Impaginato 185 Adv. Hort. Sci., 2023 37(2): 185­196 DOI: 10.36253/ahsc­12938 Effect of different nitrogen forms and bio­treatments on the growth and seed yield of downy safflower (Carthamus lanatus) H.A. Ashour (*), S.E.A. Esmail, A.B. El­Attar Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University, Giza, Egypt. Key words: Arbuscular mycorrhiza fungi, Downy safflower, nitrogen fertilization, Trichoderma viride, vermicompost. Abstract: A field experiment was carried out to investigate the effect of differ­ ent nitrogen forms and some biotreatments (Trichoderma viride, vermicompost and arbuscular mycorrhiza fungi) alone or in combination on vegetative growth, seed yield and some chemical traits of downy safflower (Carthamus lanatus L.). Nitrogen was supplied as ammonium sulfate, ammonium nitrate and urea at the rates (5, 3 and 2 g/plant, respectively). Bio treatments included Trichoderma viride, vermicompost and arbuscular mycorrhiza fungi. The results showed that all nitrogen forms significantly increased the plant growth and yield, pigments content, and total carbohydrates in leaves and seeds, as well as N, P and K%, total phenols and oil content in seeds. All bio treatments signifi­ cantly increased the tested parameters compared to control. The integration of ammonium sulfate with T. viride was the most effective treatment since deter­ mined the highest increases of the tested traits. Results showed that for enhancing downy safflower plant growth, and nutritional values of seed, the combined treatment of T. viride at 5 ml/plant and ammonium sulfate at 5 g/plant is recommended. 1. Introduction Carthamus lanatus L. (also called downy safflower, woolly distaff this­ tle or saffron thistle) is an erect spiny biennial plant native of the Mediterranean region. It is closely related to safflower, which is in the same genus. Downy safflower is reported to be sudorific (sweat inducing), fever­reducing and anthelmintic (Hellwig, 2004; DiTomaso et al., 2017; Adel El­Gazzar et al., 2019), Previous studies revealed its importance due to different components of diverse chemical nature such as flavonoids, sesquiterpenes glycosides, lipids, aromatic acids, sterols, triterpenes , volatiles alkaloids, tannins and saponins (Abu El­Khair, 2020). Plant nutrition is one of the most essential factors which increase plant production. Nitrogen (N) is the most recognized in plant as it is pre­ sent in the structure of the protein molecule and plays a vital role in syn­ (*) Corresponding author: hossam.ahmed@agr.cu.edu.eg Citation: ASHOUR H.A., ESMAIL S.E.A., EL­ATTAR A.B., 2023 ­ Effect of different nitrogen forms and bio‐treat‐ ments on the growth and seed yield of downy saf‐ flower (Carthamus lanatus). ­ Adv. Hort. Sci., 37(2): 185­196. Copyright: © 2023 Ashour H.A., Esmail S.E.A., El­Attar A.B. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 22 March 2022 Accepted for publication 23 January 2023 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-12938 http://en.wikipedia.org/wiki/Safflower http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(2): 185­196 186 thesis of plant compositions via the action of various enzymes activities and protein synthesis (Taiz and Zeiger, 2002). Nitrogen has an important role in plant metabolism that impacts quantitative and qualitative plant production by enhancing the growth and stimu­ lating the essential processes which leads to increase the active substances. Ammonium sulfate (AS), ammonium nitrate (AN) and urea are the main forms of inorganic N fertilizers and are extensively utilized in modern agriculture. AS application was better than AN and urea for increasing vegetative growth yield parameters, chlorophyll content, NPK % in seeds and seed oil % of sunflower and jojoba (El Mantawy, 2017; El Sayed, 2020; Hegab et al., 2021). Trichoderma is a genus of saprotrophic fungi and a widespread component of the soil rhizosphere; it has been reported to enhance plant growth and to control many of plant diseases (Colla et al., 2014). One of the well­known stimulatory effects of Trichoderma on plants is the ability to dissolve phos­ phate through acidification, chelation or redox activi­ ty to improving the utilization by plants (Mansour et al., 2021). The benefits of Trichoderma species in stimulating plant growth can be realized via various mechanisms including boost nutrient uptake, solubi­ lization, sequestration of inorganic nutrients and enhancement of root hair development (Harman, 2006; Lorito et al., 2010). Trichoderma spp. promote plant hormone synthesis that improve root growth and root hair formation which lead to more efficient use of nitrogen, phosphorus, potassium and micronu­ trient (Mastouri et al., 2010). Moreover, Trichoderma is able to produce metabolites with hormonal activi­ ties such as indole­3­acetic acid (Contreras­Cornejo et al., 2011). The positive impact of T. viride inocula­ tion on physiological and biochemical features of plants has been reported by many authors. The fun­ gus is able to improve growth and yield parameter (Ghoneem et al., 2019), promote photosynthetic pig­ ments (Kumar et al., 2015; Ghoneem et al., 2019), enhance nutrient status in leaves and roots (Metwally, 2020), increase essential oil and total phe­ nol content (Shaikh et al., 2019; Hassanin et al., 2020; Sanei and Razavi, 2018; Ghoneem et al., 2019) and promote peroxidase activity. Vermicompost is an organic product that is obtained from biodegradation and stabilization of organic waste via the interaction between earth­ worms and microorganisms, lead to break up organic matter residues into fine particles (Ndegwa and Thompson, 2001; Campitelli and Ceppi, 2008). It has a favorable effect on the physical and chemical struc­ ture of soil as well as plant growth (Bachmana and Metzger, 2008). Additionally, it induces and boosts the absorption of nutrients by plants and favors a biological control of bacterial and fungal plant pathogens (Rivera and Wright, 2009). It has high microbial and enzymatic activity and contains large amounts of plant growth regulators like auxins, gib­ berellins cytokinins, macronutrients and micronutri­ ents (Atiyeh et al., 2002). The favorable effect of ver­ micompost application on the growth and yield of many plants has been reported by previous studies (Adamipour et al., 2019; Levinsh, 2020; Abd El­ Hamed et al., 2021). Arbuscular mycorrhizal fungi (AMF) are soil fungi which are prevalent in most agricultural ecosystems to associate with more than 80% of plant species (Wang and Qiu, 2006). Previous studies have shown that plant inoculation with AMF improves growth, seeds yield, promotes photosynthetic pigments and carbohydrates content, enhances accumulation of macro­ and micronutrients in leaves (Amiri et al., 2017; Gashgaril et al., 2020; Mohamed, 2020), as well as increases the nutritional values of seeds like proteins and oil percentage (Ashour et al., 2021). Although the beneficial roles of nitrogen and bio treatments on medicinal and aromatic crops and their valuable effect on improving growth and pro­ duction, there are no sufficient available data about their effectiveness on the growth and yield of downy safflower plants. Therefore, this research is aimed to evaluate the influence of different nitrogen forms (ammonium sulfate, ammonium nitrate and urea) and some biotreatments (T. viride, vermicompost or arbuscular mycorrhiza fungi) on vegetative growth, seed yield and some chemical parameters of downy safflower (Carthamus lanatus) plant. 2. Materials and Methods The field experiment was conducted at the Experimental area of the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza Governorate during the two successive seasons of 2019/2020 and 2020/2021. The latitude, longitude and altitude of the experimental site was 0°01’92.70” N, 31°20’68.08” E and 22 m above sea level, respectively. Experimental procedure Seeds of Carthamus lanatus plants were acquired Ashour et al. ‐ Effect of different nitrogen forms and biotreatments on Carthamus lanatus 187 from experimental farm of Faculty of Pharmacy, Cairo University. On 1st November (of the two con­ secutive years), seeds were sown in a seedling trays (50 x 90 cm diameter) at saran house with 42% shad­ ing, 28/18°C (day/night) temperature, 14 h light con­ ditions, and 30­35% relative humidity. After 30 days from seeds sowing, uniform seedlings, with an aver­ age height of 18­20 cm, were transplanted in the experimental open field in plots (3×3 m), with a dis­ tance of 50 cm among rows, 70 cm between plants. Some physical and chemical properties of the experi­ mental soil (average value of the two seasons) were determined according to Jackson (1973), and the results are presented in Table 1. Nitrogen fertilization included ammonium sulfate (21 %N and 23­24%S) at 5 g/plant, ammonium nitrate (33 %) at 3 g/plant and urea (46% N) at 2 g/plant. Nitrogen forms were applied as two separate doses. The first addition was before transplanting and the second was before flowering. Plants treated with nitrogen forms were also inoc­ ulated with T. viride, vermicompost and arbuscular mycorrhiza fungi (Amf), the control plants were not treated. T. viride, were obtained from Pest Rearing Department, Central Agricultural Pesticides Laboratory, Agricultural Research Centre, Dokki, Giza, Egypt. 109cfu/ml conidial suspension of T. viride was diluted in 5 liters of water so as to prepare solution strength of 2X105cfu/ml. For each seedling, 100 ml of solution was used which accounted 2X107cfu of Trichoderma per seedlings. 100 ml of the solution was used to drench the soil per seedlings (Mastouri et al., 2010; Chirino­Valle et al., 2016). Vermicompost was acquired from Central Laboratory for Agricultural Climate (CLAC), Agricul­ ture Research Center, Ministry of Agriculture, Giza, Egypt. It was applied at 5 g/seedling. Chemical analy­ ses of vermicompost used in this work (average value of the two seasons) are shown in Table 2. Amf inoculum contained roots, hyphae, spores colonized by Glomus mosseae NRC31 and Glomus fasciculatum NRC15 obtained from Agricultural Microbiology Department, National Research Center, Dokki, Giza, Egypt. Inoculum material consisted of 275 spores g­1 (the infectivity 104 propagola). AMF inoculation treatments were carried out by injecting 5 g/seedling of the inoculum. The three bio treatments were applied as two doses, the first addition was after 3 weeks from trans­ planting (21th December in both seasons, respectively) and the second was after 2 months from transplanti­ ng at branching start (21th February in two seasons, respectively). Irrigation, manual weeding, pest and diseases control were done when needed. The layout of the experiment was factorial 4x4 in randomized complete blocks design with 16 treat­ ments. The first factor was 4 nitrogen forms (includ­ ing the control). The second factor was 4 biotreat­ ments (including the control) with 3 replicates, each replicate consisting of 32 plants (2 plants from each treatment). Vegetative growth and yield parameters measure‐ ment Vegetative growth parameters were registered after 120 days from transplanting (On 1st April). Two Soil characteristics Data Physicical characteristics Soil Texture Clay Clay (%) 43.30 Coarse sand (%) 4.20 Fine sand (%) 21.70 Silt (%) 30.80 Field capacity (V %) 67.85 Chemical characteristics Macro­nutrients (%) N 94.19 P 21.29 K 59.64 Organic matter (%) 1.76 CaCO3 (%) 1.54 Electrical conductivity (dS/m) 1.54 Cation exchange capacity (meq/100 g) 40.22 pH 7.27 Table 1 ­ Physical and chemical properties of experimental soil (mean of two seasons) CaCO3= calcium carbonate, pH= soil acidity. Properties pH EC (dS/m) Organic matter (%) N (%) P (%) K (%) Fe ppm Zn ppm Mn ppm Vermicompost 8.41 6.6 42.9 1.65 1.14 1.69 166 109 96 Table 2 ­ Chemical analysis of vermicompost used in this work (mean of two seasons) Adv. Hort. Sci., 2023 37(2): 185­196 188 samples of plants were taken and used to measure growth parameters including plant height (cm), num­ ber of branches/plant, stem diameter (cm, at 5 cm above the soil surface), fresh and dry weights of leaves, stems and roots as well as leaf area (cm2). At the harvesting stage (on 1st to15th May) yield parame­ ter were measured: number of flower heads/plant, weight of flower heads/plant, weight of seeds/plant and weight of 100 seeds (gr). The seed content of total carbohydrates, (N, P and K), total phenols and oil were also determined. Chemical analysis The chemical analysis were performed at the end of each season (on 1st to15th May). Chlorophyll and carotenoid contents. Chlorophyll pigments including Chl a, Chl b and carotenoid con­ tents (mg g­1) were determined according to Lichtenthaler and Buschmann (2005), leaves extract­ ed by suspending them in 5 ml of 95% aqueous ace­ tone at 60 ̊C then the total volume completed to 10 ml with 95% aqueous aceton. The aqueous acetone supernatant was then taken for spectrophotometric measurement. A blank of acetone was taken at wavelengths of 663, 645 and 452.5 nm respectively, and data were then calculated using the following equations: Chlorophyll a (mg g −1) = 0.0127 A663 − 0.00269A645 Chlorophyll b (mg g −1)= 0.0029A663 − 0.00468A645 Carotenoids (mg g −1) = 4.2 E 452.5 − 0.0264 Total carbohydrates. Total carbohydrates content in leaves and seeds (percentage of dry matter) was determined in dried samples according to Dubois et al. (1956). A known weight (0.1 g) of the dried sam­ ples was completely hydrolyzed with 10 ml sulphuric acid (67%) in a test tube on a boiling water bath for one hour. The solution was decolorized and the fil­ trate was diluted to 100 ml with distilled water. A known volume (1 ml) of the extract was taken in a test tube, to which 1 ml phenol solution (5%) was added, followed by 5 ml of concentrated sulphuric acid. The optical density of the resulting color was measured at 490 µm, using a spectrophotometer, against a blank reagent. The standard curve of glu­ cose was used to calculate the total carbohydrates concentration in the extract. N, P and K content of seeds. Half gram of dried seeds samples was digested using tertiary acid mixture (HClO4 + HNO3 + H2SO4) and the extract was analyzed to determine concentrations of N, P and K (as percent­ age of dry seeds) according to Estefan et al. (2013). Nitrogen concentration was determined by using the micro­Kjeldahl method. Phosphorus was determined calorimetrically by using the chlorostannous molyb­ dophosphoric blue colour method in sulphuric acid. Potassium was determined by using the flame pho­ tometer apparatus (CORNING M 410, Germany). Total phenolics content. Total phelolics content was determined in the seeds extract by using the Folin Ciocalteau’s reagent colorimetric method and results are expressed as milligram of gallic acid equiv­ alent per gram of seeds dry weight extract (mg GAE/g DW) (John et al., 2014). Briefly, 1 mL of seed extract was mixed with 2.5 mL of 10% (w/v) Folin­Ciocalteu reagent. After 5 min, 2.0 mL of Na2CO3 (75%) was subsequently added to the mixture and incubated at 50°C for 10 min with intermittent agitation. Afterwards, the sample was cooled and the absorbance was measured utilizing a UV Spectrophotometer (Shimazu, UV­1800) at 765 nm against a blank without extract. The outcome data were expressed as mg/g of gallic acid equivalents in milligrams per gram (mg GAE/g) of dry extract. Seed oil (%). The oil content of seeds was deter­ mined according to AOAC (1995) using soxhlet appa­ ratus using petroleum ether as a solvent. The clean air dried seeds were separately crushed in a Willey mill, then extracted in Soxhlet apparatus, samples of 10 g of seeds were moved into Soxhlet apparatus in 100 ml of N­hexane and the extraction period extended to 6 hours (30­36 syphon cycle approx.). The N­hexane extract was dried over anhydrous sodi­ um sulfate, then filtered and the oil was obtained by distillation under vacuum. oil % was calculated according to the equation: oil % = extracted oil weight (g) ̸ seeds sample weight (g) ̸ × 100. Statistical analysis Results of the two field trials performed in two different growth seasons were combined in order to obtain an average value for each parameter. The means of all results were subjected to Two­Ways analysis of variance (ANOVA) in randomized com­ plete blocks design. Means of data were compared by using Duncan’s multiple range tests at P = 5% (Snedecor and Cochran, 1989). 3. Results and Discussion Vegetative growth parameters The data in Table 3, 4 and figure 1 revealed that Ashour et al. ‐ Effect of different nitrogen forms and biotreatments on Carthamus lanatus 189 under the same level of N forms, application of T. viride, vermicompost or Amf treatments resulted in significant increase of tested vegetative growth para­ meters (viz., plant height, number of branches/plant, stem diameter, fresh and dry weights of leaves, stems and roots and leaf area) compared to control. Among the tested treatments, application of T. viride appeared to be the most effective treatment since *, **, *** significant at P≤0.05, P≤0.01, P≤0.001 respectively, (n=3). Table 4 ­ Plant height, No. of branches/plant, Stem diameter, Fresh and dry weights of leavesof Carthamus lanatus as affected by the interaction between nitrogen forms and biotreatments (mean of two seasons) Amf= Arbuscular mycorrhiza fungi, AS= ammonium sulfate, AN= ammonium nitrate. Data represent the mean value ±S.E. the mean of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan mul­ tiple rang test. Table 3 ­ Mean square for the effect of nitrogen forms and biotreatments and their interaction on vegetative growth, yield parameters of Carthamus lanatus Traits Source of variation Treatment Error CV Biotreatments (A) Nitrogen sources (B) (A × B) Plant height (cm) 4102.63 *** 3164.686 *** 133.843 *** 2.236 0.926 No. of branches/plant 123.894 *** 140.852 *** 18.727 *** 0.785 6.276 Stem diameter (cm) 0.779 *** 0.888 *** 0.058 *** 0.011 8.215 Fresh weight of leaves (g/plant) 3539.035 *** 2397.09 *** 484.993 *** 3.234 1.495 Dry weight of leaves (g/plant) 345.389 *** 253.726 *** 47.235 *** 1.86 3.728 Fresh weight of stems (g/plant) 56027.069 *** 16413.722 *** 1476.819 *** 9.154 1.383 Dry weight of stems (g/plant) 3390.971 *** 989.016 *** 74.53 *** 0.902 1.799 Fresh weight of roots (g/plant) 1451.436 *** 1788.839 *** 476.746 *** 5.688 4.887 Dry weight of roots (g/plant) 117.628 *** 128.286 *** 25.247 *** 0.611 6.305 Leaf area (cm2) 870.237 *** 1304.444 *** 115.687 *** 0.734 2.09 No. of flower heads/ plant 453.436 *** 382.616 *** 46.209 *** 1.077 6.178 weight of flower heads/ plant 388.412 *** 606.905 *** 45.35 *** 1.783 3.706 weight of seeds/ plant 964.929 *** 1057.951 *** 102.45 *** 0.736 3.534 weight of 100 seeds/ plant 0.549 *** 0.799 *** 0.072 *** 0.002 1.993 Biotreatments (A) Nitrogen forms (B) Plant height (cm) No. of branches/plant Stem diameter (cm) Fresh weight of leaves (g/plant) Dry weight of leaves (g/plant) Control Control 124.33±0.60 k 7.00±0.58 g 0.70±0.05 e 86.50±2.18 i 26.07±0.81 h AS 140.83±1.17 gh 11.33±0.33e 1.15±0.06 c 101.83±0.67 g 30.97±0.48 fg AN 130.83±0.44 j 9.33±0.44 f 0.82±0.06 dc 96.83±1.17 h 28.20±0.9 h N­urea 139.67±1.59 hi 11.50±0.29 e 1.17±0.03 c 107.50±0.76 f 34.03±0.12 e T. viride Control 148.67±0.88 f 11.00±0.29 e 0.93±0.07 d 109.17±1.01 f 34.07±0.62 e AS 189.00±0.76 a 23.17±0.17 a 1.87±0.03 a 178.33±1.42 a 54.57±1.4 a AN 175.50±1.50 d 15.50±0.29 c 1.53±0.09 b 136.17±2.6 c 41.42±0.86 c N­urea 179.50±0.29 c 17.83±0.44 b 1.53±0.06 b 136.67±1.86 c 41.63±0.78 bc Vermicompost Control 137.83±1.17 i 9.17±0.33 f 1.30±0.06 c 110.17±0.44 f 30.63±1.12 g AS 184.17±0.60 b 13.33±0.73 d 1.62±0.09 b 142.67±1.09 b 43.65±0.71 b AN 175.83±1.09 d 15.33±0.93 c 1.52±0.01 b 107.5±1.61 f 32.98±0.61 ef N­urea 179.67±0.88 c 16.50±0.87 bc 1.65±0.01 b 128.17±0.83 d 38.37±0.59 d Amf Control 142.67±1.17 g 11.17±0.33 e 0.82±0.03 de 108.33±0.88 f 32.70±0.73 e­g AS 187.00±0.76 a 21.83±0.93 a 1.52±0.07 b 121.17±0.67 e 36.48±0.81 d AN 169.50±1.04 e 15.17±0.44 c 1.15±0.01 c 118.67±0.44 e 38.02±0.46 d N­urea 179.50±0.76 c 15.33±0.33 c 1.57±0.02 b 134.67±0.83 c 41.55±0.42 bc 190 Adv. Hort. Sci., 2023 37(2): 185­196 registered the highest values. These results are in line with those findings of prior authors (Lakshman and Ghodke, 2018; Shaikh and Mokat, 2018; Ghoneem et al., 2019; Guo et al., 2020; Hassanin et al., 2020; El­ Dabaa et al., 2021), who reported increases in vege­ tative growth parameters due to T. viride inoculation. The useful effect of T. viride on vegetative growth parameters may be related to participation of such microorganisms in biotransformation of cellulose, increasing cell reproduction, nitrogen mineralization and phosphorus solubilization. They also increase the volume of roots which in turn, increases absorption of water and nutrients, consequently increasing both growth and yield of the crops (Nepali et al., 2020). The data in Table 4 and figure 1 also revealed that under the same rate of bio treatment (T. viride, ver­ micompost or Amf) treating the plants with different nitrogen forms resulted in significant increase in veg­ etative growth parameters compared to control and among the tested nitrogen forms, with the applica­ tion of ammonium sulfate leading to superior growth compared with ammonium nitrate or urea. The increases in vegetative growth parameters due to ammonium sulfate treatments are in agreement with the findings of several studies on different plants including Cynara cardunculus (Sarhan et al., 2014), Urtica pilulifera (Wahba et al., 2014), Nigella sativa (Khalid and Shedeed, 2015), Sunflower (El Mantawy, 2017; El Sayed, 2020), Thymus vulgaris (Basal et al., 2019) and Jojoba (Hegab et al., 2021). The positive effect of ammonium sulfate may be attributed to the role played by the acidic component that decrease the values of soil pH and thus simplify the uptake of nutrients by the plant roots (Fouda, 2017). Yield parameters Results of figure 2 indicated that within each level of N forms, in most cases, application T. viride, vermi­ compost or Amf treatments caused a significant increase in yield parameters (namely, No. of flower heads/plant, weight of flower heads/plant, weight of seeds/plant, weight of 100 seeds) compared to con­ trol. T. viride treatment appeared to be the most effective one since recorded the highest values. Increases in yield parameters due to T. viride treat­ ments are matched well with those of previous stud­ ies on different crops including Coriandrum sativum (Khan and Parveen, 2018), Triticum aestivum (Mahato et al., 2018), Cuminum cyminum (Ghoneem et al., 2019). Fig. 1 ­ Fresh weight of stems (g/plant) (A), dry weight of stems (g/plant) (B), fresh weight of roots (C), dry weight of roots (D), leaf area (E) of Carthamus lanatus as affected by the interaction between nitrogen forms and bio treat­ ments (mean of two seasons). Column with different let­ ters indicate a significant difference at 5% level. Data represent the mean values ± SE the mean of three repli­ cates. Ashour et al. ‐ Effect of different nitrogen forms and biotreatments on Carthamus lanatus 191 most effective one was ammonium sulfate, for which the highest mean value was found. The increases in yield parameters due to ammonium sulfate treat­ ments are the same as the results of Sarhan et al., 2014 on Cynara cardunculus, Wahba et al. (2014) on Urtica pilulifera, Khalid and Shedeed (2015) on Nigella sativa, El Mantawy (2017); El Sayed (2020) on Helianthus annuus, Prinsloo and Plooy (2017) on Sutherlandia frutescens, Hegab et al. (2021) on Simmondsia chinensis. Contents of pigments and total carbohydrates in leaves As shown in Table 5 and 6 within each rate of N forms, in most cases, application of T. viride, vermi­ compost or Amf rates resulted in significant increase in the mean values of pigments content (chlorophyll a, b and carotenoids) and total carbohydrates in leaves compared to control. The highest mean values were found for the plants treated with T. viride. Such increase in pigments content or total carbohydrates in leaves due to T. viride inoculation is in accordance with those obtained by previous reports on Salvia officinalis (Kumar et al., 2015), Cuminum cyminum (Ghoneem et al., 2019), Allium cepa (Metwally, 2020). Chlorophyll is used by plants for light­trapping and energy transduction during the anabolic process of photosynthesis. A higher content of photosynthetic pigments can be correlated to the augmentation in carbohydrates content of leaves. Results of Table 6 also pointed out that within biotreatments (T. viride, vermicompost or Amf), in most cases, the mean values of pigments content and total carbohydrates in leaves of the plants treat­ ed with various nitrogen forms were significantly higher than the control. Nitrogen in the form of ammonium sulfate was superior to the other two nitrogen sources for enhancing the value of the para­ meters considered. The results are analogy with that recorded by earlier research (Sarhan et al., 2014; Wahba et al., 2014; El Mantawy, 2017), they report­ ed increase in pigments content or total carbohy­ drates in leaves due to application of ammonium sul­ fate. The superior effect of ammonium sulfate in increasing pigments contents may be related to sul­ fur element that is a constituent of succinyl Co­A which involved in chlorophyll synthesis in leaves and its activation at cellular level enhances photosynthe­ sis that eventually boost vegetative growth. Fig. 2 ­ Evolution of total phenolic content in season 2018 (mg gallic acid/100 g of fresh fruit) of four selected farms (131, 272, 351, 432) and their average trend. Bars repre­ sent standard error of the mean (±SEM). Values followed by the same letter in every sampling point are not signifi­ cantly different from each other. Mean separation by LSD test (P≤0.05). Data in figure 2 also exhibited that within each rate of T. viride, vermicompost or Amf, the plants treated with nitrogen forms had significantly higher values of yield parameters, in most cases, than those of control. Among the tested nitrogen sources, the Adv. Hort. Sci., 2023 37(2): 185­196 192 Moreover, it is known the function of sulfur in the synthesis of proteins, oils, vitamins, and flavored compounds in plants since, it is a constituent of the three amino acids methionine (21% S), cysteine (26% S) and cystine (27% S), that are the building blocks of protein (El Mantawy, 2017). Contents of total carbohydrates, N, P and K in seeds It is clear from data reported in figure 3 that with­ in each rate of N forms, in most cases, application of T. viride, vermicompost or Amf caused significant increase in total carbohydrates, macronutrients (N, P and K %) in seeds compared to control. Among the Table 5 ­ Mean Square for the effect of nitrogen forms and bio treatments and their interaction on some chemical constituents of Carthamus lanatus Traits Source of variation Treatment Error CV bio treatments (A) Nitrogen sources (B) (A × B) Chlorophylls A content (mg/g f.w) 14.545 *** 9.932 *** 0.801 *** 0.002 2.18 Chlorophylls B content (mg/g f.w) 8.327 *** 71.103 *** 3.727 *** 0.112 2.932 Carotenoids content (mg/g f.w) 0.288 * 0.268 * 0.148 ** 0.073 7.57 Total carbohydrates [%] in leaves 35.699 *** 55.094 *** 2.029 * 0.771 4.30 Total carbohydrates [%] in seeds 22.491 *** 47.094 *** 2.901 * 1.188 4.68 N% in seeds 1.682 ** 1.761 ** 0.102 * 0.158 17.71 P% in seeds 0.036 *** 0.029 *** 0.004 ** 0.002 9.91 K% in seeds 0.078 *** 0.161 *** 0.01 * 0.004 4.58 Total phenols [%] in seeds 0.568 *** 0.795 *** 0.068 *** 0.003 1.79 Oil [%] in seeds 29.47 *** 48.638 ** 0.423 * 1.498 4.63 *, **, *** significant at P≤0.05, P≤0.01, P≤0.001 respectively, (n=3). Table 6 ­ Pigments, total carbohydrates in leaves as affected by the interaction between nitrogen forms and bio treatments (mean of two seasons) Amf= Arbuscular mycorrhiza fungi, AS= ammonium sulfate, AN= ammonium nitrate. Data represent the mean value ±S.E. the mean of three replicates. Means in a column with different letters indicate a significant difference for each variable at 5% level using Duncan mul­ tiple rang test. Bio treatments (A) Nitrogen forms (B) Chlorophylls A content (mg/g f.w.) Chlorophylls B content (mg/g f.w.) Carotenoids content (mg/g f.w.) Total carbohydrates [%] in leaves Control Control 3.68±0.03 k 7.04±0.12 i 2.64±0.57 b 15.44±0.59 g AS 5.06±0.01 i 13.74±0.07 c 3.55±0.01 a 19.88±1.15 d­f AN 4.09±0.11 j 11.06±0.33 f 3.58±0.01 a 16.22±0.58 g N­urea 5.88±0.04 g 11.27±0.1 ef 3.58±0.01 a 20.18±0.52 c­e T. viride Control 5.88±0.04 g 7.28±0.05 i 3.60±0.03 a 18.90±0.58 ef AS 8.24±0.09 a 15.26±0.14 a 3.88±0.06 a 24.37±1.15 a AN 6.11±0.09 f 12.13±0.34 d 3.63±0.02 a 21.32±0.57 b­e N­urea 6.74±0.02 d 12.26±0.21 d 3.65±0.02 a 23.02±1.73 ab Vermicompost Control 6.62±0.07 de 8.95±0.02 h 3.57±0.08 a 17.28±1.15 fg AS 7.8±0.14 b 13.78±0.04 bc 3.59±0.11 a 22.58±1.21 a­c AN 5.92±0.11 fg 11.79±0.02 de 3.63±0.09 a 21.21±0.64 bc­e N­urea 7.83±0.10 b 14.31±0.06 b 3.57±0.07 a 22.42±0.59 a­d Amf Control 5.50±0.02 h 8.78±0.35 h 3.55±0.06 a 18.90±0.58 ef AS 7.30±0.05 c 11.11±0.05 f 3.64±0.13 a 21.55±0.55 bcd AN 6.45±0.12 e 10.11±0.31 g 3.70±0.04 a 20.67±0.57 b­e N­urea 7.95±0.03 b 13.37±0.13 c 3.62±0.03 a 22.83±1.17 ab Ashour et al. ‐ Effect of different nitrogen forms and biotreatments on Carthamus lanatus 193 tested treatment T. viride appeared to be the most effective one. In this regard, Kumar et al. (2015) on Salvia officinalis stated that the plants inoculated with T. viride had higher phosphorus content in shoot and root as compared with control. Also, Metwally (2020) on Allium cepa declared that the plants inocu­ lated with T. viride improved total carbohydrates and N, P or K% in plant organs. Data in figure 3 also exhibited that within T. viride, vermicompost or Amf treatmens, the plants treated with nitrogen forms had significantly higher values of total carbohydrates, N, P and K in their seeds than the control. Among the tested nitrogen forms ammo­ nium sulfate was superior in its effect than the other two nitrogen sources. Such results confirmed the reports of prior works (Wahba et al., 2014; Khalid and Shedeed, 2015; El Sayed, 2020; Hegab et al., 2021) who showed increase in total carbohydrates or N, P and K% in seeds as result of ammonium sulfate application. The increased content of total carbohydrate in seeds may be related to the increase in chlorophyll content of plants, corresponding to improved photo­ synthesis efficiency (Khalid and Shedeed, 2015). Total phenols in seeds Data in figure 4 A displayed that within each rate of N forms, in most cases, application of T. viride, ver­ micompost or Amf treatments caused a significant increase in total phenols in seeds compared to con­ trol. The highest mean values were found to be asso­ ciated with the plants treated with vermicompost. Similar results were reported by Abd El­Hamed et al. (2021) who found that vermicompost caused increase in total phenols in leaves of Dracocephalum moldavica. Within each treatment of T. viride, vermicompost or Amf, the plants fertilized with different rates of nitrogen forms had significantly higher values of total phenols in seeds than those of control. Nitrogen as ammonium sulfate was superior to the other nitro­ gen forms in augmentation total phenols in seeds. These results confirmed the reports of earlier researches (Munene et al., 2017; Petropoulos et al., 2018; Prinsi et al., 2020; Machado et al., 2022) that reported increase in total phenols in plant organs due to ammonium sulfate treatments. Seed oil (%) It is evident from data figure 4 (B) that within each rate of N forms application of T. viride, vermicompost Fig. 3 ­ Evolution of superficial SS index of four selected farms (131, 272, 351, 432) and their average trend during stor­ age in 2018. Bars represent standard error of the mean (±SEM). Values followed by the same letter in every sam­ pling point after harvest are not significantly different from each other. Mean separation by LSD test (P≤0.05). results are in agreement with those reported by pre­ vious researches on different plants including Cynara cardunculus (Sarhan et al., 2014), Helianthus annuus (El Mantawy, 2017) and Simmondsia chinensis (Hegab et al., 2021), they indicated that ammonium sulfate treatments caused increase in oil percentage in seeds. The increments in oil content due to ammonium sulfate application may be attributed to its promoting role in the formation of amino acids methionine (21% S) and cysteine (27% S); synthesis of proteins and oil content of seeds. Also, sulfur is an important element for oil crops which a constituent of acetyl Co­A, which converted into malonyl Co­A to synthesis of fatty acid (El Mantawy, 2017). 4. Conclusions Summing up the results, it can be concluded that for enhancing growth, nutritional values of seeds, the interacted treatment of T. viride inoculation at 5ml /plant and ammonium sulfate at 5 g/plant is recom­ mended for downy safflower plants. Acknowledgements This work was carried out under the financial assistance from Faculty of Agriculture, Cairo University. References ABD EL­HAMED R.S., MOHAMMED H.F., EL­SAIED R.M. 2021 ‐ Response of dragonhead plant to vermicompost and nitrogen fertilizer. ­ World J. Agric. Sci., 17(3): 242­ 251. ABU EL­KHAIR R.M., GHANEM M.Y., OMRAN G.A., SEIF EL­ DIN A.A. 2020 ­ Qualitative GC‐MS analysis and antimi‐ crobial activity of volatiles from Carthamus lanatus (L.) growing in egypt. ­ Rec. Pharm. Biomed. Sci., 4(1): 6­12. ADAMIPOUR N., KHOSH­KHUI M., SALEHI H., RHO H., 2019 ­ Effect of vermicompost on morphological and physio‐ logical performances of pot marigold (Calendula offici­ nalis L.) under salinity condition. ­ Adv. Hort. 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Results in figure 4 also indicate that, within each treatment of T. viride, vermicompost or Amf, oil per­ centage in seeds of plants treated with different nitrogen forms were significantly higher than those of control. Ammonium sulfate was slightly better in its effect than the other two nitrogen forms. These Fig. 4 ­ Evolution of antioxidant capacity (mg ascorbic acid/100 g of fresh fruit) and SS index after 4 months of cold storage (T2) in seasons 2019 of four farmers (131, 272, 351, 432) and their average trend. Bars represent standard error of the mean (±SEM). Values followed by the same letter between four producers are not significantly different from each other considering DPPH values at T0 and T1 or SS index during storage. Mean separation by LSD test (P ≤0.05). 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