Plant Physiology and Biochemistry 74 (2014) 1e8

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Research article

Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens B. Ramos-Solano*, A. Garcia-Villaraco, F.J. Gutierrez-Mañero, J.A. Lucas, A. Bonilla, D. Garcia-Seco University CEU San Pablo, Facultad de Farmacia, Ctra. Boadilla del Monte km 5.3, 28668 Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 September 2013 Accepted 22 October 2013 Available online 4 November 2013

The aim of this study was two-fold: first, to characterize blackberry fruits from Rubus sp. var. Lochness along the year, and secondly, to evaluate the ability of a Pseudomonas strain (N21.4) to improve fruit yield and quality under field conditions in production greenhouses throughout the year. The strain was root or leaf inoculated to blackberry plants and fruits were harvested in each season. Nutritional parameters, antioxidant potential and bioactive contents were determined; total fruit yield was recorded. Blackberries grown under short day conditions (autumn and winter) showed significantly lower
Brix values than fruits grown under long day conditions. Interestingly, an increase in fruit
Brix, relevant for quality, was detected after bacterial challenge, together with significant and sustained increases in total phenolics and flavonoids. Improvements in inoculated fruits were more evident from October through early March, when environmental conditions are worse. In summary, N21.4 is an effective agent to increase fruit quality and production along the year in blackberry; this is an environmentally friendly approach to increase fruit quality. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Blackberry Bioactives Plant growth promoting rhizobacteria Elicitation Pseudomonas

1. Introduction Worldwide commercial production of blackberry (Rubus sp.) is estimated to be approximately 154,578 tons annually (Strik, 2007), North America and Europe being the biggest producers, with increasing surface all over the world. Blackberries are consumed as fresh fruits, frozen or derived products such as juices, concentrated products or for dietary supplements (Kaume et al., 2012). On the other hand, berries are gaining importance in our diet because of their benefits for health, especially in terms of preventing disease rather than healing (Paredes-Lopez et al., 2010). Such a market potential calls for sustainable agricultural practices that are environmentally friendly and safe for human health. Blackberry (Rubus sp.) is an aggregate fruit, composed of small drupelets, belonging to the Rosaceae family, especially rich in plant polyphenols (Kaume et al., 2012). One subclass of plant polyphenols are flavonoids, a large group that includes anthocyanins, known by being strong natural antioxidants, together with carotenoids, ascorbate and tocopherols (Moyer et al., 2001); however, benefits of flavonoids for health are beyond a simple antioxidant benefit (Martin et al., 2013). Due to the high polyphenol * Corresponding author. Tel.: þ34 913 726 411; fax: þ34 913 510 496. E-mail addresses: [email protected], [email protected] (B. Ramos-Solano). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.029

concentration and diversity, berry fruits including blackberries, are increasingly often referred to as natural functional foods (ParedesLopez et al., 2010) and show a broad spectrum of health-promoting effects when delivered through the diet (Kaume et al., 2012). Secondary metabolism aims to improve adaptative capacity of the plant to changing environmental conditions. Consequently, since polyphenols are secondary metabolites, their levels change according to environmental conditions (Poulev et al., 2003; Boué et al., 2008). Therefore, the dose of flavonoids present in the fruit changes, difficulting the establishment doseeresponse ratio. This lack of reproducibility in the fruit composition may be overcome by means of elicitation that is, triggering plant’s metabolism with elicitors (Capanoglu, 2010; Mañero et al., 2012). So far, elicitors have been grouped into two distinct blocks abiotic factors (light intensity, temperature, and chemicals) and biotic factors (pathogenic bacteria, beneficial bacteria, fungi and insects) (Millet et al., 2010). Biotic elicitation, with plant growthpromoting rhizobacteria (PGPR), is proposed as a useful strategy to improve biomass production and to trigger secondary metabolism at the same time (Gutiérrez Mañero et al., 2003; Zhang et al., 2004). Upon recognition of the non-pathogenic biotic agent by the means of their MAMPs (microbe associated molecular patterns) (Erbs and Newman, 2012), a series of metabolic changes are systemically initiated throughout the plant to activate the plant’s

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innate immune system (Jones and Dangl, 2006; Van Wees et al., 2008). The use of PGPR as elicitors to trigger secondary metabolism has a double advantage. First, defensive metabolites are bioactive compounds and therefore, elicited edible plant species constitute food products with an added value for human health; second, from a physiological point of view, these metabolic changes indicate that the biotic agent would be priming the plant, and therefore, it would provide protection upon stress challenge (Conrath, 2011). Despite simultaneous induction of growth and accumulation of secondary metabolites is rare in nature, the use of selected PGPR bacteria or certain bacterial components (MAMPs), to increase levels of secondary metabolites has been demonstrated in various studies as in Digitalis lanata for cardenolides (Mañero et al., 2003), in Glycine max for isoflavones (Ramos-Solano et al., 2010), or in Arabidopsis thaliana to enhance defense against pathogens (Van Wees et al., 2008), or others. In view of the above, there is a great interest in determining effectiveness of PGPRs as biological agents to obtain consistent and reproducible improvement on secondary metabolism. These improvements will result in an increased food quality, with consistent high bioactive content, together with high production rates. On the basis of the foregoing, the objective of this study was to evaluate the ability of the PGPR strain Pseudomonas fluorescens N21.4 (CECT 7620), with a well contrasted background in growth promotion and induction of secondary metabolism, to improve blackberry production and quality along the year in field conditions. This strain has already shown its ability to improve production from July through October (García-Seco et al., 2013), so the first aim was to evaluate if the effect would be consistent in the four seasons. To achieve this goal, nutritional and bioactive contents will be evaluated in root or leaf inoculated plants and compared to noninoculated controls in four different greenhouses along the year. 2. Results Since the recorded data was abundant, a multivariate analysis was carried out initially in order to identify the most relevant factors in our experiment. Blackberries have a growth cycle that includes three physiological plant status: strong vegetative growth, flowering and fruiting, each part accounting for a third of the cycle. Flowering and fruiting overlap; first fruits appear very slowly at first (T1 ¼ 2e3 weeks), then there is a maximum fruiting period that lasts an average of four weeks (T2), and the end of fruiting that strongly decreases (T3 ¼ 2e3 weeks). Ordination of samples on the principal component analysis (PCA) carried out with all data recorded in the trial (Fig. 1) revealed that the most relevant factor in this ordination was the plant’s physiological status (T1, T2, or T3), since most samples from T2 grouped towards the lower values of axis I irrespective of the bacterial treatment or the season, and T1 and T3 towards the higher values of the same axis. The second factor conditioning this ordination was the season, with Autumn (A) and Winter (W) samples grouping towards the left side of the graph (lower values of axis I) and Spring (S) and Summer (Su) samples grouping towards the higher values of this axis, hence a strong environmental influence is shown. Finally, bacterial treatments hardly influence this separation, although leaf treatments (F) separate samples towards the higher values of axis II, while root treatments do the opposite, revealing that there is a difference based on the inoculation. Axis I accounted for 79.8% of variance and axis II accounted for 18.5%, indicating that the separation was reliable. This is further supported by the db-RDA analysis that reveals statistical significance of sample distribution. The loading factors that influence Winter and Autumn sample ordination separations are DPPH and pH, while total phenolics and
Brix are responsible for separation of summer and spring samples (Table 1).

Fig. 1. Principal components analysis (PCA) performed with data from nutritional and bioactive characterization of blackberries (pH,
Brix, citric acid content, antioxidant potential, total phenolics, total flavonoids and total anthocyanins) in different seasons (A, W, S, Su) at the three physiological moments of fruit production (T1: beginning, T2: maximum, T3: end) and with different treatments with (leaf (F) or root (R)-inoculated bacteria and non-inoculated control (CQ)).

Based on the above information, data from T2 (when fruit production of the plant was at its maximum) is presented to characterize blackberries along the year, and to compare the potential improvement in fruit quality attributable to bacterial treatments. Fig. 2 shows data from blackberry nutritional (Fig. 2a) and bioactive (Fig. 2b) characterization along the year. Blackberries have a pH value of 3 along the year. Fruits grown under short day Table 1 P-values from distance-based redundancy analysis (db-RDA) carried with data from nutritional and bioactive characterization of blackberries (pH,
Brix, citric acid content, antioxidant potential, total phenolics, total flavonoids and total anthocyanins) in different seasons (A, W, S, Su) in the three physiological moment of fruit production (T1: beginning, T2: maximum, T3: end) and with different treatments (leaf (F), root (R) inoculated bacteria and noninoculated control (CQ)). P-value Seasons A vs. Su A vs. S A vs. W Su vs. S Su vs. W S vs. W

0.001* 0.001* 0.001* 0.001* 0.005* 0.001* 0.002*

Treatments CQ VS R CQ VS F R VS F

0.037* 0.139 0.192 0.017*

Plant state T1 vs. T2 T1 vs. T3 T2 vs. T3

0.001* 0.001* 0.001* 0.003*

Interactions Treatments vs. plant state Treatments vs. season Plant state vs. season

0.003* 0.001* 0.001*

Significant differences (p < 0.05) are indicated by an asterisk.

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3. Discussion

Fig. 2. A) Nutritional (pH,
Brix, citric acid content, antioxidant potential) and B) bioactive characterization (total phenols, total flavonoids and total anthocyanins) of blackberries in different seasons (A, W, S, Su at the maximum fruit production period (T2) in non-inoculated control (CQ)). Different letters indicate significant differences between seasons according to LSD test (p < 0.05).

conditions (Fall and Winter) show significantly lower
Brix values (7
Brix) than fruits grown under long day conditions (11
Brix). Citric acid content is higher in Summer and Autumn (1.61%). Antioxidant potential is determined as the ability to block free radicals of DPPH and expressed as EC50. Since EC50 is the amount of blackberry extract needed to reduce to 50% the amount of free radicals of DPPH solution, lower values indicate higher antioxidant potential. Antioxidant potential is best in Summer (EC50 ¼ 3.63 mg/ mL), when EC50 values are lowest (Fig. 2a). Autumn and Winter samples show intermediate values while the least favorable results are found in Spring samples (8.0 mg/mL). Total phenolic content (Fig. 2b) ranges from 500 GAE/100 g FW in Autumn and Spring to over 600 mg equivalents gallic acid/100 g FW in Winter (663.6 mg equivalents of gallic acid (GAE) per 100 g of fresh weigh). All other bioactives evaluated follow a similar trend (Fig. 2b), peaking in Winter. Flavonoids range from 47 mg CE/100 g FW in Spring and Autumn to 73 mg equivalents of catechin (CE) per 100 g FW in Winter. Anthocyanins range from 160 mg equivalents of C3G per 100 g FW in Spring and Summer to 251.4 mg equivalents of cyanidin-3-glucoside(C3G) per 100 g FW in Winter.

Dietary consumption of flavonoids or other polyphenolics protect against the increasing incidence of cardiovascular disease, metabolic syndrome and age-degenerative diseases (Luo et al., 2008; Martin, 2013; Seeram, 2011). Therefore, increasing polyphenols in plant foods is of great interest, and dietary intake of plant polyphenolics are amongst government’s health concerns. To achieve this goal, a great effort has been made in the field of transgenic plants (Butelli et al., 2008), which has brought a huge understanding of plant metabolism. In addition to the use of transgenic and high yield varieties, a more immediate approach to increase polyphenolics in plant foods is the use of PGPR to improve yield and quality traits in horticultural food crops (Weyens et al., 2009; Esitken et al., 2010), simultaneously reducing application rates of chemical fertilizers (Adesemoye et al., 2009). In the present study, this approach was carried out in blackberry due to its high commercial value (Luby and Shaw, 2009) and bioactive contents with potential for human health. In a previous study, the ability of strain N21.4 to improve plant production and quality in Autumn was demonstrated (García-Seco et al., 2013) and the present study reports that this effect is maintained under different environmental conditions all over the year, with more marked effects under adverse conditions. When phytonutrients or bioactive compounds in plant foods are evaluated, one of the most limiting factors is the variability due to environmental conditions. This multivariate type of analysis has allowed us to identify the factors that determine the quality of blackberries to a greater extent, selected amongst a high number of variables that range from the environmental to the molecular level. When all these factors are considered simultaneously, changes following inoculation are evaluated in a more reliable way. Characterization of blackberries throughout the production period confirmed the high quality regarding the bioactive content and antioxidant potential throughout the year, overcoming contents of Vaccinium corymbosum (blueberries) (Giovanelli & Buratti, 2009), one of the most studied berries up to date (Giacalone et al., 2011; Yousef et al., 2013). Besides, according to these data, Rubus sp. var. Lochness shows higher total phenolic contents (500e600 mg/100 g FW) and anthocyanins (160e250 mg/100 g FW) than other Rubus spp. varieties grown under natural cycle (289.3 mg/100 g FW and 90 mg/100 g FW, respectively) (Benvenuti et al., 2004), or the same variety grown at higher latitudes (Rutz, 2012; Jordheim et al., 2011). Elicitation with P. fluorescens N21.4 further improved quality, being more effective the root inoculation over leaf treatment, indicating that a systemic induction of the plant’s metabolism is involved. These improvements focused on the fruit’s antioxidant potential, and bioactive contents (total phenolics, total flavonoids and total anthocyanins) and on
Brix, which helps quantify the quality of the fruit. Since one of the most outstanding improvements for the market in nutritional quality is the increase in
Brix, it was one of the target parameters to improve with elicitation. Flavor is derived from the interactive taste and aroma of many chemical constituents whilst Soluble Solids Content (SSC) and Titratable Acidity (TA) contribute to fruit flavor. High concentration of sugars and acids are required for good berry flavor (Kader, 1991). High acid with low sugar results in a tart berry, while high sugar and low acid results in a bland taste. When both are low, the fruit is tasteless (Kader, 1991). Treating blackberry plants with N21.4 resulted in significantly increased values in
Brix from November through March, and effects were especially relevant in fruits from root inoculated plants, harvested in Autumn, Winter and early Spring (Fig. 3). This increase supports the notion of the ability of PGPR to enhance sucrose contents by

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Fig. 3.
Brix in blackberries from root or leaf inoculated plants, at the beginning, maximum and end of the producing period, in the four seasons (Autumn Au, Winter W, Spring Sp, and Summer Su). Different letters (x, y, z) indicate significant differences between treatments in each sampling moment, between sampling moments (a, b, c) within each treatment or between treatments throughout the experiment (a, b) according to LSD test (p < 0.05).

increasing the plant’s photosynthetic efficiency. Other studies with PGPR reveal an increase of sugar accumulation as well as the suppression of classic glucose signaling (Zhang et al., 2008). The effect is especially marked under adverse conditions, when the bacteria are able to improve plant fitness (Domenech et al., 2007). Leaf

Fig. 4. Fruit yield. Total fruit yield (g/plant) 1a) under the different treatments with N21.4. Treatments: C: Non-inoculated controls with chemicals; R: root inoculation; F: Leaf spray inoculations.

inoculation was not as effective increasing
Brix and produced marked fluctuations probably due to interaction with other factors like environmental pathogens. The suggested rise in photosynthesis could be interpreted as an increase in flowering buds (data not shown), which would result in enhanced fruit production per plant (Fig. 4) (þ33%). Data from total fruit production in each season shows that the beneficial effects of the inoculation is best when environmental conditions are not favorable that is when the plant needs additional support. When environmental conditions are naturally favorable, for example during Spring, the bacteria does not inhibit the plant’s genetic ability to produce fruits. The controls and inoculated plants achieve the same yields (5 kg/plant) (Fig. 4). This beneficial effect in yield, limited only to adverse conditions, extends to bioactive contents. Improvements in total phenolics (Fig. 5) and flavonoids (Fig. 6) are found from October through March in two ways: first, significantly increased values are detected in treated fruits except at T2 in Winter, and secondly, both showed milder changes along the year in inoculated plants while controls change more markedly following environmental changes. Anthocyanins were not affected by any treatment (data not shown). Leaf inoculation is not as effective increasing any of the evaluated parameters, causing even significant decreases in some of them. Although at first glance this treatment seems to be inappropriate for field inoculation, a more detailed metabolic study could reveal changes in other phytonutrients not considered in this study, as it has been reported that other metabolic pathways have

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Fig. 5. Total phenolic contents (mg of gallic acid equivalents per 100 g fresh weight (FW) in blackberries from root or leaf inoculated plants, at the beginning, maximum and end of the producing period, in the four seasons (Autumn Au, Winter W, Spring Sp, and Summer Su). Different letters (x, y, z) indicate significant differences between treatments in each sampling moment, between sampling moments (a, b, c) within each treatment or between treatments throughout the experiment (a, b) according to LSD test (p < 0.05).

been triggered following a stimuli (Davies, 2007). In addition, this suggested transformation to other phytonutrients is consistent with the role of secondary metabolism in plant defense: some compounds are accumulated as phytoanticipins to be later converted into phytoalexins upon stimulation by stress (Boué et al., 2008; Algar et al., 2012). Considering globally the changes caused by N21.4, a trend to increase and stabilize total phenolics and total flavonoids with no changes in anthocyanins is demonstrated. It is beyond any doubt that the strain has triggered plant metabolism causing a systemic induction, although the targeted control points of plant metabolism are not clear. In line with these results, studies with transgenic plants have demonstrated that significant increases in a given metabolite like flavonoids (Luo et al., 2008) or anthocyanins (Butelli et al., 2008) are achieved when the whole metabolic pathway is affected rather than a single gene; this would explain the general increase caused by N21.4 detected in blackberry. On the other hand, there are limiting steps controlled by key enzymes like PAL in tomato (Bate et al.,1994) or CHS in Arabidopsis (Burbulis and Winkel-Shirley, 1999) and tomato (Verhoeyen et al., 2002) which may be controlled by different transcription factors (Butelli et al., 2008), and the reported competition between different branches in a metabolic pathway (Davies, 2007; Ruhmann et al., 2013) support the positive effects on total phenolics and flavonoids and no effects on anthocyanins. Connecting the metabolic competition with the MAMPs (elicitors) able to differentially trigger some key enzymes (activity and expression), and the role of these compounds in plant defense, our

results support that N21.4 is able to cause a systemic induction of the plant metabolism which shows at least in the phenylpropanoid pathway, although the mechanism has not been identified yet. Practical results are high-quality blackberries with improved contents in total phenolics and flavonoids, provided within a consistent range. In addition, fruit production is increased in already high yield varieties in the framework of an environmentally friendly agriculture. 4. Materials and methods 4.1. Plant material Blackberries from the tetraploid hybrid Rubus var. Lochness, also known as Rubus sp. var. Lochness, (Jennings and McNicol, 1989) were used. This is a high yield thornless variety. All assays were carried out at production fields of the company Agricola El Bosque (Lucena del Puerto, Huelva, Spain). Plants and greenhouses were kindly provided by the company and all were handled according to regular agricultural practices. Four different groups of plants were used: plants grown from July to November 2010, fruiting in Autumn; plants grown from August 2010 to January 2011, fruiting in Winter; plants grown from October 2010 to early June 2011, fruiting in Spring; and plants grown from December 2010 to late June 2011, fruiting in Summer. All were grown under natural light conditions; all of these plants undergo an artificial cold period before transplanted to the greenhouses. Blackberries have a growth cycle that includes three physiological plant status: strong

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Fig. 6. Total flavonoid contents (mg catechin equivalents (CE per 100 g fresh weight (FW) in blackberries from root or leaf inoculated plants, at the beginning, maximum and end of the producing period, in the four seasons (Autumn Au, Winter W, Spring Sp, and Summer Su). Different letters (x, y, z) indicate significant differences between treatments in each sampling moment, between sampling moments (a, b, c) within each treatment or between treatments throughout the experiment (a, b) according to LSD test (p < 0.05).

vegetative growth, flowering and fruiting, each part accounting for a third of the cycle. Flowering and fruiting overlap; first fruits appear very slowly at first (T1 ¼ 2e3 weeks), then there is a maximum fruiting period that lasts an average of four weeks (T2), and the end of fruiting that strongly decreases (T3 ¼ 2e3 weeks). The duration of each physiological status and the duration of the fruiting period varies depending on the season, but the three stages of fruiting always appear.

4.2. Bacterial strain The bacterial strain used was P. fluorescens N21.4 (Spanish Type Culture Collection accession number CECT 7620), a Gram negative bacilli isolated from the rhizosphere of Nicotiana glauca. It releases siderophores and chitinases, triggering defensive metabolism in Solanum lycopersicum (Ramos-Solano et al., 2010), A. thaliana (Domenech et al., 2007) and G. max (Algar et al., 2012). It also increases isoflavone contents in G. max (Ramos-Solano et al., 2010) and fruit yield in blackberry (García-Seco et al., 2013).

4.3. Inoculum preparation Bacterial strain was maintained at 80
C in nutrient broth with 20% glycerol. Inoculum was prepared by streaking strains from 80
C onto plate count agar (PCA) plates, incubating plates at 28
C for 24 h, and scraping bacterial cells off the plates into sterile

10 mM MgSO4 buffer. Optical density (600 nm) was adjusted to one. Inoculum was delivered to plants at 107 c.f.u./mL. 4.4. Experimental design The experimental area was defined within a blackberry production plot, arranged in 200 m long tunnels greenhouses, and each one covers two lines. Within one tunnel, one line was marked for root treatments (6 plants) (R) (n ¼ 6), another for leaf inoculations (6 plants) (F) (n ¼ 6). In each line, the same number of plants were marked as non-inoculated controls (CQ) (n ¼ 6), with regular chemical treatments; this experimental design was carried out in the 4 greenhouses used to evaluate bacterial effects. Therefore, a total of 72 plants (6 plants  3 treatments  4 seasons) were used in the trial. The chemical treatments used were devoted to plague control and are under a non-release policy agreement (products for control of Spodoptera littoralis, Botrytis cinerea and acaricides). Root inoculations were carried out by soil drench 500 ml of inoculum at 107 c.f.u./mL in water, watering was suspended before inoculation; leaf inoculations were delivered by spraying leaves with a 107 c.f.u./ mL bacterial suspension with wetting agents. One week after transplant to production greenhouses, plants were inoculated throughout the growth and fruiting period every two weeks until production finished. Fruits were handpicked and weighed throughout the production period to evaluate effects in yield. Fruit quality was assessed at three time points of the production period (beginning, middle and

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end, T1, T2 and T3, respectively) by the following parameters: pH,
Brix, citric acid content, antioxidant potential, quantitative determination of phenols, flavonoids and anthocyanins. Yield was recorded weekly and is presented as total yield per plant. 4.5. Fruit quality and production 4.5.1. Quality of fruit Blackberries were handpicked twice a week and sent at 4
C to the lab. Upon arrival, 240 g of blackberries (around 50 fruits) from each treatment were weighed and split in three replicates (n ¼ 3). Each replicate (80 g) was then powdered with liquid nitrogen and a hand mixer (Braun Multiquick5); this pool was used for nutritional characterization (pH and
Brix) and bioactive characterization (total anthocyanins, total phenolics, total flavonoids and antioxidant potential). 4.5.1.1. Nutritional parametres. For pH, citric acid and
Brix, 40 g of each replicate were centrifuged for 10 min at 4000 rpm. Determinations were done on supernatants from fresh fruits. 4.5.1.1.a. pH. pH was measured with MicropH2001 (CRISON) pHmeter. 4.5.1.1.b.
Brix. Brix grades were measured with Portable refractometer. 4.5.1.1.c. Citric acid. Citric acid content was measured following the AOAC (2000) official method 932.05 (2000), by acid-base evaluation with NaOH 0.1 N. 4.5.1.2. Bioactive characterization. 40 g of each replicate were extracted with 80% methanol (1/10 w/v), sonicated with an ultrasonic cleaner (Selecta 50 Hz) for 5 min, and centrifuged at 3.500 rpm for 5 min at 4
C. Determinations were carried out in the methanolic extract (for anthocyanin determination methanol was acidified 0.1% HCl). 4.5.1.2.a. Total phenolics. The phenolic content (TPC) was determined quantitatively with Folin-Ciocalteau reagent (Sigmae Aldrich, St Louis, MO) by colorimetry (Xu and Chang, 2007) with modifications, using gallic acid as a standard (SigmaeAldrich, St Louis, MO). One mL aliquot of the extract solution was mixed with 0.250 mL of a 2 N Folin-Ciocalteu reagent (SigmaeAldrich, St Louis, MO) and 0.75 mL of 20% Na2CO3 solution. The mixture was allowed to stand at room temperature for 30 min and absorbance was measured at 760 nm with a UVeVisible spectrophotometer (Biomate 5). A calibration curve was constructed with gallic acid (r ¼ 0.99). Results were expressed as mg gallic acid equivalent (GAE) per 100 g of fresh weight (FW). 4.5.1.2.b. Total flavonoids. Total flavonoid content was determined quantitatively by the aluminum chloride colorimetric assay (Zhishen et al., 1999), using catechin as a standard (SigmaeAldrich, St Louis, MO). One mL aliquot of the extract solution was added to 10 mL volumetric flask containing 4 mL of distilled water. 300 mL of 5% NaNO2 was added and after 5 min, 300 mL of 10% AlCl3 was added. After 1 min, 2 mL of 1 M NaOH was added and the total volume was brought to 10 mL with distilled water. The solution was mixed thoroughly and absorbance was measured against prepared reagent blank at 510 nm. A calibration curve was constructed with catechin (r ¼ 0.99). Total flavonoid content of fruit extracts was expressed as mg catechin equivalents (CE) per 100 g FW. All samples were analyzed in triplicate. 4.5.1.2.c. Total anthocyanins. Total anthocyanin content was determined quantitatively by the pH differential method of (Giusti & Wrolstad, 2001). Extracts were diluted 1:15 with pH 1.0 buffer (0.2 M KCl pH 1) and pH 4.5 buffer (1 M CH3COONa pH 4.5). Absorbance was measured at 520 and 700 nm in a UVeVisible spectrophotometer (Biomate 5). A calibration curve was

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constructed with cyanidin-3-glucoside POLYPHENOLS (NO) (r ¼ 0.99). Results were expressed as mg of cyanidin-3- glucoside/ 100 g FW. 4.5.1.2.d. Antioxidant potential: 1,1-diphenil-2-pricrilhidrazil (DPPH). The radical scavenging activity of Rubus spp. extract against DPPH free radical (antioxidant potential) was measured using the method of (Brand-Williams et al., 1995). The antioxidant potential EC50 (amount of blackberry extract needed to reduce to 50% the amount of free radicals of DPPH solution) was determined by a curve dilution made with different blackberry extract concentrations mixed with 0.1 mM DPPH in methanol 80%. The absorbance at 517 nm was measured after standing in the dark for 30 min. The control and blank were made with methanol. Since EC50 is the amount of blackberry extract needed to reduce to 50% the amount of free radicals of DPPH solution, lower values indicate higher antioxidant potential. 4.5.2. Yield measurement Total yield over the production period was determined by weighing fruits, which were handpicked twice a week. Weight was recorded weekly and is presented as total production per greenhouse. 4.6. Statistics Two different types of multivariate analyses were performed with CANOCOTM v4.5 software to evaluate the most relevant factors in our data: principal component analysis (PCA) and dbRDA. Firstly, ordinations provided by principal components analysis (PCA) were carried out with average values of the three replicates. Secondly, distance-based redundancy analyses (dbRDA, (Legendre and Legendre, 1998), were carried out with the following parameters: pH,
Brix, citric acid content, antioxidant potential, quantitative determination of phenols, flavonoids and anthocyanins. This analysis (db-RDA) begins with the calculation of distances among replicates (BrayeCurtis index), followed by principal coordinate analysis (PCoA), and finally, by redundancy analysis (RDA). db-RDA is a multivariate test of hypothesis which tests for the effect of treatments on a multivariate data table and indicates statistical significance of the ordination. Two-way ANOVA was performed to evaluate differences among the treatments for the following parameters: i)
Brix; ii) total phenolics, iii) flavonoids and iv) anthocyanins; all of them throughout all the sampling times in which the effect of the two factors (sampling time and bacteria treatment) and their interaction were analyzed using Statgraphics 5.1. When differences were significant, the LSD post-hoc test was also performed (Sokal and Rohlf, 1981). Acknowledgments AGL2009-08324, BES-2010-038057, AGRICOLA EL BOSQUE S.L. “LA CANASTITA”. Melanie Hofte for critical reading of the article. References Adesemoye, A.O., Torbert, H.A., Kloepper, J.W., 2009. Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microb. Ecol. 58, 921e929. Algar, E., Gutierrez-Manero, F.J., Bonilla, A., Lucas, J.A., Radzki, W., Ramos-Solano, B., 2012. Pseudomonas fluorescens N21.4 metabolites enhance secondary metabolism isoflavones in soybean (Glycine max) calli cultures. J. Agric. Food Chem. 60, 11080e11087. Bate, N.J., Orr, J., Ni, W., Meromi, A., Nadler-Hassar, T., Doerner, P.W., Dixon, R.A., Lamb, C.J., Elkind, Y., 1994. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco

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