Fate of sheep urine-N applied to an upland grass sward

Summary Sheep urine was applied once in August to 1 m² plots of a N-deficientLolium perenne-dominated sward at a rate equivalent to a single urination (48 g N m^–2) at an upland site. After 17 days herbage dry matter (DM) and total N were increased 19- and 63-fold respectively compared with a control receiving water only. Soil mineral N (NH ₄ ⁺ and NO ₃ ^– ) levels in the top 20 cm were greater in urine plots until 30 days after urine application when cumulative yields of herbage DM and N were 10 and 21 times greater than those of the control. Maximum recovery of urine N by herbage was only 16% of that applied, and, although swards responded rapidly to urination there were substantial losses of N, perhaps via leaching and/or volatilisation, from the soil-plant system.     

Uptake and leaching of nitrogen from artificial urine applied to grassland on different dates during the growing season

Artificial urine, equivalent to 30 g N m^-2, was applied to replicated plots in a perennial ryegrass (Lolium perenne L.) sward, each plot receiving a single application on one of six dates between July and November 1990. Recoveries of urine-N in herbage up to the end of the growing season in November decreased linearly for consecutive application dates, ranging from 40% of the urine-N applied in July to a negligible proportion of the final application. In contrast, contents of urine-derived N remaining in the soil (to 1-m depth) in November increased from 3% of the N applied in July to 66% for the final application. Almost all of this was present as nitrate + nitrite-N. Only soils that had received urine in September or later contained significantly greater quatities of mineral-N than the control plots. The mineral-N content of soils collected the following April indicated that most of this urine-derived N had been lost from the soil over the winter. Estimates of the quantities of N leached ranged from 0.7 g N m^-2 from untreated plots to 18.6 g N m^-2 from plots treated with urine in November. Although grass yields and N uptakes in March and April provided evidence of a residual effect from the previous year's urine applications, contents of mineral-N and of potentially mineralisable N in urine-treated soils in April were not significantly different from those in untreated soils.     

基于容器分区处理探究黑麦草生长对喀斯特不同土壤生境和水分的响应

为了探究不同水分条件下喀斯特地区分布不均、厚薄不一土壤小生境对禾本科草本植物生长的影响,用3种不同深度的容器(对照深度CK,深土D和浅土S)两两组合为6种复合容器(CK-CK、CK-S、CK-D、D-D、S-D和S-S)以实现容器分区,研究了黑麦草的根系生长、生物量积累及其分配特征。结果表明:1)在水分充足(W_0)条件下,组合了浅土容器和深土容器的处理中,黑麦草的根系生长(根长、根直径、根表面积和根生物量)均低于对照容器(CK-CK),且有浅土容器的组合处理(S-S,S-D,CK-S)受抑制程度大于有深土容器的组合处理(CK-D,D-D);当水分含量降低后,即中水(W_1)和低水(W_2)条件下,有深土容器的组合[D-D和(或)CK-D]根系生长与对照相比显著增加,而有浅土容器的组合[S-S和(或)CK-S]根系生长与对照相比显著降低。2)对比同一处理不同容器分区中黑麦草生长指标发现,在水分充足情况下,深土容器和浅土容器均会抑制植物生长,而当水分减少,S区根系生长被严重抑制,但D区根系增长优势明显。3)水分充足条件下,根冠比未受到显著影响;当水分降低时,组合了深土容器的处理根冠比均有升高的趋势,组合了浅土容器的处理根冠比有降低趋势。由此可见,不同土壤生境带来的物理空间限制会影响植物根系生长和生物量积累与分配,但水分的减少会改变根系生长及生物量积累对不同土壤生境的响应:在水分充足时,土壤物理空间是影响根系生长和生物量积累与分配的主要因子,黑麦草主要发展浅层根系。而当水分减少时,黑麦草根系在浅层土壤中无法获取供给生长代谢活动的足量水分,更倾向于将有限的有机物分配给根,通过根系伸长、表面积和体积增大、直径增粗等策略加强水分吸收,从而增强对干旱的抗逆性,提高对土壤和水分异质性的适应。     

刈割后黑麦草生理保护作用对其补偿性生长的影响

通过对黑麦草(Lolium perenne L)在轻度、中度、重度、全割刈割处理6 d和12 d后,残留叶片和叶片再生部分生长速率,超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活力,丙二醛、可溶性糖、脯氨酸含量的分析以揭示在刈割胁迫后叶片抗氧护酶活力和渗透调节物含量与其补偿性生长的关系,以及牧草耐刈性的生理调控机理。结果表明,轻度和中度及全割后叶片生长速率均高于对照,重度刈割低于对照。全割后叶片补偿性生长最明显、轻度和中度次之,重度刈割无补偿性生长。对照黑麦草叶片各部位抗氧化酶和渗透调节物含量不同,叶片顶部MDA含量较高,伴随着较高的SOD、CAT活力和较高的脯氨酸含量;叶片基部MDA含量最低,SOD、CAT活力及脯氨酸含量也较低。与对照相比,不同强度刈割6 d黑麦草再生叶和叶片平均MDA含量、SOD和CAT活力、可溶性糖和脯氨酸含量均较低。而不同强度刈割12 d,黑麦草再生叶和叶片平均MDA含量仍较低,但SOD和CAT活力增高,脯氨酸含量增加,POD活力和可溶性糖含量低于对照。这表明刈割在减少了叶面积,降低光合能力的同时,刈割伤害胁迫启动了牧草补偿性生长使残留叶片快速生长,而且残留叶片面积与其补偿生长速率成正相关。另外,虽然不同强度刈割下叶片补偿性生长速率不同,但不同强度刈割(12 d)均激活残留叶片抗氧化保护酶系统和促进脯氨酸积累。在补偿生长过程中,CAT和SOD能及时清除残留叶片中积累的氧自由基,维持较低的膜脂过氧化和细胞膜完整性,积累的脯氨酸能维护细胞水分平衡。因此,抗氧化酶(SOD和CAT)和渗透调节物(脯氨酸)在黑麦草刈割后受伤部位快速自愈及残留叶片快速补偿生长中起重要生理保护作用。     

Short term effects of radiata pine and selected pasture species on soil organic phosphorus mineralisation

Silvopastoral systems comprise part of the continued expansion of conifer plantings on grassland in New Zealand. Greater understanding of the short term dynamics of soil organic P in such systems will further our knowledge about soil carbon and phosphorus relationships which will enable improved nutrient management in the field. A glasshouse experiment was carried out to examine the short-term effects (36 weeks) of combinations of radiata pine (Pinus radiata), lucerne (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) grown in the same soil type with a range of carbon (C) and phosphorus (P) levels on plant P uptake and the specific mineralisation rate (SMR). The SMR is defined as net mineralisation rate (i.e. gross mineralisation less microbial and geochemical uptake) and calculated from organic P decline as a percentage of organic P in the original soil before planting. This included an investigation of the effect of tree ectomycorrhizal (EM) hyphae on soil organic P. Plant P uptake was positively correlated with water soluble organic carbon (WSOC) and SMR, which in turn was closely related to soil C levels. The soils with high WSOC and C levels (which also contained high levels of labile inorganic and organic P) enabled high P uptake. Although P uptake was the greatest under radiata pine, the trees tended to deplete inorganic P to a lesser extent than the forages. When tree and forage species were combined, P uptake by forages was similar to when the forages were grown alone. The various soil and plant treatments significantly affected SMR. The two low C soils, showed the greatest organic P mineralisation while a high C soil, which contained significant levels of bicarbonate extracted inorganic P at planting and was under a long established undisturbed pasture, showed the least mineralisation. Trees grown alone showed the greatest SMR, EM hyphae and trees with lucerne were slightly lower than trees alone, while the forages showed the lowest SMR. The findings of this study showed that changes in organic P are strongly influenced by interactions between plant species (radiata pine, lucerne, ryegrass) and soil properties as determined by land use and management.     

Control and variation ofin vivo nitrate reductase activity inLolium perenne L. cv S24

Summary Variation in nitrate reductase activity associated with vegetative development and morphological differences inLolium perenne L. cv S24 was examined using anin vivo technique of enzyme assay. Little difference in enzyme activity per unit weight of leaf laminar tissue was found between tillers, between and within leaf laminae but activity in leaf laminae, was much higher than that of leaf sheaths; the activity of shoot material was greater than the apparent root activity. Senescence of leaf material was not accompanied by marked changes invivo nitrate reductase activity.     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

The influence of humic and fulvic acids on silver uptake by perennial ryegrass,and its relevance to the cycling of silver in soils

Summary An experiment in which perennial ryegrass (Lolium perenne L.) was grown in solution cultures containing various forms of radiosilver (^110mAg) is ptake of labelled Ag from solutions containing either AgNO₃, Ag₂SO₄, AgCH₃COO, Ag-humic acid (HA) or Ag-fulvic acid (FA) complexes is compared. In each case concentrations of Ag in the plant roots was several fold higher than in the plant shoots. However the Ag concentration in roots from the Ag-HA preparations was only about 5% of that from the AgNO₃ solutions; from Ag-FA it was 18%. Transport index values were highest for plants grown in the Ag-HA solution, indicating greater mobility of Ag within these plants. Preliminary data for a selective extraction procedure performed on Ag-HA preparations suggest sorption onto various active sites. The relevance of these findings to the cycling of Ag within soils is discussed.     

Seasonal variability of apoplastic NH4 + and pH in an intensively managed grassland

The stomatal compensation point of ammonia (_s) is a major factor controlling the exchange of atmospheric ammonia (NH₃) with vegetation. It is known to depend on the supply of nitrogen and to vary among plant species, but its seasonal variation has not yet been reported for grassland. In this study, we present the temporal variation of apoplastic NH₄ ⁺ concentration ([NH₄ ⁺]_apo) and pH (pH_apo) measured in leaves of Lolium perenne L. in a grassland, through two periods of cutting / fertilisation, followed by a livestock grazing period. The total free NH₄ ⁺ concentration measured in foliage ([NH₄ ⁺]_fol), and soil mineral NH₄ ⁺ and NO₃ ^– concentration are also presented. The value of [NH₄ ⁺]_apo varied from less than 0.01 mM to a maximum of 0.5 mM occurring just after fertilisation, whereas the apoplastic pH ranged from pH 6 to 6.5 for most of the time and increased up to pH 7.8, 9 days after the second fertilisation, when grazing started. [NH₄ ⁺]_fol varied between 20 and 50 g N-NH₄ ⁺ g^–1 f.w. The compensation point at 20°C, ranged from 0.02 g NH₃ m^–3 between the fertilisations to 10 g NH₃ m^–3 just after the second fertilisation. The reasons for these seasonal changes are discussed, with respect to plant metabolism and the concentration of ammonium and nitrate in the soil.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

氧化石墨烯对黑麦草种子内生真菌群落结构和多样性的影响

为了探究氧化石墨烯(GO)对黑麦草种子内生真菌群落结构和多样性的影响,将黑麦草种子在0.4%、0.8%和1.2%水平的GO溶液中胁迫4 d,采用高通量测序技术,分析GO胁迫下黑麦草种子内生真菌群落组成和多样性的变化。结果显示,4个样本所有样品共分离获得303种真菌,归属于10门39纲84目160科240属。在门分类水平上,子囊菌门(Ascomycota)和担子菌门(Basidiomycota)是主要的内生真菌类群;在属分类水平上,各处理的共有优势菌属为链格孢属(Alternaria)。不同GO处理黑麦草种子内生真菌群落结构存在差异,随着GO浓度的增加,子囊菌门的丰度出现下降,0.8%和1.2%GO处理较对照分别显著降低了19%和20%(P<0.05);所有GO处理的担子菌门丰度均显著高于对照(P<0.05);1.2%处理链格孢属的丰度较对照显著降低了37.36%。与对照相比,1.2%GO 处理内生真菌的丰富度和多样性显著增加,ACE、Chao1和Shannon指数分别增加了123.5%、127.4%和117.5%(P<0.05)。主坐标分析(PCoA)分析表明,1.2%GO处理内生真菌群落结构与其他处理有较大差异;线性判别分析(LEfSe)分析发现, 各处理差异指示种明显不同。可见,GO改变了黑麦草种子内生真菌群落的组成和多样性,尤其是高浓度处理(1.2%)。研究可为碳纳米材料暴露对共生物种的潜在影响研究提供参考。     

Loss of exudates from the roots of perennial ryegrass inoculated with a range of micro-organisms

To determine the effect of microbial metabolites on the release of root exudates from perennial ryegrass, seedlings were pulse labelled with [¹⁴C]-CO₂ in the presence of a range of soil micro-organisms. Microbial inoculants were spatially separated from roots by Millipore membranes so that root infection did not occur. Using this technique, only microbial metabolites affected root exudation. The effect of microbial metabolites on carbon assimilation and distribution and root exudation was determined for 15 microbial species. Assimilation of a pulse label varied by over 3.5 fold, dependent on inoculant. Distribution of the label between roots and shoots also varied with inoculant, but the carbon pool that was most sensitive to inoculation was root exudation. In the absence of a microbial inoculant only 1% of assimilated label was exuded. Inoculation of the microcosms always caused an increase in exudation but the percentage exuded varied greatly, within the range of 3–34%.     

Two stable QTL involved in adult plant resistance to powdery mildew in the winter wheat line RE714 are expressed at different times along the growing season

Powdery mildew (Blumeria graminis f. sp. tritici) is one of the major diseases of wheat (Triticum aestivum). Adult plant resistance (APR) to powdery mildew is considered more durable than resistance conferred by major race-specific resistance genes. The objective of the present study was a better understanding of the genetic basis of APR in RE714 by means of QTL analysis of several resistance scores along the growing season. A population of 160 recombinant inbred lines obtained from the cross between RE714 and Hardi (susceptible) was assessed for APR under natural infection conditions during 3 years and a genetic map with whole genome coverage was developed with microsatellite and AFLP markers in this population. Two major QTL on chromosomes 5D and 6A were detected each year, and 6 minor QTL were detected only in 1 or 2 years. The QTL on chromosome 5D was detected during all the growing season each year and its R ² value varied between 8.5 and 56.3%, whereas the QTL on chromosome 6A was detected at 1–4 scoring dates in the 3 years, and its R ² value varied between 6.1 and 20.5%. The two QTL explained between 24.4 and 52.1% of the phenotypic variance for AUDPC, depending on the year. The models including QTL and cofactors in the composite interval mapping explained between 29 and 72% of the variance. The molecular markers linked to the two major QTL could be used in marker-assisted selection for adult plant resistance to powdery mildew. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in the amide and amino acid composition of xylem exudate from perennial ryegrass (Lolium perenne L.) during regrowth after defoliation

The paper presents the results of amino acid analyses in xylem sap during leaf regrowth of ryegrass plants defoliated firstly at the 8th and secondly at the 12th week of culture. The free amino acid composition of leaves, stubble and roots was also determined and some of the results are reported. Prior to defoliation, xylem sap contained a high proportion of amides, particularly glutamine. During regrowth after defoliation, the proportion of asparagine in the xylem sap increased until the third day when the highest ratios of asparagine/glutamine appeared. The results are compared with relative amounts of free amino acids in the different plant parts and discussed in relation to source-sink nitrogen transfer.     

Nitrogen contribution of cowpea green manure and residue to upland rice

Cowpea, Vigna unguiculata (L.) Walp., is well adapted to acid upland soil and can be grown for seed, green manure, and fodder production. A 2-yr field experiment was conducted on an Aeric Tropaqualf in the Philippines to determine the effect of cowpea management practice on the response of a subsequent upland rice crop to applied urea. Cowpea was grown to flowering and incorporated as a green manure or grown to maturity with either grain and pods removed or all aboveground vegetation removed before sowing rice. Cowpea green manure accumulated on average 68 kg N ha⁻¹, and aboveground residue after harvest of dry pods contained on average 46 kg N ha⁻¹. Compared with a pre-rice fallow, cowpea green manure and residue increased grain yield of upland rice by 0.7 Mg ha⁻¹ when no urea was applied to rice. Green manure and residue substituted for 66 and 70 kg urea-N ha⁻¹ on upland rice, respectively. In the absence of urea, green manure and residue increased total aboveground N in mature rice by 12 and 14 kg N ha⁻¹, respectively. These increases corresponded to plant recoveries of 13% for applied green manure N and 24% for applied residue N. At 15 d after sowing rice (DAS), 33% of the added green manure N and 16% of the added residue N was recovered as soil (nitrate + ammonium)-N. At 30 DAS, the corresponding recoveries were 20 and 37% for green manure N and residue N, respectively. Cowpea cropping with removal of all aboveground cowpea vegetation slightly increased (p<0.05) soil (nitrate + ammonium)-N at 15 DAS as compared with the pre-rice fallow, but it did not increase rice yield. Cowpea residue remaining after harvest of dry pods can be an effective N source for a subsequent upland rice crop.     

Root exudates: a pathway for short-term N transfer from clover and ryegrass

The short-term transfer of nitrogen (N) from legumes to grasses was investigated in two laboratory studies. One study was done in pots where the roots of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were allowed to co-exist, and a second study was performed using a micro-lysimeter system designed to maintain nutrient flow from the clover to the grass, whilst removing direct contact between the root systems. The ¹⁵N-dilution technique was used to quantify the transfer of N between species. Levels of ammonia and amino acids were measured in root exudates. The amounts of N transferred were in the same order of magnitude in both the pot and micro-lysimeter experiments. In the micro-lysimeter experiment, 0.076 mg of N were transferred per plant from clover to ryegrass during the course of the experiment. Ammonium exudation was much higher than amino acid exudation. The most abundant amino acids in both clover and ryegrass root exudates were serine and glycine. However, there was no correlation between the free amino acid profile of root extracts and exudates for both plant species: Asparagine was the major amino acid in clover roots, while glutamine, glutamate and aspartate were the major amino acids in ryegrass roots. Comparison of exudates obtained from plants grown in non-sterile or axenic conditions provides evidence of plant origin of ammonium, serine and glycine.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.     

不同种植方式下两种草本营养元素对土壤厚度和水分减少的响应

为了探究不同种植方式下草本植物对喀斯特"土层浅薄"和"岩溶干旱"生境的养分调节响应,选择苇状羊茅(Festuca arundinacea)和黑麦草(Lolium perenne)为研究材料,在盆栽水分控制条件下设置了2种土壤厚度[对照土壤厚度(T_(CK))和浅土(T_S)]、2种水分处理[对照水分(W_(CK))和干旱(W_D)]和2种种植方式(单种和混种),研究土壤厚度和水分减少对混种下两种草本植物元素含量、积累和分配的影响。结果表明:(1)与对照组(CK:T_(CK)W_(CK))相比,在浅土组(S:T_SW_(CK))、干旱组(D:T_(CK)W_D)和浅土+干旱组(SD:T_SW_D),苇状羊茅和黑麦草的地上和根系C和N含量在单种和混种下(浅土除外)显著增加,P含量和各部分元素积累量显著降低;而苇状羊茅的根系各元素分配比在3种低资源水平下(S、D、SD)由单种时增加转为混种时降低,而黑麦草的根系营养元素分配比在浅土中增加,但在干旱处理下减少。(2)在对照资源水平下(CK),混种后苇状羊茅的地上部分C含量、根系P含量、地上、地下和总的元素积累量和根系元素分配比显著高于单种,而在3种低资源水平下达到各参数在单种和混种下无显著差异。(3)在各资源水平下,混种后黑麦草各部分C、N、P的含量、积累量和根系元素分配比大体上与单种无显著差异。结果表明,在低资源水平下,苇状羊茅和黑麦草通过增加C和N元素含量表现出较强的资源获取和防御能力。在混种条件下,苇状羊茅能够通过调节自身元素的积累和分配来提高竞争力,而黑麦草保持相对恒定的策略来响应竞争。     

The effects of low,regulated supplies of nitrate and ammonium nitrogen on the growth and composition of perennial ryegrass

Summary Perennial ryegrass was grown in flowing solution culture with nitrogen supplied in amounts that increased exponentially,i.e. in parallel with the rate of increase in growth. Nitrogen was supplied as either NO ₃ ⁻ or NH ₄ ⁺ , and the amounts to be added were calculated on the basis of extrapolated values for dry weights obtained from fitted curves. There were two rates of addition for each form of N aimed at providing adequate (5.0 per cent) and less than adequate (2.75 per cent) contents in the plants in each case. Measured plant weights and N concentrations were in close agreement with predicted values over a four week experimental period. There was no effect of N-form at high N, and these plants produced 46 per cent more dry matter than the plants at low N. Only minor differences in overall growth occurred with NO ₃ ⁻ or NH ₄ ⁺ plants at low N, but the NH ₄ ⁺ plants had a greater shoot:root ratio. The absorption rate (m mol Ng⁻ root d⁻¹) for NH ₄ ⁺ -N was therefore greater than for NO ₃ ⁻ -N. The cation/anion composition of the plants was affected in a predicable way, and to a greater or lesser extent at high or low N, respectively, in NO ₃ ⁻ or NH ₄ ⁺ plants. The major changes in cation composition came through effects on potassium absorption. Plants with low NO ₃ ⁻ appeared to be under greater N stress than those with low NH ₄ ⁺ because of the lower shoot:root ratio and the greater C∶N ratio in the shoots.     

Soil nitrogen availability under grassland and cultivated corn

Summary Adjacent corn and ryegrass plots were fertilized with rates of 0, 50, 100, 150, and 200 kg N as ammonium nitrate/ha. Corn growing on this soil did not respond to fertilizer N while ryegrass responded to rates of up to 200 kg N/ha. The differences in N availability was also reflected in the higher profile NO₃–N under corn than under ryegrass. The same general trends occurred on a second soil, where N availability for the hay crop was also less than for corn crop. Compared with corn, hay responded more to N fertilizer and had lower soil NO₃–N levels.Grasslands appear to respond to higher N fertilizer rates than cultivated crops on the same soil.Vermont Agricultural Experiment Station Journal Article No. 495.