PRODUCTION, CHEMICAL COMPONENTS, AND CONTENT OF BIOACTIVE COMPOUNDS OF STRAWBERRY CULTIVARS

There are only a few strawberry cultivars available in Brazil and all are imported from North America and Europe. Thus, the introduction of new materials and their evaluation under Brazilian environmental conditions is imperative. The objective of this study was to evaluate the production, chemical components, and concentration of bioactive compounds in strawberry cultivars under edaphoclimatic conditions in the municipality of Pelotas-RS. The experiment was conducted in the field with low tunnels in 2011 and 2012. The experiment in the field was arranged in a completely randomized design, with eight treatments (cultivars), four replications. Each plot consisted of nine plants. The experiment in laboratory was arranged in a completely randomized 8x3 factorial design (8 cultivars and 3 months of harvest) and 4 replications. The variables analyzed were fruit number and fruit mass per plant, production, yield, soluble solid content, titratable acidity, ascorbic acid, anthocyanins, phenolic compounds, and antioxidant activity. Cultivar and harvest month influenced acidity, and the contents of soluble solids, ascorbic acid, and bioactive compounds. Cultivar Camarosa showed higher production, and higher contents of anthocyanin, phenolic compounds, and soluble solids. Cultivars ‘Palomar and ‘Aromas’ stand out for the content of ascorbic acid.


INTRODUCTION
According to estimates made by FAO, Brazil is not among the world's leading strawberry producers: an area of only 400 hectares is reported and annual production of 3,390 tons (FAOSTAT 2019).
The domestic strawberry production is almost all directed to the fresh fruit market (98%) (ANTUNES; PERES, 2013); however, it is still insufficient to supply the internal market due to the growing annual consumption. Furthermore, the strawberry cultivation is an activity of considerable economic importance because of its high profitability per area.
The choice of cultivars is one of the factors that largely determine the success of strawberry cultivation. Cultivars must be adapted to the edaphoclimatic characteristics of each region, especially temperature and photoperiod (DUARTE FILHO et al., 2007). Flowering and fruit setting of strawberry are decisively influenced by climatic conditions, cultural practices, and genetic characteristics of the cultivars. Thus, a highyielding cultivar in a certain region may not adapt to another environment and not result in high yields (PEREIRA et al., 2013).
Flowering, fruiting, and consequently the production of strawberries, as well as the sugar and acid content of the fruits are influenced by the cultivar, among other factors (CRESPO et al., 2010;PEREIRA et al., 2013;GUNDUZ;OZDEMIR, 2014). Possibly, genetics is also linked to other fruit quality characteristics and attributes related to healthiness, which are valued by the increasingly demanding consumer market.
In this sense, identifying cultivars that adapt to the growing conditions in the region of Pelotas-RS and produce consumer-appealing fruits is a challenge to be met. In this sense, this study aimed to evaluate production, physicochemical quality, and content of bioactive compounds in strawberry cultivars produced under the edaphoclimatic conditions in Pelotas-RS.

MATERIAL AND METHODS
The experiment was conducted in the field for two consecutive years, 2011 and 2012, in an area belonging to Embrapa Temperate Climate, Pelotas-RS, 31º40'S, 52º26'W; 60m. The climate of the region is subtropical mesothermal-humid (Cfb), with no dry season and mild winters (KÖPPEN, 1931). In 2011, rainfall ranged from 96.4 to 78.7 mm from July to October. In 2012, in the same months, the rainfall ranged from 106.9 to 162.8 mm (EMBRAPA 2014). The soil in the experimental area is classified as typic eutrophic Red Argisol (EMBRAPA, 2006).
The field experiment was arranged in a completely randomized design, with eight treatments (cultivars) and four replications. Each plot consisted of nine plants. The experiment in laboratory was arranged in a completely randomized 8x3 factorial design (8 cultivars: Festival, Camarosa, Camino Real, Aromas, Portola, Albion, San Andreas, Monterey, and Palomar, and 3 months of harvest: August, September, and November), and four repetitions. A sampling was conducted by collecting 1 kg of fruit for each sample, of which four replications were used for the phytochemical analyses and three replications for the physicochemical analyses.
Analysis of Variance (ANOVA) and the F test (P≤0.05) were conducted with the data, and if significant, they were compared by the Tukey or Scott Knott test at 5% significance.
Plug strawberry transplants were purchased from the Patagonia region. Planting in 2011 included three short-day cultivars: Festival (University of Florida), Camarosa, and Camino Real (University of California); and five neutralday cultivars: Aromas, Portola, Albion, San Andreas and Monterey (University of California). In 2012, the same cultivars were evaluated, except that 'Festival' was replaced by 'Palomar'.
At the time of transplanting, in both years, the roots were pruned as described by Cocco et al. (2012). Transplanting was carried out as soon as cultivars were available, at different planting dates and, consequently, harvest dates also varied (Table  1).
Planting was done in the conventional system: in 20 m long, 1.1 m wide, and 0.2 m high raised beds, mulched by 40-µm-thick black polyethylene, for three rows of plants spaced 0.3 x 0.3 m, in a population of 62500 plants per hectare, covered by low tunnels of 100μm thick transparent lowdensity polyethylene held by polyvinyl chloride (PVC) hoops of 0.8 m maximum height.
The sides of the low tunnel were opened according to weather conditions. On sunny days, they were opened early in the morning (8:00 am) and in the late afternoon (5:00 pm) they were closed. On rainy days, the tunnels were kept closed. A drip irrigation system consisting of two drip lines was used to water the plants in the bed twice a week. Chemical fertilization was carried out according to the recommendations for the crop (SANTOS; MEDEIROS, 2003). The plants were fertigated twice a week with 50g of 18-18-18 KristalonTM. During the crop cycle, dry leaves, stolons, and fruits with disease symptoms were removed from the plants.
Phytosanitary control was carried out by monitoring plants (curative) using fungicides, insecticides, and acaricides recommended for the crop and registered with the Ministry of Agriculture and Livestock (MAPA), as symptoms appeared, throughout the crop cycle.
The harvest began in the first half of August in the 2011 crop and at the end of July in 2012 and lasted until the second half of January 2012 in the 2011 crop and until the last day of November in the 2012 crop. It was done twice per week. Strawberries were picked fully ripe, with 100% red color, weighed on a digital scale, and counted.
Production was obtained by weighing the total mass of the harvested strawberries. The mass obtained by summing all harvests during the experiment was divided by the number of plants in the experimental plot, consisting of nine plants, and the ratio between them represented the average production per plant. The results were expressed as fruit fresh mass per plant (g plant -1 ). Yield was estimated as t ha -1 and calculated as the number of plants per unit area, considering a population of 62500 plants ha -1 . The mean fruit mass (g) was calculated by dividing the total fruit mass plant -1 by the number of fruit plant -1 . The mean number of fruit was obtained by counting the strawberries and dividing by the number of plants in the plot.
The chemical evaluations, contents of ascorbic acid, phenolic compounds, anthocyanins, and the antioxidant activity were determined for strawberries produced only in 2012, in September, October, and November, with a 30-day interval between evaluations.
All analyses were performed at the Food Center of Embrapa Temperate Climate, Pelotas, RS.
Fruit were handpicked at random in the first hour of the morning. First, they were placed on a tray, then into plastic bags labeled with the cultivar name, stored in a Styrofoam box, and transported to the laboratory.
In the laboratory, initially, fruits were selected by discarding the rotten ones, and the leaves were removed. Afterwards, two samples were separated: one for the physicochemical analysis and the other for the analysis of bioactive compounds. The sample for the analysis of bioactive compounds was frozen at -18 ºC for the further analyses of anthocyanins, phenolic compounds, and determination of the antioxidant activity. Soluble solids (SS), titratable acidity (TA), and ascorbic acid content were evaluated using fresh fruit.
Soluble solids, titratable acidity, and ascorbic acid content were determined using the juice centrifuged from 100g of fresh sample, with three replications. The soluble solids content was determined in a digital refractometer, with temperature corrected to 20ºC. A drop of pure juice was read and the result was expressed as º Brix. Titratable acidity was determined by the potentiometric method, with a 0.1 N NaOH solution until reaching pH 8.1 and was expressed as the percentage of citric acid (%). All determinations followed the methods described in AOAC (2000).
The SS/TA ratio was calculated (soluble solids content divided by the titratable acidity) for the different cultivars (BRACKMANN et al., 2011). The ascorbic acid content was quantified by titration of L-ascorbic acid with a 2,6-dichloroindophenol solution, according to the methodology of AOAC (2000).
To quantify the bioactive compounds and determine the antioxidant activity, the step of total extraction of the compounds (anthocyanins and phenolic compounds) was carried out first, with four replications.
In the extraction, a solution of 95% methanol acidified with a 1.5 M HCL was used in the proportion of 85:15.
To obtain the extract, the samples were centrifuged at -4 ºC until total separation of the supernatant. Then, the content of anthocyanins, phenolic compounds and antioxidant activity were quantified using the extract.
The quantification of total anthocyanins was performed using the methodology adapted from Fuleki and Francis (1968). Samples were read at 535nm in a spectrophotometer (Thermo Spectronic, Genesys 10uv). The results were calculated using a standard curve of cyanidin-3-glycoside and expressed as mg cyanidin-3-glycoside.100 g -1 sample (FULEKI; FRANCIS, 1968).
The total phenolic compounds were determined by the method described by Swain and Hillis, 1959. The phenolic compounds were quantified by means of a colorimetric reaction using the Folin-Ciocalteau reagent. Sample readings were taken in a spectrophotometer at 765nm, after 2 hours of reaction. The results were expressed as mg chlorogenic acid.100g -1 fresh fruit, which were obtained from a standard curve (SWAIN; HILLIS, 1959) The total antioxidant capacity was measured against the stable radical DPPH (2,2-diphenyl-1picrylhydrazyl), according to the method described by Brand-Williams et al. (1995). The reaction was carried out with a DPPH solution (0.044 g L -1 ) and the absorbance was measured at 515nm after 24h of reaction in a place protected from light, at room temperature (below 25°C). The antioxidant activity was determined with a standard trolox curve and expressed as mg trolox eq .100g -1 fresh fruit (BRAND -WILLIAMS et al., 1995).

RESULTS AND DISCUSSION
In the 2011 harvest, the strawberry cultivars Festival, Camarosa, Aromas, and Monterey were the most productive (Table 2), with average production ranging from 741.89 to 876.12 g.plant -1 . They were followed by the cvs. Portola, Camino Real, and San Andreas (561.61 to 594.00 g.plant -1 ), which were also not statistically different. The least productive cultivar was Albion (420.58 g.plant -1 ).
Results of this study obtained in 2011 are similar to the reports of Vignolo et al. (2011) for cvs. Camarosa (813.2 g.plant -1 ) and Camino Real (540.4 g.plant -1 ) from a research carried out in Pelotas-RS. However, Pereira et al. (2013), in a study conducted in the south of Minas Gerais (22°28'16''S, 46°08'42''W, 1,375 m altitude), found lower production during the productive stage. The authors reported the values of 322.30 g.plant -1 for cv. Festival, 372.06 g.plant -1 for cv. Aromas, and 358.06 g.plant -1 for cv. Camarosa, which is probably due to edaphoclimatic differences. Ruan et al. (2013) evaluatead neutral day cultivars and obtained 294.8 g.plant -1 for Albion, 333.6 g.plant -1 for Monterey, 309.1 g.plant -1 for San Andreas and 524.4 g.plant -1 for Portola, which, except for the similar results of Portola, the other cvs showed productions lower than those of this work.
In 2012, the production of strawberries was much lower than in 2011, with values among cultivars ranging from 137.97 to 381.5 g.plant -1, as well as the cvs showing different behaviors (Table  2). Cultivar Camarosa was the most productive (381.5 g.plant -1 ), followed by cv. Albion (263.86 g.plant -1 ), then Portola, Aromas, Palomar, San Andreas, which were not significantly different, varying from 137.97 to 207.19 g.plant -1 . The cvs. Monterey and Camino Real were the least productive in 2012, with production ranging from 60.11 to 73.55 g.plant -1 . The year 2012 was atypical for strawberry production in the region of Pelotas, with high rainfall, from 106.9 to 162.8 mm, from July to October, while rainfall in 2011 varied from 96.4 to 78 .7 mm (Table 3). It may also have been influenced by the winds that reached 75.6 km h -1 in September 2012 (EMBRAPA, 2014).   The cultivars that stood out for mean fruit mass in 2011 were Monterey, Portola, Camino Real, San Andreas, and Albion, varying from 16.26 to 17.88 g. Cultivar Festival produced a larger number of fruits and had a greater production, however, the fruits presented less mass, around 12.92g. Genetic characteristics and the management determine fruit size; it also depends on the fruit load, but there is not always a direct relationship between increased productivity and decreased fruit size and vice versa In 2012, cv. Albion had the highest fruit mass, with 16.67 g per fruit, but the others showed masses from 11.17 to 13.6 g, which were not statistically different. These masses are in agreement with the results of Resende et al. (2010) who reported means from 8.45 to 16.09 g per fruit for the cultivars Dover, Sweet Charlie, Camarosa, and Oso Grande. These results also corroborate Vignolo et al. (2011), who found 18.5 g for cv. Camarosa and 20.2g for cv. Camino Real. This demonstrates the influence of the genotype on the productive behavior of cultivars, in addition, the productive response is affected by climatic factors such as rainfall. In addition, the year 2012 was atypical for fruit production in the region of Pelotas, not only due to rainfall that was higher from July to October ( Table  3) -months of higher production -but also due to the incoming winds, which achieved 75.6 km/h in September 2012 and harmed inflorescences, bee activity, and, consequently, pollination.
The evaluation of the quality of the fruits in terms of soluble solids content, acidity, and the balance between sweetness and acidity (SST/ATT) showed an interaction (p≤0.05) between cultivar and harvest month (Table 4). Jouquand et al. (2008) also observed variation in the content of chemical components of strawberry between harvest months, showing that climatic factors may influence the production of these compounds. However, Crespo et al. (2010) and Gunduz and Ozdemir (2014) found a greater influence of the genetic factor on the production of these compounds compared to factors such as the production system and cultivation site.
The content of soluble solids (SS) and titratable acidity (TA) in strawberries were higher in November (°Brix from 7.8 to 9.95 and acidity from 0.70 to 0.93) than in September (°Brix from 7.2 to 8.7 and acidity from 0.69 to 0.80) and October (°Brix from 5.6 to 7.4 and acidity from 0.68 to 0.94) for the cultivars evaluated (Table 4). Except for the cultivar Albion (Soluble Solids and Acidity) and Portola (Acidity). This behavior can be explained by the progressive increase in light intensity and temperature from August to December in Rio Grande do Sul. Periods of greater light intensity and higher temperatures favor the metabolism of plants, inducing accumulation of sugars. This variable may also be related to the degradation of starch into glucose at the end of ripening, favored by environmental conditions, since temperature is an effective enzyme activator (reaction catalyst). Nevertheless, it was not possible to evaluate the chemical components in the cultivars Monterey, Camino Real, San Andreas, and Camarosa because the amount of fruit produced during the 2012 productive stage was not enough for the analyses, leading the authors to ponder what could have caused this reduction?
The balance between sweetness and acidity in the strawberry decisively influences fruit acceptance and can be examined by the ratio between the content of soluble solids and titratable acidity (SST/ATT). Among the cultivars, Palomar had the highest means for this variable in September (11.69) and October (10.42). In November, when light increases and, consequently, photosynthesis is favored, SST/ATT become higher and closer among cultivars (10.77 to 11.88), except for cv. Albion (9.71), but cv. 'Aromas' stood out.
The soluble solid content and the titratable acidity found in the literature for strawberries varies according to the cultivar, cultivation system, and climatic conditions prevailing in the production year. The means obtained in this work are consistent with the literature, with soluble solids content ranging from 5.8 to 10º Brix (ANTUNES et al., 2010;MASNY;ZURAWICZ, 2010) and titratable acidity from 0.40 to 0.98% (ANTUNES et al., 2010;PORTELA et al., 2012).
Recent studies have shown that bioactive compounds such as anthocyanins and phenolic compounds have therapeutic properties and high antioxidant potential (ALVAREZ-SUAREZ et al., 2014;KRUGER et al., 2014). Our results presented in Tables 5 and 6 show the influence of the cultivar factor on the content of ascorbic acid, anthocyanins, phenolic compounds, and antioxidant activity, as well as on the content of soluble solids and organic acids.
Ascorbic acid is a water-soluble compound, has antioxidant activity and plays an important role in the human immune system. Table 5 shows the values for this compound ranging from (42.33 to 81.08 mg.100mL -1 for strawberry cultivars evaluated during the 2012 production cycle. In the same manner, Singh et al. (2011) andCrespo et al. (2010) quantified the ascorbic acid content of strawberry cultivars and found a wide range between them. Crespo et al. (2010) reported ascorbic acid from 20 to 70 mg.100g -1 In this study, cv. Palomar had the highest ascorbic acid content, ranging from 65.08 to 81.08 mg.100mL -1 in September and October, but in November, cv. Aromas showed the highest content (78.3 mg.100mL -1 ). At harvest, the fruits picked in October had the highest ascorbic acid content, followed by berries harvested in November and then in September. In the same way as the chemical components, quantification of ascorbic acid content in the cultivars Monterey, Camino Real, and San Andreas was not possible to due to lack of production in 2012, which was caused by plant depletion.
The content of anthocyanins ranged from 40.81 mg.100g -1 for cv. Aromas in October to 92.18 mg.100g -1 for cv. Camarosa in September (Table  6). Camarosa produced the greatest amount of compounds throughout the production cycle. Singh et al. (2011) also found variation in the content of anthocyanins among strawberry cultivars and reported values from 40.53 to 125.41 mg.100g -1 . Josuttis et al. (2012) recorded contents ranging from 24 to 88.1 mg.100g -1 for strawberries produced in different regions, agreeing with the findings of this study. Among the months of harvest, the highest content of this compound was found in November for most cultivars.
From September to November in southern Rio Grande do Sul there is increased sunlight incidence and plants synthesize anthocyanins to protect them from solar radiation, therefore, the increase in anthocyanin concentration. Cultivar Camorosa presented the same anthocyanin content throughout the production cycle.
The results for content of phenolic compounds and antioxidant activity are shown in Table 6. The contents of phenolic compounds ranged from 235.83 mg.100g -1 for Albion in October to 490.97 mg.100g -1 for Camarosa in September. In September and October, cv. Camarosa produced the highest concentrations of phenolic compounds, however, in November, it had the lowest concentration compared with the other cultivars. Josuttis et al. (2012) also found a wide range (152.6 to 320.95 mg.100g -1 ) for the content of phenolic compounds in strawberry cultivars produced in four regions. The antioxidant activity ranged from 842.97 mg .100g -1 for Aromas in October to 3752.62 for Albion in September.
In September, during the harvest, the content of phenolic compounds in strawberries was 20% to 40% higher than the contents measured in October and November. The antioxidant activity measured in September was 3 to 4 times higher than in October and November. However, Oliveira et al. (2009) observed different behavior for strawberries of cultivars Camarosa and Festival during the productive stage, from October to December. They found an increase from 20% to 30% in the content of phenolic compounds over the harvest period. CARVALHO, S. F. et al.

CONCLUSIONS
• Cultivar and harvest month influence fruit quality in terms of soluble solids, acidity, ascorbic acid content, and bioactive compounds content.
• The cultivars Palomar and Aromas stand out with respect to ascorbic acid content.
• In the edaphoclimatic conditions of the region of Pelotas, cultivar Camarosa stands out for the production of strawberries and the contents of anthocyanins, phenolic compounds, and soluble solids.
• The contents of ascorbic acid, phenolic compounds, and anthocyanins may vary during the strawberry production cycle depending on the climatic conditions and respond differently to environmental factors, since the highest production of phenolic compounds was found in September, of ascorbic acid in October, and anthocyanins in November.

DECLARATION OF INTERESTS
The authors declare that they have no known