Optimizing essential oil, fatty acid profiles, and phenolic compounds of dragon’s head (Lallemantia iberica) intercropped with chickpea (Cicer arietinum L.) with biofertilizer inoculation under rainfed conditions in a semi-arid region

ABSTRACT In arid and semi-arid regions, intercropping systems combined with biofertilizer application help improve the quantity and quality of food and industrial crops. This study assessed the effect of biofertilizers on the seed yield, essential oil (EO) concentration, fatty acid profile, and phenolic compounds of dragon’s head in intercropping patterns under rainfed conditions during the 2019 and 2020 growing seasons. The treatments comprised five planting patterns [sole cropping of dragon’s head (D) and chickpea (Ch) and intercropping patterns of 1D:1Ch, 2D:1Ch, 1D:2Ch] and three fertilizer sources [arbuscular mycorrhizal fungi (AMF), bacterial biofertilizers (BI), no fertilization (control)]. The most sustainable combination was 2D:1Ch with BI fertilization, with the highest EO concentration in dragon’s head aerial parts (0.94%) and seeds (0.59%). The major EO constituents of dragon’s head aerial parts were germacrene-D and (E)-caryophyllene and seeds were 1,8-cineole and O-cymene. The 2D:1Ch intercropping pattern with BI fertilization also had the highest concentrations of linoleic acid, oleic acid, and phenolic compounds (quercetin and chlorogenic acid) and the highest land equivalent ratio (1.15). These results indicate that intercropping and BI fertilization can improve dragon’s head cultivation, rendering it a sustainable strategy for EO production under rainfed conditions.


Introduction
Numerous agronomic practices increase crop production per unit area, but many can have harmful environmental impacts. For example, the excessive use of chemical fertilizers and pesticides in conventional agriculture adversely affects environmental ecosystems, including biodiversity losses, environmental pollution, especially soil and water resource pollution, and the contamination of human food resources (Tuomisto et al. 2012), threatening agrobiodiversity and human health (Zhang et al. 2018).
One step toward sustainable farming systems is using species/cultivar mixtures (Hussain et al. 2020;Wezel et al. 2020). Intercropping, where two or more plant species are cultivated simultaneously on the same piece of land (Martin-Guay et al. 2018;Guo et al. 2021), increases biodiversity and thus agroecosystem sustainability. Intercropping legumes with medicinal plants contributes to yield sustainability in low-input agriculture through optimal land use and increased soil fertility (Amani Machiani et al. 2021a;Raza et al. 2021;Rezaei-Chiyaneh et al. 2021a, b). In these systems, the nitrogen fixed by the legume component is often available for the accompanying species, contributing to system sustainability .
A key pillar for including medicinal plants in sustainable systems is biological fertilizer application, such as bacterial (N-fixing, P-and K-solubilizing bacteria) and fungal biofertilizers (arbuscular mycorrhizal fungi) and blue-green algae. Biofertilizers contain microbial communities that alleviate environmental pollution by improving plant growth and reducing chemical fertilizer use (Khalid et al. 2017). Biofertilizers increase plant growth by improving soil fertility through fixing atmospheric nitrogen, solubilizing fixed phosphates, and producing nutrients required for plant growth. Indeed, these fertilizers mobilize soil nutrients, increasing their availability to plants (Sun et al. 2020). Furthermore, biological fertilizers enhance plant growth by synthesizing growth hormones, e.g. auxin, cytokinins, and gibberellin (Singh et al. 2021). Fotohi  reported that biofertilizers significantly modified the oil concentrations, EO concentrations, and chemical constituents of ajowan (Carum copticum L.) intercropped with pea (Pisum sativum L.) and fenugreek (Trigonella foenum-graecum L.). Namazi et al. (2022) found that savory (Satureja hortensis L.)/common bean (Phaseolus vulgaris L.) intercropping with biofertilizer application improved EO content and EO chemical composition (increased concentration of carvacrol, γ-terpinene, α-terpinene, p-cymene, and β-myrcene).
Mycorrhizal fungi act as biological fertilizers, supplying plant nutrients in sustainable agriculture systems by increasing uptake through an extensive network of AMF hyphae and host plants (Rathod et al. 2011;Sheteiwy et al. 2021a). Symbiosis with mycorrhizal fungi mitigates the adverse effects of nutrient poverty and drought and salinity stress and improves plant growth, nutrient uptake (e.g. nitrogen and phosphorus), and plant tolerance to environmental stresses such as water deficiency (Begum et al. 2019). Mycorrhizal fungi increase the root system efficiency of agricultural and medicinal plant species by contributing to the active absorption and mobilization of nutrients, especially immobile minerals, e.g. phosphorus, zinc, and copper (Weisany et al. 2016;Abdel-Salam et al. 2018;Sheteiwy et al. 2021b). Studies have shown that inoculating plants roots with AMF increases EO quantity and quality of medicinal and aromatic plants such as thyme (Thymus vulgaris L.) (Amani Machiani et al. 2021a), peppermint (Mentha × piperita L.) (Ostadi et al. 2020), and holy basil (Ocimum tenuiflorum L.) (Thokchom et al. 2020).
Dragon's head (Lallemantia iberica) is a plant species from the family Lamiaceae that is highly resistant to water deficit and thus can be used in crop rotations in water-deficit regions. Dragon's head is a multi-purpose species due to its medicinal and industrial properties. The plant is used to treat cough, dysentery, and constipation, reduce fat and blood cholesterol, promote good heart and brain function, and as a tranquilizer (Rezaei-Chiyaneh et al. 2021a). The major EO constituents of dragon's head include germacrene-D, β-caryophyllene, δ-cadinene, and bicyclogermacrene (Heydari and Pirzad 2021), with the percentage extracted varying between plant organs. Dragon's head has a long history of cultivation in arid and semi-arid parts of Iran; its oil is used in the food industry, and to produce furniture polish, paint, and soap (Koohdar et al. 2018). The oil contains important unsaturated fatty acids (e.g. oleic, linoleic, and linolenic acids) and saturated fatty acids (e.g. palmitic and stearic acids) (Mohammadghasemi et al. 2021).
Chickpea is an important rainfed species of the family Fabaceae, ranked third for global production. Due to its high content of digestible proteins, carbohydrates, food fibers, vitamins, and minerals, chickpea seeds have high nutrient value (Hajjarpoor et al. 2018). Chickpea's ability to fix nitrogen is important for the soil mineral balance in agroecosystems (Gopalakrishnan et al. 2017). Chickpea can grow in nutrient-poor soils, resist drought and heat stress, and is a low-cost crop in agricultural systems in semi-arid temperate regions (Fernández-García et al. 2013).
Water shortages and low rainfall in arid and semi-arid regions have significantly reduced seed yields in recent years. In regions facing water scarcity, it makes sense to choose drought-tolerant crops that can also be grown in rainfed agriculture. No studies have investigated the effect of intercropping systems coupled with biofertilizer inoculation on the secondary metabolites in different plant organs of dragon's head, including aerial parts and seeds, under rainfed conditions in a semi-arid region. This study assesses the interactions between intercropping systems and biofertilizers on chickpea and dragon's head seed yield, dragon's head EO (in different plant organs), oil quantity and quality, and phenolic compounds.
We hypothesized that: i) AMF or bacteria inoculation as biofertilizer enhances dragon's head and chickpea productivity; ii) biofertilizer application as an eco-friendly strategy improves essential oil and fixed oil quality; iii) intercropping patterns with AMF or bacteria inoculation improve the phenolic concentration of dragon's head; iv) biofertilizer application in intercropping increases land-use efficiency.

Experimental site
The study was conducted as a factorial experiment based on a randomized complete block design with three replications in the 2019 and 2020 growing seasons at a farm (37°39′24.82″ N, 44°58′12.42″ E) in Urmia city in Western Azerbaijan province, Iran. Weather data (see Supplementary Table 1) was collected from the Iran Meteorological Organization. This region has a semi-arid climate, with a mean temperature of 12°C, annual precipitation of 390 mm, and elevation of 1,320 m above sea level. Before the study commenced, four soil samples (0-30 cm soil depth) were collected randomly across the experimental area to determine selected soil physicochemical properties (Supplementary  Table 2).
The seeds of dragon's head (landrace Shahin Dezh) and chickpea (cv. Saeed) were sown in 5 m long rows with an inter-row spacing of 40 cm. The within-row spacing of dragon's head (100 plants m -2 ) and chickpea (25 plants m -2 ) was 2.5 and 10 cm, respectively.
For the BI treatments, chickpea and dragon's head seeds were sprayed uniformly with BI solution (10 8 active bacteria g -1 ) diluted in water and dried for 1 h in the shade before sowing in the plots. The mycorrhizal inoculum (AMF) comprised a mixture of sterile sand, mycorrhizal hyphae, spores, and colonized root fragments. Each g of inoculum contained 90 living spores of Funneliformis mosseae+ Rhizophagus irregularis. The inoculum (40 g plant -1 ) was banded directly below the seeds at sowing.
Chickpea seeds were inoculated with liquid Rhizobium leguminosarum before planting. Sowing occurred on 10 March in both years. No irrigation was applied during the growing season. Weeds were removed by hand, as required. No chemical fertilizers were applied during land preparation or the growing season.

Measurements
At flowering, dragon's head was harvested from 1 m 2 in each plot to determine biological yield, EO concentration, and EO yield. At the end of the growing season on 11 July 2019 and 2 July 2020, both species were harvested from 2.4 m 2 in each plot, excluding the margins on all sides, when about 75% of the dragon's head capsule and the chickpea pods had yellowed, to calculate seed yield, EO concentration, and EO yield for dragon's head, and seed yield for chickpea.

Essential oil extraction and analysis
EO extraction used the Clevenger apparatus and an EO extractor (Rezaei-Chiyaneh et al. 2021a). For this purpose, 15 g of dragon's head seed from each plot was ground to pass through a 1 mm screen. The sieved samples were placed in a jar with 150 mL distilled water and boiled inside the Clevenger for 3 h. The extracted EO concentration was weighed (g), and the EO concentration and EO yield were calculated as follows (Nasiri 2021): The EO yield of dragon's head (kg ha -1 ) was calculated as EO concentration (%) × seed yield (Fotohi Chiyaneh et al. 2022).
Extracted EOs were dried using anhydrous sodium sulfate and stored at 4°C until analysis with a gas chromatography-mass spectrometry (GC-MS) (Agilent 7890/5975A GC/MSD) following the protocol of Faridvand et al. (2021).

Fixed oil extraction and analysis
Five grams of ground dragon's head seed from each treatment was mixed in 300 mL n-hexane to extract the fixed oil. After 6 h of extraction in a Soxhlet apparatus, the solvent was removed from the oil by rotary evaporation and stored in amber glass bottles at 4°C for later analysis by GC-MS, following the protocol of Moradzadeh et al. (2021). The oil concentration was calculated as: Oil concentration % ð Þ ¼ Extracted oil g ð Þ 5 g dragons head ground seed � 100 The oil yield of two dragon's head plants was calculated as oil concentration (%) × seed yield.

Phenolic acid extraction
Dried seeds were dissolved in 2 mL 80% MeOH and transferred to an ultrasonic bath for 30 min. The homogenates were centrifuged at 3,000 rpm for 15 min and transferred to sealed jars. The extracts were crushed through fine membrane lighters and stored at 20°C. Finally, 20 mL of the extract was injected into HPLC to separate and analyze the phenolic acid (Namazi et al. 2022).

Isolation, identification, and determination of phenolic acid quantities
Phenolic acid analysis was performed using an Agilent 1100 (HPLC), comprising a 20 μL injection loop, degasser, diode-array detector (HPLC-DAD) adjusted at 250, 272, and 310 nm, four-solvent gradient pump, octadecylsilane column, and ChemStation software for data processing. The elution process to isolate the phenolic compounds involved an initial mobile phase in a 1% solution of 10% acetonitrile and 90% acetic acid at a flow rate of 1 mL min -1 , reaching 25% acetonitrile and 75% acetic acid after 5 min, and 65% acetonitrile and 35% acetic acid after 10 min (total isolation time: 15 min) (Moradzadeh et al. 2021).

Root colonization of AMF
Young, fresh dragon's head and chickpea roots were collected from 10-20 cm soil depth to determine root colonization percentage, according to the method of Phillips and Hayman (1970). Firstly, roots were washed with water, cut into small pieces (1.5 cm long), and then washed in 10% potassium hydroxide at 90°C for 10 min. Next, the segments were placed in 2% HCl at 25°C for 10 min and stained with trypan blue (0.05%) and lactic acid (80%) for 12 h before being washed and stored in a lactic acid, glycerol, and distilled water (1:1:1 v/v/v) solution. Finally, the colonization percentage of dragon's head and chickpea roots were measured according to the method of Giovannetti and Mosse (1980).

Land equivalent ratio (LER)
The partial LER of dragon's head (LER D ) and chickpea (LER Ch ) and total LER (LER T ) were calculated as follows (Gitari et al. 2020): where Y DI and Y Ds are the seed yield of dragon's head under intercropping or sole cropping, respectively, and Y ChI and Y ChS are the seed yield of chickpea under intercropping or sole cropping, respectively.

Statistical analyses
Analysis of variance (ANOVA) using PROC mixed and mean comparisons was performed by Duncan's multiple range test (P < 0.05) using the SAS 9.4 software package (SAS Institute Inc., Cary, NC) to assess the impact of intercropping patterns and fertilizer sources on oil concentration and EO productivity of dragon's head, and yield and root colonization of dragon's head and chickpea. Intercropping pattern, bacterial fertilizer, and their interactions were considered fixed effects, while blocks were considered random effects. All graphs were drawn in MS-Excel.

Dragon's head
The ANOVA results revealed that planting pattern, fertilization source, and their interaction significantly affected root colonization, biological and seed yield, EO concentration and yield, and oil concentration and yield of dragon's head (Supplementary Table 3).

Biological yield
Sole cropping with BI fertilization produced the highest biological yield (6,890.2 kg ha -1 ) of dragon's head, followed by sole cropping with AMFfertilization (6,820.7 kg ha -1 ). Intercropping one row dragon's head + one row chickpea (1D:1Ch) without fertilization produced the lowest biological yield (3,078.6 kg ha -1 ) of dragon's head. The three intercropping patterns had 37.8% lower average biological yield than sole cropping. In addition, AMF and BI fertilization enhanced biological yield by 26.3% and 29.6%, respectively, compared with no fertilization (Figure 1a).

Essential oil concentration and essential oil yield
The intercropping systems produced higher EO concentrations in the aerial parts and seeds dragon's head than sole cropping. The 2D:1Ch intercropping pattern with BI fertilization produced the highest EO concentration in aerial parts (0.94%) and seeds (0.59%), while sole cropping without fertilization produced the lowest. In addition, AMF and BI fertilization increased EO concentrations by 25.8% and 33.9% in aerial parts and 52% and 72% in seeds, respectively, compared with no fertilization Figure 1 (c,d). The 2D:1Ch intercropping pattern with BIfertilization produced the highest EO yield in aerial parts (48.2 kg ha -1 ) and seeds (4.1 kg ha -1 ) Figure 2(a,b). The second growing year produced 10.6% higher EO yield of aerial parts than the first year (Supplementary Table 3).

Oil concentration and yield
All intercropping patterns had higher oil concentrations than sole cropping. The 2D:1Ch intercropping pattern with BIfertilization produced the highest (36.2%) oil concentration, and sole cropping without fertilization produced the lowest (31%). Sole cropping with AMF and BI fertilization produced the highest oil yields (Figure 2d). In addition, AMF and BI fertilization increased the average oil 1D:1Ch, 2D:1Ch, and 1D:2Ch, ratios of dragon's head and chickpea in the intercropping patterns) on dragon's head aerial plant essential oil yield (a), seed essential oil yield (b), seed oil content (c) and seed oil yield (d). Different lower-case letters above the bars indicate significant (P ≤ 0.05) differences. 3.98 ± 0.02 † Identification methods: MS, comparison of the mass spectrum with those of computer mass libraries Wiley, Adams, and NIST 08; RI, comparison of retention index with those reported in Adams and NIST 08; † † RI, linear retention indices on DB-5 MS column, experimentally determined using homolog series of n-alkanes; † † † fertilizer source (C, control; BI, bacteria inoculation; AMF, arbuscular mycorrhizal fungi) and cropping pattern (Ds, dragon's head sole cropping; Chs, chickpea sole cropping; 1D:1Ch, 2D:1Ch, and 1D:2Ch, ratios of dragon's head and chickpea in the intercropping patterns). 3.76 ± 0.07 † Identification methods: MS, comparison of the mass spectrum with those of the computer mass libraries Wiley, Adams, and NIST 08; RI, comparison of retention index with those reported in Adams and NIST 08; † † RI, linear retention indices on DB-5 MS column, experimentally determined using homolog series of n-alkanes; † † † fertilizer source (C, control; BI, bacteria inoculation; AMF, arbuscular mycorrhizal fungi) and cropping pattern (Ds, dragon's head sole cropping; Chs, chickpea sole cropping; 1D:1Ch, 2D:1Ch, and 1D:2Ch, ratios of dragon's head and chickpea in the intercropping patterns).

Oil composition
The main oil constituents of dragon's head included linoleic acid (42.73-55.4%), oleic acid (16.48-21.2%), linolenic acid (10.24-15.4%), and palmitic acid (11.2-16.6%). The 2D:1Ch intercropping pattern with BI fertilization produced the highest oleic and linoleic acid values, and sole cropping without fertilization produced the lowest. The 1D:2Ch intercropping pattern with AMF fertilization produced the highest linolenic acid. Sole cropping with AMF fertilization produced the highest palmitic acid concentration, and the 2D:1Ch intercropping pattern without fertilization produced the lowest. The oleic and linoleic acid concentrations increased by 15% and 6% with BI fertilization and 12% and 5% with AMF fertilization, respectively, compared with no fertilization (Table 3).

Chickpea
The ANOVA results revealed that planting pattern, fertilization source, and their interaction significantly affected chickpea root colonization and seed yield (Supplementary Table 3).

Land equivalent ratio (LER)
All intercropping patterns fertilized with BI and AMF had calculated LER values >1. The 2D:1Ch intercropping pattern with BI fertilization had the highest LER (1.15), while the 1D:1Ch intercropping pattern without fertilization had the lowest (0.78) (Figure 4). The maximum LER of 1.15 indicates that 15% more cultivation area is needed in sole cropping conditions to achieve the same yield as intercropping. In addition, the use of AMF and BI fertilization in all intercropping treatments increased the LER by 32.9% and 29.3% compared with no fertilization, respectively.

Discussion
This study demonstrated that intercropping enhanced root AMF colonization in dragon's head and chickpea. The presence of a legume species (e.g. chickpea) in intercropping patterns enhances nutrient availability through biological nitrogen fixation and soil microorganisms such as bacteria and AMF (Weisany et al. 2016;Maitra et al. 2021). Wahbi et al. (2016) noted that the presence of a legume in intercropping stimulated alkaline phosphatase activity in the soil, increasing AMF dynamics. Similarly, De Araujo Pereira et al. (2017) concluded that the presence of a legume in intercropping enhanced soil organic matter and N availability, increasing the diversity of AMF and soil bacterial communities and positively impacting root colonization. Amani Machiani et al. (2021a) reported higher AMF colonization in thyme/soybean intercropping patterns than sole cropping.
Our results showed that the various intercropping patterns had lower biological and seed yields of dragon's head and chickpea than sole cropping. That is, the intercropping patterns had higher interspecific competition for environmental resources (e.g. solar radiation, nutrition, and space) than sole cropping ). In addition, both intercropped species had lower partial plant densities than sole cropping, decreasing partial plant productivity in these cropping patterns. Song et al. (2021) noted decreased plant productivity in intercropping patterns due to higher interspecific competition between the intercropped species than intraspecific competition between plants under sole cropping. The intercropping patterns in our study produced higher total plant productivity (based on LER index) than sole cropping, indicating the advantage of these planting patterns. The highest LER (1.15) indicated that 15% more cultivation area is needed under sole cropping to achieve the same intercropping yield. Higher LER values under intercropping than sole cropping have been reported for fennel (Foeniculum vulgare L.)/common bean (Phaseolus vulgaris L.) (Rezaei-Chiyaneh et al. 2020), dragonhead (Dracocephalum moldavica)/soybean (Glycine max (L.) Merr.) , saffron-pumpkin/watermelon (Koocheki et al. 2019).
Drought stress conditions reduced plant productivity by about 30-50% due to reduced water availability and nutrient accessibility and decreasing carbon assimilation via photosynthesis (Maitra et al. 2022). Plant inoculation with bacterial biofertilizers and AMF could be an effective strategy for improving plant performance under drought stress. The results demonstrate that BI and AMF fertilization significantly enhanced plant productivity under sole cropping and intercropping conditions. In sustainable and low-input agricultural systems, soil nutrient availability plays an important role in plant performance and productivity (Duchene et al. 2017). The BI and AMF fertilization improved nutrient availability by increasing the activity of N-fixing and P-and K-solubilizing bacteria. AMF symbiosis with host plants also reduced soil acidity in the rhizosphere by releasing H + ions, increasing nutrient solubility. Amani Machiani et al. (2021b) reported that AMF inoculation of host plants enhanced macro-and micro-nutrient accessibility through the exudation of organic acids and phosphatase enzymes, enhancing plant productivity due to increased photosynthesis, increasing carbohydrate storage in plant organs for transfer (from sinks to sources) during the reproductive stage of plant growth. Similarly, Rezaei-Chiyaneh et al. (2021a) noted that AMF and biofertilizer enhanced seed yield by 19.8% and 14.8% in dragon's head and 12.4% and 26.3% in fenugreek, respectively. Essential oils (also known as volatile oils or ethereal oils) are highly volatile, aromatic, concentrated extracts from different plant organs used in many industries, including pharmaceutical and food, due to their characteristic flavor and fragrance properties and other biological activities (Bhavaniramya et al. 2019). Our results showed that the 2D:1Ch intercropping pattern with BI fertilizer produced the highest EO concentration, and the main constituents of dragon's head were germacrene-D (in aerial parts) and 1,8-Cineole (in seeds). Essential oils comprise variable mixtures of terpenoids compounds, such as monoterpenes and sesquiterpenes. Terpenes are linear or cyclic compounds comprising isoprene units. In plant cells, the isoprene concentration depends on nutrient availability and biomolecules such as ATP, NADPH, and acetyl-CoA (Ormeno and Fernandez 2012). BI fertilization likely increased nutrient availability and nitrogen fixation in chickpea, which was directly/indirectly transferred to the intercropped companion plants (dragon's head), increasing plant growth and photosynthesis. The increased carbohydrate concentration during photosynthesis increases the production of terpenes and EOs in medicinal and aromatic plants (Amani Machiani et al. 2019). In addition, Rostaei et al. (2018) noted that nutrient accessibility by plants plays an important role in the development and division of the glandular trichomes, EO channels, and secretory ducts. Vocciante et al. (2022) concluded that various mechanisms of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere increase germination and seedling growth under drought stress. Under drought stress (rainfed), where water shortage inhibits plant growth, BI application reduced adverse stress effects by increasing antioxidant activity (e.g. catalase, superoxide dismutase) (Seleiman et al. 2021) and thus decreasing reactive oxygen species (ROS) activity. In addition, the increased nutrient availability enhances photosynthesis and produces EO precursor compounds (Mohammadi et al. 2018). The EO yield of medicinal and aromatic plants positively correlated with seed yield and EO concentration. The EO yield of dragon's head plants was calculated as oil concentration (%) × seed yield. Therefore, the higher EO yield in 2D:1Ch fertilized with BI could be due to the higher EO concentration.
Our results showed that the 2D:1Ch intercropping pattern with BI fertilization had the highest oil concentration and main unsaturated fatty acids, such as oleic and linoleic acid. Oil quality directly depends on the ratio of unsaturated to saturated fatty acids (Moradzadeh et al. 2021). Thus, the oil quality of dragon's head in the 2D:1Ch intercropping pattern with BI fertilization increased due to the high concentration of unsaturated fatty acids. The improved nutrient accessibility and thus plant growth and photosynthesis would have affected the production of fatty acid precursor compounds and the activity of associated enzymes such as fatty acid synthase and acetyl-CoA carboxylase (Aid 2019). Akbari et al. (2011) also noted that biofertilizers increased the oil concentration and oil quality of sunflower by decreasing saturated fatty acids (palmitic and stearic) and increasing unsaturated fatty acids (linoleic acid and oleic acid). Other studies have reported the positive effects of intercropping systems on plant nutrient availability, oil productivity, and oil quality. For example, Rezaei-Chiyaneh et al. (2021b) reported that a 66:33 (black cumin: fenugreek) intercropping pattern with BI fertilization had the highest fixed oil concentration of black cumin (26.7%) due to increased nutrient uptake and oil quality by increasing oleic and linoleic acid concentrations.
Phenolic compounds have an aromatic benzene ring with one or more hydroxyl groups and play key roles in structural polymers (lignin), attractants (flavonoids and carotenoids), and antioxidant activity under stressful conditions (Kumar et al. 2020). This study demonstrated that the intercropping patterns under BI fertilization enhanced the concentrations of the main phenolic compounds in dragon's head, including chlorogenic acid, quercetin, and comaric acid. Fotohi  noted that the activity of enzymes (such as phenylalanine ammonia-lyase) involved in the biosynthesis of phenolic compounds depends directly on nutrient availability, especially N. Therefore, the higher phenolic compounds in chickpea/dragon's head intercropping with BI fertilization are likely due to the positive effects of biological nitrogen fixation by chickpea and transfer to companion plants, and BI fertilization increasing nutrient solubility and nutrient uptake by plants (Sinkovič and Žnidarčič 2016). Thus, chickpea/dragon's head intercropping with BI fertilizers under rainfed conditions improved plant defense mechanism systems and plant performance. Similarly, Jiménez-Gómez et al. (2020) concluded that Bacillus halotolerans biofertilizer enhanced nutrient availability and phenolic compounds in Coriandrum sativum L. plants. Khalid et al. (2017) reported that biofertilizer containing beneficial fungi and rhizospheric bacteria enhanced the phenolic and flavonoid concentrations of Spinacia oleracea L., and Salehi et al. (2019) showed that fenugreek intercropped with buckwheat had higher phenolic seed concentration than monocropping.

Conclusion
The low nutrient use efficiency of dragon's head under rainfed conditions in arid and semi-arid regions adversely affected plant productivity. Combining intercropping and biofertilizer application decreased the negative effects of stressful (drought) conditions by increasing phenolic compounds and plant performance. Among the different cropping patterns, the two rows dragon's head + one row chickpea intercropping (2D:1Ch) pattern with BI inoculation produced the highest essential oil (EO) and oil concentration in dragon's head and increased EO quality due to increased germacrene-D (in aerial parts) and 1,8-Cineole (in seeds). The fixed oil quality improved in the same treatment due to increased unsaturated fatty acid and decreased saturated fatty acid concentrations. Considering the decline in available agricultural land, especially in arid and semi-arid areas, a 2D:1Ch intercropping pattern with biofertilizer addition is a more sustainable solution for improving crop productivity, oil concentration, phenolic compounds, and EO quality under rainfed conditions than monocropping. Future research should focus on the differences between synthetic fertilizers and biofertilization in sole and intercropping systems to holistically evaluate the economic and environmental benefits of biofertilization versus synthetic fertilizer application in intercropping dragon's head and chickpea.

Disclosure statement
No potential conflict of interest was reported by the author(s).