Optimizing Intercropping Systems of Black Cumin (Nigella sativa L.) and Fenugreek (Trigonella foenum‐graecum L.) through Inoculation with Bacteria and Mycorrhizal Fungi

This study evaluates the effects of intercropping patterns, plant growth‐promoting rhizobacteria and arbuscular mycorrhizal fungi (AMF) on seed yield and yield components of black cumin (Nigella sativa L.) and fenugreek (Trigonella foenum‐graecum L.), as well as the essential oil and fatty acid profile of black cumin. A two‐year two‐factorial field experiment was conducted in 2015 and 2016 to investigate intercropping of black cumin and fenugreek in five ratios and biofertilizer application as AMF and bacteria. Intercropping reveals higher concentrations of nitrogen and phosphorus compared with monocropping, whereas monocropping inoculated with bacteria shows the highest seed yield of both fenugreek (151 g m−2) and black cumin (148 g m−2). Regarding the quality of black cumin, the combination of a black cumin:fenugreek‐intercropping pattern of 66:34 with bacteria fertilization is most promising, as it shows i) the maximum essential oil content, oil yield, and fixed oil content, ii) the highest contents of thymol and p‐cymene, iii) the highest content of linoleic acid, and iv) the maximum land equivalent ratio. Conclusively, bacteria fertilization and black cumin:fenugreek‐intercropping pattern of 66:34 helps improving essential oil, fixed oil quality, and quantity of black cumin, thus creating a more sustainable cultivation system for black cumin and fenugreek.

small-scale farmers in semiarid regions where plant production is limited by lack of water inputs, water retention, and low organic matter content. [30] The literature on the interactive effects of intercropping and biofertilizers in black cumin's yield and quality is scant. Therefore, we hypothesized that: i) all intercropping ratios can increase the medicinal properties and the fatty acid profile of black cumin compared with monocropping, ii) application of biofertilizers in intercropping system will improve seed yield of black cumin and fenugreek, and iii) combined application of biofertilizers in intercropping systems will increase N and P uptake and the land equivalent ratio (LER).

Experimental Site and Weather Condition
A two-year field trial was conducted in 2015 and 2016 growing seasons at the research farm of Naqadeh, West Azerbaijan Province, Iran (36°57″00.0″N, 45°24″00.0″E, 1330 masl). Soil samples were collected from 0 to 30 cm depth, and physical and chemical characteristics were measured. Soil type was silty clay with average pH of 7.9, organic carbon content of 9.5 g kg -1 , EC of 0.36 dS m -1 , total N of 0.8 g kg -1 , available P of 10.45 mg kg -1 , and available K of 241.22 mg kg -1 . Weather data were obtained from the Iran Meteorological Organization (IRIMO) ( Table 1).

Plant Materials and Cultural Management Practices
Before cultivation, the seeds of both species were inoculated with phosphate-solubilizing bacteria P. agglomerans and P. putida plus N-fixing bacteria A. vinelandii in the form of powder at a rate of 100 g ha -1 (Zist Fanavar Sabz Company, Iran). The bacterial population was 5 × 10 9 colony forming units (CFU) g -1 of beneficial bacteria. Bacterial fertilizer powder was mixed with water and uniformly sprayed to cover the seed, and then seeds were air-dried. The AMF inoculum was obtained from Iran Soil and Water Research Center (Tehran, Iran). At planting, 20 g of inoculum containing ≈4000 spores of the AMF mix of F. mosseae and R. irregularis were poured into each planting hole. Each gram of inoculum media contained 200 living spores of F. mosseae or R. irregularis. The origin of mycorrhizal fungi was from the soils of Tabriz plain of Iran.
No chemical fertilizers were used in this study. The seeds of BC and F were each sown at a rate of 33.3 seeds m -2 by the furrow method with inter and intrarow spacing of 40 and 7.5 cm, respectively, on March 20, 2014, andMarch 22, 2015. The rows were 4 m long for both species. Plots and blocks were separated by a buffer space of 1 and 3 m, respectively. The first irrigation was done immediately after planting to facilitate the emergence of the seedlings, and the subsequent irrigations were applied as per the climatic conditions and plant demand every 7 d until the end of the growing season. Weed infestation in this study was manually controlled.

Crop Sampling for Determination of Seed Yield and Yield Components
The black cumin and fenugreek plants were hand harvested from a 4.8 m -2 central area (i.e., 3 m length of four central rows) at each plot to eliminate the border effect July 5 and August 25, 2015 and July 8 and August 27, 2016, respectively. At harvest, the follicles of the black cumin and the pods of the fenugreek were yellow, corresponding to typical harvesting date for each crop. Harvested seeds were dried at room temperature to reach 14% moisture content.
To determine yield components for each crop, ten plants were randomly selected at each plot. For fenugreek, measurements included plant height, number of pods per plant, number of seeds per pod, and 1000-seed weight. The measured traits for the black cumin were plant height, number of follicles per plant, number of seeds per follicle, and 1000-seed weight.

Plant Nutrient Analysis
Plant material samples were digested following the method proposed by Jones and Case. [31] Following harvest, seed samples of both species were analyzed for macro-and micronutrients content. The Kjeldahl method was used to determine the N content. [32] The concentration of P was determined by the yellow method, in which vanadate-molybdate is used as an indicator. [33] Phosphorus content was measured at 470 nm using a spectrophotometer.

Fixed Oil Isolation and Analysis
The fixed oil content of the black cumin seeds was extracted according to the AOCS (1993) method. The samples were first milled and powdered at 70 °C, and then 10 g subsample was separated after 24 h, and was immersed in Soxhlet with 300 CC of diethyl ether solution. After 6 h, the desired solvent was separated from the oil by rotary. Then, the oil was stored in amber glass bottles to isolate and identify the composition. The fixed oil of black cumin was analyzed using gas chromatographymass spectrometry (GC-MS) following previously reported methods by Rezaei-Chiyaneh et al. [34]

Essential Oil Extraction and Analysis
The EO extraction was performed by water distillation. To this end, 15 g of BC seed was weighed from each plot and boiled for 3 h in a Clevenger apparatus after briefly milling in 150 mL of water to extract its EO. Then, the content of the EO was calculated by weighing. Following, the EO content and EO yield were calculated as follows [19] EO yield of black cumin (g m −2 ) = Seed yield (g m −2 ) × EO content (%) Gas chromatography-mass spectrometry analysis was done using an Agilent 7890/5975C (Santa Clara, CA) GC/MSD. For separation of EO components, and HP-5 MS capillary column (5% phenyl methyl polysiloxane, 30 m length, 0.25 mm i.d., 0.25 µm film thickness) was used. The following oven temperature was applied: 3 min at 80 °C, subsequently 8 °C min −1 to 180 °C, held for 10 min at 180 °C. Helium was used as carrier gas at a flow rate of 1 mL min -1 . The sample was injected (1 µL) in split mode (ratio, 1:50). EI mode was 70 Ev. The mass range was set to be from 40 to 550 m/z. The components were recognized by comparing the calculated Kovats retention indices (RIs), calculated with respect to a mixture of n-alkane series (C8-C30, Supelco, Bellefonte, CA), and mass spectra. [35] GC-FID analysis was done by an Agilent 7890 A instrument. The separation was performed in an HP-5 capillary column. The analytical conditions were the same as above. Quantification methods were the same as those reported in previous papers. [18,34,36]

Root Colonization
Root colonization percentage was determined using ten plants from each experimental plot. Plants were carefully uprooted, then roots were rinsed with distilled water, cleared in 10% KOH, rinsed with water again, acidified with 1% HCl, and stained in 0.05% Trypan Blue in lacto-glycerol. [37] Mycorrhizal colonization was assessed using the grid-line intersection method described by Giovannetti and Mosse. [38]

Statistical Analysis
Analysis of variance (ANOVA) was performed using PROC Mixed procedures of SAS version 9.3 (SAS Institute Inc., Cary, NC). The fertilizers application, cropping ratio, and year were considered as fixed effects, whereas blocks were considered random. Mean comparisons for each trait were performed using Duncan's multiple range test at the P < 0.05 level.

Plant Performance of Fenugreek
The ANOVA showed that the main effects of cropping patterns and biofertilizers were significant (P < 0.01) on all recorded traits (plant height, number of pods per plant, number of seeds per pod, 1000-seed weight, and seed yield) (Table S1, Supporting Information). However, the interaction of cropping patterns and biofertilizer sources was significant for all traits, except for number of seeds per pod and 1000-seed weight (Table S1, Supporting Information). Means comparison indicated that the highest plant height (54.3 cm) was related to the intercropping pattern of 66BC:F34 inoculated with the bacteria (Figure 1A). In addition, highest number of pods per fenugreek plant (19.2) was obtained from bacteria-applied monocropping ( Figure 1B). The maximum 1000-seed weight (8.3 g) and seeds per pod (10.5) were obtained from the bacteria fertilizer, respectively. The lowest mentioned attributes were achieved in monocropping without biofertilizer consumption ( Table 2). On the other hand, the highest seed yield (150 g m -2 ) was produced by a monocropping fertilized with bacteria, whereas that had no significant difference with mycorrhiza-inoculated monocropping system. However, the lowest seed yield (68 g m -2 ) was related to the intercropping pattern of 66BC:34F without biofertilizer (control) ( Figure 1C). Furthermore, the application of AMF and bacteria increased the seed yield by 19.0% and 30.2% compared with control, respectively. In addition, the seed yield in 2016 was 9.6% greater than that in 2015 (Table S3, Supporting Information).

Plant Performance
All traits of black cumin (plant height, number of follicles per plant, number of seeds per follicle, 1000-seed weight, seed yield, EOc, EO yield, fixed oil content, and oil yield) were influenced by different planting ratios and biofertilizers (Table S2, Supporting Information). In addition, the interaction of planting pattern and biofertilizers was significant for number of follicles per plant, seed yield, EOc, EO yield, fixed oil content, and oil yield (Table S2, Supporting Information).
Means comparison disclosed that the highest plant height (54.7 cm), seeds per follicle (30.4 cm), and highest 1000-seed weight (2.7 g) were related to the black cumin monocropping, respectively ( Table 3). In addition, compared to the control conditions, the application of biofertilizer improved the mentioned traits (Table 3). On the other hand, the highest number of follicles per plant (23.2) was obtained from the monocropping of black cumin fertilized with bacteria fertilization ( Figure 2B).
Besides, the highest seed yield (148 g m -2 ) was recorded from the monocropping fertilized with bacteria biofertilizers, but the latter treatment did not differ significantly from the mycorrhizainoculated monocropping system ( Figure 2A). Furthermore, inoculation of bacteria fertilization and AMF increased the seed yield by 27.9% and 19.4% compared with control, respectively. Finally, the seed yield of black cumin was 5.8% greater in 2016 compared with 2015, respectively (Table S3, Supporting Information).

Essential Oil Concentration and Yield
The black cumin EO content and EO yield in intercropping were greater than monocropping. It was found that the highest EO content (1.3%) and EO yield (1.7 g m -2 ) were recorded in the intercropping pattern of 66BC:34F fertilized with bacteria fertilization ( Figure 2C,D). Moreover, the lowest EO content (0.9%) and EO yield (0.8 g m -2 ) were obtained from a monocropping without fertilizer consumption ( Figure 2C,D). In addition, bacteria fertilization and AMF enhanced EO content of black cumin up to 14.7% and 10.8% compared with control, respectively. Furthermore, inoculation of bacteria fertilization and AMF increased the EO yield by 48.4% and 32.6% compared with control, respectively. Also, the EO yield in 2016 was 3.4% higher than that in 2015, respectively (Table S3, Supporting Information).

Fixed Oil Content and Oil Yield
The highest fixed oil content (26.7%) was obtained from the intercropping pattern of 66BC:33F fertilized with the bacteria biofertilizer. However, the lowest fixed oil content was observed in the black cumin monocropping without the application of fertilizer, which was 8.3% higher than that of the monocropping under without the application of biofertilizers ( Figure 3A). Also, the average fixed oil content in intercropping was 3.7% greater than the monocropping. Besides, bacteria fertilization and AMF enhanced fixed oil content of black cumin up to 2.98% and 1.80% compared with control, respectively.
On the other hand, the highest oil yield (52.6 g m -2 ) was related to the monocropping black cumin fertilized with bacteria fertilization. The lowest (26.4 g m -2 ) was obtained from the cropping ratio of 33BC:66F without the application of fertilizers ( Figure 3B). Furthermore, the application of bacteria fertilization and AMF increased the oil yield by 31.9 and 21.7 compared with control, respectively. In addition, the oil yield in 2016 was 4.6% higher than that in 2015, respectively (Table S3, Supporting Information).

Oil Compositions
The main fatty acids in black cumin oil included unsaturated fatty oleic acid (21.0-22.9%), linoleic acid (47.7-60.9%), and saturated fatty palmitic acid (8.8-15.1%), stearic acid (2.0-3.4%), and behenic acid (2.0-3.4%). According to Table 5, the highest content of oleic acid was obtained with the cropping ratio of 33BC:64F with inoculation of AMF and the highest linoleic acid were related to the intercropping pattern of 66BC:34F fertilized with bacteria fertilization, but the highest content of palmitic acid and stearic acid was obtained from the monocropping system that was not fertilized with biofertilizers. In addition, the maximum contents of behenic acid were observed in the intercropping pattern of 33BC:64F inoculated with AMF. Also, the average oleic acid and linoleic acid in intercropping were 3.4% and 8.1% greater than the monocropping. Besides, AMF and bacteria fertilization enhanced oleic acid and linoleic of black cumin up to 2.3-12.1% and 4.6-19.9% compared with control, respectively (Table 5).

Nutrient Content of Black Cumin
The N and P contents in both plants were influenced by cropping pattern and biofertilizers (Tables S1 and S2, Supporting Information). Also, the interaction of biofertilizer inoculation and intercropping ratio had a significant effect on N and P content  ( Table S2, Supporting Information). The results indicated that the content of both elements in different intercropping patterns after inoculated with AMF and bacteria fertilization were higher in seeds of both plants than in monocropping without biofertilizer consumption. When compared with AMF, the bacteria application strongly increased the mentioned nutrients content (p < 0.05). The highest N and P content of black cumin was observed in the intercropping pattern of 66BC:34F fertilized with bacteria fertilization (Figure 4A,C). The lowest contents of N and P in both plants were achieved in monocropping without biofertilizer consumption ( Figure 4A,C). Also, the average N and P content in intercropping was 11.0% and 19.2% higher than the monocropping of black cumin, respectively.

Nutrient Content of Fenugreek
The results indicated that the content of N and P in different intercropping ratio after biofertilization application was higher in seed of fenugreek than in monocropping without biofertilizer consumption. The highest increment level of N and P nutrients was observed in the intercropping pattern of 34BC:66F after use of bacteria ( Figure 4B,D). However, there were no significant differences in terms of P content between the intercropping pattern of 34BC:66F with AMF and bacteria application. The lowest content of N and P was achieved in monocropping without biofertilizer consumption ( Figure 4B,D). Furthermore, the average N and P, content of fenugreek in intercropping was 9.48% and 12.70% higher than the monocropping of fenugreek, respectively.

Root Colonization
In fenugreek, the highest root colonization (75.16%) was obtained from the plants inoculated with AMF in the intercropping pattern of 34BC:66F ( Figure 5A). Furthermore, the application of AMF and bacteria increased the root colonization by 83.51% and 25.56% compared with control, respectively. In black cumin, the highest root colonization (60.66%) was recorded in the intercropping pattern of 66BC:34F treated with AMF ( Figure 5B). Furthermore, the application of AMF and bacteria fertilization increased the root colonization by 80.99% and 36.95% compared with control, respectively. In addition, the average root colonization in intercropping was 19.78% and 30.95% higher than the monocropping of fenugreek and black cumin, respectively. But the lowest root colonization of both plants was observed in monocropping without the application

LER
The highest partial LER of the black cumin (0.84) and fenugreek (0.78) was obtained from the intercropping pattern of 66BC:34F and 33BC:64F treated with bacteria fertilization, respectability. The highest (1.44) and the lowest (1.20) total LER was obtained from the intercropping pattern of 66BC:34F inoculated with bacteria fertilization and unfertilized 33BC:64F intercropping ratio, respectively (Figure 6).

Plant Performance
It seems that the decrease in plant height of fenugreek in intercropping could be because of the competition with black cumin for water, minerals, solar radiation, and space, which reduced the utilization of environmental resources and subsequently reduced its height in intercropping system. On the other hand, one reason for the increase in the plant height of black cumin in the intercropping probably is because of the light competition with fenugreek, which led to increased light absorption by black cumin, which ultimately increased the plant height and also representing that black cumin has been the dominant crop in the intercropping patterns and was benefited from intercropping compared with fenugreek. [20,40] Different mechanisms have been proposed for the effect of PGPR and AMF on the growth characteristics of plants. PGPR and AMF affect the uptake of macro-and microelements and enhance the production of plant growth hormones such as gibberellin (effect on longitudinal cell growth, especially on stem internodes), auxin and cytokinin (effect on cell division), which are responsible for increasing plant growth. [41] Numerous reports have pointed out the positive effects of PGPR and AMF on height of different plants. [42,43] The higher yield and yield components in monocropping could be because of the reduction of interspecific competition in monocropping, which resulted in an increase in the seed yield of both plants compared to other different intercropping ratios. [44] On the other hand, the decrease in the yield components and seed yield of both plants in intercropping system can be attributed to the more excellent synchronization of the black cumin growth with fenugreek, which has resulted in more interspecific competition in different       intercropping ratio compared to the intraspecific competition in monocropping. [45] Also, as the plant ratios increased, the yield component decreased because of the reduction of space required for growth and subsequent increase of interspecific competition compared to intraspecific competition between the two species, which resulted in a decrease in seed yield. [19] It also seems that in intercropping with increasing plant ratios, the other plant has less access to environmental factors (light, nutrients, and moisture) and eventually transfers less photosynthate production to the seed, which leads to a decrease in the yield components. [46] Because black cumin is considered as the main plant species in this study (EO yield and quality), the intercropping with higher black cumin proportion (66BC:34F) is considered as the best performing one. However, the higher LER (1. 2-1.44) shows that all intercropping systems (50 BC:50F, 66BC:34F, 34BC:66F) allow significantly more efficient agricultural production than monocropping.
The results of this study showed that the yield and yield components significantly enhanced with the application of biofertilizers. It seems that inoculation with these biofertilizers because of enhancing nutrients availability, which is an effective factor in stimulating plant growth and photosynthesis, improves the conditions for growth, and consequently increased yield components and seed yield of both species. [47] However, it can be concluded that bacterial treatments compared to fungal treatments lead to a positive and significant effect on the yield by bringing about a proper balance between N and P and other microelements. [48] Previous research indicated that the PGPR and AMF increase yield and yield components of plants by increasing root growth and increasing plant access to nutrients and water. [49,50]

EO Content, EO Yield, and Compositions of Black Cumin
As N is one of the elements that affect the activity of photosynthetic enzymes in plants, any factor that increases N absorption can eventually lead to an increase in plant's photosynthesis, [51] which can lead to an increase in EO production as well. [52] PGPRs and AMF increase EO of medicinal plants by increasing plant access to important nutrients such as N, P, and micronutrients (iron, zinc, and Cu). [53] Therefore, inoculation with these fertilizers owing to improving availability of nutrients, which is an effective factor in stimulating plant growth and photosynthesis, improves the conditions for growth, photosynthate production, and consequently increased quantitative and qualitative production of the EOs of medicinal plant. [54,55] Vafadar-Yengeje et al. [56] concluded that the Moldavian balm-faba bean (Vicia faba L.) intercropping increased the Moldavian balm EO quality by enhancing the amount of geraniol and geranyl acetate compared with the sole cropping system. Rezaei-Chiyaneh et al. [18] reported that PGPR application in the intercropping system improved the EO quality and quantity of fennel.

Seed Fixed Oil Content, Oil Yield, and Compositions of Black Cumin
It seems that suitable conditions for the growth of black cumin plants such as optimum use of nutrients available in the intercropping pattern of 66BC:33F and better light distribution in the total canopy will improve growth and photosynthesis. Consequently, it leads to an increase in the fixed oil content and oil compositions in intercropping compared to monocropping. Moreover, the use of biofertilizers improved soil microbial activity and root system development and improved access to nutrient absorption and consequently increased fixed oil content. [57] Combined consumption increased biological fixation of N, the solubility of immobilized phosphate, a decrease in soil pH, and the production of various hormones (such as cytokinin, auxin, biotin, and pantothenic acid) because of the synergistic effects of bacteria (azotobacter and pseudomonas). In this way, intercropping stimulates nutrient absorption and improves both quality and quantity of the fixed oil of the black seeds by affecting photosynthetic processes. [58,59] These results agree with the findings of Saeidi et al. [60] in safflower-faba bean intercropping, and Rezaei-Chiyaneh et al. [ 18 ] in fennel-common bean intercropping under application of biofertilizer.

Nutrients
Obtained results demonstrated that the concentrations of nutrients in the intercropping system inoculated with AMF and bacterial biofertilizer were higher than monocropping without application of biofertilizer. Arbuscular mycorrhizal fungi dissolve immobile elements and applicable to the host plant by improving their root uptake, releasing organic acids, and acidifying the rhizosphere environment and biochemical properties of the soil. [61] Besides, AMF have a profound effect on the root physiology of the plant, which activates glutamine synthetase, arginase, and urease enzyme leading to an increase in the content of nutrients concentrations in the plants. [62] On the other hand, the increase in nutrients uptake is related to the improvement of root uptake through the infiltration of the fungal mycelium into the soil, followed by plant access to more nutrients from the soil. [63]  The flow rate of P into the mycorrhizal plant is 3-6 times higher than in non-mycorrhizal plants. [64] The increasing rate of P uptake by the host plant is because of the presence of mycorrhizal hyphae within the epidermis of the plant, which provides a large surface area for the transfer of the nutrients, especially P to the host plant. [65] Besides, the production and secretion of phosphatase enzyme by hyphae of AMF causes insoluble and stabilized phosphate in the soil to transform to soluble form and be absorbed by the root. Moreover, AMF may increase nodulation and N fixation in legumes by increasing P uptake in these plants. [47] Nitrogen-fixing bacteria can improve N availability to plants by a process of biological N fixation, and phosphate-solubilizing bacteria dissolve insoluble forms of phosphate by releasing some organic acids. As a result, the absorption of nutrients by the plant increases. These results agree with the findings of Weisany et al. [53] and Ingraffia et al. [66] who investigated AMF inoculation in dill (Anethum graveolens L.)common bean and wheat-faba bean intercropping, respectively.

Root Colonization
Based on the results, the highest root colonization of both plants was obtained from the intercropping systems when they were inoculated with AMF. The high root colonization in the intercropping system compared to monocropping under the use of biological fertilizers might be because of higher greener cover, adequate moisture, and increasing soil biological activities. [67,68] Furthermore, differences in the root system, root depth, and root biomass of two plants, root exudates, and the availability of nutrients provide favorable conditions for root colonization. [69] In addition, inoculation with biofertilizers, especially, induces the creation of a more extensive network of root fungi hyphae and causes root growth along with the increase in root colonization percentage. Hassan et al. [70] reported that the use of plant growth-promoting bacteria plays an important role in root colonization through root exudates such as amino acids, monosaccharides, and organic acids. In agreement with our results, Rezaei Chiyaneh et al., [71] in the intercropping of isabgol (Plantago ovata) and lentil (Lens culinaris) and Ingraffia et al., [66] in the intercropping of wheat/faba bean, reported that the inoculation with AMF increased root colonization.

LER
Our results indicated that total LER was greater than 1 in all treatments. Therefore, it can be concluded that the intercropping system performed better than monocropping. The higher LER of the intercropping system can be related to the correct arrangement and supplementary use of nutrients, water, and radiation by the components of the intercropping system. [72] Therefore, these conditions improved the growth and yield of both species, and LER increased compared with the plant's monocropping. These results agree with the findings of Fallah et al. [52] in dragonhead-soybean, Koocheki et al. [73] in saffron (Crocus sativus L.)-pumpkin (Cucurbita pepo L.)-watermelon (Citrullus lanatus L.) and Rezaei Chiyaneh et al. [18] in fennelcommon bean intercropping.

Conclusion
A combination of PGPR and intercropping pattern of 66BC:33F is the recommended treatment to improve LER and nutrient uptake. This is because both seed yield and seed oil quality of intercropped black cumin and fenugreek can be improved through inoculation with PGPR and AMF. This improved overall productivity not only provides economic benefits for growers but also helps to better contribute to SDG 12 (sustainable production) because of the improved LER. Against this backdrop, it can be further assumed that the increased species diversity of intercropped black cumin and fenugreek inoculated with PGPR and AMF also helps to better contribute to SDG 15 (life on land) and 13 (climate action) because of habitat diversification and an improved response diversity. Future research should focus on differences between synthetic fertilizers and biofertilizers in sole and intercropping systems to holistically evaluate the economic and environmental benefits of biofertilization versus synthetic fertilizer application in intercropping black cumin and fenugreek.