In this research, plants subjected to mycorrhizal fungi exhibited an expanded root system, enhancing the availability of water and essential nutrients, notably nitrogen, phosphorus, and potassium, crucial for plant growth and development [17]. The mycorrhizal hyphae played a role in augmenting magnesium absorption, consequently elevating the levels of total chlorophyll and flavonoids in both quinoa and maize plants [18]. The synthesis of total chlorophyll is contingent upon the adequate absorption of minerals, particularly nitrogen and magnesium [19]. Phosphorus plays a crucial physiological role in the biosynthesis pathway of biochemical compositions, as it exhibits a synergistic effect with nitrogen. Our findings indicate that the utilization of AMF resulted in an augmentation of secondary metabolite levels in quinoa compared to the control treatment. This increase may be attributed to the enhanced biochemical compositions [18]. Secondary metabolites, such as flavonoids belonging to phenylpropanoids, play a crucial role in maintaining the structural and functional integrity of cells, thereby mitigating oxidative damage caused by ROS. This protective mechanism aids plants in coping with drought stress [20, 21]. AMF has been observed to significantly enhance anthocyanin and total flavonoid concentrations in Medicago truncatula leaves [22]. It's noteworthy that these mycorrhizal effects can, in part, be attributed to the improved phosphorus (P) content [23].
Xia et al. [21] demonstrated that mycorrhizae lead to the accumulation of root metabolites. However, the exact roles of these secondary metabolites induced by AM symbiosis in enhancing plant drought tolerance remain largely unknown. One of the well-known benefits of AM symbiosis to the host plant is the significant improvement in water and nutrient availability facilitated by AM. This, in turn, can explain the changes observed in carbohydrates and certain components of secondary metabolism, which are referred to as general mycorrhizal effects [24]. While the effects of mycorrhiza on plants are frequently considered to be highly specific to host plants and/or fungal species, there are common characteristics in the alterations of mycorrhiza-associated metabolites in plant roots [25]. In line with our findings, previous studies have reported that mycorrhizal associations often lead to increased levels of bioactive phenolic and lignin metabolites compared to plants not inoculated with AM [26, 27]. Furthermore, mycorrhizal symbiosis can induce plant-specific metabolic changes, particularly in stress conditions, which cannot be fully explained solely by nutrient effects [22]. Experimental data and meta-analysis have unveiled highly specific effects of mycorrhizal fungi, plant species, and/or their interactions on plant metabolism [17].
The growth, development, and metabolic activity of plants are contingent upon their photosynthetic capacity and ability to utilize soil nutrients [28]. By introducing phosphorus supplementation in conjunction with AMF, the levels of available nutrients can be modified, thereby leading to alterations in the metabolic environment of plants. This, in turn, facilitates the enhanced absorption and assimilation of CO2 from the atmosphere. The presence of mycorrhizal fungi resulted in enhanced biochemical properties, including increased antioxidant enzymes activity, flavonoids content, and total phenol, achieved by reducing peroxidase activity [29]. The hyphae of arbuscular mycorrhizal fungi possess the capability to augment water and nutrient uptake by plants, thereby establishing a symbiotic relationship between plants and AMF [30]. Quinoa seeds are known for their abundance of bioactive compounds, which display potent antioxidant activity and contain natural antioxidant compounds [31]. The total phenolic compounds present in quinoa contribute to its antioxidant activity [32]. According to Nsimba et al. [33], the total phenol content of quinoa extract grown in Japan was measured to be 148.0 mg g-1 equivalent of tannic acid, which was higher compared to the total phenol content (94.3 mg g-1) of quinoa grown in Bolivia. The disparities in phenolic component content between these two quinoa ecotypes can be attributed to variations in environmental factors and agricultural conditions [32]. Similar findings were observed in our experiment. Our results indicated that in the Shahrood region, the highest phenolic component content was observed in the treatment involving mycorrhiza inoculation alone. However, in the Mayamey region, the application of mycorrhiza and 50 kg ha-1 of phosphorous resulted in the highest phenolic component content (Figure 3).
The total phenolic component content in quinoa seeds varied between 16.8 and 59.7 mg/100 g, whereas the content of soluble phenolic compounds in the samples ranged from 7 to 61 mg/100 g [31].While saponins present in quinoa have been identified as anti-quality factors, isolated saponins also possess intriguing biological properties [34]. Our findings revealed that the saponins content decreased as the proportion of maize in mixed crops increased (Figure 3). The application of 100 kg ha-1 phosphorus significantly reduced the saponin levels compared to the control and 50 kg ha-1 phosphorus in the 75% maize proportion. It has been reported that saponins may exhibit nutritional or pharmacological value and their levels can vary under different conditions [35].Tannins, belonging to the group of polyphenols, can form complexes with other macromolecules, leading to various biological effects, both beneficial and undesirable. Polyphenols are natural substances that are widespread and commonly distributed in the plant kingdom [36]. Phenolic compounds, flavonoids, and saponins, which are products of secondary metabolism, exhibit crucial antioxidant activities that promote cardiovascular and cerebrovascular health. Additionally, they possess antiallergenic, antibacterial, anticarcinogenic, and anti-inflammatory effects [37].
Intercropping systems in agriculture have demonstrated significant effectiveness in enhancing production, contingent upon specific ecological conditions [38]. In a study conducted by Koca [39] on the forage yield and growth parameters of the maize-quinoa intercropping system, it was observed that the amount of maize dry matter increased in nearly all mixtures. Li and Cai [28] observed that enhancing the presence of AMF led to significant improvements in various maize traits, including biomass, chlorophyll content, plant height, and leaf area. AMF mycelium plays a crucial role in enhancing phosphatase activity and microbial activity in the soil. These fungi have a significant impact on the availability of phosphorus in the soil. Through their symbiotic relationship with plants, particularly crops, AMF promotes root growth and facilitates the conversion of inorganic phosphorus into organic forms [13]. AMF can be found in symbiosis with a wide range of crops, residing on the surface of roots and within the epidermis of root cells. The hyphae of mycorrhizal fungi are finer than root hairs, allowing them to penetrate soil micropores and transport certain minerals such as nitrates and phosphates to their host plant in exchange for carbohydrates [40]. This aligns with the findings of the current study. Consequently, plants associated with mycorrhizae generally exhibit greater drought tolerance compared to those without mycorrhizal associations [41]. Phenolic compounds, as secondary metabolites, play a role in detoxification, aiding in the neutralization of active oxygen species and free radicals before they can harm plant cells. The antioxidant potential of a plant is contingent upon the concentration of phenolic and tannin compounds. This study demonstrated an increase in the amount of phenolic and tannin compounds when AMF were utilized. Under stress conditions, mycorrhizal plants respond to oxidative stress by enhancing the synthesis of phenolic compounds. Previous research has also reported an increase in phenolic and tannin compounds facilitated by AMF in various plants, including violets [42], grapes [43], and Commiphora leptophloeos [44].
In our study, variations in the saponin content of quinoa were observed. As per the results, the saponin content decreased with an increase in the proportion of maize in mixed crops (Figure 3). The application of 100 kg ha-1 phosphorus significantly reduced the saponin content compared to the control and 50 kg ha-1 phosphorus in the 75% maize proportion. Additionally, when quinoa samples underwent different pearling processes, the saponin levels decreased [45]. These findings highlight the influence of cropping proportion, phosphorus application, and processing techniques on the saponin content of quinoa. Moreover, the quantities and composition of saponins in the quinoa plant were not consistently stable due to the continual removal of seed pericarps and bran in the harvesting process to render it edible, for instance, through pressure-cooking and toasting [45]. The total saponin content in quinoa grown ranged from 3.81 to 27.1 mg gr-1 in Washington State [46, 47], aligning with our findings. An increase in the nitrogen (N) uptake rate per root length has been shown to enhance saponin accumulation in roots, indicating a significant positive relationship between N uptake rate per root length and saponin content in taproots. Sufficient nitrogen fertilizer has the potential to enhance both root structure and nutrient uptake efficiency, subsequently promoting saponin synthesis [48]. Given the crucial role of nitrogen in saponin synthesis, the reduction in saponin content observed in high concentrations of phosphorus (100 kg ha-1) in this study may be attributed to a decrease in nitrogen absorption and, consequently, a decrease in saponin contents.
Andrino et al. [49] demonstrated the presence of mono-, di-, and tricarboxylic low-molecular-weight organic acids in compartments containing orthophosphate (OP) or goethite-bound-orthophosphate (GOE-PA) and phytic acid (PA or GOE-PA), indicating the occurrence of reductive dissolution and ligand exchange/dissolution reactions. Additionally, hyphae grown in goethite loaded with OP and PA exhibited an elevated content of unsaturated lipids, suggesting increased membrane fluidity to maintain optimal hyphal functionality and facilitate phosphorus incorporation. In the presence of plants, AMF demonstrates extensive practical diversity and contributes to growth-promoting functions by producing various metabolites. These functions include the mineralization of phytate, the production of siderophores, the dissolution of phosphorus, and the production of low-molecular-weight organic acids [50]. Additionally, the mycelium of rhizobium plays a role in mobilizing phosphorus from acid phytic, as highlighted by Selvakumar et al. [51]. AMF contribute to phytate mineralization, leading to the transfer of phosphorus [52]. These studies collectively emphasize the significant contributions of AMF in promoting plant growth by facilitating the mineralization of phytate and enhancing phosphorus availability through various mechanisms. While previous studies have predominantly focused on the mechanisms of interaction between plants and beneficial microbes, such as bacteria and AMF, with an emphasis on model and crop plants [53, 25], the associated transcriptomic and metabolomic changes induced by AMF to alleviate effects of adverse environmental conditions in non-model plant species have not been fully elucidated. The photosynthetic capacity and ability to utilize soil nutrients are crucial factors that influence the growth, development, metabolic activities, and fatty acid composition of plants [28]. Zamani et al. [54] demonstrated that the amounts of phenolic compounds and fatty acid profiles in the intercropping of Lallemantia iberica and Cicer arietinum L. increased with the application of AMF.
In the 50% intercropping pattern of quinoa with the application of AMF, the levels of phytic acid in quinoa increased. This can be attributed to the improved accessibility of nutrients facilitated by AMF, which in turn affects plant growth and photosynthesis. These changes in nutrient availability and metabolic processes can influence the production of fatty acid precursor compounds and the activity of enzymes like fatty acid synthase and acetyl-CoA carboxylase [55]. Furthermore, other studies have highlighted the positive effects of intercropping systems on plant nutrient availability, oil productivity, and oil quality. For instance, Rezaei Chiyaneh et al. [56] demonstrated that a cropping ratio of 50:50 in an intercrop of black cumin with fenugreek, using biofertilizer, resulted in the highest oil concentration in black cumin. This increase in oil concentration can be attributed to the enhanced nutrient uptake facilitated by the intercropping system. These findings emphasize the potential benefits of incorporating AMF and intercropping practices with biofertilizers in enhancing nutrient availability, oil production, and oil quality in various plant species. Rezapour et al. [57] demonstrated that the joint application of phosphorus and AMF resulted in an enhanced synthesis of fatty acids in groundnut. Additionally, the application of phosphorus fertilizer in a groundnut-corn intercropping system with a ratio of 1:2 improved groundnut oil content [57]. In a parallel study, it was found that the combined use of phosphorus chemical fertilizer and biological fertilizer increased safflower oil yield. These findings highlight the potential of using phosphorus fertilizers in conjunction with AMF and intercropping systems to enhance fatty acid synthesis and improve the oil content and yield of various oilseed crops like groundnut and safflower [58].
Oil synthesis, compared to other compounds studied in this research, requires a greater amount of energy. As a result, the utilization of mycorrhiza plays a significant role in enhancing the availability and absorption of nutrients by the plant root, providing the necessary energy for oil synthesis. This increased nutrient uptake facilitated by mycorrhiza ultimately leads to an augmentation in the production of oil. Furthermore, the energy required by plant processes is supplied in the form of high-energy ATP molecules. Phosphorus plays a crucial role in the formation of these energy-rich molecules. Therefore, the availability of phosphorus in the plant system contributes to the production of more high-energy ATP molecules, consequently promoting an increase in oil synthesis [59]. In summary, the utilization of mycorrhiza enhances nutrient availability and absorption, providing the necessary energy for oil synthesis. Additionally, the availability of phosphorus facilitates the production of high-energy ATP molecules, further supporting oil synthesis in plants [60]. Additionally, intercropping groundnut with corn decreased the palmitic acid content in peanut oil compared to sole cultivation, thereby enhancing the quality of peanut oil [57]. Research indicates that elevated phosphorus fertilizer consumption led to an increase in the saturated palmitic fatty acid content in peanut oil, resulting in reduced oil quality [57] and an increase in saturated fatty acids in corn [61]. Polyphenol compounds, naturally occurring in plants, possess antioxidant potential and are capable of neutralizing free radicals [62]. The effectiveness of antioxidant activity relies on the arrangement and the number of hydroxyl groups in the phenolic compounds, which can mitigate oxidation by donating hydrogen atoms to radicals [63]. At the Mayamey location, the levels of CAT, POX, and SOD enzymes were found to be 7.50%, 6.25%, and 16.39% higher, respectively, compared to the Shahrood location. Notably, in perennial ryegrass plants, the activity of antioxidant enzymes has been observed to increase through the inoculation with mycorrhiza, indicating a positive correlation between mycorrhizal inoculation and enhanced antioxidant enzyme activity [64]. Polyphenol oxidases, which are copper-containing antioxidant enzymes abundant in plants, utilize oxygen molecules to oxidize orthodiphenolic compounds like caffeic acid and catechol, converting them into quinone [65]. Increased levels of phenolic compounds are associated with elevated antioxidant activity, and tannins specifically exhibit a positive correlation with antioxidant activities [66]. Additionally, the augmentation of catalytic enzyme activity through inoculation with symbiotic fungi has been documented in various plants, including rice [67], roses [68], and citrus [69], aligning with our research findings. Quinoa seeds, in particular, are known to be abundant in bioactive compounds with potent antioxidant properties, making them a valuable source of natural antioxidants [31]. These findings align with the results obtained from our experiment. In the Shahrood region, the highest phenolic component content was observed with mycorrhiza inoculation alone treatment. Conversely, in the Mayamey region, the application of mycorrhiza inoculation combined with phosphorus (50 kg ha-1) resulted in the highest observed phenolic component content (Figure 3). Quinoa seeds boast a wealth of protein, lipids, fiber, vitamins, and minerals. Beyond its optimal blend of essential amino acids, quinoa harbors various phytochemicals, such as saponins, phytosterols, phytoecdysteroids, phenolic compounds, polysaccharides, as well as bioactive proteins and peptides. Recent studies showcasing the positive impacts of these compounds on metabolic, cardiovascular, and gastrointestinal health have propelled quinoa into the spotlight as a recognized functional food and nutraceutical [70]. Supplementing phosphorus in conjunction with AMF can modify the availability of nutrients, influencing the metabolic environment of plants and improving their ability to assimilate CO2 from the atmosphere. For instance, in a study by Kaling et al. [71], transcriptome and metabolome analyses of poplar plants inoculated with mycorrhizae and herbivores revealed that plants exhibit a specific accumulation of specialized protective compounds, such as protease inhibitors and aldoxime, at the cost of plant constitutive phenol-based compounds, as a defense mechanism against herbivores. AMF form a symbiotic relationship with the majority of plants and are typically found on the surface of plant roots and in the vicinity of root cells' epidermis. The hyphae of these fungi have the unique ability to penetrate soil micropores, facilitated by their thinner structure compared to plant root hairs. This enables them to transport minerals, including phosphates and nitrates, from the soil to their host plant in exchange for carbohydrates, as corroborated by the findings of our study and the research conducted by van Der Heijden et al. [72].
While prior studies have noted AMF associations with quinoa, the observed colonization rates have been minimal [73]. In the study by Vestberg et al. [73], a 19% colonization rate was observed, the highest recorded under field conditions before our observation. In greenhouse studies conducted by Kellog et al. [74] on 10 different quinoa genotypes, the reported colonization of quinoa roots by AMF ranged from 0 to 3%. However, Benaffari et al. [18] reported a higher colonization rate of 50%. These findings indicate that the intensity and frequency of AMF colonization in quinoa roots are significantly reduced in a monoculture system (p< 0.05). Conversely, treatments involving intercropping with maize (75% maize: 25% quinoa) and the addition of AMF (AMF+p50 kg ha-1) exhibited the highest frequency of mycorrhizal associations. In this study, as the levels of soluble phosphorus in the soil rose, there was a corresponding decrease in the colonization of roots by AMF. These fungi possess specific biochemical and physiological characteristics that enhance the availability of phosphorus to roots. Through the release of protons, these fungi acidify the rhizosphere, thereby increasing the solubility and transfer of phosphorus, particularly in alkaline soils, as observed in the soil of the studied areas. In acidic soils where phosphorus is predominantly bound with iron or aluminum, mycorrhizal fungi's neutralization of chelating agents can enhance the root bioavailability of soil phosphorus [72].