Environmental Pollution 196 (2015) 230e238
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Effects of ozone on growth, net photosynthesis and yield of two African varieties of Vigna unguiculata Rashied Tetteh a, Masahiro Yamaguchi b, 1, Yoshiharu Wada c, Ryo Funada d, Takeshi Izuta d, * a
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, 321-8505, Japan d Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2014 Received in revised form 8 October 2014 Accepted 9 October 2014 Available online
To assess the effects of O3 on growth, net photosynthesis and yield of two African varieties of cowpea (Vigna unguiculata L.), Blackeye and Asontem were exposed as potted plants to air that was either filtered to remove O3 (FA), non-filtered air (NF), non-filtered with added O3 of approximately 50 nL L1 (ppb) from 11:00 to 16:00 (NF þ O3) for 88 days in open-top chambers. The mean O3 concentration (11:00 e16:00) during the exposure period had a range from 16 ppb in the FA treatment to 118 ppb in the NF þ O3 treatment. Net photosynthetic rate and leaf area per plant were significantly reduced by exposure to O3, reducing the growth of both varieties. Exposure to O3 significantly reduced the 100-seed weight and number of seeds per pod. As a result, cowpea yield was significantly reduced by long-term exposure to O3, with no difference in sensitivity between the varieties. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ozone Cowpea Growth Net photosynthesis Yield
1. Introduction Tropospheric ozone (O3) is a major secondary air pollutant that is formed by a complex series of photochemical reactions from primary precursor emissions of nitrogen oxides (NOx) and volatile organic compounds (VOCs). Relatively high concentrations of O3 are linked to hot sunny weather and occur over wide areas (Ashmore, 2005). The global background level of O3 has more than doubled since the Industrial Revolution, and its peak values regularly exceed the 50 nL L1 (ppb) guideline of the World Health Organization in many parts of the world (WHO, 2006). The Intergovernmental Panel on Climate Change predicted that ambient levels of O3 may increase by 20e25% between 2015 and 2050 and by a further 40e60% by 2100 (IPCC, 2007). Ozone impairs plant metabolisms, leading to yield reductions in agricultural crops (Pleijel et al., 2006). O3 pollution could potentially pose a great threat to global food security by 2030 (Royal Society, 2008). Predicted yield losses for rice and wheat, the two
* Corresponding author. E-mail address: [email protected] (T. Izuta). 1 Present address: Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. http://dx.doi.org/10.1016/j.envpol.2014.10.008 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
most important food crops globally, are 4e8% and 9e18%, respectively (Van Dingenen et al., 2009). According to Bouarar et al. (2011), Africa is an important source region for O3 precursors. Large amounts of carbon monoxide CO, NOx and VOCs are emitted from burning biomass associated with savanna and forest fires, which take place during dry and monsoon periods over West and Central Africa, respectively, and with agricultural waste and domestic biofuel combustion (Sauvage et al., 2005). In West Africa, the World Meteorological Organization modelled monthly mean O3 concentrations around 36 ppb during daytime measurements (WMO, 2011). Current ambient levels of O3 were reported by Feng and Kobayashi (2009) to be high enough to reduce yields of cereals, grain legumes and tubers. Ainsworth et al. (2012) indicated that O3 reduces plant productivity by entering leaves through the stomata; within the leaves, it generates reactive oxygen species (ROS) and causes oxidative stress, which in turn reduces the photosynthesis rate, plant growth and biomass accumulation. According to Black et al. (2000) and Fiscus et al. (2005), O3-induced yield losses are often attributed to reduced photosynthetic activity and to lower supplies of assimilates to support reproductive development and seed growth. Although numerous studies have addressed the impacts of O3 on crops worldwide, limited efforts have been made to
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investigate these effects on tropical crops such as cowpea, one of the main food security crops in Africa, where it is widely cultivated. Cowpea (Vigna unguiculata L.) is an annual grain legume indigenous to tropical Africa (Padulosi and Ng,1997). This crop is the most commonly cultivated grain legume in Africa. It is one of the most important food legume crops in the semi-arid tropics of Asia, Africa, Southern Europe, and Central and South America. Cowpea is well adapted to high temperatures (i.e. 20e35 C) and drought compared with other crop species (Hall et al., 2002; Hall, 2004). Singh (2002) and Langyintuo et al. (2003) noted that cowpea plays a crucial role in the lives of millions of people in Africa and other developing countries, where it is a major source of dietary protein that nutritionally complements staple low-protein cereals and tuber crops and provides income for farmers and traders. Worldwide, annual cowpea production is estimated to be 5.7 million tonnes (MT) of dried grains, of which over 90% are produced in Africa (FAOSTAT, 2012). On the African continent, West Africa represents the largest production zone of cowpea (Pottorff et al., 2012). In Ghana, an average (2002e2009) of 156,000 ha is produced annually making this country the fifth highest producer of cowpea in Africa (MoFA, 2010). On per hectare basis, Addo-Quaye et al. (2011) reported cowpea grain yield ranging from 0.8 to 1.3 tonnes. Ghana has a warm climate owing to its proximity to the equator and is thus favourable for O3 formation, but atmospheric O3 monitoring and related research are limited (WMO, 2008). Over the past decade, the demand for agricultural land for food production has increased dramatically with the growing population, leading to more biomass burning through deforestation and illegal mining and significantly higher concentrations of O3 precursors (Bouarar et al., 2011). Moreover, relatively high concentrations of these precursors are generated in cities by vehicular emissions due to poor vehicle maintenance, industrialisation and the use of secondhand appliances such as refrigerators. The recent discovery of oil in Ghana is also likely to further increase these precursors and threaten food security, including cowpea production. Therefore, the effects of O3 on the growth, yield and net photosynthesis of African crops such as cowpea must be clarified. Adepipe and Tingey (1979) revealed that cowpea cultivars were more sensitive to short-term exposure to O3 at the three-leaf than at the two-leaf stage, while no consistent leaf injury was observed at less than 0.50 mL L1 (ppm) of O3 as compared with 1.0 ppm. Ozone increased stomatal diffusive resistance in all cultivars. Umponstira et al. (2006) reported that the biomass of cowpea was significantly lower and visible foliar injury of the plants exposed to 40 ppb and 60 ppb of O3 was significantly greater as compared to the control plants after 7-days of exposure. Similarly, Rangserodsombat et al. (2010) observed a significant O3-induced reduction in the soluble sugar and starch contents in leaf and root tissues relative to control plants. Malaiyandi and Natarajan (2014) reported that the acute exposure (i.e.15 min twice a day) of cowpea to O3 at 60 ppb increased dry weight, shoot length, leaf area, number of epidermal cells, number of stomata and total chlorophyll content. However, Umponstira et al. (2009) found that long-term exposure (74 days) of 40 ppb and 60 ppb of O3 significantly reduced total biomass, particularly in root dry weight, number of nodules, distribution of nodule size over 2 mm and nodule dry weight. Moreover, total nitrogen in plant tissues and nitrogenase activity were significantly reduced during the vegetative, flowering and harvesting stages of growth. At the present time, however, there is little information on the long-term effects of O3 on the growth and yield of cowpea or on varietal differences. The objectives of the present study were to assess the long-term effects of different concentrations of O3 on the growth, net photosynthesis and yield of two varieties of cowpea and to ascertain whether these effects differed between the two varieties.
231
2. Materials and methods 2.1. Plant material and growth conditions Two cowpea varieties, ‘Blackeye’ the white type and ‘Asontem’, which are the most widely cultivated varieties in Ghana, were obtained from the Department of Crop Science, University of Ghana (Legon, Ghana). The seeds were sown on 19 June, 2013, in mediumsized plastic pots (2-L) filled with sandy soil (Kanuma pumice soil) in three hills (two seeds per hill) and later thinned to one plant per pot. Hyponex compound liquid fertiliser (NPK 6-10-5; 4 mL in 1 L of water) was applied at a rate of 200 mL per pot 9 days after sowing and again at 2 weeks intervals on 15 July, 30 July and 13 August during the cultivation period. Plants were watered daily throughout the cultivation period. 2.2. Experimental design and growth chamber The experiment was conducted at the Fuchu experimental farm of Tokyo University of Agriculture and Technology (Fuchu, Tokyo, Japan) from 19 June to 27 September, 2013. The experiment used a randomised complete block design with nine open-top chambers (0.6 m 0.6 m 1 m high), each assigned to one of three gas treatments (3 replications). For each variety, we used four seedlings (eight plants per chamber). 2.3. Gas treatments The seedlings in the open-top chambers were exposed to air that was either filtered to remove O3 (FA), unfiltered (NF) or unfiltered and supplemented with approximately 50 ppb O3 generated by an electrical discharge generator (SO-03UN, Hamanetsu Co., Hamamatsu, Japan) for 5 h from 11:00 to 16:00 each day (NF þ O3). These treatments lasted 88 days, from 1 July to 26 September. The generated O3 was passed through a water trap to remove nitrogen by-products before being injected into the NF þ O3 chambers. Plants of each variety were rotated within the chamber every week and among chambers at 2-week intervals to minimise variation among plants and treatments. 2.4. Monitoring of climatic parameters and ozone concentrations The concentration of O3 generated was monitored each day with an O3 monitor (model 1150; Dylec, Ibaraki, Japan), and an O3 voltage recorder (VR-71; T & D Corp., Nagano, Japan) was used for O3 concentration measurements in all the chambers throughout the cultivation period. Air temperature and relative air humidity were monitored using a thermo recorder (TR-72Ui; T & D Corp.) comprising a sensor and logger. The accumulated O3 over a threshold of 40 ppb (AOT40) was calculated as the sum of differences between the hourly mean O3 concentration and 40 ppb when the hourly mean O3 concentration exceeded 40 ppb. 2.5. Measurement of growth parameters Plant height in centimetres, number of leaves per plant and SPAD value using a chlorophyll meter (SPAD-502; Minolta, Tokyo, Japan) were measured at 10-day intervals after 20 days of exposure to O3 until pod set. Although the relationship between SPAD value and leaf chlorophyll content is non-linear (Uddling et al., 2007), SPAD measurement is non-destructive hence we can know the seasonal change of ozone effect on chlorophyll concentration. Eleven days after sowing (30 June), plants were initially sampled and were sampled again at the end of the experiment (27 September). After sampling, each plant was separated into leaves, stem and roots, and
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oven dried at 80 C for 5 days to constant weight before the determination of plant biomass (i.e. leaf, stem, root and whole-plant dry masses, plus root-to-shoot dry mass (R/S) ratio). Leaf area per plant was measured with an area meter (AAM-8; Hayashi Denko, Tokyo, Japan) at both samplings. After 44 days of O3 exposure, few leaves were sampled and its leaf area and dry weight were measured to calculate SLA using the formula of Leopold and Kriedemann (1975). Number of days from sowing to first harvest was counted to assess the impact of O3 on cowpea life cycle.
its interaction with variety, two-way ANOVA was used to test the effects of gas treatment and variety. When a significant interaction between O3 and variety was detected, the Tukey's HSD test was performed to identify significant differences among the three gas treatments.
2.6. Measurement of yield and yield components
Table 1 indicates air temperature, relative air humidity, concentration of O3 and accumulated O3 exposure over thresholds of 0 and 40 ppb (AOT0 and AOT40, respectively) inside the open-top chambers during the 88 days of gas treatment (1 July to 26 September, 2013). The highest daily maximum air temperature was detected in August (39.3 C) and the minimum in September (20.2 C) (Table 1). The average daily relative air humidity in all the chambers was in the range of 44.9e96.8% during the experimental period. The climatic condition during the experimental period was similar to that during the growing period of cowpea in African countries including Ghana (Hall et al., 2002; Hall, 2004). The highest average O3 concentration between 11:00 and 16:00 was observed in the NF þ O3 treatment in July at around 118 ppb, while the FA treatment recorded the lowest value of 16 ppb in September. This trend was similar to the average O3 concentration over 12 h (between 6:00 and 18:00). The average hourly O3 concentration was in the range of 204 ppb in the NF þ O3 treatment in August to a minimum of 1 ppb in all the treatments in September. The highest AOT40 was detected in July (14.0 ppm h) in the NF þ O3 treatment.
3. Results 3.1. Environmental parameters
Dry pods from 12 plants per treatment per variety were harvested at maturity (from 28 August to 27 September) to determine pod length per plant, number of seeds per pod, 100-seed weight, number of pods per plant, yield per plant, yield per hectare and harvest index. The harvested pods were dried to constant weight. Seeds were separated from the pods and dried again before the measurement of the yield parameters. 2.7. Measurement of leaf gas exchange rates On 8e10 August, nine plants per treatment of each variety were selected for the measurements of net photosynthetic rate (A) and stomatal diffusive conductance to H2O (Gs) of the third or fourth fully-expanded leaves from the bottom using a portable photosynthetic measurement system (LI-6400; Li-Cor, Lincoln, NE, USA). During these measurements, atmospheric CO2 concentration, air temperature, relative air humidity, flow rate and photosynthetic photon flux density in the leaf chambers were maintained at 390 mmol mol1, 25 ± 0.5 C, 70 ± 5%, 650 mmol s1 and 1500 mmol m2 s1, respectively.
3.2. Visible foliar injury Fig. 1 shows O3-induced visible foliar injury in the two cowpea varieties. In Blackeye, injury was evident as chlorosis 3 weeks after the start of O3 exposure in the NF þ O3 treatment. In Asontem, visible foliar injury appeared as browning 2 weeks after the start of the NF þ O3 treatment. Notably, in the NF treatment of both
2.8. Statistical analyses Statistical analyses were conducted using the SPSS Statistics 21 (IBM, Chicago, IL, USA). Because we focused on the effects of O3 and
Table 1 Air temperature, relative humidity, average O3 concentration and AOT of O3 in each gas treatment during the exposure period in 2013. Period (2013)
Air temperature ( C)
Relative humidity (%)
24 h-Ave
12 h-Ave
1e31 July 1e31 August 1e26 September 1 July-26 September
29.0 29.8 25.9 28.4
33.3 34.1 29.4 32.4
Period (2013)
Gas treatment
1e31 July
1e31 August
1e26 September
1 Julye26 September
FA NF NF FA NF NF FA NF NF FA NF NF
(0.2) (0.2) (0.2) (0.2)
þ O3
þ O3
þ O3
þ O3
a
(0.4) (0.2) (0.5) (0.3)
Daily max 38.4 39.3 35.2 37.8
(0.5) (0.3) (0.9) (0.4)
b
Daily min. 23.2 23.8 20.2 22.5
c
(0.1) (0.1) (0.2) (0.1)
24 h-Ave
12 h-Ave
Daily max
Daily min.
75.6 76.0 78.6 76.6
60.9 62.0 67.6 63.3
96.2 96.8 96.8 96.6
44.9 44.9 47.5 45.7
(1.4) (1.6) (4.1) (2.0)
(1.9) (1.9) (3.4) (1.9)
O3 concentration (ppb)
(0.7) (1.1) (3.2) (1.4)
(1.7) (1.9) (2.6) (1.6)
AOT (ppm h)
24 h-average
12 h-averagea
5 h-averageb
Daily max.c
Daily min.d
AOT0
AOT40
15 30 45 14 28 38 12 21 33 13 26 39
17 39 66 15 36 56 13 28 50 15 34 57
19 51 118 18 46 94 16 35 87 18 44 100
60 124 180 75 137 204 31 70 141 55 110 175
1 1 2 1 1 2 1 1 1 1 1 1
10.4 22.0 32.7 9.9 20.5 28.7 7.3 13.2 20.6 9.2 18.6 27.3
0.1 4.3 14.0 0.3 3.1 10.4 0.0 0.8 7.0 0.2 2.7 10.4
Each value is the mean of air temperature or relative air humidity in nine chambers; the standard deviation is shown in parentheses. AOT0 ¼ Accumulated exposure over a threshhold of 0 ppb. AOT40 ¼ Accumulated exposure over a threshold of 40 ppb. a 12 h ¼ 6:00e18:00. b 5 h ¼ 11:00e16:00. c Mean of daily 1-h maximum value. d Mean of daily 1-h minimum value FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone.
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233
Fig. 1. Typical O3-induced visible foliar injury in Blackeye and Asontem cowpea varieties. Foliar injuries were observable at 3 and 2 weeks, respectively, after the start of the elevated (NF þ O3) treatment.
varieties, visible foliar injury was more evident at flowering and pod set. No visible foliar injury was observed in the plants grown in the FA treatment.
Blackeye and Asontem are shown in Table 3. There was a significant difference in the effects of O3 on leaf area per plant between the two varieties. In Blackeye, leaf area per plant was significantly lower in the NF and NF þ O3 treatments than in the FA treatment. In Asontem, leaf area per plant in the NF þ O3 treatment was significantly less than that in the FA and NF treatments. NF þ O3 recorded the highest SLA in Asontem with the least being the FA treatment in both varieties. Leaf, stem, root or whole-plant dry masses and R/S ratio of both varieties were significantly reduced by exposure to O3. The varieties differed significantly in the effects of O3 on stem dry mass. In Blackeye, the stem dry mass was significantly higher in both FA and NF than in NF þ O3. In Asontem, the stem dry mass was significantly lower in the NF þ O3 than in the NF treatment.
3.3. Plant growth parameters Fig. 2 shows the effects of O3 on plant height and number of leaves per plant of Blackeye and Asontem. Although there were significant differences in plant height between the two varieties on all the measuring days, no significant effects of O3 on plant height were observed (Table 2). There were significant differences in the number of leaves per plant between the varieties on the 20th, 30th and 50th days after the start of O3 exposure. In both varieties, the number of leaves per plant was significantly different among the three gas treatments on the 30th day after the start of exposure to O3. In Blackeye, the three gas treatments did not differ significantly from the FA and NF treatments of Asontem. However, NF þ O3 treatment showed the highest reduction in number of leaves at 30 days after O3 exposure in Asontem (Tukey's HSD test, p < 0.05). The effects of O3 on leaf area per plant; specific leaf area (SLA); leaf, stem, root and whole-plant dry masses; and R/S ratio of
NF
FA
3.4. Yield and yield-related parameters Fig. 3 shows the effects of O3 on yield and yield components of Blackeye and Asontem. Exposure to O3 had no significant effect on the number of pods per plant in either variety. However, the number of seeds per pod, 100-seed weight and yield per plant were significantly reduced by exposure to O3. Blackeye produced
NF+O3
70
Asontem
60
Plant height (cm)
Plant height (cm)
70
Blackeye
60 50 40 30 20 10 0
50 40 30 20 10 0
10
20
30
40
50
60
10
60
Blackeye
30
Leaf number per plant
Leaf number per plant
35
25 20 15
20
30
40
50
60
Days after the start of exposure to O3
Days after the start of exposure to O3
*
10 5 0
Asontem
50 40 30 20 10
*
0 10
20
30
40
50
Days after the start of exposure to O3
60
10
20
30
40
50
60
Days after the start of exposure to O3
Fig. 2. Effects of O3 on plant height and number of leaves per plant of two cowpea varieties. Each value is the mean of three growth-chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05.
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Table 2 Two-way ANOVA of treatment and variety effect of plant height, number of leaves and SPAD value on the 20th, 30th, 40th and 50th day after the exposure to O3 (DAE). DAE
ANOVA
Plant height
Number of leaves
SPAD value
20
Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV
0.810 0.004 0.692 0.370 0.000 0.723 0.228 0.000 0.312 0.102 0.012 0.112
0.208 0.000 0.226 0.043 0.001 0.011 0.201 0.693 0.549 0.516 0.004 0.496
0.000 0.009 0.828 0.000 0.000 0.317 0.000 0.897 0.338 0.000 0.101 0.242
30
40
50
not differ from the NF treatment at all measurement date while a significant reduction was observed in Asontem at 40 and 50 days after O3 exposure. Besides, the NF þ O3 showed a significant reduction in SPAD value in both varieties. There was no significant difference in the effects of O3 on SPAD value between the varieties on any measurement date. The effects of O3 on A, Gs and intercellular CO2 concentration (Ci) in the leaves of Blackeye and Asontem are indicated in Fig. 5. In both varieties, A and Gs were significantly reduced by exposure to O3, but Ci was not significantly affected in either. No significant difference in the effects of O3 on Gs and Ci were found between the varieties, but a significant difference in A. In Blackeye, A in the NF and NF þ O3 treatments was significantly less than that in the FA treatment. In Asontem, A in the NF þ O3 treatment was significantly less than that in the FA and NF treatments.
Significant (p < 0.05) factors are marked in bold.
4. Discussion significantly fewer seeds per pod than Asontem but its 100-seed weight was significantly greater. In Asontem, the reduction in seed weight per pod and 100-seed weight was higher in the NF þ O3 treatment compared to the FA and NF treatments which showed no difference. The reduction in yield per plant in the NF and NF þ O3 treatment was higher in Blackeye than in Asontem. Table 4 summarises the yield-related parameters of Blackeye and Asontem. Exposure to O3 significantly reduced the number of days to pod harvest and pod length in both varieties. In Blackeye, the number of days to pod harvest was significantly less in the NF and NF þ O3 treatment than in Asontem. Pods were significantly shorter in Blackeye than in Asontem. The yield per hectare of both varieties was significantly reduced by exposure to O3, but no significant effects on the harvest index were detected in either variety. The reduction in yield per hectare in the NF and NF þ O3 treatments was higher in Blackeye than in Asontem which showed only a reduction in the NF þ O3 treatment. There was no significant difference in the effects of O3 on days to harvest, pod length, yield per hectare or harvest index between the varieties.
3.5. Physiological parameters Fig. 4 shows the effects of O3 on SPAD value in the leaves of Blackeye and Asontem. On the 20th, 30th, 40th and 50th days after the start of exposure to O3, SPAD value in both varieties was significantly reduced (Table 2). In Blackeye, the FA treatment did
4.1. Effect of ozone on growth of two cowpea varieties In the present study, exposure to O3 significantly reduced leaf area per plant in both varieties of cowpea (Table 3). Because O3 did not significantly affect leaf number per plant, the reduction in leaf area per plant could be attributed to smaller leaves and high leaf drop at maturity (data not shown). Morgan et al. (2003) observed that reduced dry matter production of soybean by exposure to a relatively low O3 concentration corresponded to lowered leaf photosynthesis, but a greater loss in dry matter production when exposed to higher O3 levels was associated with reductions in both leaf photosynthesis and leaf area. The O3-induced reduction in the leaf area per plant is considered to be a factor adversely affecting growth in these two cowpea varieties. Leaf, stem, root and whole-plant dry masses and R/S ratio of the two cowpea varieties were significantly reduced by exposure to O3 (Table 3). Such reductions in growth are reported to occur via decreased translocation of fixed carbon to edible plant parts (grains, fruits, pods and roots) because of either reduced availability at the source or lowered phloem transport capabilities; additionally, decreased carbon transport to the roots reduces nutrient and water uptake and affects plant stability which is the ability of plant to withstand adverse weather and soil conditions (Wilkinson et al., 2012). The O3-induced reduction in R/S ratio may be attributed to a decrease in root biomass, indicating that the shoot was prioritised over the root system in resource allocation (Keutgen et al., 2005). In Asontem, the O3-induced reduction in leaf dry mass was mainly
Table 3 Effects of O3 on leaf area per plant, specific leaf area (SLA), dry mass and ratio of root dry mass to shoot dry mass (R/S ratio) of two cowpea varieties. Variety
Gas treatment
Leaf area (cm2)
SLA (cm2 g1)
Dry mass (g)
Blackeye
FA NF NF þ Oз FA NF NF þ O3 Treatment (T) Variety (V) TV
838.9 442.7 377.3 846.9 915.6 687.6 ** *** *
197.4 221.1 243.8 198.5 253.1 270.6 *** * n.s.
1.99 2.01 1.18 1.45 1.50 0.87 * n.s. n.s.
Leaf
Asontem
ANOVA
(139.3) ab (18.4) c (45.9) c (189.9) a (125.6) a (95.0) b
(13.2) (10.5) (11.8) (7.3) (10.6) (27.9)
R/S ratio (g g1) Stem
(0.34) (0.57) (0.20) (0.94) (0.37) (0.15)
5.02 3.36 1.69 5.27 5.86 3.40 *** ** *
(0.83) (0.65) (0.39) (1.26) (0.64) (0.11)
Root ab b c ab a b
3.28 1.94 0.97 2.81 2.63 1.10 *** n.s. n.s.
Whole-plant (1.06) (0.22) (0.12) (0.44) (0.53) (0.15)
10.28 (2.11) 7.32 (1.07) 3.85 (0.68) 9.53 (2.62) 9.99 (1.51) 5.37 (0.15) * n.s. n.s.
0.46 0.38 0.34 0.43 0.35 0.26 * n.s. n.s.
(0.09) (0.11) (0.03) (0.07) (0.03) (0.04)
FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone. Each value is the mean of three growth-chamber replicates and the standard deviation is shown in parentheses. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant. When significant interaction between O3 and variety was detected, Tukey's HSD test was performed to identify significant differences among the 6 treatments. Values with different letters are significantly different at p < 0.05. SLA was computed on the 44th day after the exposure to O3.
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235
Fig. 3. Effects of O3 on number of pods per plant, number of seeds per pod, 100-seed weight and yield per plant of two cowpea varieties. Each value is the mean of three growthchamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. ¼ not significant.
because of high leaf drop at maturity (data not shown). The extent of O3-induced reduction in stem dry mass in the NF treatment was greater in Blackeye than in Asontem, which may be attributed to differences in O3-induced reduction in net photosynthesis between the varieties (Fig. 5). Umponstira et al. (2006) reported that the biomass of O3-fumigated cowpea was significantly lower and the extent of visible foliar injury was significantly greater than in control plants. Our results confirmed the findings of Umponstira et al. (2009), which indicated that exposure to O3 significantly reduced total biomass of cowpea, particularly root dry weight, number of nodules, distribution of nodule size over 2 mm and nodule dry weight. Interestingly, Malaiyandi and Natarajan (2014) observed increases in dry weight, shoot length and leaf area of cowpea after acute exposure to 60 ppb O3. These findings suggest that different O3 exposure types may give different results, although a great difference in the sensitivity to O3 may exist among cowpea varieties. 4.2. Effect of ozone on net photosynthesis of two cowpea varieties In the present study, exposure to O3 significantly reduced net photosynthetic rate in both varieties of cowpea (Fig. 5). This
reduction could be partially due to the reductions in SPAD value and stomatal diffusive conductance. During photosynthetic electron transport in the thylakoid membrane, chlorophyll plays a significant role in capturing light to power photosystems I and II, which provide energy-rich molecules (ATP and NADPH) to the Calvin cycle (Salvatori et al., 2013). Therefore, the reduced net photosynthetic rate of Blackeye may have occurred from thylakoid membrane dysfunction as a consequence of long-term exposure to O3. In general, the damage caused by O3 to the photosynthetic machinery may have progressively decreased the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), thereby reducing the net photosynthetic rate (Ojanpera et al., 1998; Fiscus et al., 2005). Xu et al. (2009) also indicated that the O3-induced reduction in the net photosynthetic rate result from both stomatal and non-stomatal factors, such as lowered carboxylation efficiency, loss of Rubisco activity and damage from ROS to chloroplasts and the photosynthetic electron transport. In the present study, however, no significant O3-induced change was observed in Ci, indicating that parallel reduction of stomatal and non-stomatal factors may be responsible for the reduced net photosynthetic rate (Fig. 5). Furthermore, the earlier onset of flowering and pod set in Blackeye could have reduced the net
Table 4 Effects of O3 on number of days to harvest after sowing, pod length, yield per hectare and harvest index of two cowpea varieties. Variety
Gas treatment
Days to harvest (day)
Pod length (cm)
Yield/ha (ton ha1)
Harvest index
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FA NF NF þ O3 FA NF NF þ O3 Treatment (T) Variety (V) TV
83.7 77.1 78.3 95.3 90.0 95.3 ** *** n.s.
12.7 (1.7) 11.7 (0.7) 9.9 (0.3) 15.4 (0.6) 15.9 (1.8) 13.9 (0.6) * *** n.s.
1.80 1.40 0.77 1.45 1.68 0.90 * n.s. n.s.
0.44 0.45 0.46 0.40 0.43 0.43 n.s. n.s. n.s.
Asontem
ANOVA
(2.9) (2.0) (5.3) (3.4) (0.0) (1.3)
FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone. Each value is the mean of three growth-chamber replicates and the standard deviation is shown in parentheses. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant.
(0.41) (0.51) (0.37) (0.53) (0.42) (0.07)
(0.03) (0.07) (0.07) (0.07) (0.05) (0.03)
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NF
FA 70
70
Blackeye
60 50 40
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60
SPAD value
SPAD value
NF+O3
***
***
30
***
20
***
50
***
40
***
30
***
***
40
50
20 10
10
0
0 10
20
30
40
50
10
60
20
30
60
Days after the start of exposure to O3
Days after the start of exposure to O3
Fig. 4. Effects of O3 on SPAD value of two cowpea varieties. Each value is the mean of three growth chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: ***p < 0.001.
20
b
Treat x Var
a
*
15
c
c
10 5 0
Blackeye
400
Ci (μmol CO2 mol air -1)
a
Treatment (T) *** Treatment(V) ***** Variety *** TVariety V
350
Treatment Treatment (T) n.s. Variety Variety (V) * T V Treat x Varn.s.
Asontem
Gs (mol H2O m-2 s-1)
A (μmol CO2 m-2 s-1)
a
NF+O3
NF
FA 25
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Treatment (T) Treatment Variety (V) Variety T V Treat x Var
Blackeye
*** *** ** ** n.s. n.s.
Asontem
n.s. * n.s.
300 250 200 150 100 50 0
Blackeye
Asontem
Fig. 5. Effects of O3 on the net photosynthetic rate (A), stomatal conductance to H2O (Gs) and intercellular CO2 concentration (Ci) of two cowpea varieties. Each value is the mean of three growth-chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. ¼ not significant. When significant interaction between O3 and variety was detected, Tukey's HSD test was performed to identify significant differences among the 6 treatments. Different letters above the bar indicate significant difference among the 6 treatments (p < 0.05).
photosynthetic rate by channelling photoassimilates from vegetative to reproductive organs (Table 4). In the present study, Asontem showed high stomatal diffusive conductance to H2O relative to Blackeye but less O3-induced reduction in the net photosynthetic rate (Fig. 5). In contrast, exposure to O3 reduced the net photosynthetic rate in Blackeye while significantly decreasing stomatal diffusive conductance to H2O. This decline in stomatal diffusive conductance has been linked to the effects of O3 on guard cells (Torsethaugen et al., 1999). Tripathi and Agrawal (2012) observed that a decrease in stomatal diffusive conductance significantly affected the internal CO2 concentration. According to Schulze et al. (1987) and Chaves et al. (2003), stomatal diffusive conductance is species specific, affected by leaf/plant age and mediated by a variety of concomitant environmental stimuli, such as CO2 concentration, soil moisture, vapour pressure deficit, gaseous pollutants, leaf temperature and irradia et al. (2010) indicated that greater stomatal diffusive tion. Brosche conductance leads to increased O3 deposition and injury because of higher internal doses in the leaf. Although we could not evaluate the amount of O3 absorbed into the leaves through stomata in the two varieties of cowpea in the present study, the higher stomatal
diffusive conductance observed in Asontem potentially means higher leaf O3 uptake than in Blackeye. Therefore, differences in the sensitivity of net photosynthesis to O3 between the two varieties could not be explained by variation in the cumulative O3 uptake alone. 4.3. Effect of ozone on yield and yield components of two cowpea varieties In the present study, exposure to O3 significantly reduced the number of seeds per pod, pod length, 100-seed weight and yield per plant of cowpea (Fig. 3 and Table 4). The reductions in yield per plant of Blackeye grown in the NF and NF þ O3 treatments and that of Asontem grown in the NF þ O3 treatment were considered to mainly result from the effects of O3 on pod length, number of seeds per pod and 100-seed weight (Fig. 3 and Table 4). Impaired phloem loading as a result of low stomatal diffusive conductance could have also impacted on cowpea yield during the long-term exposure to O3 (Wilkinson et al., 2012). Perhaps the reduction in seed yield was because of a higher O3 sensitivity at flowering, when photosynthate allocation patterns change and less energy is available for
R. Tetteh et al. / Environmental Pollution 196 (2015) 230e238
detoxification and repair processes (Vandermeiren et al., 1995; Tingey et al., 2002). Additionally, premature leaf senescence resulting from O3-induced foliar injury decreases light interception and net photosynthesis; consequent reductions in assimilation and alterations in assimilate partitioning also contributed to yield reduction in the two cowpea varieties (Black et al., 2000; Feng et al., 2007). Morgan et al. (2003) observed that exposure at maturity to O3 significantly reduced the seed yield of soybean plants by 24%. Huang et al. (2004) reported a 60% yield reduction in soybean plants exposed to 100 ppb O3. Heagle et al. (2002) found that a sensitive snap bean genotype (S156) had a 90.1% reduction in final pod weight under elevated O3 concentrations (72 nmol mol1). They also reported a 15.4% reduction in final pod weight for an O3tolerant snap bean cultivar (Tenderette) at the same O3 concentrations. In the present study, the yield reductions of the two cowpea varieties under elevated O3 had a range of 39e58%, indicating that cowpea sensitivity to O3 could be similar to that of soybeans. On per hectare basis, cowpea grain yield in the FA treatment was in the range of that observed in the field, although cowpea grain yield per hectare is different depending on the varieties, spacing and agronomic practices. The visible injury in the NF treatment was evident at flowering and pod set and significant O3-induced reduction in physiological parameters were observed in the present study. Therefore, it is possible that current levels of O3 adversely affect cowpea growth, yield and physiological function in the field, although the present experiment was conducted in Japan. 5. Conclusions In the present study, we found that the net photosynthetic rate and leaf area per plant were significantly reduced by long-term exposure to O3 at realistic levels, resulting in reduced growth in two cowpea varieties (Blackeye and Asontem). Yields were significantly reduced by long-term exposure to O3, with no difference in sensitivity between the two varieties. The results clearly implied that increasing O3 concentrations are likely to threaten food security in Ghana and other African countries. Our future research will focus on grain quality, nitrogen fixation and biochemical processes such as free radical scavenging to explain the differences among cowpea varieties. Acknowledgements The first author is grateful to the Education Program for FieldOriented Leaders in Asia and Africa (FOLENS) of Tokyo University of Agriculture and Technology, Japan, for financial support. I also thank Yu Taniguchi, Eri Kubota, Lijuan Sun and Mohammed Zia Uddin Kamal for their support during the experimental period. References Addo-Quaye, A.A., Darkwa, A.A., Ampiah, M.K.P., 2011. Performance of three cowpea (Vigna unguiculata (L) Walp) varieties in two agro-ecological zones of the Central Region of Ghana II: grain yield and its components. J. Agric. Bio. Sci. 6, 34e42. Adepipe, N.O., Tingey, D.T., 1979. Ozone phytotoxicity in relation to stress ethylene evolution and stomatal resistance in cowpea (Vigna unguiculata) cultivars. Z. Pflanzenphysiol. 93, 259e264. Ainsworth, E.A., Yendrek, C.R., Sitch, S., Collins, W.J., Emberson, L.D., 2012. The effects of tropospheric ozone on net primary productivity and implications for climate change. Annu. Rev. Plant Biol. 63, 637e661. Ashmore, M.R., 2005. Assessing the future global impacts of ozone on vegetation. Plant Cell. Environ. 29, 949e964. Black, V.J., Black, C.R., Roberts, J.A., Stewart, C.A., 2000. Impact of ozone on the reproductive development of plants. New. Phytol. 147, 421e447.
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Bouarar, I., Law, K.S., Pham, M., Liousse, C., Schlager, H., Hamburger, T., Reeves, C.E., de le c, P., Szopa, S., Ravegnani, F., Viciani, S., D'Amato, F., Cammas, J.-P., Ne Ulanovsky, A., Richter, A., 2011. Emission sources contributing to tropospheric ozone over Equatorial Africa during the summer monsoon. Atmos. Chem. Phys. 11, 13395e13419. , M., Merilo, E., Mayer, F., Pechter, P., Puzo ~rjova, I., Brader, G., Kangasj€ Brosche arvi, J., Kollist, H., 2010. Natural variation in O3 sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance. Plant Cell. Environ. 33, 914e925. Chaves, M.M., Maroco, J.P., Pereira, J.S., 2003. Understanding plant responses to drought, from genes to the whole plant. Funct. Plant Biol. 30, 239e264. FAOSTAT, 2012. Food and Agriculture Organization Database. Available online at: http://faostat.fao.org. Feng, Z., Kobayashi, K., 2009. Assessing the impacts of current and future concentrations of surface ozone on crop yield with meta-analysis. Atmos. Environ. 43, 1510e1519. Feng, Z.Z., Yao, F.F., Chen, Z., Wang, X.K., Zheng, Q.W., Feng, Z.W., 2007. Response of gas exchange and yield components of field-grown Triticum aestivum L. to elevated ozone in China. Photosynthetica 45, 441e446. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant Cell. Environ. 28, 997e1011. Hall, A.E., Ismail, A.M., Ehlers, J.D., Marfo, K.O., Cisse, N., 2002. Breeding cowpeas for tolerance to temperature extremes and adaptation to drought. In: Fatokun, C.A., Tarawali, S.A., Singh, B.B., Kormawa, P.M., Tamo, M. (Eds.), Challenges and Opportunities for Enhancing Sustainable Cowpea Production. International Institute of Tropical Agriculture, Ibadan, Nigeria, pp. 14e21. Hall, A.E., 2004. Breeding for adaptation to drought and heat in cowpea. Eur. J. Agron. 21, 447e454. Heagle, A.S., Miller, J.E., Burkey, K.O., Eason, G., Pursley, W.A., 2002. Growth and yield responses of snap bean to mixtures of carbon dioxide and ozone. J. Environ. Qual. 31, 2008e2014. Huang, H., Wang, C., Bai, Y., Wen, M., 2004. A diagnostic experimental study of the composite influence of increasing O3 and CO2 concentration on soybean. Chin. J. Atmos. Sci. 28, 601e612. IPCC, 2007. Climate Change 2007. Contribution of working group I to the fourth assessment report of the intergovernmental panel on climate change. Available at: http://www.ipcc.ch/. Keutgen, A.J., Noga, G., Pawelzik, E., 2005. Cultivar-specific impairment of strawberry growth, photosynthesis, carbohydrate and nitrogen accumulation by ozone. Environ. Exp. Bot. 53, 271e280. Langyintuo, A.S., Lowenberg-DeBoer, J., Faye, M., Lambert, D., Ibro, G., Moussa, B., Kergna, B., Kushwaha, S., Musa, S., Ntoukam, G., 2003. Cowpea supply and demand in West and Central Africa. Field Crops Res. 82, 215e231. Leopold, A.C., Kriedemann, P.E., 1975. Plant Growth and Development, second ed. McGraw-Hill, New York, pp. 77e105. Malaiyandi, M., Natarajan, M., 2014. Impact of ozone on morphological, physiological, and biochemical changes in cowpea (Vigna unguiculata (L.) Walp.). Ozone Sci. Eng. 36, 36e42. Ministry of Food and Agriculture (MoFA), 2010. Cowpea area, production and yield in Ghana. In: Bulletin of Tropical Legumes (2012). Morgan, P.B., Ainsworth, E.A., Long, S.P., 2003. How does elevated ozone impact soybean? A meta-analysis of photosynthesis, growth and yield. Plant Cell. Environ. 26, 1317e1328. Ojanpera, K., Patsikka, E., Ylaranta, T., 1998. Effects of low ozone exposure of spring wheat on net CO2 uptake, Rubisco, leaf senescence and grain filling. New. Phytol. 138, 451e460. Padulosi, S., Ng, N.Q., 1997. Origin, Taxonomy, and morphology of Vigna unguiculata (L.) Walp. In: Singh, B.B., Mohan Raj, D.R., Dashiell, K.E., Jackai, L.E.N. (Eds.), Advances in Cowpea Research. 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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Effects of ozone on growth, net photosynthesis and yield of two African varieties of Vigna unguiculata Rashied Tetteh a, Masahiro Yamaguchi b, 1, Yoshiharu Wada c, Ryo Funada d, Takeshi Izuta d, * a
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, 321-8505, Japan d Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2014 Received in revised form 8 October 2014 Accepted 9 October 2014 Available online
To assess the effects of O3 on growth, net photosynthesis and yield of two African varieties of cowpea (Vigna unguiculata L.), Blackeye and Asontem were exposed as potted plants to air that was either filtered to remove O3 (FA), non-filtered air (NF), non-filtered with added O3 of approximately 50 nL L1 (ppb) from 11:00 to 16:00 (NF þ O3) for 88 days in open-top chambers. The mean O3 concentration (11:00 e16:00) during the exposure period had a range from 16 ppb in the FA treatment to 118 ppb in the NF þ O3 treatment. Net photosynthetic rate and leaf area per plant were significantly reduced by exposure to O3, reducing the growth of both varieties. Exposure to O3 significantly reduced the 100-seed weight and number of seeds per pod. As a result, cowpea yield was significantly reduced by long-term exposure to O3, with no difference in sensitivity between the varieties. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ozone Cowpea Growth Net photosynthesis Yield
1. Introduction Tropospheric ozone (O3) is a major secondary air pollutant that is formed by a complex series of photochemical reactions from primary precursor emissions of nitrogen oxides (NOx) and volatile organic compounds (VOCs). Relatively high concentrations of O3 are linked to hot sunny weather and occur over wide areas (Ashmore, 2005). The global background level of O3 has more than doubled since the Industrial Revolution, and its peak values regularly exceed the 50 nL L1 (ppb) guideline of the World Health Organization in many parts of the world (WHO, 2006). The Intergovernmental Panel on Climate Change predicted that ambient levels of O3 may increase by 20e25% between 2015 and 2050 and by a further 40e60% by 2100 (IPCC, 2007). Ozone impairs plant metabolisms, leading to yield reductions in agricultural crops (Pleijel et al., 2006). O3 pollution could potentially pose a great threat to global food security by 2030 (Royal Society, 2008). Predicted yield losses for rice and wheat, the two
* Corresponding author. E-mail address: [email protected] (T. Izuta). 1 Present address: Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan. http://dx.doi.org/10.1016/j.envpol.2014.10.008 0269-7491/© 2014 Elsevier Ltd. All rights reserved.
most important food crops globally, are 4e8% and 9e18%, respectively (Van Dingenen et al., 2009). According to Bouarar et al. (2011), Africa is an important source region for O3 precursors. Large amounts of carbon monoxide CO, NOx and VOCs are emitted from burning biomass associated with savanna and forest fires, which take place during dry and monsoon periods over West and Central Africa, respectively, and with agricultural waste and domestic biofuel combustion (Sauvage et al., 2005). In West Africa, the World Meteorological Organization modelled monthly mean O3 concentrations around 36 ppb during daytime measurements (WMO, 2011). Current ambient levels of O3 were reported by Feng and Kobayashi (2009) to be high enough to reduce yields of cereals, grain legumes and tubers. Ainsworth et al. (2012) indicated that O3 reduces plant productivity by entering leaves through the stomata; within the leaves, it generates reactive oxygen species (ROS) and causes oxidative stress, which in turn reduces the photosynthesis rate, plant growth and biomass accumulation. According to Black et al. (2000) and Fiscus et al. (2005), O3-induced yield losses are often attributed to reduced photosynthetic activity and to lower supplies of assimilates to support reproductive development and seed growth. Although numerous studies have addressed the impacts of O3 on crops worldwide, limited efforts have been made to
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investigate these effects on tropical crops such as cowpea, one of the main food security crops in Africa, where it is widely cultivated. Cowpea (Vigna unguiculata L.) is an annual grain legume indigenous to tropical Africa (Padulosi and Ng,1997). This crop is the most commonly cultivated grain legume in Africa. It is one of the most important food legume crops in the semi-arid tropics of Asia, Africa, Southern Europe, and Central and South America. Cowpea is well adapted to high temperatures (i.e. 20e35 C) and drought compared with other crop species (Hall et al., 2002; Hall, 2004). Singh (2002) and Langyintuo et al. (2003) noted that cowpea plays a crucial role in the lives of millions of people in Africa and other developing countries, where it is a major source of dietary protein that nutritionally complements staple low-protein cereals and tuber crops and provides income for farmers and traders. Worldwide, annual cowpea production is estimated to be 5.7 million tonnes (MT) of dried grains, of which over 90% are produced in Africa (FAOSTAT, 2012). On the African continent, West Africa represents the largest production zone of cowpea (Pottorff et al., 2012). In Ghana, an average (2002e2009) of 156,000 ha is produced annually making this country the fifth highest producer of cowpea in Africa (MoFA, 2010). On per hectare basis, Addo-Quaye et al. (2011) reported cowpea grain yield ranging from 0.8 to 1.3 tonnes. Ghana has a warm climate owing to its proximity to the equator and is thus favourable for O3 formation, but atmospheric O3 monitoring and related research are limited (WMO, 2008). Over the past decade, the demand for agricultural land for food production has increased dramatically with the growing population, leading to more biomass burning through deforestation and illegal mining and significantly higher concentrations of O3 precursors (Bouarar et al., 2011). Moreover, relatively high concentrations of these precursors are generated in cities by vehicular emissions due to poor vehicle maintenance, industrialisation and the use of secondhand appliances such as refrigerators. The recent discovery of oil in Ghana is also likely to further increase these precursors and threaten food security, including cowpea production. Therefore, the effects of O3 on the growth, yield and net photosynthesis of African crops such as cowpea must be clarified. Adepipe and Tingey (1979) revealed that cowpea cultivars were more sensitive to short-term exposure to O3 at the three-leaf than at the two-leaf stage, while no consistent leaf injury was observed at less than 0.50 mL L1 (ppm) of O3 as compared with 1.0 ppm. Ozone increased stomatal diffusive resistance in all cultivars. Umponstira et al. (2006) reported that the biomass of cowpea was significantly lower and visible foliar injury of the plants exposed to 40 ppb and 60 ppb of O3 was significantly greater as compared to the control plants after 7-days of exposure. Similarly, Rangserodsombat et al. (2010) observed a significant O3-induced reduction in the soluble sugar and starch contents in leaf and root tissues relative to control plants. Malaiyandi and Natarajan (2014) reported that the acute exposure (i.e.15 min twice a day) of cowpea to O3 at 60 ppb increased dry weight, shoot length, leaf area, number of epidermal cells, number of stomata and total chlorophyll content. However, Umponstira et al. (2009) found that long-term exposure (74 days) of 40 ppb and 60 ppb of O3 significantly reduced total biomass, particularly in root dry weight, number of nodules, distribution of nodule size over 2 mm and nodule dry weight. Moreover, total nitrogen in plant tissues and nitrogenase activity were significantly reduced during the vegetative, flowering and harvesting stages of growth. At the present time, however, there is little information on the long-term effects of O3 on the growth and yield of cowpea or on varietal differences. The objectives of the present study were to assess the long-term effects of different concentrations of O3 on the growth, net photosynthesis and yield of two varieties of cowpea and to ascertain whether these effects differed between the two varieties.
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2. Materials and methods 2.1. Plant material and growth conditions Two cowpea varieties, ‘Blackeye’ the white type and ‘Asontem’, which are the most widely cultivated varieties in Ghana, were obtained from the Department of Crop Science, University of Ghana (Legon, Ghana). The seeds were sown on 19 June, 2013, in mediumsized plastic pots (2-L) filled with sandy soil (Kanuma pumice soil) in three hills (two seeds per hill) and later thinned to one plant per pot. Hyponex compound liquid fertiliser (NPK 6-10-5; 4 mL in 1 L of water) was applied at a rate of 200 mL per pot 9 days after sowing and again at 2 weeks intervals on 15 July, 30 July and 13 August during the cultivation period. Plants were watered daily throughout the cultivation period. 2.2. Experimental design and growth chamber The experiment was conducted at the Fuchu experimental farm of Tokyo University of Agriculture and Technology (Fuchu, Tokyo, Japan) from 19 June to 27 September, 2013. The experiment used a randomised complete block design with nine open-top chambers (0.6 m 0.6 m 1 m high), each assigned to one of three gas treatments (3 replications). For each variety, we used four seedlings (eight plants per chamber). 2.3. Gas treatments The seedlings in the open-top chambers were exposed to air that was either filtered to remove O3 (FA), unfiltered (NF) or unfiltered and supplemented with approximately 50 ppb O3 generated by an electrical discharge generator (SO-03UN, Hamanetsu Co., Hamamatsu, Japan) for 5 h from 11:00 to 16:00 each day (NF þ O3). These treatments lasted 88 days, from 1 July to 26 September. The generated O3 was passed through a water trap to remove nitrogen by-products before being injected into the NF þ O3 chambers. Plants of each variety were rotated within the chamber every week and among chambers at 2-week intervals to minimise variation among plants and treatments. 2.4. Monitoring of climatic parameters and ozone concentrations The concentration of O3 generated was monitored each day with an O3 monitor (model 1150; Dylec, Ibaraki, Japan), and an O3 voltage recorder (VR-71; T & D Corp., Nagano, Japan) was used for O3 concentration measurements in all the chambers throughout the cultivation period. Air temperature and relative air humidity were monitored using a thermo recorder (TR-72Ui; T & D Corp.) comprising a sensor and logger. The accumulated O3 over a threshold of 40 ppb (AOT40) was calculated as the sum of differences between the hourly mean O3 concentration and 40 ppb when the hourly mean O3 concentration exceeded 40 ppb. 2.5. Measurement of growth parameters Plant height in centimetres, number of leaves per plant and SPAD value using a chlorophyll meter (SPAD-502; Minolta, Tokyo, Japan) were measured at 10-day intervals after 20 days of exposure to O3 until pod set. Although the relationship between SPAD value and leaf chlorophyll content is non-linear (Uddling et al., 2007), SPAD measurement is non-destructive hence we can know the seasonal change of ozone effect on chlorophyll concentration. Eleven days after sowing (30 June), plants were initially sampled and were sampled again at the end of the experiment (27 September). After sampling, each plant was separated into leaves, stem and roots, and
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oven dried at 80 C for 5 days to constant weight before the determination of plant biomass (i.e. leaf, stem, root and whole-plant dry masses, plus root-to-shoot dry mass (R/S) ratio). Leaf area per plant was measured with an area meter (AAM-8; Hayashi Denko, Tokyo, Japan) at both samplings. After 44 days of O3 exposure, few leaves were sampled and its leaf area and dry weight were measured to calculate SLA using the formula of Leopold and Kriedemann (1975). Number of days from sowing to first harvest was counted to assess the impact of O3 on cowpea life cycle.
its interaction with variety, two-way ANOVA was used to test the effects of gas treatment and variety. When a significant interaction between O3 and variety was detected, the Tukey's HSD test was performed to identify significant differences among the three gas treatments.
2.6. Measurement of yield and yield components
Table 1 indicates air temperature, relative air humidity, concentration of O3 and accumulated O3 exposure over thresholds of 0 and 40 ppb (AOT0 and AOT40, respectively) inside the open-top chambers during the 88 days of gas treatment (1 July to 26 September, 2013). The highest daily maximum air temperature was detected in August (39.3 C) and the minimum in September (20.2 C) (Table 1). The average daily relative air humidity in all the chambers was in the range of 44.9e96.8% during the experimental period. The climatic condition during the experimental period was similar to that during the growing period of cowpea in African countries including Ghana (Hall et al., 2002; Hall, 2004). The highest average O3 concentration between 11:00 and 16:00 was observed in the NF þ O3 treatment in July at around 118 ppb, while the FA treatment recorded the lowest value of 16 ppb in September. This trend was similar to the average O3 concentration over 12 h (between 6:00 and 18:00). The average hourly O3 concentration was in the range of 204 ppb in the NF þ O3 treatment in August to a minimum of 1 ppb in all the treatments in September. The highest AOT40 was detected in July (14.0 ppm h) in the NF þ O3 treatment.
3. Results 3.1. Environmental parameters
Dry pods from 12 plants per treatment per variety were harvested at maturity (from 28 August to 27 September) to determine pod length per plant, number of seeds per pod, 100-seed weight, number of pods per plant, yield per plant, yield per hectare and harvest index. The harvested pods were dried to constant weight. Seeds were separated from the pods and dried again before the measurement of the yield parameters. 2.7. Measurement of leaf gas exchange rates On 8e10 August, nine plants per treatment of each variety were selected for the measurements of net photosynthetic rate (A) and stomatal diffusive conductance to H2O (Gs) of the third or fourth fully-expanded leaves from the bottom using a portable photosynthetic measurement system (LI-6400; Li-Cor, Lincoln, NE, USA). During these measurements, atmospheric CO2 concentration, air temperature, relative air humidity, flow rate and photosynthetic photon flux density in the leaf chambers were maintained at 390 mmol mol1, 25 ± 0.5 C, 70 ± 5%, 650 mmol s1 and 1500 mmol m2 s1, respectively.
3.2. Visible foliar injury Fig. 1 shows O3-induced visible foliar injury in the two cowpea varieties. In Blackeye, injury was evident as chlorosis 3 weeks after the start of O3 exposure in the NF þ O3 treatment. In Asontem, visible foliar injury appeared as browning 2 weeks after the start of the NF þ O3 treatment. Notably, in the NF treatment of both
2.8. Statistical analyses Statistical analyses were conducted using the SPSS Statistics 21 (IBM, Chicago, IL, USA). Because we focused on the effects of O3 and
Table 1 Air temperature, relative humidity, average O3 concentration and AOT of O3 in each gas treatment during the exposure period in 2013. Period (2013)
Air temperature ( C)
Relative humidity (%)
24 h-Ave
12 h-Ave
1e31 July 1e31 August 1e26 September 1 July-26 September
29.0 29.8 25.9 28.4
33.3 34.1 29.4 32.4
Period (2013)
Gas treatment
1e31 July
1e31 August
1e26 September
1 Julye26 September
FA NF NF FA NF NF FA NF NF FA NF NF
(0.2) (0.2) (0.2) (0.2)
þ O3
þ O3
þ O3
þ O3
a
(0.4) (0.2) (0.5) (0.3)
Daily max 38.4 39.3 35.2 37.8
(0.5) (0.3) (0.9) (0.4)
b
Daily min. 23.2 23.8 20.2 22.5
c
(0.1) (0.1) (0.2) (0.1)
24 h-Ave
12 h-Ave
Daily max
Daily min.
75.6 76.0 78.6 76.6
60.9 62.0 67.6 63.3
96.2 96.8 96.8 96.6
44.9 44.9 47.5 45.7
(1.4) (1.6) (4.1) (2.0)
(1.9) (1.9) (3.4) (1.9)
O3 concentration (ppb)
(0.7) (1.1) (3.2) (1.4)
(1.7) (1.9) (2.6) (1.6)
AOT (ppm h)
24 h-average
12 h-averagea
5 h-averageb
Daily max.c
Daily min.d
AOT0
AOT40
15 30 45 14 28 38 12 21 33 13 26 39
17 39 66 15 36 56 13 28 50 15 34 57
19 51 118 18 46 94 16 35 87 18 44 100
60 124 180 75 137 204 31 70 141 55 110 175
1 1 2 1 1 2 1 1 1 1 1 1
10.4 22.0 32.7 9.9 20.5 28.7 7.3 13.2 20.6 9.2 18.6 27.3
0.1 4.3 14.0 0.3 3.1 10.4 0.0 0.8 7.0 0.2 2.7 10.4
Each value is the mean of air temperature or relative air humidity in nine chambers; the standard deviation is shown in parentheses. AOT0 ¼ Accumulated exposure over a threshhold of 0 ppb. AOT40 ¼ Accumulated exposure over a threshold of 40 ppb. a 12 h ¼ 6:00e18:00. b 5 h ¼ 11:00e16:00. c Mean of daily 1-h maximum value. d Mean of daily 1-h minimum value FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone.
R. Tetteh et al. / Environmental Pollution 196 (2015) 230e238
233
Fig. 1. Typical O3-induced visible foliar injury in Blackeye and Asontem cowpea varieties. Foliar injuries were observable at 3 and 2 weeks, respectively, after the start of the elevated (NF þ O3) treatment.
varieties, visible foliar injury was more evident at flowering and pod set. No visible foliar injury was observed in the plants grown in the FA treatment.
Blackeye and Asontem are shown in Table 3. There was a significant difference in the effects of O3 on leaf area per plant between the two varieties. In Blackeye, leaf area per plant was significantly lower in the NF and NF þ O3 treatments than in the FA treatment. In Asontem, leaf area per plant in the NF þ O3 treatment was significantly less than that in the FA and NF treatments. NF þ O3 recorded the highest SLA in Asontem with the least being the FA treatment in both varieties. Leaf, stem, root or whole-plant dry masses and R/S ratio of both varieties were significantly reduced by exposure to O3. The varieties differed significantly in the effects of O3 on stem dry mass. In Blackeye, the stem dry mass was significantly higher in both FA and NF than in NF þ O3. In Asontem, the stem dry mass was significantly lower in the NF þ O3 than in the NF treatment.
3.3. Plant growth parameters Fig. 2 shows the effects of O3 on plant height and number of leaves per plant of Blackeye and Asontem. Although there were significant differences in plant height between the two varieties on all the measuring days, no significant effects of O3 on plant height were observed (Table 2). There were significant differences in the number of leaves per plant between the varieties on the 20th, 30th and 50th days after the start of O3 exposure. In both varieties, the number of leaves per plant was significantly different among the three gas treatments on the 30th day after the start of exposure to O3. In Blackeye, the three gas treatments did not differ significantly from the FA and NF treatments of Asontem. However, NF þ O3 treatment showed the highest reduction in number of leaves at 30 days after O3 exposure in Asontem (Tukey's HSD test, p < 0.05). The effects of O3 on leaf area per plant; specific leaf area (SLA); leaf, stem, root and whole-plant dry masses; and R/S ratio of
NF
FA
3.4. Yield and yield-related parameters Fig. 3 shows the effects of O3 on yield and yield components of Blackeye and Asontem. Exposure to O3 had no significant effect on the number of pods per plant in either variety. However, the number of seeds per pod, 100-seed weight and yield per plant were significantly reduced by exposure to O3. Blackeye produced
NF+O3
70
Asontem
60
Plant height (cm)
Plant height (cm)
70
Blackeye
60 50 40 30 20 10 0
50 40 30 20 10 0
10
20
30
40
50
60
10
60
Blackeye
30
Leaf number per plant
Leaf number per plant
35
25 20 15
20
30
40
50
60
Days after the start of exposure to O3
Days after the start of exposure to O3
*
10 5 0
Asontem
50 40 30 20 10
*
0 10
20
30
40
50
Days after the start of exposure to O3
60
10
20
30
40
50
60
Days after the start of exposure to O3
Fig. 2. Effects of O3 on plant height and number of leaves per plant of two cowpea varieties. Each value is the mean of three growth-chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05.
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Table 2 Two-way ANOVA of treatment and variety effect of plant height, number of leaves and SPAD value on the 20th, 30th, 40th and 50th day after the exposure to O3 (DAE). DAE
ANOVA
Plant height
Number of leaves
SPAD value
20
Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV Treatment (T) Variety (V) TV
0.810 0.004 0.692 0.370 0.000 0.723 0.228 0.000 0.312 0.102 0.012 0.112
0.208 0.000 0.226 0.043 0.001 0.011 0.201 0.693 0.549 0.516 0.004 0.496
0.000 0.009 0.828 0.000 0.000 0.317 0.000 0.897 0.338 0.000 0.101 0.242
30
40
50
not differ from the NF treatment at all measurement date while a significant reduction was observed in Asontem at 40 and 50 days after O3 exposure. Besides, the NF þ O3 showed a significant reduction in SPAD value in both varieties. There was no significant difference in the effects of O3 on SPAD value between the varieties on any measurement date. The effects of O3 on A, Gs and intercellular CO2 concentration (Ci) in the leaves of Blackeye and Asontem are indicated in Fig. 5. In both varieties, A and Gs were significantly reduced by exposure to O3, but Ci was not significantly affected in either. No significant difference in the effects of O3 on Gs and Ci were found between the varieties, but a significant difference in A. In Blackeye, A in the NF and NF þ O3 treatments was significantly less than that in the FA treatment. In Asontem, A in the NF þ O3 treatment was significantly less than that in the FA and NF treatments.
Significant (p < 0.05) factors are marked in bold.
4. Discussion significantly fewer seeds per pod than Asontem but its 100-seed weight was significantly greater. In Asontem, the reduction in seed weight per pod and 100-seed weight was higher in the NF þ O3 treatment compared to the FA and NF treatments which showed no difference. The reduction in yield per plant in the NF and NF þ O3 treatment was higher in Blackeye than in Asontem. Table 4 summarises the yield-related parameters of Blackeye and Asontem. Exposure to O3 significantly reduced the number of days to pod harvest and pod length in both varieties. In Blackeye, the number of days to pod harvest was significantly less in the NF and NF þ O3 treatment than in Asontem. Pods were significantly shorter in Blackeye than in Asontem. The yield per hectare of both varieties was significantly reduced by exposure to O3, but no significant effects on the harvest index were detected in either variety. The reduction in yield per hectare in the NF and NF þ O3 treatments was higher in Blackeye than in Asontem which showed only a reduction in the NF þ O3 treatment. There was no significant difference in the effects of O3 on days to harvest, pod length, yield per hectare or harvest index between the varieties.
3.5. Physiological parameters Fig. 4 shows the effects of O3 on SPAD value in the leaves of Blackeye and Asontem. On the 20th, 30th, 40th and 50th days after the start of exposure to O3, SPAD value in both varieties was significantly reduced (Table 2). In Blackeye, the FA treatment did
4.1. Effect of ozone on growth of two cowpea varieties In the present study, exposure to O3 significantly reduced leaf area per plant in both varieties of cowpea (Table 3). Because O3 did not significantly affect leaf number per plant, the reduction in leaf area per plant could be attributed to smaller leaves and high leaf drop at maturity (data not shown). Morgan et al. (2003) observed that reduced dry matter production of soybean by exposure to a relatively low O3 concentration corresponded to lowered leaf photosynthesis, but a greater loss in dry matter production when exposed to higher O3 levels was associated with reductions in both leaf photosynthesis and leaf area. The O3-induced reduction in the leaf area per plant is considered to be a factor adversely affecting growth in these two cowpea varieties. Leaf, stem, root and whole-plant dry masses and R/S ratio of the two cowpea varieties were significantly reduced by exposure to O3 (Table 3). Such reductions in growth are reported to occur via decreased translocation of fixed carbon to edible plant parts (grains, fruits, pods and roots) because of either reduced availability at the source or lowered phloem transport capabilities; additionally, decreased carbon transport to the roots reduces nutrient and water uptake and affects plant stability which is the ability of plant to withstand adverse weather and soil conditions (Wilkinson et al., 2012). The O3-induced reduction in R/S ratio may be attributed to a decrease in root biomass, indicating that the shoot was prioritised over the root system in resource allocation (Keutgen et al., 2005). In Asontem, the O3-induced reduction in leaf dry mass was mainly
Table 3 Effects of O3 on leaf area per plant, specific leaf area (SLA), dry mass and ratio of root dry mass to shoot dry mass (R/S ratio) of two cowpea varieties. Variety
Gas treatment
Leaf area (cm2)
SLA (cm2 g1)
Dry mass (g)
Blackeye
FA NF NF þ Oз FA NF NF þ O3 Treatment (T) Variety (V) TV
838.9 442.7 377.3 846.9 915.6 687.6 ** *** *
197.4 221.1 243.8 198.5 253.1 270.6 *** * n.s.
1.99 2.01 1.18 1.45 1.50 0.87 * n.s. n.s.
Leaf
Asontem
ANOVA
(139.3) ab (18.4) c (45.9) c (189.9) a (125.6) a (95.0) b
(13.2) (10.5) (11.8) (7.3) (10.6) (27.9)
R/S ratio (g g1) Stem
(0.34) (0.57) (0.20) (0.94) (0.37) (0.15)
5.02 3.36 1.69 5.27 5.86 3.40 *** ** *
(0.83) (0.65) (0.39) (1.26) (0.64) (0.11)
Root ab b c ab a b
3.28 1.94 0.97 2.81 2.63 1.10 *** n.s. n.s.
Whole-plant (1.06) (0.22) (0.12) (0.44) (0.53) (0.15)
10.28 (2.11) 7.32 (1.07) 3.85 (0.68) 9.53 (2.62) 9.99 (1.51) 5.37 (0.15) * n.s. n.s.
0.46 0.38 0.34 0.43 0.35 0.26 * n.s. n.s.
(0.09) (0.11) (0.03) (0.07) (0.03) (0.04)
FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone. Each value is the mean of three growth-chamber replicates and the standard deviation is shown in parentheses. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant. When significant interaction between O3 and variety was detected, Tukey's HSD test was performed to identify significant differences among the 6 treatments. Values with different letters are significantly different at p < 0.05. SLA was computed on the 44th day after the exposure to O3.
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235
Fig. 3. Effects of O3 on number of pods per plant, number of seeds per pod, 100-seed weight and yield per plant of two cowpea varieties. Each value is the mean of three growthchamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. ¼ not significant.
because of high leaf drop at maturity (data not shown). The extent of O3-induced reduction in stem dry mass in the NF treatment was greater in Blackeye than in Asontem, which may be attributed to differences in O3-induced reduction in net photosynthesis between the varieties (Fig. 5). Umponstira et al. (2006) reported that the biomass of O3-fumigated cowpea was significantly lower and the extent of visible foliar injury was significantly greater than in control plants. Our results confirmed the findings of Umponstira et al. (2009), which indicated that exposure to O3 significantly reduced total biomass of cowpea, particularly root dry weight, number of nodules, distribution of nodule size over 2 mm and nodule dry weight. Interestingly, Malaiyandi and Natarajan (2014) observed increases in dry weight, shoot length and leaf area of cowpea after acute exposure to 60 ppb O3. These findings suggest that different O3 exposure types may give different results, although a great difference in the sensitivity to O3 may exist among cowpea varieties. 4.2. Effect of ozone on net photosynthesis of two cowpea varieties In the present study, exposure to O3 significantly reduced net photosynthetic rate in both varieties of cowpea (Fig. 5). This
reduction could be partially due to the reductions in SPAD value and stomatal diffusive conductance. During photosynthetic electron transport in the thylakoid membrane, chlorophyll plays a significant role in capturing light to power photosystems I and II, which provide energy-rich molecules (ATP and NADPH) to the Calvin cycle (Salvatori et al., 2013). Therefore, the reduced net photosynthetic rate of Blackeye may have occurred from thylakoid membrane dysfunction as a consequence of long-term exposure to O3. In general, the damage caused by O3 to the photosynthetic machinery may have progressively decreased the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), thereby reducing the net photosynthetic rate (Ojanpera et al., 1998; Fiscus et al., 2005). Xu et al. (2009) also indicated that the O3-induced reduction in the net photosynthetic rate result from both stomatal and non-stomatal factors, such as lowered carboxylation efficiency, loss of Rubisco activity and damage from ROS to chloroplasts and the photosynthetic electron transport. In the present study, however, no significant O3-induced change was observed in Ci, indicating that parallel reduction of stomatal and non-stomatal factors may be responsible for the reduced net photosynthetic rate (Fig. 5). Furthermore, the earlier onset of flowering and pod set in Blackeye could have reduced the net
Table 4 Effects of O3 on number of days to harvest after sowing, pod length, yield per hectare and harvest index of two cowpea varieties. Variety
Gas treatment
Days to harvest (day)
Pod length (cm)
Yield/ha (ton ha1)
Harvest index
Blackeye
FA NF NF þ O3 FA NF NF þ O3 Treatment (T) Variety (V) TV
83.7 77.1 78.3 95.3 90.0 95.3 ** *** n.s.
12.7 (1.7) 11.7 (0.7) 9.9 (0.3) 15.4 (0.6) 15.9 (1.8) 13.9 (0.6) * *** n.s.
1.80 1.40 0.77 1.45 1.68 0.90 * n.s. n.s.
0.44 0.45 0.46 0.40 0.43 0.43 n.s. n.s. n.s.
Asontem
ANOVA
(2.9) (2.0) (5.3) (3.4) (0.0) (1.3)
FA: Filtered air; NF: Non-Filtered air; NF þ O3: NF: Non-filtered air plus additional ozone. Each value is the mean of three growth-chamber replicates and the standard deviation is shown in parentheses. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant.
(0.41) (0.51) (0.37) (0.53) (0.42) (0.07)
(0.03) (0.07) (0.07) (0.07) (0.05) (0.03)
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R. Tetteh et al. / Environmental Pollution 196 (2015) 230e238
NF
FA 70
70
Blackeye
60 50 40
Asontem
60
SPAD value
SPAD value
NF+O3
***
***
30
***
20
***
50
***
40
***
30
***
***
40
50
20 10
10
0
0 10
20
30
40
50
10
60
20
30
60
Days after the start of exposure to O3
Days after the start of exposure to O3
Fig. 4. Effects of O3 on SPAD value of two cowpea varieties. Each value is the mean of three growth chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: ***p < 0.001.
20
b
Treat x Var
a
*
15
c
c
10 5 0
Blackeye
400
Ci (μmol CO2 mol air -1)
a
Treatment (T) *** Treatment(V) ***** Variety *** TVariety V
350
Treatment Treatment (T) n.s. Variety Variety (V) * T V Treat x Varn.s.
Asontem
Gs (mol H2O m-2 s-1)
A (μmol CO2 m-2 s-1)
a
NF+O3
NF
FA 25
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Treatment (T) Treatment Variety (V) Variety T V Treat x Var
Blackeye
*** *** ** ** n.s. n.s.
Asontem
n.s. * n.s.
300 250 200 150 100 50 0
Blackeye
Asontem
Fig. 5. Effects of O3 on the net photosynthetic rate (A), stomatal conductance to H2O (Gs) and intercellular CO2 concentration (Ci) of two cowpea varieties. Each value is the mean of three growth-chamber replicates, and the vertical bars indicate standard error. Two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, n.s. ¼ not significant. When significant interaction between O3 and variety was detected, Tukey's HSD test was performed to identify significant differences among the 6 treatments. Different letters above the bar indicate significant difference among the 6 treatments (p < 0.05).
photosynthetic rate by channelling photoassimilates from vegetative to reproductive organs (Table 4). In the present study, Asontem showed high stomatal diffusive conductance to H2O relative to Blackeye but less O3-induced reduction in the net photosynthetic rate (Fig. 5). In contrast, exposure to O3 reduced the net photosynthetic rate in Blackeye while significantly decreasing stomatal diffusive conductance to H2O. This decline in stomatal diffusive conductance has been linked to the effects of O3 on guard cells (Torsethaugen et al., 1999). Tripathi and Agrawal (2012) observed that a decrease in stomatal diffusive conductance significantly affected the internal CO2 concentration. According to Schulze et al. (1987) and Chaves et al. (2003), stomatal diffusive conductance is species specific, affected by leaf/plant age and mediated by a variety of concomitant environmental stimuli, such as CO2 concentration, soil moisture, vapour pressure deficit, gaseous pollutants, leaf temperature and irradia et al. (2010) indicated that greater stomatal diffusive tion. Brosche conductance leads to increased O3 deposition and injury because of higher internal doses in the leaf. Although we could not evaluate the amount of O3 absorbed into the leaves through stomata in the two varieties of cowpea in the present study, the higher stomatal
diffusive conductance observed in Asontem potentially means higher leaf O3 uptake than in Blackeye. Therefore, differences in the sensitivity of net photosynthesis to O3 between the two varieties could not be explained by variation in the cumulative O3 uptake alone. 4.3. Effect of ozone on yield and yield components of two cowpea varieties In the present study, exposure to O3 significantly reduced the number of seeds per pod, pod length, 100-seed weight and yield per plant of cowpea (Fig. 3 and Table 4). The reductions in yield per plant of Blackeye grown in the NF and NF þ O3 treatments and that of Asontem grown in the NF þ O3 treatment were considered to mainly result from the effects of O3 on pod length, number of seeds per pod and 100-seed weight (Fig. 3 and Table 4). Impaired phloem loading as a result of low stomatal diffusive conductance could have also impacted on cowpea yield during the long-term exposure to O3 (Wilkinson et al., 2012). Perhaps the reduction in seed yield was because of a higher O3 sensitivity at flowering, when photosynthate allocation patterns change and less energy is available for
R. Tetteh et al. / Environmental Pollution 196 (2015) 230e238
detoxification and repair processes (Vandermeiren et al., 1995; Tingey et al., 2002). Additionally, premature leaf senescence resulting from O3-induced foliar injury decreases light interception and net photosynthesis; consequent reductions in assimilation and alterations in assimilate partitioning also contributed to yield reduction in the two cowpea varieties (Black et al., 2000; Feng et al., 2007). Morgan et al. (2003) observed that exposure at maturity to O3 significantly reduced the seed yield of soybean plants by 24%. Huang et al. (2004) reported a 60% yield reduction in soybean plants exposed to 100 ppb O3. Heagle et al. (2002) found that a sensitive snap bean genotype (S156) had a 90.1% reduction in final pod weight under elevated O3 concentrations (72 nmol mol1). They also reported a 15.4% reduction in final pod weight for an O3tolerant snap bean cultivar (Tenderette) at the same O3 concentrations. In the present study, the yield reductions of the two cowpea varieties under elevated O3 had a range of 39e58%, indicating that cowpea sensitivity to O3 could be similar to that of soybeans. On per hectare basis, cowpea grain yield in the FA treatment was in the range of that observed in the field, although cowpea grain yield per hectare is different depending on the varieties, spacing and agronomic practices. The visible injury in the NF treatment was evident at flowering and pod set and significant O3-induced reduction in physiological parameters were observed in the present study. Therefore, it is possible that current levels of O3 adversely affect cowpea growth, yield and physiological function in the field, although the present experiment was conducted in Japan. 5. Conclusions In the present study, we found that the net photosynthetic rate and leaf area per plant were significantly reduced by long-term exposure to O3 at realistic levels, resulting in reduced growth in two cowpea varieties (Blackeye and Asontem). Yields were significantly reduced by long-term exposure to O3, with no difference in sensitivity between the two varieties. The results clearly implied that increasing O3 concentrations are likely to threaten food security in Ghana and other African countries. Our future research will focus on grain quality, nitrogen fixation and biochemical processes such as free radical scavenging to explain the differences among cowpea varieties. Acknowledgements The first author is grateful to the Education Program for FieldOriented Leaders in Asia and Africa (FOLENS) of Tokyo University of Agriculture and Technology, Japan, for financial support. I also thank Yu Taniguchi, Eri Kubota, Lijuan Sun and Mohammed Zia Uddin Kamal for their support during the experimental period. References Addo-Quaye, A.A., Darkwa, A.A., Ampiah, M.K.P., 2011. Performance of three cowpea (Vigna unguiculata (L) Walp) varieties in two agro-ecological zones of the Central Region of Ghana II: grain yield and its components. J. Agric. Bio. Sci. 6, 34e42. Adepipe, N.O., Tingey, D.T., 1979. Ozone phytotoxicity in relation to stress ethylene evolution and stomatal resistance in cowpea (Vigna unguiculata) cultivars. Z. Pflanzenphysiol. 93, 259e264. Ainsworth, E.A., Yendrek, C.R., Sitch, S., Collins, W.J., Emberson, L.D., 2012. The effects of tropospheric ozone on net primary productivity and implications for climate change. Annu. Rev. Plant Biol. 63, 637e661. Ashmore, M.R., 2005. Assessing the future global impacts of ozone on vegetation. Plant Cell. Environ. 29, 949e964. Black, V.J., Black, C.R., Roberts, J.A., Stewart, C.A., 2000. Impact of ozone on the reproductive development of plants. New. Phytol. 147, 421e447.
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