The Role of Eco-Friendly Soil Amendments Co-Application on the Performance of Lingrain (Linum Usitatissimum L.) Under Late-Season Irrigation Limitation


 This study's main purpose was to investigate the probable amelioration of limited irrigation conditions by soil amendments for lingrain plants. The experiment was accomplished as a completely randomized factorial design along with three replications. The first factor was green manure (without (Gc) and with Trifolium pratense (Gr)), the second factor consisted of Rhizophagus irregularis mycorrhiza (Fm), vermicompost (Fv), both of mycorrhiza and vermicompost (Fm+v) and none of them (Fc), and also the third factor was irrigation regime (full irrigation and late-season water limitation). Green manure, vermicompost and mycorrhiza single-use enhanced the plant performance under water limitation conditions in comparison with the control. However, in the presence of vermicompost, along with green manure or mycorrhiza developed a positive synergistic effect on most of the traits. Combining green manure with the dual fertilizer (Fm+v) resulted in the vermicompost and mycorrhiza synergistic effects, especially under limited irrigation. Consequently, the triple fertilizer (Gr×Fm+v) experienced the highest amount of LRWC, root colonization, leaf nitrogen, chlorophyll a, chlorophyll b, carotenoids, antioxidant enzymes activity, grain yield and oil yield, which would lead to more resistance of plants to limited irrigation conditions.


Introduction
Climate change and water scarcity have sparked drought as the most critical environmental stress that affect the crops growth, development and productivity in all over the world 91 . Along with the increase in greenhouse gas emissions under the constant global climate change, an increase in the drought intensity and incidence is expected soon. Approximately half of the agricultural lands in all over the world are constantly endangered to water shortages, resulting in a 50% reduction in grain yield 23 . Drought stress can seriously disturb secondary metabolites concentration, also along with the morphological and physiological traits 24,64 . Plants could create different mechanisms in order to defend against water stress, for instance: root development increasing for more water absorption, increasing the free radical scavenging enzymes activity, and osmolytes accumulation for moderating the osmotic pressure 40, 95 . Plant resistance to drought can also be increased using other external methods, for instance; by different soil amendments of which the eco-friendly treatments (for example mycorrhizal inoculation and organic fertilizer like vermicompost and green manure) have great importance. Organic fertilizers increase the products yield and quality by soil structure improving and nutrient availability increasing 7 . using nutrients achieved from organic sources helps maintaining the organic matter balance along with improving the chemical, physical and biological properties of the soil 60 . Green manure plays a signi cant role in nutrients supplying for crops, decreasing dependency on chemical fertilizers, enhancing the crops yield, enriching the cultivated elds ecological environment, and lowering the soil destruction and contamination 86 .
Vermicompost, derived from organic wastes of the earthworm, has nutrients that are readily absorbable by a plant.
Vermicompost as a nutrient-rich and nature-friendly substance has several potential applications as soil conditioners 20 . It stimulates microbial growth and activity and increases the soil nutrient mineralization. It increases fertility and the soil quality and contains plant growth stimulating agents (like vitamins, hormones, and enzymes), which help the plant grow better 96 . Arbuscular mycorrhizal fungi (AMF) are omnipresent in land-dwelling ecosystems and indicates symbiotic interactions with many plant species roots 51 . The AMF can protect plants against the biotic and abiotic stresses by different mechanisms such as increasing the nutrient absorption, photosynthetic activity, osmolytes accumulation, antioxidant enzymes production, and also improving the rhizosphere environment 93 . The methods used by the AM fungi for increasing the host plants-water relationships are not clear enough, but this may be caused by the water cumulative absorption through exterior hyphae, the adjustment of the stomatal system, the increased antioxidant enzymes activity and nutrients uptake mainly phosphorus 1 .
Lingrain is one of the earliest identi ed cultivated plants, and since ancient times, it has been extensively cultivated for its ne, cellulose-rich, bast bres and its oil 48 . Lingrain oil is a superb dietary supplement being rich in omega-3 fatty acids and α-linolenic acid. The oil is also bene ted in the industrial raw materials production 58 . Although, the worldwide lingrain harvested area and its production went down from 1993 to 2014, the oil yield increased at a rate of 1.72%. Hence it was cultivated on 2.3 million hectares of lands in all over the world with 560000 tons of oil production 22 .
There is often a severe shortage of water supply for irrigation of crops in the late growing season in most semi-arid countries, including Iran, which coincides with mid and late-summer. The signi cant effect of various soil amendments, including organic and biologic fertilizers, has separately been reported on improvement of different plants performances under water de cit stress. Since Iranian and most semi-arid regions soils are poor in organic matter, and few available studies were conducted on the simultaneous integrated use of organic and biologic fertilizers, this research purpose was to investigate the effect of single and combined use of these eco-friendly fertilizers for possible mitigation of lingrain performance under late-season irrigation limitation.

Results
Leaf Relative water content (LRWC) LRWC considerably decreased in water-limited condition. However, Green manure noticeably increased the LRWC in mycorrhizal plants (Fm and Fm + v). In both irrigation conditions, single and combined use of vermicompost and mycorrhiza had the ability of increasing the LRWC signi cantly, in comparison with the control, with the highest increase (92.56%) belonging to the Fm + v as shown in Table 3.

Colonization
Sole and dual usage of mycorrhiza and vermicompost (Fv, Fm and Fm + v) would lead to more colonization in comparison with non-fertilized treatment (Fc) ( Table 3). Green manure usage increased colonization in all fertilizer treatments except Fc in both irrigation and mycorrhizal plants in water limited condition. The greatest colonization was achieved in the combination of mycorrhiza and vermicompost (Fm + v) that were co-applied with green manure as indicated in Table 3. Moreover, limited-irrigation signi cantly declined the colonization in all fertilizer treatments (Table 3).
Leaf N Water limitation decrease the levels of leaf nitrogen. The single and dual usage of vermicompost and mycorrhiza would result in increased leaf nitrogen in both irrigation conditions in comparison with Fc. Application of green manure and mycorrhiza signi cantly elevated leaf nitrogen in both irrigation conditions except fv. Green manure combination with mycorrhiza and vermicompost could generate the highest leaf nitrogen percentage as indicated in Table 3.

Leaf P
Water inadequacy could reduce the leaf P concentrations. Under normal irrigation conditions, single and double fertilizers (Fm, Fv and Fm + v) brought approximately a higher percentage of leaf P in comparison with control (Fc), but there was no signi cant difference amongst the fertilizer treatments (Table 5).
Although, dual usage of mycorrhiza and vermicompost (Fm + v) accumulated more P in the leaf, but it indicated no signi cant difference with the single applications of mycorrhiza and vermicompost (Fm and Fv) in water-limited condition ( Table 5). In second year leaf P increased signi cantly than rst year (Table 6).

Leaf K
Water stress strongly decreased the leaf K content in all fertilizer treatments. Leaf K percentage was higher in second year in comparision of rst year (Table 5). Under normal irrigation conditions Fm + v) were more effective in leaf K content increasing in comparison with single vermicompost and mycorrhiza. However, they were not signi cantly different from each other under limited irrigation conditions. On the other hand, they all produced more leaf K in comparison with the control treatment (Fc).

Photosynthetic pigments
Water limitation signi cantly reduced chlorophyll a, chlorophyll b, and carotenoids contents. Green manure application soared chlorophyll a content in both irrigation condition. Also, Fm + v had the highest content of chlorophyll a in all conditions, after that Fm and Fv were in the second and third place, respectively. In addition, the lowest chlorophyll a quantity belonged to Gc × Fc (Table 3).
Green manure increased the amount of chlorophyll b, and single use of mcorrhiza and vermicompost could not affect the chlorophyll b under full irrigation conditions, but it mycorrhizal plants resulted in a signi cant increase of chlorophyll b content under limited-irrigation (Table 3). Using green manure signi cantly increased the level of carotenoids in Fm + v in both full irrigation and late-season water stress. In both conditions, the combined treatment of vermicompost and mycorrhiza produced the highest carotenoids content as shown in Table 3.

Proline
At this stage, Water limitation signi cantly increased. green manure didn't change the proline concentration except (fm + v) in full irrigation regime. The single and dual application of vermicompost and mycorrhiza increased the proline content in water-limited conditions, also Fm + v indicated the highest proline accumulation under water limitation. Also the single usage of fertilizers had same effect on proline concentration in both irrigation regimes (Table 5).

Total soluble sugar
Total soluble sugar concentration inhanced in seconde year in comparision of rst year in both irrigation regimes (Table 6). Application solo (fm) and (fv) didn't have signi cant effec on TSS concentration in full irrigation regime. Also fm + v decreased TSS concentration in this condition. All fertilizer sources increased TSS concentraion in irrigation cut-off conditinon. So that the dual fertilizer treatment (Fm + v) produced more TSS than the others under irrigation cut-off conditinon ( Table 5).

Glycine betaine (GB)
Water limitation considerably enhanced the levels of GB in lingrain leaves. Although, the application of vermicompost and mycorrhiza increased GB content under limited-irrigation circumstance, but the highest GB was obtained from dual fertilizer treatment (Fm + v). Additionally, under full irrigation, only mycorrhizal fertilizers (fm, fm + v) led to an increase in GB content (Table 5).
Hydrogen peroxide Drought stress considerably increased the level of leaf hydrogen peroxide. However, using fertilizer treatments caused a signi cant decline in its amount, in comparison with the control (Fc). Dual fertilizer (Fm + v) brought approximately the highest reduction percentage for hydrogen peroxide. The mycorrhiza-containing treatments more effectively reduced the amount of hydrogen peroxide compared to vermicompost (Table 5). Green manure usage had signi cant effect on H2o2 concentration in all treatments except (Fv) in both irrigation regimes (Fig. 1).

Malondialdehyde (MDA)
Water limitation enormously increased the levels of MDA, which was as a result of lipid peroxidation increasing. However, single and dual usage of the fertilizers resulted in a remarkable drop in MDA content compared to the control (Fc). Dual fertilizer (Fm + v) brought approximately the maximum reduction for MDA. The mycorrhizal treatments have the ability of decreasing the level of MDA more effectively, in comparison with vermicompost. Also, the single application of green manure was helpful in the MDA level reducing. Green manure usage decreased MDA level only in mycorrhizal treatmens in irrigation cut-off irrigation (Table 4).

Catalase (CAT)
Water limitation increased CAT activity. Incorporation of green manure under water limitation increased the activity of CAT enzyme in all treatments (Fig. 2). By applying mycorrhiza, with or without vermicompost, indicated a higher in uence on the enzyme activity under water limitation, as well. Furthermore, the highest activities of CAT (8.71, 8.61. mmol min-1/g FW) were observed in mycorrhizal plants (Table 5).

Ascorbate peroxidase (APX)
Water de cit elevated the APX activity. Green manure increased the activity only under water limitation condition in all treatments excep (fm). Furthermore, vermicompost and mycorrhiza, single or combined, had a more stimulatory effect on the enzyme activity under water limitation. The mycorrhiza and vermicompost combination caused the highest activity of APX (Table 4) Superoxide dismutase (SOD) In both years, SOD activity was signi cantly increased by water limitation as shown in Table 6. The application of green manure enhanced the activity of SOD in the limited-irrigation situation (Fig. 3). The synergistic effects of the co-application and the separate usages of mycorrhiza were more than that of vermicompost under limited-irrigation (Table 5).

Guaicole peroxidase (GPOX)
Water de cit elevated the Guaicole peroxidase activity. Green manure increased the anzyme activity in all treatments except vermicompost in full irrigation regime. Furthermore, enzyme activity was equal when using mycorrhiza under both irrigation conditions. The mycorrhiza and vermicompost combination caused the highest activity of GPOX (Table 4) Grain yield Limited irrigation signi cantly reduced the grain yield compared to full irrigation; however, the application of green manure increased grain yield relative to non-application of green manure in this condition. Although, co-application of green manure with vermicompost and mycorrhiza had no in uence on grain yield in full irrigation. Green manure application under complete irrigation situation produced the maximum grain yield (4862 kg ha-1) in Fm + v treatment. In addition, using green manure reduced the adverse effects of water restriction on grain yield, and the presence of mycorrhiza and/or vermicompost will additionally mitigate these adverse effects (Table 4).

Oil yield
The sole and conjoint usage of mycorrhiza and vermicompost along with co-application of green manure under full and limited irrigation would result in an increase in oil yield during comparing them with their corresponding controls (Table 4).
Using mycorrhiza was more effective oil yield in increasing compared to vermicompost, but the dual fertilizer produced the highest oil yield in both irrigation regimes with or without green manure. However, adding green manure to them produced more oil yield than control (Gc × Fc). Furthermore, the green manure inclusion to Fm + v produced the maximum level of oil yield (1602.66 kg h-1) under full irrigation condition as displayed in Table 6.

Discussion
Water de cit signi cantly reduced the leaf relative water contents (LRWC) 59 . By Considering the high water absorption e ciency of the soil by peripheral hyphae 36 , using different types of mycorrhizal fungi could cause a substantial rise in the water status of inoculated plants, which is in association with non-mycorrhizal plants, under water-limited conditions 97 .
Using organic fertilizers improves the water potential and maintains leaf turgor pressure, which is In agreement with this study result 12 . With respect to the green manure and vermicompost roles in soil organic matter and water content enhancement, dual-fertilized plants (Fm + v) indicated high percentage of LRWC in green manured plots.
This study results demonstrated that water stress inhibited mycorrhizal colonization in lingrain roots. These results are in agreement with earlier ndings, proposing that mycorrhization will be decreased at low levels of soil moisture 79 . AMF are obligate biotrophic fungi, consequently very slow germination of spores and expansion of the hyphae or their complete inhibition under drought conditions could elucidate the drought adverse consequences on colonization 32 . Therefore, lower colonization can occur because of two reasons; the carbon obtainability reducing from host plants and also the fungal spore germination reducing in limited water conditions 92 . Up to the best of our knowledge, organic matter can affect the activity and frequency of soil microorganisms like AMF, which can increase the plant nutrients 53 . Vermicompost promotes the mycorrhiza colonization in the roots 39,49,80 . The highest percentage of AMF colonization (66%) in Ocimum basilicum roots was observed in using vermicompost + G. intraradices combination in comparison with control 89 . Therefore, the combination of vermicompost, mycorrhiza and green manure exhibit high root colonization under full irrigation condition in this research.
Nutrients absorption directly relates to the soil water status, thus the nutrient ow from soil to root decreases in moisture shortage conditions 5 . With respect to better solubility of phosphorus in irrigated soil, the phosphorus concentration of the leaves indicates a reduction along with water restriction, which is probably caused by reduced solubility, mobility (mass ow or propagation), transfer between the roots and branches, and also the absorption of P under drought conditions 43,77 . Mycorrhizal inoculation can increase the infection percentage and absorption range in the extramatrical mycelium, and after that increase the nitrogen absorption rate and phosphorus and improve the nutrition in plants 63 . However, inoculated plants under limited irrigation contained less nitrogen and phosphorus content, which may be due to drought constraints in mycelium development and reduced absorption range, in comparison with infected plants in well-watered condition 52 .
Green manure will increase the soil holding-capacity for water and nutrients content and mobility in the upper layer of soil, by providing organic matter. Plants can use these elements, and therefore increase their growth and productivity 82 .
Moreover, there is an extensive record on the green manure advantages for nitrogen nutrition of succeeding crops 21,45,68 .
Mineralized nitrogen from leguminous plant residues can provide considerable quantities of N to a subsequent crop 73 .
Green manure effect on phosphorous availability could be caused from its P uptake and unavailable inorganic P changing to more available organic forms to the subsequent crop 42 . Furthermore, biodegradation of green manure remainders can produce bicarbonate (H2CO3) that can provide higher bio-available P for the plants by dissolving mineral P of the soil 78 .
Vermicompost provided high nutrition by balancing the nutrients, due to the fact that it contains all macro and some micronutrients. In regard with our results, vermicompost treatments increased the K uptake of Setaria grass 75  Water limitation reduced the leaf chlorophyll, and also carotenoid concentrations. One of the common symptoms of oxidative stress under drought conditions is the chlorophyll content reduction, which may be due to photo-oxidation and the pigment degradation 2,66 . This study results indicated that the amount of carotenoids, chlorophyll b and a signi cantly decreased under water limitation. These result are not consistent with several studies reported that carotenoid increased under water de cit conditions 6,9 . Earlier researches 9,46,47 approved our results that vermicompost application in soil brought about a remarkable growth in carotenoids content under water de cit condition. mycorrhizal inoculation resulted in the maintenance of a very high chlorophyll content relative to non-mycorrhizal plants in those plants subjected to late-season water limitation. This study results are in agreement with those reported by (Metwally et al. 2019) 59 . Using green manure can enhance the nitrogen availability, which can be used by lingrain in the chlorophyll production. Chlorophyll plays a crucial role in photosynthesis, such that increased chlorophyll formation will improve photosynthetic reactions and nally raise plant growth and production. Also, Subaedah and Aladin (2016) 82 clari ed the enhancement of chlorophyll content by using of green manure in maize, which is in parallel with our ndings.
Amassing proline under stress conditions is identi ed as one of the plant remedies for osmotic regulation improving in the leaves. Proline plays a leading role during stress in the plant. In addition to a great osmolyte, it acts as a metal chelator, a signalling molecule and an antioxidant molecule 81 14 demonstrated a reduction in H2O2 content in vermicompost application. Moreover, using green manure would resulted in MDA and H2O2 concentrations reduction, and also along with that, enhancement of the catalase and peroxidase activities in water limitation. It appeared that green manure could impede H2O2 accumulation and lipid peroxidation in plants by increasing the water content of the soil. Limited-irrigation causes oxidative stress by increasing the production of ROS in plant cells, and oxidative damage is also eased by antioxidant enzymes like APX, CAT, and SOD 74,83 . Amongst anti-oxidants enzymes, SOD and CAT are recognized as the most frequently used detoxi cation enzymes, which play a major role in controlling excessive ROS, in collaboration with involved enzymes of the ascorbate-glutathione cycle 10,15,38 . In this study, the triple application of mycorrhiza, vermicompost and green manure increased the CAT activity in both irrigation conditions, consequently, they can facilitate quick sweeping of ROS especially H2O2, so that metabolic reactions are less affected. Earlier results con rmed that the mycorrhizal and vermicompost-applied plants indicated considerably higher enzyme activities in stress condition 13,47,61,67 .
Applying the green manure in soils at high doses increased maize grain yield in comparison with the control soil 50 . Similar results were obtained with the green manure application in wheat grain yield by Gruter et al. (2017) 37 .
Vermicompost along with biofertilizers-treated plots indicated a substantial increase in essential oil content, essential oil yield and biomass of Ocimum basilicum in comparison with control 89

Conclusions
These results demonstrated that water limitation could reduce the pigments contents, nutrient contents, LRWC, colonization percentage, and (grain and oil) yields. However, it is responsible for a signi cant increase in H2O2, MDA, osmolytes and antioxidant enzymes activities in this condition. With respect to these results, the inclusion of vermicompost increased the lingrain performance for most of the investigated traits in comparison with the control (Fc). Although, Fv had no advantage compared mycorrhizal fertilizers (Fm and Fm + v), but its presence along with green manure or mycorrhiza could develop a positive synergistic effect on these traits.
Moreover, adding green manure to vermicompost treatments (Gr × Fv) resulted in a signi cant increase of colonization percentage, oil yield in comparison with Gc × Fv. Also, Gr × Fv signi cantly increased the APX activity and grain yield under drought stress conditions. However, it caused no signi cant difference in non-stressed condition, in comparison with single vermicompost treatment (Gc × Fv). In other words, green manure increased the vermicompost e cacy under stress conditions. Also, adding the green manure could result in a remarkable grain yield superiority of Fm + v in comparison with Fm. In the green manure absence, these two fertilizer treatments effect were similar in full irrigation condition. Consequently, combining green manure with the dual fertilizer (Fm + v) led to the synergistic effects of vermicompost and mycorrhiza.
In drought stress conditions, adding the green manure improved mycorrhiza e ciency on elevating RWC, leaf nitrogen, chlorophyll a, chlorophyll b, grain yield, oil yield and antioxidant enzymes activity (catalase, ascorbate peroxidase, superoxide dismutase and Guaicole peroxidase), All fertilizer treatments (FV, Fm and Fm + v) imposed less hydrogen peroxide and malondialdehyde on lingrain in both irrigation conditions in comparison with control. The lowest amount of hydrogen peroxide and malondialdehyde was achieved from the application of dual fertilizer under normal irrigation conditions followed by mycorrhiza and vermicompost fertilizers, and also the highest amount was achieved from non-fertilized control and under the limited irrigation conditions .
The co-application of eco-friendly soil amendments would result in the pro table lingrain production under water limitation. Therefore, as a result the triple fertilizer (Gr × Fm + v) experienced the highest amount of LRWC, leaf nitrogen, chlorophyll a, chlorophyll b, carotenoids, grain yield and oil yield. In addition, GrFm + v enjoyed the maximum quantity of and also antioxidant enzymes activity under drought stress conditions. Consequently, it can be concluded that the combined usage of organic and biological fertilizers (green manure, vermicompost, and mycorrhiza) can be also applied as a solution in order to maintain the lingrain plant performance under the water-limited condition.

Soil amendments
The soil texture was salty loam with a pH of 7.8. Other soil physical and chemical properties are indicated in Table 1. Mycorrhizal inoculum included a blend of sterile sand, mycorrhizal hyphae and spores (30 spores g-1 inoculum), and colonized fragments of canola root that were isolated from an AMF community of a canola farm by plant protection department of Agricultural and Natural Resources Research Centre, West Azerbaijan province, Urmia, Iran. Vermicompost was prepared with respect to the method suggested by Ayyobi et al. (2013) 8 . Chemical properties of the vermicompost that was used in these experiments are tabulated in Table 2.

Plant culture
By passing a month from green manure integration, the land was manually prepared. At rst and second year, the measured dry biomass production by green manure was 4.72 t ha-1 and 4.95 t ha-1, respectively. Also, inoculum (300 g m − 2) was incorporated into the soil of grainbed before the grain sowing. The exact amounts of vermicompost were manually spread onto the soil surface and incorporated into the top 20 cm of the soil Lingrain Grains were achieved from the Agricultural and Natural Resources Research Centre of West Azerbaijan province, Urmia, Iran. The grains were sown in 8 rows on 21 April 2017 and 24 April 2018, respectively (with two cm intra-row and 25 cm inter-row spaces). The full irrigation treatment included ve irrigations (after sowing, at the 4-leaf stage, at stem elongation, at owering and capsule formation stages), and limited irrigation treatment (irrigation cut -off from owering stage) had three irrigations (after sowing, at 4-leaf and stem elongation stages). The irrigation water amount in each plot was measured by the use of a water meter. Moreover, the average humidity at the surface and depths of 20 and 40 cm of soil was determined using a soil moisture meter (EXTECH MO750) in each plot before each irrigation. After that, with respect to the lack of moisture under the eld capacity point, the amount of water that was required for each irrigation was calculated by following equation: Where, Vn, the volume of irrigation water (m3), Fc moisture content at the eld capacity point (volume), θ soil moisture (volume), A area of the plot (m2) and h, Effective depth of lingrain root (m).
In terms of the above equation, the amounts of water used for irrigation were 2346 and 1420 m3/ha (for the rst year), and 1750 and 600 m3/ha (for the second year) for full and limited irrigation, respectively.
All the practices were manually handled in this experiment, in order to prevent interference with all materials.

Parameters measured
Leaf relative water content (LRWC): Leaf relative water content was assayed in leaf samples with respect to Garcıá-Mata and Lamattina (2001) 27 .

AMF Colonization
The mycorrhizal root colonization percentage was determined for each plot in ve roots. In order to achieve this goal, roots were cleared by 10% KOH at 90 °C for 10 min, and after that were stained in 0.05% lactic acid-glycerol-Trypan Blue 70 . The mycorrhizal colonization was estimated with respect to Giovannetti and Mosse (1980) 31 gridline intersect method.

Nutrient content
The leaf samples were dried in an oven for 72 h at 70 °C, and after that, were grounded using an electric mill. The Kjeldahl method was used for Nitrogen measurement 65 . Spectrophotometry and ame photometry methods were applied in order to determine P and K contents, respectively.
Photosynthetic pigment content assay Approximately 10 ml of acetone 80% was added to extracts of fresh leaf tissue samples gradually (0.2 g). After that, it was centrifuged for 10 min at 400 rpm and the absorbance was recorded using a spectrophotometer at 645, 663, and 470 nm. Proline Leaf fresh tissue (1 g) was used for leaf proline extracting in sulfosalicylic acid 5% (w/v). Leaf proline was determined using spectrophotometric analysis at 520 nm of the ninhydrin reaction, with respect to (Bates et al. 1973) 11 .

Total soluble sugars (TSS)
Leaf total soluble sugar was measured by the anthrone method 56 . Also, the TSS concentration was determined using the glucose standard curve.
Hydrogen peroxide (H2O2) The fresh leaf tissue (0.5 g) was grounded in a pre-chilled mortar with 5 mL of 0.1% (w/v) TCA. After that, this homogenate was centrifuged at 12000 × g for 15 min at 4 °C. also, 0.5 ml of the supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M KI and the OD of the suspension was read at 390 nm 88 .

Malondialdehyde (MDA)
Malondialdehyde was measured with respect to the thiobarbituric acid (TBA) reaction, as it was explained by Heath and Packer (1968) 44 . Fresh leaf tissues (0.1 g) were frozen in liquid nitrogen, and homogenized in 5 ml of 0.1% trichloroacetic acid (TCA). The homogenates were transferred into tubes and centrifuged at 3600 rpm for 10 min at room temperature. Two millilitres of this extract was added to 5 ml 0.5% TBA. The mixture was incubated for 30 minutes at 95 ° C, and then was immediately placed on ice for 5 minutes and centrifuged again at 10000 × g for 10 minutes, respectively. The supernatant absorbance was determined at 532 and 600, nm (A532 and A600). The MDA concentration was calculated due to following equation: [( 532 -600)/155] X 100 Glycine betaine (GB) Glycine betaine analysis was performed with respect to the method of Grieve and Grattan (1983) 35 .

Catalase (CAT), Ascorbate peroxidase (APX) and Superoxide dismutase (SOD)
The fresh leaf tissues (5.0 g) were macerated in 10 mL potassium phosphate buffer [pH 7.8; 50 mM], by the use of an icecooled sterilized pestle and mortar. After that, the homogenates were centrifuged for 20 min at 4 °C at12000 g. The supernatant was applied to the antioxidant enzymes activity assay.
Catalase activity was assayed in regard with Maehly and Chance (1954) 55 . 50 ml enzyme extract was added to 2.5 ml of 50 mM phosphate buffer (pH 7.4), and 0.1 ml of 1% hydrogen peroxide in the ice bath, in order to determine CAT activity. The H2O2 content reduction was obtained at 240 nm for 1 min.
Ascorbate peroxidase activity was determined b absorption reducing at 290 nm due to ascorbate oxidation in 3 min, with respect to Chen and Asada (1989) 16 . The reaction mixture involved 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 1.54 mM hydrogen peroxide, and 50 ml enzyme extract. Superoxide dismutase activity was measured regarding Dhindsa et al. (1981) 18 method in terms of its ability to inhibit the nitro blue tetrazolium reduction by superoxide radicals generated by photochemical reactions. The 3 ml reaction mixture contained 50 mM phosphate buffer, pH 7-8, 13 mM methionine, 75 µM NBT, 2 µM ribo avin, 0-1 mM EDTA, and 0-50 µl enzyme extract. NBT solution, ribo avin, EDTA and 1.0 ml DDW were incubated in light under 15 W uorescent lamps for 15 min. The blank reaction was processed also for 15 min in darkness. The samples and controls absorbance was measured at 560 nm. Guaicole peroxidase was measured regarding 25 .

Grain Oil Extraction
Soxhlet technique was utilized in orderto extract grain oil 71 . Oil yield was calculated by the following formula: Oil yield = % oil X seed yield Grain yield At plant maturity, above-ground biomass was harvested from 1 m2 of each experimental plot by ignoring marginal effects. Each plot Grains were separated and weighed in order to measure grain yield.

Statistical analysis
Combined factorial analysis of variance (three factors) including those gathered data for about two years (with one year as a random factor) was performed using SAS software and by applying the general linear model (GLM) procedure to establish these three factors main and interactive in uences. After that, this model was approved by checking the residues normality and homogeneity (using the Bartlett test). Also, No data transformation was performed. The mean values were compared using the protected least signi cant difference (PLSD) at P ≤ 0.05, due to the reason that the treatment factors were qualitative. 97. Zhao, R., Guo, W., Bi, N., Guo, J., Wang, L., Zhao, J., & Zhang, J. Arbuscular mycorrhizal fungi affect the growth, nutrient uptake and water status of maize (Zea mays L.) grown in two types of coal mine spoils under drought stress.  Figure 1 Interaction effects of green manure * fertilizers on Hydrogen peroxide concenttation (Means followed by the same letter are not signi cantly different) Figure 2 Interaction of green manure and irrigation regimes on catalase activity. Figure 3