Effect of irrigation and bio-fertilizers on morphological and bio-chemical traits of milk thistle

Milk thistle (Silybum marianum L.) is an annual herbaceous plant species originated from the east of the Mediterranean region¹, but it is extensively distributed in hot and arid regions. Today, this plant species has provoked interests due to its significance in pharmaceutical industries (treatment of liver diseases) and plant oil production^2,3. It is used in the treatment of hepatic disorders, hepatitis (A), high blood cholesterol content, some toxicities, bilious precipitations and stones, migraine, spleen diseases, and alcoholic cirrhosis and has antiviral, anti-coagulation and anti-thrombotic activities⁴.

The medicinal plants the black cumin (Nigella sativa L.), the echinacea purpureae (L.) moench and the milk thistle (Silybum marianum L.) are effective in strengthening the body’s immunity and in treating hepatitis diseases. It should be noted that black cumin is not very well known in the world and has a low turnover, and the water consumption of echinacea is also higher^5,6. Due to the reasons mentioned, the milk thistle was selected for the present study. Oil, food, cosmetics, and forage uses are some other applications of thistle. Drought stress is one of the most important and destructive abiotic stresses influencing plant production. It is estimated that about 26% of all arable lands of the world are located in arid regions⁷. On the other hand, we have witnessed the loss of crop quality and soil fertility in the recent decades due to the inappropriate and excessive use of fertilizers and chemical pesticides and the crisis of the environment pollution, especially the pollution of water and soil resources, which entails the contamination of food and endangers the safety of human communities. Furthermore, the inadequate supply of nutrients⁸ and irrigation water⁹ dramatically disturb plant growth and physiology. Hence, reductions in yields and quality associated the unsuitable water and fertilization management¹⁰. So, the focus has dramatically been shifted towards finding approaches to improving soil and crop quality and removing pollutants. The prevention of the excessive application of chemical inputs and sustainable food production along with environmental conservation are issues that have drawn the attention of most researchers and crop producers^11,12. In this respect, biofertilizer-based sustainable farming has been considered more than ever to stop or reduce the consumption of chemical inputs^13,14.

In a study on the combined effect of drought stress and biofertilizer inoculation on the quantitative and qualitative traits of thistle, Mohammadpour Vashvaei et al.¹⁵ reported that the simple and interactive effects of drought stress and biofertilizers were significant for all traits. It has also been reported that the use of biofertilizers induced plant growth by increasing nutrient availability and thereby it increased yield components (capitule number per plant, seed number per capitule, and 1000-seed weight), which resulted in the enhancement of thistle yield¹⁶. Volaii et al.¹⁷ found the positive impact of biofertilizer and vermicompost on yield and yield components of thistle and stated that the concurrent use of the two biofertilizers was most effective.

In another study, Eskandari Nasrabadi et al.¹³ revealed promising results as to the reduction of chemical fertilizer use in the cultivation of thistle and reported the need to consider more use of biological and organic fertilizers. Based on the results of a four-year experiment by Haban et al.¹⁸ about the negative impact of chemical fertilizers on thistle seed yield in the long run, it was recommended to use biofertilizers. However, Yazdani Biuki et al.¹⁹ reported that none of the morphological traits and yield components of thistle was influenced by different fertilizer treatments. They hypothesized that thistle may not easily lend itself to the effect of fertilizers.

The milk thistle plant strengthens the body’s immune system and is one of the top-selling medicinal plants in the world in terms of turnover. Considering the water crisis in Iran and especially in the northwest of Iran, which has led to the drying up of Lake Urmia, the cultivation of crops with low water requirements deficit irrigation tactic is considered important under the conditions of water scarcity²⁰. However, irrigating plants with water less than normal level undoutly results in decline in crop productivity and quality²¹. Therefore, under these circumstances, unconventional strategies should be adopted. The cultivation of such crops will solve the environmental problems of the region, change the cropping pattern from high-consumption to low-consumption crops, increase farmers’ income and ultimately improve the living conditions of farmers in the region. If this scientific research is not carried out, the northwestern region of Iran will be deserted and devoid of inhabitants in the next 40 years due to salt storms in Lake Urmia. This is because toxic salt particles are dispersed in the atmosphere and are the most important factor in the occurrence of respiratory diseases, various types of cancer and skin and digestive problems. These particles also have an impact on eye health, heart health and high blood pressure. Since milk thistle is of high importance due to its drought stress adaptation and resistance and nowadays the use of biofertilizer to curb on the use of chemical fertilizer and to increase crop yields is important for the movement towards sustainable agriculture, which can be much more important under no-irrigation conditions²² and since little research has addressed the effect of combined use of no-irrigation conditions and biofertilizers on medicinal plants²³ including milk thistle, the present study focused on the effect of bio-fertilizers on the quality and quantity of thistle under no-irrigation conditions. Bio-fertilizers effectively increase the plant’s tolerance to drought stress, prevent a reduction in grain yield and improve the plant’s antioxidant properties (phenolic and flavonoid compounds).

The results of ANOVA are presented in Table 1 for morphological traits and in Table 2 for biochemical traits. Accordingly, the interaction of irrigation × biofertilizer was significant for biological yield, capitule number, and DPPH radical scavenging at the P < 0.01 level and for seed yield per plant and plant height at the P < 0.05 level. But, it was not significant for the other traits including harvest index, crown diameter, branch number, oil yield, phenol and flavonoid content, superoxide radical scavenging, nitric oxide radical scavenging, and chain breaking.

Seed and biological yield per plant

Based on the comparison of the means, the treatment of the P biofertilizer to the fully-irrigated plants was related to the highest seed yield (20.74 g), and the non-irrigated plants that were not treated with a biofertilizer produced the lowest one (10.99 g). The interaction of ‘P biofertilizer × full irrigation’, which produced the highest seed yield per plant, did not differ significantly only from the treatments of the NPK biofertilizer and the N biofertilizer to the full irrigation conditions. Among the fully-irrigated plants, those treated with the P biofertilizer exhibited the highest seed yield (20.74 g) and those not treated with a biofertilizer exhibited the lowest one (17.68 g) so that only P biofertilizer was grouped with the control in a separate statistical group, and the other treatments did not show a significant difference from the control. Under the no-irrigation conditions, the highest and lowest seed yields were 16.78 and 10.99 g obtained from the NPK biofertilizer and no-biofertilizer, respectively. In general, the bio-fertilizer treatment with full irrigation and without irrigation increased grain yield by about 15 and 35%, respectively, compared to the control treatment. In these conditions, in addition to the NPK biofertilizer, the P biofertilizer differed from the control significantly too whereas the N biofertilizer and the K biofertilizer were in the same statistical group with the control. So, it can be concluded that the NPK fertilizer could partially alleviate the adverse impacts of drought stress. In other words, although when less water is consumed, which naturally induces drought stress and seed yield per plant is decreased, the application of the NPK biofertilizer can partially mitigate the negative impacts on the yield. This may partially be attributed to the positive effect of biofertilizers on improving plant nutritional status under stressful conditions^24,25. It seems that the separate use of the N and K biofertilizers entailed responses similar to the control.

In a similar finding, Mohammadpour Vashvaei et al.¹⁵ attributed the decline in seed yield of milk thistle under water deficit conditions to the reduction of photosynthesis and assimilation in the plants and the decline in the nutrients mobilized from the leaves to the seeds. Volaii et al.¹⁷ reported the positive effect of biofertilizer and vermicompost on yield and yield components of milk thistle and stated that the combined use of the two biofertilizers had the strongest impact on the traits. In another study, Eskandari Nasrabadi et al.¹³ found promising results as to the decrease in chemical fertilizer use in the cultivation of milk thistle. They emphasized that more attention should be given to the use of biofertilizers and organic fertilizers.

The interaction of ‘NPK biofertilizer × full irrigation’ had the highest and ‘no biofertilizer × no irrigation’ had the lowest biological yield per plant (132 and 86.2 g plant⁻¹, respectively). Under the full irrigation conditions, in addition to the NPK biofertilizer, the N biofertilizer produced a considerably high biological yield of 127 g plant⁻¹. As expected, the lowest biological yield (116 g plant⁻¹) was obtained from the full irrigation of the control plants, which was in the same statistical group with the P and K biofertilizers. Under no irrigation conditions, the NPK biofertilizer had the highest biological yield of 118 g plant⁻¹ followed by the N biofertilizer, which produced 102 g plant⁻¹ of biological yield. The other treatments did not differ from the control significantly (Fig. 1). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased biological yield by about 11.5 and 27%, respectively, compared to the control treatment. It has been documented that low water supply reduced the availability of nutrients and uptake²⁶, while reactive oxygen species are stimulated which damage the cell membrane with reduction of photosynthesis efficiency²⁷.

Sanchez et al.²⁸ reported for Plantago major L. and P. lanceolota L. that the application of biofertilizers enhanced biological yield. Rodriguez et al.²⁹ attributed the decline in biological yield of the stressed plants to the dry weight of their roots, stems, and leaves. Consistently, Anwar et al.³⁰ observed that the application of NPK fertilizer + vermicompost increased the biological yield of basil versus the control. Yazdani Biuki et al.¹⁹ reported that the changes in the biological yield and harvest index of milk thistle in response to chemical, biological, and organic fertilizers versus the control were statistically significant.

It was found that the P biofertilizer had the highest harvest index of 16.3% and the N biofertilizer had the lowest one of 13.8%. The other treatments were placed in the same statistical group with the N biofertilizer despite some small variations (Fig. 2). This is consistent with the results of Darzi et al.³¹ for fennel and Rezaei-Chiyaneh et al.³² for Lallemantia as to the increase in yield and yield components of these plant species in response to the treatment of the biological phosphate fertilizer.

The treatment of N biofertilizer to the fully-irrigated plants was associated with the highest plant height of 178 cm and the control treatment under no-irrigation conditions was associated with the lowest plant height of 111 cm. Under the full irrigation conditions, the NPK and P biofertilizers were grouped in the same statistical category with the N biofertilizer, whereas the treatment of no biofertilizer had the lowest plant height of 153 cm and was significantly different from the other treatments. Under no irrigation conditions, the NPK biofertilizer had the highest plant height (138 cm) and the N biofertilizer had the second highest plant height, but the other treatments did not differ from the control significantly (Fig. 3a). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased plant height by about 14 and 20%, respectively, compared to the control treatment. The morphological changes in medicinal plants in response to drought stress have been subject to extensive studies. Our findings are in agreement with the findings as to the decline in the plant height of basil³³ and savory³⁴ under drought stress. The significant impact of fertilizer treatments on plant height of milk thistle has already been reported³⁵.

The mean crown diameter was higher in the full irrigation conditions (23.3 mm) than in the no-irrigation conditions (19.3 mm). The NPK biofertilizer exhibited the highest crown diameter of 22.6 mm followed by the N biofertilizer in the second rank (22.4 mm). These two treatments were significantly different from the control (19.3 mm) whereas this difference was not observed in the P and K biofertilizers (Fig. 3b). The quantity and quality of a plant’s vegetative growth depend on cell division, elongation, and differentiation, and all these are influenced by drought stress³⁶. For the same reason, the crown diameter was lower in the no-irrigation conditions than in the full-irrigation conditions. Since the crown is a sink of assimilates, the improvement of nutritional conditions by the studied fertilizers increased assimilates and this increased stem diameter.

Based on the results of the comparison of means, when drought stress was applied, almost five capitules were decreased from all plants. All treatments, including the control and biofertilizers, produced their highest capitule number (15 capitules) in the full irrigation conditions and they were all categorized in the same statistical group. However, the differences between the fertilizer treatments with one another and with the control were more considerable in the no-irrigation conditions so that the NPK biofertilizer was related to the highest number of capitules (13.3 capitules) and the control to the lowest number (8.7 capitules). Also, the N and P biofertilizers were placed in the same group and had an intermediate number of capitules. Only, the K biofertilizer did not differ from the control significantly (Fig. 3c). In general, the bio-fertilizer treatment with full irrigation and without irrigation increased capitule number by about 2 and 35%, respectively, compared to the control treatment. Irrigation is effective in increasing the number of capitules per plant due to its effect on increasing moisture and water uptake by the plants and improving nutrient uptake and retention capacity. On the other hand, drought stress limited plant growth and reduced the number of auxiliary branches, thereby reducing the number of capitules per plant.

Volaii et al.¹⁷ concluded that organic and biological fertilizers were influential on most growth traits of milk thistle including the number of capitules per plant and capitule diameter. Mohammadpour Vashvaei et al.¹⁵ mentioned the number of capitule per plant to be the most important seed component. They revealed that this trait was significantly (P < 0.01) influenced by drought stress, biofertilizer, and their interaction. Most researchers agree that this trait is genetic and is influenced by genotype³⁷.

Number of auxiliary branches

The plants fully irrigated produced more branches (3 auxiliary branches) than those not irrigated (2.69 auxiliary branches). On the other hand, the application of the NPK and N biofertilizers was related to the highest number of auxiliary branches (3.11 and 3 auxiliary branches, respectively) and the control had the lowest number (2.46 auxiliary branches). But, the P and K biofertilizers were in between these two extremes (Fig. 4a). The increase in the number of auxiliary branches in the full irrigation conditions is related to the improved vegetative growth of the plant. The increased number of auxiliary branches in the fertilizer treatments can be caused by the improved growth conditions due to the uptake of nutrients, especially N. A study on the effect of biofertilizers (nitrogen and phosphate solubilizing microorganisms) on the quantitative and qualitative yield of milk thistle revealed that although biofertilizers enhanced some recorded traits, there were no significant differences among the fertilizer treatments in the number of branches per plant, capitule diameter, the number of capitule per plant, and 1000-seed weight¹⁷.

According to Fig. 4b, oil yield was higher under the full irrigation conditions (5.2 g plant⁻¹) than the no-irrigation conditions (3.5 g plant⁻¹). Among the biofertilizer treatments, the NPK biofertilizer (4.94 g plant⁻¹) and the P biofertilizer (4.83 g plant⁻¹) had the maximum and the no-biofertilizer treatment had the minimum oil yield (3.68 g plant⁻¹). A study reported that the soil application of various organic and chemical fertilizers was not influential on yield components and morphological traits of milk thistle, but they influenced oil percentage and seed silymarin and silybin contents so that the plants treated with compost, Azotobacter, and their mixture produced the highest seed oil percentage. In addition, the plants treated with chemical fertilizer had the lowest silybin content¹⁹. Finally, the acceptable oil percentage of milk thistle implies its high medicinal and nutritional significance. Based on our results, it can be considered a source of plant oil production.

In the present study, the null hypothesis as to the equal total phenol content of the different treatments was supported. In contrast, some research has shown than the accumulation of phenol compounds is very sensitive to nutrient stress so that total phenol content usually increases with the decrease in N content of the medium, but higher amounts of N in the medium usually stimulate plant growth and hinder phenol synthesis³⁸. Mazarie et al.³⁹ found that as drought stress was intensified, the phenol content of common sage was increased. The mean phenol content of milk thistle in the present study was 30.3 mg quercetin per g dry matter.

Based on the observations and the comparison of means, flavonoid content was the maximum in the plants treated with the NPK biofertilizer (2.59 mg quercetin g⁻¹ DM) and then the K biofertilizer (2.56 mg quercetin g⁻¹ DM), but the N and P biofertilizers did not differ from the control (2.27 mg quercetin g⁻¹ DM) significantly (Fig. 5). Since silymarin, the active ingredient of milk thistle, is a flavonoid compound composed of five flavonolignans, it can be concluded that the application of NPK and K biofertilizers increased silymarin content. Also, studies on the relationship between the accumulation of flavonolignans and the vegetative traits of milk thistle revealed a positive significant relationship between the accumulation of these compounds and plant height. Similarly, the increase in plant height and flavonoid content in the plants treated with the NPK biofertilizer in the present study supports it.

DPPH radical scavenging

According to the results of the means comparison in Fig. 6a, the capacity of inhibiting DPPH radical was increased by almost 5% under the no-irrigation conditions. Also, the results showed that under the full irrigation conditions, this capacity did not vary among the fertilizer treatments significantly. However, under the no-irrigation conditions, the treatments of K (58.8%), NPK (57.6%), and P biofertilizers (55.7%) had the highest inhibiting capacity and they were in the same statistical group, the N biofertilizer (51.6%) was in the intermediate group, and no-biofertilizer application (44.3%) exhibited the lowest DPPH radical scavenging capacity. The difference in antioxidant activity may be associated with the difference in phenol content and other active compounds. The percent scavenging of DPPH radical in thyme was measured by Mehran et al.⁴⁰ to be 66.6%. The results of a study showed that drought stress increased DPPH free radicals scavenging⁴¹. Studies have generally reported the positive effect of biofertilizer on increasing antioxidant properties, e.g. DPPH free radicals scavenging⁴².

Superoxide radical scavenging

Superoxide radical scavenging was higher under the full irrigation conditions (77.2%) than the no-irrigation conditions (72.3%). Also, among the biofertilizer treatments, it was the highest (81.4%) in the plants treated with the NPK biofertilizer followed by those treated with the K biofertilizer (79.7%). The plants treated with the P biofertilizer were at an intermediate level (75.3%), and those treated with the N biofertilizer (70.3%) and those not treated at all (70.0%) had the lowest scavenging capacity (Fig. 6b). Superoxide anion (O₂⁻) is a reduced form of molecular oxygen that is a free radical composed of the electron transport systems of mitochondria. Some electrons that pass through the mitochondria chain reaction react with oxygen directly and form superoxide anion. Free radicals are highly reactive and mostly damage proteins and break down DNA strings⁴³. With respect to the effect of biofertilizers on superoxide radical scavenging, it has been shown that biofertilizers increase this antioxidant trait⁴².

Nitric oxide radical scavenging

Based on the comparison of the means (Fig. 6c), the full irrigation conditions had higher nitric oxide radical scavenging (26.9%) than the no-irrigation conditions (27.3%). In addition, the trend of nitric oxide radical scavenging capacity for different biofertilizers (Fig. 6c) was similar to that of superoxide radical scavenging capacity. Nitric oxide radical scavenging capacity was the highest in the plants treated with the K biofertilizer (28.1%) and the lowest in those not treated with a biofertilizer (26.2%). Except for the NPK biofertilizer, all treatments were in the same statistical group with the control. In general, the bio-fertilizer treatment with full irrigation and without irrigation increased DPPH radical scavenging by about 7 and 25%, respectively, compared to the control treatment. Marcocci et al.⁴⁴ concluded that nitric oxide radical scavengers compete with conducting oxygen to reduce nitrite oxide generation not only in the plants of the Lamiaceae but also in other plant species.

Chain-breaking activity

The treatments of the NPK (16.0%), K (15.8%), and P biofertilizers (15.7%) had the highest chain-breaking activity with slight differences, respectively. They were in the same statistical group. But, the treatments of the N biofertilizer (13.6%) and no biofertilizer (13.0%) had the minimum chain breaking and were placed in the same statistical group (Fig. 7). In a study on the effect of humic acid on the antioxidant properties of thyme under the ecological conditions of Urmia, Iran, Taghipour et al.⁴⁵ reported that the highest total phenol content, DPPH radical scavenging percentage, and chain-breaking activity were obtained in the first harvest from the application of humic acid and the highest DPPH radical scavenging percentage and chain-breaking activity were obtained in the second harvest from the treatment of humic acid.

The present study was carried out at the agricultural research station of Miandoab city located in the south of West Azarbaijan province in Iran (Lat. 36°58’ N., Long. 46°06’ E., Alt. 1314 m.) during 2017. The climate of the Miandoab region is relatively hot in the summer, and relatively cold in the winter. The average rainfall in the region amounts to 289 mm. Table 3 contains meteorological parameters information related to the months of the experiment. To determine its physical and chemical characteristics and estimate fertilizer requirements^46,47, the soil at the study site was randomly sampled from a depth of 0–30 cm before sowing. Then, they were mixed and sent to a laboratory. The results are presented in Table 4.

The field trial was carried out a split-plot experiment based on a randomized complete block design with 10 treatments and 3 replications. The main plot was assigned to irrigation at two levels (I₁: irrigation water with crop water requirement (CWR) of 100% and I₂: no-irrigation conditions) and the sub-plot was assigned to bio-fertilizer at five levels (control, nitrogen bio-fertilizer- N(b), phosphate bio-fertilizer- P(b), potash bio-fertilizer- K(b), and complete bio-fertilizers- NPK(b)). In this study, the irrigation technique employed was the surface irrigation (furrow method) three irrigations with a water depth of 100 mm were conducted in the sensitive areas of the milk thistle plant, including the vegetative period, the pre-flowering period, and the seed filling period. As a result, the water requirement of this plant is 300 mm or 3000 m³ per ha. On May 15, May 30, and June 15, irrigation was done with an interval of 15 days. Furthermore, the growth period for this plant was four months (late March to mid-July). It should also be mentioned that the milk thistle plant from the research farm of Urmia University named after Dr. Amir Rahimi has been registered in the herbarium of the Urmia Agricultural Research Centre of West Azerbaijan Province with the international code 11,071-WESTA. In general, previous research experiments have shown that the amount of heavy elements in agricultural soils in the current research area is not too critical^48,49.

Azotobacter 1 contains the bacteria of the O4 strain of Azotobacter vinelandii, which fixes atmospheric N actively into the forms that are absorbable by plants. One 100-g container of Azotobacter 1 can be an effective replacement for 30–50 kg chemical N fertilizer. The phosphate biofertilizer contained two phosphate-solubilizing bacteria that decompose insoluble phosphorus compounds of soil by two mechanisms – the secretion of organic acids and enzyme phosphatase. Then, this nutrient becomes available to plants. Based on the amount of soil absorbable phosphorus, each package of this biofertilizer can replace 50–100% of the chemical phosphate fertilizer demand of plants. The biofertilizer Pota-Barvar-2 contains two potassium solubilizing bacteria that decompose insoluble potassium in the root zone and release its ions, thereby optimizing potassium uptake. So, it can be a replacement for at least 50% of potassium chemical fertilizers.

The biofertilizers used in the study were applied to the seeds during sowing, for which the biofertilizers were diluted to the amount required to dampen the seeds for 1 ha. Then, the seeds were spread on a piece of plastic or a clean surface, and the diluted solution was applied to them with a sprayer. Then, the seeds were mixed with the solutions well. The seeds used in the experiment were from the local landrace of Western Europe, which has been domesticated and is extensively produced at the farms of this region. After a tillage operation in the autumn and land preparation in February 2017, a total of 20 plots were built, each with an area of 6 m². The seeds were sown on rows with an inter-row spacing of 60 cm and an on-row spacing of 25 cm. They were sown at a depth of 3–5 cm in Late-March, 2017 after inoculation with the biofertilizers. After the germination and growth of the plants, the extra plants were thinned at the 2-4-leaf stage and the empty parts of the plots were re-planted on April 10, 2017. At the same time, the weeds were removed by hand. Each plot contained five sowing rows spaced by 2 m.

It should be noted that in the full irrigation treatment, the irrigation of the plots was initiated one month after sowing and repeated every 15 days. Also, the plants were sown at the physiological maturity stage four months after sowing. Since the maturity of the plants and plant constituent parts was not concurrent, the harvest was performed at several stages after the drying and yellowing of the plants. The harvested plants were sun-dried for several days until they lost their moisture. Then, the plants of each plot were weighed and crushed to have their seeds separated. Then, the remaining foliage was separated from the seeds by screening and airing and the crop of each plot was poured into separate packages and weighed.

To determine morphological traits and yield components, 10 plants were randomly harvested from each plot to measure the traits like plant height, crown diameter, capitule number, and the number of auxiliary branches. Also, to determine seed yield, two marginal rows and 0.5 m from each side of the plots were eliminated as the marginal effect and the plants of the remaining area were harvested to find out their seed yield. The ratio of the economical part (seed) to total dry weight (biological yield) was taken as the seed harvest index. Indeed, the harvest index was calculated by the following equation and expressed in percent:

To find out the oil yield, the ground seeds were oven-dried at 75 °C for 24 h. Then, 2.5 g of each sample was wire-wrapped in a piece of thin cloth and was then placed in an oil extraction device. The samples were boiled in 300 cc of n-hexane for 6 h. Then, the seeds wrapped in the cloth were oven-dried again at 75 °C for 24 h. Then, its weight was placed in the equation of oil calculation to determine the oil percentage. Afterward, the oil samples were poured into micro-tubes and were sent to a laboratory to be analyzed. Oil yield per plant was calculated by the following equation:

To estimate the flavonoid content of different parts, 10 µL of the plant extract was first diluted with 1 mL of distilled water and it was then added with 0.075 mL of sodium nitrite (5%). Five minutes after the reaction, 0.15 mL of aluminum chloride (10%) was added, and 6 min after the reaction, 0.5 mL of sodium hydroxide (1 mol L⁻¹) was incorporated and was adjusted to a final volume of 3 mL. Finally, its absorption was read at 510 nm and the total flavonoid content was determined by the quercetin standard curve. To find out DPPH stable radical scavenging rate, firstly 10 µL of the extract was mixed with 2 mL of methanol solution (0.004%). Then, the absorption of the solution was read at 517 nm after 30 min of incubation (at the darkness at room temperature). DPPH scavenging activity was calculated by the following Eqs^50,51.:

In which A_blank represents the extract-free reaction mixture absorption and A_sample represents the extract-containing reaction mixture absorption.

Superoxide free radical scavenging was measured by the procedure described in Beauchamp and Fridovich⁵². So, 9 mL of tris-HCL buffer solution (pH = 8.2, 50 mmol L⁻¹) was poured into a test tube and it was placed in a water bath at 25 °C for 20 min. Then, 40 µL of pyrogallol solution (45 mmol L⁻¹ pyrogallol in 10 mmol L⁻¹ hydrochloric acid), already incubated at 25 °C, was injected into the upper part of the test tube with a µL syringe, and they were mixed. The solution was incubated at 25 °C for 3 min, and immediately after that, one drop of ascorbic acid was poured into the mixture to stop the reaction. The mixture absorbance was read at 320 nm as A₀ after 5 min. A₀ represents the pyrogallol auto-oxidation rate. The auto-oxidation rate A₁ was calculated by the same procedure with the only difference being the addition of 50 µL of tris buffer to the extract. At the same time, a control blank of the reaction compound was considered as A₂. Percent radical scavenging was calculated by the following equation:

To collect free nitric radicals, 10 µL of the extract was added with 0.5 mL of phosphate-buffered saline (10 mmol) and 2 mL of sodium nitroprusside (10 mmol). It was, then, incubated at 25 °C for 150 min. Then, 0.5 mL of this solution was mixed with 1 mL of sulfanilic acid (0.33% in glacier acetic acid 10%) and was rested for 5 min for the reaction to complete. Then, 1 mL of naphthyl ethylene diamine dihydrochloride (0.1%) was added and the mixture was kept at 25 °C for 30 min during which a pink color formed in the solution. Next, the absorbance was read at 540 nm to determine percent inhibition by the following Eqs^44,53.:

In which A_blank represents the extract-free reaction mixture absorbance and A_sample represents the extract-containing reaction mixture absorbance.

Chain breaking activity was measured by the DPPH reagent and the method described in Brand-Williams et al.⁵⁴ with slight modifications. So, 10 µL of the extract was mixed with 1.9 mL of methanol solution (0.004%) of DPPH. Then, its absorbance was read at time zero and again 30 min after incubation at the darkness and room temperature at 515 nm. The reaction speed was calculated by the following equation:

There were more than two treatments groups in the present study. Therefore, an analysis of variance was performed to investigate the presence or absence of treatment differences overall. If treatment differences were significant, a comparison of means was performed based on Duncan’s test. The test of normality of experimental error distribution and the analysis of variance (ANOVA) for all traits were performed in the SAS 9.4 software package. The comparison of the means for all traits was carried out by Duncan’s multiple range test at the P < 0.05 probability level. Finally, all graphs were drawn in MS-Excel.

The milk thistle is of high importance due to its drought stress adaptation and resistance and nowadays the use of biofertilizer to curb on the use of chemical fertilizer and to increase crop yields is important for the movement towards sustainable agriculture, which can be much more important under no-irrigation conditions and since little research has addressed the effect of combined use of no-irrigation conditions and biofertilizers on medicinal plants including milk thistle, the present study focused on the effect of bio-fertilizers on the quality and quantity of thistle under no-irrigation conditions. Bio-fertilizers effectively increase the plant’s tolerance to drought stress, prevent a reduction in grain yield. In the present study, bio-fertilizers were examined to determine whether they influence the plant’s tolerance to drought stress and whether, on the other hand, the yield of the plant is reduced.

The results showed that most morphological and biochemical traits of milk thistle were affected by the irrigation conditions and biofertilizers. The use of biofertilizers improved these traits versus the control. Given the significant role of biofertilizers in the development of sustainable agriculture, they can be a good candidate in case there is a need for nutrient supplements for this plant. Under drought stress conditions, the combination of bio-fertilizers (NPK(b)) and irrigation helps the plant to tolerate drought stress easily and also improves the plant’s antioxidant properties (phenolic and flavonoid compounds). Although the absence of irrigation results in a decrease in milk thistle yield, the utilization of bio-fertilizers, specifically complete bio-fertilizer and phosphate, can effectively compensate for the decrease in yield by enhancing nutritional conditions. It is recommended that the milk thistle plant be considered for cultivation and exploitation in arid and semi-arid areas and rain-fed areas. In addition to its medicinal properties, it is recommended to utilize the milk thistle plant in the food industry and animal husbandry.