Integrated effects of bacteria and fungi biofertilizers on morphological traits, antioxidants indices, and polyphenol compounds of quinoa (Chenopodium quinoa Willd.) under salinity condition

It is imperative to assess the potential of halophyte plant species, such as quinoa, in resisting high salinity levels in arid and semi-arid regions where the productivity of crops is dramatically affected. A factorial experiment based on a completely randomized design with three replications was conducted to explore the effect of integrated biofertilizer, on morphological traits, antioxidants, and polyphenol compounds of quinoa under salinity stress. The studied factors included NaCl salinity stress at three levels of 0, 150, and 300 mM (S 0 , S 150 , and S 300 , respectively), Trichoderma (T) fungus at two levels (its use and non-use), and biofertilizer at three levels (control, nitrogen (F N ), and phosphorus (F P ). The means of the studied traits showed that the highest shoot length and dry weight was related to S 0 T 0 F N , the highest root length to S 150 TF N , the highest root dry weight to S 0 T 0 F N , and the highest phenol and avonoid contents to S 300 TF P and S 0 TF N treatments. Among polyphenols, the highest caffeic acid, rutin, coumaric acid, and quercetin were observed in S 0 TF P, and the highest levels of chlorogenic, rosmarinic, cinnamic acids, and apigenin (mg/kg) were observed in S 0 TF N . To sum up, Bacteria and fungi biofertilizers were effective on the studied traits at the three salinity levels.


Introduction
Quinoa (Chenopodium quinoa willd) is an annual dicotyledon pseudocereal from the sub-family spinach that is native to South America and the Andes where its cultivation dates back to thousands of years ago and it is used as an essential source of nutrient 67 . The young leaves of quinoa are consumed fresh as a leafy vegetable like spinach 45 . It is also used as an ingredient in the recipes of many foods. The seeds of quinoa are rich in free gluten and nutritional value, and their protein content is greater than corn and wheat. They also contain a great deal of methionine and lysine when compared to cereals 37 . Quinoa is also known as a halophyte cereal that is more resistant to environmental stresses, including salinity and water de cit, than barley, wheat, and corn 25,56 .
Saline soils severely inhibit crop growth and soil productivity 43 . The best approach to combating soil degradation and saline water is the cultivation of halophyte plant species 48 . Salinity hinders many metabolic processes in plants and affects crop production 20 . A major adverse impact of salinity is the over-accumulation of Na + and Cl − within cells, which leads to ion imbalance and physiological irregularities. Salinity stress brings about several biochemical, physiological, and morphological changes in plants and affects their growth, photosynthesis, protein synthesis, respiration, and energy production 26 .
Today, researchers are increasingly paying more attention to the use of microorganisms, such as growthpromoting bacteria, to increase yields 23 . Rhizobacters in uence plant development and yields and mitigate the risks arising from salt stress in addition to contributing to nitrogen biosynthesis, siderophore production, the dissolution of mineral phosphate, and the production of plant hormones 37 . As such, it is of crucial importance to develop such techniques as the use of biofertilizers that contain bacteria that biologically enhance stress resistance of plants 46 . It was reported that quinoa had a high tolerance to salinity in the germination stage, and the application of N and P biofertilizers increased its salinity tolerance so that all recorded morphological traits of the quinoa plants treated with biofertilizers were signi cantly increased compared to the control 7 . It has been found that biofertilizers, in addition to playing a role in the uptake of speci c elements, contribute to absorbing other elements, reducing diseases, improving soil structure, further stimulating plant growth, increasing crop quantity and quality, and enhancing plant resistance to environmental stresses including drought and salinity 42 .
Trichoderma is a soil-borne microorganism, some species of which are known to stimulate plant growth 32 . (There is a report as to the increased growth of Trichoderma-treated quinoa plants 47 .
Trichoderma protects plants from environmental stresses such as salinity and improves their growth and development 12 . The fungal strains of the genus Trichoderma are known to produce secondary metabolites with different biological activities. Trichoderma can produce fungal materials that are capable of stimulating plants to produce their own defense metabolites 62 . Trichoderma spp. also synthesizes many biologically active compounds, including cell wall-decomposing enzymes 63 .
By establishing and abundantly sporing in the soil environment, especially in the root zone of most crops, Trichoderma species not only reduce pathogens in the soil but also stimulate the growth of plant roots and shoots by biochemical mechanisms 31 . Antioxidants are essential for coping with oxidative stress. A health bene t of quinoa is related to its nutritional properties, as well as the content of biologically active molecules such as phenolic compounds and their antioxidant activity 5 .
This study aimed to investigate the tolerance of quinoa to different salinity levels along with the effect of biofertilizers and Trichoderma on improving morphological parameters, antioxidants, and leaf polyphenolic compounds and to make some practical recommendations for the development of its cultivation in saline regions.

Materials And Methods
The study was conducted as a factorial experiment based on a completely randomized design with three replications under controlled conditions at the research greenhouse of Agriculture Faculty, Urmia University in 2018. The treatments included salinity at three levels (0, 150, and 300 µM NaCl), Trichoderma at two levels (no fungus and with fungus), and biofertilizer at three levels (0, N, P). The soil was sampled and analyzed before the experiment was initiated (Table 1). Fifty-four 6.5-kg pots containing sterilized soil and sand at a 3:1 ratio were prepared and the soil sample was tested for its composition. Trichoderma was added to each pot at a rate of 7.5 g, and the seeds of quinoa cv. 'Titicaca' were cultivated. The seeds that were supposed to be treated with biofertilizer were impregnated with fertilizer for half an hour. Azotobaror-1 biological fertilizer, which contains the bacteria of Azotobacter vinelandii strain O4, was used as N biofertilizer, which actively xes atmospheric nitrogen in an absorbable form for plants. Barvar-2 phosphate biofertilizer was used as P biofertilizer. It contains two types of phosphate-solubilizing bacteria from the genus Pantoea (strain P5) and Pseudomonas putida (strain P13). After the seeds germinated, six plants were nally kept in each pot. The different rates of salt stress were applied to the plants at the one-month (6-leaf) stage, and 10 days later, the plants were harvested. Total phenolic content was determined by Folin-Ciocalteu reagent 60 . The avonoid content of the extract was estimated by Zhishen et al.'s (1999) 71 procedure. Also, the HPLC values were determined using a high-performance liquid chromatography device (model series 1100, Agilent, USA) equipped with a 20-µl injection loop, a four-solvent gradient pump, a degassing system, an oven stone, and a diode array detector, adjusted at 250, 272, and 310 nm 58 . The data were statistically analyzed using SAS software version 9.4. The means were compared using the PLSD test at the P < 0.05 level, and MS-Excel software was used to draw the graphs.

Morphological parameters
The analysis of data variance showed that the morphological and antioxidant parameters of quinoa were signi cant at the P < 0.01 level ( Table 2). ns: non-signi cant; * and **: signi cant at the P < 0.05 and P < 0.01 levels, respectively.

Shoot length
The highest shoot length (cm) was obtained from the treatment of S 0 T 0 F N , which was different from the other treatments signi cantly. The lowest was related to S 300 T 0 F P , but it did not differ from that of S 300 TF N and S 300 T 0 C signi cantly. The plants treated with P biofertilizer (F P ) at the salinity level of 300 µM grew longer shoot when they were exposed to Trichoderma than when they were not (Fig. 1).

Shoot dry weight
The highest shoot dry weight (g) was obtained from the plants treated with S 0 T 0 F N , which was different from the other treatments signi cantly. The lowest one was related to S 0 T 0 C and S 300 T 0 C, but they did not differ from S 300 T 0 F N and S 150 T 0 F P signi cantly (Fig. 2).

Root length
The plants treated with S 150 TF N grew the longest roots (cm) and differed from the plants exposed to the other treatments signi cantly. The lowest root length was observed in S 300 TF N . In the salinity levels of 0 and 300 µM treated with the biofertilizer F N and T, root length was decreased (Fig. 3).

Root dry weight
The S 0 T 0 F N -treated plants showed the highest root dry weight (g), differing from the other treatments signi cantly, and the plants treated with S 0 T 0 F P and S 300 TF N exhibited the lowest one. The plants treated with the biofertilizer N (F N ) at the salinity level of 300 µM (S 300 ) exhibited lower root dry weight when they were exposed to Trichoderma (T) than when they were not (Fig. 4).

Antioxidants Phenol
The highest phenol content (mg gallic acid per g dry matter) was related to the treatment of S 300 TF P , but it did not differ from the treatments of S 300 TF N , S 150 T 0 C, and S 0 T 0 C signi cantly. The lowest was related to S 0 T 0 F N (Fig. 5).

Flavonoid
The treatment of S 0 TF N gave rise to the highest avonoid content (mg gallic acid per g dry matter), not differing from S 0 TF P signi cantly. The lowest was obtained from S 150 T 0 C, which was not different from S 300 TC signi cantly (Fig. 6).

HPLC
The highest amounts of caffeic acid, rutin, coumaric, and quercetin were related to S 0 TF P and the highest amounts of chlorogenic, rosmarinic acid, cinnamic acid, and apigenin (mg/kg) were observed din S 0 TF N ( Fig. 7).

Morphological traits
The results show that salinity stress at the rate of 150 mM salt (S 150 ) 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 (S 300 ) treatment, shoot growth was decreased, which is supported by the reports of  48 . The signi cant reduction in shoot length at high salinity (S 300 ) seems to be associated with different responses, including changes in nutrient uptake, cellular homeostasis, and metabolic pathways of plants exposed to salinity stress 49 . 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 nitrate 70 . 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 slowly 41 .
The highest shoot and root length and dry weight were observed in the treatment of S 0 T 0 F N at the salinity level of 0 mM (S 0 ) and in the treatment of S 300 T 0 F N at the salinity level of 300 mM (S 300 ). 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 (S 0 T 0 F N ). The biofertilizer Azotobarvar-1 (containing Azotobacter) is a selective molecular nitrogen xer that can synthesize and secrete some biologically active substances in the root zone and improve the root system, thereby in uencing the uptake of water and nutrients, biological N xation, crop yields, and soil properties 2 . Shoot and root length and dry weight were signi cantly increased by the treatment of S 150 TF N compared to other treatments at the salinity level of 150 mM. In addition to xing 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 parts 7 .
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 (S 0 ) and 300 mM (S 300 ) had an antagonistic effect on Azotobacter activity, which eventually reduced the growth of quinoa. The antagonistic ability of Trichoderma with bacteria has been reported 64 . It has been suggested that the acidi cation of the buffer by Trichoderma inhibits plant growth 50 . 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 signi cant 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 crops 55 .
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 development 69 . The application of Trichoderma along with P biofertilizer signi cantly reduced root length increase, had no signi cant effect on shoot length, but increased shoot dry weight signi cantly. It can be inferred from these results that at high salinity (300 mM), the application of Trichoderma along with phosphate-solubilizing bacteria in uenced plant growth positively, and this effect was signi cant 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 form 28 . The interaction of a plant with bene cial microbes varies greatly depending on the genotypes and may range from growth inhibition to growth enhancement and vigorous growth 54 . Different species of Trichoderma are used for their ability to enhance plant growth and development and their ability to grow in adverse conditions 66 . 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 (S 150 TC) decreased root length signi cantly compared to the control (S 150 T 0 C). Trichoderma had no signi cant effect on shoot growth and root biomass. At 0 and 300 mM salinity levels, Trichoderma without the presence of bacteria (S 300 TC) increased shoot length and dry weight signi cantly 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 Trichoderma 47 .
By secreting fungal metabolites, activating growth regulating signals and plant growth-responsible phytohormones, and increasing the solubility of soil nutrients, Trichoderma can increase plant growth 6,16 .

Total phenol
At the salinity level of 0 mM, the control treatment (S 0 T 0 C) and at the salinity level of 150 mM, the treatment of S 150 T 0 C 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 enzymes 9 or indirectly alleviate the damages of oxidative stress by modulating the function of several proteins associated with this stress 30 . 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 S 150 T 0 F P in which biomass was signi cantly reduced, the plants spent most of their photosynthates on shoot growth. In the treatment of S 150 T 0 F P , 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 treatment 15 . In the treatment of S 0 T 0 F N , 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 signi cant 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 signi cantly and remarkably in the treatments of S 300 TF N and S 300 TF P .
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 NH 4 + carrier cation channels and subsequently, Na + uptake 44 . 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 exposed 29,39 . The use of a combination of seaweed and cyanobacteria as biofertilizer agents improved the growth and phenol content of fava beans 52 . In a study, it was found that an increase in phenol synthesis in Trichoderma-inoculated tomato plants improved their 40 . growth under drought stress, thereby protecting them against oxidative stress by ROS scavenging The increased synthesis of phenols and avonoids is involved not only in the formation of cell walls, which protects plants against biotic stresses but also in antioxidant activity directly 61 .
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 salinity 8 . It seems that in the treatment of S 300 TF P , 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 cells 24 . Unlike the treatment of S 300 TF P , in the treatment of N biofertilizer along with Trichoderma at high salinity (S 300 TF N ), there was no increase in shoot and root length and biomass, but root growth was signi cantly 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 growth 1 .

Total avonoid
The results showed that avonoid content did not increase under stress and the production of avonoids from photosynthates was decreased. Although the application of Trichoderma and biofertilizer alone or in combination reduced the amount of avonoid reduction in salinity (150 mM salt), an increase was observed in avonoid content in all treatments of the 150 mM salinity level versus the control of this salinity level. This increase was more signi cant and pronounced in the treatments of S 150 TF P and S 150 T 0 F N . It seems that quinoa's defense system in salinity stress was more based on the increase in other phenols. The highest amount of avonoids was observed in the treatments of S 0 TF N and S 0 TF P .
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 damage 59 . Flavonoid content can be increased by applying biological fertilizers alone or a combination with chemical fertilizers such as nitrogen fertilizers 22 . Factors such as genotype (cultivar), soil, and environment seem to in uence biochemical processes that happen during germination versus the primary and secondary metabolites and phenol compounds pro le of quinoa 14 . By imposing salinity stress on Chichorium spinosum, researchers observed that it increased avonoid content and antioxidant activity 51 . Flavonoids are often induced by abiotic stress and are involved in plant protection 38 . Accumulation of avonoids due to salinity can indicate that the plant needs large amounts of avonoids to counteract the harmful effects of salinity 3 . Flavonoids make membranes resistant to oxidative agents by reducing uidity and preventing the release of free radicals 38 . Organic-grown cabbage, spinach, and green peppers generally had higher levels of avonoid and antioxidant activity 19 . The increased synthesis of avonoids by Trichoderma-inoculated plants act as endogenous regulators of auxin motions and growth regulator, and these plants may exhibit ne-regulation of growth hormone and photoprotection in photosynthesis pathway 13 .

Polyphenols (HPLC)
Quinoa leaf is a potentially rich source of phenolic and avonoid compounds 33 . Gawlik-Dziki et al.
(2013) 21 , who analyzed HPLC to identify ChL aglycones of quinoa leaves, detected ten main phenolic acids and four avonoids in the chemical polyphenolic fraction. The main phenolic acids included ferulic, sinapinic, and gallic acids, whereas kaempferol and isoramentine were the most abundant avonoid.
Also, a great deal of rutin was observed. Quercetin has been reported in the shoot of C. album 36 . 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 signi cant increase in the treatments of S 0 TF N and S 0 TF P . Apigenin was present in small amounts in all treatments but showed a large increase in the treatments of S 0 TF N and S 0 TF P . Rutin was found in almost the same amount and quercetin in different amounts in all treatments, but the amount of these two avonoids was also increased signi cantly in the treatments of S 0 TF N and S 0 TF P . Apigenin content was also increased slightly in these two treatments. On the other hand, total avonoid content was remarkably enhanced in these two treatments. The results are consistent with one another. The stressful conditions along with F P and F N 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 in uence 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, avonoids, and tannins 35 . The increase in total phenol in the treatments of S 300 TF N and S 300 TF P 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 species 53 . An increased level of phenolic acids has been reported with increasing NaCl concentrations in barley 57 . 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 salinity 34 . In Nigella sativa, salinity stress increased the biosynthesis of some speci c phenolic compounds such as quercetin, apigenin, and trans-cinnamic acid 11 . There is another report that salinity stress can cause the accumulation of phenolic compounds in plant tissues 68 .
A study on artichoke revealed that the amount of polyphenolic compounds was decreased with increasing salinity 27 . The results on olives showed that the use of Trichoderma increased the concentration of polyphenols in olive leaves 17 .

Conclusions
The present study investigated the effect of salinity stress, bacteria biofertilizers, and Trichoderma on morphological traits, antioxidants, and polyphenol compounds of quinoa. The results showed that the application of biofertilizers and Trichoderma under salinity stress in uenced morphological traits, antioxidants, and polyphenol compounds of this plant species. At the salinity level of 300 mM, the application of Trichoderma had an increasing impact on the activity of phosphate solubilizing bacteria and nally induced resistance and increased the growth of the quinoa plants under severe salinity levels.
The results revealed that at severe salinity levels, biofertilizers along with Trichoderma were effective in improving total phenols. Flavonoid content was not increased in stressful conditions, and the defensive system of this plant in salinity stress is more based on increasing other phenols. In total, it is recommended to apply bacterial biofertilizers along with Trichoderma to increase quantitative and qualitative traits of quinoa.