Drought is one of the main devastating environmental factors limiting crops’ development and productivity. This study investigated the role of combining intercropping and co-inoculation of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) to protect barley and alfalfa against drought damage. The experiment design consisted of four inoculation treatments: (1) non-inoculated plants (C), (2) plants inoculated with AMF consortium (AMF), (3) plants inoculated with the bacterial consortium (PGPR), and (4) plants co-inoculated with AMF + PGPR (AMF + PGPR), and two irrigation regimes: (i) well-watered, equivalent to 75% field capacity (FC), and (ii) drought, where watering was maintained at 35% FC. For each treatment (inoculated or not inoculated and stressed or not stressed), the plants of barley and alfalfa were monocropped and intercropped. Growth (shoots and roots dry weight), physiological (stomatal conductance and chlorophyll fluorescence), and biochemical (stress markers, osmolytes contents, and antioxidant enzyme activities) parameters were all measured. The results showed that applying intercropping and microbial inoculation AMF or/and PGPR enhanced the tolerance of plants to drought stress. The most pronounced effect was displayed by combining intercropping system and co-inoculation of AMF + PGPR, which improved shoot and root dry weight by 141 and 280% in barley and by 512 and 533% in alfalfa, respectively, compared to their respective uninoculated monocultures. Similarly, combining intercropping and co-inoculation with AMF + PGPR enhanced acid phosphatase, superoxide dismutase, and catalase activities by 125%, 161%, and 58% in barley and by 114%, 311%, and 112% in alfalfa, respectively, compared to their respective uninoculated monocultures. Furthermore, the thousand-seed weight was increased by 73% in barley intercropped and inoculated with AMF +PGPR. These findings revealed that intercropping barley and alfalfa and co-inoculation of AMF +PGPR may provide a sustainable approach to enhance drought tolerance, increase crop productivity, and promote food security.
