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Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 5 (2017) pp. 147-159 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.605.018 Effect of Moisture Stress on Key Physiological Parameters in Sunflower Genotypes A. Geetha*, A. Sivasankar, P. Saidaiah and Lakshmi Prayaga Department of Plant Physiology, College of Agriculture, ANGRAU, Rajendranagar, Hyderabad (A.P.) – 500 030, India *Corresponding author ABSTRACT Keywords Drought, Sunflower, RWC, Photosynthetic rate, SPAD readings, Article Info Accepted: 04 April 2017 Available Online: 10 May 2017 A study was conducted to investigate the effect of drought on physiological characteristics in twelve sunflower genotypes. Moisture stress treatment were imposed at flower bud initiation stage (irrigation withheld from 40 DAS to 60 DAS) whereas, Control plots were irrigated at 10 days intervals throughout the crop growth period. Results revealed that water stress showed repressing effect on Relative water content, Photosynthetic rate, leaf fluorescence, membrane leakage, chlorophyll content and specific leaf area in all the genotypes examined. Decline in Specific leaf area under water stress is considered as adaptation to water stress. However, genotypic variation was significant for characters studied. Genotypes SH-177, SH-491 and DSF-111 was considered as promising lines by maintaining higher RWC, photosynthetic rates, leaf fluorescence and SPAD chlorophyll meter readings wither lower membrane leakage and Specific leaf area. Introduction Sunflower (Helianthus annuus L.) occupies a prominent place among oilseed crops as it contributes about 12 % to the world edible oil production. In India, sunflower is cultivated in an area of 21.6 m ha with an annual production 1.32 m tones. In fact, large area under sunflower is cultivated under rainfed situation, where intermittent moisture stress is most prevalent. The decrease in productivity in oilseeds in general and in sunflower in particularly is mainly due to abiotic stresses. Drought is the most limiting of all abiotic stresses as it causes more than 70% reduction in biomass and seed yield in sunflower (Umashaanker, 1991). Although sunflower has good potential to tolerate drought because of well developed root system. The productivity is still affected by drought. If drought tolerant sunflower hybrids/ varieties are developed, sunflower can be grown successfully under water limiting conditions. Researchers have linked various physiological traits of plants to drought with their tolerance mechanisms. Among this, relative leaf water contents (RWC) is best measure to level the water deficit in the plant at a specific point of time. As RWC is related to cell volume, it may closely reflect the balance between water supply to the leaf and transpiration rate (Sinclair and Ludlow, 1985). The measurement of solute leakage from plant tissue is a long standing method for estimating membrane integrity in relation to 147 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 environmental stresses, growth and development, and genotypic variation. In this regard, the degree of stability of cell membrane is considered to be one of the best physiological indicators of drought stress tolerance. were followed to raise a healthy crop. In each entry, five plants were tagged randomly. Observations were recorded at 45,60 and 75 DAS i.e., during 5 days after imposition of stress, twenty days after imposition of stress and 15 days of stress recovery period on RWC, photosynthetic rate, Leaf fluorescence, membrane integrity, SPAD and SLA. The use of chlorophyll fluorescence from intact attached leaves proved to be a reliable, non intrusive method for monitoring photosynthetic events and for judging the physiological status of the plant. Fluorescence induction patterns and derived indices have been used as empirical diagnostic tools in stress physiology. Photosynthesis response to water stress is poorly understood in sunflower. The extent to which photosynthetic capability is maintained during periods of water stress and the ability for rapid recovery of photosynthesis after re watering is important in crop adaptation to drought environments. Hence, Present investigation was carried out to study physiological traits like RWC, photosynthetic rate, Leaf fluorescence, membrane integrity, SPAD and SLA in twelve genotypes of sunflower under drought conditions. Leaf disc of approximately 4 cm2 area in rectangle shape was taken from plant under irrigated and stressed regimes and fresh weight was measured. Discs were then dipped in glass vials containing 20 ml of deionized water. These veils were left for four hours at room temperature. After four hours, leaf discs were blotted and their turgid weight was recorded by formula as given below. RWC = (Fresh weight-Dry weight / Turgid weight –Dry weight) X 100 Photosynthetic rate is calculated using is portable infrared gas analyzer (IRGA). The optimal and effective quantum yields of PSII were measured using the fluorometer OS-500 (Opti-Science, USA). Membrane leakage was measured using automatic conductivity meter. Single leaf disks 1cm diameter were excised with a leaf punch from the fourth main stem leaf, one disc per variety per treatment from similar interveinal areas were taken, and placed into trays with individual cells containing 2ml double de- ionized water. The electrical conductivity as a measure of cell leakage was read 48 hrs after the leaf disks were placed in double de- ionized water at room temperature. The resulting electrical conductivity of the ion concentration in the solution depended on the leakage from the leaf disk. Chlorophyll concentration was assessed using a chlorophyll meter (SPAD502, Minolta).Measurements being taken at three points of each leaf (upper, middle and lower part).Average of these three readings was considered as SPAD reading of the leaf. Recording of SPAD readings was carried out Materials and Methods The experiment was laid out in factorial Random Block Design with two factors and 12 treatments which were replicated thrice during rabi, 2010-11 at College Farm, College of Agriculture, ANGRAU, Rajendranagar, Hyderabad. Control (irrigated) and water stress was used as factors. Control plots were irrigated at 10 days intervals throughout the crop growth period whereas, in stress treatment irrigation withheld from 40 DAS to 60 DAS. The treatments comprised of 12 lines. Each genotype was sown in two rows at 5 m length with spacing of 60 x 30 cm. Two to three seeds were sown per hill to achieve uniform stand. Thinning was done at two weeks after sowing to retain one seedling per hill. Recommended package of practices 148 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 fortnight intervals starting from 45DAS, in the (3rd, 5th and 7th) leaf to the top of the plant. The mean of SCMR reading was taken out in the end and presented as average SPAD value. Specific leaf area (SLA), an indication of leaf thickness was measured by taking the fully expanded leaf, more specifically the fifth leaf from the top, the leaf area was measured using leaf area meter. Later, the leaf was kept for drying at 80◦ C for 3-4days and once the leaf was dried, leaf weight was taken and SLA was computed as per the equation given below. showed highest photosynthetic rate followed by TSF -103, DSF-111, RSF-106 and RSF101 recorded on par and were significantly superior over other genotypes at recovery period (75 DAS). Decrease in photosynthetic rate is due to increase in stomatal resistance due to partial closure of stomata as well as difference in activation states of photosynthetic enzymes (Lawlor, 2002). The limitation of photosynthesis under drought through metabolic impairment is more complex phenomenon than stomatal limitation and mainly it is through reduced photosynthetic pigment contents in sunflower (Reddy et al., 2004). SLA (cm2/g) = Leaf area/ Leaf weight. Stress imposition at flower bud initiation stage resulted in significant difference in initial fluorescence between treatments throughout stress period. Maximum initial fluorescence was recorded at 5 days after initiation of stress (89.29) compared to control (96.14) (Table 3). At 15 days after release of stress, genotype DSF-104 recorded highest initial fluorescence in control, stress and interaction of genotype x treatments. Maximum fluorescence (Fo) was initially high at 5 days after imposition of stress, thereafter decrease was seen at 20 days after stress imposition and 15 days after release of stress. The maximum fluorescence differed significantly from 45 DAS onwards (Table 4). Among the stress treatments, the reduction in maximum fluorescence was more at 15 days after release of stress (17.39%) than at remaining stages compared to their controls. Among genotypes, significant difference was found in maximum fluorescence from 45 DAS onwards. At recovery period (75 DAS), genotype ASF-107 under control and DSF114 followed by SH-177 under stress exhibited high maximum fluorescence among genotypes. But in combined effect, SH-177 recorded maximum (Fm) value followed by DSF-114. The decrease of Fv/Fm after severe water stress was recently reported by Miyashita et al., (2004). Results and Discussion Relative water content (RWC) decreased with increase in stress duration. Relative water content was reduced from (15%) at 45 DAS to (26%) at 60 DAS compared to their controls (Table 1). At 15 days after release of stress, genotypes varied significantly in RWC content.SH-491 followed by SH-177 under control and only SH-177 under stress recorded higher RWC % over remaining genotypes, while, SH-177 retained highest relative water content than rest of cultivars in interaction. Under water stress some genotype maintains its RWC at par with that of non stress conditions due to production of osmoprotectants or Compatible solutes and this compound reduces osmotic potential (Jha and Singh, 1997). Photosynthetic rate was significantly reduced under stress conditions compared to control (Table 2). Percent reduction of photosynthetic rate was highest (11.68%) at 75 DAS than at 60 DAS (10.68%) and 45 DAS (10.52%) compared to controls (Table 2). Among sunflower genotypes DSF-111 under control, whereas SH-177 followed by TSF-103 and RSF-106 under stress showed superior photosynthetic rates than other genotypes at 75 DAS. However, in interaction SH-177 149 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.1 Mean of relative water content (RWC) (%) of sunflower cultivars during stress and after stress influenced by moisture stress Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Control Stress RSF-101 73.50 70.84 TSF-103 85.13 75.64 ASF-107 82.15 64.61 DSF-114 74.70 65.23 SH-177 91.52 69.93 DSF-104 72.20 61.73 RSF-106 81.30 69.93 DSF-111 83.00 71.40 RSF-107 78.07 66.20 ASF-104 79.85 71.20 TSF-106 80.85 76.32 SH-491 88.55 62.33 Mean 80.90 68.78 CD at 5% for treatments CD at 5% for genotypes CD at 5% for TxG Mean 72.17 80.38 73.38 69.97 80.72 66.97 75.62 77.20 72.14 75.53 78.59 75.44 74.84 % decrease 3.61 11.14 21.35 12.67 23.58 14.50 13.98 13.98 15.20 10.83 5.60 29.61 14.98 Twenty days after imposition of stress % Control Stress Mean decrease 74.10 64.30 69.20 13.23 84.03 64.43 74.23 23.32 80.83 54.43 67.63 32.66 72.57 50.93 61.75 29.81 86.83 49.50 68.17 42.99 75.83 63.10 69.47 16.79 79.07 66.13 72.60 16.36 79.80 67.53 73.67 15.37 83.40 54.80 69.10 34.29 79.67 54.00 66.83 32.22 79.87 64.50 72.18 19.24 90.50 58.07 74.28 35.84 80.54 59.31 69.93 26.36 Fifteen days after release of stress % Control Stress Mean decrease 75.90 72.20 74.05 4.87 87.17 73.57 80.37 15.60 80.67 65.80 73.23 18.43 73.83 59.80 66.82 19.01 90.03 78.23 84.13 13.11 88.50 70.23 79.37 20.64 75.53 71.23 73.38 5.69 80.53 73.03 76.78 9.31 85.37 60.80 73.08 28.78 80.37 58.60 69.48 27.08 79.23 70.73 74.98 10.73 91.40 65.17 78.28 28.70 82.38 68.28 75.33 17.11 1.17 0.84 0.78 2.88 2.05 1.92 4.07 2.9 2.72 150 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.2 Mean of photosynthetic rate (μ mol m-2 s-1) of sunflower cultivars during stress and after stress as influenced by moisture stress Twenty days after imposition of stress % % decrease Control Stress Mean decrease 15.49 22.87 20.40 21.63 10.79 9.67 25.00 23.00 24.00 8.00 3.18 23.57 22.20 22.89 5.83 17.93 19.40 17.17 18.28 11.51 2.36 26.10 23.94 25.02 8.28 4.76 22.27 18.80 20.53 15.57 15.44 25.33 23.53 24.43 7.11 4.97 26.57 21.00 23.78 20.95 25.05 21.83 21.27 21.55 2.60 6.79 25.16 20.00 22.58 20.51 16.14 19.10 17.93 18.52 6.11 6.61 23.07 21.10 22.08 8.53 10.52 23.36 20.86 22.11 10.68 Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Control Stress RSF-101 21.30 18.00 TSF-103 20.33 18.37 ASF-107 20.97 20.30 DSF-114 13.20 10.83 SH-177 19.80 19.33 DSF-104 17.23 16.41 RSF-106 18.13 15.33 DSF-111 16.10 15.30 RSF-107 17.70 13.27 ASF-104 16.20 15.10 TSF-106 19.17 16.07 SH-491 19.67 18.37 Mean 18.32 16.39 CD at 5% for treatments CD at 5% for genotypes CD at 5% for TxG Mean 19.65 19.35 20.63 12.02 19.57 16.82 16.73 15.70 15.48 15.65 17.62 19.02 17.35 Fifteen days after release of stress % Control Stress Mean decrease 28.33 22.30 25.32 21.29 27.73 25.80 26.77 6.97 25.84 22.83 24.34 11.64 20.67 19.67 20.17 4.84 27.50 26.13 26.82 4.97 23.67 20.33 22.00 14.08 26.50 25.43 25.97 4.03 30.67 22.57 26.62 26.41 24.33 22.00 23.17 9.59 26.03 21.87 23.95 15.98 20.00 19.00 19.50 5.00 24.37 22.00 23.18 9.71 25.47 22.49 23.98 11.68 0.23 0.19 0.66 0.56 0.47 1.63 0.79 0.67 2.3 151 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.3 Mean of initial fluorescence (Fo) of sunflower cultivars during stress and after stress as influenced by moisture stress Five days after imposition of stress % S.No. Genotype Control Stress Mean decrease 1 RSF-101 86.67 84.67 85.67 2.31 2 TSF-103 99.33 92.17 95.75 7.21 3 ASF-107 120.00 90.67 105.33 24.44 4 DSF-114 83.33 80.67 82.00 3.20 5 SH-177 92.67 87.17 89.92 5.94 6 DSF-104 104.50 100.50 102.50 3.83 7 RSF-106 88.20 85.33 86.77 3.25 8 DSF-111 95.17 91.00 93.08 4.38 9 RSF-107 95.17 84.53 89.85 11.17 10 ASF-104 97.33 92.67 95.00 4.79 11 TSF-106 94.00 87.00 90.50 7.45 12 SH-491 97.33 95.17 96.25 2.23 Mean 96.14 89.29 92.72 7.12 CD at 5% for treatments 0.87 CD at 5% for genotypes 2.07 CD at 5% for TxG 2.93 Twenty days after imposition of stress % Control Stress Mean decrease 76.33 76.00 76.17 0.44 90.33 82.00 86.17 9.23 84.00 83.33 83.67 0.79 81.00 78.67 79.83 2.88 84.33 81.33 82.83 3.56 90.87 87.33 89.10 3.89 84.67 82.00 83.33 3.15 88.20 87.00 87.60 1.36 94.83 77.67 86.25 18.10 84.00 80.33 82.17 4.37 93.67 80.33 87.00 14.23 93.00 88.33 90.67 5.02 87.10 82.03 84.57 5.83 Fifteen days after release of stress % Control Stress Mean decrease 72.33 69.67 71.00 3.69 79.67 70.67 75.17 11.30 79.00 66.67 72.83 15.61 77.00 73.53 75.27 4.50 79.33 73.83 76.58 6.93 88.00 85.67 86.83 2.65 79.67 68.33 74.00 14.23 83.00 81.67 82.33 1.61 76.67 64.67 70.67 15.65 80.33 77.67 79.00 3.32 86.33 74.00 80.17 14.29 83.67 79.67 81.67 4.78 80.42 73.84 77.13 8.18 0.34 0.49 0.84 1.21 1.18 1.71 152 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.4 Mean of maximum fluorescence (Fm) of sunflower cultivars during stress and after stress as influenced by moisture stress Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Control RSF-101 237.33 TSF-103 313.00 ASF-107 307.00 DSF-114 295.33 SH-177 321.00 DSF-104 373.67 RSF-106 296.67 DSF-111 318.00 RSF-107 294.67 ASF-104 283.00 TSF-106 265.33 SH-491 347.67 Mean 304.39 CD at 5% for treatments CD at 5% for genotypes CD at 5% for T x G Stress 220.00 277.83 281.67 244.67 283.00 251.67 283.67 252.33 264.67 266.00 206.00 218.33 254.15 Mean 228.67 295.42 294.33 270.00 302.00 312.67 290.17 285.17 279.67 274.50 235.67 283.00 279.27 5.16 12.65 17.88 % decrease 7.30 11.24 8.25 17.16 11.84 32.65 4.38 20.65 10.18 6.01 22.36 37.20 16.50 Twenty days after imposition of stress % Control Stress Mean decrease 208.33 194.33 201.33 6.72 246.33 220.67 233.50 10.42 297.33 237.67 267.50 20.07 276.00 233.67 254.83 15.34 280.00 225.67 252.83 19.40 258.00 195.00 226.50 24.42 253.00 213.67 233.33 15.55 271.00 241.33 256.17 10.95 217.67 211.67 214.67 2.76 235.00 208.00 221.50 11.49 241.67 197.33 219.50 18.34 222.00 211.67 216.83 4.65 250.53 215.89 233.21 13.83 1.49 3.66 5.17 153 Fifteen days after release of stress % Control Stress Mean decrease 192.83 148.00 170.42 23.25 211.00 187.00 199.00 11.37 266.67 160.00 213.33 40.00 232.00 221.33 226.67 4.60 242.00 219.00 230.50 9.50 233.33 193.00 213.17 17.29 210.33 195.83 203.08 6.89 189.87 186.83 188.35 1.60 205.33 125.00 165.17 39.12 218.33 186.00 202.17 14.81 222.33 161.33 191.83 27.44 196.67 181.67 189.17 7.63 218.39 180.42 199.40 17.39 1.79 4.39 6.21 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.5 Mean of Maximum quantum efficiency of PS –II Photo chemistry (Fv/Fm) of sunflower cultivars during stress and after stress as influenced by moisture stress Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype RSF-101 TSF-103 ASF-107 DSF-114 SH-177 DSF-104 RSF-106 DSF-111 RSF-107 ASF-104 TSF-106 SH-491 Mean CD at 5% for treatments CD at 5% for genotypes CD at 5% for TxG % Control Stress Mean decrease 0.74 0.70 0.72 6.28 0.72 0.66 0.69 8.33 0.70 0.65 0.68 8.23 0.73 0.70 0.71 4.57 0.73 0.72 0.73 1.82 0.75 0.63 0.69 15.44 0.72 0.68 0.70 6.45 0.71 0.67 0.69 6.10 0.67 0.65 0.66 3.23 0.69 0.67 0.68 3.85 0.67 0.66 0.67 0.50 0.58 0.53 0.56 9.14 0.70 0.66 0.68 6.19 Twenty days after imposition of stress % Control Stress Mean decrease 0.68 0.53 0.61 22.06 0.71 0.61 0.66 14.08 0.68 0.61 0.65 10.29 0.68 0.67 0.68 1.47 0.73 0.64 0.69 12.33 0.67 0.60 0.64 10.85 0.69 0.64 0.67 7.25 0.60 0.56 0.58 7.13 0.65 0.59 0.62 9.23 0.63 0.56 0.60 11.11 0.66 0.60 0.63 9.09 0.56 0.55 0.56 1.79 0.66 0.60 0.63 9.89 Fifteen days after release of stress % Control Stress Mean decrease 0.62 0.50 0.56 19.35 0.63 0.55 0.59 12.70 0.65 0.58 0.61 10.63 0.64 0.62 0.63 2.60 0.64 0.60 0.62 6.25 0.57 0.50 0.54 12.28 0.60 0.57 0.59 5.00 0.52 0.49 0.51 5.77 0.49 0.35 0.42 28.57 0.60 0.56 0.58 6.67 0.56 0.49 0.53 12.50 0.55 0.54 0.55 1.82 0.59 0.53 0.56 10.12 0.004 0.001 0.002 0.01 0.002 0.005 0.014 0.003 0.007 154 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.6 Mean of membrane leakage (μA/cm2) of sunflower cultivars during stress and after stress influenced by moisture stress Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Control Stress Mean RSF-101 7.31 8.37 7.84 TSF-103 5.92 7.51 6.71 ASF-107 7.58 9.77 8.68 DSF-114 6.20 7.50 6.85 SH-177 6.42 10.63 8.53 DSF-104 5.60 6.87 6.23 RSF-106 6.02 7.49 6.76 DSF-111 7.41 8.87 8.14 RSF-107 7.26 9.48 8.37 ASF-104 3.90 6.90 5.40 TSF-106 2.90 4.72 3.81 SH-491 2.34 6.97 4.65 Mean 5.74 7.92 6.83 CD at 5% for treatments 0.06 CD at 5% for genotypes 0.14 CD at 5% for T x G 0.2 % decrease -14.40 -26.87 -28.79 -21.03 -65.71 -22.62 -24.41 -19.71 -30.62 -76.92 -62.76 -197.72 -38.07 Twenty days after imposition of stress % Control Stress Mean decrease 10.97 13.50 12.23 -23.10 10.03 12.96 11.50 -29.21 11.54 15.07 13.31 -30.52 10.67 13.93 12.30 -30.63 8.83 11.07 9.95 -25.28 9.20 9.87 9.53 -7.25 9.43 10.99 10.21 -16.58 11.80 13.27 12.53 -12.43 11.70 13.60 12.65 -16.27 10.37 13.97 12.17 -34.73 4.87 8.03 6.45 -65.07 6.03 8.83 7.43 -46.41 9.62 12.09 10.86 -25.69 155 Fifteen days after release of stress % Control Stress Mean decrease 13.27 16.00 14.63 -20.60 12.80 14.60 13.70 -14.06 15.53 16.27 15.90 -4.72 15.97 17.97 16.97 -12.53 11.23 12.37 11.80 -10.09 11.73 12.80 12.27 -9.09 11.97 13.07 12.52 -9.19 16.93 19.67 18.30 -16.14 16.77 19.63 18.20 -17.10 16.03 19.03 17.53 -18.71 9.03 10.07 9.55 -11.44 9.47 11.03 10.25 -16.55 13.39 15.21 14.30 -13.54 0.067 0.07 0.164 0.231 0.18 0.26 Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 147-159 Table.7 Mean of SPAD meter readings and initial fluorescence (Fo) of sunflower cultivars during stress and after stress as influenced by moisture stress Five days after imposition of stress S.No. 1 2 3 4 5 6 7 8 9 10 11 12 Genotype Control Stress RSF-101 39.67 32.37 TSF-103 40.00 39.17 ASF-107 41.47 40.10 DSF-114 39.87 39.83 SH-177 45.07 36.00 DSF-104 40.80 36.33 RSF-106 42.50 36.37 DSF-111 38.43 26.60 RSF-107 39.47 39.43 ASF-104 40.37 36.80 TSF-106 36.67 36.50 SH-491 44.60 36.97 Mean 40.74 36.83 CD at 5% for treatments CD at 5% for genotypes CD at 5% for T x G Mean 36.02 39.58 40.78 39.85 40.53 38.57 39.43 32.52 39.45 38.58 36.58 40.78 38.79 0.31 0.75 1.06 % decrease 18.40 2.08 3.30 0.08 20.12 10.96 14.43 30.79 0.08 8.84 0.45 17.12 9.59 Twenty days after imposition of stress % Control Stress Mean decrease 44.93 33.30 39.12 25.89 42.53 35.70 39.12 16.07 40.4 39.00 39.70 1.24 40.23 37.30 38.77 7.29 41 39.00 40.00 4.88 38.85 34.00 36.43 12.48 38.5 35.33 36.92 8.23 36.35 35.20 35.78 3.16 38.15 36.40 37.28 4.59 38.00 35.80 36.90 5.79 35.23 35.00 35.12 0.66 42.00 39.85 40.93 5.12 39.68 36.78 38.23 7.30 0.28 0.68 0.96 156 Fifteen days after release of stress % Control Stress Mean decrease 37.83 31.90 34.87 15.68 33.67 30.67 32.17 8.91 32.33 31.60 31.97 2.27 39.60 35.93 37.77 9.26 38.97 37.50 38.23 3.76 35.10 32.10 33.60 8.55 34.57 33.00 33.78 4.53 33.33 29.20 31.27 12.40 33.87 29.53 31.70 12.80 32.50 31.57 32.03 2.87 34.50 30.10 32.30 12.75 36.63 32.73 34.68 10.65 35.24 32.15 33.70 8.76 0.32 0.78 1.11