Morphological traits
The results show that salinity stress at the rate of 150 mM salt (S150) increased quinoa growth, which is the normal halophytic behavior of this plant and is consistent with the report of Parvez et al. (2020)48. But, at 300 mM salt (S300) treatment, shoot growth was decreased, which is supported by the reports of Becker et al. (2017)10 and Parvez et al. (2020)48. The significant reduction in shoot length at high salinity (S300) seems to be associated with different responses, including changes in nutrient uptake, cellular homeostasis, and metabolic pathways of plants exposed to salinity stress49. Osmotic damage, ion toxicity, and changes in the balance of available nutrients are some factors involved in the loss of plant height in saline environments. The decline of plant height under salinity has also been ascribed to physiological dryness of the root zone and competition between the ions of chlorine, sulfate, and nitrate70. Salinity stress in the early stages causes osmotic stress, which reduces the water content of cells and inhibits their elongation, and even after osmotic balance and cell turgor are restored, they expand and elongate slowly41.
The highest shoot and root length and dry weight were observed in the treatment of S0T0FN at the salinity level of 0 mM (S0) and in the treatment of S300T0FN at the salinity level of 300 mM (S300). The results indicate that at the salinity levels of 0 and 300 mM, the N biofertilizer increased growth when the plants were not treated with Trichoderma (S0T0FN). The biofertilizer Azotobarvar-1 (containing Azotobacter) is a selective molecular nitrogen fixer that can synthesize and secrete some biologically active substances in the root zone and improve the root system, thereby influencing the uptake of water and nutrients, biological N fixation, crop yields, and soil properties2. Shoot and root length and dry weight were significantly increased by the treatment of S150TFN compared to other treatments at the salinity level of 150 mM. In addition to fixing N and making a balance in nutrient uptake, bacteria of biofertilizers synthesize plant growth promoters and different acids, thereby enhancing root and shoot growth and development, and this, in turn, contributes to more assimilation and its mobilization to other parts7.
Trichoderma seems to have an increasing effect on Azotobacter activity at the salinity level of 150 mM, which is normal salinity for quinoa as a halophyte plant, but Trichoderma at the salinity levels of 0 mM (S0) and 300 mM (S300) had an antagonistic effect on Azotobacter activity, which eventually reduced the growth of quinoa. The antagonistic ability of Trichoderma with bacteria has been reported64. It has been suggested that the acidification of the buffer by Trichoderma inhibits plant growth50. This can affect the interaction of Trichoderma with other organisms and bacteria and even with the plant itself too. At the salinity level of 0 mM, the application of P biofertilizer increased shoot dry weight and root length and decreased root dry weight compared to the control at the same salinity level. The interactive effect of Trichoderma and P biofertilizer was not significant on morphological characteristics at the salinity level of 0 mM. In summary, inhibition of plant growth and lack of lateral root growth during simultaneous cultivation of C. quinoa with biological control strains of Trichoderma under axenic conditions indicates that Trichoderma, especially growth regimes, can damage plants. The mechanisms of this injury may explain the exceptional cases of increased growth observed in the treated soil-grown crops55.
However, at 150 mM salinity, the application of P biofertilizer reduced shoot length and dry weight and root dry weight and increased root length versus the control at the same salinity level. Biofertilizers have been reported to increase uptake by root through increasing root development69. The application of Trichoderma along with P biofertilizer significantly reduced root length increase, had no significant effect on shoot length, but increased shoot dry weight significantly. It can be inferred from these results that at high salinity (300 mM), the application of Trichoderma along with phosphate-solubilizing bacteria influenced plant growth positively, and this effect was significant on length. The phosphate biofertilizer Barvar-2 contains phosphate-solubilizing bacteria that secrete organic acids and acid phosphatase and convert insoluble phosphorus in soil (especially in areas with high soil calcium) into a plant-absorbable form28. The interaction of a plant with beneficial microbes varies greatly depending on the genotypes and may range from growth inhibition to growth enhancement and vigorous growth54. Different species of Trichoderma are used for their ability to enhance plant growth and development and their ability to grow in adverse conditions66. It seems that Trichoderma in the salinity level of 300 mM salt had an increasing effect on the activity of phosphate-solubilizing bacteria and ultimately increased the growth of quinoa in high salinity.
Trichoderma treatment without the presence of bacteria at the salinity level of 150 mM salt (S150TC) decreased root length significantly compared to the control (S150T0C). Trichoderma had no significant effect on shoot growth and root biomass. At 0 and 300 mM salinity levels, Trichoderma without the presence of bacteria (S300TC) increased shoot length and dry weight significantly compared to the control treatment at the same salinity levels. There are reports as to the increased growth of some plant species, including quinoa, when they were treated with Trichoderma47.
By secreting fungal metabolites, activating growth regulating signals and plant growth-responsible phytohormones, and increasing the solubility of soil nutrients, Trichoderma can increase plant growth6,16.
Antioxidants
Total phenol
At the salinity level of 0 mM, the control treatment (S0T0C) and at the salinity level of 150 mM, the treatment of S150T0C was related to the highest total phenol. This shows that at the salinity level of 0 mM, the application of biofertilizers and Trichoderma alone or in combination reduced total phenol content. It seems that the plant spent most of its photosynthates on the growth of plant shoots, which is consistent with the results of this study. Phenolic compounds are secondary plant metabolites that act as substrates for many antioxidant enzymes9 or indirectly alleviate the damages of oxidative stress by modulating the function of several proteins associated with this stress30. The application of biofertilizers and Trichoderma alone or in combination reduced the amount of total phenol at the salinity level of 150 mM salt. Except for the treatment of S150T0 FP in which biomass was significantly reduced, the plants spent most of their photosynthates on shoot growth. In the treatment of S150T0FP, the P biofertilizer increased root length, which could be related to the non-selective uptake of elements by carriers and ion channels at low salinity conditions. Decreased pH due to the use of biosulfur biofertilizer can be the reason for the decline of polyphenols in this treatment15. In the treatment of S0T0FN, total phenol shows the lowest quantity. It can be said that Azotobacter contributed to increasing nitrogen uptake and subsequently increasing protein synthesis and cell growth and proliferation, resulting in a significant increase in shoot and root length and biomass. It seems that an increase in nitrogen uptake is the mechanism of the bacteria itself, which has led to no increase in Na+ uptake, a reduction of stressful conditions for the cell, and the lack of a need for the increase in total phenol content of the leaves. Total phenol content was increased significantly and remarkably in the treatments of S300TFN and S300TFP. The treatment of N or P biofertilizers with Trichoderma may have increased the uptake of Na+ by the roots and its accumulation in the leaves. The absorption of nutrients by cation channels is not selective, and the interaction of Trichoderma and bacteria is likely to increase the activity of NH4+ carrier cation channels and subsequently, Na+ uptake44. Plants exposed to oxidative stress use special defense mechanisms, such as increasing the concentration of total phenol. Studies have shown that different levels of salinity treatment increased total phenol content by 8–35%4. Also, phenol compounds accumulated in the leaves of corn and chickpea to a much greater extent in the plants exposed to salinity stress than those not exposed29,39. The use of a combination of seaweed and cyanobacteria as biofertilizer agents improved the growth and phenol content of fava beans52. In a study, it was found that an increase in phenol synthesis in Trichoderma-inoculated tomato plants improved their40. growth under drought stress, thereby protecting them against oxidative stress by ROS scavenging The increased synthesis of phenols and flavonoids is involved not only in the formation of cell walls, which protects plants against biotic stresses but also in antioxidant activity directly61.
The increase in phenolic compounds is due to the fact that free hydroxyl groups attached to the aromatic ring alleviate the oxidative damages caused by ions through scavenging radicals and other mechanisms, e.g. singlet oxygen reduction and metal chelating by bonding to toxic ions, thereby protecting cytoplasm and chloroplast structures against the negative impacts of salinity8. It seems that in the treatment of S300TFP, proper Na+ compartmentation was performed in leaf cells so that the phenols were chelated with Na+ and transferred it into the vacuole. By the reduction of cytosolic Na+ and the creation of suitable cellular conditions, photosynthetic materials were used to synthesize proteins and materials necessary for growth and increased shoot and root length, as well as shoot and root biomass in this treatment. There are reports as to Na+ compartmentation and transfer of cytosolic Na+ into the vacuole to prevent the destructive effects of sodium in the cytosol of plant cells24. Unlike the treatment of S300TFP, in the treatment of N biofertilizer along with Trichoderma at high salinity (S300TFN), there was no increase in shoot and root length and biomass, but root growth was significantly reduced. It seems that most photosynthates are used to synthesize secondary metabolites, the major of which are phenols. The increase in phenol content under osmotic stress in different tissues of many plants can occur due to the role of total phenols in regulating the important mechanism of plant metabolic processes, the overall result of which is the effect on plant growth1.
Total flavonoid
The results showed that flavonoid content did not increase under stress and the production of flavonoids from photosynthates was decreased. Although the application of Trichoderma and biofertilizer alone or in combination reduced the amount of flavonoid reduction in salinity (150 mM salt), an increase was observed in flavonoid content in all treatments of the 150 mM salinity level versus the control of this salinity level. This increase was more significant and pronounced in the treatments of S150TFP and S150T0FN. It seems that quinoa’s defense system in salinity stress was more based on the increase in other phenols. The highest amount of flavonoids was observed in the treatments of S0TFN and S0TFP. Accumulation of phenolic compounds in salinity-tolerant plants is a solution to inhibit the activity of reactive oxygen radicals and protect cell membranes from salinity stress damage59. Flavonoid content can be increased by applying biological fertilizers alone or a combination with chemical fertilizers such as nitrogen fertilizers22. Factors such as genotype (cultivar), soil, and environment seem to influence biochemical processes that happen during germination versus the primary and secondary metabolites and phenol compounds profile of quinoa14. By imposing salinity stress on Chichorium spinosum, researchers observed that it increased flavonoid content and antioxidant activity51. Flavonoids are often induced by abiotic stress and are involved in plant protection38. Accumulation of flavonoids due to salinity can indicate that the plant needs large amounts of flavonoids to counteract the harmful effects of salinity3. Flavonoids make membranes resistant to oxidative agents by reducing fluidity and preventing the release of free radicals38. Organic-grown cabbage, spinach, and green peppers generally had higher levels of flavonoid and antioxidant activity19. The increased synthesis of flavonoids by Trichoderma-inoculated plants act as endogenous regulators of auxin motions and growth regulator, and these plants may exhibit fine-regulation of growth hormone and photoprotection in photosynthesis pathway13.
Polyphenols (HPLC)
Quinoa leaf is a potentially rich source of phenolic and flavonoid compounds33. Gawlik-Dziki et al. (2013)21, who analyzed HPLC to identify ChL aglycones of quinoa leaves, detected ten main phenolic acids and four flavonoids in the chemical polyphenolic fraction. The main phenolic acids included ferulic, sinapinic, and gallic acids, whereas kaempferol and isoramentine were the most abundant flavonoid. Also, a great deal of rutin was observed. Quercetin has been reported in the shoot of C. album36. Among the phenolic compounds studied in this research, rosemaric and coumaric acids were the predominant phenolic compounds in quinoa leaves. Caffeic acid was observed in lower amounts. The amounts of cinnamic and chlorogenic acids were slight, all of which showed a significant increase in the treatments of S0TFN and S0TFP. Apigenin was present in small amounts in all treatments but showed a large increase in the treatments of S0TFN and S0TFP. Rutin was found in almost the same amount and quercetin in different amounts in all treatments, but the amount of these two flavonoids was also increased significantly in the treatments of S0TFN and S0TFP. Apigenin content was also increased slightly in these two treatments. On the other hand, total flavonoid content was remarkably enhanced in these two treatments. The results are consistent with one another. The stressful conditions along with FP and FN and Trichoderma were effective in increasing total phenol. The results seem to depend upon not only the plant’s response to different levels of salt but also the interaction among antioxidants. Phenolic compounds can influence one another antagonistically. These changes in the amount of the studied phenols can indicate the different antioxidant potential of various compounds in dealing with salinity stress and the interaction between microorganisms. Phenolic compounds encompass a wide range of compounds, including phenolic acids, flavonoids, and tannins35. The increase in total phenol in the treatments of S300TFN and S300TFP could be related to other phenolic compounds that have not been studied and measured in this study. In plants, the biological synthesis of polyphenols and their accumulation is generally stimulated in response to biotic/abiotic stress such as salinity. Plants exposed to salinity stress may provide potential sources of polyphenols by increasing the concentration of polyphenols in their tissues and limiting biomass production. The optimal performance of polyphenols has been suggested to be accomplished by using stress-tolerant species53. An increased level of phenolic acids has been reported with increasing NaCl concentrations in barley57. El-Din et al. (2009)18 reported that salinity stress exposure of thyme increased such compounds as caffeic, chlorogenic, ferulic, and rosemaric acids. In M. chamomilla, the accumulation of phenolic acids such as chlorogenic and caffeic acids was increased with increasing salinity34. In Nigella sativa, salinity stress increased the biosynthesis of some specific phenolic compounds such as quercetin, apigenin, and trans-cinnamic acid11. There is another report that salinity stress can cause the accumulation of phenolic compounds in plant tissues68. A study on artichoke revealed that the amount of polyphenolic compounds was decreased with increasing salinity27. The results on olives showed that the use of Trichoderma increased the concentration of polyphenols in olive leaves17.