on physiological and biochemical characteristics of two maize genotypes cultivated under controlled growth conditions. The selected maize genotypes being salt-tolerant and salt-sensitive were respectively designated as Sahwal-2002 and Sadaf. The experiment was conducted in triplicates, two varieties, three priming treatments and two salinity levels, in the Government College University, Faisalabad. The antioxidants activity was measured by comparing the tolerance in response to acute and prolonged salinity treatment. The difference of genotype with salinity tolerance as well as seed priming with phenylalanine were not dependent on antioxidant activity when salt exposure was prolonged. The results show that an indirect relationship was present for PAL seed priming and oxidative damage due to salt. The antioxidant enzymes present in plant effectively reduced the oxidative damage of salt and thus, increased the overall crop yield.     

Kinetics of 15N-labelled nitrate uptake by maize (Zea mays L.) root segments

Abstract

Isotherms and kinetic constants of nitrate uptake by excised root segments from the apical root zone of 6-d-old maize seedlings pretreated with nitrate were investigated using ¹⁵N-labelled nitrate. The isotherms were resolved into two systems namely a multiphasic saturable system at substrate concentrations lower than 2 mol m^?-3 and a linear system at higher concentrations. The detailed analysis of the multiphasic saturable system suggested the existence of at least three phases, which followed the Michaelis-Menten kinetics. The I _max and K _m of each phase increase from the lower phase to the upper phase. The distance from the root tip and the presence of stele affected considerably the linear system but only slightly the saturable system.     

Small amounts of ammonium (NH$ _4^+ $) can increase growth of maize (Zea mays)

Nitrate (NO$ _3^ - $ ) and ammonium (NH$ _4^+ $ ) are the predominant forms of nitrogen (N) available to plants in agricultural soils. Nitrate concentrations are generally ten times higher than those of NH$ _4^+ $ and this ratio is consistent across a wide range of soil types. The possible contribution of these small concentrations of NH$ _4^+ $ to the overall N budget of crop plants is often overlooked. In this study the importance of this for the growth and nitrogen budget of maize (Zea mays L.) was investigated, using agriculturally relevant concentrations of NH$ _4^+ $ . Maize inbred line B73 was grown hydroponically for 30 d at low (0.5 mM) and sufficient (2.5 mM) levels of NO$ _3^ - $ . Ammonium was added at 0.05 mM and 0.25 mM to both levels of NO$ _3^ - $ . At low NO$ _3^ - $ levels, addition of NH$ _4^+ $ was found to improve the growth of maize plants. This increased plant growth was accompanied by an increase in total N uptake, as well as total phosphorus, sulphur and other micronutrients in the shoot. Ammonium influx was higher than NO$ _3^ - $ influx for all the plants and decreased as the total N in the nutrient medium increased. This study shows that agriculturally relevant proportions of NH$ _4^+ $ supplied in addition to NO$ _3^ - $ can increase growth of maize.     

Effects of soil bulk density on seminal and lateral roots of young maize plants (Zea mays L.)

It is well established that increasing soil bulk density (SBD) above some threshold value reduces plant root growth and thus may reduce water and nutrient acquisition. However, formation and elongation of maize seminal roots and first order lateral (FOL) roots in various soil layers under the influence of SBD has not been documented. Two studies were conducted on a loamy sand soil at SBD ranging from 1.25 g cm^–3 to 1.66 g cm^–3. Rhizotrons with a soil layer 7 mm thick were used and pre‐germinated plants were grown for 15 days. Over the range of SBD tested, the shoot growth was not influenced whereas total root length was reduced by 30 % with increasing SBD. Absolute growth rate of seminal roots was highest in the top soil layer and decreased with increasing distance from the surface. Increasing SBD amplified this effect by 20 % and 50 % for the top soil layer and lower soil layers, respectively. At the end of the experiment, total seminal roots attributed to approximately 15 % of the total plant root length. Increasing SBD reduced seminal root growth in the lowest soil layer only, whereas FOL root length decreased with SBD in all but the uppermost soil layer. For FOL, there was a positive interaction of SBD with distance from the soil surface. Both, increasing SBD and soil depth reduced root length by a reduction of number of FOL roots formed while the length of individual FOL roots was not influenced. Hence, increasing SBD may reduce spatial access to nutrients and water by (i) reducing seminal root development in deeper soil layers, aggravated by (ii) the reduction of the number of FOL roots that originate from these seminal roots.     

Nitrogen and NaCl salinity effects on the growth and nutrient acquisition of the grasses Aeluropus littoralis,Catapodium rigidum,and Brachypodium distachyum

Salinity and low nitrogen availability are important growth‐limiting factors for most plants. Our objective was to assess the influence of nitrogen (N) and salt levels on the growth and mineral nutrition of three forage grasses of varying salt resistance which are widely found in Tunisian salt lands, Aeluropus littoralis, Catapodium rigidum, and Brachypodium distachyum. Their response to salt and N interaction has not been studied and further investigations are necessary. Twenty day–old plantlets were hydroponically cultivated in Hewitt's nutrient solution. Half the plants were then exposed to 100 mM NaCl and the other half to no NaCl, and N was supplied at 0.5 or 5.0 mM N as NH₄NO₃. Plants were harvested after 60 d growth. Saline treatment (100 mM NaCl) decreased growth of B. distachyum (a relatively salt‐sensitive plant), but no significant effect was noted for A. littoralis (a relatively salt‐resistant plant) in both low– and high–N availability treatments. However, the effect of 100 mM NaCl on growth of C. rigidum (a moderately salt‐resistant plant) depended on N level. Increasing N availability and NaCl did not influence phosphate, sulfate, calcium, and magnesium concentrations in both A. littoralis and C. rigidum, but increased N supply reduced shoot sodium and chloride (Cl^–) accumulation. Potassium acquisition in A. littoralis and C. rigidum plants was severely depressed by increasing N availability under saline and nonsaline conditions, respectively. In these species, the increase of nitrate accumulation via N was attenuated by salinity. In contrast, total N content and allocation toward shoots were enhanced in these conditions, especially in A. littoralis, the most resistant species. It appears that increasing N availability at moderate salt levels has a beneficial effect on growth of species with high and moderate salt resistance, but not on species with low resistance to salinity.     

Analysis of the lectins from teosinte (Zea diploperennis) and maize (Zea mays) coleoptiles

To identify molecular evidence of the common origin of maize and teosinte, a lectin from teosinte coleoptile (TCL) was purified, through affinity chromatography on a lactosyl-Sepharose column, and some of the physicochemical parameters were compared with those from the maize coleoptile lectin (CCL). TCL is a 92 kDa glycoprotein constituted mainly by aspartic, glutamic, glycine, leucine, and lysine residues; in minor proportion, methionine and cysteine were also found. The glycannic portion of the lectin, which corresponds to 10% w/w, is composed by Gal, Man, and GlcNAc. CCL is an 88.7 kDa glycoprotein that contains 12% sugars by weight; its sugar and amino acid compositions are similar to those of TCL. TCL is formed by two isoforms identified through acidic electrophoresis, whereas CCL is constituted by a single molecular form. The NH(2) termini of both TCL isoforms are blocked, but their amino acid sequences determined from tryptic peptides by matrix-assisted laser desorption ionization time-of-flight) indicated that TCL isoforms have no homology with other mono- or dicotyledonous lectins, including CCL. TCL, just as CCL, showed hemagglutinating activity toward animal erythrocytes, including human A, B, and O. Hapten inhibition assays indicated that although TCL shows broader sugar specificity than CCL, it recognizes Gal in O- and N-glycosidically linked glycans. Both lectins are equally well recognized by antibodies against TCL.     

Effects of exogenously applied salicylic acid on the induction of multiple stress tolerance and mineral nutrition in maize (Zea mays L.)

Abstract

It has been proposed that salicylic acid (SA) acts as an endogenous signal molecule responsible for inducing environmental stress tolerance in plants. In this study, the effects of seed soaked (1.0 mM for 24 h) and soil incorporated (0.1 mM and 0.5 mM) salicylic acid (SA) supply on growth and mineral concentrations of maize (Zea mays L., Hamidiye F₁) grown under either salt, boron toxicity or drought-stressed conditions were investigated. Exogenously applied SA either with seed soaked (SS) or soil incorporated (SI) increased plant growth significantly in all the stresses conditions. Salicylic acid inhibited Na and Cl accumulation in saline conditions, and 0.5 mM of soil incorporated SA decreased B significantly in boron toxicity treatment. Except in drought condition, SA treatments stimulated N accumulation in plants. And P, K, Mg and Mn concentrations of SA received plants were increased in the stress conditions. These results suggest that SA regulates the response of plants to the environmental stresses and could be used as a plant growth regulator to improve plant growth and stimulate mineral nutrient concentrations under stress conditions.     

Root size and nitrogen‐uptake activity in two maize (Zea mays) inbred lines differing in nitrogen‐use efficiency

Considerable differences in response to nitrogen (N) availability among plant species and cultivars have been well documented. Focusing on the uptake of N, it is not clear which factor or factors determine efficient N acquisition. Two maize (Zea mays L.) inbred lines (478, N‐efficient; W312, N‐inefficient) were used to compare the relative contribution of root uptake activity and root size to N acquisition. Nitrogen‐efficient inbred 478 had higher yields and accumulated more N under field conditions than W312 under both high‐ (135 kg N ha^–1) and low‐N (no N supplied) conditions. The root system of 478, as indicated by total root length, root biomass, and root‐to‐shoot ratio, was larger and more responsive to low N stress. Especially, 478 developed more and longer axial roots at low N stress. On the contrary, the average N‐accumulation rate in 478 was lower than that of Wu312. In solution culture, ¹³NO₃^– influx in 478 was lower than in W312 after 8 h of nitrate provision. The expression of nitrate‐transporter genes ZmNRT1.1, ZmNRT2.1, ZmNRT2.2, and ZmNAR2.1 was stronger and lasted for a longer time after NO induction in W312. It is concluded that the efficient N acquisition in 478 is due to (1) a larger root system and (2) a stronger response of root growth to low N induction.     

Corn (Zea mays L.) Grain and Stover Yield as Affected by Soil Residual Mineral Nitrogen

Evaluation of nitrogen (N) dynamic in soil using regression equations is important for proper determination of N fertilization. A 3-year field experiment was conducted to (1) develop the best-fitted regression model relating corn grain and stover yield to soil residual ammonium (NH₄)-N and nitrate (NO₃)-N for corn yield prediction and (2) evaluate how such a model can be beneficial to the health of ecosystem by predicting the appropriate rates of N fertilization for corn production. Soil NH₄-N and NO₃-N were determined at corn harvest at the depths of 0–30 and 30–60 cm. Nitrogen fertilizer rates and soil mineral N accounted for a maximum of 93% variation in corn grain yield. Soil mineral N enhanced corn yield more than N fertilizer. Totals of 63.1 and 14.1 kg/ha of soil residual NO₃-N and NH₄-N were found in the 0- to 60-cm depth, indicating the importance of performing soil N tests.     

Denitrification with and without maize plants (Zea mays L.) under irrigated field conditions

The study was conducted under irrigated field conditions to examine the effect of maize plants on denitrification. Both planted and unplanted field plots received 150kgNha^–1 as urea. In a third treatment, which was also planted and received urea at 150kgNha^–1, the soil nitrate N content was brought up to equal to that in the unplanted plots by applying additional doses of N as calcium nitrate. Soil cores were collected 24 and 72h after irrigation and the denitrification rate was measured by the acetylene inhibition method. Nitrate-N content, aerobically mineralizable C, microbial biomass carrying capacity and denitrification potential were also studied on field-moist soil. Maize plants grown under field conditions always had the potential to increase denitrification in conditions of both high and low water-filled porosity. When nitrate-N content of the planted soil decreased due to plant uptake, denitrification was reduced in the planted soils. However, when nitrate-N uptake by plants was compensated through additional doses of nitrate fertilizer, denitrification was always higher in planted than unplanted soil. The stimulatory effect of plants on denitrification was observed at both high and low soil nitrate-N concentrations, though it was more pronounced at high nitrate-N levels. The effect of plants on denitrification and related parameters was confined to the root zone. Received: 15 April 1996     

Evaluation of an acid phosphatase assay for detection of phosphorus deficiency in leaves of maize (Zea mays L.)

Assays based on acid phosphatase activity in plant tissue show promise for rapid diagnosis of phosphorus deficiency. This study was undertaken to evaluate a simple leaf disc assay for the detection of the phosphorus deficiency in maize leaves. Leaf discs were excised from 36 to 48 day old greenhouse‐grown maize plants which had been supplied with phosphate in solution culture at levels of 5, 25 or 75 yM and which had total P concentrations in the leaves ranging from 26 to 309 mmol kg^‐1 dry wt. For acid phosphatase activity determinations, discs were incubated 15 min. at 30°C with 5 mM paranitrophenol phosphate in a pH 5.8 acetate buffer. In plants which showed visual symptoms of P‐deficiency, acid phosphatase activity was 2 to 3 times as great as in P‐suf‐ficient plants. Results were inconsistent in plants which were only moderately P‐deficient. This study indicates that the leaf disc acid phosphatase assay should be useful in confirming visual diagnosis of acute phosphorus deficiency in maize, but may not be adequately sensitive to detect moderate P deficiency.     

Improving iron bioavailability and nutritional value of maize (Zea mays L.) in sulfur-treated calcareous soil

Soil factors such as pH, calcium, carbonate, and bicarbonate precipitation products in calcareous soils reduce iron (Fe) availability to crops and limit grain Fe concentrations. In the present greenhouse study, we evaluated the potential of Fe fertilizer amendments combined with organic amendments, like biochar (BC) and poultry manure, in sulfur (S)-treated low pH calcareous soils (pH_S1) to assess Fe biofortification of maize. Elemental sulfur (S) was used both for lowering soil pH and Fe solubilization. Soil pH was successfully lowered down from 7.8 to 6.5 by S application at the rate of 2.5 g kg^?1 soil. Pot experiment results revealed that Fe fertilizer combined with BC and S (pH_S1) significantly increased root and shoot dry weight, grain weight, photosynthetic rate, transpiration rate, and stomatal conductance by 69%, 86%, 28%, 74%, 57%, and 33%, respectively, relative to the control. Similarly, combined application of Fe + BC in S-amended (pH_S1) soil increased starch (34%), protein (64%), and fat (1 fold) while antinutrient phytate and polyphenols were decreased up to 29% and 40%, respectively, over control. Regarding the maize nutrients profiles, application of Fe with BC gave the maximum increase of Fe and ferritin was increased 1.7 fold at pH_S1. The results of this study showed that Fe fertilization with BC at pH_S1 soil is beneficial for crop growth and Fe bioavailability.     

Response of maize (Zea mays L.) to potassium nano-silica application under drought stress

Abstract

To investigate the influence of potassium nano-silica (PNS) on maize plant under drought stress including non-stress (NS), moderate drought stress (MDS) and severe drought stress (SDS), a factorial experiment was conducted with completely randomized blocks with three replications. Drought stress decreased the concentrations in the shoot of phosphor (P), calcium (Ca), iron (Fe), zinc (Zn), manganese (Mn) and silica (Si) and nitrogen (N), P, Ca, Fe, Zn, copper (Cu), Mn and Si concentrations of seed. There was an increase in the concentration in the N seed and shoot potassium (K) concentration under drought stress. It was observed that applying PNS increased nutrient absorption. The highest concentration of N in the seed was obtained at 100?ppm PNS. The highest concentrations of seed K and N, Cu, Mn and Si in the shoot were found when 200?ppm of PNS was applied. Applying PNS had no significant effect on the concentrations of P, Ca, sodium (Na) and Cu in the seed, and of Ca and Na in the shoot. These findings demonstrate that the application of PNS can limit the negative effects of drought stress and improve plant’s resistance against drought stress.     

Diversification of maize (Zea mays L.) through teosinte (Zea mays subsp. parviglumis Iltis & Doebley) allelic

Genetic Resources and Crop Evolution - Zea mays ssp. parviglumis is the progenitor of maize and assume to have tolerance against various biotic and abiotic stresses. It...     

Nitrogen use efficiency and performance of maize (Zea mays L.) cultivars as influenced by calcium carbide and inorganic nitrogen application rates in a derived savanna

Abstract

Field experiments were conducted to investigate nitrogen use efficiency and performance of maize (Zea mays L.) cultivars as influenced by calcium carbide (CaC₂) and nitrogen (N) rates in a derived Savanna (2016 and 2017). Maize cultivars {SUWAN-I [open pollinated variety (OPV)] and OBA SUPER II (hybrid)}, rates of N (0, 60 and 90?kg ha^?1) and CaC₂ (0, 30 and 60?kg ha^?1), were arranged in split-split plot respectively, fitted into a randomized complete block design in three replicates. N Partial factor productivity (PFP_N), Agronomic Use Efficiency (both years) and Apparent recovery of N (2017) increased in the order 60?>?90?>?0?kg N ha^?1, except N Internal use efficiency which was in the order 0?>?60?>?90?kg N ha^?1 (2017). Grain yield increased with increasing rates of N in both years. OBA SUPER-II had significantly higher grain yield than SUWAN-I (2017). Similar pattern was observed on number of grains per cob, dry cob weight, PFP_N and plant height (2017). Conversely in 2016, grain, total and shoot N uptakes were significantly higher in SUWAN-I than OBA SUPER-II. Increasing application of CaC₂ increased grain N uptake and number of grains per cob. Number of leaves and stem girth increased in the order of 60?>?0?>?30?kg?CaC₂ ha^?1. Increased grain yield with N rates could be associated with NHI and N use efficiency. These evidences suggested that hybrid maize performed better than OPV in a derived Savanna.     

Maize (Zea mays L.) Grain Yield Response to Methods of Nitrogen Fertilization

ABSTRACT

In the developing world, fertilizer application is commonly achieved by broadcasting nutrients to the soil surface without incorporation. A commonly used nitrogen (N) source is urea and if not incorporated, can sustain N losses via ammonia volatilization and lower crop yields. This study evaluated the effect of planting, N rate and application methods on maize (Zea mays L.) grain yield. An experiment with a randomized complete block design (nine treatments and three replications) was established in 2013 and 2018 in Oklahoma. The planting methods included; farmer practice (FP), Oklahoma State University hand planter (OSU-HP), and John Deere (JD) mechanical planter. Side-dress N application methods included; dribble surface band (DSB), broadcast (BR), and OSU-HP. Nitrogen was applied at the rate of 30 and 60 kg ha^?1 as urea and UAN at V8 growth stage. On average, planting and applying N at 60 kg ha^?1 using OSU-HP resulted in the highest yield (11.4 Mg ha^?1). This exceeded check plot yield (5.59 Mg ha^?1) by 104%. Nitrogen application improved grain yield by over 57% when compared to the 0-N check (8.77 Mg ha^?1). Mid-season N placement below the soil surface using OSU-HP makes it a suitable alternative to improve grain yield.