The trial was conducted over two growing seasons (2021 and 2022) in an experimental strawberry field managed by the Laimburg Research Centre and located in the municipality of Martell (46° 33′ 30.618^′′ N; 10° 46′ 53.649^′′ E; 1,361 m a.s.l.) in Alto Adige/South Tyrol, Italy. The Martell Valley, historically renowned for berry production, is a side valley of the Venosta Valley and is characterized by a typical alpine-mountain climate. The Martell Valley is part of the Stelvio National Park, which is listed in the Official List of Protected Natural Areas (EUAP), established under Italian Law 394/1991 (Framework Law on Italian Protected Areas).

The transplanting of cold-stored strawberry plants in spring (especially in May) in soil conditions is currently the most popular cultivation strategy adopted by farmers in the valley. Flowering begins in June (about a month after transplanting) and fruiting in July–August, depending on the cultivars and weather conditions. Some farmers opt for a second harvest the following year using the same plants. In this case, the harvest will occur earlier than in the first year, and precisely in late June-July.

Meteorological trends during the growing seasons (from May to August 2021 and 2022) were recorded by an iMETOS^® weather station using the cloud platform “Field-Climate” (Pessl Instruments, Weiz, Austria) and the data are reported in Table 1.

The soil properties of the 0–20 cm layer were as follows: humic loamy sand, pH = 5.1, no free carbonate, organic carbon expressed as humus of 7.3%, phosphorus = 5.0 mg 100 g⁻¹, potassium = 8.0 mg 100 g⁻¹, and magnesium = 18.0 mg 100 g⁻¹. For P and K, the plant-available fraction was determined with Method (ÖNORM L 1087:2019 A.5, 2019). Field preparation took place in April with the incorporation of soil amendments (e.g., manure from local livestock farms, the application dosage was 20.0 t ha⁻¹). Tillage was performed at the beginning of May 2021, followed by the formation of raised beds and covering with white plastic mulch film. The beds were spaced 1.1 m apart (distance measured from the centerline of the beds) with a length of 22.0 m. Poly-covered high tunnels with a structure of stainless-steel rods were built (22.0 m length, 5.5 m width, and 3.0 m height). Given the width of 5.5 m, five beds per tunnel were covered. Each bed consisted of a staggered double row with strawberry plants 0.3 m apart in each row and 0.2 m between rows, in order to reach 60,000 plants ha⁻¹ (an average plant density as indicated by Soppelsa et al., 2023).

The main crop represented by strawberry plants (Fragaria × ananassa Duch.) cv. Roxana (frigo plants A++ from the nursery: Geoplant Vivai s.r.l. Soc. Agr., Italy) were transplanted on the 1st of June 2021. Roxana is a rustic variety (e.g., low susceptibility to leaf pathogens) and performs particularly well in an organic farming system. Strawberry plants were subjected to different intercropping combinations with herbs (companion crops) such as Allium schoenoprasum L. (chives), Calendula officinalis L. var. Daisy Golden (marigold), Mentha x piperita L. var. Multimentha (peppermint), Mentha suaveolens Ehrh. (strawberry mint), Salvia officinalis L. var. Extrakta (common sage) which were planted within the double row (strawberry-herb combination ratio of 2:1) (Figure 1). A. schoenoprasum, C. officinalis, and S. officinalis were sown in a greenhouse at Laimburg Research Centre in March 2021. A standard substrate was used, and plants were transplanted about 4 weeks after sowing. Plantlets were transplanted again after 4 weeks in multipots with a diameter of 10 cm. Peppermint was propagated from root stolons and the plantlets were transplanted in multipots (10 cm diameter). Plantlets of M. suaveolens were purchased from a local nursery. The aromatic plants were planted in the field at the same time as the strawberry plants. Considering the plant life cycle, C. officinalis is an annual plant and was therefore replanted during the second cropping year (mid-May). Plants were managed in the same way in terms of fertilization and watering. No pesticides were used throughout the experiment. The experimental design was organized as a completely randomized block design. Each intercropping combination was replicated six times. Each replicate represented an experimental unit, consisting of 20 strawberry plants (main crop) and 10 plants (companion crop) (Supplementary Figure S1). The bed was subdivided into three experimental units (each 6.0 m long, separated by 1.0 m buffer zone). Monocropped crops were also included in the cropping system design (also 6 replicates). This means that strawberry plants were planted staggered in double rows, while complementary crops in single rows.

Strawberry plant growth as affected by intercropping regime was determined at the end of the second cropping year by randomly selecting four plants per replicate. Plants in monoculture were also collected. Each selected plant was separated into roots and aerial part and weighted fresh (g fresh weight (FW) plant⁻¹). Then, plants were put in an oven (ED 56, Binder GmbH, Tuttlingen, Germany) at 65 °C until they reached a stable weight, and the dry mass was recorded (g dry weight (DW) plant⁻¹). This evaluation was not carried out in the first year as we wanted to preserve the plots for the following year.

Ripe strawberry fruits (uniformly red) were harvested every 3 days during the period from the end of July to mid-August 2021 (first harvest year) and throughout the month of July 2022 (second harvest year). Data were expressed as total production per plant (g fruit plant⁻¹), calculated by dividing the harvested total fruit weight by the number of plants (considered 20 plants per experimental unit). The plant organs of companion crops with significant commercial interest are aerial parts including stems, leaves and flowers. Marigold flowers were taken starting from July to August at regular intervals (every 5 days) for a total of 12 times per year. The other companion crops were harvested at pre-flowering stage in late July and late August, precisely on 26th July, 30th August 2021, and 21st July, 30th August 2022. Only for chives a further harvest was carried out on 9th June 2022.

Strawberries were sampled at two intermediate picking times for each harvest year. Twenty healthy fruits per replicate were analyzed to determine parameters such as: -Flesh firmness, expressed with the Durofel index (ID) which represents the elasticity of the skin of the fruit (Agrosta^® Winterwood instrument, Agrosta Sàrl, Serqueux, France); -Total soluble solids (°Brix), determined with a refractometer (RFM840, Bellingham-Stanley Ltd., Kent, UK); -Titratable acidity (g L⁻¹ of citric acid), measured with a titrator (Flash Automatic Titrator, Steroglass, Perugia, Italy) by titrating strawberry pulp to pH 8.2 using 0.1 M NaOH; -External fruit color, assessed with a colorimeter (CR-400, Konica Minolta, Tokyo, Japan) by measuring the same fruits at three different positions around the equatorial side of each fruit. The colorimetric coordinates (L^*, a^*, b^*) were used to calculate the color index (Equation 1; according to Tessmer et al., 2016).

The nutrient content analyses were carried out on samples of stems, leaves, flowers and fruits, randomly collected from the main and companion crop plants during the central harvest phase of each year. The dried samples were ground into fine powder and homogenized for elemental characterization. Nitrogen content was determined with an elementary analyzer method according Dumas DIN EN ISO_16634_1:2009 (2009) (LECO Mod. Truspec) and the other macro (P, K, Ca, Mg, S) and microelements (Fe, Cu, B, Zn, Mn) were analyzed with microwave-assisted acid digestion [EPA 3052 1996 (1996); Milestone Mod. UltraWave] using the inductively coupled plasma optical emission spectrometry [ICP-OES; EPA 6010D 2014 (2014); Agilent Model 720].

The Land Equivalent Ratio (LER) indicates the relative area of the monoculture crops required to produce an equivalent yield in an intercropping system. As proposed by Mead and Willey (1980), LER was assessed as indicated in Equation 2.

Where “Y” indicates the individual crop yield, “I” the crop production in intercropping, “M” the production in monocropping, “f ” the target crop (i.e., strawberry), “k” the companion crop (i.e., chives, marigold, mint, or sage). An LER of 1 indicates that the intercropped crops have similar production level whether they are intercropped or monocultured. If the LER is greater than 1, it means that the intercrop needs less land area than the monoculture to produce equal yields of the different products. On the contrary, if lower than 1, the intercropping system has a worsening effect on the crops and pure cultivation of the respective crops should be preferred (Bybee-Finley and Ryan, 2018).

As duration of the land occupation by a cropping system is not considered in the LER calculation, Hiebsch and McCollum (1987) proposed an Area Time Equivalency Ratio (ATER) in which the time factor of crops was included (Equation 3).

Where t_f and t_k refer to the period (days) between planting and harvesting for strawberries (90 days) and herbs (120 days), respectively. T indicates the duration of the whole intercropping system (120 days is the duration period of an intercropping combination per year).

According to Mason et al. (1986), the Land Utilization Efficiency (LUE) provides a more accurate assessment than LER and ATER taken individually, because they may lead to an over- or underestimation of the cropping system, respectively. LUE was computed as shown in Equation 4.

The System Productivity Index (SPI) allows the yield of complementary crop to be standardized in terms of the main crop, and to identify the crop combinations that use resources more efficiently (Odo, 1991). SPI was calculated by the following Equation 5 and expressed as megagrams per hectare.

Percentage Yield Difference (PYD) is related to the yield difference between a monocrop (100%) and an intercropping system (expressed in percentage) (Afe and Atanda, 2014). It presumes that yield reduction of one crop should be compensated by an increase in yield of the other crop. The lower the PYD value, the more efficient the intercropping system, and vice versa. Equation 6 was proposed by Afe and Atanda (2014).

Aggressivity (A) was proposed by McGilchrist (1965) as a measure of how much the relative yield of the main crop in the mixture was greater than that of a companion crop. This competitive index was computed as shown in Equations 7a and 7b (Willey, 1979).

Where Z_f indicates the planting proportion (%) of strawberry crop (67%) in an intercropping system whereas Z_k is the proportion of companion crop (33%). The value of aggressivity (A) zero means both crops are equally competitive. A positive value of A indicates the dominant crop, and a negative value the dominated one.

Competition Ratio (CR) is an indicator of competitive ability of intercropping components. It denotes the number of times by which one component crop is more competitive than the other, as expressed in Equations 8a and 8b (Willey and Rao, 1980).

If the value CR_f is < 1, there is a positive benefit of combining intercrops, indicating that the main crop can be cultivated in combination with the companion crop. If CR_f > 1, the competitive ability of the main crop is higher than that of the companion crop in the mixture.

The index named Actual Yield Loss (AYL) proposed by Banik (1996) represents the proportionate yield loss or gain of intercrops in comparison to the respective monoculture. Compared to the LER, the AYL considers the actual planting proportion of component crops. It was calculated according to Equation 9.

AYL_f and AYL_k indicate the actual yield loss of the main crop and companion crop, respectively. The value (positive or negative) of AYL denotes an advantage/disadvantage in adopting the consociation system (Banik et al., 2000).

The above indices do not take the economic aspect into account. Through the Monetary Advantage Index (MAI) proposed by Ghosh (2004) (see Equation 10), an economic approach was introduced to assess the intercropping combination.

Where P_f refers to the commercial value of strawberries (a plausible direct sales price on the local farmers' market is 5 EUR per kg) and P_k to the unit price of the companion crop (i.e., chives 10 EUR kg⁻¹, marigold flowers 20 EUR kg⁻¹, mint 5 EUR kg⁻¹, sage 12 EUR kg⁻¹ fresh weight). The higher the MAY value, the more profitable the mixture.

According to Edje (1982), the income equivalent ratio (IER) was the conversion of LER in monetary terms. In other words, it corresponds to the area under monocropping required to produce the equivalent gross income achieved in the intercropping system (Equation 11; Gitari et al., 2020). If the IER is greater than 1, it means that the intercropping system is advantageous.

Pests and beneficial arthropods—including pollinators, predators, and parasitoids—were monitored on both the main crop and companion crops from May to August each year. Observations were conducted every 3 days between 09:00 and 12:00 using visual inspection of the aerial parts of the plants. Some arthropods are very sensitive to movement and the presence of an observer, so we avoided any contact with plants. Each experimental unit was inspected (approximately 2 min) considering all the plants (strawberries and/or complementary crops). Visual monitoring is a widely accepted method for assessing taxa that are identifiable in the field (Ambrosino et al., 2006; Cardona et al., 2021; Dalton et al., 2024; Montgomery et al., 2021; Thompson et al., 2021). This approach is particularly suitable within protected areas such as the Stelvio National Park, as it minimizes disturbance and avoids biodiversity impoverishment. The presence of arthropod specimens was documented through photography for subsequent taxonomic identification to the order, family, genus, and species levels.

Data normality and homoscedasticity were examined with the Shapiro-Wilk test and the Flinger–Killeen's test, respectively. A two-way ANOVA was performed on data collected from both years and mean separation of the dependent variables obtained with the post-hoc Tukey HSD test (p < 0.05). In case of significant interaction between “treatments” and “years”, results were presented separately for the 2 years in dedicated figures or tables. In the case of non-normal data, Kruskal-Wallis test was applied. A one-way ANOVA was performed on data (e.g., plant biomass) collected only in the last year of cultivation (2022). For comparisons between two groups, a two-tailed unpaired Student's t-test was performed. All analyses were carried out in R v. 4.4.2. (R foundation for statistical computing, Vienna, Austria). Values were expressed as mean ± standard deviation (SD).

Total strawberry production resulted significantly affected by the factor “year” (Figure 2). During the first year (Figure 2A), the amount of strawberries reached an average of 150 g per plant, with no significant differences among the cropping patterns (mono- and inter-cropping). In contrast, in 2022 (Figure 2B), strawberry plants cultivated in sole cropping were characterized by the highest fruit production (282 g plant⁻¹). Overall, FRA-ALL, FRA-CAL, and FRA-MENp combinations significantly reduced strawberries by −56%, −34%, −27%, respectively, as compared to respective monoculture production. Conversely, FRA-MENs and FRA-SAL exhibited non-significant difference compared to strawberry in pure cropping (Figure 2B).

Strawberry qualitative traits assessed as flesh firmness (FF), total soluble solid (TSS), titratable acidity (TA), and color index (CI) were not significantly affected by treatments (Table 2). The factor “year” was the only significantly relevant for the quality parameters. In general, FF, TSS and TA values were observed to be higher during the second cropping year, indicating more consistent, sweeter, and more acid-containing fruits. Fruit coloration appeared more intensely red in the first year (Table 2).

Total biomass of strawberry plants under different cropping patterns at the end of the second year is presented in Figure 3. Intercropping with species from the genera Allium, Calendula, and Mentha (specifically M. piperita) resulted in a significant reduction in biomass, with an average decrease of 33% compared to monoculture (Figure 3). Consistent with the trends observed for fruit production data, the intercropping combinations FRA-MENs and FRA-SAL did not differ significantly from monoculture, indicating minimal impact on plant biomass.

Generally, intercropping combinations have almost always generated a significant reduction in production compared to the monoculture of those companion crops (xlimited in specialized farming systemsFigure 4). During the first year, the graph shows a deterioration in the performance of plants under a mixture system, with an average of −40% (Figure 4A). The gap between the two cultivation systems increased in the second year, except for peppermint (around 135 g in both mono- and inter-cropping systems) (Figure 4B).

Chives was the crop with the highest growth potential, quintupling production from the first to the second year. Marigold flower production, on the other hand, declined dramatically in 2022, as well as for peppermint in monoculture (−70%). The amount collected from strawberry mint and sage did not differ appreciably between cropping years.

Peppermint and chives were the companion crops with the highest production under pure stand in 2021 and 2022, respectively. Conversely, the companion crop with the lowest growth capacity in both harvest years was strawberry mint (less than 50 g per plant).

The concentrations of certain nutrients in leaves, stems and flowers of the companion crops were significantly (p < 0.05) influenced by the cropping year and the intercropping treatment (Figure 5). When compared with pure stands, intercropping treatments significantly increased the plant content of K (+50%) and S (+30%) in marigold and chives leaves, respectively, in both years (Figures 5A, B). In addition, the sulfur content also increased in other interplant treatments, e.g., in marigold (Figures 5B, C) and peppermint (Figure 5D), but this was significant only in the second year. Similarly, in 2022, the phosphorus content increased by +68, +28, and +39% in the leaves (Figure 5B) and flowers (Figure 5C) of marigold, and peppermint (Figure 5D), respectively, under a mixture regime. A higher magnesium content (+41%) was observed in the flowers of C. officinalis intercropped with strawberry in the second year (Figure 5C). The N and Ca contents of intercropped plants showed no significant differences compared to the respective monocultures (Figure 5). A high Ca content was evident in the marigold leaves, with values tending to exceed 5% DW (Figure 5B). For strawberry mint and sage treatments, macronutrient content in plant tissues was not significantly affected by cultivation regimes (Figures 5E, F).

Intercropping patterns notably influenced manganese (Mn) accumulation in plant tissues (Figure 6). Overall, Mn content was reduced under intercropping for all companion crops, with the exception of S. officinalis. Particularly striking was the high Mn concentration in marigold (C. officinalis) leaves under sole cropping, where levels exceeded 1,300 mg kg⁻¹ dry weight (DW). In contrast, intercropped marigold exhibited a significant reduction in leaf Mn content, with decreases exceeding 70% (Figure 6B). A similar trend was observed in marigold flowers, although absolute Mn values were substantially lower, ranging from 33 to 125 mg kg⁻¹ DW (Figure 6C). In 2021, intercropping significantly increased boron (B) and iron (Fe) concentrations in strawberry mint (M. suaveolens), by 45% and 18%, respectively, compared to monoculture (Figure 6E). In 2022, B content in marigold leaves doubled under intercropping (Figure 6B). No significant differences in micronutrient content were detected between sole and intercropped systems for chives, peppermint, and sage across both years (Figures 6A, D, F).

The data reported in Figure 7 and Table 3 were presented separately for the 2 years, as the interaction between the factor “treatment” and the factor “year” was significant. LER, ATER, LUR, SPI, and PYD were significantly (p < 0.05) influenced by intercropping combinations with a substantial effect attributable to crop years. Generally, analyzing the indices separately for each year, we observed that during the first year the partial LER of target crop (i.e., strawberry) tended to be close to 1, corresponding to the theoretical value assumed for the pure stand (Figure 7A), while the partial LER values for companion crops ranged between 0.46 and 0.71. As consequence, the total LER 2021 values were higher than 1 in all cases (increased from +43 to +62% compared to sole cropping treatment), without significant differences among the intercropping treatments. On the contrary, intercropping combinations with total LER values around one occurred in 2022 (e.g., in FRA-ALL, FRA-CAL, and FRA-MENs) (Figure 7B). However, the highest LER value (above 1) in 2022 was recorded in FRA-MENp mix, with a value (1.64) significantly higher than the other intercropping combinations. A similar trend was shown for the ATER and LUE (Table 3). Regarding the SPI and PYD (Table 3), extreme values were recorded in the second cropping year, because the highest significant value was obtained in FRA-MENp combination, while the lowest in FRA-MENs.

As previously observed for the biological efficiency indices, significant differences emerged for competition indices depending on the factor “year” (Table 4). In our 2-year experiment, the aggressivity of companion crops exhibited consistently positive (up to +0.02) or equal to 0, and consequently Fragaria values were negative (up to −0.02) or zero, suggesting that strawberry was the dominated crop, or that both crops were equally competitive (A = 0), respectively. In only one combination was strawberry dominant (e.g., in FRA-MENs during the second year).

In almost all cropping systems, the partial CRk was greater than one, meaning that companion crops were characterized by a higher ability of competition than Fragaria (Table 4). The highest and lowest CRf values were recorded in 2022 with the FRA-MENs and FRA-ALL combinations, respectively. In addition, the highest and the lowest CRk were obtained in the same intercropping combinations, but in the opposite way. That means that strawberry mint appeared as a poor competitor, while chives were a strong competitor.

In all treatments, an advantage in intercropping system was achieved as confirmed by the positive value of the AYL index, except for FRA-MENs (−0.63) and FRA-CAL (−0.13) in the second year (Table 4).

The calculation of the Income Equivalent Ratio (IER) and the Monetary Advantage Index (MAI) offers an effective means of assessing the economic efficiency of cropping systems tested (Table 5). The total IER, resulting from the sum of partial IERf and IERk, showed values above 1 in all intercropping combinations except in FRA-MENs.

In the second year, the MAI value decreased dramatically in 3 out of 5 combinations. For instance, FRA-CAL reached the highest MAI value in 2021 (EUR 48,833.86), while a negative value was observed in the following year. Similar differences were recorded for FRA-MENs (EUR−10,664.81, corresponding to the lowest MAI value), and for FRA-ALL (the MAI value in 2022 was lower than in the first year but with a positive sign). The MAI values remained stable (above EUR 20,000) for the combinations FRA-MENp and FRA-SAL (both years).

The visual survey conducted during the experimental period recorded the highest species richness in the blocks subjected to intercropping treatments (+100% as compared to strawberry in pure crop; Figure 8). The number of individuals observed varied depending on the seasons. Arthropods belonging to six insect orders, including Coleoptera, Diptera, Hymenoptera, Lepidoptera, Thysanoptera, and Hemiptera, were identified and assigned to species level.

Bees (especially Apis mellifera L.) were the most frequently observed insects throughout the blooming period, both on target and companion plants. In the group of pollinators, long hoverfly (Sphaerophoria scripta L.), thick-legged hoverfly (Syritta pipiens L.), common drone fly (Eristalis tenax L.) bumblebee (Bombus terrestris L.), small tortoiseshell (Aglais urticae L.) and peacock butterfly (Aglais io L.) were also detected but only on companion crops, particularly on chives and marigold.

Since no pest control treatments were applied, harmful insects such as strawberry-blossom weevil (Anthonomus rubi Herbst), garden foliage beetle (Phyllopertha horticola L.), western flower thrips (Frankliniella occidentalis Pergande), black vine weevil (Otiorhynchus sulcatus Fabricius), strawberry aphids (Chaetosiphon fragaefolii Cockerell) were observed on strawberry plants.

A well-known predator such as the common ladybug (Coccinella septempunctata L.) was found throughout the vegetative and productive phases of the plants.

Other arthropods identified were green tortoise beetle (Cassida viridis L. on peppermint), longhorn beetle (Leptura quadrifasciata L. on strawberry), bordered straw (Heliothis peltigera Denis and Schiffermüller on sage), click beetles (Athous haemorrhoidalis Fabricius on strawberry), and European garden spider (Araneus diadematus Clerck on peppermint).