Journal of Applied Botany and Food Quality 90, 165 - 173 (2017), DOI:10.5073/JABFQ.2017.090.021 1Agricultural Botany Department, Faculty of Agriculture, Fayoum University, Fayoum, Egypt 2 King Khalid University, Faculty of Science, Biology Department, Saudi Arabia 3 Research Center for Advanced Materials Science (RCAMS) Seed inoculation with Azospirillum lipoferum alleviates the adverse effects of drought stress on wheat plants Ramadan A. Agami1*, Hamed A. Ghramh2, 3, Mohamed Hashem2, 3 (Received November 12, 2016) * Corresponding author Summary Drought is one of the major environmental stresses that adversely affects crop growth and productivity worldwide. The effect of inoculation with Azospirillum lipoferum on growth, yield, water status, osmoprotectant, antioxidant system and grain anatomy of wheat plants under drought stress conditions was investigated. The plants exposed to the drought stress exhibited a significant reduction in growth, grains yield, relative water content and leaf photosynthetic pigments, as well as alterations in grain anatomy. However, the treatment with A. lipoferum alleviated the stress generated by drought and improved the above-mentioned parameters. Drought stress increased proline, protein, soluble carbohydrates, relative membrane permeability and activities of antioxidant enzymes (SOD and POX). The antioxidant enzymes, phenols and grain anatomy exhibited changes in response to A. lipoferum inoculation in the absence or presence of drought stress. Our data suggest that inoculation with A. lipoferum could protect wheat plants from the harmful effects of drought stress through changes in the antioxidant defense system. Keywords: Triticum aestivum, Drought stress, Azospirillum lipo- ferum, Antioxidant defense system, Anatomy, Compatible solutes Introduction Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world, however this crop is exposed to a variety of abiotic stresses, such as drought, salt loading and freezing that in- fluence its development, growth and productivity. Drought stress is among the most destructive abiotic stresses that increased in inten- sity over the past decades affecting world’s food security. It is ex- pected to cause serious plant growth problems for more than 50% of the arable lands by 2050 (Kasim et al., 2013). Drought affects plant water potential and turgor, enough to interfere with normal func- tions (Hsiao, 2000), and induces changes in physiological and mor- phological characters in the plants (RaHdari and Hoseini, 2012). Common plant symptoms after water stress are: stunting, limiting in CO2 diffusion to chloroplasts by stomatal closure, reduction in photosynthesis rate, and acceleration of leaf senescence. Moreover, in wheat, a severe drought stress during the late growth stages (an- thesis − post anthesis) induces chlorophyll degradation, cell solute leakage, and accelerated spike and grain maturation (Beltrano et al., 1999). Drought stress also causes severe alterations in cell membrane selective permeability (leakage of cell solutes), fluidity and microviscosity (Navarri-Izzo et al., 1993; Beltrano et al., 1999). It induces free radicals affecting antioxidant defenses and Re- active Oxygen Species (ROS) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals resulting in oxidative stress. High concentrations of ROS can cause damage to various levels of organi- zation (Smirnoff, 1993), like initiate lipid peroxidation, membrane deterioration and degrade proteins, lipids and nucleic acids in plants (Hendry, 2005; SgHerrI et al., 2000; Nair et al., 2008). Much of the injury in plants under abiotic stress is due to oxidative damage at the cellular level, which is the result of imbalance between the formation of reactive oxygen species (ROS) and their detoxification. Plant cells produce different antioxidant enzymes such as catalase (CAT), peroxides (POX), superoxide dismutase (SOD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX) that scavenge the reactive free radicals (Simova-Stoilova et al., 2008). Generally, drought negatively affects quantity and quality of the plant growth. Therefore, to produce more food, the alleviation of drought stress is important to achieve the designated goals. Globally, an extensive research is being carried out to develop strate- gies to cope with drought stress through development of drought tolerant varieties, shifting the crop calendars, resource manage- ment practices and others and other means (VenKateswarlu and SHanKer, 2009) and most of these technologies are cost intensive. Recent studies indicate that microorganisms can also help plants to cope with drought stress. Bacteria of the genus Azospirillum are among the best investigated plant growth promoting rhizobacteria (PGPR) detected in the rhizosphere of many crop plants. They are able to produce plants hormones such as auxin, and proteins like polyamines, fix N2, increase root growth and control pathogens. Such abilities collectively result in the enhanced growth of plants under stress (Ramos et al., 2002; BHasKara rao and CHaryulu, 2005; Russo et al., 2008; Cassan et al., 2009). Many researchers have in- dicated that Azospirillum spp. can mitigate the unfavorable effects of water stress on plant growth (ArzanesH et al., 2009; Pereyra et al., 2009). The present study was designed with the objective to examine chan- ges in the antioxidant defense system of wheat plants under the effect of Azospirillum lipoferum applied by seed inoculation and exposed to drought stress. The tested hypothesis is that A. lipoferum will posi- tively modify the level of antioxidant system that will mitigate the injuries generated by drought stress. Consequently, A. lipoferum will enhance the wheat performance under drought stress. Materials and methods Plant materials and bacterial strain Seeds of wheat (Triticum aestivum L. cv. Giza 168) were obtained from the Crop Institute, Agricultural Research Center, Giza, Egypt. Bacterial strain Azospirillum lipoferum N040 was obtained from Agricultural microbiology department, Faculty of Agriculture, Cairo University for research purpose. Inoculum preparation and bacterial growth Bacterial culture was prepared by growing the Azospirillum li- poferum N040 in liquid nitrogen free biotin based (NFB) medium (Piccoli et al., 1997) [5 g l-1 peptone and 3 g l-1 beef PH 7.0] at 25 ± 1 °C with shaking (150 rpm) for 48 h. The bacterial cells were 166 R.A. Agami, H.A. Ghramh, M. Hashem pelletized by centrifugation (5000 rpm) for 10 min and re-suspen- ded in sterilized tap water containing 0.025% (v/v) Tween-20 to the desired concentration (108 CFU ml-1). Experimental design and treatments Two pot experiments were carried out at Demo Experimental Farm, Faculty of Agriculture, Fayoum University (Southeast Fayoum; 29° 17’N; 30° 53’E), during the two successive seasons of 2014 and 2015. Wheat seeds were surface-sterilized with 70% ethanol (3 min), treated with 2% sodium hypochlorite (NaClO) (5 min), and followed by repeated washing with sterile distilled water (3 times for 1 min). Then surface-disinfected seeds were incubated in sterilized liquid nitrogen free biotin based medium (as control) or in bacterial sus- pension (108 CFU ml-1) for 2 h on a rotary shaker at 81 rpm. Ten inoculated seeds (107 bacteria per seed) or non-inculcated seeds (control) were sown in each plastic pot (32 cm in diameter, 25 cm in deep) containing 15 kg of soil and were thinned to five plants, one week after germination. Soil used in the pots had the following physic-chemical characteristics: sand, 2.7%; silt, 28.7%; clay, 68%; pH, 7.28 (1:2, w/v, soil and water solution); EC, 3.49 dS m-1 (1:2, w/v, soil and water solution); CaCO3 10.81% and organic matter 3.39%; total nitrogen, 39.6 (mg/kg dry soil); available phosphorus, 32.9 (mg/ kg dry soil); extractable potassium, 8.33 (mg/kg dry soil). The first experiment was conducted on 15 November, 2014 and the second one was conducted on 19 November, 2015 in an open greenhouse. The average day and night temperatures were 22 ± 3 oC and 11 ± 2 oC, respectively. The relative humidity ranged from 38.1 to 69.8%, and day-length from 10 to 11 h. Pots were arranged in the greenhouse in a complete randomized block design with four replications for each treatment. Recommended doses of N, P, and K fertilizers (150-100- 60 kg ha-1) were applied to each pot and equal amounts of tap water was added to the pots to maintain the optimal soil moisture depen- ding on plant and soil conditions (up to 1000 ml). Drought stress was applied after 30 days of planting to grain ripening. The two soil water conditions were either well-watered (100% of crop evapotranspira- tion (ETc)) or dried up (60% of crop evapotranspiration). Crop evapo- transpiration was determined using gravimetrical method described by Maoa et al. (2014). Irrigation was applied twice a week during the experimental period. Samples of wheat plants (36 per each treatment) were collected after 90 days from sowing to assess morphological data. Length of shoots and spikes (cm) was measured by using a meter scale. Numbers of fertile tillers per plant and numbers of spikelets per spike were coun- ted. Flag leaf area (cm2) was measured using a digital leaf meter (LI- 3000 Portable Area meter Produced by LI-COR Lincoln, NE, USA). Samples of flag leaves were collected to estimate the concentration of total chlorophylls and total carotenoids, proline, total soluble protein, total soluble carbohydrates and total soluble phenols, relative mem- brane permeability, relative water content and activities of antioxi- dant enzymes. The experiment was terminated after 130 days from sowing after exposing the plants to water stress for 100 days. The 130-day-old plants from each treatment were collected for various measurements. The 130-day-old wheat plants were removed from the pots and moved smoothly to remove the adhering soil particles by dipped them in a bucket filled with water. Roots and straw were weighed to record their fresh weight, then were placed in an oven at 70 °C to reach a constant dry weight (DW). Grain yield per plant and 1000-kernel weight was also estimated. The powder of dried grains was used to determine the concentration of total soluble protein and total soluble carbohydrates. Anatomical study For observation of grain anatomy, samples were taken from the main spike at the age of 110 days and fixed in FAA solution (containing 50 cm3 of 95% (v/v) ethanol + 10 cm3 of formaldehyde + 5 cm3 of glacial acetic acid + 35 cm3 of distilled water) for 48 h. Thereafter, the samples were washed in 50% ethanol, dehydrated and cleared in tertiary butanol series, and embedded in paraffin wax. Cross sec- tions, 25 μm thick, were cut by a rotary microtome (Leitz, Wetz- lar, Germany), adhered by a Haupt’s adhesive, stained with a crystal violet-erythrosin combination (Sass, 1961), cleared in carbol xylene, and mounted in Canada balsam. The sections were observed and documented using an upright light microscope (AxioPlan, Zeiss, Jena, Germany). Measurements were done using a micrometer eye- piece and average of five readings was calculated. Photosynthetic pigments determination Total chlorophyll and carotenoids concentration (mg g-1 FW) were estimated according to the procedure given by Arnon (1949). Flag leaves discs (0.2 g) of 90-day-old plants were homogenized with 50 ml 80% acetone. The slurry was strained through a cheese cloth and the extract was centrifuged at 15,000 × g for 10 min. the optical density of the acetone extract was measured at 663, 645 and 470 nm using a UV-160A UV Visible Recording Spectrometer, Shimadzu, Japan. Free proline determination Proline concentration in wheat flag leaves was measured following the rapid colorimetric method of Bates et al. (1973). Proline was extracted from 0.5 g of dry leaf samples by grinding in 10 ml of 3% sulpho-salicylic acid. The mixture was then centrifuged at 10,000 × g for 10 min. Two ml of the supernatant was added into test tubes and 2 ml of freshly prepared acid-ninhydrin solution was added. Tubes were incubated in a water bath at 90 °C for 30 min. The reac- tion was terminated in ice-bath. The reaction mixture was extracted with 5 ml of toluene and the vortex process was done for 15 s. The tubes were allowed to stand at least for 20 min in the dark at room temperature to allow the toluene and aqueous phases to be separated. The toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm using a UV-160A UV Visible Re- cording Spectrometer, Shimadzu, Japan. The proline concentration in the sample was determined from a standard curve using analytical grade proline. Total soluble proteins determination The total soluble proteins concentration of the dry flag leaves and grains was determined according to the method described by Brad- ford (1976) with bovine serum albumin as a standard. An amount of 0.2 g of samples was ground in a mortar with 5 ml of phosphate buffer (pH 7.6) and was then transformed to the centrifuge tubes. The homogenate was centrifuged at 8000 rpm for 20 min. The su- pernatant of different samples was put in separate tubes. The volume of the samples in tubes was then made equal by adding a phosphate buffer solution and the extraction were stored in the refrigerator at 4 °C for further analysis. After extraction, 30 μl of different samples were taken out in separate tubes and were mixed with 70 μl of dis- tilled water. Then, 2.9 ml of the Coosmassic Brilliant Blue solution was added to each sample tube and mixed thoroughly. The total vol- ume was 3 ml in each tube. All tubes were incubated for 5 min at room temperature and then, the absorbance was recorded at 600 nm against the Blank. A standard curve of absorbance (600 nm) versus concentration (μg) of total soluble proteins was calculated. Total soluble carbohydrates determination Leaf and grains total soluble carbohydrates concentration were as- sessed by the method recommended by the Association OF Of- Azospirillum lipoferum alleviates drought stress on wheat plants 167 ficial agricultural cHemists (1990) using phenol sulphuric acid reagent method. Total soluble phenols determination The soluble phenol concentration in wheat flag leaves was extracted as described by Hsu et al. (2003). 0.2 g of dry leaves were homo- genized in 80 ml methanol and kept overnight. The filtrates were diluted to 100 ml, and served as a stock solution. According to slinKard and Singleton (1997), 200 μl of the stock solution was added to 1.4 ml distilled water, and 0.1 ml of 50% (1N) Folin-Cio- calteu phenol reagent. After three min., 0.3 ml of 20% (w/v) sodium carbonate was added. The mixture was allowed to stand for 2 h. Af- ter gentle vortex, the absorbance was determined at 765 nm. Total soluble phenol concentration was standardized against tannic acid. Relative water content determination Relative water content (RWC) was determined in midrib excluding- fresh flag leaf discs of 2 cm2 area. Discs were weighed quickly and immediately floated on double distilled water in Petri dishes to satu- rate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor mass was noted. Dry mass of the discs was recorded after dehydrating them at 70 °C for 48 h. By pla- cing the values in the following formula (Hayat et al., 2007), RWC was calculated: RWC (%) = (Fresh mass – dry mass) / (Turgid mass – dry mass) × 100 Relative membrane permeability determination For the RMP measurement, the flag leaves were cut into equal pieces and transferred to test tubes containing 20 ml of deionized distilled water. The test tubes were vortexed for 10 s and the solution was as- sayed for initial electrical conductivity (EC0). These tubes were kept at 4 °C for 24 h and then assayed for EC1. The same samples were autoclaved at 121 °C for 20 min to determine EC2. Percent RMP was calculated as following the formula described by Yang et al. (1996). RMP (%) = (EC1−EC0) / (EC2−EC0) × 100 Enzyme assays Flag leaves were excised from wheat plants and rapidly weighed. Each 1.0 g sample was ground with a pestle in an ice-cold mortar containing 10 ml of 50 mM phosphate buffer, pH 7.0. The homo- genate was centrifuged at 20,000 × g for 30 min at 4 °C. The super- natant was then filtered through two layers of cheese-cloth and used to measure various anti-oxidant enzyme activities. Superoxide dismutase (SOD) activity was determined according to the method of FridovicH (1975). One Unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of oxidation of nitroblue tetrazolium (NBT) at 560 nm. Each 3 ml reaction mixture contained 50 mM phosphate buffer pH 7.0, 200 mM methionine, 1.125 mM NBT, 1.5 mM EDTA, 75 mM riboflavin, and 10-40 μl of crude enzyme extract. The riboflavin was added as the last component. The tubes were shaken and placed 30 cm below two 15 W fluorescent lights. The reaction was started by switching on the light, and allowed to run for 10 min. Switching-off the light stopped the reaction. The tubes were then covered immedi- ately with a black cloth and the absorbance was measured spectro- photometrically at 560 nm. A non-irradiated reaction mixture was set to zero absorbance as the blank. The volume of enzyme extracts that produced a 50% inhibition of the oxidation (color reaction) was read from the resulting graph. Peroxidase (POX) activity in flag wheat leaves was measured using the method of THomas et al. (1981). POX was assayed using guaiacol as the substrate. The crude enzyme extract was prepared in a similar way to that used for SOD. Each reaction mixture consisted of 3 ml of 0.1 M phosphate buffer pH 7.0, 30 μl of 20 mM H2O2, 50 μl of crude enzyme extract, and 50 μl of 20 mM guaiacol. The reaction mixture was incubated for 10 min at room temperature in a cuvette. The ab- sorbance was then measured at 436 nm. POX activity was expressed in A 436 Units g -1FW leaf min-1. Statistical analysis All the pots for two experiments (288) were arranged in a complete randomized design with nine pots per replicate and four replicates per treatment. Analysis of variance was performed using the SPSS software package to determine the least significant difference (LSD) among treatments at P ≤ 0.05, and the Duncan’s multiple range test was applied for comparing the means. Results Growth traits Azospirillum lipoferum inoculation significantly improved the plant growth, straw and the grain yields of wheat in presence or absence of drought stress under open greenhouse conditions. However, drought stress significantly decreased the length of shoot, number of fertile tillers per plant, flag leaf area, length of spike, number of spiklets per spike, root fresh and dry weights (Tab. 1). Comparing to the con- trol (100% ETc), the above-mentioned parameters were decreased under 60% of ETc by 27.17, 63.76, 64.85, 28.08, 19.95, 64.56 and 72.52%, respectively. After the inoculation of the wheat grains with Tab. 1: Effect of Azospirillum lipoferum inoculation on the growth traits [length of shoot (cm), No of fertile tillers per plant, flag leaf area (cm2), length of spike (cm), No of spikelets per spike, root fresh mass (g) and root dry mass (g)] of wheat (Triticum aestivum L., cv. Giza-168) plants grown under non-stressed and drought-stressed condition. Treatments Parameters Irrigation levels Inoculation Length of shoot No of fertile Flag leaf area Length of spike No of spikelets Root fresh Root dry weight tillers per plant per spike weight 100% ETc No-inoculated 44.17±1.80b 3.67±0.58a 16.30±1.21b 9.83±0.20a 13.33±0.58b 0.790±0.06b 0.473±0.03b Inoculated 50.00±0.58a 4.67±0.58a 27.60±2.52a 9.87±0.35a 15.33±0.58a 1.600±0.48a 1.090±0.06a 60% ETc No-inoculated 32.17±0.58d 1.33±0.58c 5.73±0.66c 7.07±0.12c 10.67±0.58c 0.280±0.02c 0.130±0.02c Inoculated 39.17±0.76c 1.67±0.58c 8.44±0.65c 8.20±0.75b 11.67±0.58c 0.280±0.03c 0.160±0.01c Values are means ± SD (n=9) and differences between means were compared by the Duncan’s multiple range test (LSD; P ≤0.05). Mean pairs followed by different letters are significantly different. 168 R.A. Agami, H.A. Ghramh, M. Hashem A. lipoferum N040 and in absence of the water stress, the growth in most cases was significantly higher than that of the control (P ≤ 0.05). Inoculation with the bacteria also improved the growth of the plants grown under the water stress and the values in some cases were significantly higher than those of the plants grown under the stress alone. Yield components Data in Tab. 2 show that wheat plants growing in the presence of water stress significantly decreased the yield components. However, the reductions were 52.45, 45.28, 71.97, 73.12, 38.49 and 19.27% for straw fresh weight per plant, straw dry weight per plant, spike fresh weight per plant, spike dry weight per plant, grain yield per plant and 1000-kernel weight respectively, compared to non-stressed and non-inoculated plants. Inoculation of grains of Triticum aestivum cv. Giza 168 with A. lipoferum N040 alleviated these deleterious effects of drought stress. In the presence of water stress A. lipoferum treat- ment showed 24.79 and 11.31%, 54.33 and 58.67%, 79.35 and 3.32%, increases in straw fresh and dry weights per plant, spike fresh and dry weights per plant, grain yield per plant and 1000-kernel weight respectively, compared to non-inoculated control. However, incuba- tion the wheat grains in A. lipoferum suspensions significantly in- creased the above-mentioned parameters especially in the absence of the water stress (100% ETc) compared to the non-inoculated plants. Anatomy of grain As concerns the grain anatomical structure, drought stress decreased height and width of the grain by 9.09 and 9.63%, height and width of the endosperm by 13.78 and 10.82% as well as pericarp and aleurone layer by 23.08, and 17.22%, respectively, in comparison to the control (Tab. 3 and Fig. 1). However, incubation the seeds in A. lipoferum suspension caused positive changes in the above-mentioned charac- teristics in absence or presence of the water stress. For example, the maximum increase was achieved in A. lipoferum pretreated plants in absence of the water stress which recorded increments of 16.49 and 14.66, 41.67, 17.66 and 15.53 and 24.49% for length and width of grain, pericarp, length and width of endosperm and aleurone layer, as compared to non-inoculated and water-stressed plants. Photosynthetic pigments The water deficiency stress significantly decreased the concentration of total chlorophyll and carotenoids (Tab. 4). Comparing to non-in- oculated control, the decrease reached 46.09 and 22.58% for total chlorophyll and carotenoids, respectively. After inoculation com- bined with drought stress the total chlorophyll and carotenoids con- tent of wheat significantly increased compared to the non-inoculated plants (60% ETc). In absence of the drought stress, A. lipoferum inoculation significantly promoted the increases in total chloro- phyll and carotenoids by 23.48 and 16.13% for total chlorophyll and carotenoids, respectively compared to non-inoculated plants (100% ETc). Compatible solutes Wheat plants subjected to drought stress exhibited a significant in- crease in the content of free proline, total soluble protein in leaves, total soluble carbohydrates in leaves and grains but showed a slight Tab. 2: Effect of Azospirillum lipoferum inoculation on the yield component [straw fresh weight per plant (g), straw dry weight per plant (g), spike fresh weight per plant (g), spike dry weight per plant (g), grain yield per plant (g) and 1000-kernel weight (g)] of wheat (Triticum aestivum L., cv. Giza-168) plants grown under non-stressed and drought-stressed condition. Treatments Parameters Irrigation levels Inoculation Straw fresh weight Straw dry weight Spike fresh weight Spike dry weight Grain yield 1000-kernel per plant per plant per plant per plant per plant weight 100% ETc Non-inoculated 9.60±0.40b 3.07±0.21b 11.95±1.00b 5.58±0.55b 2.52±0.03c 38.40±0.61b Inoculated 18.32±0.21a 6.03±0.64a 15.95±0.80a 7.07±0.64a 6.51±0.11a 40.80±0.26a 60% ETc Non-inoculated 4.67±0.15d 1.68±0.03c 3.35±0.09d 1.50±0.10c 1.55±0.06d 31.00±2.00c Inoculated 5.85±0.35c 1.87±0.06c 5.17±0.40c 2.38±0.18c 2.78±0.03b 32.03±0.84c Values are means ± SD (n=9) and differences between means were compared by the Duncan’s multiple range test (LSD; P ≤0.05). Mean pairs followed by different letters are significantly different. Tab. 3: Effect of Azospirillum lipoferum inoculation on the height and width of grain and endosperm, thickness of percarp and aleurone layer [μm] of wheat (Triticum aestivum L., cv. Giza-168) plants grown under non-stressed and drought-stressed condition. Treatments Parameters Irrigation levels Inoculation Dimensions of grain Dimensions of endosperm Percarp Aleurone layer thickness thickness Height Width Height Width 100% ETc Non-inoculated 2062.4±11.6b 3873±12.6b 1884.8±6.9b 3646.3±18.7b 52.0±2.0a 60.0±1.0a Inoculated 2184.3±3.9a 4013±15.3a 1912.0±11.8a 3756.7±7.6a 56.7±3.1a 61.0±1.0a 60% ETc Non-inoculated 1875.0±10.0c 3500±20.0d 1625.0±7.0d 3251.7±12.6c 40.00±2.0c 49.7±1.5b Inoculated 1875.2±13.9c 3752±17.6c 1711.8±11.5c 3619.0±16.8b 46.00±1.0b 57. 7±2.5a Values are means ± SD (n=9) and differences between means were compared by the Duncan’s multiple range test (LSD; P ≤0.05). Mean pairs followed by different letters are significantly different. Azospirillum lipoferum alleviates drought stress on wheat plants 169 decrease in total soluble protein in grains compared to the non- inoculated and non-stressed plants (Tab. 4). The increases were 31.86, 97.22, 35.82 and 18.61%, respectively. However, in presence of drought stress inoculation of grains with A. lipoferum alleviated these effects of drought stress on the previous compatible solutes and significantly decreased these parameters compared to non-inocu- lated and water-stressed plants. The reductions were 12.08, 16.20, 7.31 and 11.35%, for leaf free proline, leaf total soluble protein, total soluble carbohydrates in leaves and grains, respectively. Total soluble phenols The stress generated by water deficit resulted in a slight decrease in total soluble phenols compared to non-water stressed plants (Tab. 5). A B C D Fig. 1: Photographs of grain section of Azospirillum lipoferum N040 inoculated Triticum aestivum L. plants grown under water stress. A) Non-inoculated + 100% ETc; B) Inoculated + 100% ETc; C) Non-inoculated + 60% ETc; D) Inoculated + 60% ETc; al, aleurone layer; en, endosperm and pe, percarp, bars = 200 μm. Tab. 4: Effect of Azospirillum lipoferum inoculation on total chlorophylls (mg g−1 FW), total carotenoids (mg g−1 FW), proline (mg g−1 DW), protein in leaves and grains (mg g−1DW) and total soluble carbohydrates in leaves and grains (mg g−1 DW) of wheat (Triticum aestivum L., cv. Giza-168) plants grown under non-stressed and drought-stressed condition. Treatments Parameters Irrigation levels Inoculation Total Total Proline Protein Protein Total soluble Total soluble chlorophylls carotenoids in leaves in grains carbohydrates carbohydrates in leaves in grains 100% ETc Non-inoculated 1.15±0.03 0.31±0.02b 1.13±0.07c 2.16±0.05d 18.75±0.48b 112.8±0.67d 623.23±0.96d Inoculated 1.42±0.10 0.36±0.01a 1.19±0.03c 3.05±0.13c 20.34±0.17a 131.7±0.47c 651.97±0.95c 60% ETc Non-inoculated 0.62±0.03 0.24±0.01d 1.49±0.07a 4.26±0.04a 17.85±0.87b 153.2±1.48a 739.20±0.61a Inoculated 1.03±0.06 0.28±0.01c 1.31±0.03b 3.57±0.04b 18.10±0.21b 142.0±10.37b 655.27±1.01b Values are means ± SD (n=6) and differences between means were compared by the Duncan’s multiple range test (LSD; P ≤0.05). Mean pairs followed by different letters are significantly different. 170 R.A. Agami, H.A. Ghramh, M. Hashem However, this attribute was significantly improved by A. lipoferum inoculation in presence or absence of the drought stress. In presence of the water deficiency stress, A. lipoferum-inoculated grains had a 36.37% increase in the total soluble phenols over its non-inoculated control. Relative water content (RWC %) and relative membrane perme- ability (RMP %) Data shown in Tab. 5 reveal that the RWC% of wheat plants was significantly reduced by 29.20% in presence of the water deficien- cy stress, but RMP% was significantly increased by 97.47% com- pared to the non-inoculated control (100% ETc). In presence of the drought stress, A. lipoferum inoculation reduced the injurious effects of drought stress on wheat plants, and maintained their RMP% and RWC% values at the near levels as in control plants. Antioxidant enzyme activities The activities of superoxide dismutase (SOD) and peroxidase (POX) are shown in Tab. 5. Growing wheat plants in presence of the water deficiency stress significantly increased SOD and POX activities by 19.22% and 51.58%, respectively, compared to the non-inoculated control (100% ETc). In addition, A. lipoferum inoculation of grains further increased these enzyme activities in presence of the drought stress by 8.82% and 31.25%, respectively compared to the control (i.e. non-inoculated and drought-stressed plants). Even in the absence of water deficiency stress A. lipoferum inoculation also significantly increased the activity of the two enzymes compared to the control (100% ETc). Discussion Water stress is one of the most adverse factors affecting plants growth and productivity. In addition, water stress causes over-pro- duction of reactive oxygen species (ROS) that can pose a threat to cells by causing oxidization of lipids, DNA, RNA and proteins, leading ultimately to cell death (smirnoff, 1995; mittler, 2002; cruz de carvalHo, 2008; Kar, 2011; sHarma et al., 2012). A ba- lance between the generation and degradation of ROS is required to avoid oxidative injury and to maintain metabolic functions under stress conditions. In plant tissues, the level of ROS is controlled by an antioxidant system that consists of antioxidant enzymes and non- enzymatic low molecular weight antioxidant molecules, including proline, ascorbic acid and carotenoids (scHutzenduBel and Polle, 2002; semida and rady, 2014; agami, 2016). In this study, water deficiency stress significantly reduced the growth of wheat plants, in terms of reduced length of shoot, number of fertile tillers per plant, flag leaf area, length of spike, number of spikelets per spike, root fresh and dry weights per plant, straw fresh and dry weights per plant, spike fresh and dry weights per plant, 1000-kernel weight and grain yield per plant. The observed reduction in the above-mentioned growth parameters under drought stress condition in this study may be due to the disturbance in metabolic process of the plant including chlorophyll destruction and the cell division (Tab. 3 and 4). Water deficiency stress causes losses in tissue water content, which reduce turgor pressure in the cell, thereby inhibiting enlargement and divi- sion of the cells causing a reduction in plant growth (sHao et al., 2007). Moreover, water stress decreased the growth rate, stem elon- gation and leaf expansion (Hale and orcutt, 1987). The decline in fresh weight may be due to the decrease in water content of the stressed plant cells and tissues which lose their turgor and thus shrink (Boyer, 1988; soHa E. KHalil and el-noemani, 2012). The de- crease in dry weights of the stressed plants could be attributed to the disturbances in metabolic processes, which lead to decreases in meristematic activity, thereby inhibiting division of cells causing a reduction in dry mass production. Drought stress impairs mitosis, cell elongation and expansion result in reduced plant height, leaf area and crop growth (nonami, 1998; Kaya et al., 2006; Hussain et al., 2008). The deleterious effects of drought stress on growth were re- ported by several researcher i.e. Beltrano and ronco (2008) they found that, dry weight per plant was decreased in wheat plants sub- jected to severe drought stress, arzanesH et al. (2011) they found that, straw yield and grain weight per ear were decreased under drought stress, Agami (2013) who reported that, drought stressed plants showed a significant reduction in growth traits and yield of let- tuce comparison to non-water- deficiency stressed plants, naveeda et al. (2014) they found that, drought stress had drastic effects on growth of maize plants; wang et al. (2016) they found that severe stress had a negative impact on growth of Heteropogon contortus plants. It has been shown from the results of this study that A. lipoferum inoculated grains in presence or absence of water deficit stress sig- nificantly improved plant growth characteristics and productivity of wheat plants. We have found that our bacterial strain has ameliora- tive effects on wheat growth grown in presence of water stress. This indicates that A. lipoferum is able to tolerate the drought stress and become active after the stress. This is very interesting regarding this strain, because there are so many situations in which the agricultural soils are subjected to moisture fluctuations. With such kind of ability of Azospirillum strain can alleviate the drought stress on plant growth and yield through stabilizing the plant growth conditions including plant water characters. These observations are in accordance with previous reports on the potential of endophytic bacteria having mul- tiple beneficial traits in improving plant productivity and to enhance drought tolerance in plants (sandHya et al., 2010; vardHarajula Tab. 5: Effect of Azospirillum lipoferum inoculation on relative water content (RWC %), relative membrane permeability (RMP %), total soluble phenols (mg g−1 DW) and the activities of superoxide dismutase (Units g−1 FW leaf min−1) and peroxidase (Units g−1 FW leaf min−1) of wheat (Triticum aestivum L., cv. Giza-168) plants grown under non-stressed and drought-stressed condition. Treatments Parameters Irrigation levels Inoculation Relative water content Relative membrane Total soluble phenols SOD activity POD activity permeability 100% ETc Non-inoculated 71.23±1.50a 6.33±0.25c 3.40±0.10b 14.93±0.61d 0.95±0.03d Inoculated 73.50±0.61a 6.57±0.31c 4.57±0.21a 16.07±0.38c 1.61±0.03b 60% ETc Non-inoculated 50.43±1.80c 12.50±0.78a 3.30±0.10b 17.80±0.40b 1.44±0.10c Inoculated 63.00±5.52b 8.03±0.32b 4.50±0.20a 19.37±0.42a 1.89±0.03a Values are means ± SD (n=9) and differences between means were compared by the Duncan’s multiple range test (LSD; P ≤0.05). Mean pairs followed by different letters are significantly different. Azospirillum lipoferum alleviates drought stress on wheat plants 171 et al., 2011). PGPR including Azospirillum spp. affect plant growth through different activities including production of plant hormones such as IAA, N2-fixation and controlling pathogens (spaepen et al., 2008; Jalili et al., 2009). Production of plant hormones by Azospi- rillum brasilense increased root growth through enhancing nutrient uptake (pereyra et al., 2009). In this study, the drought stress markedly reduced dimension of wheat grain. This was mainly due to the reduction in height and width of endosperm, pericarp and aleurone layer thickness (Tab. 3 and Fig. 1). Tissues exposed to environments with low water availability have generally shown reduction in cell size, and increase in vascular tissue and cell wall thickness (guerfel et al., 2009). In contrast, A. lipoferum was found to be more efficient in mitigating the adverse effects of stress by inducing positive changes the grain anatomy. The beneficial effect of A. lipoferum on wheat grain structure may be due to the crucial role of A. lipoferum in improving N2-fixing potential, and plant growth regulators such as auxins, gibberellins and cyto- kinins which play a role in cell division and expansion. Drought stress caused a significant reduction in the total chlorophylls and carotenoids concentration (Tab. 4). This reduction may be at- tributed to the increase in activity of chlorophyll-degrading enzyme chlorophyllase under stress conditions (reddy and vora, 1986). A. lipoferum inoculation could alleviate the reduction in total chloro- phylls and carotenoids concentration under water deficiency stress. A similar result was reported by Heidari et al. (2011), who stated that inoculation of bacterial strain like Pseudomonas sp., Bacillus lentus, Azospirillum brasilens, increased chlorophyll content in basil (Ociumum basilicm L.) under drought stress. Proline concentration in leaves of wheat plants were significantly increased under water deficiency stress (Tab. 4) which may be due to up regulation of proline biosynthesis pathway to keep proline in high levels, which helps in maintaining cell water status, protects membranes and proteins from stress (YosHiBa et al., 1997). The A. lipoferum inoculation in presence of drought stress showed lower values for the proline concentration than those in the water stress alone, suggesting that if proline is a stress indicator, wheat plants treated with A. lipoferum should have better drought tolerance. Be- cause of A. lipoferum in presence of drought stress supported the antioxidant system in wheat plants to enable them to tolerate drought stress. Therefore, it could be expected that, the level of proline de- clined as result of recovery from stress. The reduced accumulation of proline may result in mitigating the stress effects of drought on wheat plants. This could be because proline is essential in proteins biosynthesis that necessary for cell division. Soluble sugars are key osmolytes contributing towards osmotic adjustment. In our study, the concentration of total soluble carbohydrates significantly increased in leaves of wheat plants subjected to drought stress. The increase in sugar concentration may be a result from starch degradation (eneBaK et al., 1997). The increment in sugar concentration may be also a result of an interrupted starch metabolism to gain osmolytes for coping with the osmotic stress arising from water deficit. A. li- poferum inoculation decreased total soluble carbohydrates concen- tration in leaves of wheat plants under drought stress. This reduction in soluble carbohydrates concentration may be resulted from miti- gating the stress generated by water stress. In this study inoculation also decreased total soluble proteins concentration in drought stress plants over than non-inoculated plants indicating A. lipoferum helps in alleviating the stress induced by water deficit. The relative water content is a good indicator of drought stress (fisHer, 2000) and in this study, we noticed that drought stress caused a decrease in relative water content in both inoculated and non-inoculated plants compared to well watered plants, however, the inoculation of wheat stressed plants with the bacterium A. lipoferum N040 significantly increased the relative water content compared to the non-inoculated controls. This may be due to a reduction in the inhibitory effect of drought on roots and the development of a more effective root system in the inoculated plants (dodd et al., 2010). Drought stress accelerated relative membrane permeability (RMP) in the inoculated and non-inoculated plants compared to well- watered plants (100% ETc). However, bacterial inoculation helped wheat plants to maintain the RMP and reduced leaf damage com- pared to non-inoculated plants under drought stress. A positive cor- relation between drought stress sensitivity and membrane damage were observed by VardHarajula et al. (2011) and sandHya et al. (2010), and the bacterial inoculation reduced the membrane damage in plants stressed by drought stress. Antioxidant enzymes are very good biochemical markers of stress and increasing their activities could be a potential to alleviate the oxidative stress-induced by water stress. In our study, drought in- duced activities of the two enzymes and this may be attributed to the generation of ROS. Consequently, the plant tries to force this by stimulation of antioxidants defense system (li et al., 2011). The inoculation showed further induction in activities of both SOD and POD (Tab. 5), increasing the antioxidant defense system efficiency against superoxide (O2-) radicals produced under water stress. It is most probable that A. lipoferum improved plant defense enzymes such as superoxide dismutase, peroxidase or phenolic compounds, to mitigate the oxidative damage elicited by drought stress. More work is necessary to find exactly the protecting roles of Azo- spirillum sp. on antioxidant enzymes system under drought stress, to provide potential new mechanisms of a plant’s tolerance to drought stress, and to define the physiological roles of Azospirillum sp. in relation to environmental stresses, including drought stress. Conclusions The inoculation of wheat plants with the bacterium A. lipoferum N040 increased the activities of some key anti-oxidative enzymes (super-oxide dismutase and peroxidase) and the concentrations of non-enzymatic antioxidants such as total carotenoids and total phe- nols as well as the concentration of compatible solute such as proline, soluble carbohydrates and soluble proteins in wheat plants grown under drought stress. 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