Acta Botanica 2-2016 - za web.indd ACTA BOT. CROAT. 75 (2), 2016 253 Acta Bot. Croat. 75 (2), 253–259, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0023 ISSN 0365-0588 eISSN 1847-8476 The effect of biostimulant and fertilizer on “low input” lettuce production Slavica Dudaš1, Ivana Šola2,*, Barbara Sladonja3, Renata Erhatić4, Dean Ban3, Danijela Poljuha3 1 Polytechnic of Rijeka, Poreč Agricultural Department, Karla Huguesa 6, Poreč, Croatia 2 Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, Zagreb, Croatia 3 Institute of Agriculture and Tourism, Karla Huguesa 8, Poreč, Croatia 4 Križevci College of Agriculture, Milislava Demerca 4, Križevci, Croatia Abstract – Lettuce production in the winter on the Adriatic coast, especially in a non-heated plastic tunnel, requires longer cultivation and is characterised by lower head mass and yield. In these conditions, the effect of biostimulant Bio-algeen S-90 and fertilizer Megagreen on the production of the traditional winter lettuce cultivar ‘Four Seasons’ was tested. Both treatments showed a positive effect on the growth and total yield of winter lettuce, and decreased the share of non-marketable yield. Bio-algeen S-90 treatment increased the plant height by 61.5%, and foliar treatment with Megagreen by 60.9%, as compared to the control treatment. Equally, both treatments resulted in higher leaf numbers (47.7% for Bio-algeen S-90 and 37.2% for Mega- green). The head mass of lettuce treated with Bio-algeen S-90 and Megagreen was 30.3% and 25.0% higher than in the control treatment, respectively. Megagreen contributed more to chlorophyll and carotenoid con- tent, while Bio-algeen S-90 elevated the amount of vitamin C and dry matter. The pH value of lettuce juice decreased after Bio-algeen S-90, while the mineral content (N, P and K) did not differ between the tested treatments. Lower nitrate content was detected after both treatments. The obtained results elucidate the effect of Bio-algeen S-90 and Megagreen on “low input” lettuce production. Key words: Bio-algeen S-90, carotenoids, chlorophyll a and b, Megagreen, mineral content, non-heated plastic tunnel, vitamin C * Corresponding author, e-mail: ivana.sola@biol.pmf.hr Slavica Dudaš and Danijela Poljuha have contributed equally to this work. Introduction Growing vegetables such as winter lettuce, rocket, leek, onion, leafy brassicas or radishes, often of local types and traditional cultivars, in semi-Mediterranean climatic condi- tions during the winter period with poorer light and milder temperature conditions, extends the production season, pro- vides fresh vegetables for local markets and ensures addi- tional income for local producers. Such production is com- monly carried out in non-heated high plastic tunnels without heating and lightning. Traditional varieties are to- day in increasing demand not only in produce markets but also in hotels and in the local gastronomy for tourist pur- poses, while worldwide they are being increasingly includ- ed in revitalisation and protection programmes for growing traditional varieties, for regional supply as niche products (Lissek-Wolf et al. 2009), but also in larger variety trials (Wenz and Wenger 2012) and in organic farming. Traditional cultivars are cultivated in Istria (northern coastal Croatia) on small areas in response to the needs of local markets, where this type of cultivar is highly appreci- ated, recognizable and achieves good prices. One of the widely spread traditional lettuce cultivars in Istria is ‘Four Seasons’. This cultivar belongs to the class of butterhead lettuce (Lactuca sativa var. capitata L.), which is predomi- nantly well suited for protected cultivation during winter because of its ability to develop heads of acceptable quality and mass in poorer light and temperature conditions. Its leaves are dark reddish-brown, and it is one of the darkest butterhead types of lettuce when grown in optimal growing conditions, though the colour intensity as well as the com- pactness of the heads depends on the weather and tempera- ture (Wenz and Wenger 2012). The ‘Four Seasons’ cultivar has a long growing tradition in organic farming in early spring or for autumn and winter harvest, due to its good cold-hardiness, and as its name says, it can be cultivated all year round. However, the heat and water shortage in sum- mer, as well as the defi ciency of light and emergence of frost in the winter on the northern Adriatic coast restrict the optimal yield of the lettuce. DUDAŠ S., ŠOLA I., SLADONJA B., ERHATIĆ R., BAN D., POLJUHA D. 254 ACTA BOT. CROAT. 75 (2), 2016 The use of biostimulants and fertilizers to enhance plant yield nowadays is a regular farming practice. Biostimulants include diverse substances and microorganisms that enhan- ce plant growth (Calvo et al. 2014). The effects of several biostimulants on plant growth from spring until autumn in the Mediterranean region have been investigated so far (Vernieri et al. 2002, Amanda et al. 2009, Lucini et al. 2015). Regarding the winter season, Johnstone et al. (2005) and Tahsin (2010) investigated the effect of preplant phospho- rous and nitrogen application, respectively, on lettuce yield, but this was not in the Mediterranean region. Organic extracts of seaweed bioactive substances have been used for ages to enhance soil fertility and crop produc- tivity. From among biostimulants prepared from seaweed extracts, Crouch et al. (1990) investigated the effect of Eck- lonia maxima (Osbeck) extract application on L. sativa L. cv. Winter Crisp. One of the most commercial seaweed extracts is made from brown seaweed Ascophyllum nodo- sum (L.) Le Jolis. However, the extract composition may vary according to the A. nodosum source, the season of co- llection and the extraction process used (Calvo et al. 2014). The A. nodosum extract often used in Croatia is Bio-algeen S-90. Mineral fertilizers are products containing nutrients essential for the normal growth and development of plants (Isherwood 2000). For some period in Croatia NPK (nitro- gen, phosphorus, potassium) were the most used fertilizers; however new preparations with other elements alongside N, P and K emerged and gave promising results as well. One such fertilizer is Megagreen, a Ca fertilizer with micro- nutrients. Bio-algeen S-90 and Megagreen, which are both imple- mented in this study, are on the list of permitted fertilizers and soil conditioners for organic farming (Ministry of Agri- culture RC 2013). So far, Bio-algeen S-90 has been applied on several crops, among them: cereals (Kolbe and Blau 1998), soybean (Redžepović et al. 2007), basil (Kwiat kow- ski and Juszczak 2011) and tomato (Mikiciuk and Dobro- milska 2014). The effect of Megagreen was also studied on several cultures, like potatoes (Horvat et al. 2014) and sug- ar beet (Artyszak et al. 2014). Both products were revealed to have a positive effect on the investigated crops. As far as we know, the possible application of these two preparations in the winter production of leafy vegetables, including let- tuce has not been tested so far. As for the production of let- tuce, it has been proven that organic fertilizers are more suitable than inorganic ones in lettuce cultivation in river sand (Masarirambi et al. 2010), that toxic nitrate concentra- tion in romaine-type lettuce increases after fertilization with sewage sludge (Castro et al. 2009), and that mineral fertili- zation increases the nitrate concentration in the lettuce cul- tivar Mantecosa (Premuzic et al. 2001). The aim of this study was to examine the effects of Bio- algeen S-90 biostimulant and the Megagreen fertilizer on winter production of the traditional lettuce cultivar ‘Four Seasons’. For that purpose we determined the effects of both treatments on: a) plant height, leaf number, head mass, marketable and non-marketable yield of lettuce, b) chloro- phyll a and b content, c) carotenoid content, d) vitamin C content, e) pH of lettuce leaf juice, f) nitrate content, and g) mineral content (N, P, K). Materials and methods Plant material and growth condition The experiment was carried out during 2012 – 2013 in a non-heated high plastic tunnel at the Agricultural Depart- ment in Poreč, Polytechnic of Rijeka, Croatia. The tra dition- al cultivar of lettuce ‘Four Seasons’ (seeds Franchi, Italy) was sown in Klasmann 1 horticultural substrate (Klas mann- Deilmann, Germany) and transplanted into 60 cell fl ats (532 mm x 323 mm x 60 mm, Koplast, Croatia). The experiment included three treatments: double wate- ring with 1.0% Bio-algeen S-90 (200 mL per plant), double foliar treatment with 0.3% Megagreen (100 mL m–2) and control. The basic unit in the experiment was the plot, 3.5 m long and 2.5 m wide (8.75 m2), and each treatment was re- presented by three replicates, i.e. three plots. Plots were arranged according to a random schedule. The experiment consisted of 9 plots (in total, 78.75 m2), the distance be- tween plots (the track) was 0.5 m, while the total area of the experiment amounted 99 m2. In total, 112 lettuce plants per plot were planted at a distance of 0.30 m x 0.30 m, and the total number of plants in the trial was 1008. The soil was terra rossa whose nutrient content and pH values were de- termined according to standard soil analysis methods. Soil was additionally enriched with 2.5 kg per 10 m2 of pelleted organic compost Hortyfl or Stallatico (Fomet, Italy). All the treatments were carried out manually, using small bottles with sprayers and a container for watering with a dosing cup. Every plant in the plot was treated sepa- rately, watered or foliar treated according to the planned tri- als and treatments. The fi rst application was done 6 days after planting and the second 14 days later. During frost, all variants of lettuce treatments and the control variants were additionally protected with agrotextile (17 g m–2). Bio-algeen S-90 and Megagreen preparations Bio-algeen S-90 (Shulze & Hermsen GmbH, Germany) is an organic biostimulant derived from marine alga Asco- phyllum nodosum, a natural algae extract, with macro- and micronutrients, containing natural chemical compounds in- cluding vitamins, amino acids and alginic acid. According to the label, Megagreen (Velebit Inf. International d.o.o., Croatia) is a Ca fertilizer with micronutrients for foliar im- plication: 44.1% CaO, 2.2% MgO, 1.2% Fe2O3, 0.7% Al2O3, 9.1% SiO2, 0.11% SO4, 132 mg kg–1 Mn, 60 mg kg–1 Zn, 22.5 mg kg–1 Cu, 11.5 mg kg–1 Pb, 3.3 mg kg–1 Ni, 3.25 mg kg–1 Cr, 0.8 mg kg–1 Cd and Hg in traces. Analysis of qualitative and quantitative yield The following parameters were monitored 82 days after the second application: plant height, leaf number, head mass, total yield, marketable yield, the share (%) of non- marketable yield (head mass lower than 100 g, visible symptoms of diseases and/or physiological disorders), chlo- LETTUCE BIOSTIMULATION AND FERTILIZATION ACTA BOT. CROAT. 75 (2), 2016 255 rophyll a and b content, carotenoid content, nitrate content in fresh leaves, pH value of fresh lettuce juice, vitamin C content in fresh leaves, dry matter content of leaves, and total N, P and K content in leaves. Chlorophyll a, chlorophyll b and carotenoid content were analysed spectrophotometrically by measuring absor- bance at 665, 649 and 480 nm, respectively, according to Wellburn (1994). Nitrate content was determined spectro- photometrically according to Cataldo et al. (1975). The pH value was determined in fresh lettuce juice using a pH meter (Sartorius Croatia – Libra Elektronik d.o.o., Zagreb, Croatia), vitamin C content using 2,6-dichloroindophenol titrimetric method and dry matter content gravimetrically at 105 °C until the mass became constant. Total N, P, K con- tents were determined according to standard ISO 11261 (2004), ISO 6878 (1998) and ISO 9964-3 (1993) proce- dures, respectively. Statistical analysis Obtained data were statistically analysed using ANOVA and post hoc Tukey test for multiple comparisons between groups on the basis of p ≤ 0.05. Due to homogeneity of er- ror variance, a synthesis of the results of two year studies was carried out. The obtained data was processed using SPSS v. 17.0, and using IBM SPSS Statistics Software Ver- sion 22.0 for principal component analysis. Results Climatic conditions in the cultivation period at the loca- tion in Poreč were characterised by mild temperatures and the emergence of frost, mostly of short duration. The aver- age air temperature was in the range 4.8 – 7.8 °C, while soil temperature was between 5.0 and 8.7 °C (Fig. 1). Nutrient content and pH values of terra rossa soil are presented in Tab. 1. The soil contained approximately 69 mg kg–1 K2O, 50 mg kg–1 P2O5 and 4 mg kg–1 CaCO3. The pH value of soil water was slightly alkaline, approximately 7.8; and of KCl approximately 7.3. The share of humus in the soil was around 4.0%. Double watering at the start of cultivation with Bio-al- geen S-90 increased the plant height of the lettuce by 61.5%, and double foliar treatment with Megagreen by 60.9%, compared to the control treatment (Tab. 2). Equally, the lettuce treated with Bio-algeen S-90 and Megagreen yielded a signifi cantly higher leaf number compared to the control treatment (increase by 47.7% and 37.2%, respec- tively). The difference in leaf number between Bio-algeen S-90 and Megagreen applications was on average 7.1% and was not signifi cantly confi rmed. Lettuce reached a head mass between 255 and 330 g (Tab. 2). Application of either Bio-algeen S-90 or Mega- green signifi cantly increased lettuce head mass. The highest mass of lettuce head was reached after Bio-algeen S-90 treatment. It was 30.3% higher than in the control treat- ment, while Megagreen treatment resulted in head mass in- crease of 25.0%. There was no signifi cant difference be- tween Bio-algeen S-90 and Megagreen effects on head mass (Tab. 2). Biostimulant and fertilizer treatment signifi cantly af- fected total yield as well as marketable yield and share (%) of non-marketable lettuce heads, compared to the control variant. Control treatment had a signifi cantly lower total yield and marketable yield as well as a signifi cantly higher percentage of non-marketable heads in total yield (Tab. 3). The infl uence of biostimulant and fertilizer on pigment content is shown in the Tab. 4. Chlorophyll a and b and ca- rotenoid content varied signifi cantly between Bio-algeen S-90 and Megagreen treatments. Compared to control, chlo- rophyll b level was signifi cantly higher after Megagreen treatment, but chlorophyll and carotenoids did not differ sig- nifi cantly. Bio-algeen S-90 treatment did not change signifi - cantly the amount of any of the tested pigments. Fig. 1. Average air and soil temperature for cultivation period on location in Poreč, Croatia (December – March), (Croatian Meteo- rological and Hydrological Service, 2013). SD – standard devia- tion. Tab. 1. Analysis of minerals (P2O5, K2O and CaCO3), pH value and humus content in terra rossa soil. Results are mean values of three replicates ± standard deviation (SD). Depth 0–30 cm Values±SD P2O5 (mg kg–1) 49.98±0.45 K2O (mg kg–1) 68.80±1.21 CaCO3 (mg kg–1) 3.61±0.70 pH H2O 7.77±0.01 pH KCl 7.26±0.01 Humus (%) 4.06±0.29 Tab. 2. The effect of Bio-algeen S-90 and Megagreen treatments on plant height, leaf number and head mass of ‘Four Seasons’ let- tuce. Results are mean values of three replicates ± standard devia- tion (SD). Different letters in the same column indicate signifi cant difference (Tukey’s test, p ≤ 0.05). LSD – least signifi cant differ- ence. Treatment Plant height (cm) Leaf number Head mass (g) Bio-algeen S-90 10.87±2.97 a 19.20±4.52 a 332.10±30.87 a Megagreen 10.83±3.19 a 17.83±3.43 a 318.74±29.33 a Control 6.73±2.11 b 13.00±4.07 b 254.92±21.71 b LSD, p ≤ 0.05 3.21 4.53 35.96 DUDAŠ S., ŠOLA I., SLADONJA B., ERHATIĆ R., BAN D., POLJUHA D. 256 ACTA BOT. CROAT. 75 (2), 2016 The amount of nitrates in lettuce fresh leaves signifi - cantly decreased after both Bio-algeen S-90 and Megagreen treatment (Fig. 2) and was 47.5% and 34.0%, respectively of the value in control plants. There was no signifi cant dif- ference in treatments with Bio-algeen S-90 and Megagreen preparations with respect to their effectiveness on the level of nitrate decrease. The pH value of fresh lettuce juice was signifi cantly de- creased only after Bio-algeen S-90 treatment (Tab. 5). Vita- min C and dry matter content increased signifi cantly after both of the treatments (Tab. 5); however a higher increase was caused by Bio-algeen S-90 treatment. The content of N, P and K minerals was not affected by either of the treatments (Tab. 5). A biplot constructed by two principal components showing treatments and tested yield components is present- ed in Fig. 3. Principal component 1 (PC1) and principal component 2 (PC2) provided signifi cant indications of 100% of the total variance in the data and showed clear sep- aration of the treatments into three groups (Fig. 3). Discussion Agricultural growing practices have been evolving to- wards organic, sustainable or environmentally friendly sys- tems. Fertilizers and lately biostimulants have increasingly been used as a tool with the potential to enable a more sus- tainable agriculture production (Bulgari et al. 2015). The global market for plant biostimulants and fertilizers is growing fast; according to the study mentioned by Calvo et al. (2014) the largest market for biostimulants in 2012 was Europe. The greatest increase in fertilizer consumption in Europe occurred in the second half of the 20th century (Ish- erwood 2000). About ten years ago the biostimulant Bio- Fig. 3. Biplot of the principal component analysis based on ten tested compounds and three yield components. Fig. 2. Nitrate content (ppm) in fresh leaves of lettuce treated with Bio-algeen S-90 and Megagreen. The means labelled by different letters are signifi cantly different (Tukey’s test, p ≤ 0.05). Tab. 3. The effect of Bio-algeen S-90 and Megagreen treatments on yield, marketable and non-marketable yield of ‘Four Seasons’ lettuce. *Non-marketable lettuce heads with head mass lower than 100 g, visible symptoms of diseases and/or physiological disor- ders like loose heads or bolting. Results are mean values of three replicates ± standard deviation (SD). Different letters in the same column indicate signifi cant difference (Tukey’s test, p ≤ 0.05). LSD – least signifi cant difference. Treatment Yield (kg m–2) Marketable yield (kg m–2) Non-marketable yield* (%) Bio-algeen S-90 3.1±0.12 a 2.54±0.32 a 18.02±0.61 a Megagreen 2.9±0.08 a 2.31±0.11 a 20.22±1.11 a Control 2.0±0.07 b 1.26±0.13 b 36.91±1.19 b LSD, p ≤ 0.05 0.56 0.99 12.30 Tab. 5. The effect of Bio-algeen S-90 and Megagreen treatment on pH value of fresh lettuce juice, vitamin C (Vit C) content, percentage of dry matter and mineral content in ‘Four Seasons’ lettuce leaves. Results are mean values of three replicates ± standard deviation (SD). Different letters in the same column indicate signifi cant difference (Tukey’s test, p ≤ 0.05). Treatment pH juice Vit C mg100 g–1FM Dry matter % N % P2O5 % K2O % Bio-algeen S-90 6.27±0.05 b 17.38±2.45 a 5.93±0.82 a 3.76±0.75 a 1.09±08 a 8.42±1.67 a Megagreen 6.43±0.03 a 15.48±1.11 b 4.43±1.41 b 4.24±0.83 a 1.23±04 a 6.66±1.55 a Control 6.40±0.08 a 13.90±0.51 c 3.93±2.12 c 4.13±0.09 a 0.98±01 a 4.85±1.39 a Tab. 4. The effect of Bio-algeen S-90 and Megagreen treatments on chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) contents in ‘Four Seasons’ lettuce leaves. Results are mean values of three replicates ± standard deviation (SD). Different let- ters in the same column indicate signifi cant difference (Tukey’s test, p ≤ 0.05). FM – fresh mass. LSD – least signifi cant difference. Treatment Chl a Chl b Car mg g–1FM Bio-algeen S-90 0.725±0.08 b 0.670±0.09 b 0.155±0.03 b Megagreen 1.011±0.02 a 1.038±0.05 a 0.216±0.00 a Control 0.849±0.01 ab 0.829±0.07 b 0.182±0.02 ab LSD, p ≤ 0.05 0.167 0.199 0.043 LETTUCE BIOSTIMULATION AND FERTILIZATION ACTA BOT. CROAT. 75 (2), 2016 257 algeen S-90 and the fertilizer Megagreen appeared on the market; however there are no data about their effects on the qualitative and quantitative yields of leafy vegetables, in- cluding lettuce. Therefore, in this work the biological ef- fects of the two preparations on “low input” lettuce cultiva- tion in winter season were investigated. The obtained positive effect of both Bio-algeen S-90 and Megagreen on winter production of lettuce is in accord- ance with similar studies on lettuce. Wenz and Wenger (2012) accordingly have obtained a positive effect of the organic chitin fertilizer Biosol®, produced on the basis of Penicillium chrysogenumon, on lettuce head mass, and also reported the relationship between head mass and seed ori- gin in the same cultivar. The application of NPK-based fer- tilizer ONE® in the study of Bulgari et al. (2014) was as ef- fi cient in plant weight increase of lettuce as the applications we investigated. In the experiment conducted on lettuce cv. Mathilda, Sternecker and Balas (2014) achieved a head mass increase of 31.0% by using the biostimulant Plan- tasalva, extract from 21 plant species with associated Lacto- bacillus and yeast, which is in good accordance with our results. Amanda et al. (2009) confi rmed the increase of baby leaf lettuce yield after treatment with Actiwave® bio- stimulant with betaine, alginic acid, and caidrine, a deriva- tive of vitamin K1. Recently, Lucini et al. (2015) have shown that L. sativa L. cv. Regina di Maggio treated with Trainer, a biostimulant with plant (L) amino acids, was more resistant to yield and biomass reduction caused by stress conditions. Treatments with both Bio-algeen S-90 or Megagreen in- creased plant height and number of leaves. Since both prep- arations affect plant height almost twice as much as head mass and leaf number, we assume that their effects could be associated with synthesis, transport and/or accumulation of auxins in lettuce. Other studies also confi rmed the positive effect of biostimulants on plant growth. Vernieri et al. (2002) showed that the application of the biostimulant Ra- difarm, a complex of plant extracts with polysaccharides, aminoacids, betains and enriched in vitamins and micronu- trients, had a positive effect on plant growth in lettuce and spinach and improved the root/shoot ratios. In lettuce, the biostimulant strongly stimulated the root growth and showed also an increase of the leaf area. We found that Bio-algeen S-90 and Megagreen effects on chlorophyll and carotenoid contents differ signifi cantly. Namely, Megagreen yielded higher amounts of pigments than Bio-algeen S-90 treatment. This is not surprising if we take into account that the core of each chlorophyll molecule is the Mg2+ ion for which Megagreen is a far better source than Bio-algeen S-90 (Megagreen contains 2.2% MgO, but Bio-algeen S-90 only 0.021% Mg; see http://www.agro- klub.com/gnojiva/shulze-i-hermsen-gmbh-284/bio-algeen- s-92-313/). Compared to control plants, only plants treated with Megagreen had increased chlorophyll b content. Therefore, we would recommend the use of Megagreen for the production of lettuce with higher pigment content. With regard to the treatments with Megagreen and Bio-algeen S-90, there are no comparable results in the literature for lettuce production. However, some other studies revealed the effect of the same preparations on pigment content in other cultures, e.g. the effect of the fertilizer Megagreen on potato (Horvat et al. 2014) and the effect of the seaweed extract Bio-algeen S-90 on tomato (Mikiciuk and Dobro- milska 2014). Although Megagreen treatment did not infl u- ence total chlorophyll content, it increased the photosyn- thetic intensity and larger sized tuber yield. Mikiciuk and Dobromilska (2014) proved the increased chlorophyll and carotenoid content in small-sized tomato leaves after multi- ple spraying with Bio-algeen S-90 during summer cultiva- tion in a high plastic tunnel. It also contributed to early har- vest and increased the clusters’ length and the number of tomato fruits in the cluster. Although results from tomato cultivation are not directly comparable with lettuce, they can serve as an orientation. Furthermore, they are not in ac- cordance with our results observed in winter cultivation, probably due to different light conditions. As previously mentioned, the chlorophyll content depends on the environ- mental conditions, especially light quality and quantity (Dias et al. 2007, Kosma et al. 2013). In suboptimal condi- tions, those environmental factors could infl uence the chlo- rophyll content more than the treatment with biostimulants. Other reasons for the different results obtained could be the method and frequency of biostimulant application. The presence of nitrates in vegetables, water and other food is a serious threat for human health, especially the part converted to the more toxic nitrites (Santamaria 2006), which account for approximately 5.0% of all ingested ni- trates (Spiegelhalder et al. 1976, Pannala et al. 2003) but can reach to 20% for individuals with a high rate of conver- sion (Thomson et al. 2007, Correia et al. 2010). In our ex- periment, we found that nitrate content in the control plants, with a value of 6,446 ppm, exceeded the permissible limit of nitrate in leafy vegetables (Commission Regulation 1258/ 2011). However, after either Bio-algeen S-90 or Megagreen treatment of fresh lettuce leaves, the amount of nitrates was signifi cantly decreased, to 34.0 and 47.5% of the control value, respectively (Fig. 2). Since in lettuce L. sativa, pro- tected or open grown, harvested in the period from October 1 to March 31 the limit amount of nitrates is 4,500 ppm expressed on the fresh weight basis, Bio-algeen S-90 or Megagreen are obviously great treatments for nitrate-relat- ed toxicity reduction of lettuce. Compared to the sew- age sludge used by Castro et al. (2009), and mineral (94% Ca(NO3)2 + 6% NH4NO3) and organic (biostabilised com- post) fertilizers used by Premuzic et al. (2001), in terms of nitrate concentration, Megagreen fertilizer would be a bet- ter choice for lettuce production. The pH value of fresh lettuce juice, as expected, varied within the slightly acidic range, between 6.27 and 6.43. US FDA/CFSAN (2007) indicates approximate pH values for lettuce in general between 5.80 – 6.15, noting that pH val- ues of vegetable juices can vary according to the cultivar, season, growing conditions or processing methods. In our experiment, the pH value of fresh lettuce juice was de- creased after Bio-algeen S-90 treatment, and we presume this could be due to amino acids and alginic acid present in this preparation (see “Materials and methods” section), as well as due to a signifi cantly higher amount of vitamin C (ascorbic acid) detected after this treatment (Tab. 5). DUDAŠ S., ŠOLA I., SLADONJA B., ERHATIĆ R., BAN D., POLJUHA D. 258 ACTA BOT. CROAT. 75 (2), 2016 Both of the treatments signifi cantly increased the amounts of vitamin C and dry matter content, however a higher in- crease was caused by Bio-algeen S-90 treatment. This was expected since Bio-algeen S-90 preparation already con- tains natural chemical compounds, including vitamins. Thus, even though after Bio-algeen S-90 and Megagreen treatments the total yield increased similarly, in case of need for higher dry mass, we would recommend the use of Bio-algeen S-90 preparation. While vitamin C differed signifi cantly between the test- ed treatments, mineral contents in lettuce leaves did not change (Tab. 5) and were in the range indicated in literature (Ismail and Fun 2003, Koudela and Petříková 2008, Masa- mba and Nguyen 2008). Since Bio-algeen S-90 preparation contains amino acids, we expected that this treatment would increase the concentration of N in lettuce leaves; however, this was not the case. We speculate this could be due to lim- ited root absorption of amino acids from the soil and/or re- tention of amino acids in the lettuce root. Compared to the result of Premuzic et al. (2001) who found that N or biosta- bilised compost fertilization does not change lettuce vita- min C content, we found that Megagreen fertilizer increased vitamin C content in lettuce and in this term suggest it as a better choice for lettuce treatment. The principal component analysis (PCA) based on ten tested compounds and three yield components enabled vi- sualization of correlations between analysed data. Principal component 1 (PC1) and principal component 2 (PC2) anal- yses provided signifi cant indications of 100% of the total variance in the data and showed clear separation of the treatments into three groups (control, Bio-algeen S90 and Megagreen). Based on the obtained results we conclude that applica- tion of either Bio-algeen S-90 or Megagreen in winter culti- vation conditions showed a signifi cant positive effect on the growth, head mass and yield of lettuce fresh leaves com- pared to the control treatment. The content of lettuce chlo- rophylls a and b and carotenoids was signifi cantly higher after Megagreen treatment compared to Bio-algeen S-90 treatment; however, compared to control plants, Megagreen treatment signifi cantly increased only chlorophyll b amount. The content of lettuce vitamin C and dry matter was signifi - cantly increased after both treatments (more after Bio-alge- en S-90 and less after Megagreen treatment). Both treat- ments signifi cantly decreased toxic nitrate content and the share of non-marketable yield in total lettuce yield. The pH value of lettuce juice decreased after Bio-algeen S-90 treat- ment. Mineral content (N, P, K) of lettuce leaves was not affected by either biostimulant or fertilizer application. Fi- nally, since the potent biostimulant Bio-algeen S-90 is a preparation derived from the northern Atlantic Ocean alga A. nodosum, and Croatia is a maritime state with various types of algae in its Adriatic sea, we propose investigation of Adriatic algae extracts as possible biostimulants. Acknowledgements The present research was not funded by any national or international institution, but the authors are grateful to the Polytechnic of Rijeka, Poreč Agricultural Department, to the Institute of Agriculture and Tourism in Poreč, Depart- ment of Biology at Faculty of Science, University of Za- greb and to the College of Agriculture in Križevci for mate- rial and infrastructural support given during the research. References Amanda, A., Valagussa, M., Piaggesi, A., Ferrante, A., 2009: Effect of biostimulants on quality of baby leaf lettuce grown under plastic tunnel. Acta Horticulturae 807, 407–412. 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