responses of winter wheat to ascophyllum nodosum (l.) le jol

Responses of winter wheat to Ascophyllum nodosum (L.) Le Jol.

extract application under the effect of N fertilization and water supply

Administration Board Director General: Konstantina Glampedakis, MA Project Director: Michalis Glampedakis, PhD Project Assistant Director: Antonis Glampedakis. MSc Scientific Committee Stamatis Stamatiadis, Lefteris Evangelou, Jean-Claude Yvin, Christos Tsadilas, José Maria Garcia Mina, Florence Cruz

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Journal of Applied Phycology ISSN 0921-8971 J Appl PhycolDOI 10.1007/s10811-014-0344-0

Responses of winter wheat to Ascophyllumnodosum (L.) Le Jol. extract applicationunder the effect of N fertilization and watersupply

Stamatis Stamatiadis, LefterisEvangelou, Jean-Claude Yvin, ChristosTsadilas, José Maria Garcia Mina &Florence Cruz

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Responses of winter wheat to Ascophyllum nodosum (L.)Le Jol. extract application under the effect of N fertilizationand water supply

Stamatis Stamatiadis & Lefteris Evangelou &

Jean-Claude Yvin & Christos Tsadilas &José Maria Garcia Mina & Florence Cruz

Received: 6 April 2014 /Revised and accepted: 11 May 2014# Springer Science+Business Media Dordrecht 2014

Abstract A greenhouse experiment was conducted to evalu-ate the effects of foliar application of an Ascophyllumnodosum seaweed extract (AZAL5) on the growth, nutrientuptake, and yield of winter wheat in a surface soil of theThessaly Plain classified as TypicXerorthent. Twelve treat-ment combinations in a randomized complete block designwith a factorial arrangement were composed of two rates ofinorganic fertilizer (0 and 50 ppm N), three rates of AZAL5(0, 1.5, and 3 % diluted extract), and two levels of watersupply (75 and 45 % of field capacity). Under soil P and Ksufficiency, the addition of fertilizer N greatly increased grainyield and nutrient uptake in the shoots (N and K) and grain (N,P, and K). Reduced water supply decreased grain yield andcaused water stress as evidenced by decreasedΔ13C in the N-deficient treatments and decreased nutrient uptake. AZAL5application caused increased grain K uptake and a 25 %increase in yield only when mineral N was added.Differences in the efficacy of the two AZAL5 concentrationsindicated that optimal dilution ratios were directly or indirect-ly dependent on soil water content. Complex interaction ef-fects between AZAL5 and water supply on grainΔ13C could

not be explained by conventional physiological response towater stress. The lack of biomass, nutrient content, andΔ13Cdifferences between AZAL5 and control treatments in theshoot indicated that the reproductive organs of wheat werethe main site of biostimulant action. Overall, the results un-derline the potential of this product to enhance the effective-ness of inorganic N fertilizers in intensively managedcropping systems under optimal irrigation, deficit irrigation,or rainfed conditions.

Keywords Nutrient uptake . Stable isotopes . Carbondiscrimination .Δ13C . δ15N . Grain yield . Shoot biomass .

Water stress . Foliar application . Seaweed

Introduction

Seaweed extracts have been used for decades in agricultureand horticulture as biostimulants to promote plant growth andincrease crop yields. When applied in small amounts, thebeneficial effects of seaweed extracts on crop growth havebeen attributed to plant growth regulators, and possiblymicronutrients, that stimulate root growth, mineral uptake,photosynthetic capacity, and stress tolerance (Khan et al.2009). Under conditions of water stress, only few studies haveprovided evidence of improved plant performance upon treat-ment with seaweed extracts (Mooney and van Staden 1985;Temple et al. 1989; Zhang and Ervin 2004). Antistress effectsmay be related to bioactive chemicals contained in the extractsand increased antioxidant enzyme activity (Fike et al. 2001),but their physiological mechanism of action is largelyunknown.

Concerning mineral uptake, the mode of action inwheat and barley is thought to implicate cytokinins orcytokinin-like molecules that regulate spikelet and

S. Stamatiadis (*)Soil Ecology and Biotechnology Laboratory, Goulandris NaturalHistory Museum, 13 Levidou Str., Kifissia, 14562 Athens, Greecee-mail: [email protected]

L. Evangelou :C. TsadilasInstitute of Soil Mapping and Classification, National AgriculturalResearch Foundation, 1 Theophrastos Str, 413 35 Larissa, Greece

J.<C. Yvin : F. CruzTIMAC AGRO INTERNATIONAL—CRIAS, 55 Boulevard JulesVerger, 35800 Dinard, France

J. M. G. MinaTIMAC AGRO, España SA Pol. Arazuri-Orcoyen, Calle C, n 32,31160 Orcoyen, Navarra, Spain

J Appl PhycolDOI 10.1007/s10811-014-0344-0

Author's personal copy

flower number through mobilization of nutrients to thereproductive organs (Featonby-Smith and van Staden1987; Beckett and van Staden 1989). Whether seaweedextracts act better under a balanced crop mineral nutri-tion or are better suited to overcome the effects ofnutrient deficiency is still under debate. In some studies,seaweed extracts from Ecklonia maxima increased yieldsunder adequate nutrient supply (Mooney and van Staden1985; Featonby-Smith and van Staden 1987), therebydemonstrating a capacity to enhance the effectivenessof conventional fertilizers. In contrast, other studiesindicated that extracts from the same seaweed speciesincreased yield only in nutrient-stressed wheat (Beckettand van Staden 1990) or even in the absence of mineralfertilizer (Nelson and van Staden 1986). Recently, Khanet al. (2011) demonstrated that the extract of a differentseaweed species, Ascophyllum nodosum, inducedcytokinin-like activity in Arabidopsis thaliana when ap-plied as a liquid culture or foliar spray. The addition ofa similar extract, coded as AZAL5, in a nutrient solu-tion increased rapeseed seedling growth of shoots androots and the uptake of nitrogen and sulfate (Janninet al. 2013). Transcriptomic analysis indicated that aplasmid division regulator was responsible for anincrease of chloroplast number and supported thefindings of Okazaki et al. (2009) that cytokinin-likeactivity induced plants to enhance the rate of chloroplastdivision. However, increased cell chloroplast numberand starch content did not increase the net rate ofphotosynthesis (Jannin et al. 2013).

The use of balanced nutrient solutions in the above studiesdid not allow the investigation of seaweed extract effects onspecific nutrient deficiencies. Beckett and van Staden (1989)reported beneficial seaweed extract effects on K-stressedwheat, and Papenfus et al. (2013) detected improved growthof okra seedlings under K or P, but not under N, deficiency.Nitrogen, along with water, is a major limiting and economicfactor in crop production that is also associated with environ-mental degradation resulting from excessive or untimely fer-tilizer application. Draught stress and N deficiency areconstraining winter wheat production and yield stability underrainfed conditions worldwide (Shangguan et al. 2000). Morefrequent episodes of severe drought are likely to occur withclimate change in the coming decades, with potentially dev-astating impact on the world’s ability to feed a growingpopulation (Nature 2013). We therefore need crop productionsystems that make more efficient use of water and nitrogen.This study was conducted in order to investigate the effect ofAZAL5, an A. nodosum seaweed extract, on the nutrientuptake and yield of winter wheat under two levels of watersupply and fertilizer N. Wheat was grown in the greenhousefor 190 days under P and K sufficiency in a common mineralsoil of the Thessaly Plain which is an intensively managed

agricultural region of Central Greece with increasingly limitedavailability of irrigation water.

Materials and methods

The greenhouse experiment was established at the NationalAgricultural Research Foundation (Institute of Soil Mappingand Classification) located in the city of Larissa, CentralGreece, in the winter and spring of 2012–2013. Fresh algae(Ascophyllum nodosum (L.) Le Jol.) were harvested on theshore of Brehat Island, France, in October 2012; they werewashed, shredded, and added to water. The solution wasacidified with concentrated 95 % sulfuric acid to pH 3. Themixture was homogenized to microrupture the algal cells andthen centrifuged and filtered. The final experimental solutionwas then concentrated as described by Jannin et al. (2013) topH 10 and provided by TIMAC AGRO International(Roullier Group, Dinard, France) under the code name“AZAL5.” The amount of biologically active extracted com-pounds varies with season and also with environmental con-ditions. Except for C, H, and O, which are the main compo-nents, AZAL5 extract was found to contain Ca, K, Mg, Na, S(Jannin et al. 2013), and small amounts of auxin (IAA),abscisic acid, and cytokinins (Table 1). While the concentra-tion of cytokinins (iP and iPR) is similar to that of otherseaweed commercial products, AZAL5 differs by containinga higher concentration of IAA and ABA. The concentratedsolution was diluted to 1.5 and 3 % by volume and wasadjusted to pH 7.5 by titration with diluted formic acid priorto application.

The surface soil collected from the area was aTypicXerorthent soil (USDA 1985) with a bulk density of1.13 g cm−3 and field water holding capacity of 0.70 m3 m−3.Soil texture and chemical properties are presented in Table 2.The soil was air dried, screened to pass a 2-mm sieve, and2,230 g (dry weight equivalent) were placed in plastic-linedcontainers (17-cm height×18-cm top diameter×11-cm bot-tom diameter) with four holes of 1-cm diameter at the bottom.Soils were wetted with deionized water to 75 % of fieldcapacity, and 20 seeds of winter wheat Triticum durum var.simetowere planted in each container on November 23, 2012.Seedlings were thinned to eight per container 20 days afterplanting. Twelve containers were randomly assigned within

Table 1 Concentration of main phytohormones contained in AZAL5extract

Component Concentration (pmol g−1 dry wt.)

N6-isopentenyladenine (iP) 26.9

N6-isopentenyladenosine (iPR) 31.5

Indoleacetic acid (IAA) 1,832.0

Abscisic acid (ABA) 139.4

J Appl Phycol


each of four blocks to designate 12 treatment combinations ina randomized complete block design with a 2×3×2 factorialarrangement composed of two rates of inorganic fertilizer (0and 50 ppm N), three rates of AZAL5 (0, 1.5, and 3 % dilutedextract), and two levels of water supply (75 and 45 % of fieldcapacity equivalent to 46 and 28 % gravimetric watercontent).

In the N fertilizer treatments, inorganic fertilizer was ap-plied in the form of ammonium nitrate and P2O5 (625 mg of20-10-0 and 27.5 mg of 0-46-0) that equate to 50 ppm N and16 ppm P per container. In the treatments without N fertilizer,

70 mg of 0-46-0 was applied to equate the 16 ppm P percontainer. The amount of P represents the conventional rate ofP applied in the area (Tsadilas et al. 1996). Potassium fertilizerwas not applied because the exchangeable K of the soil (1.2cmol kg−1, Table 2) was sufficient for wheat growth. AZAL5dilutions were sprayed on the leaves until the first drops ofdiluted extract fell in the pot soil. Spraying of the foliage wasperformed three times within the growing season at 6, 8, and18 weeks after planting which corresponded to the growthstages of 3–4 unfolded leaves (Zadok scale 14–15), mainshoot with 3–4 tillers (Zadok scale 23–24), and early bootstage (Zadok scale 38–41), respectively. The AZAL5 concen-trations chosen reflect those commercially applied in the areafor similar products. During the experimental period, thecontainers were watered two times a week with deionizedwater to account for water evaporation losses and air temper-ature was monitored with a digital thermometer.

Plant growth was monitored by measuring plant height(height of two tallest leaves) six times during the growingseason. The aboveground biomass was harvested 190 daysafter planting, and shoots (stems + leaves) and ears wereseparated for each pot. The number of ears and grains perear was counted before the plant fractions were dried at65 °C for 2 days. Shoots, ears, and grains were weighedbefore shoots, and grains were ground to a powder with astainless steel mill. Ground samples were analyzed fortotal nitrogen and carbon content, as well as isotopiccomposition (δ15N and δ13C), with an automated combus-tion elemental analyzer interfaced with a continuous-flowisotope ratio mass spectrometer (PDZ Europa, UK).Samples were prepared as described by Schepers et al.(1989). The isotopic signature of the leaves providedinformation of plant stress relative to water shortage(δ13C) and fertilizer N uptake (δ15N). Carbon isotopediscrimination (Δ, ‰) of shoots and grains was calculated

Table 2 Soil texture and chemical properties

Soil property Value

Sand (%) 30

Clay (%) 44

Silt (%) 26

pH 1:1 7.7

EC 1:1 (μS cm−1) 455

Equivalent CaCO3 (%) 0.9

Soil organic matter (%) 1.1

P Olsen (mg kg−1) 8.2

Exchangable Κ (cmol+kg−1) 1.2

Exchangable Mg (cmol+kg−1) 9.0

Cation exchange capacity (cmol+kg−1) 26.6

Extractable Fe (DTPA) (mg kg−1) 4.4

Extractable Cu (DTPA) (mg kg−1) 1.3

Extractable Zn (DTPA) (mg kg−1) 0.86

Extractable Mn (DTPA) (mg kg−1) 6.3

Extractable B (HwsB) (mg kg−1) 0.8

NO3-N (mg kg−1) 13

DTPA diethylene triamine pentaacetic acid

Table 3 Analysis of variance of wheat growth and yield at harvest

Effect F values

Height (cm) Shoot (g plant−1) Ears (g plant−1) Grains (g ear−1) Grains (no. ear−1) Grains (g grain−1)

Block 1.3 5.9* 1.4 1.3 0.9 0.2

Nitrogen (N) 199.6** 421.4** 63.1** 58.7** 34.0** 0.5

Water (W) 12.2* 30.0** 7.6* 11.1* 8.3* 0.0

N×W 2.2 1.0 0.3 0.9 0.0 2.4

Extract (E) 0.2 0.3 0.4 0.8 0.3 0.0

N×E 0.1 0.2 0.7 0.6 0.5 2.6

W×E 3.5* 0.8 3.6* 3.9* 2.3 0.5

N×W×E 2.3 1.5 2.4 2.8 3.1 1.2

*Significant effect at the 0.05 probability level according to LSD

**Significant effect at the 0.0001 probability level according to LSD

J Appl Phycol


from their δ13C values by the following formula (Farquharet al. 1989) where air δ13C was taken as −8.0 ‰:

Δ ¼ δ13C air‐δ13C plant

1þ δ13C plant

K and P concentrations were determined with a flamephotometer and phasmatophotometer, respectively, afterground samples were heated at 500 °C for 5 h and ash wasdigested with 1 N HCl (Benton Jones J.Jr et al. 1991).

The main effects (irrigation, fertilizer, and AZAL5) andtheir interactions were analyzed by a general linear model fora randomized complete block factorial design using theStatistical Analysis System software, version 9 (SASInstitute, Cary, NC). The LSD test was used to detect differ-ences between means at P<0.05. Standard errors were com-puted by root mean square errors. The slice option was used totest for differences between AZAL5 concentrations withineach fertilizer × irrigation combination.

Results and discussion

Wheat growth and yield

Effects of nitrogen and water supply Analysis of varianceindicated two significant main effects on wheat growth and

grain yield at harvest, those of N fertilization and water supply(Table 3). The effects of N fertilization were greatest on shootbiomass, grain yield, and grain number with an 84, 75, and69 % average increase compared to unfertilized treatments,respectively (Table 4). In addition, plants of the fertilizedtreatments had a greater number of tillers at 83 days afterplanting (2.2 vs 1.4 tillers in the unfertilized treatments) andhad greater development as per Zadok scale at 131 days afterplanting. The great plant response to fertilizer N and the lowsoil organic matter content (1.1 %, Table 1) indicated that themineralization potential of this soil was far below the Nrequirement of the crop. Water supply had greatest effects ongrain number and weight with 31 and 29 % increase underoptimal water supply relative to reduced supply, respectively(Table 4). The effects of water supply were less pronouncedthan those of fertilization because of the possibly mild waterstress imposed by the high water holding capacity of this soil.The weight of grains was not affected by any of the factorsinvestigated (Table 2), and yield increases were associatedwith greater number of grains per ear.

Seaweed extract effects As a main effect, AZAL5 concentra-tion was insignificant on growth and productive parameters ofwheat, but a significant irrigation × seaweed extract interac-tion emerged (Table 3). Investigation of this interaction effectrevealed that differences between AZAL5 concentrations forear weight, grain weight, and grain number were significantwithin each fertilizer-irrigation combination only when Nfertilizer was added (Table 5). In fertilized treatments of

Table 4 Means of wheat growth and yield for the four fertilizer-irrigation combinations at harvest (n=12)

Fert N (ppm) WFC (%) Height (cm) Shoot (g pot−1) Ears (g pot−1) Grains (g pot−1) Grains (no. pot−1) Grains (g grain−1)

0 45 63.3 c 6.1 d 4.8 c 2.7 c 72 c 0.039

0 75 64.9 c 7.4 c 5.7 c 3.5 c 101 bc 0.036

50 45 73.8 b 11.5 b 7.7 b 4.8 b 130 b 0.037

50 75 78.0 a 13.2 a 9.0 a 6.1 a 160 a 0.040

Means followed by the same letter (or no letter) are not significantly different at the 0.05 probability level according to LSD. Height indicates meanheight of two tallest plants per pot, and shoot indicates stem + leaves

Fert N fertilizer N added,WFC water field capacity

Table 5 Testing for AZAL5 concentration effects on wheat productive parameters by slicing within each fertilizer × water supply combination

Fert N (ppm) WFC (%) F values

Height (cm) Shoot (g pot−1) Ears (g pot−1) Grains (g pot−1) Grains (no. pot−1) Grains (g grain−1)

0 45 0.54 0.41 0.01 0.01 0.07 0.34

0 75 0.25 0.16 0.28 0.12 0.13 0.87

50 45 2.43 0.75 6.08* 2.62 1.52 0.00

50 75 2.89 1.19 6.15* 5.30* 4.53* 2.99


*Significant effect at the 0.05 probability level according to LSD

J Appl Phycol


75 % water field capacity (WFC), only the 1.5 % AZAL5concentration increased wheat yield, but a higher concentra-tion of 3 % was apparently required under reduced watersupply (45 % WFC) to show similar stimulatory effects(Fig. 1). In both cases, there was a significant 25 %increase of grain yield in comparison to that attained bythe respective 0 % extract controls. The positive effects ofseaweed extracts on crop growth have been attributed toplant growth regulators, and possibly micronutrients, thatstimulate root growth, mineral uptake, photosynthetic

capacity, and stress tolerance as summarized from thebody of literature by Khan et al. (2009). The mode ofaction in wheat and barley is thought to implicate cytoki-nins or cytokinin-like molecules that regulate the selectivesurvival of dominant tillers and spikelets through mobili-zation of nutrients. Thus, if the cytokinin supply to thereproductive organs is augmented either through an im-proved nutritional status or by feeding exogenous cytoki-nins, more and/or larger nutrient sinks could result(Featonby-Smith and van Staden 1987).

N fertilizer applied

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Fig. 1 Treatment effects on ear, grain and shoot (stem + leaf) weight. Means followed by the same letter (or no letter) are not significantly different at the0.05 probability level within each fertilizer-water supply combination. Bars denote standard errors

J Appl Phycol


The fact that increased yield appeared only under N fertiliza-tion (Fig. 1) indicated that foliar AZAL5 applications stimu-lated the plants to utilize soil mineral N and possibly otheravailable nutrients more efficiently. We are unaware of sea-weed extract studies focusing on wheat N nutrition, and mostprevious studies used some kind of nutrient solution withouttesting for specific nutrient effects. Of those, some at leastagree with our results in that seaweed extracts can enhance theeffectiveness of conventional fertilizers because extracts, ap-plied as foliar spray or soil drench, increased yields underadequate nutrient supply of barley and wheat (Mooney andvan Staden 1985; Featonby-Smith and van Staden 1987).Similar stimulatory effects under adequate nutrient supplyare reported for other crops such as rye (Kotze and Joubert1980), beans (Featonby-Smith and van Staden 1984; Beckettet al. 1994), cucumber (Nelson and van Staden 1984), andsoybeans (Rathore et al. 2009). The latter author obtained anear-linear positive response of soybean height and yield withincreasing foliar application of seaweed extract in a fieldexperiment where farm yard manure was applied underrainfed conditions in the humid subtropics. Other studies,however, seem to contradict our results because Kelpak (acommercial South African kelp concentrate) had no signifi-cant effect on yield when wheat received an adequate supplyof nutrients. Instead, foliar application increased yield only innutrient-stressed and K-stressed plants (Beckett and vanStaden 1989, 1990) or even in the absence of fertilizer whenseaweed extract was applied as a soil drench (Nelson and vanStaden 1986). In part, the findings of Papenfus et al. (2013)appear to agree with those of Beckett and van Staden (1989) inthat shoot and root biomass increased under K or P deficiencywhen okra seedlings were grown for 8 weeks in a Kelpak-containing nutrient solution. In contrast, Kelpak did not have

Fertilizer N under optimal water sypply

40 60 80 100 120 140 160 180 200

Pla

nt h

eigh

t (cm

)

20

30

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0% extract

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Early bootstage

Fertilizer N under reduced water supply

Days after planting

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0% extract

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3% extract

Early bootstage

Fig. 2 Monitoring of plant height during the growing season for theseaweed extract treatments receiving N fertilizer. Vertical grey arrowsindicate the timing of foliar applications

Table 6 Effect of fertilizer N and water supply on wheat nutrient content

Fert N (ppm) WFC (%) Concentration (%) Content (mg plant−1)

N P K N P K

Grain

0 45 2.04 a 0.29 0.42 b 6.91 c 0.98 c 1.45 c

0 75 1.84 ab 0.32 0.43 b 8.04 bc 1.36 bc 1.91 c

50 45 1.71 b 0.27 0.43 b 10.16 b 1.55 ab 2.57 b

50 75 1.74 b 0.27 0.50 a 13.71 a 2.07 a 3.85 a

Shoot

0 45 0.27 ab 0.12 b 1.21 2.05 c 0.94 b 9.24 c

0 75 0.30 a 0.16 a 1.23 2.73 b 1.48 a 11.25 c

50 45 0.28 a 0.04 c 1.17 3.98 a 0.61 c 16.79 b

50 75 0.21 b 0.06 c 1.18 3.50 a 0.96 b 19.58 a

Within columns, means followed by the same letter (or no letter) are not significantly different at the 0.05 probability level according to LSD


J Appl Phycol


any effect on seedling biomass under N deficiency whichagrees with the results of our study.

The differences in the efficacy of the two AZAL5 seaweedconcentrates indicated that optimal dilution ratios were direct-ly or indirectly dependent on soil water content. It is notewor-thy that the application of 3 % AZAL5 increased grain yieldunder reduced water supply to levels equal to those attained byoptimal water supply (Fig. 1). Mooney and van Staden (1985)obtained a significant yield increase when a relatively highseaweed extract concentration (1:250 dilution) was applied towheat that was supplied with a nutrient solution and subjectedto water stress for 14 days during the flowering stage. Lowerextract concentrations did not have an effect on yield underthese conditions of water stress. The lower extract concentra-tion of 1.5 % was similarly ineffective under lower watersupply in our study (Fig. 1). The efficacy of foliar applicationunder different soil moisture regimes is also influenced by thetype of seaweed product as was found to be the case withbeans grown in dry field capacity and wet sandy loam soilfertilized with N and P (Temple et al. 1989). In the more recentliterature, enhanced plant performance under conditions ofwater stress has been attributed to stress tolerance as inducedby bioactive chemicals contained in the seaweed extracts.Cytokinin activity mitigates stress-induced free radicals thatmay explain drought tolerance in creeping bent grass (Zhangand Ervin 2004) and increased antioxidant enzyme activity ofturf grasses (Fike et al. 2001).

Monitoring of plant height during the growing season(Fig. 2) indicated that the two early foliar applications didnot have a direct effect on wheat growth. This is because plantheight of the fertilized AZAL5 treatments was similar to orlower than the control treatment during 80 days followingapplication. Plant height increased at least 2 weeks after thethird foliar application at the early boot stage under both levelsof water supply (Fig. 2). At those times, the plants hadprogressed to the heading and flowering stage. The apparentlate onset of stimulatory effects may explain the lack ofdifferences in shoot biomass between fertilized treatmentswith or without seaweed application (Table 5, Fig. 1).Previous studies on wheat and barley have predominantlyfocused on Kelpak applications with mixed results. Unlikeour results, they showed that early seaweed application in-creased vegetative growth or grain yield either under fertili-zation (Featonby-Smith and van Staden 1987) or withoutadded fertilizer (Nelson and van Staden 1986). Others obtain-ed no effects when wheat was provided with an adequatesupply of nutrients, but yield increased only in nutrient-stressed plants with early seaweed application (Beckett andvan Staden 1989, 1990).

The apparent disagreement of previous studies on the ef-fects of seaweed extracts on wheat growth and yield undervarying conditions of nutrient and water supply demonstratesthe complex interactions between a multitude of factors, and

caution should be given to direct comparisons. Such factorsare the different seaweed products tested, their method ofextraction and the resulting dilution ratios, the crop-specificmode of action, the timing and type of application, the

Amount of N (mg ear-1)

0 5 10 15 20 25 30

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y=0.15 + 0.04xR2=0.71

y=0.10 + 0.18xR2=0.93

Fig. 3 The relationship of grain nutrients to grain yield

J Appl Phycol


different nutrient media used versus potting soils of oftenunknown residual nutrients, and mineralization potential.

Elemental composition and nutrient uptake

Effects of nitrogen and water supply The effects of fertilizer Nand water supply on the concentration of grain and shootnutrients were variable (Table 6). Noteworthy were the increaseof grain K concentration in the fertilizer N treatments of 75 %WHC and the threefold decrease of shoot P concentration withN application. However, the total amounts of grain N, P, and Kincreased in the fertilizer and 75 % WFC treatments (Table 6)due to their large yield response effects (Table 4). Among thethree nutrients, N and K had the greatest uptake increase in thegrain and shoots (Table 6, Fig. 5). Shoot P was the exceptionwith higher content in the 75 % WFC treatments without Napplication. K uptake alone explained 93 % of the total varia-tion of grain yield. The actual strength of the relationships ofgrain K, N, and P contents with grain yield is shown in Fig. 3.

ANOVA indicated that water supply strongly affected grainand shoot Δ13C (P<0.001). In the N-deficient treatments,reduced water supply decreased grain Δ13C by 2.4 ‰

(Fig. 4) and provided evidence of reduced transpiration andgreater water-use efficiency (WUE) which is a sign of greaterwater stress (Farquhar et al. 1989). Many studies have report-ed the effect of water stress on WUE and Δ13C. Under waterstress, stomatal conductance probably becomes the majorfactor driving carbon assimilation and discrimination andmay lead to a positive correlation between grain Δ13C andyield (Monneveux et al. 2006; Zhu et al. 2009; Wang et al.2013). Shoot Δ13C, mainly composed of mature stems, wasless sensitive to water supply in the N-deficient treatments(Fig. 4). The higher Δ13C values of the shoots relative to thegrains (Fig. 4) are in agreement with the literature (Shaheenand Hood-Nowotny 2005; Zhu et al. 2009).

Nitrogen application decreased shoot Δ13C but had noapparent effect on grain Δ13C because of a significant cross-over interaction between N and water supply (P<0.01). Infact, N application decreased grainΔ13C under optimal watersupply but increased grain Δ13C under reduced water supply(Fig. 4, open bars). In the scientific literature, the reports onhow the amount of N fertilization, or its interaction with watersupply, affects wheatΔ13C are contradictory. Negative effectsof N supply onΔ13C have been obtained by Cabrera-Bosquet

No fertilizer

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Δ g

rain

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a a

b

Fig. 4 Water supply x AZAL5 interaction effects on grain and shoot Δ13C (‰). Means followed by the same letter (or no letter) are not significantlydifferent at the 0.05 probability level within each fertilizer-water supply combination. Bars denote standard errors

J Appl Phycol


et al. (2009), Wang et al. (2013), and Clay et al. (2001). Thelatter detected these effects only under water stress and ap-pears to contradict our findings. Cabrera-Bosquet et al. (2009)explained the decreasedΔ13C in plants with high N supply bya reduction in stomatal conductance that decreased both netCO2 assimilation rate andΔ13C. Others contradict these find-ings in that N supply increased grain or leafΔ13C (Shangguanet al. 2000; Dalal et al. 2013.) The effects of N application onΔ13C of plant organs remain unclear.

Seaweed extract effects As in the case of grain yield, signif-icant water supply×AZAL5 interaction effects on grain

nutrients andΔ13C occurred only with fertilizer N application(Figs. 4 and 5). The lack of such effects on shoot Δ13C andnutrients (Figs. 4 and 6) points to earlier research that thereproductive organs of wheat are the main site of biostimulantactivity (Featonby-Smith and van Staden 1987). The stem isassimilating C at the early stages of growth, and stem C issubjected to isotopic fractionation when partitioning fromstem to grain at later growth stages (Zhu et al. 2009). Foliarapplication of 1.5 % AZAL5 increased the uptake of grain Kunder optimal water supply (Fig. 5). Awell-balanced K nutri-tion is required to attain optimum wheat yields, and this wasevident with our data (Fig. 3). An increased N uptake by 3 %

No fertilizer

45 75

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in K

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)

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0% extract1.5% extract3% extract a

b b

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aa

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in P

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Fig. 5 Treatment effects on grain nutrient content. Means followed by the same letter (or no letter) are not significantly different at the 0.05 probabilitylevel within each fertilizer-water supply combination. Bars denote standard errors

J Appl Phycol


AZAL5 concentration under reduced water supply was notstatistically significant (Fig. 5). Beckett and van Staden (1989)reported increased uptake of nutrients (K and N) and increasedwheat yield under K deficiency when roots were flushed with0.25%Kelpak solution. However, Kelpak was not effective inincreasing yield when wheat was supplied with adequatenutrients and contradicts our results in this respect. In addition,our foliar applications were not effective in increasing wheatyield and nutrient uptake under conditions of N deficiency(Figs. 1 and 5). Caution should be given to direct comparisons

due to the differences between our experiments in terms ofpotting media, extract products, i.e., greater amounts of auxinsand abscisic acid in AZAL5, and their mode of application.

The interactive effect of AZAL5 and water supply on grainΔ13C in the fertilized N treatments is as difficult to explain asthat of N and water supply. Only the high extract concentra-tion (3 %) caused changes in grain Δ13C which decreasedunder reduced water supply and increased under optimalwater supply (Fig. 4). The decrease of grain Δ13C in theformer would suggest that, under conditions of water stress,

No fertilizer

45 75

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Fig. 6 Treatment effects on shoot nutrient content. Bars indicate standard errors.

J Appl Phycol


AZAL5 induced stress tolerance by reducing stomatal con-ductance and transpiration thereby increasing WUE.However, it is unclear how such a mechanism explains thehigh grain yield attained by this treatment (Fig. 1). An alter-native hypothesis is thatΔ13C decreased by greater photosyn-thetic capacity or improved carboxylation efficiency inducedby bioactive chemicals contained in the extract, thereby stim-ulating ear growth and grain yield. AZAL5 or cytokinin-likecompounds were recently shown to enhance chloroplast ac-tivity (Okazaki et al. 2009; Jannin et al. 2013). The increase ofgrain Δ13C in the 3 % AZAL5 (Fig. 4) under optimal watersupply was not associated with an increase in grain yield butwith a reduced N uptake (Fig. 5).

When a crop like wheat exhibits a strong preference fornitrate, the isotopic signal of applied fertilizer N may becomedistinct enough to reveal its contribution in the plant tissue(Shearer and Legg 1975). Such an approach has been used tocompare fertilizer and soil N contribution to crop N uptake indifferent management practices and across years (Bort et al.1998; Dalal et al. 2013 and references therein). Interpretationof crop δ15N data for fertilizer N uptake in such studies ofnatural abundance assumes the similarity of other N transfor-mation processes and a large difference between the isotopicsignal of applied fertilizer (near zero δ15N) and soil (δ15N 8 to16). Unfortunately, this was not the case in our study as thelow grain δ15N values of the unfertilized treatments (meanδ15N=3.15, data not shown) implied a relatively low soilisotopic signal and constrained further interpretations.

Conclusions

While fertilizer N was the most important factor in determin-ing wheat yield, yield increases caused by AZAL5 applicationwere of the order of 25 % and comparable to those resultingfrom optimal water supply (29 % increase relative to reducedwater supply). AZAL5 stimulated wheat nutrient uptake andincreased yield only under N fertilization. AZAL5 effectswere insignificant under N deficiency despite the adequate Pand K content of the soil. These effects underline the potentialof this product to increase nutrient uptake efficiency of inten-sively managed conventional cropping systems with strongincentives to maximize yields and reduce environmental con-tamination. The effect of extract application on crop perfor-mance under suboptimal levels of N and P fertilization shouldbe explored in order to evaluate the fertilizer replacementvalue of this product. AZAL5 also increased wheat yieldunder conditions of water stress and signifies its importancein promoting crop growth in semiarid Mediterranean climateswhere wheat crops are often subject to drought. The fact that agreater AZAL5 concentration was required to increase grainyield in reduced soil moisture conditions needs further

investigation in order to determine the underlying cause andoptimal dilution ratios. Contrary to the findings of previousstudies, early foliar application of this product appeared tohave no direct effect. The lack of biomass, nutrient content,andΔ13C differences between AZAL5 and control treatmentsin the shoot indicated that the reproductive organs of wheatwere the main site of biostimulant activity. Further research isneeded in order to verify AZAL5 effects under field condi-tions and their association to physiological plant responsessuch as rate of photosynthesis, chloroplast number, and starchcontent.

Acknowledgments We would like to thank TIMAC AGRO Interna-tional (Roullier Group, Dinard, France) for donating the AZAL5 productand supporting this research project, Eleftheria Tsadila for performing thestable isotope analysis of the plant samples, the laboratory staff of theInstitute of Soil Mapping and Classification for soil-plant analyses, andDr. Dimitris Taskos for a critical review of the manuscript.

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responses of winter wheat to ascophyllum nodosum (l.) le jol

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