Increase in anthocyanins and their relationship with nutrition, stomatal density, and yield of purple lettuce (Lactuca sativa L.) nourished with humus based on vegetable waste

Aumento de antocianinas e sua relação com nutrição, densidade estomática e produtividade da alface roxa (Lactuca sativa L.) adubada com húmus à base de resíduos vegetais

Abstract

Global food security faces challenges due to the geopolitical situation, climate change, unhealthy diets, and the post-pandemic period. In Peru, the effects of the socioeconomic crisis were also felt, especially in the increase in food prices. For this reason, research was conducted on the increase of anthocyanins and their relationship with nutrition, stomatal density, and the yield of purple lettuce nourished with humus based on vegetable waste (HBVW). The objective was to determine the increase in anthocyanins in relation to nutrition, stomatal density, and the yield of purple lettuce nourished with HBVW. The methodology is based on applied research with an experimental approach; for this, a completely randomized block design was used, consisting of 3 blocks and 5 treatments, which were T₁ with 0, T₂ with 6, T₃ with 8, T₄ with 10, and T₅ with 12 t ha^-1 of HBVW. The doses were applied 15 days after transplanting, and physical characteristics were evaluated (total plant length, weight of purple lettuce, diameter of purple lettuce, and commercial yield), nutrient concentration in leaves (nitrogen, potassium, phosphorus, calcium, magnesium, sulfur, molybdenum, iron, manganese, copper, zinc, boron, chlorides, and sodium), anthocyanin concentration, total nitrogen, and phosphorus consumption. Also the stomatal density and profitability. Results determined that T₅ stood out in total plant length with 28.90 cm, lettuce weight with 96.33 g, diameter with 17.07 cm, and commercial yield with 19.387 t ha^-1. Total nitrogen consumption with 291.44 kg ha^-1 and total phosphorus pentoxide consumption with 412.98 kg ha^-1 and profitability with 88.4%. Nutrient concentration in T₄ leaves with sulfur, molybdenum, manganese, copper, zinc, and boron. Anthocyanin concentration T₂ with 364.62 mg/100 g and stomatal density T₁ with 460 stomata/mm². He concluded that the appropriate dose (T₄) significantly stood out in anthocyanin concentration and yield. This dose provided nutrients to the soil that improved availability for greater absorption by the plant, which influenced higher nutrient concentration and an adequate number of stomata in the leaves. These biochemical characteristics optimized the reactions of photosynthesis, carbohydrate formation, and translocation and increased anthocyanin synthesis. Resulting in a strengthening against nutritional and environmental stress, pests, and reactive oxygen species (ROS).

Keywords:
compost; dosage; nutrition; anthocyanins; yield; purple lettuce

Resumo

A segurança alimentar global enfrenta desafios decorrentes da situação geopolítica, das mudanças climáticas, das dietas pouco saudáveis e do período pós-pandemia. No Peru, os efeitos da crise socioeconômica também foram sentidos, especialmente com o aumento dos preços dos alimentos. Diante disso, foi realizada uma pesquisa sobre o aumento de antocianinas e sua relação com a nutrição, densidade estomática e produtividade da alface roxa adubada com húmus à base de resíduos vegetais (HBRV). O objetivo foi determinar o aumento de antocianinas em relação à nutrição, densidade estomática e produtividade da alface roxa adubada com HBRV. A metodologia é baseada em pesquisa aplicada, com abordagem experimental, utilizando um delineamento em blocos casualizados, composto por 3 blocos e 5 tratamentos, sendo eles T1 com 0, T2 com 6, T3 com 8, T4 com 10 e T5 com 12 t ha^-1 de HBRV. As doses foram aplicadas 15 dias após o transplantio, e foram avaliadas as características físicas (comprimento total da planta, peso da alface roxa, diâmetro da alface roxa e produtividade comercial), concentração de nutrientes nas folhas (nitrogênio, potássio, fósforo, cálcio, magnésio, enxofre, molibdênio, ferro, manganês, cobre, zinco, boro, cloretos e sódio), concentração de antocianinas, nitrogênio total e consumo de fósforo. Também a densidade estomática e a rentabilidade. Os resultados determinaram que T5 se destacou em comprimento total da planta com 28,90 cm, peso da alface com 96,33 g, diâmetro com 17,07 cm e produtividade comercial com 19,387 t ha^-1. Consumo total de nitrogênio de 291,44 kg ha^-1 e consumo total de pentóxido de fósforo de 412,98 kg ha^-1, com rentabilidade de 88,4%. Concentração de nutrientes nas folhas T4 com enxofre, molibdênio, manganês, cobre, zinco e boro. Concentração de antocianina T2 com 364,62 mg/100 g e densidade estomática T1 com 460 estômatos/mm². Concluiu-se que a dose adequada (T4) se destacou significativamente na concentração de antocianina e na produtividade. Essa dose forneceu nutrientes ao solo que melhoraram a disponibilidade para maior absorção pela planta, o que influenciou em maior concentração de nutrientes e número adequado de estômatos nas folhas. Essas características bioquímicas otimizaram as reações de fotossíntese, formação e translocação de carboidratos e aumentaram a síntese de antocianina, fortalecendo a planta contra estresse nutricional e ambiental, pragas e espécies reativas de oxigênio (EROs).

Palavras-chave:
composto orgânico; dosagem; nutrição; antocianinas; produtividade; alface roxa

1. Introduction

Global food security faces a serious threat due to interrelated economic and social factors, such as the trade confrontation between the United States and China, the war between Russia and Ukraine, climate change, the aftermath of the pandemic, and the inadequate management of natural resources. These elements have increased the costs of products, including energy and fertilizers necessary for agricultural production. According to Abay et al. (2023), the crisis between Russia and Ukraine poses serious challenges to global and regional food security, as it has displaced millions of people and disrupted agricultural production and trade in one of the main exporting regions. Similarly, Maqbool et al. (2024) mention that agricultural activity, food supply, food security, and environmental sustainability are in a precarious phase, which exacerbated the community and ecological position during the pandemic. Zhang et al. (2023) state that future climate change may have substantial impacts on both water resources and food security in the black soil region of China.

In this context, Peru also felt the effects of the economic crisis, which led to a significant increase in fertilizer and energy prices compared to pre-pandemic levels. This situation affected farmers in the region, increasing production costs, exacerbating indebtedness, and creating economic instability. In this regard, Zegarra and Vásquez (2023) argue that the agricultural crisis caused by the pandemic increased fertilizer prices, which severely affected corn, potato, rice, and other crop producers, as the price of a 50 kg bag of urea increased drastically, from S/60 or S/65 to almost S/300, with the added problem of an initial shortage. Likewise, Diez Pérez (2022) states that the Peruvian state has been threatened by the international crisis and reveals vulnerabilities in the strategic management of its supply chain, showing a strong dependence on strategic products such as urea and other fertilizers, which are key to maintaining the agro-industrial production chain.

Due to this situation, it is necessary to innovate sustainable alternatives to reduce production costs and, at the same time, decrease environmental pollution. One option is to take advantage of the resources available in the field, such as the excess cow manure and vegetable (pea) waste that are generated and are sources of nutrients. By adding value to them as compost and using it for the breeding of the California worm (Eisenia foetida), humus is obtained from these residues. Applied to lettuce cultivation, this humus can improve soil properties, increasing its anthocyanin content and thus obtaining an ecological and healthy lettuce, which will help minimize the food crisis and improve health. In this regard, Solaiman et al. (2019) mention that humus-rich compost increased the colonization of arbuscular mycorrhizal fungi in the roots, which significantly influenced the growth of the shoots, roots, and overall development of the lettuce. Similarly, Gurmessa et al. (2021) found that cow manure has a pH of 7.95, 3.14% total nitrogen, 47% total carbon, a C/N ratio of 154, 940 ds/m electrical conductivity (EC), 9 mg kg^-1 (ppm) of manganese, 10 ppm of zinc, 1.52 ppm of nickel, 0.5 ppm of chromium, 3 ppm of copper, 20 ppm of iron, 6 g kg^-1 of potassium, 1.91 g kg^-1 of calcium, 2.38 g kg^-1 of magnesium, 1.77 g kg^-1 of phosphorus, and other elements. Moreover, Arce-Insuasty et al. (2019) determined that pea pods, with a dry weight of 30 g, contain 39.6% carbon, 3.77% nitrogen, and 5.25% hydrogen, while the foliage, also with a dry weight of 30 g, contains 40.5% carbon, 3.05% nitrogen, and 5.25% hydrogen.

It is necessary to mention that HBVW improves the physical, chemical, and biological properties of the soil, as it forms aggregates, adds nutrients such as nitrogen, phosphorus, potassium, calcium, magnesium, manganese, boron, etc., and promotes the development of beneficial microorganisms that are involved in the mineralization process. This fertilizer improves the availability of nutrients so that plants can absorb them efficiently. Therefore, nutrition with this fertilizer can optimize biochemical reactions that strengthen plants against nutritional, environmental, and pest stress. As a result, purple lettuces are obtained, succulent and with a high anthocyanin content that protects the plants. According to Theunissen et al. (2010), vermicompost contains humic acids and essential nutrients such as N, P, K, Ca, Mg, S, Fe, Mn, Zn, Cu, and B, which improve nutrition, photosynthesis, and chlorophyll content, and promote the synthesis of phenolic compounds like anthocyanins and flavonoids that enhance plant quality and act as pest deterrents. Likewise, Pandelea et al. (2023) determined that the application of compost at rates of 30 to 40 t ha^-1 contains total phenols and provides adequate nutrient levels that strengthen plants against environmental stress, significantly improving berry quality, increasing dry matter and vitamin C content, and boosting berry weight.

On the other hand, it is noteworthy that the application of HBVW can increase the anthocyanins present in purple lettuces. These pigments, biosynthesized in the vacuoles of plant cells, stand out for their dark blue-purple color and their flavonoid content. Moreover, the higher concentration of these compounds acts as a defense mechanism against nutritional, environmental, pest, and ROS stress, protecting cells from damage caused by free radicals by neutralizing their harmful effects. For this reason, it is essential to establish a balance between crop yield and anthocyanin content to achieve optimal performance with high levels of these compounds, which is beneficial for consumer health. According to Zhao et al. (2021), anthocyanins, as soluble phenolic compounds, are mainly found in the vacuoles of the epidermis and subcutaneous tissues; their cellular localization influences the color of the tissue, generating shades of purple-red in the vacuoles or brown in the cytoplasm due to oxidation. Similarly, according to Naing and Kim (2021), under extreme stress conditions, the excess of ROS causes oxidative damage, which induces plants to produce anthocyanins and activate the transcription of genes associated with their biosynthesis, allowing anthocyanins to act as antioxidants and neutralize the excess ROS. Sunil and Shetty (2022) argue that, to improve yields in large-scale production, it is possible to optimize parameters such as production media, light, temperature, agitation, and pH, in addition to employing tools like precursor feeding, genetic engineering, and synthetic biology, which represent promising approaches to increase anthocyanin production in plants and microorganisms.

For this reason, an investigation was conducted on the increase of anthocyanins and their relationship with nutrition, stomatal density, and the yield of purple lettuce cultivated with HBVW. The main objective of this study was to determine the correlation between the increase in anthocyanins and their relationship with nutrition, stomatal density, and the yield of purple lettuce nourished with HBVW. To this end, a completely randomized block design was implemented, consisting of three blocks and five treatments, including a control and the standard dose.

Finally, the purpose of this research is to utilize the waste generated in the field, such as cow manure and vegetable residues (pea). Through the breeding of California worms under optimal conditions, humus is produced, an organic fertilizer that, when applied in adequate doses to the cultivation of purple lettuce, can increase both the yield and the concentration of anthocyanins. These results can benefit the consumer both economically and health-wise.

2. Materials and Methods

2.1. Location of the experiment

The experiment is located in the Tres Piedras sector, in the Supe Puerto district, Barranca province, Lima. The coordinates were recorded as latitude south 10°47'19.916” and longitude west 77°44'8.539”, with an altitude recorded at 75 meters above sea level. The weather conditions were recorded with temperatures between 20 and 23°C, relative humidity of 75 to 80%, and a 2% chance of precipitation.

2.2. Type of research

This is applied research with an experimental approach, as the samples were continuously evaluated during the experiment, and the data obtained from the physical characteristics were processed through statistical analysis. In this way, the appropriate dosage was determined and recommended to the farmers in the area.

2.3. Population

The population is composed of red lettuce cultivation, which is grown from 0 to 150 meters above sea level; therefore, the data obtained were validated in this experiment.

2.4. Sample

50% of the plants from each plot were taken for the sample. These plants were marked with tape in the center of the twin furrows on each side in order to avoid edge effects, and physical characteristics such as total plant length, plant weight, equatorial diameter, and commercial yield were evaluated.

2.5. Study factor

The doses of HBVW were established considering the soil analysis conducted by the National Institute of Agricultural Innovation (INIA) of Huaral, the amount applied by the farmers in the area (between 8 and 10 t ha^-1 for lettuce cultivation), and the recommendations from scientific studies. According to Hirzel and Salazar (2016), it is recommended that fertilization with compost made from plant residues and/or by-products or mixtures of animal and plant by-products be between 6 and 10 t ha^-1. Consequently, the standard dose of 8 t ha^-1 was defined, including a control (see Table 1). Additionally, it is worth noting that fieldwork, such as weeding, irrigation, and pest control, was carried out uniformly for all treatments.

Table 1
HBVW doses for purple lettuce cultivation.

Treatment	HBVW dosage (t ha-1)
T1	0
T2	6
T3	8
T4	10
T5	12

2.6. Nitrogen determination in soil

2.6.1. Calculation of soil weight

Nitrogen in the soil was calculated using the following Formula 1 for soil weight per hectare (Ipanaqué Roña, 2023).

Formula for soil weight per hectare.

$\left[WeightHa\right]=\left(Soild.\right)\left(Apparentd.\right)\left(Ha\right)$ (1)

where: Weight Ha: Weight of hectare (2800 t ha^-1 of soil); Soil d.: Soil depth (0.20 m); Apparent d.: Apparent density (1.4 g/cm³); Ha: hectare (10000 m²).

2.6.2. Quantification of organic carbon

The Van Bemmelen formula (Formula 2) was then used, which is [organic carbon]=(M.O.x 0.58) (Vela Correa et al., 2012).

Formula of organic carbon.

$\left[Carbonorg.\right]=\left(O.M.\right)\left(0.58\right)$ (2)

where: Carbon org.: Organic Carbon (0.8236%); O.M.: Organic matter (1.42%) (Table 2) (INIA, 2023a).

Table 2
Soil analysis of the purple lettuce experimental area.

N° Lab.	CE 1:2:5 mS/cm	pH 1:2:5	OM %	N %	P mg kg-1	K mg kg-1	CaCO3%	Cation exchange (mEq/100 g soil)				CEC
								Ca	Mg	Na	K
SU422-DO-23	0.228	8.31	1.42	0.07	14.25	241.37	1.81	6.36	0.71	0.29	0.62	7.98

• CEC = Cation exchange capacity; O.M.: Organic matter. Source: INIA (2023a).

2.6.3. Quantification of the carbon to nitrogen ratio (C/N)

Next, the C/N ratio was determined, for which the following Formula 3 was used.

C/N ratio formula

$\left[C/N\right]=\frac{\left(Carbonorg.\right)}{N}$ (3)

where: Carbon org.: Organic Carbon (0.8236%); N: 0.07% nitrogen (Table 2) (INIA, 2023a); C/N: C/N ratio is 11.76.

2.6.4. Conversion from total to available nitrogen factor

The value of the C/N ratio was obtained, which is 11.76; it was taken as an indicator of the C/N ratio ranges indicated in Table 3, highlighting the equivalence of 140 ppm of nitrogen.

Table 3
Conversion of total nitrogen factor to available in ppm in relation (C/N).

Margin C/N ratio	Conversion factor of Total Nitrogen (T.N.) in percentage to Nitrogen in ppm
Greater than 12	11.2
From 10 to 12	140
Less than 10	225

• Source: Kass (1998).

Then, the available nitrogen (AN) was calculated by multiplying the 140 ppm by the 0.07% soil nitrogen (see Table 2) (INIA, 2023a), resulting in 9.8 ppm AN. Next, the crop weight (2800 t ha^-1 of soil) was projected with 9.8 ppm ND, resulting in 27.44 kg ha^-1 SDN (soil nitrogen available).

2.7. Determination of the standard dosage of HBVW in relation to nitrogen

The determination of the standard HBVW dose was carried out as follows:

Based on the recommendation for fertilizing purple lettuce, which establishes 170 kg ha^-1 of nitrogen (see Table 4) (INIA, 2023b), 27.44 kg ha^-1 of NDS were subtracted, resulting in a total of 142.56 kg ha^-1 of NDA (applied available nitrogen).

Table 4
Recommended nutrition for purple lettuce cultivation.

Nutrients (kg ha-1)
N	P2O5	K2O
170	90	160

• Source: INIA (2023b).

It was taken into account that 100 kg of HBVW contain 2.2 kg of nitrogen (see Table 5, INIA, 2023c). From this amount, the nitrogen content was projected for applications of 6 to 10 t ha^-1 of HBVW, which corresponds to a range of 132 to 220 kg ha^-1 of nitrogen.

Table 5
Chemical characteristics of HBVW.

N° Lab.	ID Sample	pH	CE (ms/cm)	Humidity (%)	OM (%)	N (%)	P2O5 (%)	K2O (%)	CaO (%)	MgO (%)	C/N
AO 143 – DO -23	Humus	7.68	1.94	32.70	45.68	2.20	2.68	2.12	6.85	1.00	10.13

• Source: INIA (2023c)

Compared to the fertilization recommendation for purple lettuce, the amount of nitrogen present in the soil was deducted, resulting in a total of 142.56 kg ha^-1 of NDA. Additionally, the nitrogen contribution from HBVW was projected, with its application varying between 6 and 10 t ha^-1, which corresponds to a range of 132 to 220 kg ha^-1 of nitrogen. Based on these values, a standard dose of 8 t ha^-1 of HBVW was determined, justified as it is a midpoint within the established ranges.

2.8. Determination of phosphorus in soil

The amount of Diphosphorus Pentoxide (P₂O₅) in the soil was calculated using the following procedures.

2.8.1. Calculation of soil weight

Soil weight per hectare formula (Ipanaqué Roña, 2023) (Formula 4).

Formula for soil weight per hectare.

$\left[WeightHa\right]=\left(Soild.\right)\left(Apparentd.\right)\left(Ha\right)$ (4)

where: Weight Ha: Weight of hectare (2800 t ha^-1 of soil.); Soil d.: Soil depth (0.20 m); Apparent d.: Apparent density (1.4 g/cm³); Ha: hectare (10000 m²).

2.8.2. Calculation of phosphorus weight in soil

Then, the weight of the arable layer was projected with phosphorus (P₂) from the soil analysis (Bello and Pino, 2000).

Projection formula for the arable layer with soil phosphorus (Formula 5).

$\left[Weight{P}_2\left(kgh{a}^{-1}\right)\right]=\frac{\left[SoilW.(kgh{a}^{-1})\right]Weight{P}_{2}inS.\left(mg\right)}{kg}$ (5)

where: Weight P₂ (kg ha^-1): Weight of phosphorus (39.9 kg ha^-1 of P₂); Soil W. (kg ha^-1): Soil weight (2800000 kg ha^-1 of soil); Weight P₂ in S. (mg kg^-1): Weight of phosphorus in the soil 14.25 ppm of P₂ (See Table 2) (INIA, 2023a).

2.8.3. Calculation of P₂O₅ weight

Then, the following projection Formula 6 was applied with moles of P₂O₅ and P₂ (Carrasco and Aguirre, 2023).

Formula for projection of P₂ to P₂O₅ kg ha^-1.

$\left[P_2{O}_5\left(kgh{a}^{-1}\right)\right]=\frac{\left[nmoles{P}_2{O}_5\right]P_2\left(kgh{a}^{-1}\right)}{\left[nmoles{P}_2\right]}$ (6)

where: P₂O₅ (kg ha^-1): 91.38 kg ha^-1 P₂O₅; n moles P₂O₅: number of moles of P₂0₅ (142 moles); n moles P₂: number of moles of P₂ (62 moles); P₂ (kg ha^-1): Phosphorus (kg ha^-1) (39.9 kg ha^-1 of P₂).

2.9. Statistical analysis

2.9.1. Analysis of variance

Once the data on the physical characteristics of the purple lettuce were obtained, they were processed using analysis of variance, which allowed the determination of the calculated F value. This value was compared with the corresponding tabulated F, considering a 5% margin of error (F. cal. > F. tab. 5% error). In this way, it was determined whether the doses had an effect, that is, whether the HBVW influenced the plant height, weight, equatorial diameter, and commercial yield or not. Additionally, it is mentioned that the tabulated F values were obtained from the Fisher table with a 5% margin of error.

2.9.2. Duncan's test

After conducting the analysis of variance, the data on physical characteristics were processed using the Duncan test at a 5% error rate, which determined which treatment stood out compared to the others. The averages of the treatments were also rated and grouped with letters; in this way, it was determined whether there was homogeneity when the letters were the same or differentiation when the letters were different.

2.9.3. Statistical processing of the amount of total nitrogen

2.20% nitrogen from HBVW was taken (see Table 5) (INIA, 2023c). This amount was projected in the treatments as T₁ with 0, T₂ with 6, T₃ with 8, T₄ with 10, and T₅ with 12 t ha^-1 of HBVW, which correspond to equivalent values of T₁ = 0, T₂ = 132, T₃ = 176, T₄ = 220, and T₅ = 264 kg of nitrogen. Subsequently, each of these treatments was ordered from lowest to highest and added to the nitrogen available in the soil, equivalent to 27.44 kg ha^-1 of AVN. In this way, the total amount of nitrogen per treatment was calculated, which was linked to the commercial yield of purple lettuce (see Table 6).

Table 6
Total nitrogen consumption for purple lettuce yield.

Treatment	HBVW dosage (t ha-1)	* HBVW nitrogen (kg ha-1)	Soil nitrogen (kg ha-1)	Total nitrogen (kg ha-1)	Anthocyanin concentration (mg/ 100 g)	Commercial yield (t ha-1)
T1	0	0	27.44	27.44	213.63	11.756
T2	6	132	27.44	159.44	364.62	14.322
T3	8	176	27.44	203.44	202.10	16.147
T4	10	220	27.44	247.44	330.36	18.008
T5	12	264	27.44	291.44	207.09	19.387

• ^*
  The HBVW contains 2.20% nitrogen (see Table 5) (INIA, 2023c). This value was projected for an application of 10 t ha^-1, which is equivalent to 220 kg ha^-1 of nitrogen. This procedure was done for all treatments.

2.9.4. Statistical processing of the amount of total phosphorus

The 2.68% of P₂O₅ content in the HBVW was taken (see Table 5, INIA, 2023c), and its application was projected for the treatments: T₁ with 0, T₂ with 6, T₃ with 8, T₄ with 10 t ha^-1, and T₅ with 12 t ha^-1 of HBVW. This is equivalent to T₁ with 0, T₂ with 160.8, T₃ with 214.4, T₄ with 268, and T₅ with 321.64 kg ha^-1 of P₂O₅. Subsequently, the values were arranged from lowest to highest, and each treatment was supplemented with 91.38 kg ha^-1 of P₂O₅ from the soil. In this way, the total phosphorus consumption was calculated, which was linked to the commercial yield of the purple lettuce (see Table 7).

Table 7
Total Consumption of Diphosphorus Pentoxide for the Yield of Purple Lettuce.

Treatment	HBVW dosage (t ha-1)	* Diphosphorus Pentoxide in HBVW (kg ha-1)	Diphosphorus pentoxide in soil (kg ha-1)	Total Diphosphorus Pentoxide (kg ha-1)	Commercial yield (t ha-1)
T1	0	0	91.38	91.38	11.756
T2	6	160.8	91.38	252.18	14.322
T3	8	214.4	91.38	305.78	16.147
T4	10	268.0	91.38	359.38	18.008
T5	12	321.6	91.38	412.98	19.387

• ^*
  The HBVW contains 2.68% Diphosphorus Pentoxide (see Table 5) (INIA, 2023c); then it was projected at a dose of 10 t ha^-1, equivalent to 268 kg ha^-1 of P₂O₅. This procedure was carried out for all treatments.

2.9.5. Statistical processing of anthocyanin concentration

200 g of purple lettuce leaves representative of any block were taken, and the treatments were randomly selected without repeating them. Subsequently, the five samples were taken to the laboratory of the Institute of Nutritional Research (IIN), where the concentration of anthocyanins in 100 g was determined. Then, the results were organized in a table, ordering the treatments from lowest to highest and including the anthocyanin concentrations along with the yield. Next, the results were analyzed (see Table 8).

Table 8
Anthocyanin concentration in purple lettuce leaves by treatment.

Treatment	HBVW dosage (t ha-1)	Anthocyanin concentration (mg/ 100 g)	Commercial yield (t ha-1)
T1	0	213.63	11.756
T2	6	364.62	14.322
T3	8	202.10	16.147
T4	10	330.36	18.008
T5	12	207.09	19.387

• Source: IIN (2025).

2.9.6. Statistical processing of stomata density

The leaves were observed under a Quanta model scanning electron microscope at a scale of 100 μm (micrometers). The resulting image was printed on A4 sheets, and the length and width were measured with a ruler; by projecting them using this scale, an area of 0.08039 mm² was obtained. Subsequently, the stomata on the printed sheets were counted, and this value was divided by the mentioned area to determine the stomatal density. This procedure was carried out for all treatments. Then, in a table, the treatments were arranged from lowest to highest, and the data on stomatal densities were placed, including the anthocyanin concentration and the commercial yield of purple lettuce (see Table 9). Next, the Formula 7 for stomatal density is detailed.

Table 9
Stomata density in purple lettuce leaves by treatment.

	T1	T2	T3	T4	T5
Number of stomata /0.08039 mm2 (lens area)	37	34	29	16	14
Stomatal density (number of stomata/mm2)	460.3	422.9	360.7	199.0	174.2
Result of the amount of anthocyanin (mg/100 g)	213.63	364.62	202.10	330.36	207.09
Commercial yield of purple lettuce (t ha-1)	11.756	14.322	16.147	18.008	19.387

• The area of the projected lens is equivalent to 0.08039 mm².

Stomatal density formula.

$Sd=\frac{Ns}{La.}$ (7)

where: Sd = Stomatal density; Ns = Number of stomata; La. = Lens area (0.08039 mm²).

2.10. Data collection techniques

2.10.1. Data collection techniques for purple lettuce characteristics

For data collection in the experiment, observation and measurement techniques were employed. Evaluation instruments were also used: for the physical characteristics, precision materials such as a digital scale, a measuring tape, and a winch were used. For the analysis of the chemical characteristics of the leaves, laboratory materials from AGQ Peru S.A.C. were used. In the case of soil analysis, resources from INIA-Huaral were used. For the determination of anthocyanin concentration, materials from the IIN were used, and for the observation and quantification of stomata, a Quanta model scanning electron microscope was used.

2.10.2. Anthocyanin determination techniques

Regarding the determination of anthocyanins in purple lettuce leaves, the differential pH method was employed. This method, detailed by Giusti and Wrolstad (2001) and cited by León Minchán (2024), allows for the estimation of total anthocyanin content even in the presence of polymerized pigments and other interferences through the use of buffer systems, such as bisulfite bleaching, and UV-visible spectroscopy measurement. The components of the Formula 8 for the concentration of monomeric anthocyanins proposed by Paredes (2018) are detailed below.

Formula for determining anthocyanin concentration

$\text{Monomeric anthocyanins}\left(\frac{mg}{100g}\right)=\frac{AxMMxDF}{ϵx1}x100$ (8)

where: A = Absorbance; MM = Molar Mass; DF = Dilution factor; ε = Molar absorptivity.

2.10.3. Techniques for the determination of nutrients in soil

A 1 kg soil sample was taken and sent to INIA-Huaral for nutrient analysis using the following methods: The pH was measured using method 9045D designed for soils and waste. Organic matter was evaluated using the AS-07 method by Walkley and Black, from which nitrogen (N) was calculated. Phosphorus (P) was determined using the method proposed by Olsen and collaborators. For potassium (K), the AS-29 method, based on acid neutralization, was used to identify calcium carbonates. Finally, the exchangeable cations, including Ca, Mg, Na, and K, were measured by analyzing the cation exchange capacity and the exchangeable bases of the soil using ammonium acetate.

2.11. Procedures

Compost was made using 60 kg of cow manure and 40 kg of pea residues. During the first few weeks, it was watered weekly by spraying to ensure uniform irrigation and eliminate salts. In the second month, irrigation was carried out every two weeks by sprinkling, along with continuous mulching, and this process was maintained for two more months. In the fourth month, the mature compost was harvested and placed in a pit for the production of humus based on California worms. Subsequently, it was watered twice a week using the sprinkler method until the sixth month.

Then, the land was prepared conventionally, following the common practices of the farmers in Supe Puerto. Afterwards, the zig-zag method was employed, which involved taking soil samples at a depth of 0.20 m throughout the entire experimental area. These samples were placed on a blanket, where they were mixed, and 1 kg of soil was taken and sent to INIA - Huaral for analysis.

The seedbed for purple lettuce was established, ensuring appropriate conditions for soil preparation and using certified seeds, characterized by their high germination rate, varietal purity, and absence of damage. Additionally, we consistently carried out weeding, irrigation, and pest monitoring tasks.

The transplanting of the seedlings was carried out on December 4, 2024, when the seedlings reached a height of 0.15 m, after 28 days from sowing. The task was carried out, leaving a distance of 0.30 m between plants, which were placed on both sides of the twin furrows. The plot had an area of 3 m² and was composed of furrows 3 m long and 1 m wide, including twin furrows with a width of 0.5 m. The experiment was structured into three blocks, each separated from the next by 0.5 m.

Next, irrigation and weeding tasks were carried out continuously, with a frequency of every 7 to 10 days. Fifteen days after transplanting, the doses of HBVW indicated in Table 1 were applied. Additionally, the physical characteristics of the plants were evaluated until the harvest, which took place on December 29. These were processed using statistical analysis.

During the harvest, 200 g samples of purple lettuce leaves were randomly collected from the blocks and treatments, avoiding the repetition of treatments. These samples were sent to the AGQ Peru S.A.C. laboratory and the IIN, where the nutrient concentration and anthocyanin concentration were determined, respectively. Additionally, fresh and undamaged samples were sent to the Quanta model scanning electron microscopy laboratory, where the stomatal density was observed and quantified.

Finally, the yield and cost data per plot were collected and extrapolated per hectare to give the commercial yield and production cost. The commercial yield was then multiplied by the unit price per kilogram to obtain the total income. The production costs were then subtracted from the total income to calculate the profit. This profit was divided by the production cost and multiplied by 100 to obtain the percentage of profitability. This was done for all treatments. The Formula 9 used to calculate the profitability is detailed below.

Formula of profitability.

$P.=\frac{U}{Cp}*100$ (9)

where: P.: Profitability; U: Utility; P.C.: Production cost.

3. Results

3.1. Soil analysis of the experimental area

After the soil analysis conducted at the INIA–Huaral laboratory, it was determined that the pH indicates a moderately alkaline level. The EC classifies the soil as very slightly saline, with a low percentage of organic matter and nitrogen, medium phosphorus concentration, and high potassium content, according to the ranges established by Prialé (2016). Regarding the exchangeable cations, such as calcium, magnesium, and sodium, these are found at medium levels, while potassium is at a high level, according to the ranges established by Agrolab (2005) (See Table 2) (INIA 2023a). Therefore, it is interpreted that the soil is suitable for the cultivation of purple lettuce. However, it is necessary to apply organic matter such as HBVW to improve soil properties and, in this way, increase yield.

Regarding the recommendation for the cultivation of purple lettuce given in Table 4 (INIA, 2023b), it was determined that the soil requires a considerable application of nitrogen and phosphorus to achieve the highest yield. This amount of nitrogen was used as the basis to establish the standard dose.

3.2. Analysis of HBVW characteristics

In the analysis of HBVW, it was determined that it has a slightly alkaline pH, accompanied by low electrical conductivity (EC) and low levels of organic matter, nitrogen, phosphorus, potassium, calcium, and magnesium. However, the C/N ratio is within the appropriate ranges, classifying it as mature compost (see Table 5, INIA, 2023c). This result is related to the studies conducted by Del Castillo and Díaz (2021), who, when producing humus with California red worms from municipal waste in the town of San Roque, recorded 33.61% organic matter, 1.96% total nitrogen, 0.46% phosphorus, 0.77% potassium, 5.63% calcium, and 0.65% magnesium. For their part, Román et al. (2013) mention that the ideal ranges for mature compost (between 3 and 6 months) include a C/N ratio of 10:1 to 15:1 and a moisture content between 30% and 40%. Consequently, the comparison of these fertilizers suggests that the use of HBVW is suitable for the cultivation of purple lettuce.

In the analysis of the microelements present in HBVW, detailed in Table 10 (INIA, 2023d), a high concentration of iron, zinc, copper, and manganese was observed compared to other organic fertilizers. According to De-La-Cruz Chicaiza (2023), 100% cattle manure contains 46.5 ppm of iron, 20.6 ppm of zinc, 2.10 ppm of copper, and 10.6 ppm of manganese, while a mixture composed of 50% market waste and 50% manure contains 44.20 ppm of iron, 20.70 ppm of zinc, 2.90 ppm of copper, and 7.30 ppm of manganese. Therefore, compared to other fertilizers, HBVW represents a significant source of micronutrients for the soil, which enhances its nutritional aspect and contributes to the commercial yield of purple lettuce.

Table 10
Microelements of the HBVW.

Elements	Iron	Zinc	Copper	Manganese
Quantity (mg kg-1)	486.00	68.50	15.20	186.00

• Source: INIA (2023d).

3.3. Physical characteristics of purple lettuce

After analyzing the data using analysis of variance, it was determined that there were no significant differences between the treatments. When processing the results with the Duncan test, it was observed that there were no statistical differences between the averages, classified as (ab). However, it is noteworthy that T₅ stood out compared to the other doses (see Table 11 and Figure 1). This result indicates that the HBVW dose had no effect, as it was observed that the physical characteristics of the treatments were homogeneous, although T₅ stood out compared to the other treatments.

Table 11
Physical characteristics of purple lettuce by treatment.

Treatment	HBVW dosage (t ha-1)	Total plant length (cm)	Weight of purple lettuce (g)	Diameter of purple lettuce (cm)	Commercial yield (t ha-1)
T5	12	28.902 a	96.33 a	17.068 a	19.387 a
T4	10	27.544 a	85.00 a	16.033 a	18.008 a
T3	8	24.815 a	72.86 ab	15.128 ab	16.147 ab
T2	6	22.441 a	57.50 ab	14.057 ab	14.322 ab
T1	0	22.037 a	43.17 b	10.708 b	11.756 b
Significance		**	**	**	**
Coefficient of variation (%)		13.44%	27.62	15.77	19.45

• Note: (*) Significant and (**) not significant. Additionally, the ratings (a) and (ab) indicate that there is a homogeneous relationship between the treatments, according to the processing with the Duncan test.

Figure 1
Comparison of purple lettuce development according to HBVW doses.

3.4. Total nitrogen consumption for the yield of purple lettuce

In the total nitrogen consumption for the yield of purple lettuce, it was determined that T₅ stood out with 291.44 kg ha^-1 of nitrogen. This result indicates that, by increasing the HBVW dose, a 90.58% difference was achieved compared to T₁. However, an adequate dose, such as that of T₄, which reached 247.44 kg ha^-1 of nitrogen, stood out considerably for its higher anthocyanin concentration and commercial yield (see Table 6).

3.5. Total phosphorus consumption for the yield of purple lettuce

Regarding the total phosphorus consumption detailed in Table 7, it is observed that T₅ stood out with 412.98 kg ha^-1 of P₂O₅ compared to the others. This result indicates that the highest dose of HBVW showed a 77.87% difference compared to T₁. However, an adequate dose, such as that of T₄, with 359.38 kg ha^-1 of P₂O₅, influenced a considerable concentration of anthocyanin and the commercial yield of purple lettuce.

3.6. Nutrient concentration in purple lettuce leaves

In the evaluation of nutrients in purple lettuce leaves, T₅ stood out for its high concentration of nutrients such as calcium, molybdenum, iron, manganese, and boron, which influenced a higher yield. On the other hand, T₄ excelled in most elements, such as sulfur, molybdenum, manganese, copper, zinc, and boron, which are within the normal ranges established according to AGQ Peru S.A.C. (2025) (see Table 12). This treatment significantly influenced the increase in anthocyanin concentration, as well as in commercial yield. Therefore, it is interpreted that the application of T₄ had a balanced response, with a notable increase in both the anthocyanin concentration and the commercial yield of the purple lettuce.

Table 12
Concentration of nutrients in purple lettuce leaves by treatment.

Elements	T1	T2	T3	T4	T5	Normal range according to AGQ Peru S.A.C. (2025)
Macronutrients (%)
Total Nitrogen	3.4	3.4	2.7	2.7	2.5	4.00 – 5.00
Potassium	3.90	4.51	3.01	3.32	3.29	6.00 – 7.00
Phosphorus	0.671	0.691	0.559	0.399	0.417	0.40 – 0.60
Calcium	0.80	0.79	1.20	1.20	1.43	2.30 – 3.50
Magnesium	0.309	0.266	0.426	0.381	0.385	0.50 – 0.80
Sulfur	0.28	0.29	0.35	0.27	0.29	0.20 – 0.60
Micronutrients (mg kg-1)
Molybdenum	1.00	< 0,10	1.00	1.00	1.98	0.20 – 1.00
Iron	199	217	236	134	263	50.0 – 100
Manganese	29.9	29.7	49.8	47.0	55.5	15.0 – 250
Copper	14.0	13.9	14.9	8.99	10.9	8.00 – 25.0
Zinc	52.8	49.5	71.8	44.0	51.5	25.0 – 250
Boron	32.9	31.7	50.8	48.0	51.5	25.0 – 60.0
Phytotoxic elements (%)
Chlorides	0.38	0.42	0.28	0.32	0.29	0.40 – 0.60
Sodium	0.50	0.43	1.44	1.15	1.33	0.20 – 0.30

• Source: AGQ Peru S.A.C. (2025).

3.7. Anthocyanin concentration in purple lettuce leaves

In the analysis of anthocyanins in purple lettuce leaves, detailed in Table 8 (IIN, 2025), a variation in the concentration of anthocyanins among the treatments was observed. It is noteworthy that the lowest dose of HBVW, represented by T₂, achieved the highest concentration, recording 364.62 mg of anthocyanins/100 g, standing out compared to the other treatments. This result is interpreted as T₂ surpassing T₁ by 41.41%, which obtained 213.63 mg of anthocyanins/100 g. However, this dose did not affect the commercial yield. On the contrary, an adequate dose, such as that represented by T₄, achieved balance by reaching a concentration of 330.36 mg of anthocyanins/100 g, also standing out for a considerable commercial yield of 18.008 t ha^-1 of purple lettuce.

3.8. Stomata density in purple lettuce leaves

Regarding the stomatal density in the leaves of purple lettuce, as indicated in Table 9 and Figure 2, it is noted that T₁, with 460 stomata/mm², stood out compared to the other treatments. Additionally, as the doses of HBVW increase, a decrease in stomatal density is observed. Therefore, it is interpreted that T₁ shows a difference of 62.15% compared to T₅, with 174 stomata/mm². However, it is necessary to highlight that T₄, with 199 stomata/mm², achieved a considerable increase, both in anthocyanin content with 330.36 mg/100 g and in commercial yield, reaching 18.008 t ha^-1 of purple lettuce.

Figure 2
Micrograph of stomata in purple lettuce leaves by treatment.

3.9. Economic profitability analysis

The economic profitability analysis, based on the HBVW doses detailed in Table 13, shows that T₅, with 88.4%, is the most profitable. This result indicates that, by increasing the fertilizer dose, a 44% increase is achieved compared to T₁. A benefit close to 90% is also obtained, including the initial investment, which confirms that the investment is viable.

Table 13
Profitability of purple lettuce by treatment.

Treatment	HBVW dosage (t ha-1)	Commercial yield (kg ha-1)	Unit value ($)	Total income ($)	Cost of production ($)	Utility ($)	Profitability (%)
T1	0	11756	0.32	3761.9	2516.79	1245.1	49.5
T2	6	14322	0.32	4583.0	2904.63	1678.4	57.8
T3	8	16147	0.32	5167.0	3033.91	2133.1	70.3
T4	10	18008	0.32	5762.6	3163.19	2599.4	82.2
T5	12	19387	0.32	6203.8	3292.47	2911.4	88.4

• Note: 1 $ equals S/. 3.75 soles.

4. Discussion

4.1. Physical characteristics of purple lettuce

After analyzing the physical characteristics of the purple lettuce, it was observed that the doses of HBVW did not have a significant effect. However, T₅ showed a remarkable improvement in all evaluations, with a 39.3% increase in commercial yield compared to T₁ (see Table 11). This result suggests that the application of higher doses of fertilizer incorporated elements into the soil, such as nitrogen, phosphorus, magnesium, calcium, boron, and other essential elements. In addition to beneficial microorganisms, this improved the availability of nutrients and their absorption by the plant, optimizing biochemical reactions such as photosynthesis, carbohydrate formation, and translocation, and strengthening the plant against environmental stress and pests. In this way, greater yield and quality were achieved in the cultivation of purple lettuce. The aforementioned is supported by Anielak et al. (2025), who mention that humic substances improve soil structure and increase its capacity to retain water and essential nutrients such as N, P, K, S, Ca, and Mg, and also in carbon storage. Guamán-Rivera et al. (2024) demonstrate that the use of biofertilizers, such as humus and mycorrhizae, significantly improves the physical and nutritional conditions of the soil, which in turn promotes the production of crops for human consumption and fodder. Nikolaidou et al. (2021) indicate that inoculation with beneficial microbes is a key strategy for sustainable agricultural production; studies demonstrate how the arbuscular mycorrhizal fungus Rhizophagus irregularis and the plant growth-promoting rhizobacterium Bacillus subtilis act together to influence lettuce growth.

4.2. Total nitrogen consumption for purple lettuce yield

The analysis of total nitrogen consumption for the yield of purple lettuce highlighted T5 with 291.44 kg ha^-1 of nitrogen, representing a 90.58% difference compared to T1 and influencing a higher yield. However, the balanced dose applied in T4, with 247.44 kg ha^-1, stood out significantly by contributing to the increase in anthocyanin concentration and commercial yield (see Table 6). This result suggests that T4 provided an adequate amount of nitrogen to the soil, improving its availability for absorption by the plants, which optimized biochemical processes such as the formation and translocation of carbohydrates, promoting an increase in anthocyanin production. These biochemical reactions strengthened the plants against nutritional, environmental, pest, and ROS stress, resulting in a notable commercial yield and a higher concentration of anthocyanins. These findings are supported by Cruz Nieto et al. (2022), who highlighted that the highest dose of compost (T₅) from market waste increased the total nitrogen consumption by 234.7 kg ha^-1 in the soil, improving its availability and favoring lettuce yield. Likewise, Solaiman et al. (2019) demonstrated that humus-rich compounds, such as Humicarb with high humic acid content, increased mycorrhizal colonization, which favored the absorption of nitrogen and phosphorus, stimulating lettuce growth. Finally, Barzegar et al. (2021) determined that the application of humic acid and kinetin increased the growth and yield of radish roots, improving biochemical attributes such as firmness, vitamin C, total soluble solids, and anthocyanin content.

4.3. Total phosphorus intake for purple lettuce yield

Regarding the total phosphorus consumption, it was specified that T₅ reached 294.44 kg ha^-1 of P₂O₅, which represented a 90.58% difference compared to T₁. However, an adequate dose, such as T₄ with 247.44 kg ha^-1 of P₂O₅, significantly influenced the concentration of anthocyanins and the commercial yield of purple lettuce (see Table 7). This result suggests that the optimal dose of T₄ provided an adequate amount of phosphorus, improving its absorption by the plant. This, in turn, optimized biochemical processes such as photosynthesis, carbohydrate formation and translocation, and the increase of anthocyanins. Thus, this amount of phosphorus strengthened the plant's response to environmental stress, pests, nutritional challenges, and ROS, resulting in an increase in anthocyanin concentration and the commercial yield of lettuce. This is supported by Neocleous and Savvas (2021), who found that phosphorus levels in recirculating solutions did not equally affect the photosynthetic metabolism of the distinct lettuce varieties; this suggests that phosphorus is fundamental in the biochemical processes of photosynthesis. Therefore, phosphorus had a significant response in the considerable increase of anthocyanin in purple lettuce. Jezek et al. (2018) state that in crops, the accumulation of anthocyanins in leaves is usually due to nitrogen or phosphorus fertilization deficiency; however, it is little known that the nutritional status of the plant also influences the synthesis of anthocyanins in the fruits, making a strategic supply of nutrients necessary to balance anthocyanin content and crop quality.

4.4. Nutrient concentration in purple lettuce leaves

In the nutrient analysis of purple lettuce leaves, T₅ stood out for its high concentration of nutrients such as calcium, molybdenum, iron, manganese, and boron, which resulted in a higher yield. However, at an appropriate dose, T₄ excelled in most elements, such as sulfur, molybdenum, manganese, copper, zinc, and boron, within normal ranges, according to AGQ Perú S.A.C. (2025) (see table 12), which significantly contributed to the increase in anthocyanin concentration and commercial yield. This result indicates that the appropriate dose (T₄) of HBVW provided essential nutrients to the soil, which the plant utilized to optimize biochemical reactions such as photosynthesis, carbohydrate formation, and translocation. These reactions strengthened and increased the concentration of anthocyanins, allowing the plant to respond to environmental stress, pests, and ROS. In this way, a considerable yield was obtained with an increase in anthocyanin concentration. The results correspond with those of Kumar et al. (2018), who mention that micronutrients are essential for plant growth but require relatively small amounts, such as boron, copper, iron, manganese, molybdenum, and zinc; among them, iron and zinc play a very important role in biochemical processes such as photosynthesis and others that influence the production of horticultural crops. On the other hand, Za et al. (2017) determined in their research that there is a positive and significant correlation between the anthocyanin content in grapes, boron availability, and the boron and potassium content in the petiole, but no significant relationships were found with other nutrients.

4.5. Anthocyanin concentration in purple lettuce leaves

In the evaluation of anthocyanins in purple lettuce leaves, it was determined that T₂ reached the highest concentration, 364.62 mg/100 g, surpassing T₁ by 41%. However, T₄, with 330.36 mg of anthocyanin/100 g, significantly influenced both the anthocyanin concentration and the yield (see Table 8). This result is analyzed to show that, with an adequate dose (T₄), the anthocyanins increased their concentration. This increase favored biochemical aspects by promoting the production of phenolic and antioxidant compounds, which acted as a defense against environmental stress, pests, and ROS. As a result, a considerable increase was observed both in yield and in anthocyanin content. This result is supported by Villamarín-Raad et al. (2023), who determined that the use of fertilizers in combination with bacterial inoculants increases the phenolic compound content in the leaves of Guadua angustifolia. Likewise, Nicolaescu and Biþã (2009) mention that humic substances such as fulvic humic acids participate in redox processes and free radical elimination; their antioxidant capacity allows for the evaluation of these properties, indicating their effectiveness in inactivating ROS. Li and Ahammed (2023) mention that the intensity and quality of light, as well as the photoperiod, have regulatory effects on the production of anthocyanins; therefore, in red leaf lettuce, the anthocyanin pathway remains inactive under low light conditions.

4.6. Stomata density in purple lettuce leaves

In the evaluation of stomatal density in purple lettuce leaves, it was determined that T₁, with 460 stomata/mm², showed a 62% difference compared to T₅, which stood out for its yield. However, T₄, with 199 stomata/mm², stood out by recording a considerable concentration of anthocyanins and an increase in yield (see Table 9 and Figure 2). This result suggests that the appropriate dose applied in T₄ added nutrients to the soil, which positively influenced the number of stomata and optimized key biochemical processes, such as transpiration, photosynthesis, carbohydrate formation, and translocation, as well as the increase in anthocyanin production. These reactions strengthened the plant against nutritional and environmental stress, pests, and reactive oxygen species (ROS), resulting in a notable increase in both yield and anthocyanin concentration. Vaičiulytė et al. (2022) reported that, although humus increased stomatal density in the lower epidermis of the leaves, the results were not significant (p > 0.05) compared to the control. On the other hand, Tanaka et al. (2013) demonstrated that the increase in stomatal density improved CO₂ exchange and raised the photosynthesis rate by 30% under constant growth conditions, confirming the positive influence of stomatal density on the photosynthetic capacity of leaves through the regulation of gas diffusion. Finally, Cirillo et al. (2021) noted that the accumulation of anthocyanins, in addition to providing the characteristic red color to the leaves, plays key roles in protecting them from excess sunlight and controlling water loss by reducing transpiration and stomatal density.

4.7. Economic profitability analysis

Regarding the economic profitability analysis, it was determined that T₅, with 88.4%, surpasses T₁ by 44% (see Table 13). This result is analyzed to show that the application of a higher dose of HBVW (T₅) provided a greater amount of nutrients to the soil, which significantly influenced the increase in yield. In this way, a profitability of 88.4% was achieved, which implies the recovery of almost 90% of the total investment, including the initial amount. Therefore, this application dose of HBVW proves to be a beneficial option, as it ensures superior profitability, and, consequently, its use is recommended in the district of Supe Puerto.

5. Conclusion

It was determined that T₅ achieved the highest yield, with 19,387 t ha^-1, but this dose did not significantly increase the anthocyanin concentration. Therefore, it is established that an adequate dose (T₄) improved nutrient availability for efficient absorption, which contributed to optimal biochemical reactions, such as photosynthesis, carbohydrate formation and translocation, and a considerable increase in anthocyanin concentration. This resulted in an increase in both anthocyanin and commercial yield.

It was also determined that, in the nutrient analysis of the leaves, T₄ stood out in most elements, such as sulfur, molybdenum, manganese, copper, zinc, and boron. This influenced the considerable increase in anthocyanin concentration and yield. Therefore, nutrients were added to the soil at this dosage, which improved their availability for the plant. This influenced a higher concentration of micronutrients in the leaves, such as boron and other elements, which contributed to the increase of anthocyanins. These compounds strengthened the plants against environmental, nutritional, pest, and ROS stress.

Regarding the anthocyanin concentration, T₂ stood out with 364.62 mg per 100 g, surpassing T₁ by 41%. However, T₄, with 330.36 mg of anthocyanins per 100 g, had a considerable increase in both anthocyanin concentration and yield. This dose added nutrients to the soil, which favored their availability and efficient absorption by the plant. These processes optimized various biochemical reactions, such as the increase of anthocyanins in the leaves, strengthening the defense mechanisms against nutritional and environmental stress, as well as against pests and ROS.

Regarding the appropriate stomatal density, T₄, with 199 stomata/mm², achieved a considerable increase in anthocyanin concentration and yield. Therefore, at this dosage, the stomatal density was established as an indicator that optimized essential biochemical processes, such as transpiration, photosynthesis, carbohydrate formation, and translocation, in addition to contributing to the increase in anthocyanin synthesis. As a result, the plant's defense mechanisms against nutritional and environmental stress, as well as against pests and ROS, were strengthened.

Data Availability Statement

I mention to you that the entire data set supporting the results of this study was published in the article itself.

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Edited by

• Editor:
  Jairo Lizandro Schmitt

Publication Dates

• Publication in this collection
  29 Sept 2025
• Date of issue
  2025

History

• Received
  24 Mar 2025
• Accepted
  21 July 2025