Effects of Local CO2 Enrichment on Strawberry Cultivation during the Winter Season

Carbon dioxide (CO2) concentration is one of the most important environmental factors in crop production. Within the range of suitable CO2 concentration for crops, an increase of CO2 concentration results in an increase in net crop photosynthesis (Cure and Acock, 1986; Keutgen et al., 1997) and thereby promotes photosynthate translocation, which directly controls the crop yield and quality (Troughton and Currie, 1977; David et al., 2014). Therefore, in greenhouse horticulture, CO2 enrichment is commonly used to realize high crop productivity (Kawashima et al., 2008). In strawberry production, CO2 enrichment has been practiced since the 1970s (Kato et al., 2015), and is used widely today in strawberry-cultivation greenhouses (Wada et al., 2010). In areas of scant solar radiation during the winter season, daytime enrichment of CO2 increased crop yield by 40% (Kawashima, 1991), while in areas of abundant solar radiation, early morning enrichment of CO2 increased crop yield by 16% (Shigeno et al., 2001). The effect of CO2 enrichment on increasing yield is well established; however, a normative CO2 enrichment strategy has not been determined (Mizukami et al., 2011). Moreover, a previous study revealed the ineffectiveness of industrystandard methods for enriching CO2 (Miyoshi et al., 2013). At our experimental site in the northern Kyushu area during the winter strawberry production season, CO2 was generally enriched in the greenhouse using a fuel-burning CO2 generator only early in the morning. CO2 concentrations inside the greenhouse began to increase with the beginning of CO2 enrichment and reached a maximum of approximately 2,000 ppm. However, beginning greenhouse ventilation to cool the greenhouse rapidly decreases the CO2 concentration inside the greenhouse. Consequently, the CO2 concentration decreases to outdoor levels before noon, when photosynthetic photon flux density (PPFD) becomes sufficient for strawberry crop photosynthesis. Thus, greenhouse ventilation renders CO2 enrichment ineffective with regard to increasing photosynthesis in the strawberry crop. In this study, we propose a new local CO2 enrichment system. The system enriches CO2 in close proximity to the crops and thereby enables efficient control of CO2 concentration even when the greenhouse ventilation system is active. We applied the system to a strawberry greenhouse with ventilation windows to examine the effect of this local CO2 enrichment. We also examined the effects of CO2 enrichment system on physiological functions such as photo-


INTRODUCTION
Carbon dioxide (CO2) concentration is one of the most important environmental factors in crop production. Within the range of suitable CO2 concentration for crops, an increase of CO2 concentration results in an increase in net crop photosynthesis (Cure and Acock, 1986;Keutgen et al., 1997) and thereby promotes photosynthate translocation, which directly controls the crop yield and quality (Troughton and Currie, 1977;David et al., 2014). Therefore, in greenhouse horticulture, CO2 enrichment is commonly used to realize high crop productivity (Kawashima et al., 2008).
In strawberry production, CO2 enrichment has been practiced since the 1970s (Kato et al., 2015), and is used widely today in strawberry-cultivation greenhouses (Wada et al., 2010). In areas of scant solar radiation during the winter season, daytime enrichment of CO2 increased crop yield by 40% (Kawashima, 1991), while in areas of abundant solar radiation, early morning enrichment of CO2 increased crop yield by 16% (Shigeno et al., 2001). The effect of CO2 enrichment on increasing yield is well established; however, a normative CO2 enrichment strategy has not been determined (Mizukami et al., 2011). Moreover, a previous study revealed the ineffectiveness of industrystandard methods for enriching CO2 .
At our experimental site in the northern Kyushu area during the winter strawberry production season, CO2 was generally enriched in the greenhouse using a fuel-burning CO2 generator only early in the morning. CO2 concentrations inside the greenhouse began to increase with the beginning of CO2 enrichment and reached a maximum of approximately 2,000 ppm. However, beginning greenhouse ventilation to cool the greenhouse rapidly decreases the CO2 concentration inside the greenhouse. Consequently, the CO2 concentration decreases to outdoor levels before noon, when photosynthetic photon flux density (PPFD) becomes sufficient for strawberry crop photosynthesis. Thus, greenhouse ventilation renders CO2 enrichment ineffective with regard to increasing photosynthesis in the strawberry crop.
In this study, we propose a new local CO2 enrichment system. The system enriches CO2 in close proximity to the crops and thereby enables efficient control of CO2 concentration even when the greenhouse ventilation system is active. We applied the system to a strawberry greenhouse with ventilation windows to examine the effect of this local CO2 enrichment. We also examined the effects of CO2 enrichment system on physiological functions such as photo- In strawberry production, carbon dioxide (CO2) enrichment is widely used to promote crop photosynthesis and to improve growth and yield of strawberry fruits. However, a standard CO2 enrichment method has not been established clearly. For sustainable strawberry production with high profitability, it is essential to establish an efficient system for CO2 enrichment. Therefore, in this study, we newly propose a local CO2 enrichment system. The system enriches CO2 only ambient to strawberry crops, not a whole greenhouse, and thereby enables efficient control of CO2 concentration. We applied this system in a strawberry cultivation greenhouse with ventilating windows and examined the performance of system. During the daytime on clear days with opened greenhouse ventilation windows, the proposed system stabilized the ambient CO2 concentration to strawberry crops at 800 ppm. Furthermore, when the day after CO2 enrichment was begun, strawberry crop photosynthesis and photosynthate translocation from source leaves were promoted as the effects of short-term CO2 enrichment. Nevertheless, the yields and quality of fruits was not affected by the system. It is considered that long-term CO2 enrichment thickened source leaves and thereby caused down-regulation of photosynthesis. Further consideration will be needed on effect of long-term CO2 enrichment on physiological function of crops.

System for local CO2 enrichment
For efficient CO2 enrichment inside the greenhouse, we propose a new local CO2 enrichment system utilizing the existing system described in our previous study for local environment control ; Figure 1 shows a schematic of this local CO2 enrichment system. The system was installed in a strawberry-cultivation greenhouse (11 m long 5 m wide 3 m high) located at the Kyushu University, Fukuoka, Japan, and consists of interrow air ducts (IAD; 0.1 m in diameter, 6 m in length, made of aluminum) that are connected to a blower (35 m 3 /min). CO2 is added to the IAD, which are punctured to disperse the CO2-enriched air in close proximity to the crops. The CO2 was supplied to the IAD by a gas cylinder of liquefied carbon dioxide, and CO2 concentrations inside the IAD were maintained at 1,000 ppm. Two treatments, CO2 enrichment (CO2 Duct) and natural CO2 conditions (No Duct), were carried out from January 27, 2015, when the primary axillary inflorence was flowered, to March 1, 2015, when the first flowering of the primary axillary inflorence was harvested. CO2 enrichment was applied from 7:00 to 17:00 under the control of timer switches (TB22101, Panasonic Co., Ltd., Japan). On clear days, the greenhouse ventilation windows were opened from 11:00 to 18:00.
Plant materials Strawberry plants (Fragaria ananassa Duch. 'Benihoppe') were grown in cultivation beds (6 m long 0.3 m wide 0.5 m high) in the greenhouse. At the beginning of June 2014, seedlings were planted in plastic pots (0.15 m diameter; 2.6 L volume) filled with mixed substrate (peat moss: coconut shells: charcoal 3:5:2 [v/v/v]) and placed on a seedling bench for growth. OK-F-1 nutrient solution (OAT Agrio Co., Ltd., Japan) with an electrical conductivity of 0.6 dS m 1 was supplied at a rate of 300 mL d 1 per plant at 9:00, 11:00, 13:00, 15:00, and 17:00. In the summer season, nutrient supplementation was halted to induce anthesis, with only water supplied. Seedlings were transplanted on October 19, 2014 to substrate-filled cultivation beds, with 20 cm intervals between plants and inter-row spacing of 15 cm, and were then grown under nutrient solution supplementation. Substrates and nutrient solutions were the same as those used for seedling cultivation, with approximately 3 L of substrate used per plant.

Measurement of environmental conditions
To analyze effectiveness of the local CO2 enrichment system during greenhouse ventilation, CO2 concentrations near the strawberry crops were measured during CO2 Duct and No Duct treatments. A sensor system (TR-76Ui; T&D Corporation, Nagano, Japan) containing a non-dispersive infrared (NDIR) CO2 analyzer and a built-in data logger with 10-min intervals was used to measure CO2 concentration in each cluster of strawberry crops during each treatment.

Measurement of leaf photosynthesis
To analyze the effect of the local CO2 enrichment system on leaf photosynthesis, we measured leaf photosynthetic rates during CO2 Duct and No Duct treatments. The measurement were carried out using a portable photosynthesis and fluorescence system (LI-6400XT, LI-COR Inc., USA) using a chamber head with a natural light window under ambient air temperature, CO2 concentration, and relative humidity conditions. Six plants were selected from each treatment for photosynthetic measurement, and the photosynthetic rates were measured for the third expanded leaf. Differences between treatments were tested for statistical significance using the Student's t-test at P 0.05.

Measurement of sucrose concentrations
To analyze the effect of local CO2 enrichment system on photosynthate translocation of strawberry crops, we measured the amount of change in leaf sucrose concentration, which represents the translocated photosynthate (Sutsawat et al., 2008), during the daytime from 6:00 to 18:00 and during the nighttime from 18:00 to 6:00 (Miyoshi et al., 2017). We collected samples at the source leaves of the strawberry, sampling from each third expanded leaf of the six plants in each treatment using a 6 mm diameter punch, while avoiding the major vein. We performed quantitative analysis on the collected samples using high-performance liquid chromatography (LC-20A, SHIMADZU CORPORATION, Japan); yielding the sucrose concentration per 1 mL of extracted solution. The molar concentration of sucrose per unit area of the source leaf was calculated as the acquired sucrose Environ. Control Biol. Fig. 1 Schematic diagram of the local CO2 enrichment system, which consists of inter-row air ducts (IAD) connected to a blower. CO2 is added into the IAD, which are perforated to disperse air into the ambient environment around the crops. CO2 enrichment was applied from 7:00 to 17:00.
concentration value divided by the molar mass of sucrose (342.3 g mol 1 ) and the area of the collected samples. Differences between treatments were tested for significance using the Tukey-Kramer test at P 0.05.

Analysis of growth and yield
To analyze the long-term effect of CO2 enrichment on strawberry growth and yield, we calculated the leaf area of fully expanded third leaves for six plants under each treatment, using the length (LL) and width (WL) of leaflets and the following equation described previously by : We also calculated growth characteristic parameters including specific leaf weight (SLW: an index of leaf thickness) for six plants under each treatment using the following equation described previously by Hidaka et al. (2015): Furthermore, to analyze the effect on yield, was harvested from six plants for each treatment. Differences between treatments were tested for significance using the Tukey-Kramer test at P 0.05. Figure 2 shows diurnal changes in CO2 concentration within strawberry crop clusters during CO2 Duct and No Duct treatments during clear days (Feb. 3, 2015) when the CO2 enrichment was begun before one week. During CO2 Duct treatment, CO2 concentration began to increase with the commencement of CO2 enrichment (7:00), and remained around 800 mol mol 1 in the proximity of the strawberry crop. At 11:00, when greenhouse ventilation windows were opened, CO2 concentration decreased rapidly to 600 mol mol 1 , but quickly recovered to 800 mol mol 1 . Then, after 11:00, while ventilation windows remained open, CO2 concentrations remained around 800 mol mol 1 . After the cessation of CO2 enrichment (17:00), CO2 concentrations decreased to 400 mol mol 1 .

Performance and effectiveness of the system
During the No Duct treatment, CO2 concentrations gradually decreased due to crop photosynthesis after sunrise, reaching a minimum of approximately 300 mol mol 1 at 11:00. After ventilation windows were opened, CO2 concentrations remained around 400 mol mol 1 . From these results, it is suggested that efficient CO2 enrichment can be made possible by using local CO2 enrichment systems in strawberry greenhouses in which window ventilation is necessary for avoiding air temperature increases. Figure 3 shows photosynthetic rates of fully expanded third leaflets under CO2 Duct treatment and No Duct treatment at 12:00 on a clear day (Feb. 3, 2015) one week after the beginning of CO2 enrichment. Photosynthetic photon flux density, air temperature, and relative humidity were relatively similar for both treatments at approximately 600 mol m 2 s 1 , 28°C, and 60%, respectively. CO2 concentrations were 800 mol mol 1 during the CO2 Duct treatment and 400 mol mol 1 during the No Duct treatment. Photosynthetic rates were 15 mol CO2 m 2 s 1 during the CO2 Duct treatment and 10 mol CO2 m 2 s 1 during the No Duct treatment. Photosynthetic rate was Vol. 55, No. 4 (2017) Leaf area 1.658 (LL WL) 2.495 SLW leaf dry weight per plant/leaf area per plant Fig. 2 Diurnal changes in CO2 concentration in close proximity to strawberry crops on a cultivation bed with CO2 enrichment (CO2 Duct) and on a cultivation bed without enrichment (No Duct) during a clear day (Feb. 3, 2015). The ventilation window of the greenhouse was opened at 11:00.  significantly higher during the CO2 Duct treatment, measuring 1.5 times the rate measured during the No Duct treatment. It is suggested that CO2 enrichment accelerated leaf photosynthesis. Figure 4 shows the daily amount of sucrose loading, which is the photosynthate translocation from leaves to phloem (Sylvie et al., 1999), under CO2 Duct and No Duct treatments on a clear day (Feb. 3, 2015) calculating by the equation described previously (Miyoshi et al., 2017). Daily amounts of sucrose loading were significantly higher during the CO2 Duct treatment, measuring 1.5 times the amounts measured during the No Duct treatment, similar to the differences in photosynthetic rate discussed above. High CO2 concentrations promote higher photosynthetic rate, causing an increase in photosynthate concentration in source leaf (Lemoine et al., 2013), and, in turn, high concentrations of photosynthate promote photosynthate translocation from source leaf (Ainsworth and Bush, 2011). Therefore, it is suggested that CO2 enrichment will promote sucrose loading from the source leaves of strawberry crops. Figure 5 shows the leaf area of the fully expanded third leaf under CO2 Duct treatment and No Duct treatment on March 1, 2015, one month after the beginning of CO2 enrichment. Leaf area was significantly larger during the CO2 Duct treatment, measuring 1.3 times the rate measured during the No Duct treatment. Figure 6 shows the specific leaf weight (SLW) of the fully expanded third leaf under each treatment on same day. SLW was significantly greater during the CO2 Duct treatment, measuring 2 times the rate measured during the No Duct treatment. On the other hand, the Brix of harvested fruit of first flower at apical inflorescences showed no significant difference between CO2 Duct and No Duct treatments (Fig. 7). Similarly, yield and number of fruits showed no significant difference between treatments (Fig. 8). Poorter (1993) reported that plant biomass increases under CO2 enrichment treatment. Lemoine et al. (2013) also reported that an increase in CO2 concentration should enhance photosynthetic rates and carbohydrate production, and have a positive effect on photosynthate transport and growth. In fact, most plants grown in high CO2 environments exhibit increased carbohydrate accumulation in leaves with biomass partitioning between source and sink organs (Makino and Mae, 1999). However, classically, there are two high-CO2 acclimation steps-short-term and long-term acclimation -and physiological functions such as photosynthetic rate and photosynthate translocation are initially enhanced only during short-term exposure; the boost of growth under high CO2 concentrations is not sustained long-term (Drake et al., 1997;Cheng et al., 1998). The short-term response to high CO2 concentration is an acclimation process whereby net photosynthesis, net carbon assimilation, and photosynthate translocation are enhanced. Excess photosynthate in source organs is only partly exported to sink organs via the phloem, and the resulting carbohydrate accumulation in leaves decreases the photosynthesis rate (Bae and Sicher, 2004). Furthermore, Hidaka et al. (2014) reported that long-term exposure of strawberry plants to the optimal environment for leaf photosynthesis induced down-regulation of leaf photosynthesis as plants limited the sink demand. From these previous researches, it is considered that one of the reasons for ineffectiveness of CO2 enrichment on Brix and yields is down-regulation of photosynthesis in source leaf caused by soluble sugar storage which is led to long-term enrichment of CO2.
In this study, we proposed a new local CO2 enrichment Environ. Control Biol.   system. The system enabled the efficient enrichment of CO2 in proximity to the crops during ventilation of greenhouse. Furthermore, photosynthesis of strawberry crop and sucrose loading were promoted because of high CO2 concentration immediately after the start of CO2 enrichment. However, marketable yield and degree Brix of harvested fruit showed no significant difference between CO2enriched and un-enriched conditions. As one of the reasons for ineffectiveness of CO2 enrichment on yiled and Brix, it is possibly considered that long-term CO2 enrichment leads to storage of excessive soluble sugar in source leaf, and, therefore, photosynthesis is down-regulated. In an effort to avoid the down-regulation of photosynthesis, previous studies have shown that the down-regulation of leaf photosynthesis in tomato plants only occurred when the number of sinks was considerably reduced under elevated CO2 treatment (Qian et al., 2012). Furthermore, Hidaka et al. (2016) suggested that adjusting the timing of initiation of CO2 enrichment to correspond with the sink strength of plants can increase fruit yield. For this new local CO2 enrichment system, further studies are necessary to adjust the timing and strength of CO2 enrichment and to promote photosynthate translocation to sink organs in order to increase fruit yield.