The effect of phosphorus and nitrogen deficiency on growth of seedlings of spring barley in dependence on irradiance: Growth analysis

Seedlings of spring barley,Hordeum vulgare L. cv. Mirena, were grown in a controlled environment chamber at high (HI: 122 Wm^?2) and low (LI: 28 Wm^?2) irradiance in the complete Richter’s nutrient solution (R) or in solution lacking either phosphorus (R -P) or nitrogen (R -N). The experiment was terminated 15 days after sowing when plants (R-N) at HI ceased to grow. At that time the dry mass of one plant was 449.8 mg, 145.7 mg and 116.8mg at HI and 203.4 mg, 110.1 mg and 91.0 at LI for R, (R-P) and (R-N), respectively. Deficiency of P and especially N reduced the size of loaf area more under HI than under LI conditions. Specific dry mass of leaves was the highest in R-N plants. The values of relative growth rate and assimilation rate are presented. Interaction of the effects of deficiency of mineral nutrients and irradiance during cultivation should be analyzed in further experiments for determination of optimum conditions for utilization of mineral nutrients.     

氮磷添加对荒漠草原植物-凋落物-土壤生态化学计量特征的影响

为明确植物、凋落物和土壤养分含量及化学计量比对土壤中添加多种限制性养分的响应，阐明"植物-凋落物-土壤"连续体化学计量动态及各组分之间的协同作用，以宁夏荒漠草原为研究对象，于2018年开始进行氮（N）、磷（P）养分添加控制试验。试验处理包括对照（CK）、N添加、P添加、NP共同添加4个处理。结果表明：（1） NP共同添加显著增加了荒漠草原植物N和P含量、以及凋落物和土壤P含量，显著降低了荒漠草原植物C：N和C：P、以及土壤和凋落物C：P和N：P。P添加显著增加了荒漠草原植物、凋落物和土壤P含量，显著降低了植物、凋落物、土壤C：P和N：P。N添加分别增加了植物、凋落物N含量和N：P，但对植物N含量影响未达到显著水平。（2） C、N、P含量和N：P大小均表现为植物 > 凋落物 > 土壤，C：N和C：P均表现为凋落物 > 植物。（3） N添加提高了荒漠草原植物对P再吸收效率，降低了荒漠草原植物对N利用效率；P添加提高荒漠草原植物对N再吸收效率，降低荒漠草原对P的利用效率；NP共同添加提高了荒漠草原植物对N和P再吸收效率，降低了荒漠草原植物对N和P利用效率。（4）植物-凋落物-土壤的N、P含量和化学计量比之间显著相关，其中植物N、P、N：P与凋落物和土壤N、P、N：P显著正相关，凋落物P、C：N与植物和土壤C：P、N：P显著负相关。（5）荒漠草原植物和凋落物N较稳定（1/H=0.45和1/H=0.48），而植物和凋落物P、N：P较敏感（1/H=0.80、0.73和1/H=0.81、0.78）。荒漠草原植物生长受N限制，N添加缓解荒漠草原植物N限制，P添加和NP添加加剧荒漠草原植物N限制，荒漠草原植物通过改变养分利用策略和再吸收利用效率适应土壤中N、P含量的变化。     

Effects of nitrogen supply and irradiance on growth and nitrogen status in the moss Dicranum majus from differently polluted areas

Abstract

A growth experiment was undertaken to study the effects of nitrogen supply and irradiance on growth and nitrogen status in the moss Dicranum majus Sm. from two areas receiving different amounts of atmospheric nitrogen deposition. Intact samples of D. majus carpets were taken from two Picea abies forests, one located in southern Norway (high-N site) and the other in central Norway (low-N site). The moss carpets were grown for 120 days at three irradiance levels (PPFD: 20,40 or 80 μmol m^?2 s^?l) and sprayed daily with equal amounts of a nutrient solution containing 30, 180 or 330 μM nitrogen as NO₃ - and NH₄ +. Concentrations and total amounts of nitrogen, soluble proteins and chlorophyll were highest in moss plants from the high-N site, both at the start and the end of the experiment. The elongation growth was highest at the lowest irradiance level. As total biomass production did not differ between nitrogen and light treatments, moss growth was presumably limited by other factors, even at the lowest supply rates. Concentrations and total amounts of nitrogen increased with increasing nitrogen supply in moss plants from both sites. Accumulated nitrogen was partly stored as protein and chlorophyll. Recycling of nitrogen from old to young tissues is discussed as a possible explanation for the rather low nitrogen demand in D. majus and the persistently higher nitrogen contents in moss plants from the high-N site.     

Distribution of Chlorophyll between the Two Photoreactions in Photosynthesis of the Blue-Green Alga Anacystis nidulans Grown at Two Different Light Intensities

Anacystis nidulans was grown in white light of two different intensities, 7 and 50 W ·m^?2. The in vivo pigmentations of the two cultures were compared. The ratio phycocyanin/chlorophyll a was 0.96 for cells grown at 7 W · m^?2 and 0.37 for cells grown at 50 W · m^?2. Phycocyanin-free photosynthetic lamellae (PSI-particles) were prepared, using French press treatment and fractionated centrifugation. Algae grown in the irradiance of 50 W · m^?2 showed a chlorophyll a/P700 ratio of 260, while algae grown at 7 W · m^?2 had a value of 140. Corresponding PSI-particles showed values of 122 and 109 respectively. Light-induced absorption difference spectra measured between 400–450nm indicated different ratios between cytochrome f and P700 in the two algal cultures. Enhancement studies of photosynthetic oxygen evolution were carried out. When a background beam of 691 nm was superimposed upon a signal beam of 625 nm, good enhancement was observed for both cultures. With the wavelengths 675 and 691 nm together a pronounced enhancement could be detected only in algae grown at the higher light level. Absorption spectra recorded on whole cells at 77°K revealed a small shift of the main red chlorophyll a absorption peak caused by light intensity. It is proposed that the reduction of the phycocyanin/chlorophyll a ratio in high light-grown cells is accompanied by an increased energy distribution by chlorophyll a into PSII.     

High Irradiance Induced Pigment Degradation and Loss of Photochemical Activity of Wheat Chloroplasts

Loss of chlorophyll (Chl) and carotenoids (Car) of leaves and changes in Chl fluorescence emission and polarisation, malondialdehyde (MDA) accumulation, and 2,6-dichlorophenol indophenol (DCPIP) photoreduction in chloroplasts of wheat seedlings grown under different irradiance and subsequently exposed to high irradiance stress (HIS; 250 W m^–2) were studied in mature and senescent primary wheat leaves. Faster rate of pigment loss was observed in leaves of moderate irradiance (MI; 15 W m^–2) grown plants, compared to high irradiance (HI-1 and HI-2; 30 and 45 W m^–2) ones when exposed to HIS. A relatively lower loss of Car in the plants grown in HI-1 and HI-2 exposed to HIS suggests HI adaptation of these seedlings. The slower rate of increase in the ratio of Chl fluorescence emission (F₆₈₅/F₇₃₅) also may suggest photoprotective strategy of HI grown seedlings. There was a positive correlation between MDA accumulation and Chl fluorescence polarisation. The DCPIP photoreduction activity in chloroplasts isolated from HI-1 and HI-2 grown plants exposed to HIS showed slower loss of electron transport activity compared to MI grown plants. These observations suggest that plants grown under higher irradiance have capacity to manage the excess quanta better than those grown under lower irradiance.     

Effect of irradiance during acclimatization on content of proline and phytohormones in micropropagated Ulmus minor

This study aimed to investigate the effects of irradiance on plant growth and content of proline and phytohormones during ex vitro acclimatization of micropropagated Ulmus minor plants. In vitro rooted plants were acclimatized to ex vitro conditions in a climate chamber with two irradiances, 200 μmol m^?2 s^?1 (high irradiance, HI) and 100 μmol m^?2 s^?1 (low irradiance, LI) for 40 d. Immediately after the ex vitro transfer, the plants experienced a water deficit [wilting leaves with the reduced relative water content (RWC)], but following the experiment, the recovery of the RWC was more pronounced in the HI treatment. Also, the content of proline, ABA, and JA-Ile were higher in HI treatment. Growth analyses revealed that HI improved growth and biomass production.     

Photosynthetic parameters of Ulmus minor plantlets affected by irradiance during acclimatization

In order to set up large-scale acclimatization protocols of micropropagated plants, an in-depth knowledge of their physiological responses during in vitro to ex vitro transfer is required. This work describes the photosynthetic performance of Ulmus minor micropropagated plants during acclimatization at high irradiance (HI; 200 ± 20 μmol m^?2 s^?1 or low irradiance (LI; 100 ± 20 μmol m^?2 s^?1). During this experiment, leaf pigment content, chlorophyll a fluorescence, gas exchange, stomata morphology, the activity of the Calvin cycle enzymes and saccharides were measured in persistent and new leaves. The results indicated that HI induces a higher photosynthetic performance compared to LI. Therefore, plants acclimatized under HI are likely to survive better after field transfer.     

Defence response produced during photodynamic damage in transgenic rice overexpressing 5-aminolevulinic acid synthase

Photodynamic and photoprotective responses at different irradiances were investigated in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum 5-aminolevulinic acid synthase (ALA-S). With high irradiance (HI) of 350 µmol m^?2 s^?1, transgenic lines P5 and P14 showed a decrease in contents of chlorophyll (Chl) and the chloroplast-encoded gene psbA mRNA, whereas a decrease in light-harvesting Chl-binding proteins was observed only in P14. These effects were not observed in the wild-type (WT) line treated with HI or all of the lines treated with low irradiance (LI) of 150 µmol m^?2 s^?1. HI resulted in a greater decrease in the quantum yield of photosystem 2 and a greater increase in non-photochemical quenching (NPQ) in the transgenic lines, particularly in P14, compared to WT. Photoprotective zeaxanthin contents increased at HI, even though carotenoid contents were lower in the transgenic lines compared to WT. When exposed to HI, superoxide dismutase greatly increased in transgenic lines P5 and P14, but peroxidase and glutathione reductase increased only in P14, in which more photodynamic damage occurred. Thus the greater expression of ALA-S in the transgenic plants developed the stronger protective functions, i.e. the increased values of NPQ and zeaxanthin, as well as more photodynamic reactions, i.e. decreased photosynthetic component and efficiency, in the photosynthetic complexes. However, the photodynamic reactions indicate that the antioxidant capacity was insufficient to cope with the severe stress triggered by photoactive porphyrins in the transgenic rice expressing ALA-S.     

Influence of Cotyledon Excision and Sucrose on Root Formation in Pea Cuttings

Sucrose was supplied to stock plants of Pisum sativum L. cv. Alaska grown at different levels of irradiance. There was no significant effect on the rooting of the cuttings by sucrose supply to intact plants regardless of the irradiance. However, an increase in the number of roots per cutting was obtained at increasing concentrations of sucrose when the stock plants had been grown at 4 W m^?2 and their cotyledons had been removed two days before the cuttings were excised. Cotyledons were removed from stock plants at different times before the excision of cuttings with the intent to regulate the endogenous supply of carbohydrate. The number of roots per cutting was reduced by removal of the cotyledons and this reduction was correlated to the number of days the stock plants had grown without cotyledons as well as to the irradiance pre-treatment. A greater reduction occurred in cuttings from plants grown under 4 W m^?2 than from those grown under 38 W m^?2. The growth of the stock plants and the subsequent stem growth of the cuttings was determined by the irradiance to the stock plants and by the time of removal of the cotyledons. Exogenous supply of sucrose had no effect on the stem growth of the cuttings.     

The fate of 15N-nitrate in a northern peatland impacted by long term experimental nitrogen, phosphorus and potassium fertilization

Information about the impact of nitrogen (N) deposition on the fate of deposited N in peatland ecosystems is lacking. Thus we investigated the fate of experimentally added ¹⁵N in long-term N-fertilized treatments in a Sphagnum-dominated ombrotrophic bog. Fertilization significantly stimulated vascular plant and suppressed Sphagnum and Polytrichum moss growth. N content in peat, mosses, and vascular plants was raised by the fertilizer addition and reached a maximum at 3.2 g m^?2 N input level with phosphorus (P) and potassium (K) addition. Most of N was retained in the vegetation and upper 10 cm of the peat. When N deposition equalled 1.6 g m^?2 and less, or 3.2 g m^?2 N with P and K addition, no inorganic N leaching was observed on the plots. This result indicates that co-fertilization with P and K raised the N retention capacity and that critical N loads with respect to N saturation depend on P and K availability. Most of the deposited ¹⁵N was recovered in the bulk peat, which may be related to a rapid immobilization of inorganic N by microorganisms and mycorrhizal assimilation. Increase of N, P, and K fertilization increased the contribution of vascular plants to N retention significantly and reduced those of mosses. The increase was mainly related to enhanced productivity, vascular biomass and N content in tissues; the reduced retention by mosses resulted from both reduced moss biomass and assimilation. The study shows that the N filter function of ombrotrophic bogs will be influenced by interactions with other nutrients and shifts in plant community structure.     

Chemical oxygen demand,nitrogen and phosphorus removal by subsurface wetlands with Phragmites vegetation in different models

To improve the removal efficiency of subsurface wetlands vegetated mainly by Phragmites, pilot‐scale gravel‐based wetlands were used to treat sewage characterized by chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) pollution. For Phragmites vegetation, COD, TP and TN removal loads of wetland vegetation with Phragmites australis–Typha angustata–Scirpus validus as main species reached 0.517 g m^?2 d^?1, 0.277 g P m^?2 d^?1 and 0.023 g N m^?2 d^?1. The COD removal loads in pilot‐scale and medium‐scale (260 m² in area) wetlands with Phragmites‐monoculture vegetation were 0.62–0.64 g m^?2 d^?1, while that of P. australis–T. angustata–S. validus wetland reached 0.974 g m^?2 d^?1. Thus, the preferable poly‐culture model for Phragmites wetland vegetation was P. australis, T. angustata, S. validus and Zizania latifolia with stem density ratio of 8:1:5:1. After harvest, nitrogen and phosphorus standing stocks of wetland vegetations ranged only 2.2–9.93 g N m^?2 and 5.39–13.5 g P m^?2, respectively, as both the above ground biomass and the nitrogen and phosphorus contents of the wetland vegetation harvested in late autumn were low.     

Bioassays with Stigeoclonium Kütz. (Chlorophyceae) to identify nitrogen and phosphorus limitations

Eleven strains of the filamentous algae genus Stigeoclonium Kütz. (Chlorophyceae) were tested as bioassay organisms to identify nitrogen and phosphorus limitations to growth in a variety of waters. The assay results were related to the nutrient status of the waters during the year. Optimal growth was obtained at an inorganic phosphorus concentration of 0.65 mg P 1^?1 and an inorganic nitrogen concentration of 3.75 mg N 1^?1. The algal growth potentials were generally higher in November and February than in August. In summer, nitrogen became important as a limiting nutrient. When ratios of inorganic nitrogen to inorganic phosphorus (N/P) were above 6.2, phosphorus was primarily limiting and at lower N/P ratios nitrogen became the important limiting factor. Stigeoclonium proved to be a suitable bioassay organism to identify nutrient limitation in freshwaters.     

Effect of photosynthetically active radiation, salinization, and type of nitrogen nutrition on growth of Salicornia europaea plants

Effects of various combinations of nutrient solution salinity (0.3, 171, and 342 mM NaCl), photosynthetically active radiation (PAR) of 600 or 1150 μmol/(m² s), and type of nitrogen nutrition (amide-N or nitrate-N) on the productivity and the content of accumulated mineral nutrients and free amino acids were studied in Salicornia europaea plants. At PAR of 600 μmol/(m² s), plant productivity increased with elevation of salinity level; at 1150 μmol/(m² s), the maximum productivity was observed in the plants grown at 171 mM of NaCl. The content of free amino acids in shoots, regardless of PAR, decreased with growing salinity level, whereas Na content, on the opposite, increased. Glutamic acid, rather than proline, was shown to be the main organic osmolyte in this plant species. Comparison of the productivity of plants grown on solutions with amide (urea) or nitrate nitrogen showed that higher biomass accumulation was achieved in the former case.     

Photosynthetic acclimation of plants to growth irradiance: the relative importance of specific leaf area and nitrogen partitioning in maximizing carbon gain

Changes in specific leaf area (SLA, projected leaf area per unit leaf dry mass) and nitrogen partitioning between proteins within leaves occur during the acclimation of plants to their growth irradiance. In this paper, the relative importance of both of these changes in maximizing carbon gain is quantified. Photosynthesis, SLA and nitrogen partitioning within leaves was determined from 10 dicotyledonous C₃ species grown in photon irradiances of 200 and 1000 µmol m^?2 s^?1. Photosynthetic rate per unit leaf area measured under the growth irradiance was, on average, three times higher for high‐light‐grown plants than for those grown under low light, and two times higher when measured near light saturation. However, light‐saturated photosynthetic rate per unit leaf dry mass was unaltered by growth irradiance because low‐light plants had double the SLA. Nitrogen concentrations per unit leaf mass were constant between the two light treatments, but plants grown in low light partitioned a larger fraction of leaf nitrogen into light harvesting. Leaf absorptance was curvilinearly related to chlorophyll content and independent of SLA. Daily photosynthesis per unit leaf dry mass under low‐light conditions was much more responsive to changes in SLA than to nitrogen partitioning. Under high light, sensitivity to nitrogen partitioning increased, but changes in SLA were still more important.     

Effects of nitrogen and phosphorus imbalance on photosynthetic traits of poplar Oxford clone under ozone pollution

Ozone (O₃) pollution and the availability of nitrogen (N) and phosphorus (P) in the soil both affect plant photosynthesis and chlorophyll (Chl) content, but the interaction of O₃ and nutrition is unclear. We postulated that the nutritional condition changes plant photosynthetic responses to O₃. An O₃-sensitive poplar clone (Oxford) was subject to two N levels (N0, 0 kg N ha^??1; N80, 80 kg N ha^??1), two P levels (P0, 0 kg P ha^??1; P80, 80 kg P ha^??1) and three levels of O₃ exposure (ambient concentration, AA; 1.5?×?AA; 2.0?×?AA) over a growing season in an O₃ free air controlled exposure (FACE) facility. The daily change of leaf gas exchange and dark respiration (R_d) were investigated at mid-summer (August). Chl a fluorescence was measured three times in July, August and September. At the end of the growing season, Chl content was measured. It was found that Chl content, the maximum quantum yield (F_v/F_m), Chl a fluorescence performance index (PI) and gas exchange were negatively affected by elevated O₃. Phosphorus may mitigate the O₃-induced reduction of the ratio of photosynthesis to stomatal conductance, while it exacerbated the O₃-induced loss of F_v/F_m. Nitrogen alleviated negative effects of O₃ on F_v/F_m and PI in July. Ozone-induced loss of net photosynthetic rate was mitigated by N in medium O₃ exposure (1.5?×?AA). However, such a mitigation effect was not observed in the higher O₃ level (2.0?×?AA). Nitrogen addition exacerbated O₃-induced increase of R_d suggesting an increased respiratory carbon loss in the presence of O₃ and N. This may result in a further reduction of the net carbon gain for poplars exposed to O₃.     

Evaluating potential of green alga <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> to accumulate phosphorus and to fertilize nutrient-poor soil substrates for crop plants

Algae are capable of accumulating nutrients from aqueous waste, which makes them a potential fertilizer. The ability of the fast growing Chlorella vulgaris strain IPPAS C1 to accumulate phosphorus (P) was probed in V-shaped plastic foil photobioreactors. The P uptake was 0.13–0.53 g(P)·m^?2·day^?1 when the algal culture densities were kept between 0.1 and 1.0 g(DW)·L^?1 in a typical summer irradiance of Central Europe. The algal biomass can be effectively utilized for soil fertilization only if the algal cells release nutrients into the soil in a form that would be available to roots and at a rate sufficient to support plant growth. To examine this, we compared the growth of wheat, Triticum aestivum L., in two nutrient-deficient substrates: “Null Erde” and sand, with and without fertilization by wet and spray-dried algae. Plants grown in the two nutrient-deficient substrates supplemented by mineral fertilizer served as a control representing optimal nutrient supply. Plants grown in a high-nutrient substrate (SoMi 513) were used as an additional reference representing the maximum growth potential of wheat. Wheat growth was monitored for 8 weeks and measured, including the increase of the leaf area as well as shoot and root dry weight in 10 randomized replicates for each substrate and fertilization variant. After harvest, the biomass and N, P, and C contents of the plant shoots and roots were recorded. Algae fertilization of “Null Erde” led to wheat growth, including root hair production, which was similar to mineral-fertilized “Null Erde” and only slightly less vigorous than in the nutrient-rich SoMi 513 substrate. The plants grown in sand were smaller than the plants in “Null Erde” but fertilization by algae nevertheless led to growth that was comparable to mineral fertilizer. These results unambiguously demonstrate that algal biomass is a viable option for delivering nutrients to support agriculture on marginal soils.     

Effect of phosphorus and nitrogen on growth,chlorophyll, amino nitrogen,soluble sugar contents and algal heterocysts of water fernAzolla pinnata

The P uptake inAzolla pinnata in mineral medium increased linearly with increasing phosphate doses and optimum growth and chlorophyll content of fern were recorded at 1.2 mM of K₂HPO₄. The higher doses of phosphate reduced growth and chlorophyll, but an increase in soluble sugar and amino nitrogen was noticed. Addition of combined nitrogen sources decreased the fern growth, soluble sugar, chlorophyll and heterocyst frequency but amino nitrogen was comparatively higher. The growth and chlorophyll content were always high when the fern was grown in the absence of added nitrogen sources; this supports the thesis about the specific importance of symbiosis for the nutrition of fern with nitrogen which cannot be replaced by mineral compounds.     

Adventitious Root Formation Induced by Gibberellic Acid and Regulated by the Irradiance to the Stock Plants

Pea plants (Pisum sativum L. cv. Alaska) were grown from seeds for eleven days at different irradiances. Cuttings were then excised and rooted at 16 W × m^?2. Gibberellic acid (GA₃, 10^?11 to 10^?3M) was applied to the cuttings immediately after excision. Cuttings from stock plants grown at the highest level of irradiance (38 W × m^?2) formed the lowest number of roots. An increasing number of roots per cutting was obtained by decreasing the irradiance to the stock plants. In cuttings from stock plants grown at low irradiances, low concentrations of GA₃ (10^?8 and 10^?7M) promoted root formation further. No effect on rooting by these GA₃ concentrations was observed when applied to cuttings originating from stock plants grown at the high irradiances. Root formation in all cuttings was inhibited by GA₃ at concentrations higher than 10^?6M. The degree of inhibition by GA₃ was influenced by the irradiance pretreatment and was increased with an increase in the irradiance during the stock plant growth. Seeds from different years produced cuttings with different response patterns regarding the irradiance and GA₃ effects on rooting.     

Effects of nitrogen and phosphorus starvation on nucleic acid and protein content of Heterocapsa sp.

Changes in the protein, RNA and DNA content related to nitrogen(N) and phosphorus (P) starvation were studied in the marinedinoflagellate Heterocapsa sp. grown in batch cultures. In bothcases of nutrient starvation, metabolic adaptations affectedprotein and RNA pools, while the DNA content per cell remainedapproximately constant. N starvation led to a parallel decreasein protein and RNA concentration which caused the protein/RNAratios to remain constant. A dramatic decrease in the RNA contentcharacterized the P-starved cultures, although protein synthesiscontinued. The ribosomal RNA content was lower than expectedgiven the continuation of protein synthesis. It is suggestedthat protein/RNA ratios could be used as an indicator of P starvation,while protein/chlorophyll ratios would characterize N starvation ¹Present address: University of Hawaii at Manoa Soest, BiologicalOceanography, 1000 Pope Road, Honolulu, HI 96822, USA     

Photosynthetic Characteristics of Chloroplasts of Primary Wheat Leaves Grown under Different Irradiance

The rate of accumulation of total chlorophyll (Chl) and carotenoids (Car) of leaves grown under high irradiance, HI (30 and 45 W m^–2) was faster than at moderate irradiance, MI (15 W m^–2). However, the senescence phase started earlier in the samples and proceeded at a faster rate. Chl a/b and Chl (a+b)/Car values showed faster loss of Chl a (compared to Chl b) and Chl (a+b) (compared to Car) in HI leaves. Protein accumulation and loss were also similar to that of Chl (a+b) content. Increase in Chl fluorescence during the development phase may suggest a gradual change in thylakoid organisation, however, the temporal kinetics were different in HI and MI samples. Increase in fluorescence polarisation during senescence of HI leaves compared to the control (MI) suggests conversion of thylakoid membranes to gel phase. Chloroplasts prepared from HI seedlings showed higher rate of photochemical activities, however, the activity declined earlier and at faster rate compared to the control.