Bio fertilizers and zinc effects on some physiological parameters of triticale under water-limitation condition

ABSTRACT In order to study bio fertilizers and zinc effects on some physiological parameters of triticale under a water-limitation condition, a factorial experiment was conducted based on randomized complete block design with three replications in 2014 and 2015. Experimental factors consisted of three irrigation treatments [normal irrigation (W0); moderate water limitation (W1) and severe water limitation (W2)]; four bio fertilizers’ levels [(no bio fertilizer (F0), application of mycorrhiza (F1), plant-growth-promoting rhizobacteria (PGPR) (F2) and both application PGPR and mycorrhiza (F3)] and four nano zinc oxide levels [(without nano zinc oxide (Zn0) as control, application of 0.3 (Zn1), 0.6 (Zn2) and 0.9 (Zn3) g L−1)]. Results showed that water limitation decreased chlorophyll content, relative water content, stomatal conductance, quantum yield and grain yield of triticale, whereas electrical conductivity and the activity of catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7) and polyphenol oxidase (PPO, EC 1.14.18.1) enzymes were increased. Inoculation of plants with bio fertilizers and zinc application improved these traits (except electrical conductivity) under water-limitation condition as well as normal irrigation. Based on the results, it was concluded that bio fertilizers and nano zinc oxide application can be recommended for profitable triticale production under water-limitation condition.


Introduction
Triticale is a human-made crop, being a hybrid formed by cross-fertilization of wheat (Triticum spp.) and rye (Secale spp.). In general, triticale combines the high-yield potential of wheat with the biotic and abiotic stress tolerance of rye, making it more suitable for the production in marginal areas (acidic, saline or soils with heavy metal toxicity) (Cantale et al. 2016). Triticale can act as a soil improver, as its extensive root system binds erosion-prone soil and provides a good substrate for conversion into subsoil organic carbon by soil microbes (Salmon et al. 2004). Although a new crop, the benefits of triticale production are enormous, and this is the reason for its adoption in more than 30 countries with an ever increasing acreage. The leading producers of triticale worldwide are Germany, France, Poland, Australia, China and Belarus. In 2005, according to the Food and Agriculture Organization (FAO), 13.5 million tons of triticale grain was harvested in 28 countries across the world. In recent years, triticale has received attention as a potential energy crop, and research is currently being conducted on the use of the crops' biomass in bioethanol production. Interest in triticale has developed around two areas of potential use for the grain and its use as forage crop (Mergoum et al. 2009).
Drought stress is the most influential factors affecting crop yield particularly in irrigated agriculture in arid and semi-arid regions. This stress induces various biochemical and physiological responses in plants as a survival mechanism (Tas and Tas 2007). Drought stress has a direct impact on the photosynthetic apparatus, essentially by disrupting all major components of photosynthesis, including the thylakoid electron transport, the carbon reduction cycle and the stomatal control of the CO 2 supply, together with an increased accumulation of carbohydrates, peroxidative destruction of lipids and disturbance of water balance (Allen and Ort 2001). It breaks down the balance between the productions of reactive oxygen species (ROS) and the antioxidant defense system causing the accumulation of ROS, which induces oxidative stress to protein, membrane lipids and disruption of DNA strands (El Tayeb 2006).
Among the numerous microorganisms in the rhizosphere, some have positive effects on plant growth promotion. These microorganisms are bio fertilizers such as plant-growth-promoting rhizobacteria (PGPR), which colonize the rhizosphere and roots of many plant species and confer beneficial effects to plants (Gusain et al. 2015). The mechanisms by which PGPRs promote plant growth are not fully understood, but are thought to include: the ability to produce phytohormones against phytopathogenic microorganisms by production of siderophores, asymbiotic N 2 fixation, the synthesis of antibiotics, enzymes and/or fungicidal compounds and also solubilization of mineral phosphates and other nutrients (Ahmad et al. 2006). Using rhizosphere microorganisms (particularly beneficial bacteria) is an alternative strategy that can improve plant performance under stress environments and, consequently, enhance plant growth through different mechanisms (Dimkpa et al. 2009).
Mycorrhiza is a symbiotic association composed of plant roots (both terrestrial and aquatic plants) and fungi. They also impart other benefits to them, including production/ accumulation of secondary metabolites, osmotic adjustment under osmotic stress, improved nitrogen fixation, enhanced photosynthesis rate and increased resistance against biotic and abiotic stresses (Willis et al. 2013). The mechanisms used by mycorrhiza to enhance the water relations of host plants are not amply clear; however, this may occur by increasing water absorption by external hyphae, regulation of stomatal apparatus, increase in activity of antioxidant enzymes and absorption of nutrients, particularly phosphorus (Birhane et al. 2012). A study conducted on wheat under water stress environment showed that mycorrhizal inoculation enhanced the activities of antioxidant enzymes such as peroxidase (POD) and catalase (CAT) compared to those in un-inoculated control plants (Khalafallah and Abo-Ghalia 2008). Mycorrhizae are able to promote the water and physiological status of the host by altering the rate of water movement in the host plant. Gong et al. (2013) showed that mycorrhizal seedlings improved their water uptake compared to non-mycorrhizal seedlings under drought stress condition. It is likely that the hyphae of the arbuscular mycorrhizae (AM) expanded the absorption region of the host plant roots and enhanced the absorption of water by the roots.
Zinc is an essential micronutrient for biological systems and plays a crucial physiological role in enzymes and proteins involved in many biochemical pathways, photosynthesis, activation of enzyme systems and protein synthesis (Tsonev and Lidon 2012). Application of Zn enhances the photochemical reactions occurring in thylakoid membrane, electron transport through PSII and increases photosynthetic rate and chlorophyll content (Roach and Liszkay 2014). Qiao et al. (2014) observed that foliar application of Zn enhanced carbonic anhydrase activity in rice leaves and hence, increased photosynthesis. Carbonic anhydrase is considered as an Zn-containing enzyme involved in photosynthesis. Similarly, in Zn-treated Solanum lycopersicum, the superoxide dismutase, CAT and ascorbate peroxidases activities were found to increase (Cherif et al. 2011).
Drought is the most severe abiotic stress factor limiting plant growth and crop production. Many physiological processes in plants are impaired by drought stress, including photosynthesis, enzyme activity and membrane stability. One billion hectares are hyper arid and 5.45 billion hectares are made up of arid, semi-arid and sub-humid areas. There are 70% (5.2 billion hectares) of dry lands around the world that are used for agriculture with a limited productivity, where crop yield depends on the mode of drought (Rezaul Karim and Ataur Rahman 2015). Since, in arid and semiarid regions, the part of growth period of triticale confronted with water-limitation condition, which can affect biochemical and physiological responses such as changes in antioxidant enzyme activities, photosynthetic efficiency of PSII, chlorophyll content and stomatal conductance. Also, application of bio fertilizers and zinc is one of the most important strategies for alleviation of drought stress effects. Bio fertilizers can improve plant performance under non-stress and stress conditions (Dimkpa et al. 2009). Triticale is sensitive to Zn deficiency, which is needed for chlorophyll formation, growth hormone stimulation, carbohydrate synthesis, enzymatic activity and reproductive processes (Cakmak et al. 1997). Therefore, the aim of this study was to evaluate the effects of bio fertilizers and zinc on some of the physiological responses of triticale under water-limitation conditions.

Materials used in experiment
A factorial experiment was conducted based on randomized complete block design with three replications in 2014 and 2015. The area is located at 38°15 ′ N latitude and 48°15 ′ E longitude with an elevation of 1350 m above mean sea level. Climatically, the area is situated in the semi-arid temperature zone with cold winter and moderate summer in north-western Iran. Experimental factors consisted of three irrigation treatments [normal irrigation (W 0 ) as control, moderate water limitation (W 1 ) or irrigation withholding at 50% of heading stage and severe water limitation (W 2 ) or irrigation withholding at 50% of booting stage]; four bio fertilizers levels [(no bio fertilizer (F 0 ), application of mycorrhiza (F 1 ), PGPR (F 2 ) and both application PGPR and mycorrhiza (F 3 )] and four nano zinc oxide levels [(without nano zinc oxide as control (Zn 0 ), application of 0.3 (Zn 1 ), 0.6 (Zn 2 ) and 0.9 (Zn 3 ) g L −1 )]. The soil was silty loam, total organic C -5.27 g kg −1 soil, with pH about 7.8 and electrical conductivity (EC) about 2.68 dSm −1 . In each plot, there were 5 rows 2 m long. Plots and blocks were separated by 1 m unplanted distances. All phosphorous (60 kg ha −1 in the form of super phosphate) and potassium (60 kg ha −1 in the form of potassium sulfate) fertilizers were applied as basal dose at the time of seedbed preparation. Nitrogen fertilizer was applied as ½ at sowing, ½ at 6-8 leaf. The triticale cultivar 'Joanilo' was used in the experiment with plant density of 400 seeds m −2 . The plots were immediately irrigated after planting. It was planted in 16th of May in 2014 and 23th of May in 2015. Mycorrhiza fungi (mosseae) were purchased from the Zist Fanavar Turan corporation, and soils were treated based on the method of Gianinazzi et al. (2001). Psedomunas putida strain 186 and Azotobacter chrocoococum strain 5 were isolated from the rhizospheres of wheat by Research Institute of Soil and Water, Tehran, Iran. For inoculation, seeds were coated with gum Arabic as an adhesive and rolled into the suspension of bacteria until uniformly coated. The strains and cell densities of microorganisms used as PGPR in this experiment were 1 × 10 7 colony forming units. The application rates of Nano-ZnO were 150 g ha −1 . Nano zinc oxide was with the average of particle size less than 30 nm and special surface of particle more than 30 m 2 g −1 . Nano zinc oxide powder added to deionized water and was placed on ultra-sonic equipment (100 W and 40 kHz) on a shaker for better solution. Foliar application with nano zinc oxide was done in two stages of period growth (4-6 leaf stage and before booting stage). Mean temperature and precipitation for cropping season at 2014-2015 is presented in Table 1.

CAT, POD and polyphenol oxidase assay
At the mid of the booting stage, the flag leaves of plants were separated for measuring the CAT, POD and polyphenol oxidase (PPO) activity. Samples were placed in aluminum foil and transported from the field on ice bath.
To measure the enzyme activity, 0.2 g of fresh tissue of flag leaf was crushed by using liquid nitrogen and then 1 ml of buffer Tris-HCl (0.05 M, pH = 7.5) was added. Obtained mixture was centrifuged for 20 min (13,000 rpm and 4°C), and then supernatant was used for enzyme activity measurements. CAT, POD and PPO activity was assayed according to Kar and Mishra (1976). Also, in the evaluation of protein carried out by Bradford (1976) method, 0.2 g of plant tissue was squashed with 0.6 ml extraction buffer and was centrifuged at 11,500 rpm for 20 min at 4°C. The supernatant was transferred to the new tubes and centrifuged for 20 min at 4000 rpm. To measure the protein amount, 10 µl of obtained extract was added to 5 µl Bradford solution and 290 µl extraction buffer and the absorbance rate was read at 595 nm.

Relative water content
At the mid of flowering, heading and grain filling stages, the flag leaves of plants were selected for measuring the relative water content, chlorophyll content, stomatal conductance, quantum yield and electrical conductivity.
Weight of fresh tissue of flag leaves was measured just after detached from the plants then taken turgid weight after leaf was incubated in distilled water for 24 h to obtain a full turgidity. Dry weight of leaf was measured after it was dried at 60°C for 24 h in an oven. Relative water content was measured according to the following formula (Chelah et al. 2011).
where, RWC, FW, DW and TW are relative water content, fresh weight, dry weight and turgid weight, respectively.

Chlorophyll content
A portable chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc., Japan) was used to measure the leaf greenness of the triticale plants. The SPAD-502 chlorophyll meter can estimate the total chlorophyll amounts in leaves of a variety of species with a high degree of accuracy, which is a nondestructive method (Neufeld et al. 2006). For each plant, measurements were taken at three locations on each leaf, two on each side of the midrib on flag leaves, and then averaged.

Stomatal conductance
Stomatal conductance was measured by using a porometer system (Porometer AP4, Delta-T Devices Ltd., Cambridge, U.K.) following the user manual instructions. Stomatal conductance measurements were taken in the flag leaves from four different plants from each treatment.

Quantum yield
The quantum yield was measured on flag leaves by the uppermost fool expanded leaf using a fluorometer (chlorophyll fluorometer; Optic Science-OS-30 U.S.A.). For this purpose, the plants were adapted to darkness for 20 min by using one special clamp and then the fluorescence amounts were measured in 1000 µM photon m 2 s, and calculation was performed using following formula (Arnon 1949): where ØPSII represents quantum yield amount of photosystem II; F m , maximum fluorescence after a saturated light pulse on plants adapted to darkness and F 0 , the minimal fluorescence in the light adapted, which was determined by illumination with far-red light.

Electrical conductivity
Electrical conductivity of flag leaves was calculated by following the standard method of Jodeh et al. (2015). EC values were measured at room temperature of 23 ± 1°C using an electrical conductivity meter. In order to measure grain yield, each plot was harvested three central rows, each 1 m long.

Statistical analysis
Analysis of variance and mean comparisons were performed using SAS ver 9.1 computer software packages. The main effects and interactions were tested using the least significant difference (LSD) test at the .05 probability level.
It seems that when plants are subjected to various abiotic stresses, some ROS such as superoxide (O 2− ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals ( · OH) and singlet oxygen ( 1 O 2 ) are produced. These ROS may initiate destructive oxidative processes such as lipid peroxidation, chlorophyll bleaching, protein oxidation and damage to nucleic acids. Zahedi and Tohidi Moghadam (2011) reported that antioxidant enzymes' activity was increased when plants were exposed to water stress. Improvement of plant growth under stressful environments could be due to the significant role of the enzymatic antioxidant system in alleviation of oxidative impact, and this mechanism has been generally pointed out in wheat (Mandhania et al. 2006). Inoculation with the PGPR, mycorrhiza and both application PGPR and mycorrhiza under water limitation significantly increased CAT, POD and PPO enzymes activity of triticale. Our results indicated that there was an increase of about 46.4%, 27.6% and 10.3% in activity of CAT, PPO and POD, respectively, with bio fertilizer application as F 3 in comparison with F 0 (Figure 1(b)). Ma et al. (2011) reported that bio fertilizers can improve plant tolerance to salinity, drought and flooding, and enable plants to survive under unfavorable environmental conditions. There are many reports which show that AM fungi can increase enzyme activities, and/or protect the plants from ROS produced under stress conditions (Gamalero et al. 2009). . Means with similar letters are not significantly different. W 0 , W 1 and W 2 are normal irrigation (control), moderate and severe water limitation, respectively. F 0 , F 1 , F 2 and F 3 are no bio fertilizer, application of mycorrhiza, PGPR, and mycorrhiza+ PGPR, respectively. Zn 0 , Zn 1 , Zn 2 and Zn 3 are without nano zinc oxide as control, application of 0.3, 0.6 and 0.9 g L −1 , respectively. . Means with similar letters are not significantly different. W 0 , W 1 and W 2 are normal irrigation (control), moderate and severe water limitation, respectively. F 0 , F 1 , F 2 and F 3 are no bio fertilizer, application of mycorrhiza, PGPR and both applications PGPR and mycorrhiza, respectively.
The antioxidant enzymes' activity increased when nano zinc oxide is applied as Zn 3 in comparison with Zn 0 . So, there was an increase of about 24.4%, 25.2% and 10.7% in activity of CAT, PPO and POD, respectively by nano zinc oxide foliar spraying as Zn 3 in comparison with Zn 0 (Figure  1(c)). Zinc is known to have a stabilizing and protective effect on bio membranes against oxidative and peroxidative damage (Cakmak 2000). The balance between free radical generation and free radical defense determines the survival of the system. Therefore, Zn may have a role in modulating free radicals and their related damaging effects by enhancing plants antioxidant systems (Zago and Oteiza 2001). In our study, application of Zn increased antioxidant enzymes activity, which indicates the Zn's impact on relieving stress effect. Jain et al. (2010) indicated that activity of POD and CAT enzymes increased in high levels of Zn application.
Effects of water limitation × bio fertilizers showed that the maximum of CAT (71.94 OD µg protein min −1 ) and PPO (89.73 OD µg protein min −1 ) activities were obtained in severe water limitation and bio fertilizer application as F 3 (Figure 2). The lowest of CAT (12 OD µg protein min −1 ) and PPO (19.32 OD µg protein min −1 ) was obtained in W 0 F 0 ( Figure  2). On the other hand, there was an increase of about 45.1% and 20.6% in the activity of CAT and PPO enzymes, respectively, in the highest levels of water limitation and bio fertilizers (W 2 F 3 ) in comparison with (W 2 F 0 ) ( Figure 2).
Also, effects of water limitation × nano zinc oxide showed that the highest of CAT enzyme activity (66.78 OD µg protein min −1 ) was obtained in W 2 Zn 3 (Figure 3). While the minimum of it (12.13 OD µg protein min −1 ) was observed in W 0 Zn 0 . There was an increase about 18.8% in activity of CAT enzyme in W 2 Zn 3 treatment in comparison with W 2 Zn 0 .
A continued increase in CAT, POD and PPO activity indicates that these enzymes are a major enzymes detoxifying hydrogen peroxide in triticale under drought stress. All plant species naturally have various defensive networks to prevent their cells from the deleterious effects of ROS, which include enzymatic and non-enzymatic antioxidants (Ahmad and Prasad 2012). CAT and POD are two enzymes that constitute main H 2 O 2 scavenging system in cells. Under stress conditions, activity of these enzymes is altered and the degree of alteration may be linked to stress tolerance of the plant (Ahmad and Prasad 2012).

Relative water content
The relative water content significantly decreased under water-limitation condition. Inoculation with PGPR, mychorrhiza and both these bio fertilizers under water limitation increased the relative water content of triticale. In addition, the relative water content significantly increased when nano zinc oxide was applied. The highest values of relative water content (84.1%, 78.1% and 75% in flowering, heading and grain filling stage stages, respectively) were obtained in normal irrigation, application of bio fertilizer as F 3 and nano zinc oxide as Zn 3 (Table 2). Of these, the minimum values (60%, 48% and 42.1% in flowering, heading and grain filling stage stages, respectively) were obtained in W 2 , F 0 and Zn 0 ( Table 2). There were an increase of about 22.5%, 32.5% and 40.6% respectively at flowering, heading and grain filling stages in content of relative water content in the W 2 F 3 Zn 3 in comparison with W 2 F 0 Zn 0 (Table 2).
To evaluate plant water status, the leaf water potential and relative water content are essential parameters that are generally used to study plant physiological responses to drought stress (Silva et al. 2010). Silva et al. (2010) stated that these parameters decrease in most plants under water deficit. Decrease in RWC in plants under drought stress may depend on plant vigor reduction and have been observed in many plants (Liu et al. 2002). The decrease in leaf relative water content could be related to low water availability under stress conditions or to root systems, which are not able to compensate for water lost by transpiration through a reduction of the absorbing surface (Gadallah 2000). Guo et al. (2010) reported that mycorrhizal roots can explore more soil volume due to their extra matrical hyphae that facilitate them for absorption and translocation of more nutrients than by non-mycorrhizal plants. Moreover, better water status might result in the increased activity and hydraulic conductivity of the roots. Previous studies have found that mycorrhizal plants often show higher leaf RWC and WUE compared to non-mycorrhizal plants. Shaharoona et al. (2006) reported that the inoculation treatment with PGPR Figure 3. Effects of water limitation × nano zinc oxide on CAT activity (mean of two years, 2014-2015). Means with similar letters are not significantly different. W 0 , W 1 and W 2 are normal irrigation (control), moderate and severe water limitation, respectively. Zn 0 , Zn 1 , Zn 2 and Zn 3 are without nano zinc oxide as control, application of 0.3, 0.6 and 0.9 g L −1 , respectively. are normal irrigation (control), moderate and severe water limitation, respectively. F 0 , F 1 , F 2 and F 3 are no bio fertilizer, application of mycorrhiza, PGPR and both applications PGPR and mycorrhiza, respectively. Zn 0 , Zn 1 , Zn 2 and Zn 3 are without nano zinc oxide as control, application of 0.3, 0.6 and 0.9 g L −1 , respectively (LSD test, P < .05).  (control), moderate and severe water limitation, respectively. F 0 , F 1 , F 2 and F 3 are no bio fertilizer, application of mycorrhiza, PGPR and both applications PGPR and mycorrhiza, respectively. Zn 0 , Zn 1 , Zn 2 and Zn 3 are without nano zinc oxide as control, application of 0.3, 0.6 and 0.9 g L −1 , respectively. (LSD test, P < .05). W 0 , W 1 and W 2 are normal irrigation (control), moderate and severe water limitation, respectively. F 0 , F 1 , F 2 and F 3 are no bio fertilizer, application of mycorrhiza, PGPR and both applications PGPR and mycorrhiza respectively. Zn 0 , Zn 1 , Zn 2 and Zn 3 are without nano zinc oxide as control, application of 0.3, 0.6 and 0.9 g L −1 , respectively (LSD test, P < .05).
isolates increased RWC from 5% to 16% under normal and 21.7-28.4% under stress conditions when compared to the un-inoculated control. The amelioration role of Zn in maintenance of relative water content might be attributed to improvement of vascular tissue (Gadallah 2000).

Chlorophyll content
The water limitation, bio fertilizers and nano zinc oxide significantly affected the chlorophyll content. The highest chlorophyll contents (61.6, 59.5 and 56.7 in flowering, heading and grain filling stages, respectively) were obtained in W 0 F 3 Zn 3 , while the lowest (44.5, 39.1 and 32.7 respectively) were obtained in W 2 F 0 Zn 0 (Table 3). Water limitation caused the reduction in chlorophyll content, while application of bio fertilizers and nano zinc oxide increased these trait values.
Results showed that under the severe water limitation, application of bio fertilizers as F 3 and foliar spraying as Zn 3 increased about 23.5% in flowering stage, 32.7% in heading stage and 33.3% in grain filling stage in content of chlorophyll, in comparison with F 0 and Zn 0 in the same water-limitation level ( Table 3). Application of bio fertilizers and nano zinc oxide increased the chlorophyll contents, which indicates the bio fertilizers' and zinc's impact on relieving stress effect. It seems that the main reason for the decrease in chlorophyll may be degradation by ROS. Another reason for the decline in chlorophyll is the application of a glutamate precursor for the biosynthesis of proline (Navari-Izzo et al. 1990). Also the decrease in chlorophyll content has been considered a typical symptom of oxidative stress and chlorophyll degradation under water stress condition (Oraki et al. 2012). Severe drought stress also inhibits the photosynthesis of plants by causing changes in chlorophyll content, by affecting cholorophyll components and by damaging the photosynthetic apparatus. Sannazzora et al. (2005) reported that plants inoculated with Glomus intraradices had higher chlorophyll density than non-mycorrhiza inoculated plants. Vivas et al. (2003) showed that inoculation of bacterial strain increased chlorophyll content of lettuce when compared to control. Apparently zinc is involved in the production of chlorophyll. Zinc deficiency can cause a drastic decrease in chlorophyll content as well as a severe damage to the fine structure of chloroplasts (Chen et al. 2007).

Stomatal conductance
The results of measurement of stomatal conductance showed that the stomatal conductance decreased under water limitation. The highest stomatal conductance (35.2 mmol m −2 s −1 at flowering stage, 27.9 mmol m −2 s −1 at heading stage and 22.7 mmol m −2 s −1 at grain filling stage) was obtained in normal irrigation, application bio fertilizers as F 3 and nano zinc oxide as Zn 3 (Table 4). The minimum of it (18.2, 14 and 11 mmol m −2 s −1 respectively in flowering, heading and grain filling stages) was observed in W 2 F 0 Zn 0 . Results showed that under the severe water limitation, application of bio fertilizers as F 3 and nano zinc oxide as Zn 3 increase stomatal conductance about 34.6% in flowering stage, 42.1% in heading stage and 35.4% in grain filling stage in comparison with F 0 and Zn 0 in the same water-limitation level (Table 4).
Under water deficit, most plants will close their stomata to decrease their transpiration rate, further limiting the water lost to the environment a process mediated by the signaling molecule abscisic acid, produced in the roots. Limiting transpiration slows the production of ROS (Monneveux et al. 2006). Stomata closure in response to drought and salinity stress generally occurs due to decreased leaf turgor and atmospheric vapor pressure along with root-generated chemical signals (Chaves et al. 2008). Thus, the decrease in photosynthetic rate under stressful conditions (salinity, drought and temperature) is normally attributed to suppression in the mesophyll conductance and the stomata closure at moderate and severe stress (Chaves et al. 2008). Plants grown under drought condition have a lower stomatal conductance in order to conserve water. A number of reports showed that stomata usually close during the initial stages of drought stress, resulting in increased WUE (net CO 2 assimilation rate/transpiration). Stomata closure is known to have a more inhibitory effect on transpiration of water than that on CO 2 diffusion into the leaf tissues (Sikuku et al. 2010). Auge et al. (1986) pointed out that AM symbiosis always affects the stomatal behavior of host plants, which is regulated by both internal physiological and external environmental conditions. Mycorrhizal colonization enhances the stomatal control in snap dragon plants and reduces the water loss during drought (Auge et al. 1986). Inoculation of PGPR increased stomatal conductance to improve leaf water potential in adverse conditions (Mia et al. 2010). Wang and Jin (2005) found that Zn deficiency depressed photosynthetic capacity because of decrease in stomatal conductance.

Quantum yield
Water limitation, bio fertilizers and nano zinc oxide application significantly affected the quantum yield. The highest quantum yield (0.864, 0.845 and 0.833 in flowering, heading and grain filling stages, respectively) was obtained in W 0 F 3-Zn 3 , while the lowest of it (0.702, 0.605 and 0.521 at flowering, heading and grain filling stage, respectively) was determined in W 2 F 0 Zn 0 (Table 5). Results showed that under severe water limitation, application of bio fertilizers as F 3 and nano zinc oxide as Zn 3 increased about 14.8%, 28.7% and 43.7% respectively in flowering, heading and grain filling stages in content of quantum yield, in comparison with F 0 and Zn 0 (Table 5).
Chlorophyll fluorescence measurements have become a widely used method to study the functioning of the photosynthetic apparatus and a powerful tool to study the plant's response to environmental stress. A fast and simple way to probe environmental stress is by the chlorophyll fluorescence parameter F v /F m , a very useful relative measurement of the maximum quantum yield of PSII primary photochemistry (Baker 2008). According to Mamnouie et al. (2006), the photochemical efficiency of photosystem II is determined by the F v /F m ratio, which is decreased significantly during drought stress. Injury to PSII can lead to a change in chlorophyll fluorescence. Thus, chlorophyll fluorescence has been used as a powerful and reliable non-invasive method for assessing the changes in the function of PSII and for reflecting the primary photosynthetic processes under environmental stress conditions (Maxwell and Johnson 2000). Photosynthetic pigments present in the photosystems are believed to be damaged by stress factors resulting in a reduced lightabsorbing efficiency of both photosystems (PSI and PSII) and hence a reduced photosynthetic capacity. Moreover, the fluorescence induction parameters (such as F o , F m , F v , F p ) and, in particular, their ratios are commonly used to determine a number of metabolic disorders in the leaves of many species subjected to a variety of stresses (Baker 2008).
According to the results of experiments, quantum yield of PSII photochemistry (F v /F m ) increases by mycorrhizae fungi and despite destruction of the PSII reaction center of plant, growth increases in the presence of mycorrhizae (Sayar et al. 2008). AMF symbiotic relationship can enhance the efficiency of excitation energy capture by chloroplasts and increase the photochemical capacity of PSII in light-adapted leaves (Gong et al. 2013). Generally, arbuscular mycorrhizal fungus plays a role in F v /F m increase by improving plants nutritional status and activating mediated genes (Sayar et al. 2008). PGPR can improve plant growth by removing of pathogenic microorganisms, dissolving of insoluble phosphate and production of plant growth regulators. Thus, F v / F m increases by improving the nutritional status of the plant, especially the available phosphorus (P) element for plant photosynthesis and also improving photosynthesis and plant growth under water stress (Kloepper et al. 1989).

Electrical conductivity
The electrical conductivity significantly increased under water-limitation condition. Inoculation with PGPR, mychorrhiza and both these bio fertilizers under water limitation decreased electrical conductivity of triticale. In addition, the electrical conductivity content significantly decreased when nano zinc oxide was applied. The highest content of electrical conductivity (149.6, 192.9 and 227.6 µS cm −1 in flowering, heading and grain filling stages, respectively) was obtained in W 2 F 0 Zn 0 ( Table 6). The minimum of electrical conductivity (70.6 µS cm −1 in flowering stage, 85.5 µS cm −1 in heading stage and 126 µS cm −1 in grain filling stage) was obtained in W 0 , F 3 and Zn 3 (Table 6). There was a decrease of about 28.4%, 29.5% and 25% respectively in flowering, heading and grain filling stages in content of electrical conductivity in the W 2 F 3 Zn 3 in comparison with W 2 F 0 Zn 0 (Table 6). Under environmental stresses, plant membranes are subject to changes often associated with the increases in permeability and loss of integrity (Blokhina et al. 2003). Cell membrane is one of the first targets of plant stresses and the ability of plants to maintain membrane integrity under drought is what determines tolerance towards drought. Quan et al. (2004) found higher electrolyte leakage in drought-stressed maize (Zea mays L.) plants than in plants grown under control conditions. Naghashzadeh (2014) showed that the mycorrhizal biofertilizer application improved cell membrane stability in maize plant as a consequence of enhancing nutrient uptake, extension of the root system and water status of the plants. Inoculation with PGPR decreased electrolyte leakage compared to un-inoculated seedlings under drought stress (Sandhya et al. 2010). Zn is also known to have a stabilizing and protective effect on the bio membrane against oxidative and peroxidative damage, loss of plasma membrane integrity and also alteration of the permeability of the membrane (Cakmak 2000).

Grain yield
Our results have also indicated that there was significant difference among irrigation regimes on grain yield (Table  7). Grain yield decreased as result of moderate and severe water limitation, when compared with data from normal irrigation treatment. Grain yield increased as result of application of bio fertilizers and nano zinc oxide under normal irrigation and water limitation. A comparison of means showed that maximum of grain yield (663.2 g m −2 ) was observed in normal irrigation and application of bio fertilizer as F 3 and nano zinc oxide as Zn 3 (Table 7). The lowest of yield (198.4 g m −2 ) was obtained in W 2 F 0 Zn 0 (Table 7). Azcon and Barea (2010) have been proposed co-inoculation PGPR and AM fungi as an efficient procedure to increase yield and plant growth. Vivas et al. (2003) suggested that there are synergistic effects on plant growth when bacteria (PGPR) and AM fungi are inoculated, particularly under growth-limited conditions. Many researchers have indicated that bio fertilizers can alleviate the unfavorable effects of drought stress on plant growth (Khalafallah and Abo-Ghalia 2008). Significant increases in growth and yield of agronomical important crops in response to inoculation with PGPR have been reported (Sandhya et al. 2010). Significant increases in grain yield with foliar Zn application have been reported in other crops (Cakmak 2000).

Conclusion
The results showed that water limitation reduced yield, quantum yield, stomatal conductance, chlorophyll content and relative water content of the plants. But the activity of CAT, POD and PPO enzymes, and electrical conductivity increased. Also, application of bio fertilizer and nano zinc oxide increased grain yield, chlorophyll content, the activity of antioxidant enzymes, quantum yield, stomatal conductance and relative water content under water-limitation conditions, while electrical conductivity decreased. It seems that plants apply defensive mechanisms such as synthesis of antioxidant enzymes and decreased stomatal conductance to improve effects of stress and application of bio fertilizer, and nano zinc oxide can be recommended for profitable triticale production under water-limitation condition.

Disclosure statement
No potential conflict of interest was reported by the authors.