Evaluation of proline，chlorophyll，soluble sugar content and uptake of nutrients in the German chamomile（Matricaria chamomilla L.）under drought stress and organic fertilizer treatments

Amin Salehi，Hamidreza Tasdighi，Majid Gholamhoseini

1Agronomy and Plant Breeding Department，Yasouj University，Yasouj，Iran

2Oil Seed Crops Department，Seed and Plant Improvement Institute，Agricultural Research，Education and Extension Organization（AREEO），Iran



Evaluation of proline，chlorophyll，soluble sugar content and uptake of nutrients in the German chamomile（Matricaria chamomilla L.）under drought stress and organic fertilizer treatments

Amin Salehi1*，Hamidreza Tasdighi1，Majid Gholamhoseini2

1Agronomy and Plant Breeding Department，Yasouj University，Yasouj，Iran

2Oil Seed Crops Department，Seed and Plant Improvement Institute，Agricultural Research，Education and Extension Organization（AREEO），Iran

ARTICLE INFO

Article history：

in revised form 10 Dec

2015，2nd revised form 11 Jan，3rd revised form 15 Jan，4th revised form 27 Jan 2016

Accepted 15 Apr 2016

Available online 26 Aug 2016

German chamomile（Matricaria chamomilla）

Drought stress

Osmotic adjustment

Organic fertilizer

Vermicompost

Objective：To investigate the effect of drought stress and organic fertilizer on German chamomile（Matricaria chamomilla）nutrient uptake，leaf chlorophyll content and osmotic adjustment under field conditions.

Methods：This experiment was carried out through a randomized complete block design with a split factorial arrangement of treatments in three replications.The main plots were subjected to the following irrigation treatments:irrigation after 60，90，120，150 and 180 mm evaporation from Class A pan.The sub-plots were treated with three vermicompost doses（0，5 and 10 t/ha）.

Results：Although drought stress reduced the nutrient percentages in the shoots，application of vermicompost enhanced the nutrient percentages，particularly when the plants were subjected to moderate to severe drought stress conditions.Moreover，the results of this study showed that the interaction between irrigation treatments and vermicompost rates on leaf chlorophyll content was significant.Comparison between the combined treatments indicated that under normal irrigation and moderate drought stress conditions chamomile plants received 5 and 10 t/ha vermicompost showed significantly higher leaf chlorophyll content comparing to the control treatment.

Conclusions：Totally，organic fertilization by vermicompost could partly alleviate the effect of drought stress on chamomile by increasing N，P and K uptake and leaf soluble sugar，especially in stressed treatments.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2016.08.009

1.Introduction

Two species of chamomile，German chamomile（Matricaria recutita）and Roman chamomile（Chamaemelum nobile）are the most important medicinal plants worldwide.This plant has been used for thousands of years in traditional Egyptian，Roman and Greek medicine to treat different diseases such as chest colds，anxiety，insomniaandpsoriasis［1］.Nowadays，onethirdofhuman demands for drugs is acquired from plants［2］.Increasing demand of pharmaceutical factories for primary materials and，more importantly，conservation of natural genetic resources，lay emphasis on the production as well as research on production and processing of medicinal and spice plants.

Plants confront several environmental stresses which can affect their growth，metabolism，and function depending on the sensitivity of species and the stage of development.As one of the most important environmental aspects，drought is responsible for the majority of global yield loss，especially in regions with low and erratic rainfall［3］.Irrespective of the grain yield loss，drought stress amplifies adverse effects of other abiotic stresses such as nutrientdeficiency.Waterdeficitadverselyaffectsmany physiologicalprocessessuchasphotosynthesis，assimilate transmission into the grain，cell expansion，and division and nutrient accumulation and transfer［4］.When soil water potential decreases，plants should be able to reduce the water potential so that they reach to the desired slope to retain water absorption. Osmotic regulation mechanism，moving the flow of water fromthe soil into the roots，through which plants reduce osmotic potential in the cells by accumulating some active ions or organic compounds plays significant roles.Sodium，potassium，calcium and chloride are the most abundant electrolytes in osmo-regulator solutions.Additionally，organic compounds such as proline and carbohydrates play a crucial role in cell osmoregulation［5］.It should be taken into account that water crisis is becoming more serious due to population growth and global climate change which are the major threats for food security and environmental sustainability［6］.Since significant amount of water is consumed in agriculture，more attention should be paid to increasing water use efficiency［7］.

Recently，low input cropping systems and innovation of modern management of resources are important objectives of sustainable agriculture，therefore，the use of organic fertilizers instead of chemical fertilizers is one step towards sustainability. Vermicomposting is a natural process in which certain species of earthworms（especially Eisenia fetida or Eudrilus eugeniae）are used for efficient decomposition of organic waste.Application of vermicompost to soil not only provides the required nutrients to plant but also boosts soil water content and biological processes. Organicmatter，especiallyvermicompost，affectscropgrowthand yield directly by supplying nutrients and indirectly by modifying soilphysicalpropertiesthatcanimprovetherootenvironmentand stimulate plant growth［8］.According to previous studies，the use of vermicompost offers a great source of available elements and also enhances nutrientuptake by roots［9，10］.Generally，vermicompost application promotes humus formation in the soil.

Recently，medicinal plants cultivation has been increased eight times during the last two decades across the world.However，there is not enough information about the effects of irrigation regimes and organic fertilizer on proline，chlorophyll and soluble sugar content as well as nutrient uptake by German chamomile.Therefore，this experiment was carried out to explore the effects of organic fertilizer application under different irrigation regimes on some traits of German chamomile.

2.Materials and methods

The field experiment was carried out at the Eghlid Agriculture Center，Fars Province，Iran（31°13′N，52°55′E，2300 m above sea level），in 2014.The study site has an average annual rainfall of 300 mm and average annual temperature of 25°C based on long-term meteorological data（30 years）.The soil of experimental site was classified as clay loam based on the soil texture triangle.More details of the soil and vermicompost properties are presented in Table 1.

First，seedbedwaspreparedusingplowanddisk.Afterthat，the experimentalplotswereestablished.Eachplotconsistsoffourrows with 30 cm distance between rows.Prior to seed sowing，certain amounts of vermicompost were spread onto the soil surface and incorporated into the top 15 cm of the soil manually.Chamomile seedsweresownduringthefirst weekofMay.Theplant densityof 33 plants per square meter was achieved by sowing seeds on the rows 10 cm.Irrigation was performed immediately after seed sowing.A closed irrigation system was implemented to avoid runoff.The treatments were randomized based on randomized complete block design arranged in split-plot with three replicates. Irrigation regimes（irrigation after 60，90，120，150 and 180 mm evaporation from Class A pan）and vermicompost rates（0，5 and 10 t/ha）were allocated to main and sub-plots，respectively.Weeds were controlled manually during the growing season.Drought stress continued until 80%flowering of the field.To determine nutrient concentrations［nitrogen（N），phosphorus（P）and potassium（K）］in shoots，20 g of sample was taken from each plot.The plantsampleswereoven-driedat70°Cfor72handthenpowdered by an electric mill.To determine N content in shoot，Kjeltec Auto 1030 Analyser（Tecator，Sweden）was used.In addition，P content wasdeterminedusinga6505JenWayspectrophotometerfollowing colorimetrically method，K content was determined using flamephotometer（JenWay PFP7 flame-photometer）.

Chlorophyll content was measured at the flowering stage using Arnon proposed method［11］.Soluble sugar was determined at flowering stage using anthrone method［12］.About 0.5 g of fresh sample were placed in a 25 mL of cuvette and then 10 mL distilled water was added.Samples were heated at 100°C for 1 h，and then filtered into 25 mL volumetric flasks.Reaction mixture（7.5 mL）contained 0.5 mL extracts，0.5 mL mixed reagent（1 g anthrone+50 mL ethyl acetate）and 5 mL H2SO4（98%），plus 1.5 mL distilled water.The mixture was heated at 100°C for 1 min and absorbance was read at 630 nm［13］. Sucrose solutions were used as standard samples.

Proline accumulation was determined by extracting fresh samples in 3%sulfosalicylic acid.The extract was heated inwater bath for 10 min and then filtered through filter paper.Two milliliters of extract was mixed into 6 mL assay media containing 2 mL ninhydrinsolutionand2mLaceticacid.Afterthat，allsampleswere incubated at 100°C for 30 min and cooled by room temperature. Thecoloredproductwasextractedbyadding4mLtoluene.Finally absorbance of organic layer was measured at 520 nm［14］.

The data were subjected to SAS 8.1 and analyzed using ANOVA.Probability levels of 1%and 5%（P≤0.01 or 0.05）were used to test the significance among the treatments.When a F-test indicated statistical significance，the protected least significant difference（LSD）was used to split the means of the main effect.Interaction effects were divided by slicing method.

Table 1 Soil and vermicompost properties.

3.Results

3.1.Shoot N content

Effects of drought stress and vermicompost were significant on the N content of shoot；furthermore，the drought stress× vermicompost interaction was significant for this trait（Table 2）.A comparison of means showed that irrespective of the irrigation regimes，application of 10 t/ha vermicompost caused a significant increase in the N content of shoot.The maximum content of N（1.30%in dry matter）was observed for those plots that received I1irrigation treatment with the highest vermicompost rate，and the minimum content of N（0.55%in dry matter）was obtained from application of the lowest levels of irrigation（I5）without vermicompost（Figure 1）.

Table 2ANOVA for the effects of different treatments on the measured traits.

Figure 1.Interaction effect of irrigation treatments×vermicompost rates on chamomile shoot N concentration. Means followed by the different letter are significantly different（P≤0.05）.

3.2.Shoot P content

P content of shoot was significantly affected by irrigation regimes and the vermicompost rates（Table 2）.Mean comparisons showed that the reduced water availability in the I3-I5irrigation treatmentscausedsignificantdecreaseinPconcentration（Table3）. Inaddition，accordingtotheresults，shootPcontentwasfoundtobe higher in V10treatment than V0and V5treatments（Table 3）.

3.3.Shoot K content

Drought stress and vermicompost application led to a decrease and increase，respectively，in the shoot K concentration.However，drought stress×vermicompost application interaction was not significant on K content of shoot（Table 2）. Compared to well-irrigated treatment（I1），K concentration of the stressed plants decreased and the relative percentages of reduction were 16%，42%，46%and 47%in I2-5irrigation treatments，respectively（Table 3）.Drought stress diminished K content of shoot，by contrast vermicompost application increased the K concentration（Table 3），particularly when the plants were irrigated with I1treatment（data not shown）.

3.4.Leaf chlorophyll a and b contents

As expected，a reduction in water availability（from I1to I5treatment）led to significant reduce in leaf chlorophyll a content（Table 3）.In contrast，the V10fertilizer treatment（10 t/ha vermicompost）had the highest leaf chlorophyll a content（11.64 mg/g freshleafweight），whereastheV0treatment（withoutvermicompost application）had the lowest leaf chlorophyll a content（11.01 mg/g fresh leaf weight）.On the other hand，leaf chlorophyll a content in the V10treatment was 2%and 6%higher than in the V0and V5treatments，respectively（Table 3）.

Table 3 Mean comparisons of irrigation and vermicompost treatment main effects on chamomile traits.

Moreover，the interaction between drought stress and vermicompost rates on the leaf chlorophyll b content was significant.Under well-irrigated treatment（I1treatments）and moderate drought stress（I2and I3treatments），when plants received 5 and 10 t/ha vermicompost（V5and V10treatments），chlorophyll b content was significantly increased compared with control treatment（Figure 2）.

Figure 2.Interaction effect of irrigation treatments×vermicompost rates on chamomile leaf chlorophyll b content. Means followed by the different letter are significantly different（P≤0.05）.

3.5.Leaf proline content

A decrease in water availability under low irrigation regime（I2-I5irrigation treatments）enhanced the leaf proline content（Figure 3）.Although，the leaf proline content increased due to vermicompost was less affected by water deficiency and low irrigation，which increased the content of leaf proline only by 6%，8%，40%and 37%in I2-I5treatments，respectively（Figure 3）.In all irrigation treatments，the leaf proline content was significantly higher in the V0treatment than V5and V10treatments（Figure 3）.

3.6.Leaf soluble sugar content

Results of this study indicated that an increase in drought stress intensity（from I1to I5irrigation treatment）led to increase leaf soluble sugar content（Table 3）.In addition，the obtained results showed that V0treatment had the lowest leaf soluble sugar content，being 5%lower than for V10（which had a maximum leaf soluble sugar content）and 1%lower than for V5（Table 3）.

Figure 3.Interaction effect of irrigation treatments×vermicompost rates on chamomile leaf proline content. Means followed by the different letter are significantly different（P≤0.05）.

moderate and severe drought stress，its impact was more noticeable under severe and very severe stress conditions（I4and I5irrigation treatments）.Drought stress（I2-5irrigation treatments）compared with well-irrigated treatment（I1treatment），in whichplotsdidnotreceivevermicompost，significantly enhanced the content of leaf proline by 11%，19%，47%and 63%in I2-I5treatments，respectively.In contrast，the leaf prolinecontentofthechamomileplantsreceived10t/ha

4.Discussion

In spite of the drought stress，vermicompost could increase N content，particularly when I1-I4irrigation treatments were applied. It can be concluded that reduction in transpiration on account of water shortage，especially due to I3-I5irrigation treatments，could either reduce water mass flow through the soil or N uptake by plants.Also，it seems that the application of vermicompost byincreasing water retention and sustaining release of nutrients，especially N［15］，improves soil physicochemical properties and reduces the stress effects and therefore increases the absorption of N.It has been reported that vermicompost application could enhance N uptake by plants even under water deficit stress conditions［16］.As a consequence of the enhanced and steady uptake of N due to vermicompost application，plants which received organic fertilizers frequently provide greater yields than those which even received chemical fertilizers.

Mass flow，diffusion and contact exchanges are three major mechanisms of element absorption in higher plants.Among them，diffusion is the most important for P absorption.Diffusion coefficient highly depends on soil water potential and decreases while soil water potential reduces.Thus，P uptake is highly affected by soil water potential and under water shortage conditions，P uptake by plants will be decreased.

Previous findings suggest that the presence of organic matters in the soil increases P solubility［17，18］.There are several mechanisms which can lead to enhanced availability and absorption of P due to organic matters:（i）the application of organic matters rich in P can increase P content in soil；（ii）organic matter decomposition helps to increase organic acids content in soil by which P fixation is reduced［19］；（iii）increase in microbial activity on account of organic matters can increase Pavailabilityintothesoilthroughformingweak-acids（H2CO3）and releasing P from primary phosphorus-containing minerals.It has also been stated that leaf P content affects the stomatal behavior under stress conditions，possibly by affecting the osmotic potential of guard cell or by wall stiffening governing the stomatal movements［20］.Therefore，it is not surprising if vermicomposttreatedplantsshowhigherperformance compared to control plants，even under stressful environments.

Drought stress diminished K concentration in the shoots，by contrast vermicompost application increased the K concentration，particularly when the plants were irrigated with I1treatment. Reduced transpiration rate due to water shortage conditions decreasedwatermassflowintothesoil，prohibitingtheKuptakeby plants.On the other hand，it is widely stated that organic fertilizer application provides positive effects on the physicochemical characteristicsofsoilandenhancesnutrientsuptakebyplants［9，10］.The favorable effects of organic fertilizers，especially vermicompost，might be due to their potential to encourage microbial activity，enhance nutrient availability and increase plant photosynthesis. Moreover，it seems that adding vermicompost to the soil not only increases nutrients availability directly，but also operates as a slow-release fertilizer to provide N，P and K to the chamomile steadily.Potassium plays a crucial role in plant metabolism，specifically where drought stress is the main issue.Potassium actuates different enzymes involved in plant growth and plays a key role in stomatal movements and protein synthesis［21］.Therefore，it is reasonable that improved nutrient uptake（P and K）by using vermicompost，especially V10treatment，has been considered as a practical approachforamplifyingdrought resistanceinchamomile.

Reducing the amount of chlorophyll a affected by the drought stress is related to increase oxygen radicals in cells.Free radicals cause peroxidation and therefore chlorophyll pigments degradation.It seems that the reduction of chlorophyll a concentration under drought conditions is mainly because of the chlorophylls enzyme activity，peroxidase and phenolic compounds，resulting in degradation of chlorophyll［22］.

Results showed that with increased drought stress，leaf chlorophyll b content decreased and by using of vermicompost the leaf chlorophyll b content increased.According to previous findings，there is a significant correlation between leaf N content and chlorophyll［23，24］.Leaf chlorophyll content is a good index to detect N statusinplantsinordertodeterminerequiredamountofNfertilizer togainhighNuseefficiencywithmaximumplantperformance［13］. In general，it can be supposed that the vermicompost as organic fertilizer can increase chlorophyll and carotenoids content by increasing the amount of N availability for plant and followed by that the ability to absorb more sunlight and produce more assimilates and finally enhancing plant growth and yield.Higher leaf chlorophyll content in vermicompost plots compared with control plots after water deficiency conditions，suggests that higher photosynthetic capacity，attributed to better drought resistance of chamomile treated with organic fertilizers.

Accumulation of different active ions，sugars and amino acids like proline is responsible for osmotic adjustment in plant cells［25］.Osmotic adjustments maintain turgor pressure，control cell expansion and growth as well as stomatal aperture，photosynthesis，and water flow during water shortage periods［26］.Our results showed that drought stress compared to the wellwatered treatment，in which plots did not receive vermicompost，significantly enhanced chamomile leaf proline content.In contrast，the proline content of the chamomile plants received 10 t/ha vermicompostwaslessaffectedbywaterdeficiencyandlowirrigation. In all irrigation treatments，leaf proline content was significantly higher in the V0treatment than V5and V10treatments.It has been reported that high levels of leaf proline can protect plants against severe drought stress and increase drought stress tolerance［26，27］. The obtained results showed that vermicompost application decreased the leaf proline content of chamomile plants compared with non-vermicompost treatment.The results indicated that vermicompost application improved chamomile drought resistance，whichdid not associate with leafproline concentration，by contrast with increasing uptake of nutrients（N，K and P）.Moreover，it should be noted that the increase in leaf proline concentration may beanincidentalchangeassociatedwithtissueinjury.Itappearsthat decline in soil bulk density［28］，enhancement in soil water holding capacity（data not shown）and improvement of soil microbial liveliness［29］due to use of vermicompost can also account for chamomile drought tolerance enhances.

Leaf osmotic adjustment by organic solutes such as soluble sugar in the stressed plants has been previously known as a resistancemechanismtowaterdeficitstress［13］.Theresultsofthis study indicated that an increase in drought stress intensity led to increase leaf soluble sugar concentration.The accumulation of soluble sugars in drought stressed plants is controlled by several mechanisms affecting soluble sugar formation and transfer in leaves［30］.Increasing the leaf soluble sugars concentration has been found to be correlated with enhancement of the relative leaf water content［25，31］.In addition，soluble sugars play a pivotal role in osmotic adjustment in plants［32］.Also，greater sugarconcentrationinleavesofplantstreatedwith vermicompost，especially 10 t/ha，might be due to increased leaf water potential and leaf area as well as reduced chlorophyll photooxidation activity.No one has reported the influence of vermicompost application on the leaf soluble sugar content of chamomile leaf under stress and non-stress conditions.In addition，higherleafsolublesugarsconcentrationintreatedplantswith the highest rate of vermicompost（V10treatment）than other treatments（V0and V5）following water deficiency conditions，suggesting conservation of better photosynthetic capacity，has been associated with more drought resistance of the plantsreceived organic fertilizers.The treated plants with 10 t/ha vermicompostalsoshowedlessstressed-inducedaccumulationofthe proline thanother plants，therefore，theywere able to keep normal nitrogen metabolism and photosynthesis.

Drought stress in addition to the negative effects on grain yield，causes institution or exacerbation of other stresses such as nutrient deficiency.According to the results，drought stress and vermicompost application had a significant impact on proline，chlorophyll，carbohydrate and nutrient uptake in German chamomile.It can be possible that with proper nutrient management and application of organic fertilizers，particularly vermicompost，medicinal plant resistance to drought stress will be improved by increasing nutrient uptake.It seems that due to exposure to the country's arid and semi-arid region，cultivation of German chamomile in drought conditions can be extended by vermicompost fertilization.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

［1］Andrzejewska J，Woropaj-Janczak M.German chamomile performance after stubble catch crops and response to nitrogen fertilization.Ind Crops Prod 2014；62:350-8.

［2］Agatonovic-Kustrin S，Babazadeh Ortakand D，Morton DW，Yusof AP.Rapid evaluation and comparison of natural products and antioxidant activity in calendula，feverfew，and German chamomile extracts.J Chromatogr A 2015；1385:103-10.

［3］Kadam NN，Xiao G，Melgar RJ，Bahuguna RN，Quiñones C，Tamilselvan A，et al.Agronomic and physiological responses to high temperature，drought，and elevated CO2interactions in cereals.Adv Agron 2014；127:111-56.

［4］Devnarain N，Crampton BG，Chikwamba R，Becker JVW，O'Kennedy MM.Physiological responses of selected African sorghum landraces to progressive water stress and re-watering.S Afr J Bot 2016；103:61-9.

［5］Gholamhoseini M，Ghalavand A，Dolatabadian A，Jamshidi E，Khodaei-Joghan A.Effects of arbuscular mycorrhizal inoculation on growth，yield，nutrient uptake and irrigation water productivity of sunflowers grown under drought stress.Agric Water Manag 2013；117:106-14.

［6］Qiu GY，Yin J，Geng S.Impact of climate and land-use changes on water security for agriculture in Northern China.J Integr Agric 2012；11:144-50.

［7］Huang J，Ridoutt BG，Xu CC，Zhang HL，Chen F.Cropping pattern modifications change water resource demands in the Beijing metropolitan area.J Integr Agric 2012；11（11）:1914-23.

［8］Singh R，Gupta RK，Patil RT，Sharma RR，Asrey R，Kumar A，et al.Sequential foliar application of vermicompost leachates improvesmarketablefruityieldandqualityofstrawberry（Fragaria×ananassa Duch.）.Sci Hortic 2010；124:34-9.

［9］Karmegam N，Daniel T.Effect of biodigested slurry and vermicompost on the growth and yield of cowpea［Vigna unguiculata（L.）］.Environ Ecol 2000；18（2）:367-70.

［10］Zhang X，Dong W，Dai X，Schaeffer S，Yang F，Radosevich M，et al.Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.Sci Total Environ 2015；536:59-67.

［11］Arnon DT.Copper enzymes in isolation chloroplast phenoloxidase in Beta vulgaris.Plant Physiol 1949；24:1-15.

［12］Li HS.Principles and techniques of plant physiological biochemical experiment.Beijing:Higher Education Press；2000.

［13］Wu QS，Xia RX.Arbuscular mycorrhizal fungi influence growth，osmotic adjustment and water stress conditions.J Plant Physiol 2006；163:417-25.

［14］Troll W，Lindsley J.A photometric method for the determination of proline.J Biol Chem 1955；215:655-60.

［15］GuoL，WuG，LiY，LiC，LiuW，MengJ，etal.Effectsofcattlemanure compost combined with chemical fertilizer on topsoil organic matter，bulk densityand earthworm activityina wheat-maize rotation system in Eastern China.Soil Till Res 2016；156:140-7.

［16］Yang L，Zhao F，Chang Q，Li T，Li F.Effects of vermicomposts on tomatoyieldandqualityandsoilfertilityingreenhouseunderdifferent soil water regimes.Agric Water Manag 2015；160:98-105.

［17］HerenicaJF，Ruiz-PorrasJC，MeleroS´anchezS，Morillo Gonz´alez E，Maqueda Porras C.Comparison between organic and mineral fertilization for soil fertility levels，crop macronutrient concentrations，and yield.Agron J 2007；99:973-83.

［18］GholamhoseiniM，GhalavandA，Khodaei-JoghanA，DolatabadianA，Zakikhani H，Farmanbar E.Zeolite-amended cattle manure effects on sunflower yield，seed quality，water use efficiency and nutrient leaching.Soil Till Res 2013；126:193-202.

［19］Wen Z，Shen J，Blackwell M，Li H，Zhao B，Yuan H.Combined applications of nitrogen and phosphorus fertilizers with manure increase maize yield and nutrient uptake via stimulating root growth in a long-term experiment.Pedosphere 2016；26:62-73.

［20］Aug´e RM.Water relations，drought and vesicular-arbuscular mycorrhizal symbiosis.Mycorrhiza 2001；11:3-42.

［21］Evelin H，Kapoor R，Giri B.Arbuscular mycorrihizal fungi in alleviation of salt stress:a review.Ann Bot 2009；104:1263-80.

［22］Ramírez DA，Yactayo W，Guti´errez R，Mares V，De Mendiburu F，Posadas A，et al.Chlorophyll concentration in leaves is an indicator of potato tuber yield in water-shortage conditions.Sci Hortic 2014；168:202-9.

［23］Feng W，He L，Zhang HY，Guo BB，Zhu YJ，Wang CY，et al. Assessment of plant nitrogen status using chlorophyll fluorescence parameters of the upper leaves in winter wheat.Euro J Agron 2015；64:78-87.

［24］Hermanson RW，Pan C，Perillo R，Stevans R，Stockle C.Nitrogen use by crops and the fate of nitrogen in the soil and vadose zonea literature search.Pullman:Washington State University；2000.

［25］K¨onigshofer H，L¨oppert HG.Regulation of invertase activity in different root zones of wheat（Triticum aestivum L.）seedlings in the course of osmotic adjustment under water deficit conditions. J Plant Physiol 2015；183:130-7.

［26］Ruiz-Lozano JM.Arbuscular mycorrhizal symbiosis and alleviation of osmotic stress.New perspectives for molecular studies. Mycorrhiza 2003；13:309-17.

［27］Cvikrov´a M，Gemperlov´a L，Martincov´a O，Vankov´a R.Effect of drought and combined drought and heat stress on polyamine metabolism in proline-over-producing tobacco plants.Plant Physiol Biochem 2013；73:7-15.

［28］Zhang Y，Li C，Wang Y，Hu Y，Christie P，Zhang J，et al.Maize yield and soil fertility with combined use of compost and inorganic fertilizers on a calcareous soil on the North China Plain.Soil Till Res 2016；155:85-94.

［29］Xin X，Zhang J，Zhu A，Zhang C.Effects of long-term（23 years）mineral fertilizer and compost application on physical properties of fluvo-aquic soil in the North China Plain.Soil Till Res 2016；156: 166-72.

［30］Arabzadeh N.The effect of drought stress on soluble carbohydrates（sugars）in two species of Haloxylon persicum and Haloxylon aphyllum.Asian J Plant Sci 2012；11:44-51.

［31］Karimi M，Ahmadi A，Hashemi J，Abbasi A，Tavarini S，Guglielminetti L，et al.The effect of soil moisture depletion on Stevia（Stevia rebaudiana Bertoni）grown in greenhouse conditions:growth，steviol glycosides content，soluble sugars and total antioxidant capacity.Sci Hortic 2015；183:93-9.

［32］Zhou Q，Yu BJ.Accumulation of inorganic and organic osmolytes and their role in osmotic adjustment in NaCl-stressed vetiver grass seedlings.Russ J Plant Physiol 2009；56:678-85.

18 Nov 2015

s are highly grateful to Yasouj University for providing funding resources for conducting this study（Grant No.88/8705）.

Amin Salehi，Agronomy and Plant Breeding Department，Yasouj University，Yasouj，Iran.

Tel:+98 912 6776836

Fax:+98 74 3322484

E-mail:aminsalehi@yu.ac.ir

Foundation Project:Supported by Yasouj University（Grant No.88/8705）.

Peer review under responsibility of Hainan Medical University.The journal implements double-blind peer review practiced by specially invited international editorial board members.

2221-1691/Copyright©2016 Hainan Medical University.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license（http:// creativecommons.org/licenses/by-nc-nd/4.0/）.