Intercropping of Cauliflower with Lettuce Is More Effective for Sustainable Fertilizer Management and Minimizing Environmental Risks

Intercropping systems are one of the sustainable agricultural models as they play an important role in protecting soil fertility, efficient use of resources, maintaining stable yields, and reducing the effectiveness of diseases and pests. The aim of this study was to investigate the effects of intercropping (IC) cauliflower (Brassica oleracea L. var. botrytis) with leaf lettuce (Lactuca sativa L. var. crispa) on the yield and quality parameters of cauliflower and to evaluate the overall productivity of the system under different nitrogen fertilization rates (160, 200, and 240 kg N ha−1). Our results showed that the leaf chlorophyll value (SPAD), plant weight, leaf weight, head diameter, head height, head weight, and total yield of cauliflower were found to increase as the nitrogen dose increased in both the monocropping (MC) and IC systems. The most efficient nitrogen fertilizer doses for cauliflower were 234.7 kg ha−1 for MC and 176.6 kg ha−1 for IC, respectively. When the intercropping system was used the total yield (cauliflower and lettuce) was higher than the yield of cauliflower (MC) for the same total area and fertilizer amount. The land equivalent ratio (LER) values were greater than 1 in the intercropping system at all fertilization rates, which indicated that the IC system was more productive than the MC system for the same unit of land. Our findings also showed that intercropping was an effective method to increase fertilizer use efficiency and the soil organic matter, nitrogen content, plant available P, K, Mg, Zn, Fe, Mn, and Cu. In conclusion, while intercropping cauliflower with lettuce did not adversely affect the yield of cauliflower, it enabled harvesting more plants (cauliflower and lettuce) from the same land area by using the same amount of fertilizer, which makes intercropping a sustainable, economical, and ecological model that increases the land-use and fertilizer-use efficiencies.


Introduction
The amount of arable land all over the world is constantly decreasing due to increasing population and industrialization [1]. Approximately 70% more food is needed to feed the overpopulation [2]. Especially in Asia and Africa, where producers own small lands, agricultural lands are under pressure to produce for human nutrition [3]. As global food demand increases, stricter environmental protection regulations and sustainability rules are put forward to prevent agricultural expansion and deforestation [4]. Today, there is no opportunity to increase the agricultural areas, and the per capita agricultural area is decreasing due to the increase in human population.
To date, many methods such as increasing the yield with breeding techniques, using genetically modified organisms, and application of chemical fertilizers and pesticides are used in order to provide the food needed for the increasing world population. While each altitude of 1850 m above sea level. Weather data (precipitation, mean temperature and mean humidity) were obtained from the Erzurum Meteorological Station, which is five kilometers away from the experimental site. The meteorological data recorded over the two growing seasons (2014 and 2015) are shown in Figure 1. The following properties of the soil in the experimental area were determined: soil texture (clay loam), pH (7.5), EC (0.015 dS cm −1 ), total N (0.0091%), exchangeable Ca (1062.50 mg kg −1 ), Mg (42.64 mg kg −1 ), K (205.20 mg kg −1 ), Na (10.10 mg kg −1 ), plant-available P (16.56 mg kg −1 ), available Fe (4.13 mg kg −1 ), Mn (4.56 mg kg −1 ), Zn (3.45 mg kg −1 ), Cu (7.86 mg kg −1 ), and B (0.48 mg kg −1 ).

Experimental Set-Up
Cauliflower and leaf lettuce seedlings were grown separately in the greenhouse of Atatürk University. Cauliflower seeds and lettuce seeds were sown in 216-celled seedling growing trays (peat-perlite (2:1, v/v)). Cauliflower was intercropped with leaf lettuce in the field after 3-4 true leaves stage was reached (Table 1; Figure 1). Cauliflower seedlings were planted in 0.60 × 0.50 m in sole and intercropping cropping systems. In intercropping treatments, one row of leaf lettuce (within-row plant spacing 0.30 m) was planted between cauliflower rows simultaneously in separate plots. In sole planting, leaf lettuce spacing was 0.30 × 0.20 m (Figure 2). Drought stress was avoided by irrigation. Hoeing was done 3 times for weed control. There was no pesticide or herbicide application. The experiment was conducted in randomized complete block design with two factors (cropping systems and nitrogen levels) with four replicates (25 plants per replicate). Two treatment effects were studied in this research: cropping system (monocropping and intercropping) and nitrogen doses (160, 200 and 240 kg N ha −1 ). Basal fertilizer application was 200 P2O5 ha −1 and 120 kg K2O ha −1 for cauliflower + lettuce

Experimental Set-Up
Cauliflower and leaf lettuce seedlings were grown separately in the greenhouse of Atatürk University. Cauliflower seeds and lettuce seeds were sown in 216-celled seedling growing trays (peat-perlite (2:1, v/v)). Cauliflower was intercropped with leaf lettuce in the field after 3-4 true leaves stage was reached (Table 1; Figure 1). Cauliflower seedlings were planted in 0.60 × 0.50 m in sole and intercropping cropping systems. In intercropping treatments, one row of leaf lettuce (within-row plant spacing 0.30 m) was planted between cauliflower rows simultaneously in separate plots. In sole planting, leaf lettuce spacing was 0.30 × 0.20 m (Figure 2). Drought stress was avoided by irrigation. Hoeing was done 3 times for weed control. There was no pesticide or herbicide application. randomized complete block design with four replications per treatment. The first factor (main factor-cropping systems) had two levels (monocropping and intercropping), and the second one (subfactor-nitrogen levels) had three levels (160, 200 and 240 kg N ha −1 ). Before the ANOVA test, the normality test was performed. Data was subjected to analysis of variance (two-way ANOVA) to compare the effects of cropping systems, nitrogen level treatments, and interactions. Means were separated by Duncan's multiple range tests (DMRT) (p < 0.05).  The experiment was conducted in randomized complete block design with two factors (cropping systems and nitrogen levels) with four replicates (25 plants per replicate). Two treatment effects were studied in this research: cropping system (monocropping and intercropping) and nitrogen doses (160, 200 and 240 kg N ha −1 ). Basal fertilizer application was 200 P 2 O 5 ha −1 and 120 kg K 2 O ha −1 for cauliflower + lettuce intercropping, and nitrogen doses were 160, 200, and 240 kg N ha −1 . For leaf lettuce alone, basal fertilizer application was 100 kg N ha −1 , 80 P 2 O 5 ha −1 , and 120 kg K 2 O ha −1 [19]. There were seven treatments: cauliflower sole (160, 200, and 240 kg N ha −1 ), cauliflower + leaf lettuce (160, 200, and 240 kg N ha −1 ), and leaf lettuce alone. Treatments were tested with 28 plots in total.
The cauliflower plants were harvested when they reached marketable size in early August. The inner rows were used for sampling and harvest. The plants were cut at ground level to determine the yield, the head weight, the plant weight, the leaf number, the leaf weight, the stem diameter, the head diameter, and the head dry matter ratio. The plant materials were kept in an incubator at 68 • C for two days to determine dry weights. The leaf lettuce plants were harvested to determine the yield, the stem diameter, and the plant weight 30 days after transplanting. A chlorophyll meter (SPAD-502, Konica Minolta Sensing, Inc., Sakai, Japan) was used to determine the chlorophyll content in the leaves of cauliflower and leaf lettuce.

Nitrate Analysis
The method by Cataldo et al. [20] was used for nitrate analysis.

Extraction and Quantification of Vitamin C
High performance liquid chromatography (HPLC) was used for Vitamin C analysis [21].

Soil and Plant Analyses
Soil samples were air dried, crushed, and passed through a 2-mm sieve before physical and chemical analysis. The Kjeldahl method [22] was used to determine organic N while plant-available P was determined by using the sodium bicarbonate method of Olsen et al. [23]. Electrical conductivity (EC) was measured in saturation extracts according to Rhoades [24]. Soil pH was determined in 1:2 extracts, and calcium carbonate concentrations were determined according to McLean [25]. Soil organic matter (OM) was determined using the Smith-Weldon method according to Nelson and Sommers [26]. Ammonium acetate buffered at pH 7 [27] was used to determine exchangeable cations. Microelements in the soils were determined by diethylenetriaminepentaacetic acid (DTPA) extraction methods [28].

Yield
The yield of cauliflower (ton ha −1 ) was calculated by multiplying the head weight (g plant −1 ) by the number of plants in 1 ha (33,333 plants).
The yield of lettuce (ton ha −1 ) was calculated by multiplying plant weight (g plant −1 ) by the number of plants in 1 ha (166,666 plants for sole, 83,333 plants for intercropping).

Land Equivalent Ratio (LER)
The LER values were calculated as follows: LER = LA + LB = AI/AS + BI/BS, where LA and LB are the individual LERs of two crops A and B; AI is the yield of crop A in intercropping, AS is the yield of crop A in sole cropping, and LA is the ratio of AI to AS; BI is the yield of crop B in intercropping, BS is the yield of crop B in sole cropping, and LB is the ratio of BI to BS.

Statistical Analysis
The experimental design was hierarchical with respect to two factors arranged in a randomized complete block design with four replications per treatment. The first factor (main factor-cropping systems) had two levels (monocropping and intercropping), and the second one (subfactor-nitrogen levels) had three levels (160, 200 and 240 kg N ha −1 ). Before the ANOVA test, the normality test was performed. Data was subjected to analysis of variance (two-way ANOVA) to compare the effects of cropping systems, nitrogen level treatments, and interactions. Means were separated by Duncan's multiple range tests (DMRT) (p < 0.05).

Results
Growth and yield parameters of cauliflower were found to be significantly affected by nitrogen fertilization, while cropping treatments had no significant effect (p > 0.05) on these parameters (Tables 2 and 3). Nitrogen application very significantly affect the plant weight (p < 0.001) and the highest plant weight was achieved when 240 kg ha −1 nitrogen was applied in both monocropping (MC) and intercropping (IC) systems in both years. Nitrogen × cropping system interaction was found to be not significant for plant weight. When 240 kg ha −1 nitrogen was applied, two-year average plant weight values of the cauliflower for the MC and IC treatments were 1127.01 g plant −1 and 11,120.19 g plant −1 , respectively. Although plant weight slightly decreased in the IC system (by 0.61%) compared to MC for cauliflower, the IC system may still be considered more productive since the total aboveground biomass was higher in IC (1599.35 g plant −1 : cauliflower (1120.19 g plant −1 ) and lettuce (389.16 g plant −1 )) than in the MC cauliflower (1127.07 g plant −1 ) and MC lettuce (440.14 g plant −1 ) for the same total area and fertilizer amount.  Similarly, the highest leaf weight, head diameter, head height and head weight for cauliflower were obtained when 240 N kg ha −1 was applied for both MC and IC treatments (Tables 2 and 3). Overall, the highest head weights were obtained from 240 kg N ha −1 application dose in both systems and for both years. Nitrogen × cropping system interaction Sustainability 2022, 14, 7874 7 of 15 was found not significant for head weight for both years. The cauliflower head weight was used for the yield calculations and the yield values for both MC and IC treatments are given in Figure 3. For the 1st year, the highest yield (18.1 ton ha −1 ) was achieved when 240 kg N ha −1 was applied in the MC system while the lowest yield (13.7 ton ha −1 ) for cauliflower was obtained in the IC system with 160 kg N ha −1 application. On the other hand, for the 2nd year, the highest yield was achieved when 240 kg N ha −1 was applied and there was no significant difference between cropping systems (18.9 ton ha −1 for both). When two-year average yield values are considered, the regression analysis shows that when 234.7 kg N ha −1 nitrogen is applied a yield of 18.5 ton ha −1 can be achieved for the MC system and a nitrogen application of 176.6 kg N ha −1 results in a yield value of 18.2 ton ha −1 for the IC system ( Figure 4). For lettuce the yield was calculated from the plant weight. Nitrogen applications significantly affected the plant weight (p < 0.001) of leaf lettuce in intercropping system for both years ( Table 4). The highest plant weights for lettuce were obtained from the MC system for both years (542.65 g plant −1 in 1st year and 337.63 in 2nd year). Cropping methods did not significantly affect the plant weight, leaf dry matter ratio, and stem diameter. Nitrogen × cropping system interaction was not significant for these parameters. Figure 5 shows that the effect of different nitrogen dose applications and cropping methods on leaf lettuce yield were statistically significant (p < 0.001) in both years. Yield of leaf lettuce was the highest in the MC system in both years (90.5 ton ha −1 and 56.3 ton ha −1 ). Although the plant weight was found to be not affected by the cropping system (Table 4), the yield was lower in the case of IC since the number of plants per unit area decreased. However, the IC system is still an economical, ecological, and sustainable model when the total yield is considered. For the same total area and fertilizer amount (240 kg N ha −1) , two-year average yield for MC cauliflower was 18.5 ton ha −1 ; on the other hand, the total yield for the IC system was 50.7 ton ha −1 (18.2 ton ha −1 (cauliflower) + 32.5 ton ha −1 (lettuce)) (Figures 3 and 5). An even higher yield (53.4 ton ha −1 (18.2 ton ha −1 (cauliflower) + 35.2 ton ha −1 (lettuce)) could be achieved if the optimum fertilizer amount for cauliflower (176.6 kg N ha −1 ) was applied ( Figure 4). As seen in the results of the research, only 18.5 tons of cauliflower product is obtained from the area where cauliflower is grown MC, while a total of 50.7 tons of product is obtained for 32.5 tons of lettuce plant in addition to the cauliflower product with the same fertilizer application in IC. When you take into account the nitrogen fertilizer used per hectare, 18.2 tons of product is obtained with 240 kg of nitrogenous fertilizer, while an additional 35.2 tons of product is taken with the same fertilizer without any change in cauliflower yield, the use of nitrogen fertilizer causes about 2.71 times more effective use. Especially considering that nitrogen fertilizer is washed away from the soil, co-sowing and washing loss are reduced, as well as effective use of nitrogen fertilizer, resulting in a reduction in production costs. Considering the two-year soil results, the increase in soil organic matter causes an increase in the bacterial activity of the soil, and this positive effect causes an increase in the usefulness of the nutrients. As seen in the results, we can see that the biodiversity increases and the availability of nutrients (P, K, Mgi Zn, Fe, Mn, Cu) increases as it is directly proportional to the increase in the number of plants per unit area and the total amount of C.

Sole
Intercrop+160 kg N Intercrop+200 kg N Intercrop+240 kg N  Table 2 shows that chlorophyll reading value of cauliflower was significantly affected by the dose of nitrogen in both IC and MC. When 240 kg ha −1 nitrogen was applied two-year average chlorophyll values of cauliflower were 66.17 and 66.95 for MC and IC treatments, respectively. On the other hand, no significant difference was observed in the head dry matter ratio of cauliflower when different nitrogen doses or cropping system was applied (Table 3). Vitamin C content of cauliflower significantly differed between treatments (Table 3). In both MC and IC systems, the highest vitamin C amount was obtained when 200 kg N ha −1 was applied. Two-year average vitamin C contents were 110.25 mg 100 g −1 and 94.5 mg 100 g −1 when 200 kg N ha −1 nitrogen was applied in MC and IC systems, respectively.
The leaf dry matter ratio of lettuce was significantly (p < 0.001) affected by nitrogen fertilization in both years whereas the effect of cropping system was not significant ( Table 4). The highest two-year average leaf dry matter ratio (5.55%) and the lowest two-year average leaf dry matter ratio (4.40%) were obtained in the MC system and the 200 kg N ha −1 nitrogen-applied IC system, respectively. The highest amounts of vitamin C in leaf lettuce (74.33 mg 100 g −1 and 79.75 mg 100 g −1 ) were found in the IC system when 160 kg N ha −1 nitrogen fertilizer was applied for both years (Table 4). Vitamin C content decreased with the increased nitrogen doses in the IC system. Nitrogen × cropping system interaction was not significant. Increasing nitrogen dose significantly increased the chlorophyll reading value of leaf lettuce. Among all treatments, the highest two-year average SPAD value (53.78) was obtained when 240 kg N ha −1 fertilizer was applied (Table 4). Tables 5-7 show the micro-and macro-element content of cauliflower. Our findings show that both nitrogen doses and cropping systems significantly (p < 0.001) affected the N, NO 3 , P, K, and Ca contents of cauliflower. NO 3 , N, P, K, Ca, Mg, Mn, and B contents of cauliflower leaves increased with increasing nitrogen doses in both cropping systems. However, intercropping decreased the content of NO 3 , N, Ca, Mg, and Mn content of cauliflower leaves. P, K, Fe, Cu, and B content of cauliflower leaves changed based on years. The highest NO 3 , N, and Ca contents were obtained when 234.7 kg N ha −1 nitrogen was applied in MC according to regression analysis. Nitrogen *** *** *** *** *** *** *** *** Cropping *** *** *** *** *** ** *** ** N × C *** *** *** *** *** *** *** *** Nitrogen *** *** *** *** * ns ns * Cropping ** *** ns ns ns ns *** ns N × C *** *** ns ns ns ns ns ns § The mean values with the same letter within the same column are not significantly different (p > 0.05). ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Soil parameters were analyzed and presented in Table 8. The effect of different nitrogen doses and cropping methods on soil macro and micronutrients were found statistically (p < 0.001) significant for both years. According to two-year average values, organic matter, N, P, K, Ca, Mg, Zn, Fe, Mn, Cu, and B of cauliflower growing soil were the highest in IC growing medium when 176.6 kg N ha −1 was applied. Our findings showed that intercropping was an efficient method to increase the soil quality index (organic matter, N, P, K, Ca, Mg, Zn, Fe, Mn, Cu, and B contents). Land equivalent ratios (LER) were found higher than 1 in intercropping system at all nitrogen fertilizer rates ( Figure 6). Land equivalent ratios (LER) were found higher than 1 in intercropping system at all nitrogen fertilizer rates ( Figure 6).

Discussion
We have investigated the effects of nitrogen fertilization doses and cropping systems on the yield and yield parameters of cauliflower. The leaf chlorophyll value (SPAD), plant weight, leaf weight, head diameter, head height, head weight, and total yield of cauliflower were found to increase as the nitrogen dose increased in both MC and IC systems. Moreover, when intercropping system was used the total yield (cauliflower and lettuce) was higher than the yield of cauliflower (MC) for the same total area and fertilizer amount (Figures 3 and 5). Nitrogen fertilization has an important role in increasing the yield and growth parameters of cauliflower, as in other Cruciferous vegetable species. Similar to our findings, earlier studies have also reported that nitrogen fertilization significantly increased the plant growth and chlorophyll content in cauliflower [17,[30][31][32] since nitrogen plays important roles in the meristematic development, chlorophyll formation, and photosynthesis [30]. On the other hand, increasing N doses lowered the vitamin C content of cauliflower heads. The vitamin C content of plants is affected by many pre-harvest factors such as genotype, fertilization, and irrigation [33]. Since excess use of nitrogen fertilizers increases the nitrate concentration in plants, the vitamin C content decreases. A similar study conducted by Chinese cabbage [34] also reported that the lowest amount of vitamin C in cabbage heads was found when the highest dose of nitrogen was applied. Our results showed that the most efficient nitrogen fertilizer doses for cauliflower were 234.7 kg ha −1 for MC and 176.6 kg ha −1 for IC, respectively (Figure 4). Although the yield of cauliflower increases with the increase in N fertilization dose, it is crucial to find the optimum dose for the growth since high doses of nitrogen activate some physiological and pathological disorders. Our findings showed that the yield of cauliflower was not affected significantly by intercropping. Similarly, Yildirim and Güvenç [18] reported that intercropping cauliflower with vegetables such as onions, lettuce and beans did not adversely affect the yield. Other studies also stated that intercropping late maturing species with early harvesting species does not have a negative effect on the yield of either species [8,10] since the species with short vegetation are harvested before competition for resources such as light, water, and nutrients begins [35,36]. While intercropping cauliflower with lettuce did not adversely affect the yield of cauliflower, it enabled harvesting more plants (cauliflower and lettuce) from the same land area by using same amount of fertilizer, which makes intercropping a sustainable, economical, and ecological model that increases the land-use and fertilizer-use efficiencies. Nitrogen fertilization had a positive effect on micro-and macronutrient content of cauliflower (Tables 5-7). The NO 3 , N, P, K, Ca, Mg, Fe, Mn, and B content of cauliflower leaves generally increased with the increasing fertilization dose. Similarly, earlier studies reported that nitrogen fertilization improved plant and root growth enhancing the plant nutrient uptake [16,17]. On the other hand, intercropping decreased NO 3 , N, Ca, Mg, and Mn contents of cauliflower leaves and the highest amounts were achieved when 240 kg N ha −1 fertilizer was used for both years. However, since the amount of decrease in plant nutrients in cauliflower leaves did not adversely affect the growth parameters, most importantly the head weight, the yield of cauliflower was not significantly affected from intercropping (Table 3 and Figure 3).
The nitrogen rate and cropping system also had significant effect on the growth and yield parameters of leaf lettuce such as plant weight, stem diameter, vitamin C content, chlorophyll reading value, and dry matter ratio (Table 4). In intercropping systems, high nitrogen fertilization treatments negatively affected plant weight, leaf dry matter ratio, and stem diameter of leaf lettuce (Table 4). This may be due to the fact that cauliflower was more competitive than leaf lettuce at high nitrogen doses [1]. Although the plant weight of leaf lettuce was not significantly affected from intercropping, the yield of leaf lettuce was significantly reduced in intercropping system compared to sole cropping ( Figure 5), since the number of lettuce in the intercropping system (83,333 plants) was half of the sole cropping system (166,666 plants).
The land equivalent ratio (LER) values were greater than 1 in the intercropping system at all fertilization rates ( Figure 6). A LER > 1 indicates that the intercropping (IC) system is more productive than the monocropping (MC) system for the same unit of land, whereas LER < 1 indicates a non-profitable intercropping system supporting a high level of interspecific competition [37][38][39]. In previous studies, it was stated that intercropping might provide productivity and profitability by high total yields obtained per unit area under field and greenhouse conditions [8,18,40]. This can be explained by the more efficient use of available resources per unit area for different crops [41]. More efficient crop production in limited areas also increases the income of farmers [40,42,43].

Conclusions
Improving vegetable production through suitable intercrop combinations and nitrogen fertilization has not been fully explored yet. The aim of this study was to investigate the effects of intercropping cauliflower with lettuce on the yield and quality parameters of cauliflower and to evaluate the overall productivity of the system under different nitrogen fertilization rates.
The yield and growth parameters were positively affected by the increase in nitrogen doses. Our results showed that the most efficient nitrogen fertilizer rates for cauliflower were 234.7 kg ha −1 for MC and 176.6 kg ha −1 for IC, respectively. When the IC system was used, the total aboveground biomass was higher compared to the MC cauliflower system for the same total area and fertilizer amount. The land equivalent ratio (LER) values were greater than 1 in the intercropping system at all fertilization rates which indicates that the IC system was more productive than the MC system for the same unit of land.
In conclusion, while intercropping cauliflower with lettuce did not adversely affect the yield of cauliflower, it enabled harvesting more plants (cauliflower and lettuce) from the same land area by using the same amount of fertilizer, which makes intercropping a sustainable, economical, and ecological model that increases the land-use and fertilizer-use efficiencies. Intercropping methods, which are among the sustainable agricultural methods, are diversified farming methods using natural resources more effectively, thus minimizing environmental risks and improving livelihood of farmers.