A novel type of seaweed extract as a natural alternative to the use of iron chelates in strawberry production

Scientia Horticulturae 125 (2010) 263–269

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A novel type of seaweed extract as a natural alternative to the use of iron chelates in strawberry production Francesco Spinelli ∗ , Giovanni Fiori, Massimo Noferini, Mattia Sprocatti, Guglielmo Costa Dipartimento di Colture Arboree, Alma Mater Studiorum, Università di Bologna, Italy

a r t i c l e

i n f o

Article history: Received 30 July 2009 Received in revised form 28 January 2010 Accepted 31 March 2010 Keywords: Plant biostimulant Soil amendment Sustainable horticulture Rhizosphere

a b s t r a c t The new generation of seaweed extracts, such as Actiwave® , may represent a promising strategy to reduce the use of phytochemicals in agriculture. Actiwave® is a metabolic enhancer derived by the algae Ascophillum nodosum, but differently from some older seaweed extracts, it has a constant and balanced formulation containing kahydrin, alginic acid and betaines which synergistically contribute to the efficacy of the product. Actiwave® has been proposed to increase the mineral nutrient uptake and the abiotic stress tolerance. The aim of this work was to evaluate, under a multidisciplinary approach, the effect of the biostimulant on the vegetative and productive performances of strawberry plants grown on a lime inducing iron chlorosis substrate. This biostimulant increased the vegetative growth (10%), the leaf chlorophyll 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 dry matter was increased up to 27% and root dry matter up to 76%. Finally, preliminary experiments showed that Actiwave® positively influenced also the root-associated microbial biocoenosis. These results are discussed in relation to the physiological and ecological mechanisms proposed to explain the beneficial effects 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. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Consumers and society increasingly value the production of high quality, healthy fruit and vegetables that, at the same time, ensures a minimal or non-adverse impact on the environment. These aspects are especially important on those horticultural crops, such as strawberry, which require highly specialized knowledge and high external inputs (Tagliavini et al., 1996, 2005). In particular, soil management, irrigation and fertilizations are all vital interventions to obtain profitable strawberry productions, though an excess of nutrients transported to the drainage area results in environmental problems such as eutrophication in rivers, lakes and sea (Neeteson and Carton, 2001). In addition, these activities may also negatively influence the root biocoenosis by reducing beneficial microorganisms and mycorrhizal fungi (Plencette et al., 2005). Due to the numerous concerns about a massive and indiscriminate irrigation and fertilization, the present tendency is to reduce significantly their use in horticulture (Van Noordwijk and Cadisch, 2002). Actiwave® (Valagro SpA, Piazzano di Atessa, and

∗ Corresponding author. Tel.: +39 0512096447; fax: +39 0512096401. E-mail addresses: [email protected], [email protected] (F. Spinelli). 0304-4238/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.03.011

CH – Italy) is a promising biostimulant which may contribute to this objective. Biostimulants are environmental friendly, natural substances able to promote vegetative growth, mineral nutrient uptake, plant fitness to different pedoclimatic conditions and tolerance to abiotic stresses (Vernieri et al., 2006). In particular, Actiwave® is able to enhance the nutrient uptake by roots and the plant tolerance to drought and saline stress (Spinelli et al., 2006). Actiwave® is a seaweed extract derived by the brown algae Ascophillum nodosum. Seaweeds are a known source of plant growth regulators (Jameson, 1993), organic osmolites (e.g. betaines), aminoacids, mineral nutrients, vitamin and vitamin precursors (Berlyn and Russo, 1990; Blunden et al., 1985). In particular, Actiwave® contains kahydrin, alginic acid and betaines which synergistically contribute to the efficacy of the formulation (Vernieri et al., 2006). Kahydrin is a derivate of the K vitamin. The exogenous application of K vitamin induces the secretion of H+ in the apoplast and the 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 et al., 1972). In addition, kahydrin has been proposed to interfere with the tricarboxylic acid cycle, thus promoting the formation of long chain molecules such as proteins and polysaccharides (Sportelli, 2005).

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On the other hand, alginic acid acts as soil conditioning agent: it combines with metallic radicals to form cross-linked polymers which increase the water-holding characteristics of the rhizosphere (Verkleij, 1992; Lattner et al., 2003). The increase of moisture in the rhizosphere contributes to create an environment more suitable for the growth of roots and root-associated beneficial microorganisms (Chen et al., 2003). Finally, betaines are compatible solutes that act as omoprotectants, thus enhancing the plant resistance to drought and salinity stress (Huang et al., 2000). Betaines also present a cytokinin-like activity (Blunden and Wildgoose, 1977) and their exogenous application increases the chlorophyll content in leaves (Whapham et al., 1993; Blunden et al., 1996) and the growth of shoots and roots (Blunden et al., 1996; Vernieri et al., 2006). Actiwave® has been applied on many crop plants and, as far as fruit trees, on apple, pear and grape (Spinelli et al., 2006). Due to the partially unknown mode of action, the complex formulation and the high variability among the environmental conditions, the effect of the Actiwave® in open field experiments was inconstant and erratic (Vernieri et al., 2006; Spinelli et al., 2006). On apple, the application of Actiwave® , in combination with a standard fertilization, increased the shoot growth, the leaf chlorophyll content, the plant productiveness and the fruit sugar content. However, the application of Actiwave® alone on nutrient depleted plants did not positively affected any of the mentioned vegetative and reproductive parameters (Spinelli et al., 2006). In addition, on apple trees showing a biennial bearing behaviour, Actiwave® , by reducing the oscillation between the “on” and “off” years, moderated the negative effects of this productive disorder (Spinelli et al., 2009). On pear, Actiwave® increased the yield and the average fruit weight, but nor the chlorophyll content, neither the qualitative parameters of fruits (Spinelli et al., 2006). Finally, on grape (cv. Sangiovese), Actiwave® increased the leaf chlorophyll concentration more than the traditional iron chelate FeEDDHA (Spinelli et al., 2006). In the present research, experiments have been performed to investigate the effects of Actiwave® on the main vegetative and productive parameters of strawberry plants grown in iron deficiency conditions. Strawberry was chosen as a model crop for berry plants because the whole biological cycle, from sprouting to fruit ripening, can be followed under highly controlled conditions, thus minimizing the influence of the environmental variations. In addition, strawberry has been widely studied and the plants are easily available (Walter et al., 2008). Iron deficiency has been observed in many crops when grown in high pH calcareous soils (Miller et al., 1984). In these soils, the iron deficiency is the most important abiotic stress limiting the strawberry production (Zaiter et al., 1993; Lieten, 2000; Kafkas et al., 2007). In fact, iron deficiency results in extensive fruit abortion with a consequent reduction of yield and fruits weight (Lieten and Baets, 1991; Zaiter et al., 1993; Almaliotis et al., 2002). In addition, also strawberry fruit quality is directly dependent on iron (Karp et al., 2002; Erdal et al., 2004). Many studies have been conducted to determine the most appropriate strategy to overcome the iron deficiency chlorosis in strawberry (Erdal et al., 2006; King et al., 1950; Zaiter et al., 1993; Türemis¸ et al., 1997). Nowadays, the use of iron chelates and the foliar sprays are the most widely accepted methods for correcting this micronutrient problem (Lucena et al., 1990; Zaiter et al., 1993; Abadia et al., 2002). In our experiments, the adaptability to iron deficiency conditions was evaluated by monitoring other parameter than the mere iron concentration in leaves. In fact, the chlorosis associated with iron deficiency is not usually a direct consequence of an absolute lack of this element as in the case of other micronutrient elements, but rather a secondary effect resulting from the complex interactions of Fe with other elements and various soil and environmental factors (Erdal et al., 2004). For example, in experi-

ments conducted under uncontrolled conditions in calcareous soils, it was found that the concentration of Fe in chlorotic leaves is similar to or even higher than that in green leaves (Erdal et al., 2004). For the mentioned reasons, the effects of Actiwave® and sequestrene were evaluated primarily on vegetative growth, leaf chlorophyll content, stomata density, yield and fruit weight. In addition, since in several plant species grown in iron deficient conditions, photosynthetic rates were depressed, these parameters were monitored on entire plants (Larbi et al., 2006). Finally, considering that rhizobacteria, such as Pseudomonas fluorescent, Bacillus sp. and streptomycetes, plays a crucial role in determining the plant fitness to the environment (De Weger et al., 1995; Gerhardson, 2002; Vessey, 2003), the effect of Actiwave® on the root-associated microbial biocoenosis was also studied. 2. Materials and methods 2.1. Plant material, experimental treatments and growing conditions Experiments were performed on strawberry plants [Fragaria ananassa (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]. The cultivar has been developed for cultivation in temperate-cold climates (Faedi and Baruzzi, 2004). Strawberry plants were planted as cold stored trayplants on a substrate obtained by mixing 1:1 (v/v) peat and sand. The peat mineral concentration declared by the manufacturer was: NH4 + 25 g m−3 , NO3 − 35 g m−3 , P2 O5 104 g m−3 , K2 O 120 g m−3 , MgO 12 g m−3 , micronutrients 25 g m−3 . Standard irrigation and fertilization was applied. As mineral fertilizer, Poly-Feed (16N-8P-32K) by Fertica S.A. was used (6 g plant−1 applied two times at BBCH 15, 41 – Stauss, 1994). To verify the efficacy of Actiwave® to increase the strawberry tolerance to iron deficiency, 1 week before its application, the plants were transplanted in a lime inducing iron chlorosis medium. This medium 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: Fe 7.8 ± 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 commercial product dissolved in 20 ml of tap water. Iron chelate sequestrene [sodium ferric ethylenediamine di (ohydroxyphenylacetate) or NaFeEDDHA containing 6% Fe] and water treated plants were used as controls. Sequestrene was applied by dissolving 1 g of commercial product (sequestrene 138 Fe – Syngenta S.p.A.) in 30 ml of 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 chamber kept at room temperature under natural daylight conditions. A constant flow of filtered air (16 l min−1 ) was maintained inside the chamber. The photon flux density in the photosynthetically active wavelength range was measured using a Quantum PAR light sensor (Spectrum Technologies Inc., Plainfield, IL, USA) and the air temperature was measured using a copper constant thermocouples (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 were carried out over eight consecutive days. Net photosynthesis was calculated in relation to the total leaf area and biomass of the plant. To determine the biomass of the enclosed plants, the dry

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weight (DW) of individual leaves was determined after desiccation in a oven for 48 h at 70 ◦ C. Leaf areas were determined by scanning all the leaves and processing the obtained files by an image analysis programme (GIMP 2.6.6. – GNU Image Manipulation Program).

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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 coating the leaf surface with clear cellulose acetate (nail polish). To calculate the stomata density, leaf samples (4 mm2 ) were collected

Fig. 1. Photosynthesis (A, C and E) and transpiration (B,D and F) rates of strawberry plants grown on an iron chlorosis inducing soil. The effect of Actiwave® (dashed line) was compared with water (solid) and sequestrene (dotted). The photosynthetically active photon flux density is also reported (grey line). The gas exchange rates were monitored for three consecutive days: September 14 (A and B), 15 (C and D) and 16 (E and F).

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between the second and third veins from the stalk, halfway between the main nerve and the leaf edge. The number of stomata was counted under a light microscope at 250× magnification from the imprints which were mounted on a glass slide a stained with a water solution of toluidine blue (1%, w/v). Stomata density was expressed as per 1 mm2 of leaf area.

given as the standard error (S.E.) of the means. As far as the data regarding the gas exchange rates and the PAR, each of the values reported in the graphs results from the average of the 3 repetitions. 3. Results 3.1. Gas exchange measurements and stomata density

2.3. Vegetative and reproductive performances Three weeks after treatment, on 12 plants per test, divided in 3 repetitions, the following parameters were measured: number of leaf per plant, average leaf area, chlorophyll content (SPAD), leaf fresh and dry matter, number of stolon per plant, average stolon length, stolon fresh and dry matter, shoot length, shoot fresh and dry matter, root fresh and dry weight. The average chlorophyll content in leaves was measured using a portable chlorophyll meter (SPAD-502, Minolta, Konica Minolta Holdings Inc., Tokyo, Japan). At harvest, on a similar set of plants, the fruit production per plant, average fruit weight, soluble solids content (SSC), flesh firmness, superficial firmness, and titratable acidity of the fruits were evaluated. These determinations were obtained by destructive analyses using a refractometer (Atago, Co., Ltd, Tokyo, Japan), a penetrometer (Effe.gi, Ravenna, Italy), a durometer Shore A Durofel® (Copa–Technologie S.A. 13150 Tarascon, France) and a titrator (Crison Instruments, SA, Barcelona, Spain) performed according to established methodologies (Costa et al., 2003). 2.4. Root-associated microbial biocoenosis A qualitative analysis of the microbial biocoenosis of the rhizosphere was performed. For the evaluation of the different culturable microorganisms, an entire root was gently washed of the adhering soil by using a sterile MgSO4 solution (10 mM). Successively, 1 mg of root system (the proper root plus the remained adhering soil) was aseptically placed in a sterile vial containing 10 ml of MgSO4 solution and vigorously shacked for 2 min on a vortex mixer. Tenfold sequential dilutions of the supernatant were plated on the appropriate agar medium. The total bacterial population was evaluated on Luria Agar (LA), whereas the presence of putatively beneficial bacteria (Pseudomonas spp., Bacillus subtilis and related species and Streptomyces spp.) was evaluated on different selective and semi-selective media. The King B medium (KB) was used for fluorescent Pseudomonas (King et al., 1954), the casein digestmannitol agarose (CM) for Bacillus (Fall et al., 2004) and the STR medium for Streptomyces spp. (Conn et al., 1998). All the media were amended with cycloheximide (50 mg l−1 ) to prevent fungal growth. Plates were incubated at 22 ◦ C till the bacterial colonies were easily distinguishable. Finally, the isolated were identified by traditional phenotypic methods (Reva et al., 2001, 2004). The whole procedure was repeated for three separate samples taken from each plant. At the same time, also the soil pH was measured. For this purpose, samples of bulk and root-adhering soil were collected, weighted and mixed in a vial with distilled water with a proportion of 1:2.5 (w/w). The pH was measured in the liquid suspension thus obtained (Lab pH-meter basic20 Crison-Instruments S.A., Barcelona, Spain). Soil pH was monitored during the first 96 h after treatment. 2.5. Statistical analysis The statistical analysis was performed using Stat software (STATISTICA version 5.0 Statsoft, Inc. 1995, Tulsa, USA). The analysis of variance and the Student Newman and Keuls test (SNK) of comparison between means were performed using the ANOVA procedure of the Stat software at P = 0.05. In the graphs, sample variability is

Under our experimental conditions, treatment with Actiwave® consistently increased photosynthetic activity of strawberry plants grown on iron chlorosis inducing soil (Fig. 1A, C and E). In addition, the effect Actiwave® was comparable with the iron chelate sequestrene. Only during the second day of the experiment, no significant effect of the treatments was observed. In that date, the photosynthetic rates of all plants showed an unusual trend that differed from what has been observed during the other days and from what has previously been reported in literature for other plants (Sabatini et al., 2003). The biostimulant also increased transpiration rates, but its effect was less consistent than on photosynthetic activity (Fig. 1B, D and F). Moreover, no substantial differences among the different days were observed. As far as the sharp decrease of the transpiration rate of sequestrene-treated plants observed during the first day, it was due to a transient leaking of the experimental chamber (Fig. 1B). In our experiments, the effect of the biostimulant on the photosynthetic 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 effect was consistent, but not statistically significant, and the highest increase was observed in the plants treated with sequestrene. In these plants, in fact, the application of the iron chelate resulted in a 10% increase of the stomata density. 3.2. Vegetative and reproductive performances The treatment with Actiwave® positively influenced the vegetative performances of strawberry plants (Table 1). More in details, the biostimulant significantly increased sprout length and dry weight and root growth. Also sprout fresh weight and average leaf area were increased, but the differences were not statistical significant. On the other hand, neither the total number of leaves, nor their average fresh weight was influenced by the treatments. Finally, the effect of Actiwave® or sequestrene on the different vegetative parameters was generally comparable. As far as the leaf chlorophyll content is concerned, 2 weeks after the transplant on iron chlorosis inducing soil, the leaves from treated and control plants were easily distinguishable by visual evaluation. The non-invasive evaluation of leaf chlorophyll content confirmed that Actiwave® significantly increase the concentration of this photosynthetic pigment (Table 1). Nonetheless, the highest Table 1 Vegetative parameters of strawberry plants grown on iron chlorosis inducing soil. Average values in each column labelled with the same letter do not differ according to SNK test (P < 0.05).

Sprout length (mm) Sprout fresh weight (g) Sprout dry weight (g) Number of leaves per plant Average leaf area (cm2 ) Average leaf fresh weight (g) Average leaf dry weight (g) Chlorophyll content (SPAD Value) Stomatal density (n mm−2 ) Total root fresh weight (g plant−1 ) Total root dry weight (g plant−1 )

Control

Actiwave

Sequestrene

25.7b 7.3a 1.1b 10.3a 55.8a 17.9a 3.8a 36.9b 183a 8.26b 1.32b

28.3a 8.8a 1.4a 9.7a 67.3a 18a 3.6a 41.0ab 195a 11.65a 2.33a

27.5a 7.7a 1.0b 10.2a 64.8a 18.2a 3.6a 47.3a 202a 10.12a 2.01a

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Table 2 Yield 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 differ according 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 ) Water Sequestrene Actiwave

Fruit weight (g) 17.6 ± 0.82 18.3 ± 0.82 19.7 ± 0.84

114b 144.5a 148.2a

Flesh firmness (g cm−2 ) 505.4 ± 18.1 520.2 ± 17.9 518 ± 18.7

Superficial firmness (g cm−2 ) 260.2 ± 5.05 269.1 ± 5.2 267.3 ± 5.21

Sugar content (◦ brix) 7.4 ± 0.18 7.1 ± 0.19 7.6 ± 0.19

Tritable Acidity (acid g/l)

pH

88.5 ± 2.03 90.2 ± 2.11 81.5 ± 1.87

3.7 ± 0.08 3.6 ± 0.08 3.7 ± 0.08

Table 3 Total root-associated bacteria, fluorescent pseudomads, Bacillus subtilis and streptomycetes found in the rhizosphere of strawberry plants treated with Actiwave® , water or sequestrene. 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 column labelled with the same letter do not differ according to SNK test (P < 0.05).

Water Sequestrene Actiwave

pH

Total bacterial population

Pseudomonas fluorescens

Bacillus subtilis

Streptomyces spp.

7.9a 7.9a 7.7b

8.23a 8.21a 7.98a

5.35b 5.29b 6.02a

5.33a 5.27a 5.62a

6.55a 6.89a 7.20a

values were observed in sequestrene-treated leaves. Also in this case, no statistical difference was observed between Actiwave® and sequestrene (Table 1). The effects of Actiwave® on fruit productivity and berry quality parameters were also evaluated. Fruit yield, expressed as grams of berry per plant, was substantially increased by the biostimulant (up to 26%) and sequestrene (up to 21%). No significant difference was observed between the yield of plants treated with Activave® or sequestrene (Table 2). Since Actiwave® and sequestrene did not increase also the average weight of fresh berry, the increment in fruit yield was due to the higher berry weight and the increased number of fruits borne by treated plants (Table 2). As far as fruit quality, Actiwave® did not significantly increase any of the parameters studied (Table 2). On plants treated with Actiwave® or sequestrene, only a 4% increment in flesh firmness was reported, but this increase was not statistically significant. The effects of Actiwave® and sequestrene were comparable on all the fruit quality traits. 3.3. Root-associated microbial biocoenosis Actiwave® increased the population of fluorescent pseudomonads associated with roots. In comparison with water control plants, the ones treated with the biostimulant harboured a 3.7 times higher pseudomonads population (Table 3). On Bacillus subtilis and streptomycetes populations only a minimal increment was observed after the treatment. On the other hand, sequestrene did not alter the population of none of the bacterial groups taken in account in this trial (Table 3). Also soil pH was monitored after the treatment with the biostimulant. In the bulk soil, the pH was 8.3, whereas, the addition of Actiwave® , which has pH 5.8, lowered the pH to 7.7–7.9. This effect rapidly faded out with time and, 24 h after the treatment, the soil pH returned to the initial values. In the rhizosphere, the pH was 7.9 and the addition of Actiwave® lowered it to 7.7. In this case the effect was more constant and also after 72 h the pH in the root system of treated plants was 0.2 units lower than control. Sequestrene did not significantly modify the pH neither in the bulk soil, nor in the root system. 4. Discussion Actiwave® significantly increased sprout length and dry weight, root weight, chlorophyll contents, photosynthesis and transpiration rates, yield and berry quality. The better vegetative growth and a higher yield indicate that, in the treated plants, more mineral nutrients and photoassimilates are available for the different plant organs.

As far as the mineral nutrients, the treated plants showed a more developed root system that might have positively influenced the nutrients uptake. In addition, the presence of kahydrin and alginic acid in the Actiwave® formulation, by acidifying rhizosphere, contribute to a more efficient mobilization and assimilation of the acid-soluble ions (Lüthje and Böttger, 1995; Vernieri et al., 2006). Concerning the increased availability of photoassimilates, iron chlorosis reduces markedly stomata aperture, chlorophyll contents and net photosynthesis (Di Martino et al., 2003). The biostimulant contrasted these negative effects. As for the higher chlorophyll contents, apart from the assumption of better iron uptake, as iron is an essential element for chlorophyll biosynthesis, this may be a direct effect of the betaines contained in the biostimulant (Whapham et al., 1993; Blunden et al., 1996). Betaines may also regulate the tissues dehydration, maintain cell turgor and stomatal conductance despite the low water potential (Borowitzka, 1981; Mäkelä et al., 1998; Huang et al., 2000). The consequent protracted stomata aperture contributes to the higher photosynthetic and transpiration rates. However, the improved water status of treated plants might be due, at least partially, to the effects of and alginic acid (Spinelli et al., 2006). Finally, Actiwave® also enhanced stomata densities. Stomata control gas exchange between the interior of a leaf and the atmosphere. Therefore they mainly contribute to the ability of plants to control their water relations and to gain carbon (Hetherington and Woodward, 2003). Different environmental factors, such as light intensity ad quality, UV-B radiation, CO2 availability and soil moisture, affect stomata density (Knapp et al., 1994; Nautiyal et al., 1994; Heijari et al., 2006). For example, the lack of water in growing medium results in a decrease of density and reduction of dimensions of stomata on strawberry leaves (Klamkowski and Treder, 2006). On the other hand, fertilization seems to not affect the stomata frequency (Lovelock and Feller, 2003). However, little information is available on the influence of iron deficiency on stomata frequency and development. The fact that in Fe chlorotic peach leaves leaf expansion and the absolute number of stomata per leaf is reduced, whereas stomatal density was not changed significantly, may suggest that Fe shortage affects stomatal differentiation (Fernández et al., 2008). However, in our research the application of sequestrene did not increase the stomata frequency. Therefore, on the lack of further experimentation, we can assume that the higher stomata density was due mainly due to the improved water status of Actiwave® -treated plants. As far as the fruiting performances, Actiwave® increased yield and berry size. Increases in fruit weight and yield quality, such as those observed in the present experiment after application of biostimulant have been also observed in other researches (Amoros et al., 2004; Roussos et al., 2009).

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Seaweed extracts have been found to contain significant amounts of cytokinins, auxins and betaines, which influence cell division during the early stages of growth along with the induction of flower formation (Roussos et al., 2009). The presence of betaines in Actiwave® formulation has promoted cell division, thus fruit size in later stages. Fruitlet growth is the result of dry matter and/or water accumulation and it depends on the sink capacity and/or on assimilates supply to the fruit (Roussos et al., 2009). Actiwave® could have increased both water and photoassimilate supply for growing fruitlets. Finally, Actiwave® influenced the bacterial biocoenosis of the root system mainly by increasing the population of fluorescent Pseudomodas. Interestingly, Urashima et al. (2005) reported that the application of organic materials reach in betaines enhance the root colonization by fluorescent Pesudomonads. Bacteria belonging to the Pseudomonas genus have been extensively studied for their ability to act as biological control agents (BCA) of broad spectrum of plant pathogens (Wilson et al., 1992; Carisse et al., 2003; Avis et al., 2008). In addition, some of these BCA strains were also shown to be plant growth promoting rhizobacteria (Howie and Echandi, 1983; Kloepper et al., 1988; Vessey, 2003; Avis et al., 2008). According to Avis et al. (2008), the growth promoting ability of bacteria from the genus Pseudomonas results from the synergic effect of different physiological and ecological mechanisms. Among them the solubilisation of insoluble P sources (Richardson, 2001) and/or regulation of the concentration of plant growth regulators (Glick et al., 1998; Vessey, 2003) seems to play an important role. For example, different Pseudomonads produce the root promoting hormone IAA (Glick et al., 1997, 1998) or interfere with its degradation (Leveau and Lindow, 2005). On the other hand, other Pseudomonads prevent the synthesis of plant growth inhibiting levels of ethylene in the roots (Penrose et al., 2001). The level of these plant hormones within the root system plays a crucial role in the growth promoting ability of a microorganism (Avis et al., 2008). For these reasons, some of the observed effects on plants growth, such as the increase of root growth observed after Actiwave® treatment, might be due to a bacterial-mediated action of this biostimulant. In conclusion, Actiwave® may represent a valid alternative to synthetic iron chelates in organic strawberry production. Nonetheless, the use of biostimulants must be combined with all the modern agronomic practices, such as precision agriculture and innovative decision-making systems, to create novel approaches to cultivation aimed at maximizing the potential of a crop plant, to boost fruit production, quality and safety and, at the same time, to minimise human impacts on the environment. However, because of Actiwave® complex formulation, a precise and unequivocal mode of action cannot be hypothesised. More likely, several direct and indirect actions contribute to the overall effect of this biostimulant. For this reason, further research is needed to clarify the relative weight of the different mechanisms underlying the mode of action of Actiwave® . Acknowledgments This research has been co-supported by the Department of Fruit Tree and Woody Plant Sciences, Alma Mater Studiorum, Università di Bologna and the Valagro SpA. We thank Mr. Roberto Vanicini and Fabio Pelliconi for the help in the management of the practical trials. References Abadia, J., Álvarez-Fernández, A., Morales, F., Sanz, M., Abadia, A., 2002. Correction of iron chlorosis by oliar sprays. Acta Hortic. 594, 115–121.

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