domain: sdv sde

text:
Maroua Jerbi

Sonia Labidi

Frédéric Laruelle

Benoit Tisserant

Yolande Dalpé

Anissa Lounès -Hadj

Faysal Ben Sahraoui

Jeddi

• Benoit Tisserant

Anissa Lounès-Hadj Sahraoui

• Faysal

Ben Jeddi

Anissa Lounès
email: anissa.lounes@univ-littoral.fr

Hadj Sahraoui

Biochemical Response of Hulless Barley (Hordeum vulgare ssp. nudum L.) to Drought

Keywords: Arbuscular mycorrhizal fungi, Biofertilization, Water stress, Hulless barley, Antioxidant activity, Nutrition

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Abiotic stresses, such as drought, have become more common due to climate change, especially in arid and semi-arid areas. The Mediterranean area, and especially Tunisia, was identified as one of the regions most exposed to global climate change. Indeed, Tunisia has experienced 23 dry years from 1974 to 1997 [START_REF] Giorgi | Climate change hot-spots[END_REF]USDA 2019). Drought is considered as one of the major factors decreasing crop productivity [START_REF] Lambers | Plant Physiological Ecology[END_REF]) and subsequently threatening food security [START_REF] Dono | Winners and losers from climate change in agriculture: Insights from a case study in the Mediterranean basin[END_REF]. In fact, drought impairs various physiological processes in plant tissues as well as causes changes in both primary and secondary metabolites [START_REF] Itam | Metabolic and physiological responses to progressive drought stress in bread wheat[END_REF]. It is well known that water stress induces oxidative damage in plant cells due to an overproduction of reactive oxygen species (ROS) such as: superoxide anion (O 2 ˙-), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (˙OH) [START_REF] Sachdev | Abiotic Stress and Reactive Oxygen Species: Generation, Signaling, and Defense Mechanisms[END_REF][START_REF] Sharma | Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions[END_REF]. Under normal conditions, the ROS are scavenged by different antioxidant mechanisms and therefore they are unable to cause any damage [START_REF] Foyer | Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface between Stress Perception and Physiological Responses[END_REF][START_REF] Silva | Photosynthetic changes and protective mechanisms against oxidative damage subjected to isolated and combined drought and heat stresses in Jatropha curcas plants[END_REF]. Oxidative stress occurs due to the imbalance between ROS generation and ROS scavenging, which reduces physiological and antioxidant defense systems [START_REF] Dar | An Introduction to Reactive Oxygen Species Metabolism Under Changing Climate in Plants[END_REF]. Moreover, the over-accumulation of ROS leads to a multitude of processes, such as lipid peroxidation and oxidative modification of proteins and nucleic acids, which extensively damage the normal cellular functioning of plants [START_REF] Sallam | Drought Stress Tolerance in Wheat and Barley: Advances in Physiology, Breeding and Genetics Research[END_REF].

Barley (Hordeum vulgare L.) is one of the oldest domesticated cereal crops [START_REF] Zohary | Domestication of Plants in the Old World: The Origin and Spread of Cultivated Plants in West Asia, Europe, and the Nile Valley, 2nd edn[END_REF] and is widely cultivated all over the world (48 million hectares in 2019), ranking fourth in cereal production (FAO 2020). Barley is a versatile crop, used for animal feed, malting and human food [START_REF] Elke | Cereal grains for the food and beverage industries[END_REF], yet is primarily used as grain or forage for livestock due to its higher fiber, protein and mineral nutrient contents [START_REF] Rogers | Grain Yield, Quality, and Nutrient Concentrations of Feed, Food, and Malt Barley[END_REF]. It has plenty of health benefits for human nutrition due to its high content of beta-glucan, tocols (including tocopherols and tocotrienols) and phenolic compounds, which are known to reduce the risk of cardiovascular disease by lowering blood cholesterol and the glycemic index [START_REF] Baik | Barley for food: Characteristics, improvement, and renewed interest[END_REF]. In Tunisia, barley is the second most cultivated cereal after durum wheat with more than 0.5 million hectares, representing 46.4% of the total area of cereals. It is frequently cultivated in semi-arid regions of the country, under rainfed conditions (DGPA 2020). Water availability is considered to be one of the main factors affecting wheat and barley production [START_REF] Hossain | High temperature combined with drought affect rainfed spring wheat and barley in South-Eastern Russia: I. Phenology and growth[END_REF]. New strategies to overcome drought are needed, especially in the context of food sustainability and security.

Global climate models in semi-arid regions suggest that water scarcity is expected to intensify in the future (IPCC 2013). To cope with this environmental stress, soil microbiota may play a key role in improving plant growth and productivity [START_REF] Berruti | Arbuscular mycorrhizal fungi as natural biofertilizers: Let's benefit from past successes[END_REF][START_REF] Grover | Role of microorganisms in adaptation of agriculture crops to abiotic stresses[END_REF]). Among these plant-growth promoting microorganisms, arbuscular mycorrhizal fungi (AMF) are the most widespread plant symbionts [START_REF] Smith | Mycorrhizal Symbiosis, 3 rd edn[END_REF]. Several studies have shown the beneficial effect of mycorrhizal symbiosis on plant growth by improving nutrient uptake efficiency under water deficit conditions [START_REF] Al-Karaki | Field response of wheat to arbuscular mycorrhizal fungi and drought stress[END_REF]Begum et al. 2019a;[START_REF] Bowles | Effects of arbuscular mycorrhizae on tomato yield, nutrient uptake, water relations, and soil carbon dynamics under deficit irrigation in field conditions[END_REF]Gholamhoseini et al. 2013;[START_REF] Jayne | Influence of arbuscular mycorrhiza on growth and reproductive response of plants under water deficit: a meta-analysis[END_REF]. Moreover, it has been reported in several studies that AMF could improve the root surface area [START_REF] Augé | Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis[END_REF] and thus increase plant water and nutrient uptake [START_REF] Berruti | Chapter 8: Arbuscular mycorrhizal fungi and their value for ecosystem management[END_REF][START_REF] Marulanda | Contribution of six arbuscular mycorrhizal fungal isolates to water uptake by Lactuca sativa plants under drought stress[END_REF]. Many studies dealing with abiotic stresses have shown that mycorrhizal symbiosis had the ability to enhance plant growth properties as well as the photosynthesis and antioxidant enzymes activities of many species such as tomato, lettuce, citrus and maize [START_REF] Mathur | Arbuscular Mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress[END_REF][START_REF] Rouphael | Arbuscular mycorrhizal fungi act as biostimulants in horticultural crops[END_REF]Xu et al. 2018a). Hence, few studies have been concerned with the physiological and the biochemical properties of barley subjected to drought stress.

Thus, the current research represents a first attempt to compare the potential of native and commercial AMF inoculations, under water stress conditions during tillering and grain filling development stages, in improving the growth of hulless barley (Hordeum vulgare ssp. nudum L.), one of the most important species for animal feed in semi-arid regions. To study the effect of AMF as biofertilizers, under water deficiency conditions, leaf water status, chlorophyll contents, antioxidant enzyme catalase and peroxidase activities, plant biomass and nutrient status were assessed.

Material and methods

Native AMF trapping and inoculant production

The soil sample used to trap and to multiply the native AMF was collected from the rhizospheric zone of durum wheat (Triticum turgidum L.) plants cultivated under rainfed conditions in Siliana, in the northern region of Tunisia (36°23′47.24′′N 10°11′8.01′′E). Soil chemical properties were presented in Table S1. For the trap culture, the substrate was composed of one volume of soil mixed with one volume of autoclaved sand and perlite mixture (1:1 v/v). Common vetch (Vicia sativa L.) was the host plant. Pots were placed under a shelter during the period from January to August 2016. Plants were irrigated with Hoagland nutrient solution [START_REF] Hoagland | The water-culture method for growing plants without soil[END_REF]. The mycorrhizal inoculant produced contains a mixture of inert substrate, mycorrhizal roots, hyphae and spores of the different native AMF species. After the trapping period, spores were extracted from the soils using the method of wet-sieving (sieves of size 250, 125 and 40 μm) and decanting described by [START_REF] Gerdemann | Spores of mycorrhizal endogone extracted from soil by wet sieving and decanting[END_REF], followed by the sucrose centrifugation method of [START_REF] Daniels | Methods for the recovery and quantitative estimation of propagules from soil[END_REF]. The spores were identified based on their morphological characteristics [START_REF] Dalpé | Arbuscular mycorrhizae. In: Manual of soil sampling and methods of analysis[END_REF]; https:// ww. zor. zut. edu. pl/ Glomeromycota). Five AMF species were isolated from the prospected site: Pacispora franciscana, Funneliformis 1 3 mosseae, Funneliformis geosporum, Rhizophagus irregularis and Glomus tenebrosum.

Experimental design

The experiment was conducted under shelter at the National Agronomic Institute of Tunisia (36°55′35.10′′N 10°11′8.01′′E). The plant species cultivated in this study was Hulless barley (Hordeum vulgare ssp. nudum L.). The experimental design was a split-plot arrangement in a completely randomized design (CRD) (Figure 1) with four mycorrhizal treatments: native inoculant containing a mixture of five AMF species as described previously (NI), commercial inoculant containing six species of Glomus sp. (CI), and two controls without inoculation (control NI and control CI) as main-plots and two water regimes: well-watered (WW) and drought (D) as sub-plots. Each treatment was replicated three times and, in each pot, four plants were maintained. For non-inoculated control plants, the same quantity of sterilized inoculant was added. Plastic pots (5 L) were surfacesterilized with sodium hypochlorite (NaCIO) followed by ethanol and then filled with air-dried soil collected from the experiment station of the institute. Chemical and physical characteristics of the soil were presented in Table S1. The CI and the NI with an average of 500 propagules per pot were added homogeneously just below the seeds. Seeds of hulless barley were surface-sterilized with 1% (v/v) NaCIO for 5 min and washed several times with distilled water and were then sown on 13 January 2018. Gravimetric water contents were measured at the start of the experiment to determine the soil field capacity. The pots were arranged randomly with four plants per pot and were watered daily to maintain the soil at 70% of water holding capacity. Two water regimes were applied: well-watered, maintaining a holding capacity at 70%, and drought condition, maintaining the water holding capacity at 30-20%. The irrigation regimes were applied by weighing each pot and adding water to the weight calculated for the desired water regime. In fact, drought condition was induced twice by withholding the irrigation for 10 days at two critical growth stages: the tillering stage (68 days after sowing equivalent to the Zadoks stage Z20) and the grain filling stage (112 days after sowing equivalent to the Zadoks stage Z65). The drought period span was for 10 days (maintaining 30-20% of the holding capacity). Plants were then re-irrigated when the holding capacity of the pot reached 20%. The crop was harvested on 5 June 2018.

Plant biomass and mineral nutrient analysis

At harvest maturity, plants from each treatment (3 pots of four plants each) were hand-harvested and the shoot biomass was weighed. Then the shoot part was dried, crushed and then divided into two subsamples for mineral analysis. The first subsample (100 mg) was used to determine the total nitrogen (N) concentration using the Kjeldahl method [START_REF] Jones | Nitrogen: Kjeldahl Nitrogen Determination-What's in a Name[END_REF]). The second one (100 mg) was ashed at 450 °C for 5 h in a muffle furnace. The ash was extracted in 20 mL of nitric acid (0.1 N) and then was digested for 10 min at 100 °C. After filtration, extracts were analyzed with a colorimetric method [START_REF] Pauwels | Manuel de laboratoire de pédologie : Méthodes d'Analyses de sols et de plantes, Equipement, Gestion de stocks de Verrerie et de Produits chimiques[END_REF] to determine the total phosphorus (P) concentration at 840 nm. Total iron (Fe), copper (Cu) and zinc (Zn) concentrations were measured at 248.3, 324.8 and 213.5 nm, respectively, with an atomic absorption spectrophotometer and calcium (Ca), sodium (Na) and potassium (K) at 422.7, 589 and 769.9 nm, respectively, with a flame photometer according to the method described by [START_REF] Pauwels | Manuel de laboratoire de pédologie : Méthodes d'Analyses de sols et de plantes, Equipement, Gestion de stocks de Verrerie et de Produits chimiques[END_REF].

Mycorrhizal root colonization assessment

Fine roots of harvested barley plants were cleared in 10% (w/v) potassium hydroxide (KOH), acidified in 1 N hydrochloric acid (HCl) and stained with 0.05% (w/v) trypan blue as described by [START_REF] Phillips | Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection[END_REF]. The percentage of mycorrhizal root colonization was determined according to the method of [START_REF] Mcgonigle | A method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi[END_REF].

Soil AMF biomass quantification

After harvesting, the AMF biomass in the soil was determined through the evaluation of their specific fatty acid biomarker amounts according to the method of [START_REF] Frostegård | Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress[END_REF] and [START_REF] Labidi | Fly ashaided phytostabilisation of highly trace element polluted topsoils improves the telluric fungal biomass: A long-term field experiment[END_REF]. From each sample, lipids were extracted from 3 g of lyophilized soil with a mixture of chloroform, methanol and citrate buffer (1:2:0.8), then the lipids were fractioned into neutral lipids, glycolipids and phospholipids in a silica acid column. The phospholipid fatty acid (PLFA) and the neutral fatty acid (NLFA) were transformed into free fatty acid methyl esters by transesterification. The final extracts were analyzed using a Perki-nElmer Autosystem gas chromatograph (GC) equipped with a flame-ionization detector (Norwalk, CT) with hydrogen as the carrier gas at a flow rate of 40 mL min -1 . The peak areas of fatty acid were quantified by adding nonadecanoic acid methyl ester (C19:0) as an internal standard. Their identification relied on the retention times of fatty acids standard (Bacterial Acid Methyl Ester CP Mix in methyl coproate SUPELCO) and its mass analysis. The system was operated in the split mode (200 as split ratio) and helium was used as the carrier gas at a linear velocity of 37 cm s -1 . Fatty acids were identified by comparing their mass spectra with the standard mass spectra in the NIST11s library. The amounts of the PLFA 16:1ω5 and NLFA 16:1ω5 representing membrane lipids and storage lipids of the AMF, respectively, were used as indicators of the AMF biomass [START_REF] Frostegård | Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress[END_REF]. Since the PLFA 16:1ω5 is not only specific for AMF but also for some Gram-negative bacteria, the ratio between NLFA and PLFA 16:1ω5 is always calculated. This ratio is high in AMF (1-200) and low in bacteria (< 1) (Olsson 1999).

Physiological and biochemical parameters measurements

During the two growth stages-tillering and grain fillingthe plant height (PH) was measured from the stem base to the crossing point of the last leaf for the four barley plants per treatment. Then, the plant leaf samples were used to determine the effect of the water regime and mycorrhizal inoculation on the physiological and the biochemical response of barley plants.

Leaf relative water content

The relative water content (RWC) was determined as described by [START_REF] Turner | Crop water deficits: a decade of progress[END_REF]. From each treatment, leaf tissues were cut out from the most expanded leaf and weighed immediately to obtain the leaf fresh weight. The leaves were then placed in distilled water for 24 h at 10 °C in the dark and then the saturated weight, or turgid weight, was measured. After that, the leaves were dried in the oven at 80 °C for 48 h to obtain their dry weight. The RWC was calculated using the following equation:

Chlorophyll fluorescence analysis

Chlorophyll fluorescence (Fv/Fm) was measured using a portable multi-mode chlorophyll fluorometer (OS5p, Opti-Sciences, INC, Hudson, USA). The leaves were darkadapted with a leaf clip holder for 15 min and were then illuminated with a saturating light. The measured Fv/Fm which corresponded to the potential quantum of photosystem II (PSII) was determined as follows: Fv/Fm = (Fm-F0) / Fm, where Fm and F0 are maximum and initial fluorescence yields of dark-adapted leaves, respectively [START_REF] Maxwell | Chlorophyll fluorescence: A practical guide[END_REF]. Six replications per leaf were taken for each treatment.

Chlorophyll a, chlorophyll b and carotenoid contents

The photosynthetic pigments chlorophyll a (chl a), chlorophyll b (chl b) and carotenoid (Car) were determined according to the method of [START_REF] Torrecillas | Determinaciòn rápida de clorofila en discos foliares de limonero[END_REF]. Chlorophyll was extracted from 100 mg fresh leaf tissues in 5 mL of 80% (v/v) acetone buffer for 72 h in darkness at 4 °C. The RWC = (fresh weightdry weight)∕(saturated weightdry weight) × 100 1 3

extract absorbance was then measured at 645, 663 and 460 nm using a UV spectrophotometer (Cary 100/300 series, Agilent Technologies, France). The concentrations of chl a, chl b and carotenoid were calculated by the equations of [START_REF] Mackinney | Absorption light by chlorophyll solutions[END_REF] and [START_REF] Arnon | Copper enzymes isolated chloroplasts. Polyphenoloxidase in Beta vulgaris[END_REF].

Antioxidant enzymes activities

Peroxidase (POX) and Catalase (CAT) activities were assessed in mycorrhized and non-mycorrhized plants, 68 and 112 days after seeding. Leaf tissues (100 mg) were crushed and homogenized in 1 mL of 100 mM phosphate buffer (pH 7.0). After centrifugation, the supernatant was used to measure the absorbance of the protein content and the activity of CAT and POX enzymes at 750, 240 and 412 nm, respectively, with a UV/Visible spectrophotometer (Cary 100/300 series, Agilent Technologies, France). The POX assay was determined according to the [START_REF] Mitchell | Elicitor-lnduced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum) Leaves[END_REF] method and the CAT assay was carried out using the method described by García-Limones et al. ( 2002) by following the decomposition of H 2 O 2 . Total proteins concentrations in the enzyme extracts were quantified according to the method of [START_REF] Lowry | Protein measurement with the folin phenol reagent[END_REF] using a commercial kit (BSA, Sigma, SaintQuentin Fallavier, France).

Statistical analysis

The effect of AMF inoculation (4 levels), water regime (2 levels) and their interactions on measured parameters was performed using the two-way analysis of variance (ANOVA) followed by least significant difference (LSD) Fisher's test at P < 0.05. Before statistical analyses, data were checked for normality and homogeneity of variance using the Shapiro-Wilk test. The principal component analysis (PCA) was performed using the package 'factoextra' [START_REF] Kassambara | Factoextra: extract and visualize the results of multivariate data analyses[END_REF]. All statistical analyses were performed using the RStudio software version 3.6.0 (R Core Team 2019).

Results

Plant biomass and mineral nutrient contents

The analysis of variance showed that the AMF inoculation, as well as the water regime levels, significantly affected the shoot biomass of barley (Table S2). The results clearly indicated that the shoot biomass of AMF-inoculated plants by either NI or CI were higher compared to those of noninoculated ones under both well-watered and drought regime (Fig. 2). Water stress significantly decreased the shoot biomass (P < 0.001, Table S2) by 41.7% compared with the well-watered regime. However, in AMF-inoculated plants, the barley shoot biomass significantly increased by 26.0%

with NI and by 41.4% with CI in comparison with non-inoculated control plants, under drought regime (Fig. 2).

The nutrient concentrations of N, P, K, Cu, Fe and Zn in barley shoot were significantly and negatively affected by the water regime (P < 0.001, Table S2). In most cases, AMFinoculated plants had a higher nutrient uptake as compared with non-inoculated ones. Under a drought regime, AMF inoculation with NI significantly improved N, P, K, Cu and Fe concentration by 1.0, 3.0, 3.5, 1.6 and 1.3 times, respectively, compared with their control plants. As for AMF-inoculated plants with CI, only N, P, Cu and Fe concentrations were increased by 1.2, 1.9, 1.7 and 1.4 times, respectively, in comparison with non-inoculated plants (Table 1). However, the K concentration showed no significant difference between AMF-inoculated plants with CI and their respective control plants. As for the Zn concentration, the interaction effect between the AMF inoculation and the water regime was not significant (Table S2). Unlike the other nutrients, the concentration of Na was significantly increased (P < 0.001, Table S2) by 38.8% with induced water stress. Under the drought regime, AMF-inoculated plants with CI showed a 31.8% lower Na concentration than non-inoculated plants. In contrast, the Na concentrations were similar between plants inoculated with NI and their respective controls (Table 1).

Mycorrhizal root colonization and soil AMF biomass

The AMF root colonization was significantly affected by the application of AMF inoculants and the interaction between all the factors (AMF inoculants × water regime) (P < 0.001, Table S3). Under well-watered condition, AMF inoculation significantly enhanced the mycorrhizal root colonization of barley 4.7 and 4.8 times for NI and CI, respectively, in 2). A drought regime significantly decreased the AMF root colonization of plants inoculated with CI by 32.7% and non-inoculated ones by 58.6% (Table 2). However, the root colonization rate reached a peak in AMFinoculated plants with NI and was higher by 42.3% compared with a well-watered regime (Table 2). In order to evaluate the effect of mycorrhizal inoculation with NI and CI on the AMF biomass in the soil, the PLFA and the NLFA 16:1ω5 were determined in inoculated and non-inoculated treatments. It was found that the addition of NI and CI significantly increased the soil NLFA 16:1ω5 content, which represents mainly the vesicle storage structures, 12.3 and 8.0 times, respectively, compared with the non-inoculated treatments at both water conditions (Table 2). The highest amounts of NLFA 16:1ω5 were observed in soils inoculated with NI (6.8 and 6.03 μg g -1 of soil under drought and wellwatered regimes, respectively) (Table 2). Similarly, the soil PLFA 16:1ω5, used here to quantify the biomass of AMF in soils, showed a similar trend as the soil NLFA 16:1ω5 content (Table 2). On the other hand, the NLFA/PLFA 16:1ω5 ratio in the soil of inoculated barley plants ranging from 1.38 to 5.86 (> 1), which indicates the AMF predominance. However, low values (< 1) of the NLFA/PLFA 16:1ω5 ratio (range 0.20-0.35), were observed in the non-inoculated soil (Table 2). For AMF-inoculated plants with NI and CI, the NLFA 16:1ω5 was significantly and positively correlated with total root mycorrhizal colonization (R 2 = 0.81***). However, there was a low and not significant correlation (R 2 = 0.16) between the concentration of NLFA 16:1ω5 in the soil and total root colonization in non-inoculated plants (Fig. 3).

Plant height

The analysis of variance (Table 3a and b) showed that PH at both growth stages (tillering and grain filling) were significantly affected by the AMF inoculants, the water regime and their interactions (P < 0.001, Table S4).

The drought regime reduced the PH of barley by 23.0% at both growth stages, compared with the well-watered regime (Table 3). Under the drought regime, the PH in 3a) and by 10.1% and 27.3%, respectively, at the grain filling stage (Table 3b) compared with those in the non-inoculated plants.

Leaf relative water content

The leaf RWC was significantly influenced by the AMF inoculants, the water regime and by the interaction between the two factors (P < 0.001, Table S4) at both growth stages. The RWC in leaves was significantly diminished in response to drought stress (Table 3). In comparison with AMF-inoculated plants with NI and CI, the RWC in non-inoculated control plants were significantly reduced by 10.1% and 16.5%, respectively, at the tillering stage (Table 3a) and by 58.1% and 48.8%, respectively, at the grain filling stage (Table 3b) under a drought regime. The results revealed that Chl a, Chl b, Car concentrations and Fv/Fm were negatively and significantly (P < 0.001) affected by the drought regime (Table 3). As for the noninoculated plants, Chl a, Chl b, and Car contents and Fv/ Fm were significantly (P < 0.001, Table S4) lower compared with the AMF-inoculated plants at both growth stages (Table 3). At the tillering stage and under a drought regime, the AMF-inoculated plants with NI and CI showed a 3.3 and 4.3 times increase, respectively, in Chl a, 1.9 and 2.6 times increase, respectively, in Chl b, 3.0 and 2.2 times increase, respectively, in Car, and 1.1 and 1.3 times increase, respectively, in Fv/Fm (Table 3a). At the grain filling stage, the same pattern was observed: AMF-inoculated plants presented the highest values under a drought regime (Table 3b).

Significance

Antioxidant Enzyme activities

The CAT and the POX activities were significantly affected by the water regime, the AMF inoculants and the interaction of both factors (P < 0.001), as shown by the twoway ANOVA results (Table S4). Under a drought regime, CAT and POX activities were significantly increased in AMF-inoculated plants at both growth stages. However, a significant decrease in the activity of these antioxidant enzymes was observed in non-inoculated plants subjected to a drought regime (Table 3). Compared with the non-inoculated plants, AMF inoculation with NI and CI improved CAT activity by 6.3 and 7.1 times, respectively, at the tillering stage (Table 3a), and 7 and 5.8 times, respectively, at the grain filling stage (Table 3b) under a drought regime. Similarly, POX activity in AMF-inoculated plants with NI and CI significantly increased by 4.8 and 5.2 times, respectively, at the tillering stage, and by 8.5 and 4.4 times, respectively, at the grain filling stage compared with those in non-inoculated ones (Table 3a andb).

Relationships between the different AMF inoculants and the measured parameters under well-watered and drought regimes

A PCA was carried out to describe the relationships between the different AMF inoculants and the measured parameters under well-watered and drought regimes. The first two components (PC1 and PC2) explained 85.7% of the total variation (Fig. 4). In fact, the first component, PC1, explained the majority of the variation (66.7%), and was closely related to the relative water content, leaf photosynthetic pigments Chl a, chl b and Car contents and chlorophyll fluorescence value at both the tillering and grain filling stages (Table S5). Also, most of the mineral nutrients were strongly and positively correlated with PC1 such as N. shoot (r 2 = 0.86, P < 0.0001), P. shoot (r 2 = 0.76, P < 0.0001) and K. shoot (r 2 = 0.85, P < 0.0001). However, the Na. shoot was negatively correlated with PC1 (r 2 = -0.84, P < 0.0001). The plant height and biomass were positively correlated with PC1 (r 2 = 0.94, P < 0.0001), as well as the total mycorrhizal root colonization (r 2 = 0.66, P < 0.001). The results showed that PC2, which explained 19% of the total variation, was predominantly influenced by the enzymatic activities such as POX (r 2 = 0.91, P < 0.0001) and CAT (r 2 = 0.93, P < 0.0001) at the grain filling growth stage, as well as the mycorrhizal root colonization (r 2 = 0.68, P < 0.001). Under a drought regime, the PCA indicated positive correlations with the AMF inoculation, plant height and biomass, nutrient concentrations of N, P, K, Cu, Fe and Zn, relative water content, photosynthetic pigments, chlorophyll fluorescence parameters and enzymatic activities. However, the concentration of Na increased during a drought regime, especially in non-inoculated plants. Plants inoculated with NI and CI tend to have higher values, especially for nutrient uptake, photosynthetic and enzymatic activities and biomass. The PCA results also confirmed the negative impact of water stress on these parameters; however, the inoculation with AMF mitigated these effects (Fig. 4, Table S5).

Discussion

Hulless barley (Hordeum vulgare ssp. nudum L.) is a very promising crop, as it can be used for food and feed, especially in semi-arid areas. However, there have been relatively few studies on it. This study is among the first ones analyzing in detail the effects of native AMF species on physiological and biochemical responses to water stress in hulless barley. In this regard, a pot experiment using native inoculant containing five AMF species of Pacispora franciscana, Funneliformis mosseae, Funneliformis geosporum, Rhizophagus irregularis and Glomus tenebrosum and commercial inoculant containing six species of Glomus sp. was carried out under well-watered and drought regimes. Total mycorrhizal colonization, plant growth parameters, mineral nutrient contents and antioxidant enzyme activities were investigated.

AMF inoculation increased mycorrhizal colonization of barley

In the present study, AMF inoculation significantly increased mycorrhizal root colonization of barley under both well-watered and drought conditions. We found that root colonization with the CI was greater in barley plants grown under well-watered rather than under drought conditions. This result is in agreement with previous studies, which reported that AMF root colonization decreased under water stress condition in Triticum aestivum cultivars inoculated with G. mosseae and with G. etunicatum [START_REF] Al-Karaki | Field response of wheat to arbuscular mycorrhizal fungi and drought stress[END_REF]) and in Cyclobalanopsis glauca using G. mosseae (actually F. mosseae (Nicol. and Gerd.)) and G. intraradices (actually R. intraradices (Schenck and Smith) Walker and Schüßler comb. nov.) [START_REF] Zhang | Effects of arbuscular mycorrhizal fungi on the drought tolerance of Cyclobalanopsis glauca seedlings under greenhouse conditions[END_REF]. Furthermore, the NI showed the highest root colonization rate when barley plants were exposed to drought conditions. The results were in line with the amounts of the NLFA 16:1ω5 and the PLFA 16:1ω5 quantified in the rhizospheric soil. The same responses were obtained in Zea mays with F. mosseae [START_REF] Ren | Arbuscular mycorrhizal fungus alters root-sourced signal (abscisic acid) for better drought acclimation in Zea mays L. seedlings[END_REF]) and in Cucumis melo with native consortium containing Glomus sp., Sclerocystis sp. and Acaulospora sp. [START_REF] Meddich | Role of mycorrhizal fungi in improving the tolerance of melon (Cucumus melo) under two water deficit partial root drying and regulated deficit irrigation[END_REF]. Similarly, [START_REF] Chareesri | Increased arbuscular mycorrhizal fungal colonization reduces yield loss of rice (Oryza sativa L.) under drought[END_REF] found that mycorrhizal colonization in rice roots was higher under dry conditions as compared with well-watered conditions due to the aerobic nature of AMF. In fact, the greater mycorrhizal root colonization with NI in comparison with CI, in addition to their high tolerance to water stress, may be related to the origin of the AMF. Similar results of the effectiveness of native AMF species in Mediterranean areas were reported in previous studies [START_REF] Caravaca | Survival of inocula and native AM fungi species associated with shrubs in a degraded Mediterranean ecosystem[END_REF][START_REF] Marulanda | Drought Tolerance and Antioxidant Activities in Lavender Plants Colonized by Native Drought-tolerant or Drought-sensitive Glomus Species[END_REF][START_REF] Marulanda | Stimulation of Plant Growth and Drought Tolerance by Native Microorganisms (AM Fungi and Bacteria) from Dry Environments: Mechanisms Related to Bacterial Effectiveness[END_REF][START_REF] Pellegrino | Field inoculation effectiveness of native and exotic arbuscular mycorrhizal fungi in a Mediterranean agricultural soil[END_REF]. It has been suggested by [START_REF] Pellegrino | Enhancing ecosystem services in sustainable agriculture: Biofertilization and biofortification of chickpea (Cicer arietinum L.) by arbuscular mycorrhizal fungi[END_REF] and [START_REF] Querejeta | Differential modulation of host plant δ13C and δ18O by native and nonnative arbuscular mycorrhizal fungi in a semiarid environment[END_REF] that native AMF species had better adaptability to local climatic and edaphic conditions in semi-arid areas than non-native species. Thus, the mixture of native AMF species may be more beneficial to the plant growth due to the intra-specific synergism and the functional complementarity among locally adapted AMF species [START_REF] Koch | The effects of arbuscular mycorrhizal (AM) fungal and garlic mustard introductions on native AM fungal diversity[END_REF][START_REF] Koide | Functional Complementarity in the Arbuscular Mycorrhizal Symbiosis[END_REF].

AMF inoculation enhanced plant growth and mineral nutrition of barley under water stress through physiological and biochemical responses

In this study, the plant height and biomass production of barley were significantly higher in plants inoculated with NI as well as with CI as compared with non-inoculated ones. These results were in agreement with previous findings reported by Begum et al. (2019b) for maize, [START_REF] Bernardo | Metabolomic responses triggered by arbuscular mycorrhiza enhance tolerance to water stress in wheat cultivars[END_REF] for wheat and [START_REF] Hashem | Arbuscular mycorrhizal fungi and biochar improves drought tolerance in chickpea[END_REF] for chickpeas. In response to stress, abscisic acid (ABA) and strigolactones were induced which may actively promote AMF functioning and therefore plant performance [START_REF] Pozo | Phytohormones as integrators of environmental signals in the regulation of mycorrhizal symbioses[END_REF].

In response to drought, the biosynthesis of strigolactones was increased in the presence of R. irregularis through the upregulation of gene SlCCD7 expression leading to better development of lettuce and tomato [START_REF] Ruiz-Lozano | Arbuscular mycorrhizal symbiosis induces strigolactone biosynthesis under drought and improves drought tolerance in lettuce and tomato[END_REF]. The improvement in barley growth in inoculated plants under water stress conditions could be related to the well-developed root system and the increase in nutrient and water uptake [START_REF] Hashem | Chapter 4-Arbuscular Mycorrhizal Fungi and Plant Stress Tolerance[END_REF] via the establishment of an extensive extra-radical hyphae of AMF [START_REF] Pagano | Drought Stress and Mycorrhizal Plant[END_REF][START_REF] Wu | Arbuscular Mycorrhizal Fungi and Tolerance of Drought Stress in Plants[END_REF]. Moreover, AMF symbiosis can increase the biosynthesis of cellulose, hemicellulose, lipid molecules and carbohydrates, which lead to plant growth enhancement [START_REF] Tarnabi | Impact of arbuscular mycorrhizal fungi (AMF) on gene expression of some cell wall and membrane elements of wheat (Triticum aestivum L.) under water deficit using transcriptome analysis[END_REF]. The boosting of plant growth observed with the AMF inoculation could be explained by the greater absorption of several nutrients from the soil by the plants, in particular nitrogen and phosphorus [START_REF] Anli | Biofertilizers as strategies to improve photosynthetic apparatus, growth, and drought stress tolerance in the date palm[END_REF]. Many studies also confirmed the correlation of growth enhancement with increased soil water level [START_REF] Wu | Arbuscular Mycorrhizal Fungi and Tolerance of Drought Stress in Plants[END_REF]. Our results revealed that plants inoculated with a combination of native AMF species and commercial AMF had considerably higher mineral nutrient N, P, K, Cu and Fe contents under drought conditions, compared with control plants. Similar results were reported by [START_REF] Al-Karaki | Field response of wheat to arbuscular mycorrhizal fungi and drought stress[END_REF] with Glomus sp., by [START_REF] Chen | Combined Inoculation with Multiple Arbuscular Mycorrhizal Fungi Improves Growth, Nutrient Uptake and Photosynthesis in Cucumber Seedlings[END_REF] with different AMF species from different genera such as Claroideoglomus sp., Funneliformis sp., Diversispora sp., Glomus sp., and Rhizophagus sp. and by [START_REF] Boutasknit | Arbuscular Mycorrhizal Fungi Mediate Drought Tolerance and Recovery in Two Contrasting Carob (Ceratonia siliqua L.) Ecotypes by Regulating Stomatal, Water Relations, and (In) Organic Adjustments[END_REF] with a mixture of 25 species of Glomus, Gigaspora, Acaulospora and Entrophospora.

The enhancement in mineral nutrition of inoculated plants could be linked to the ability of AMF to develop a hyphal network in order to access the unexplored region beyond the root depletion zone and thus to increase nutrient acquisition [START_REF] Smith | Mycorrhizal Symbiosis, 3 rd edn[END_REF]. Nitrogen and phosphorus are considered to be the most important elements for plant growth. Under water deficit condition, increased P and N in inoculated plants subsequently improved photosynthetic activities, which reflect the enhancement of plant growth by AMF (Begum et al. 2019a;[START_REF] Hameed | (ed) Use of microbes for the alleviation of soil stresses: Volume 2: Alleviation of Soil Stress by PGPR and Mycorrhizal Fungi[END_REF][START_REF] Zhu | Effects of arbuscular mycorrhizal fungi on photosynthesis, carbon content, and calorific value of black locust seedlings[END_REF].

Here, the increase in biomass and growth rates in AMFinoculated plants can be directly linked to an increase of the P content in leaves, the major component in many metabolic processes, especially those related to nucleic acid synthesis, maintenance of membrane structures, cell division, energy metabolism and many other physiological processes during plant development [START_REF] Malhotra | Phosphorus Nutrition: Plant Growth in Response to Deficiency and Excess[END_REF]. Also, the current work pointed out that barley plants inoculated with native AMF species had a higher K concentration but a lower Na concentration as compared with the non-inoculated ones. These results are in line with those of [START_REF] Al | Arbuscular mycorrhizal fungi modulates dynamics tolerance expression to mitigate drought stress in Ephedra foliata Boiss[END_REF] and Begum et al. (2019b). This might indicate the ability of AMF to pre-select elements for plants, such as K, which act as osmotic equivalents while they limit the uptake of toxic Na [START_REF] Hammer | Elemental composition of arbuscular mycorrhizal fungi at high salinity[END_REF]) by retaining these ions in their intra-radical hyphae structures [START_REF] Al-Karaki | Nursery inoculation of tomato with arbuscular mycorrhizal fungi and subsequent performance under irrigation with saline water[END_REF]. In addition, [START_REF] El-Mesbahi | Plant potassium content modifies the effects of arbuscular mycorrhizal symbiosis on root hydraulic properties in maize plants[END_REF] demonstrated that the plant K concentration influences the regulation of root hydraulic properties via AMF symbionts. Furthermore, an increased K concentration in barley can indirectly regulate osmotic adjustment and reduce the excessive accumulation of ROS in cell tissues (Begum et al. 2019b).

In the present study, mycorrhizal inoculation enhanced the leaf RWC of barley plants under well-watered and drought conditions, which is in agreement with the results of Aalipour et al. (2020a), Gholamhoseini et al. (2013), [START_REF] Mathur | Arbuscular Mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress[END_REF] and [START_REF] Zhu | Arbuscular mycorrhizae improve photosynthesis and water status of Zea mays L. under drought stress[END_REF]. This could be related to the ability of AMF to enhance the stomatal conductance capacity [START_REF] Augé | Hydraulic conductance and water potential gradients in squash leaves showing mycorrhiza-induced increases in stomatal conductance[END_REF][START_REF] Symanczik | Effects of two contrasted arbuscular mycorrhizal fungal isolates on nutrient uptake by Sorghum bicolor under drought[END_REF]), osmotic adjustment [START_REF] Augé | Arbuscular mycorrhizae and soil/plant water relations[END_REF][START_REF] Ruiz-Lozano | Hyphal contribution to water uptake in mycorrhizal plants as affected by the fungal species and water status[END_REF] and water retention [START_REF] Augé | Moisture retention properties of a mycorrhizal soil[END_REF] under such osmotic stress. Under drought conditions, mycorrhizal symbiosis regulates the level of ABA and cytokinin in roots in order to modulate better stomatal behavior by establishing a balance between root water uptake and leaf water transpiration [START_REF] Ouledali | Influence of arbuscular mycorrhizal fungi inoculation on the control of stomata functioning by abscisic acid (ABA) in drought-stressed olive plants[END_REF]. Xu et al. (2018b) demonstrated that R. intraradices upregulated the expression of 14-3-3 genes TFT2 and TFT3 through ABA signaling which resulted in enhanced stomatal behavior and water use efficiency in tomato plants subjected to drought stress. What was demonstrated is the implication of AMF in the regulation of plant aquaporin genes in order to increase the leaf water potential and reduce leaf transpiration [START_REF] Aroca | Induction of Plant Tolerance to Semiarid Environments by Beneficial Soil Microorganisms -A Review[END_REF]. Furthermore, [START_REF] Li | First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices[END_REF] identified two aquaporin genes GintAQPF1 and GintAQPF2 in R. irregularis (formerly known as G. intraradices) which exhibit a higher capacity to transport water to plants via mycorrhizal hyphae under drought stress. Also, [START_REF] He | Mycorrhizas induce diverse responses of root TIP aquaporin gene expression to drought stress in trifoliate orange[END_REF] demonstrated that inoculation with F. mosseae upregulated the expression of aquaporin genes (such as PtTIP1;2, PtTIP1;3 and PtTIP4;1) that led to a significant increase in leaf water potential of trifoliate orange seedlings under drought stress. Both AMF inoculants improved the Fv/Fm in barley leaf, which is considered to be a good indicator of the potential of the photosynthetic system to respond to abiotic stresses [START_REF] Araus | Chlorophyll fluorescence as a selection criterion for grain yield in durum wheat under Mediterranean conditions[END_REF][START_REF] Krause | Chlorophyll Fluorescence and Photosynthesis: The Basics[END_REF]. This result is consistent with Ceratonia siliqua under drought conditions [START_REF] Meddich | Role of mycorrhizal fungi in improving the tolerance of melon (Cucumus melo) under two water deficit partial root drying and regulated deficit irrigation[END_REF]. The increase in chlorophyll fluorescence value could be linked to the stimulation of photosynthesis by AMF [START_REF] Kaschuk | Are the rates of photosynthesis stimulated by the carbon sink strength of rhizobial and arbuscular mycorrhizal symbioses?[END_REF]). In fact, mycorrhizal symbiosis could mitigate the negative effect of water stress on PSII by inducing the activation of reaction centers, resulting in an increase of electron transport between PSII and PSI [START_REF] Pinior | Mycorrhizal impact on drought stress tolerance of rose plants probed by chlorophyll a fluorescence, proline content and visual scoring[END_REF]. [START_REF] Mathur | Arbuscular Mycorrhizal fungi (AMF) protects photosynthetic apparatus of wheat under drought stress[END_REF] revealed that inoculation with R. intraradices, F. mosseae and F. geosporum help Triticum aestivum plants to maintain the structure and the function of PSI and PSII under drought condition. Water stress induced significant declines in photosynthetic pigments Chl a, Chl b and Car. This result could be explained by the fact that thylakoid membranes, which contain protein photosynthetic complexes that play a critical role in light absorption and conversion in chloroplast, are destroyed under water stress [START_REF] Fang | General mechanisms of drought response and their application in drought resistance improvement in plants[END_REF]. Our findings showed that under water stress condition, barley plants inoculated with a mixture of native AMF species and a commercial AMF inoculant are able to maintain high chlorophyll contents compared with non-inoculated plants. This result is consistent with previous studies of Begum et al. (2019b) and [START_REF] Chen | Combined Inoculation with Multiple Arbuscular Mycorrhizal Fungi Improves Growth, Nutrient Uptake and Photosynthesis in Cucumber Seedlings[END_REF] carried out on other plant species such as cucumber (Cucumis sativus L. cv. Zhongnong No. 106) and maize (Zea mays L.), respectively. In fact, AMF enhance the rate of photosynthesis by stimulating rubisco activity, electron transport rates, adenosine tri-phosphate (ATP) production and even the ATP/ (adenosine diphosphate) ADP ratio in the chloroplast through increases in N and P mass fractions in leaves (Bechtaoui et al. 2021;[START_REF] Kaschuk | Are the rates of photosynthesis stimulated by the carbon sink strength of rhizobial and arbuscular mycorrhizal symbioses?[END_REF]). Thus, the results obtained were in agreement with other studies, suggesting that the higher water content in AMF mycorrhized plants led to a higher photosynthetic activity under stressful conditions [START_REF] Boutasknit | Arbuscular Mycorrhizal Fungi Mediate Drought Tolerance and Recovery in Two Contrasting Carob (Ceratonia siliqua L.) Ecotypes by Regulating Stomatal, Water Relations, and (In) Organic Adjustments[END_REF][START_REF] Meddich | Role of mycorrhizal fungi in improving the tolerance of melon (Cucumus melo) under two water deficit partial root drying and regulated deficit irrigation[END_REF]. Interestingly, there was an increase in Car contents in the leaves of barley inoculated with AMF as compared with non-inoculated barley under both well-watered and drought conditions. It's well-known that carotenoid has an important photoprotective role against photoinhibition [START_REF] Young | The photoprotective role of carotenoids in higher plants[END_REF]. The increase in carotenoid content in AMFinoculated plants may also protect the plant system from being damaged by ROS [START_REF] Krishna | Biochemical changes in micropropagated grape (Vitis vinifera L.) plantlets due to arbuscular-mycorrhizal fungi (AMF) inoculation during ex vitro acclimatization[END_REF]. Carotenoid represent a group of lipophilic antioxidants involved in detoxifying various forms of ROS. They are able to scavenge singlet molecular oxygen ( 1 O 2 ) and triplet chlorophyll in order to protect the photosynthetic apparatus [START_REF] Sharma | Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions[END_REF]). To cope with the oxidative damage, plant cells induced the production of antioxidant enzymes [START_REF] Farooq | Plant drought stress: effects, mechanisms and management[END_REF]) such as POX and CAT playing a critical role in quenching H 2 O 2 under water deficit stress [START_REF] Frostegård | Involvement of antioxidants and lipid peroxidation in the adaptation of two cool-season grasses to localized drought stress[END_REF]. In the present study, AMF inoculation, in particular with native AMF species, exhibited a more efficient antioxidant defense system as indicated by the increased POX and CAT concentrations under drought conditions, which may regulate the ROS level, thus reducing the oxidative damage. Similar results were observed by Aalipour et al. (2020b), [START_REF] Anli | Biofertilizers as strategies to improve photosynthetic apparatus, growth, and drought stress tolerance in the date palm[END_REF], [START_REF] Boutasknit | Impact of arbuscular mycorrhizal fungi and compost on the growth, water status, and photosynthesis of carob (Ceratonia siliqua) under drought stress and recovery[END_REF] and [START_REF] Mo | Regulation of Plant Growth, Photosynthesis, Antioxidation and Osmosis by an Arbuscular Mycorrhizal Fungus in Watermelon Seedlings under Well-Watered and Drought Conditions[END_REF]. It is well recorded that drought stress induces the accumulation of ROS within the cell, mainly O 2 ˙-and H 2 O 2 , due to the disruption of cellular homeostasis [START_REF] Sharma | Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions[END_REF]. In order to avoid the extensive oxidative damage of ROS, which can even cause cell death, plants use their antioxidant defense system using enzymatic and non-enzymatic components [START_REF] Das | Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants[END_REF]. Catalases and peroxidases represent the most important antioxidant enzymes, which are able to regulate the intracellular level of H 2 O 2 . Catalases are considered to be the most efficient enzyme in H 2 O 2 -quenching since they convert two molecules of H 2 O 2 directly into water (H 2 O) and molecular oxygen and play a key role in dioxygen (O 2 ) removal, while peroxidases reduce H 2 O 2 to H 2 O and inhibit cell peroxidation of membrane lipids by decreasing the malondialdehyde (MDA) content [START_REF] Ahmad | Drought Stress Induced Oxidative Damage and Antioxidants in Plants[END_REF][START_REF] Dumanović | The Significance of Reactive Oxygen Species and Antioxidant Defense System in Plants: A Concise Overview[END_REF]. Under stress conditions, maintaining a high level of antioxidant enzymes, in particular catalase, may help plants to prevent cell damage through regulating the ROS level [START_REF] Pedranzani | Arbuscular mycorrhizal symbiosis regulates physiology and performance of Digitaria eriantha plants subjected to abiotic stresses by modulating antioxidant and jasmonate levels[END_REF][START_REF] Ye | Effects of Arbuscular Mycorrhizal Fungi on Watermelon Growth, Elemental Uptake, Antioxidant, and Photosystem II Activities and Stress-Response Gene Expressions Under Salinity-Alkalinity Stresses[END_REF]. The contribution of AMF in regulating the ROS homeostasis through antioxidant enzyme activity has already been reviewed [START_REF] Wu | Mycorrhizal association and ROS in plants[END_REF]. Our results revealed that inoculation with either the native inoculant or the commercial one significantly enhanced the antioxidant defense system of hulless barley subjected to drought stress. These findings corroborate those of [START_REF] Gholinezhad | Effect of mycorrhizal inoculation in reducing water stress in sesame (Sesamum indicum L.): the assessment of agrobiochemical traits and enzymatic antioxidant activity[END_REF] who found that CAT and POX activity, as well as other antioxidant enzymes, were improved in sesame inoculated with R. irregularis and F. mosseae under water stress. The study of [START_REF] Zou | Mycorrhiza-induced lower oxidative burst is related with higher antioxidant enzyme activities, net H 2 O 2 effluxes, and Ca 2+ influxes in trifoliate orange roots under drought stress[END_REF] showed that inoculation with R. irregularis increased calcium (Ca 2+ ) influxes resulting in less H 2 O 2 production in trifoliate orange roots under drought stress. Langeroodi et al. ( 2020) demonstrated that R. irregularis increased the level of superoxide dismutase (SOD), peroxidase and other antioxidant enzymes in chicory, as well as reduced H 2 O 2 levels under drought stress. Similarly, [START_REF] Huang | Mycorrhizal-induced calmodulin mediated changes in antioxidant enzymes and growth response of drought-stressed trifoliate orange[END_REF] suggested that inoculation with F. mosseae helps trifoliate orange to maintain higher antioxidative enzyme activities, such as SOD isozymes and CAT, with lower H 2 O 2 accumulation. The improved tolerance of AMF-inoculated plants could be related to the up-regulated expression of antioxidants enzyme genes such as leaf PtMn-SOD, PtPOD and PtCAT1 [START_REF] He | Mycorrhizas enhance drought tolerance of trifoliate orange by enhancing activities and gene expression of antioxidant enzymes[END_REF]. Overall, AMF inoculation could enhance plant growth and photosynthesis through the up-regulation of the antioxidant plant system.

Conclusion

This study shows that arbuscular mycorrhizal fungi (AMF) inoculation, especially with the native inoculant, may be efficient in enhancing the growth and the biomass production of hulless barley under water stress condition during the tillering and grain filling growth stages. Our results demonstrate the beneficial role of AMF symbionts in mitigating the adverse effects of drought. The increase in plant growth was related to (i) an improvement of mycorrhizal root colonization, (ii) an increase in mineral nutrient nitrogen (N), phosphorus (P), potassium (K), copper (Cu) and iron (Fe) uptake and (iii) an enhancement of photosynthesis and antioxidant enzyme activities. In order to better understand the potential of AMF, field trials should be carried out in the future. The use of native or exotic AMF strains as biofertilizer could represent a promising approach in assuring the sustainability of agricultural cropping systems in semi-arid Mediterranean regions, which are vulnerable to water deficit.

Fig. 1

 1 Fig. 1 Experimental design layout of the shelter pot experiment using a split-plot arrangement in a completely randomized design (CRD); the main plot treatments consist of four arbuscular mycorrhizal fungi (AMF) inoculants: native inoculant containing a mixture of five AMF species Pacispora franciscana, Funneliformis mosseae, Funneliformis geosporum, Rhizophagus irregularis and Glomus tenebrosum (NI), commercial inoculant containing six species of Glomus sp. (CI), and two respective controls without inoculation (control NI and control CI); the subplot treatments consist of two water regimes: Well-watered (WW) and drought (D) with three replicates

Fig. 2

 2 Fig. 2 Effect of arbuscular mycorrhizal fungi (AMF) inoculants and water regime on shoot biomass of hulless barley. NI, native inoculant containing a mixture of five AMF species (NI); CI, commercial inoculant; control NI and control CI, two controls without inoculation. Bars indicate standard error of the mean (n = 3). Statistically significant effect at *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

 3 Fig. 3 The correlation between mycorrhizal root colonization and soil NLFA 16:1ω5 content of inoculated and non-inoculated plants with native inoculant (NI) and with commercial inoculant (CI) under the two different water regimes, ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001

  were grown in agricultural soil (non-sterile) inoculated with native inoculant containing a mixture of five AMF species (NI); with commercial inoculant (CI); or non-inoculated with sterilized inoculants (control NI and control CI). Different letters in the same line indicate significant differences at 0.05 by Fisher's LSD test. Asterisks indicate significance at *P < 0.05, **P < 0.01 and *** P < 0.001, and ns indicates not significant (P > 0.05)

Table 1

 1 Effect of arbuscular mycorrhizal fungi (AMF) inoculation and water regime on nitrogen (N), phosphorus (P), potassium (K), sodium (Na), copper (Cu), iron (Fe) and zinc (Zn) contents of barley (Hordeum vulgare ssp. nudum L.) shoots Well-watered 1.71 ± 0.06 c 0.31 ± 0.04 bc 28.35 ± 2.94 c 77.16 ± 12.29 bc 6.40 ± 0.46 c 27.03 ± 2.82 c 21.82 ± 2.64 a control CI × Well-watered 2.09 ± 0.02 b 0.26 ± 0.03 cd 21.33 ± 1.94 c 68.83 ± 7.49 c 5.13 ± 0.70 cd 23.73 ± 4.98 cde 22.46 ± 1.22 a NI × Well-watered 2.29 ± 0.03 a 0.33 ± 0.04 bc 46.65 ± 7.14 ab 48.11 ± 7.12 d 10.13 ± 1.00 a 48.53 ± 3.37 a 37.63 ± 10.61 a CI × Well-watered 2.13 ± 0.02 b 0.56 ± 0.06 a 51.18 ± 6.89 a 50.24 ± 13.19 d 8.00 ± 1.51 b 36.70 ± 2.19 b 48.17 ± 14.18 a control NI × Drought 1.45 ± 0.05 f 0.11 ± 0.06 e 11.37 ± 1.51 d 88.42 ± 3.54 ab 2.57 ± 0.65 f 18.63 ± 1.03 e 13.59 ± 2.08 a control CI × Drought 1.31 ± 0.01 g 0.20 ± 0.07 de 21.02 ± 2.95 c 97.39 ± 5.25 a 3.33 ± 0.67 ef 19.20 ± 4.19 de 14.90 ± 1.34 a NI × Drought 1.55 ± 0.04 e 0.33 ± 0.09 bc 40.22 ± 6.78 b 86.87 ± 3.29 ab 4.20 ± 0.62 de 24.70 ± 2.15 cd 20.75 ± 3.12 a CI × Drought 1.61 ± 0.03 d 0.38 ± 0.05 b 24.23 ± 2.76 c 66.44 ± 10.35 c 5.80 ± 0.72 c 26.20 ± 3.67 c 27.31 ± 11.10 a

	001, and ns indicates not

Plants were grown in agricultural soil (non-sterile) inoculated with native inoculant containing a mixture of five AMF species (NI); with commercial inoculant (CI); or non-inoculated with sterilized inoculants (control NI and control CI). Different letters in the same line indicate significant differences at 0.05 by Fisher's LSD test. Asterisks indicate significance at *P < 0.05, **P < 0.01 and *** P < 0.AMF-inoculated plants with NI and CI were increased by 11.2% and 19.8%, respectively, at the tillering stage (Table

Table 2

 2 Effect of arbuscular mycorrhizal fungi (AMF) inoculation and water regime on total root colonization (Tot Myc), soil neutral lipid fatty acid (NLFA 16:1ω5), phospholipid fatty acid(PLFA 16:1ω5) amounts and ratio between NLFA and PLFA (NLFA/PLFA 16:1ω5) of hulless barley (Hordeum vulgare ssp. nudum L.

		Tot Myc (%)	NLFA 16:1ω5 (μg g -1	PLFA 16:1ω5 (μg g -1	NLFA / PLFA 16:1ω5
			dry soil)	dry soil)
	AMF inoculation
	control NI	6.66 ± 4.86 c	0.39 ± 0.26 c	1.71 ± 0.70 bc	0.23 ± 0.13 c
	control CI	7.27 ± 3.07 c	0.47 ± 0.23 c	1.58 ± 0.84 c	0.34 ± 0.18 c
	NI	59.25 ± 11.97 a	6.41 ± 0.72 a	2.83 ± 1.89 a	3.61 ± 1.80 a
	CI	37.77 ± 9.96 b	4.01 ± 1.08 b	2.26 ± 1.14 ab	2.01 ± 0.62 b
	Water regime
	Well-watered	28.45 ± 19.95 a	2.98 ± 0.79 a	3.01 ± 1.10 a	0.86 ± 0.14 b
	Drought	27.03 ± 18.17 a	2.66 ± 1.05 a	1.17 ± 0.21 b	2.23 ± 0.92 a
	AMF inoculation × Water regime
	control NI × Well-watered	10.4 ± 3.4 d	0.49 ± 0.31 d	2.31 ± 0.42 c	0.20 ± 0.10 c
	control CI × Well-watered	9.4 ± 3.0 d	0.60 ± 0.23 d	2.12 ± 0.93 cd	0.35 ± 0.24 c
	NI × Well-watered	48.9 ± 5.9 b	6.03 ± 0.56 a	4.42 ± 0.49 a	1.38 ± 0.20 bc
	CI × Well-watered	45.2 ± 7.1 b	4.84 ± 0.91 b	3.23 ± 0.64 b	1.52 ± 0.32 bc
	control NI × Drought	3.0 ± 2.6 d	0.30 ± 0.21 d	1.13 ± 0.14 e	0.26 ± 0.17 c
	control CI × Drought	5.2 ± 1.3 d	0.36 ± 0.20 d	1.05 ± 0.22 e	0.33 ± 0.16 c
	NI × Drought	69.6 ± 1.3 a	6.79 ± 0.74 a	1.23 ± 0.33 e	5.86 ± 2.13 a
	CI × Drought	30.4 ± 5.7 c	3.18 ± 0.11 c	1.29 ± 0.18 de	2.50 ± 0.40 b
	Significance
	AMF inoculation	***	***	**	***
	Water regime	ns	ns	***	***
	AMF inoculation × Water regime	***	**	**	***

) Plants were grown in agricultural soil (non-sterile) inoculated with native inoculant containing a mixture of five AMF species (NI); with commercial inoculant (CI); or non-inoculated with sterilized inoculants (control NI and control CI). Different letters in the same line indicate significant differences at 0.05 by Fisher's LSD test. Asterisks indicate significance at *P < 0.05, **P < 0.01 and *** P < 0.001, and ns indicates not significant (P > 0.05)

Table 3

 3 Effect of arbuscular mycorrhizal fungi (AMF) inoculation and water regime on plant height (PH), leaf relative water content (RWC), leaf chlorophyll fluorescence (Fv/Fm), leaf chloro-

	L.) at tillering (a) and grain ssp. nudum and carotenoid (Car) contents, catalase (CAT) and peroxidase (POX) activities of hulless barley (Hordeum vulgare				POX (nkat g -1 FW) FW) Leaf RWC (%) Fv/Fm Chl a (mg g -1 FW) Car (mg g -1 FW) CAT (nkat g -1 Chl b (mg g -1 FW)		7.62 ± 1.78 c 59.36 ± 19.42 c 0.62 ± 0.07 c 1.52 ± 0.64 b 0.78 ± 0.65 c 0.21 ± 0.18 b 1.06 ± 0.27 c	7.24 ± 3.33 c 49.80 ± 0.31 d 0.62 ± 0.1 c 1.26 ± 0.55 c 0.66 ± 0.47 c 0.19 ± 0.15 b 0.83 ± 0.36 c	20.88 ± 8.71 a 81.84 ± 7.27 a 0.72 ± 0.09 b 3.94 ± 0.94 a 1.44 ± 0.95 b 1.50 ± 0.99 a 2.24 ± 0.80 a	18.14 ± 4.66 b 72.88 ± 14.15 b 0.75 ± 0.05 a 3.78 ± 0.48 a 1.67 ± 1.21 a 1.46 ± 1.02 a 1.47 ± 0.14 b		11.59 ± 2.12 b 77.01 ± 13.01 a 0.75 ± 0.05 a 3.21 ± 1.36 a 1.88 ± 0.73 a 0.31 ± 0.19 b 1.70 ± 0.74 a	15.35 ± 10.97 a 54.93 ± 14.44 b 0.60 ± 0.07 b 2.04 ± 1.25 b 0.40 ± 0.22 b 1.37 ± 1.07 a 1.09 ± 0.53 b		9.23 ± 0.08 d 77.04 ± 2.00 c 0.68 ± 0.01 b 2.11 ± 0.15 d 1.37 ± 0.05 c 0.04 ± 0.01 c 1.27 ± 0.22 bcd		10.22 ± 1.09 d 56.83 ± 1.79 e 0.71 ± 0.01 b 1.75 ± 0.25 e 1.07 ± 0.19 d 0.041 ± 0.02 c 1.14 ± 0.07 cd		88.43 ± 0.56 a 0.80 ± 0.00 a 4.77 ± 0.06 a 2.83 ± 0.47 a
	(Chl b)
	phyll a (Chl a), chlorophyll b	filling (b) growth stages	a.	Tillering	PH (cm)	AMF inoculation	control NI 26.62 ± 3.38 c	control CI 28.22 ± 5.05 b	NI 31.21 ± 5.43 a	CI 31.31 ± 3.43 a	Water regime	Well-watered 33.23 ± 2.55 a	Drought 25.46 ± 2.21 b	AMF inoculation × Water regime	control NI × Well-29.62 ± 0.66 d	watered	control CI × Well-32.83 ± 0.44 c	watered	NI × Well-watered 36.12 ± 0.44 a

Acknowledgements

This study was supported by the Tunisian Ministry of Higher Education and Scientific Research. This work has been carried out in the framework of the ALIBIOTECH project which is financed by the European Union, the French State and the French Region of Hauts-de-France as well as TRIPLET project financed by A2U. We are grateful for the English language revision provided by Stephanie Alouache and Nick Dettman. We thank Kalthoum Sifaoui and Ines Essid from the Soil Direction (National Institute of agronomic research of Tunisia) and Natacha Bourdon from UCEIV for the technical support.

Supplementary Information

The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s42729-022-00802-2.

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Authors and Affiliations

Maroua Jerbi 1 • Sonia Labidi 1 • Frédéric Laruelle 2 • Benoit Tisserant 2 • Yolande Dalpé 3 • Anissa Lounès-Hadj Sahraoui 2 • Faysal Ben Jeddi 1