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Scientia Horticulturae 125 (2010) 263–269

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

novel type of seaweed extract as a natural alternative to these of iron chelates in strawberry production

rancesco Spinelli ∗, Giovanni Fiori, Massimo Noferini, Mattia Sprocatti, Guglielmo Costaipartimento di Colture Arboree, Alma Mater Studiorum, Università di Bologna, Italy

r t i c l e i n f o

rticle history:eceived 30 July 2009eceived in revised form 28 January 2010ccepted 31 March 2010

eywords:lant biostimulantoil amendmentustainable horticulturehizosphere

a b s t r a c t

The new generation of seaweed extracts, such as Actiwave®, may represent a promising strategy toreduce the use of phytochemicals in agriculture. Actiwave® is a metabolic enhancer derived by the algaeAscophillum nodosum, but differently from some older seaweed extracts, it has a constant and balancedformulation containing kahydrin, alginic acid and betaines which synergistically contribute to the efficacyof the product. Actiwave® has been proposed to increase the mineral nutrient uptake and the abioticstress tolerance. The aim of this work was to evaluate, under a multidisciplinary approach, the effectof the biostimulant on the vegetative and productive performances of strawberry plants grown on alime inducing iron chlorosis substrate. This biostimulant increased the vegetative growth (10%), the leafchlorophyll content (11%), the stomata density (6.5%), the photosynthetic rate and the fruit production

(27%) and berry weight. The most significant result was the increase of the plant biomass: the shoot drymatter was increased up to 27% and root dry matter up to 76%. Finally, preliminary experiments showedthat Actiwave® positively influenced also the root-associated microbial biocoenosis. These results arediscussed in relation to the physiological and ecological mechanisms proposed to explain the beneficialeffects of this seaweed extract. Finally, the effects of Actiwave® and sequestrene were significantly similar,thus showing that this biostimulant may represent an environmental-friendly substitute of the iron chelates.

. Introduction

Consumers and society increasingly value the production ofigh quality, healthy fruit and vegetables that, at the same time,nsures a minimal or non-adverse impact on the environment.hese aspects are especially important on those horticultural crops,uch as strawberry, which require highly specialized knowledgend high external inputs (Tagliavini et al., 1996, 2005). In par-icular, soil management, irrigation and fertilizations are all vitalnterventions to obtain profitable strawberry productions, thoughn excess of nutrients transported to the drainage area results innvironmental problems such as eutrophication in rivers, lakesnd sea (Neeteson and Carton, 2001). In addition, these activitiesay also negatively influence the root biocoenosis by reducing

eneficial microorganisms and mycorrhizal fungi (Plencette et al.,

005). Due to the numerous concerns about a massive and indis-riminate irrigation and fertilization, the present tendency is toeduce significantly their use in horticulture (Van Noordwijk andadisch, 2002). Actiwave® (Valagro SpA, Piazzano di Atessa, and

∗ Corresponding author. Tel.: +39 0512096447; fax: +39 0512096401.E-mail addresses: francesco.spinelli3@unibo.it, spinelli@agrsci.unibo.it

F. Spinelli).

304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2010.03.011

© 2010 Elsevier B.V. All rights reserved.

CH – Italy) is a promising biostimulant which may contribute tothis objective. Biostimulants are environmental friendly, naturalsubstances able to promote vegetative growth, mineral nutri-ent uptake, plant fitness to different pedoclimatic conditions andtolerance to abiotic stresses (Vernieri et al., 2006). In particu-lar, Actiwave® is able to enhance the nutrient uptake by rootsand the plant tolerance to drought and saline stress (Spinelli etal., 2006). Actiwave® is a seaweed extract derived by the brownalgae Ascophillum nodosum. Seaweeds are a known source ofplant growth regulators (Jameson, 1993), organic osmolites (e.g.betaines), aminoacids, mineral nutrients, vitamin and vitamin pre-cursors (Berlyn and Russo, 1990; Blunden et al., 1985). In particular,Actiwave® contains kahydrin, alginic acid and betaines which syn-ergistically contribute to the efficacy of the formulation (Vernieriet al., 2006).

Kahydrin is a derivate of the K vitamin. The exogenous applica-tion of K vitamin induces the secretion of H+ in the apoplast andthe consequent acidification of the rizosphere (Lüthje and Böttger,1995). The lower pH in the soil enhances the reduction of Fe(III) to

the soluble Fe(II) which can be uptaken by plant roots (Chaney etal., 1972). In addition, kahydrin has been proposed to interfere withthe tricarboxylic acid cycle, thus promoting the formation of longchain molecules such as proteins and polysaccharides (Sportelli,2005).

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64 F. Spinelli et al. / Scientia H

On the other hand, alginic acid acts as soil conditioning agent:t combines with metallic radicals to form cross-linked polymers

hich increase the water-holding characteristics of the rhizosphereVerkleij, 1992; Lattner et al., 2003). The increase of moisture in thehizosphere contributes to create an environment more suitable forhe growth of roots and root-associated beneficial microorganismsChen et al., 2003).

Finally, betaines are compatible solutes that act as omoprotec-ants, thus enhancing the plant resistance to drought and salinitytress (Huang et al., 2000). Betaines also present a cytokinin-likectivity (Blunden and Wildgoose, 1977) and their exogenous appli-ation increases the chlorophyll content in leaves (Whapham etl., 1993; Blunden et al., 1996) and the growth of shoots and rootsBlunden et al., 1996; Vernieri et al., 2006).

Actiwave® has been applied on many crop plants and, as far asruit trees, on apple, pear and grape (Spinelli et al., 2006). Due tohe partially unknown mode of action, the complex formulationnd the high variability among the environmental conditions, theffect of the Actiwave® in open field experiments was inconstantnd erratic (Vernieri et al., 2006; Spinelli et al., 2006).

On apple, the application of Actiwave®, in combination with atandard fertilization, increased the shoot growth, the leaf chloro-hyll content, the plant productiveness and the fruit sugar content.owever, the application of Actiwave® alone on nutrient depletedlants did not positively affected any of the mentioned vegetativend reproductive parameters (Spinelli et al., 2006). In addition, onpple trees showing a biennial bearing behaviour, Actiwave®, byeducing the oscillation between the “on” and “off” years, mod-rated the negative effects of this productive disorder (Spinelli etl., 2009). On pear, Actiwave® increased the yield and the averageruit weight, but nor the chlorophyll content, neither the qualita-ive parameters of fruits (Spinelli et al., 2006). Finally, on grape (cv.angiovese), Actiwave® increased the leaf chlorophyll concentra-ion more than the traditional iron chelate FeEDDHA (Spinelli et al.,006).

In the present research, experiments have been performed tonvestigate the effects of Actiwave® on the main vegetative androductive parameters of strawberry plants grown in iron defi-iency conditions. Strawberry was chosen as a model crop for berrylants because the whole biological cycle, from sprouting to fruitipening, can be followed under highly controlled conditions, thusinimizing the influence of the environmental variations. In addi-

ion, strawberry has been widely studied and the plants are easilyvailable (Walter et al., 2008). Iron deficiency has been observed inany crops when grown in high pH calcareous soils (Miller et al.,

984). In these soils, the iron deficiency is the most important abi-tic stress limiting the strawberry production (Zaiter et al., 1993;ieten, 2000; Kafkas et al., 2007). In fact, iron deficiency results inxtensive fruit abortion with a consequent reduction of yield andruits weight (Lieten and Baets, 1991; Zaiter et al., 1993; Almaliotist al., 2002). In addition, also strawberry fruit quality is directlyependent on iron (Karp et al., 2002; Erdal et al., 2004).

Many studies have been conducted to determine the mostppropriate strategy to overcome the iron deficiency chlorosis intrawberry (Erdal et al., 2006; King et al., 1950; Zaiter et al., 1993;üremis et al., 1997). Nowadays, the use of iron chelates and theoliar sprays are the most widely accepted methods for correctinghis micronutrient problem (Lucena et al., 1990; Zaiter et al., 1993;badia et al., 2002). In our experiments, the adaptability to ironeficiency conditions was evaluated by monitoring other parame-er than the mere iron concentration in leaves. In fact, the chlorosis

ssociated with iron deficiency is not usually a direct consequencef an absolute lack of this element as in the case of other micronu-rient elements, but rather a secondary effect resulting from theomplex interactions of Fe with other elements and various soil andnvironmental factors (Erdal et al., 2004). For example, in experi-

turae 125 (2010) 263–269

ments conducted under uncontrolled conditions in calcareous soils,it was found that the concentration of Fe in chlorotic leaves is sim-ilar to or even higher than that in green leaves (Erdal et al., 2004).

For the mentioned reasons, the effects of Actiwave® andsequestrene were evaluated primarily on vegetative growth, leafchlorophyll content, stomata density, yield and fruit weight. Inaddition, since in several plant species grown in iron deficient con-ditions, photosynthetic rates were depressed, these parameterswere monitored on entire plants (Larbi et al., 2006).

Finally, considering that rhizobacteria, such as Pseudomonas flu-orescent, Bacillus sp. and streptomycetes, plays a crucial role indetermining the plant fitness to the environment (De Weger et al.,1995; Gerhardson, 2002; Vessey, 2003), the effect of Actiwave® onthe root-associated microbial biocoenosis was also studied.

2. Materials and methods

2.1. Plant material, experimental treatments and growingconditions

Experiments were performed on strawberry plants [Fragariaananassa (cv Queen Elisa)] grown in greenhouse under natural light(25 ◦C day/18 ◦C night). Queen Elisa is short day, early ripening,not re-blooming cultivar derived by the crossing between USB35(Lateglow × Seneca) and cv Miss [(Comet × Honeoye) × Dana]. Thecultivar has been developed for cultivation in temperate-cold cli-mates (Faedi and Baruzzi, 2004). Strawberry plants were planted ascold stored trayplants on a substrate obtained by mixing 1:1 (v/v)peat and sand. The peat mineral concentration declared by the man-ufacturer was: NH4

+ 25 g m−3, NO3− 35 g m−3, P2O5 104 g m−3, K2O

120 g m−3, MgO 12 g m−3, micronutrients 25 g m−3. Standard irriga-tion and fertilization was applied. As mineral fertilizer, Poly-Feed(16N-8P-32K) by Fertica S.A. was used (6 g plant−1 applied twotimes at BBCH 15, 41 – Stauss, 1994).

To verify the efficacy of Actiwave® to increase the strawberrytolerance to iron deficiency, 1 week before its application, the plantswere transplanted in a lime inducing iron chlorosis medium. Thismedium presented the following characteristics: total carbonate(CaCO3) 72.9%, active lime 12.0%, pH 8.3, texture fraction: 50% sand,23% silt and 27% clay. The micronutrient concentrations were: Fe7.8 ± 0.3 ppm, Cu 59.1 ± 1.0 ppm and Zn 5.9 ± 0.1 ppm.

Actiwave® was applied by watering plants with 10 ml of com-mercial product dissolved in 20 ml of tap water. Iron chelatesequestrene [sodium ferric ethylenediamine di (ohydroxypheny-lacetate) or NaFeEDDHA containing 6% Fe] and water treated plantswere used as controls. Sequestrene was applied by dissolving 1 g ofcommercial product (sequestrene 138 Fe – Syngenta S.p.A.) in 30 mlof tap water. The experiments were repeated twice.

2.2. Gas exchange measurements and stomata density

Fourteen days after each treatment, four plants, for each test,were transferred individually to a 12 l flow-through plastic cham-ber kept at room temperature under natural daylight conditions.A constant flow of filtered air (16 l min−1) was maintained insidethe chamber. The photon flux density in the photosyntheticallyactive wavelength range was measured using a Quantum PAR lightsensor (Spectrum Technologies Inc., Plainfield, IL, USA) and the airtemperature was measured using a copper constant thermocou-ples (RS components; Vimodrone, Milan, Italy). CO2 assimilation

was measured using an infra-red gas analyser EGM-4 (PPsystems,Boston, MA, USA) operating in a closed mode. Measurements werecarried out over eight consecutive days. Net photosynthesis wascalculated in relation to the total leaf area and biomass of theplant. To determine the biomass of the enclosed plants, the dry

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eight (DW) of individual leaves was determined after desiccation

n a oven for 48 h at 70 ◦C. Leaf areas were determined by scan-ing all the leaves and processing the obtained files by an imagenalysis programme (GIMP 2.6.6. – GNU Image Manipulation Pro-ram).

ig. 1. Photosynthesis (A, C and E) and transpiration (B,D and F) rates of strawberry plantsompared with water (solid) and sequestrene (dotted). The photosynthetically active phoor three consecutive days: September 14 (A and B), 15 (C and D) and 16 (E and F).

turae 125 (2010) 263–269 265

Also the stomata density was measured. For this purpose, two

fully expanded, mature leaves were sampled from each plant.Imprints of the abaxial side of the leaves were obtained by coatingthe leaf surface with clear cellulose acetate (nail polish). To cal-culate the stomata density, leaf samples (4 mm2) were collected

grown on an iron chlorosis inducing soil. The effect of Actiwave® (dashed line) waston flux density is also reported (grey line). The gas exchange rates were monitored

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after the transplant on iron chlorosis inducing soil, the leaves fromtreated and control plants were easily distinguishable by visualevaluation. The non-invasive evaluation of leaf chlorophyll contentconfirmed that Actiwave® significantly increase the concentrationof this photosynthetic pigment (Table 1). Nonetheless, the highest

Table 1Vegetative parameters of strawberry plants grown on iron chlorosis inducing soil.Average values in each column labelled with the same letter do not differ accordingto SNK test (P < 0.05).

Control Actiwave Sequestrene

Sprout length (mm) 25.7b 28.3a 27.5aSprout fresh weight (g) 7.3a 8.8a 7.7aSprout dry weight (g) 1.1b 1.4a 1.0bNumber of leaves per plant 10.3a 9.7a 10.2aAverage leaf area (cm2) 55.8a 67.3a 64.8aAverage leaf fresh weight (g) 17.9a 18a 18.2a

66 F. Spinelli et al. / Scientia H

etween the second and third veins from the stalk, halfwayetween the main nerve and the leaf edge. The number of stomataas counted under a light microscope at 250× magnification from

he imprints which were mounted on a glass slide a stained withwater solution of toluidine blue (1%, w/v). Stomata density was

xpressed as per 1 mm2 of leaf area.

.3. Vegetative and reproductive performances

Three weeks after treatment, on 12 plants per test, divided in 3epetitions, the following parameters were measured: number ofeaf per plant, average leaf area, chlorophyll content (SPAD), leafresh and dry matter, number of stolon per plant, average stolonength, stolon fresh and dry matter, shoot length, shoot fresh andry matter, root fresh and dry weight.

The average chlorophyll content in leaves was measured usingportable chlorophyll meter (SPAD-502, Minolta, Konica Minoltaoldings Inc., Tokyo, Japan).

At harvest, on a similar set of plants, the fruit production perlant, average fruit weight, soluble solids content (SSC), flesh firm-ess, superficial firmness, and titratable acidity of the fruits werevaluated. These determinations were obtained by destructivenalyses using a refractometer (Atago, Co., Ltd, Tokyo, Japan), a pen-trometer (Effe.gi, Ravenna, Italy), a durometer Shore A Durofel®

Copa–Technologie S.A. 13150 Tarascon, France) and a titrator (Cri-on Instruments, SA, Barcelona, Spain) performed according tostablished methodologies (Costa et al., 2003).

.4. Root-associated microbial biocoenosis

A qualitative analysis of the microbial biocoenosis of the rhizo-phere was performed. For the evaluation of the different culturableicroorganisms, an entire root was gently washed of the adher-

ng soil by using a sterile MgSO4 solution (10 mM). Successively,mg of root system (the proper root plus the remained adher-

ng soil) was aseptically placed in a sterile vial containing 10 mlf MgSO4 solution and vigorously shacked for 2 min on a vortexixer. Tenfold sequential dilutions of the supernatant were plated

n the appropriate agar medium. The total bacterial population wasvaluated on Luria Agar (LA), whereas the presence of putativelyeneficial bacteria (Pseudomonas spp., Bacillus subtilis and relatedpecies and Streptomyces spp.) was evaluated on different selec-ive and semi-selective media. The King B medium (KB) was usedor fluorescent Pseudomonas (King et al., 1954), the casein digest-

annitol agarose (CM) for Bacillus (Fall et al., 2004) and the STRedium for Streptomyces spp. (Conn et al., 1998). All the media were

mended with cycloheximide (50 mg l−1) to prevent fungal growth.lates were incubated at 22 ◦C till the bacterial colonies were easilyistinguishable. Finally, the isolated were identified by traditionalhenotypic methods (Reva et al., 2001, 2004). The whole procedureas repeated for three separate samples taken from each plant.

At the same time, also the soil pH was measured. For thisurpose, samples of bulk and root-adhering soil were collected,eighted and mixed in a vial with distilled water with a propor-

ion of 1:2.5 (w/w). The pH was measured in the liquid suspensionhus obtained (Lab pH-meter basic20 Crison-Instruments S.A.,arcelona, Spain). Soil pH was monitored during the first 96 h afterreatment.

.5. Statistical analysis

The statistical analysis was performed using Stat software (STA-ISTICA version 5.0 Statsoft, Inc. 1995, Tulsa, USA). The analysis ofariance and the Student Newman and Keuls test (SNK) of compar-son between means were performed using the ANOVA proceduref the Stat software at P = 0.05. In the graphs, sample variability is

turae 125 (2010) 263–269

given as the standard error (S.E.) of the means. As far as the dataregarding the gas exchange rates and the PAR, each of the valuesreported in the graphs results from the average of the 3 repetitions.

3. Results

3.1. Gas exchange measurements and stomata density

Under our experimental conditions, treatment with Actiwave®

consistently increased photosynthetic activity of strawberry plantsgrown on iron chlorosis inducing soil (Fig. 1A, C and E). In addi-tion, the effect Actiwave® was comparable with the iron chelatesequestrene. Only during the second day of the experiment, no sig-nificant effect of the treatments was observed. In that date, thephotosynthetic rates of all plants showed an unusual trend that dif-fered from what has been observed during the other days and fromwhat has previously been reported in literature for other plants(Sabatini et al., 2003).

The biostimulant also increased transpiration rates, but its effectwas less consistent than on photosynthetic activity (Fig. 1B, D andF). Moreover, no substantial differences among the different dayswere observed. As far as the sharp decrease of the transpiration rateof sequestrene-treated plants observed during the first day, it wasdue to a transient leaking of the experimental chamber (Fig. 1B).

In our experiments, the effect of the biostimulant on the photo-synthetic and transpiration does not seem increase over time.

As far as the leaf surface anatomy, the treatment with Actiwave®

induced an increase of the stomata density (Table 1). This effectwas consistent, but not statistically significant, and the highestincrease was observed in the plants treated with sequestrene. Inthese plants, in fact, the application of the iron chelate resulted ina 10% increase of the stomata density.

3.2. Vegetative and reproductive performances

The treatment with Actiwave® positively influenced the vege-tative performances of strawberry plants (Table 1). More in details,the biostimulant significantly increased sprout length and dryweight and root growth. Also sprout fresh weight and average leafarea were increased, but the differences were not statistical signifi-cant. On the other hand, neither the total number of leaves, nor theiraverage fresh weight was influenced by the treatments. Finally,the effect of Actiwave® or sequestrene on the different vegetativeparameters was generally comparable.

As far as the leaf chlorophyll content is concerned, 2 weeks

Average leaf dry weight (g) 3.8a 3.6a 3.6aChlorophyll content (SPAD Value) 36.9b 41.0ab 47.3aStomatal density (n mm−2) 183a 195a 202aTotal root fresh weight (g plant−1) 8.26b 11.65a 10.12aTotal root dry weight (g plant−1) 1.32b 2.33a 2.01a

F. Spinelli et al. / Scientia Horticulturae 125 (2010) 263–269 267

Table 2Yield and fruit quality parameters of strawberry plants grown on a iron chlorosis inducing soil. As far as the fruit yield, values labelled with the same letter do not differaccording to SNK test (P < 0.05). For all the other characteristics, the average values are followed by the standard error of the mean.

Yield (g plant−1) Fruit weight (g) Flesh firmness(g cm−2)

Superficialfirmness (g cm−2)

Sugar content(◦brix)

Tritable Acidity(acid g/l)

pH

Water 114b 17.6 ± 0.82 505.4 ± 18.1 260.2 ± 5.05 7.4 ± 0.18 88.5 ± 2.03 3.7 ± 0.08Sequestrene 144.5a 18.3 ± 0.82 520.2 ± 17.9 269.1 ± 5.2 7.1 ± 0.19 90.2 ± 2.11 3.6 ± 0.08Actiwave 148.2a 19.7 ± 0.84 518 ± 18.7 267.3 ± 5.21 7.6 ± 0.19 81.5 ± 1.87 3.7 ± 0.08

Table 3Total root-associated bacteria, fluorescent pseudomads, Bacillus subtilis and streptomycetes found in the rhizosphere of strawberry plants treated with Actiwave® , water orsequestrene. Bacterial populations are expressed as log10 cfu. The pH of the rhizosphere has been measured for the first 72 h after the treatments. Values in each columnlabelled with the same letter do not differ according to SNK test (P < 0.05).

pH Total bacterial population Pseudomonas fluorescens Bacillus subtilis Streptomyces spp.

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alues were observed in sequestrene-treated leaves. Also in thisase, no statistical difference was observed between Actiwave® andequestrene (Table 1).

The effects of Actiwave® on fruit productivity and berry qualityarameters were also evaluated. Fruit yield, expressed as grams oferry per plant, was substantially increased by the biostimulantup to 26%) and sequestrene (up to 21%). No significant differenceas observed between the yield of plants treated with Activave®

r sequestrene (Table 2). Since Actiwave® and sequestrene did notncrease also the average weight of fresh berry, the increment inruit yield was due to the higher berry weight and the increasedumber of fruits borne by treated plants (Table 2).

As far as fruit quality, Actiwave® did not significantly increaseny of the parameters studied (Table 2). On plants treated withctiwave® or sequestrene, only a 4% increment in flesh firmnessas reported, but this increase was not statistically significant. The

ffects of Actiwave® and sequestrene were comparable on all theruit quality traits.

.3. Root-associated microbial biocoenosis

Actiwave® increased the population of fluorescent pseudomon-ds associated with roots. In comparison with water control plants,he ones treated with the biostimulant harboured a 3.7 times higherseudomonads population (Table 3). On Bacillus subtilis and strep-omycetes populations only a minimal increment was observedfter the treatment. On the other hand, sequestrene did not alterhe population of none of the bacterial groups taken in account inhis trial (Table 3).

Also soil pH was monitored after the treatment with the bios-imulant. In the bulk soil, the pH was 8.3, whereas, the addition ofctiwave®, which has pH 5.8, lowered the pH to 7.7–7.9. This effectapidly faded out with time and, 24 h after the treatment, the soilH returned to the initial values. In the rhizosphere, the pH was.9 and the addition of Actiwave® lowered it to 7.7. In this case theffect was more constant and also after 72 h the pH in the root sys-em of treated plants was 0.2 units lower than control. Sequestreneid not significantly modify the pH neither in the bulk soil, nor inhe root system.

. Discussion

Actiwave® significantly increased sprout length and dry weight,

oot weight, chlorophyll contents, photosynthesis and transpira-ion rates, yield and berry quality. The better vegetative growthnd a higher yield indicate that, in the treated plants, more mineralutrients and photoassimilates are available for the different plantrgans.

b 5.33a 6.55ab 5.27a 6.89aa 5.62a 7.20a

As far as the mineral nutrients, the treated plants showed amore developed root system that might have positively influencedthe nutrients uptake. In addition, the presence of kahydrin andalginic acid in the Actiwave® formulation, by acidifying rhizo-sphere, contribute to a more efficient mobilization and assimilationof the acid-soluble ions (Lüthje and Böttger, 1995; Vernieri et al.,2006).

Concerning the increased availability of photoassimilates, ironchlorosis reduces markedly stomata aperture, chlorophyll contentsand net photosynthesis (Di Martino et al., 2003). The biostimulantcontrasted these negative effects. As for the higher chlorophyll con-tents, apart from the assumption of better iron uptake, as iron is anessential element for chlorophyll biosynthesis, this may be a directeffect of the betaines contained in the biostimulant (Whapham etal., 1993; Blunden et al., 1996). Betaines may also regulate thetissues dehydration, maintain cell turgor and stomatal conduc-tance despite the low water potential (Borowitzka, 1981; Mäkelä etal., 1998; Huang et al., 2000). The consequent protracted stomataaperture contributes to the higher photosynthetic and transpira-tion rates. However, the improved water status of treated plantsmight be due, at least partially, to the effects of and alginic acid(Spinelli et al., 2006). Finally, Actiwave® also enhanced stomatadensities. Stomata control gas exchange between the interior ofa leaf and the atmosphere. Therefore they mainly contribute tothe ability of plants to control their water relations and to gaincarbon (Hetherington and Woodward, 2003). Different environ-mental factors, such as light intensity ad quality, UV-B radiation,CO2 availability and soil moisture, affect stomata density (Knappet al., 1994; Nautiyal et al., 1994; Heijari et al., 2006). For example,the lack of water in growing medium results in a decrease of den-sity and reduction of dimensions of stomata on strawberry leaves(Klamkowski and Treder, 2006). On the other hand, fertilizationseems to not affect the stomata frequency (Lovelock and Feller,2003). However, little information is available on the influence ofiron deficiency on stomata frequency and development. The factthat in Fe chlorotic peach leaves leaf expansion and the absolutenumber of stomata per leaf is reduced, whereas stomatal densitywas not changed significantly, may suggest that Fe shortage affectsstomatal differentiation (Fernández et al., 2008). However, in ourresearch the application of sequestrene did not increase the stom-ata frequency. Therefore, on the lack of further experimentation,we can assume that the higher stomata density was due mainlydue to the improved water status of Actiwave®-treated plants.

As far as the fruiting performances, Actiwave® increased yieldand berry size. Increases in fruit weight and yield quality, such asthose observed in the present experiment after application of bios-timulant have been also observed in other researches (Amoros etal., 2004; Roussos et al., 2009).

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Seaweed extracts have been found to contain significantmounts of cytokinins, auxins and betaines, which influence cellivision during the early stages of growth along with the inductionf flower formation (Roussos et al., 2009). The presence of betainesn Actiwave® formulation has promoted cell division, thus fruit sizen later stages.

Fruitlet growth is the result of dry matter and/or water accu-ulation and it depends on the sink capacity and/or on assimilates

upply to the fruit (Roussos et al., 2009). Actiwave® could havencreased both water and photoassimilate supply for growingruitlets.

Finally, Actiwave® influenced the bacterial biocoenosis of theoot system mainly by increasing the population of fluorescentseudomodas. Interestingly, Urashima et al. (2005) reported thathe application of organic materials reach in betaines enhance theoot colonization by fluorescent Pesudomonads. Bacteria belongingo the Pseudomonas genus have been extensively studied for theirbility to act as biological control agents (BCA) of broad spectrum oflant pathogens (Wilson et al., 1992; Carisse et al., 2003; Avis et al.,008). In addition, some of these BCA strains were also shown to belant growth promoting rhizobacteria (Howie and Echandi, 1983;loepper et al., 1988; Vessey, 2003; Avis et al., 2008). According

o Avis et al. (2008), the growth promoting ability of bacteria fromhe genus Pseudomonas results from the synergic effect of differenthysiological and ecological mechanisms. Among them the solubil-

sation of insoluble P sources (Richardson, 2001) and/or regulationf the concentration of plant growth regulators (Glick et al., 1998;essey, 2003) seems to play an important role.

For example, different Pseudomonads produce the root pro-oting hormone IAA (Glick et al., 1997, 1998) or interfere with

ts degradation (Leveau and Lindow, 2005). On the other hand,ther Pseudomonads prevent the synthesis of plant growth inhibit-ng levels of ethylene in the roots (Penrose et al., 2001). The levelf these plant hormones within the root system plays a crucialole in the growth promoting ability of a microorganism (Avist al., 2008). For these reasons, some of the observed effects onlants growth, such as the increase of root growth observed afterctiwave® treatment, might be due to a bacterial-mediated actionf this biostimulant.

In conclusion, Actiwave® may represent a valid alternative toynthetic iron chelates in organic strawberry production. Nonethe-ess, the use of biostimulants must be combined with all the moderngronomic practices, such as precision agriculture and innovativeecision-making systems, to create novel approaches to cultivationimed at maximizing the potential of a crop plant, to boost fruitroduction, quality and safety and, at the same time, to minimiseuman impacts on the environment.

However, because of Actiwave® complex formulation, a pre-ise and unequivocal mode of action cannot be hypothesised. Moreikely, several direct and indirect actions contribute to the over-ll effect of this biostimulant. For this reason, further research iseeded to clarify the relative weight of the different mechanismsnderlying the mode of action of Actiwave®.

cknowledgments

This research has been co-supported by the Department of Fruitree and Woody Plant Sciences, Alma Mater Studiorum, Universitài Bologna and the Valagro SpA. We thank Mr. Roberto Vanicinind Fabio Pelliconi for the help in the management of the practicalrials.

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