Interactions between changing climate conditions in a semi-closed greenhouse and plant development, fruit yield, and health-promoting plant compounds of tomatoes

Scientia Horticulturae 138 (2012) 235–243

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Interactions between changing climate conditions in a semi-closed greenhouse and plant development, fruit yield, and health-promoting plant compounds of tomatoes Dennis Dannehl a,∗ , Christian Huber a , Thorsten Rocksch a , Susanne Huyskens-Keil b , Uwe Schmidt a a b

Humboldt-Universität zu Berlin, Faculty of Agriculture and Horticulture, Division Biosystems Engineering, Albrecht-Thaer-Weg 1, 14195 Berlin, Germany Division Urban Plant Ecophysiology, Section Quality Dynamics/Postharvest Physiology, Lentzeallee 55/57, 14195 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 28 September 2011 Received in revised form 12 January 2012 Accepted 16 February 2012 Keywords: Carotenoids Phenolic compounds Antioxidant activity Fruit quality Fog cooling Solanum lycopersicum L

a b s t r a c t Climate change will lead to an excessive change in climate conditions in greenhouses, particularly during the summer. Therefore, a new climate strategy for greenhouses was developed to avoid plant damages. In this context, interactions between changing microclimatic conditions depending on different climate strategies and plant growth, fruit yield as well as secondary plant compounds were investigated between 2008 and 2009. The results showed that a combined application of a high pressure fog system and CO2 enrichment can be applied to decrease the inside temperature and to increase the levels of relative humidity and CO2 concentrations at a high ambient temperature, accompanied by an increase in mean temperature. Such microclimate in the greenhouse were sufficient to accelerate plant growth, to increase dry matter in leaves, and to promote the formation of fruit set per truss in comparison to those grown under conventional climate conditions. Furthermore, the algorithm of the new climate strategy led to a maximum total yield increase by 20%, to a reduction of blossom-end rot in tomatoes and to a pronounced increase in fruit size during the spring experiments. The climate conditions caused by the new technology significantly promoted secondary metabolism, resulting in a maximum increase in contents of lycopene (by 49%), ␤-carotene (by 35%), and phenolic compounds (by 16%) as well as associated antioxidant activity in the water-insoluble (by 18.5%) and water-soluble (by 35.4%) fraction compared to the conventional treated plants. Therefore, the new climate strategy may be appropriate to increase the total yield and to improve the fruit quality as well as the health-promoting properties of tomatoes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The tomato is exported all over the world and has enormous economic value for producers as well as numerous health benefits for consumers (Leoni, 2003). Epidemiological studies have demonstrated that the consumption of tomatoes can reduce the occurrence of human prostate cancer (Kotake-Nara et al., 2001) and several risk factors which are attributed to high levels of total cholesterol, e.g. cardiovascular diseases (Jacob et al., 2008). The reasons for these positive effects are associated with numerous different secondary plant compounds with antioxidant properties, which have a free binding character and can detoxify reactive oxygen species (ROS) (Bazzano et al., 2002). One of the most abundant secondary plant compounds in fully matured tomatoes is lycopene representing approximately 90% of the total

∗ Corresponding author. Tel.: +49 30 2093 46414; fax: +49 30 2093 46415. E-mail address: [email protected] (D. Dannehl). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2012.02.022

carotenoid content (Koskitalo and Ormrod, 1972), followed by ␤-carotene (7%) (Hermann, 2001). In addition, tomatoes contain other broad classes of substances with antioxidant properties, such as flavonoids and other phenolic compounds, particularly chlorogenic acid (Martinez-Valverde et al., 2002). These secondary plant compounds benefit human health and play a major role in plant responses to stress as well, i.e. they are involved in the plant defence against biotic stress (e.g. herbivore) and abiotic stress (e.g. UV-B irradiation and electric current). These sources of stress can lead to an accumulation of various phytochemicals in plants, e.g. phenolic compounds (Dannehl et al., 2009; Padmavati et al., 1997; Schreiner et al., 2009) and carotenoids (Becatti et al., 2009; Dannehl et al., 2011). However, it is well known that high temperatures can induce an overheating in tomatoes, which inhibits the lycopene biosynthesis (Dumas et al., 2003). Recently, a report from the German Advisory Council on Global Change was published with the prediction that the mean global temperature will be increased by 2 ◦ C by the end of this century (WBGU, 2008). This temperature increase will be accompanied by temperature extremes in summer and

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winter. Such climate change will lead to a change in climate conditions in greenhouses, particularly during the summer. That means that the mean temperature will excessively increase, whereas the relative humidity will be simultaneously reduced. On the other hand, the enhancement of carbon dioxide (CO2 ) in greenhouses will become less important, mainly because the ventilation have to be opened earlier to reduce the inside temperature. These future climate conditions in greenhouses could have an adverse effect on tomato plants, e.g. on photosynthesis, fruit yield, and phytochemical compounds with antioxidative properties (Camejo et al., 2005; Dumas et al., 2003; Van Der Ploeg and Heuvelink, 2005). To counteract these predictions, an innovative climate control strategy for greenhouses, named Descending-Fog-system (DescFog), with a new algorithm for the ventilation and for the combined application of a high pressure fog system and CO2 enrichment was developed at the Humboldt-Universität zu Berlin (Schmidt et al., 2008a). Until now, there is little information available on changes to plant growth and secondary plant compounds of tomatoes as affected by higher temperatures and higher levels of relative humidity in combination with high CO2 levels during growth are scant. Thus, the main objective of this study was to investigate the effects of an established climate control strategy and DescFog on vegetative growth, fruit yield, carotenoids, phenolic compounds, and antioxidant activity of tomatoes during several growing seasons.

2. Materials and methods 2.1. Experimental set-up The experiments were conducted with tomatoes in two separate cabins (115 m2 ) of an experimental Venlo-type greenhouse at the Humboldt-Universität zu Berlin (Germany), where the plants were grown under the same light conditions. One cabin, operated by conventional climate control strategy (reference) as used in practice (heating day/night, <18 ◦ C/<16 ◦ C; ventilation opening >25 ◦ C) was compared with a DescFog controlled cabin (heating day/night, <18 ◦ C/<16 ◦ C). In all experimental periods, the enrichment of CO2 was set at 900 ppm from 05.00 a.m. to 11.00 a.m. and at 700 ppm from 11.00 a.m. to 06.00 p.m. in both cabins. Additionally, only the DescFog system was equipped with a high pressure fog system (150 bar), including six fog nozzles which were fixed to the side walls at a height of 4 m. As such, the system produces very small droplets of demineralised water (10 ␮m) above the plants and thus ensures a uniform evaporative cooling. When the droplets evaporate, the cold air descended into the plants, whereas the energy-rich water vapour ascended to the roof. In order to maintain droplet evaporation, a coupled control for fog and ventilation was conducted by a microcontroller. The ventilation was opened with short time pulses at a relative humidity (RH) of 80% measured above plants and a minimum aperture (max. 10%) to remove the water vapour from the roof region. The fog system was deactivated at a RH of 80% in the plant population, in order to protect the plants against phytosanitary problems. When temperatures between 25 ◦ C and 27 ◦ C were measured in the plant population, the DescFog control was activated to maintain the high levels of CO2 . The highest limit for the semi-closed operation mode of DescFog was set at a temperature of 27 ◦ C to avoid plant damage (Camejo et al., 2005). The microclimatic data were recorded separately for each cabin and measured in 30 seconds intervals. The mean CO2 concentration, the mean RH, and the mean temperature in the plant population were calculated per week (wk) and expressed as ppm/wk, %/wk, and ◦ C/wk, respectively.

2.2. Plant cultivation, assessment of plant development and yield determination The investigations were conducted between 2008 and 2009. Each year was split into two experimental periods, i.e. spring (from March to June) and autumn (from August to November). Per cabin and experiment, 128 tomato plants (Solanum lycopersicum L. cv. Pannovy) were cultivated in rock-wool cubes and irrigated via drip irrigation. Before the plants were transferred to the respective experimental cabin, they were grown under the same conditions for a two month period. During the first six weeks, the plant development (n = 20 per cabin) was recorded weekly in the experimental cabins. The leaf length and leaf width were measured non-destructively with a folding ruler to determine the average leaf area growth per plant and week. The leaf area of each individual leaf was calculated using an exponential function which was developed in a series of separate experiments. The results are stated in square centimetre (cm2 ). Furthermore, the number of flowers and harvested fruit per truss of each plant was evaluated to investigate the effects of different climate conditions on the development of fruit set. The average fruit set per truss were calculated according to the ratio of the number of harvested fruit against the number of flowers; the results are stated in percent (%). The total fruit yield of each plant was weighed weekly to calculate the total yield for the respective experimental cabin. Another main objective of this study was to evaluate the quality of the tomatoes. As such, the weight of each fruit was categorised according to different weight classes: A-fruit > 70 g; 70 g ≥ B-fruit ≥ 50 g; C-fruit < 50 g. The weekly total yield per plant was used to determine the yield for the respective weight class. At the end of each experimental period, the total yield and their individual fruit weight characteristic are expressed as kilogram per plant (kg plant−1 ).

2.3. Sampling and analysis To investigate the effects of different climate strategies on secondary plant compounds during several growing seasons, tomatoes (>70 g) were randomly harvested with three replicates (n = 45) per cabin at ripening stage 9 (according to the Organisation for Economic Co-operation and Development, OECD colour gauge) from the fifth truss. As such, it was ensured that possible changes in the secondary metabolism in tomatoes were not affected by different light intensities as demonstrated by Brandt et al. (2006). Each tomato was quartered to determine different secondary plant compounds in the same fruit; one part thereof was immediately used to calculate the dry matter. The remaining tomato quarters were shock-frozen and stored at −20 ◦ C. One part of the frozen tomatoes was mixed to obtain a homogenous starting material and used to determine lycopene, ␤-carotene, and the antioxidant activity of water-soluble and the water-insoluble fraction. Another quarter was freeze dried for 48 h (Christ Alpha 1-4, Christ; Osterode, Germany) and afterwards ground and mixed into a fine, homogenised powder, which was used to analyse the total phenol content. To investigate the plant response to different climate conditions, the dry matter of the tomato leaves was determined. Therefore, the first fully developed leaf from the shoot tip of each tomato plant was cropped at the same time as the tomatoes were harvested. Three different sample collections of the cropped leaves (n = 80) per experimental cabin were dried with five replications (each 20 g) at 105 ◦ C for 24 h. Subsequently, the dry matter was calculated by the ratio of the dry weight to the fresh weight of the leaf samples and is expressed as % dry matter.

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2.3.1. Extraction and determination of the total phenol content Three replications of each freeze dried sample (0.2 g, n = 45) were used for the analysis of total phenol content in tomatoes. The samples were extracted following the method described by Connor et al. (2002) using acetic acetone (50% distilled water, 49.5% acetone, and 0.5% pure acetic acid; v,v,v) and subsequently analysed using the Folin–Ciocalteu method according to Slinkard and Singleton (1977). The modified procedure of this analysis was exactly described in detail by Dannehl et al. (2011). The results of total phenol content are expressed as milligram gallic acid per gram of dry matter (mg GAE g−1 DM). 2.3.2. Extraction and analysis of carotenoids The lycopene and ␤-carotene content were extracted following the method described by Fish et al. (2002). Five ml of 0.05% (w/v) butylated hydroxytuloene in acetone, 5 ml of 96% ethanol, 10 ml hexane, and 0.1 g CaCO3 were added to each sample (0.5 g, n = 45). This mixture was combined with 3 ml distilled water, homogenised (Ultra-Turrax T 25, Janke & Kunkel, IKA-Labortechnik; Staufen, Germany) for 1 min at 16,000 rpm and afterwards shaken for phase separation. The hexane layer was used to measure the carotenoid content by a spectrophotometer (Model 690, Gamma Analysen Technik GmbH; Bremerhaven-Lehe, Germany) at wavelengths of 505 nm (lycopene) and 453 nm (␤-carotene). The total lycopene and ␤-carotene contents were calculated as reported by Nagata and Yamashita (1992) and the results are expressed as milligram per gram dry matter (mg g−1 DM). 2.3.3. Sample preparation and analysis of the antioxidant activity The homogenous starting material described above was used to determine the presence of antioxidants in the extracts of phenolic compounds and carotenoids using the Trolox equivalent antioxidant activity (TEAC) assay, as described by Rohn et al. (2004). The phenolic compounds and carotenoids were extracted according to the modified method by Wang et al. (1996). To extract the phenolics, an aliquot of 0.5 g sample material (n = 45) was mixed vigorously for 20 s with 2 ml distilled water and centrifuged for 10 min at 4000 rpm. This procedure was repeated three times and the supernatants were collected and standardized to a final volume of 10 ml. In the following text, the phenolic extract is referred to as water-soluble fraction. The residue of the phenolic extract was used again to extract the carotenoids. The extraction was carried out as mentioned above, but with 7 ml acetone and with a final volume of 25 ml. Based on the solvent, the sample extract contains carotenoids as well as polyphenols and is in the following sections called as waterinsoluble fraction. To generate the ABTS radical cation and to start the reaction, 200 ␮l potassium persulfate (10 mmol/l in phosphate buffer, pH 7.2) was added to the sample mixture, containing an aliquot of 500 ␮l working solution ABTS (2,2 -azino-bis[3ethylbenzothiazoline-6sulfonic acid] diammonium salt, 0.5 mmol/l in phosphate buffer, pH 7.2; Sigma–Aldrich, Taufkirchen, Germany) and 100 ␮l diluted sample extracts. After exactly 6 min, the absorbance of all mixtures was measured at 734 nm. Trolox was used as standard and antioxidant activity in the water-soluble and water-insoluble fraction is expressed as millimol Trolox per gram dry matter (mmol Trolox g−1 DM).

Fig. 1. Influence of climate control strategies on microclimate conditions in greenhouses depending on different ambient conditions. Displayed parameters: global radiation, CO2 concentration, and ventilation (a); temperature and relative humidity (b).

prerequisites were not given, e.g. homogeneity of variance. All tests were performed at a significant level of p < 0.05. The principal component analysis (PCA) of several data sets with different examination parameters were evaluated with XLSTAT 2010. PCA is a helpful mathematical tool which reduces the dimensionality of a transformed data set. The results elucidate the connection of observations and relation of parameters generally by using only the first few principal components. The score plot was used to map the distribution of different observations of the data set (e.g. growing seasons), whereas the loading plot was used to explain a possible grouping as well as the importance and interaction of the variables (e.g. total yield and CO2 ). The score plot and the loading plot were presented in the same figure. One advantage of this multivariate data analysis is that the influence of all parameters can be observed simultaneously (Abdi and Williams, 2010). To provide comparable weights for all parameters, the collected data were autoscale preprocessed. Therefore, each variable was meancentred and variance was scaled to unity. For the PCA, the total yield and yield of different weight classes per week and the mean values of CO2 concentration, RH, and temperature per week had to be taken into consideration. 3. Results and discussion

2.4. Statistical analysis The effects of different climate strategies on tomato plants were calculated by using analysis of variance (ANOVA) with SPSS, package version 19.0. Significant differences were calculated using Tukey tests or using Games–Howell tests if the statistical

3.1. Microclimatic changes depending on different climate strategies Depending on different ambient conditions, the operating modes of the applied climate strategies are shown in Fig. 1. Based

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Fig. 2. Effects of climate control strategies on mean temperature, relative humidity, and CO2 concentration per week during different experimental periods: spring 2008 (a), autumn 2008 (b), spring 2009 (c), and autumn 2009 (d).

on the lower level of ventilation in the DescFog cabin, the temperature values were higher compared to the reference cabin during the morning and evening hours. Nevertheless, DescFog positively influenced the microclimatic conditions for plant growth at high ambient temperatures compared to the reference cabin. This means that the temperature could be lowered from 30 ◦ C to 27 ◦ C, whereas the RH was increased up to 30% and CO2 concentration up to 120 ppm at a maximum ambient temperature (Fig. 1 a,b). The new algorithm for the combined application of a high-pressure fog system and CO2 enrichment operated most effectively when the maximum global radiation did not exceed 700 W m−2 and the ventilation opening was temporarily opened up to a maximum of 40% (Fig. 1). In general, an increase in mean temperature per week was observed in the DescFog cabin compared to the reference cabin (Fig. 2). In this context, a maximum temperature difference of 1.7 K was found. In more detail, however, the prevailing daily mean temperatures in the cabins differed more from each other: the maximum difference ranged between 2.1 K and 2.4 K during the series of spring experiments as well as 1.4 K and 1.7 K during the autumn experiments, respectively. It was concluded that these effects were mainly caused by temporary temperature differences within both cabins, especially in the morning and evening hours. Furthermore, the mean RH was continuously maintained at 80% by the operation mode of DescFog, whereas the RH in the conventionally controlled cabin varied widely, especially during the experimental periods in spring (54–80%). At the same time, similar values were recorded in terms of CO2 levels (Fig. 2a and c) with a maximum mean CO2 difference of 177 ppm during the fruit production cycle in spring 2008. It was concluded that, based on the ventilation behaviour of the conventional climate strategy, the levels of RH and CO2 were

lowered. In contrast, the calculated levels of RH and CO2 for both cabins differed insignificantly during the autumn experiments, especially from October to the end of the productive period (Fig. 2b and d). The almost parallel climate conditions of the cabins resulted from infrequently periods of ventilation. 3.2. Effects of different climate conditions 3.2.1. Plant growth, fruit set, and fruit yield No significant differences were found with respect to the average leaf area growth per plant, which were grown under different climate conditions (Table 1). The results were calculated weekly for each experimental period and have shown that different levels of CO2 did not affect the already mentioned examination parameter. This fact is supported by an insignificant correlation between the respective average leaf area growth per plant and the mean CO2 levels in the conventional cabin (r = 0.20, p < 0.05) as well as in the DescFog cabin (r = 0.17, p < 0.05). In this context, the presented results were consistent with those of Reinert et al. (1997), who reported that different levels of CO2 had only a marginal impact on the leaf area per plant. Nevertheless, the average leaf area growth per plant and week tended to decrease from the third week until the end of the assessments under the influence of DescFog, where the leaves of tomato plants were decreased in size, but a higher number of trusses per plant were found (data not shown). Therefore, it was concluded that more leaves with a reduced leaf area were formed and this resulted in accelerated plant growth. It could be that higher RH and mean temperatures in the DescFog cabin were responsible for the described growth characteristics of tomato plants. Similar results were reported in other studies, which demonstrated a reduced leaf size and a faster plant growth of tomatoes under the

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Table 1 Average leaf area growth per plant and week depending on different climate strategies. Trial year

Climate strategy

Average leaf area growth per plant and week (cm2 )a Week 1

Week 2

Week 3

Week 4

Week 5

Spring 2008

Reference DescFog

1882 ± 731a 1797 ± 272a

2318 ± 461b 2751 ± 511b

2501 ± 779a 2169 ± 522a

2437 ± 464a 1713 ± 605a

2980 ± 977b 2243 ± 812ab

Autumn 2008

Reference DescFog

1902 ± 443a 1895 ± 465a

1906 ± 511ab 1953 ± 707ab

2765 ± 568a 2107 ± 340a

1889 ± 362a 1804 ± 648a

1908 ± 509ab 1689 ± 544a

Spring 2009

Reference DescFog

1951 ± 428a 1986 ± 676a

1796 ± 668ab 2002 ± 650ab

2579 ± 756a 2067 ± 410a

2287 ± 970a 1628 ± 207a

2282 ± 984ab 1847 ± 354a

Autumn 2009

Reference DescFog

1452 ± 257a 1507 ± 243a

1159 ± 318a 1282 ± 283a

2670 ± 728a 2150 ± 258a

2698 ± 690a 2030 ± 582a

2987 ± 942b 1970 ± 726ab

a The values were tested using Tukey-test and represent the mean of the leaf growth per plant and week (n = 20 per cabin) ± standard deviation. Different small letters indicate significant differences (p < 0.05).

influence of a low water vapour pressure deficit (VPD) (Armstrong and Kirkby, 1979; Cockshull, 1998). Adams et al. (2001) have found that an elevated mean temperature in greenhouses (22 ◦ C), which is comparable to that in the DescFog cabin, led to faster plant development and more produced trusses. Such observations could also explain the higher average leaf area growth per plant and week in response to a higher RH during the experimental periods in spring caused by the conventional climate strategy (r = 0.55, p < 0.05). In contrast, the examined vegetative growth parameter tended to decrease in correlation with increasing mean temperatures (r = −0.48, p < 0.05) in respect to the DescFog cabin, especially in the spring experiments. Therefore, it is assumed that the temperature was the limiting factor for the leaf area growth per plant and week when all measured environmental factors were considered. In general, the dry matter of the leaves was higher during the experimental periods in spring than in autumn (Fig. 3). Although the average leaf area growth per plant and week differed only to a small extent, it was found that the dry matter was significantly increased in all trial periods by influence of DescFog in comparison to CO2 enrichment without fog (Fig. 3). The highest dry matter increase was found during the experimental period in spring 2009 (113%), followed by autumn 2009 (110%), autumn 2008 (108%), and spring 2008 (107%). It is assumed that the photosynthesis and other metabolic activities, including an increased carbohydrate supply were benefited from higher mean temperatures and higher levels of CO2 as well as RH in the DescFog cabin resulting in an increased dry matter of leaves. These assumptions are supported by Schmidt et al. (2008a,b), who found highest photosynthetic activity at elevated levels of CO2 (700 ppm) and RH (80–90%) in a temperature

Fig. 3. DescFog induced changes of dry matter of tomato leaves and fruit (DM leaves: n = 80 and DM fruit: n = 45 per climate strategy). The calculated dry matter of leaves was tested using Games–Howell test and of tomatoes using Tukey-test and represent the mean of three replicates ± standard deviation. Different small and large letters indicate significant differences (p < 0.05).

range from 24 ◦ C to 27 ◦ C by using the same experimental set-up. Other studies showed a high photosynthesis and an increased dry matter per tomato plant at an elevated CO2 environment (Reinert et al., 1997; Vanoosten et al., 1995), where no adaptation of photosynthesis occurred (Dieleman et al., 2006).Regarding the effects of different climate controls on fruit set per truss, no significant differences were found within each experimental period, including year-to-year comparisons (Table 2). To be more specific, the climate conditions in the DescFog cabin positively influenced the formation of fruit set per truss compared to the prevailing conditions in the reference cabin, resulting in an average increase by 6% per truss and without the formation of parthenocarpic fruit. These results contradicted the findings from other scientists, who state that with increasing levels of RH, the pollination of the flowers is suppressed and the flower abortion is increased (Heuvelink et al., 2008; Mulholland et al., 2001). In the present study, it is assumed that a high level of RH can be considered critical if the temperature of the flowers falls below the dew-point temperature of the environmental air and droplets nebulised by the fog system do not evaporate completely before they reach the plant canopy. These conditions can lead either to a condensate formation on different segments of the flower or to a strong wetting of the whole flower, resulting in reduced pollination success. Furthermore, it might be possible that the reduced fruit set per truss, as developed in the reference cabin, was partly evoked by the temporarily higher temperature which increased up to 30 ◦ C (Fig. 1b). This explanation supports the results published by Sato et al. (2006), who demonstrated a reduced number of fruit set per truss at a temperature of 32 ◦ C compared to 28 ◦ C. PCA was used to investigate the influence of different environmental factors on total yield and different weight classes of tomatoes in more detail. The main objective was to compare different climate strategies depending on different experimental periods (Fig. 4). The results were displayed by principal component (PC) 1 and PC 2 and explain 67% of the data in the reference and 70% in the Descfog cabin. In both cases, a clustering of the experimental periods in spring and autumn was observed. The average total yields per plant depending on both climate strategies were on average twofold higher during the experimental periods in spring compared to those in autumn (Fig. 5). These results were associated with sub-optimal conditions of light and temperature during the autumn experiments, resulting in a reduced assimilate partitioning between the vegetative and generative parts (Adams et al., 2001; Newton et al., 1999). However, no year-to-year effect occurred in respect to the applied climate strategies (Fig. 4). The grouping of the growing seasons was only caused by the environmental factors temperature and CO2 . Regarding the Descfog and conventional cabin, the observations of the experimental period in autumn were described by high CO2 levels and in spring by high temperatures. These two environmental factors correlated negatively (Descfog:

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Table 2 Effects of different climate strategies on average fruit set per truss. Average fruit set per truss (%)a

Trial year

Climate strategy

First truss

Second truss

Third truss

Fourth truss

Fifth truss

Sixth truss

Spring 2008

Reference DescFog

94.5 ± 6.0a 97.5 ± 4.1a

92.1 ± 12.0a 91.6 ± 13.6a

90.8 ± 11.1a 93.8 ± 10.4a

93.6 ± 6.9a 96.0 ± 4.6a

92.3 ± 9.9a 96.6 ± 5.1a

94.0 ± 8.0a 95.1 ± 8.0a

Autumn 2008

Reference DescFog

96.5 ± 5.1a 98.3 ± 4.0a

93.8 ± 15.1a 98.3 ± 4.0a

79.3 ± 25.3a 94.6 ± 13.0a

98.0 ± 4.8a 97.5 ± 6.1a

93.6 ± 9.8a 96.6 ± 5.1a

86.5 ± 15.0a 90.1 ± 12.4a

Spring 2009

Reference DescFog

89.0 ± 6.6a 98.5 ± 3.6a

84.3 ± 7.0a 95.0 ± 8.9a

94.5 ± 10.0a 95.8 ± 7.0a

91.8 ± 9.8a 97.3 ± 4.1a

81.1 ± 9.4a 98.3 ± 4.0a

97.0 ± 4.6a 97.0 ± 7.3a

Autumn 2009

Reference DescFog

90.8 ± 7.8a 98.3 ± 4.0a

93.0 ± 10.8a 96.8 ± 4.9a

80.1 ± 22.2a 97.0 ± 7.3a

83.6 ± 24.4a 92.0 ± 6.7a

90.5 ± 23.2a 98.3 ± 4.0a

83.3 ± 16.1a 94.3 ± 8.7a

a The calculated fruit set per truss were tested using Tukey-test and the values represent the mean of 20 tomato plants ± standard deviation. Different small letters indicate significant differences (p < 0.05).

Fig. 4. Differences in PCA plots of the first two PCs between the weekly total yields and total yields of A-, B-, and C-fruit (>70 g, 50–70 g, and <50 g) influenced by conventional control (a) and DescFog control (b) based on their responses to the mean temperature, CO2 concentration, and RH per week (Ø Temperature, Ø CO2 , and Ø RH week−1 ). Conventional conditions (Ref); DescFog conditions (DF); experimental period in spring (Sp) as well as in autumn (A); years 2008 (8) and 2009 (9).

r = −0.72, p < 0.05; Reference: r = −0.88, p < 0.05) as a result of the ventilation opening at higher temperatures. In this context, it was found that the temperature affected the total yield and Afruit per week to a small extent, but comparable for both climate systems (ranging between: r = 0.46 and r = 0.56, p < 0.05), i.e. the yields tended to increase with the increasing mean temperature. In contrast, a high and positive correlation between temperature and B-Fruit was observed only through the application of Descfog (r = 0.73, p < 0.05). The RH influenced the yields in different ways

Fig. 5. Effect of different climate strategies on tomato yield at the end of each trial year (n = 128 per cabin). The yields were tested using Games–Howell tests and the bars represent the mean of yields per plant ± standard deviation. Different small and large letters printed as bold, bold/italic as well as plain/italic indicate significant differences (p < 0.05).

with respect to the applied climate strategies. In contrast to the reference cabin, the total yields and yields of A-Fruit per week tended to increase with high levels of RH in the Descfog cabin. This was derived from the correlation between RH and total yield (r = 0.51, p < 0.05) and A-Fruit (r = 0.45, p < 0.05) respectively. Furthermore, it was found that the weekly total yield and the yield of all weight classes correlated negatively with the CO2 concentration per week (Fig. 4a and b), however, to a lower extent by DescFog compared to the conventional climate strategy. These results were found due to the facts that: the yields and mean temperatures per week increased and the mean CO2 concentration decreased during the spring experiments (Fig. 2a and c); the yields and mean temperatures per week decreased and the mean CO2 concentration increased during the autumn experiments (Fig. 2b and d). These results indicated that the CO2 concentration in the DescFog cabin was the main limiting factor for a possible yield increase, whereas all investigated environmental factors in the reference cabin were below the optimal conditions to produce more tomatoes (particularly B-fruit) mainly during the experimental periods in spring. It was concluded that the combination of higher levels of mean temperatures, RH, and CO2 concentration in the DescFog cabin promoted photosynthesis, resulting in an enhanced assimilate supply, formation of fruit set, and a pronounced increase in fruit size as well as total yield. These assumptions were confirmed by the results at the end of each fruit production cycle. In all investigations, the total yield of the plants grown in the DescFog cabin was either significantly increased or showed at least an upward trend compared to the control plants, where the maximum differences occured in spring 2008 (20%) and in autumn 2009 (17%) (Fig. 5). Similar plant responses in terms of yield were

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demonstrated by the influence of a lower VPD (Bertin et al., 2000) and by an elevated mean temperature (Adams et al., 2001) as well as CO2 concentration (Reinert et al., 1997), which were similar to the climate conditions prevailing in the DescFog cabin. In addition, the DescFog system partially, but significantly, affected the quality of tomatoes, especially those which were produced in the spring experiments. The total yield of the weight class A (up to 17%) as well as B (up to 39%) was increased and the yield of the weight class C was decreased up to 32% compared to the reference cabin. Similar changes in volume of fruit growth were found when higher levels of RH (Leonardi et al., 2000; Mulholland et al., 2001) and an elevated mean temperature of 22 ◦ C (Adams et al., 2001) were present; this was comparable to the climate conditions in the DescFog cabin. The yields of blossom-end rot fruit were adversely affected by the influence of the conventional climate strategy (data not shown) and could be induced by low levels of RH accompanied by a calcium deficiency in the plant cells during the season with higher solar radiation (De Kreij, 1996). Regarding both climate strategies, only trend differences were found between different weight classes from October to the end of the autumn experiments due to minor deviations in climate conditions (Fig. 5).Finally, the fruit dry matter ranged between 4.2% and 6.3%. For all investigations, the DM of DescFog treated tomatoes was decreased significantly by 8% on average compared to the fruit grown under conventional conditions (Fig. 3). These results contradict those concerning the DM of leaves. Morandi and Grappadelli (2009) reported that the water content in fruit may decrease by epidermal transpiration and xylem backflow from the fruit to the leaves based on the water potential gradients. If large amounts of substances are removed from the fruit during these processes, then fruit shrinking may occur in different plants such as peaches (Morandi et al., 2007) and tomatoes (Leonardi et al., 2000). Leonardi et al. (2000) have demonstrated that a higher VPD led to a decrease in fresh weight by up to 10% due to fruit shrinkage which resulted in a reduction of fruit water content. Therefore, in the present study it was found that a lower VPD (DescFog control) increased the fresh weight of tomatoes with a higher water accumulation, but at the same time this process was accompanied by a decrease in DM (Bertin et al., 2000). However, a greater VPD (conventional control) might have induced small amounts of water stress resulting in a reduced fresh weight and an increased DM in the tomato fruit (Rosales et al., 2011). 3.2.2. Carotenoids, phenolic compounds, and antioxidant activity In the present study, lycopene, ␤-carotene, and phenolic contents as well as the antioxidant activity in the water-soluble and water-insoluble fraction in tomatoes were consistent with results reported in other literature (Bahorun et al., 2004; Frusciante et al., 2007; Raffo et al., 2002). In general, it was found that the contents of the studied parameters were higher in the spring experiments compared to the production period in autumn; this observation has already been discussed in Section 3.2.1. At the end of each experiment, the DescFog system significantly increased all analysed secondary plant compounds compared to the conventional operated system (Table 3). A multifactorial analysis of variance showed that the experimental period (p = 0.001, p < 0.05) as well as the applied climate strategy (p = 0.001, p < 0.05) were significantly responsible for all increasing values, but it could not be clearly determined which variable had the greater impact. In comparison to the conventional climate strategy, the maximum increase in lycopene (by 49%), ␤-carotene (by 35%), and phenolic compounds (by 16%) was found in tomatoes treated with DescFog during the experimental period in spring 2008, followed by spring 2009, autumn 2008, and autumn 2009 (Table 3). In this context, a high correlation was found between lycopene and ␤-carotene (r = 0.87, p < 0.05), which indicated that the biosynthesis of these

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two carotenoids was coherent. It is assumed that the higher daily mean temperature in the DescFog cabin (Fig. 2) essentially led to an increase in the biosynthesis of carotenoids, particularly that of lycopene in tomatoes. This supports Brandt et al. (2006) and Krumbein et al. (2006), who found that the lycopene content in tomatoes increased with increasing mean temperature and reached its maximum concentration at 25 ◦ C. On the other hand, it could also be possible that the temporary increase in temperature in the reference cabin evoked stress leading to a decrease in the biosynthesis of carotenoids. Similar results were reported by Dumas et al. (2003) and Lurie et al. (1996) at temperatures exceeding 32 ◦ C, where the ripening process, including the biosynthesis of lycopene, were strongly inhibited. In the present study, similar explanation with regards to the increase of carotenoids was given for the higher accumulation of phenolic compounds caused by DescFog. Raffo et al. (2006) and Toor et al. (2006) demonstrated that phenolic compounds (e.g. chlorogenic acid, rutin, and naringenin) increased with increasing mean temperatures from 22.4 ◦ C to 23.6 ◦ C or with mean temperatures ranging between 18 ◦ C and 19.8 ◦ C. These results once again support those found in the present study – an increase in mean temperature by 1 ◦ C or 2 ◦ C can affect the biosynthesis of phenolic compounds. However, the information regarding the effects of RH and CO2 on carotenoids and total phenol content in tomatoes is hard to come by. Leonardi et al. (2000) found that different levels of VPD did not affect the colour intensity and therefore, most likely did not affect the carotenoids in tomatoes. On the other hand, it might be possible that a much lower mean RH in the reference cabin led to a reduction in transpiration as well as a calcium deficit resulting in a decreased carotenoid content. In this context, it was shown that the lycopene content in tomatoes was increased when a high concentration of calcium was sprayed (Subbiah and Perumal, 1990), whereas no affect was detected regarding total phenol content in response to different calcium concentrations in the nutrient solution (Fanasca et al., 2006). Furthermore, the influence of different CO2 concentrations on secondary plant compounds remains controversial in literature (Islam et al., 1996; Krumbein et al., 2006; Wang et al., 2003), as it is difficult to determine a comparison of the various investigations. These results indicated that the enriched CO2 atmosphere in the DescFog cabin could also be responsible for an increase in carotenoid and total phenol content. Islam et al. (1996) have found that tomato fruit, which were exposed to a CO2 enrichment of 800 ppm, developed a more intense red colour than those exposed to only 400 ppm. In this context, the red colour of tomatoes and the amount of lycopene correlated highly during the ripening stage (Arias et al., 2000). In contrast, Krumbein et al. (2006) found that different levels of CO2 concentrations (348–997 ppm) did not affect the quantity of antioxidants (e.g. lycopene) in tomatoes. They concluded that no oxidative stress in plants occurred at an elevated CO2 supply, whereas a similar experiment showed that strawberries grown under CO2 enrichment conditions between 600 ppm and 900 ppm had higher contents of phenolic compounds and higher oxygen radical absorbance activity (Wang et al., 2003). Finally, the carotenoids and phenolic compounds are mainly responsible for antioxidant activity in tomatoes (Raffo et al., 2002). Therefore, there were good correlations between antioxidant activity in the water-insoluble fraction and lycopene (r = 0.74, p < 0.01) as well as ␤-carotene (r = 0.73, p < 0.01), including antioxidant activity in the water-soluble fraction and total phenol content (r = 0.71, p < 0.01). In comparison to the conventional climate strategy, the antioxidant activity in the water-soluble and in the water-insoluble fraction of tomatoes was significantly promoted by DescFog during the experimental periods in spring (Table 3). However, the accumulation of carotenoids and phenolic compounds caused by DescFog could not significantly increase the antioxidant activity during the autumn experiments. It may be possible that the latter

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Table 3 Effects of different climate conditions on secondary plant compounds and associated antioxidant activity in tomatoes. Trial year

Secondary plant compounds * (mg g−1 DM), ** (mg GAE g−1 DM)

TEAC (␮mol TROLOX g−1 DM)

Climate strategy

*

Lycopene

*

ß-carotene

**

Water-insoluble fraction

Water-soluble fraction

Spring 2008

Reference DescFog

1.18 ± 0.08bc 1.76 ± 0.06f

0.23 ± 0.02c 0.31 ± 0.02e

3.71 ± 0.10b 4.29 ± 0.09d

58.21 ± 2.11d 68.99 ± 1.85e

14.53 ± 0.99d 19.67 ± 0.66f

Autumn 2008

Reference DescFog

1.03 ± 0.03b 1.20 ± 0.09d

0.24 ± 0.01c 0.28 ± 0.01d

3.69 ± 0.11b 3.87 ± 0.09c

46.57 ± 0.67b 49.50 ± 0.80b

9.42 ± 0.63bc 10.57 ± 1.13c

Spring 2009

Reference DescFog

1.09 ± 0.08ab 1.44 ± 0.07e

0.23 ± 0.02c 0.27 ± 0.02d

3.73 ± 0.09b 4.23 ± 0.06d

53.64 ± 1.22c 63.08 ± 2.23d

14.30 ± 0.67d 17.33 ± 0.58e

Autumn 2009

Reference DescFog

0.91 ± 0.08a 1.03 ± 0.05b

0.17 ± 0.01a 0.20 ± 0.01b

3.44 ± 0.07a 3.65 ± 0.09b

32.53 ± 1.00a 34.17 ± 0.80a

Phenolics

7.32 ± 0.50ab 8.22 ± 1.30b

Contents are means of 45 tomatoes and three replicates ± standard deviation. Different small letters within a column indicate significant differences (p < 0.05). * unit of carotenoids and ** unit of the total phenol content.

result occurred due to the small differences in amounts of secondary plant compounds with respect to both climate strategies during this production cycle. Nevertheless, the highest antioxidant activity in the water-insoluble fraction was obtained using DescFog, which were increased by 18.5% in spring 2008, followed by spring 2009 (17.6%), autumn 2008 (by 6.3%), and autumn 2009 (by 5%) compared to the reference cabin. In consideration of DescFog, the antioxidant activity in the water-soluble fraction followed the same pattern, as previously discussed, resulting in an increase by 35.4%, 21.2%, 12.2%, and 12.1%, respectively. These results indicated that the tomatoes produced under the DescFog climate conditions possess more health-promoting properties as those produced by a conventional climate control strategy. 4. Conclusion The results showed that DescFog is a useful tool in greenhouses to counteract the predictions for climate change. As such, the new algorithm for the combined application of a high pressure fog system and CO2 enrichment can be applied to decrease the inside temperature and to increase the levels of RH and CO2 concentrations at a high ambient temperature, accompanied by a slight increase in mean temperature compared to a conventional climate strategy. In consideration of DescFog, it is assumed that the photosynthesis and other metabolic activities, including an increased carbohydrate supply were promoted by changes in microclimate conditions resulting in an accelerated plant growth and an increased dry matter in leaves. The new technology applied to tomato plants in greenhouses did not adversely affect the formation of fruit set per truss. The changed climate parameters caused by DescFog led to an increase in total yield as well as fruit size, whereas the occurrence of blossom-end rot in tomato fruit was reduced. This indicated that the quality of the fruit was improved compared to fruit grown under the conventional climate conditions. Furthermore, DescFog promoted the biosynthesis of carotenoids and phenolic compounds in tomatoes which most likely benefits human health. However, it could not be clearly determined which climate parameter had a greater impact on the secondary plant compounds. Thus, more detailed, further studies will have to be conducted in order to investigate the interactions between microclimate conditions and phytochemical compounds in tomatoes. Acknowledgement We would like to thank all the scientists and co-workers of Division Biosystems Engineering and Division Urban Plant Ecophysiology who supported our scientific findings during the experiments.

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