NITROGEN, PHOSPHORUS AND POTASSIUM CONTENT IN DIFFERENT ORGANS OF PINEAPPLE CULTIVARS AT DIFFERENT PLANTING DENSITY

'Champaka', and 'MD-2' were established at 30000, 45000, and 60000 plants ha -1 . Eight samples were taken to determine the N, P, and K content in the organs and the total plant. Results. The highest and lowest N, P, and K contents were detected in the leaf and root, respectively. The highest N and K contents occurred at 441 - 506 days after planting. Higher P content occurred close to harvest. The highest N, P, and K contents per plant were at 30000 plants ha -1 (14.86, 1.52, and 16.29 g plant -1 , respectively) and the lowest at 60000 plants ha -1 (10.16, 1.13, and 14.6 g plant -1 , respectively). Higher N, P, and K contents per hectare were detected with 60000 plants ha -1 (609, 68, and 875 kg ha -1 , respectively). At harvest, ‘Smooth Cayenne’ at 60000 plants ha -1 accumulated the highest amount of N, P, and K (147, 37, and 306 kg ha -1 , respectively). Implications. The changes that can occur in the nutrient requirements of pineapple as a function of cultivar, planting density, and stage of plant development were identified. This information will be useful for producers, agricultural technicians, and researchers in Mexico and the world, to generate fertilization programs or establish new research. Conclusion. At the beginning of plant growth, a higher N, P, and K contents in the leaf, this amount decreases as the fruit harvest approaches. Regardless of cultivar, the highest nutrient content per plant occurs at the lowest planting density, however, the highest content per hectare occurs at the highest planting density. At harvest time, fewer nutrients are removed from the soil with 'Champaka' and 'MD-2' fruit.


INTRODUCTION
The nitrogen (N), phosphorous (P) and potassium (K) are nutrients required by the pineapple along its production cycle. K is reported as the nutrient extracted in greater amount, it is associated with weight gain and improve fruit quality. N is the second nutrient more required by the plant, it is associated with the amino acid synthesis and protein formation; it had a positive influence in plant development, yield, and fruit quality. P is between the third and the fourth nutrient more required, it participates in energy transfer as part of the ATP, it is a component of nucleic acids, nucleotides, coenzymes, phospholipids and phytic acid (Maia et al., 2020). Besides as the plant growths, the nutritional status of the plant change and those changes can be perceived in the concentration and accumulation of nutrients in different parts of the plant (Ahmed et al., 2007).
The cultivar used influences plant size, nutrient absorption, dry matter production, fruit yield and dry matter accumulation in the different organs of the plant (Hanafi et al., 2009;Neri et al., 2021); which affects the concentration and accumulation of nutrients in each part of the plant (Sampaio et al., 2011;Trejo et al., 2020). Planting density is another factor that can affect the concentration of nutrients because it modifies the size of the plant; as the plantation density increases, the number of leaves emitted, the size of the stem and the size of the fruit are reduced (Cardoso et al., 2013;Souza et al., 2019). Although a smaller size and amount of dry matter is obtained from the plant in the highest planting density, the yield, and the amount of dry matter per hectare is greater as the planting density increases, consequently, the amount of nutrient extracted per hectare increases as the amount of dry matter produced increases (Souza et al., 2019).
The fruit and the crown are removed from the field during harvest, which means that with its biomass, 55-78% of the N and 39-60% of the total P absorbed by the plant can be removed from the production system (Ahmed et al., 2007;Salgado García et al., 2017). After the harvest, the residues that remain in the field: root, leaf, stem, and peduncle; can represent 64-86% of the total biomass produced (Hanafi et al., 2009). This biomass represents a source of organic matter and nutrients that can be used in a second production cycle or in the subsequent crop.
In Mexico, before the year 2000, low planting densities (< 30000 plants ha -1 ) were used on commercial farms. Considering the above, an experiment was established to evaluate three planting densities in three pineapple cultivars. The first two cultivars were 'Smooth Cayenne' and 'MD-2', in 2021, these varieties represented 67% and 29% of the total area planted in Mexico (SIAP, 2023). The third cultivar was 'Champaka', a promising genotype for new commercial plantations (Uriza-Ávila et al., 2018). The results showed the effect of planting density and cultivar on plant growth, yield, and fruit quality (Rebolledo-Martínez et al., 2005;Rebolledo Martínez et al., 2006), however, it is unknown how these factors affect the behavior of nutrients during plant development. For this reason, the objective was to determine the effect of planting density and cultivar on the N, P and K content during plant development, as well as the amount accumulated in the fruit at harvest time. This information will be useful to agricultural technicians and pineapple producers around the world, as well as to agricultural researchers, to generate fertilization programs or to establish new experimental works.

MATERIALS AND METHODS
An experiment was stablished in the Papaloapan Basin, within the facilities of the 'Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias' (INIFAP). The 'Papaloapan' Experimental Station (INIFAP) is located in a region with an Aw0 climate and it is characterized by having dystric cambisol soils, poor in organic matter and nutrients: 4.8 pH; 0.043 dS m -1 EC; 0.92 and 0.053 % of organic matter and N, respectively; 3 mg kg -1 P; 0.6 and 0.74 meq 100 g -1 of K and Ca, respectively; 0.08 cmol kg -1 Mg; 0.34, 50, 1.23, 0.90 and 16 mg kg -1 of Na, Fe, Cu, Zn and Mn, respectively.
Pineapple shoots 'MD-2', 'Smooth Cayenne' and 'Champaka' weighing 400 to 500 g were selected from the gene bank of 'Papaloapan' Experimental Station. A split plots treatment arrangement with a randomized block design and four repetitions was used. Planting density (30000, 45000 and 60000 plants ha -1 ) was considerate as a large plot and the small plot was the pineapple cultivar. The experimental unit was 120 plants. The experimental unit consisted of three 9 m long planting beds (distance of 1.25 m from center to center of the beds) with two rows per bed spaced 45cm.
The flower induction treatment was carried out with calcium carbide dissolved in water at 2%. Three applications were made, at 704, 707 and 710 DAP. At each date, 60 ml of the solution was applied to each plant. The application was made between 18:00 and 22:00 hours.
Eight plant samplings were made during the entire production cycle. On each sampling date, one fully competent plant was obtained from each experimental unit. Sampling was carried out at 108,153,202,278,321,442,506 and 551 DAP, which were segmented into roots, stems, leaves (Only green leaves), peduncles, and fruits. The total fresh weight of each plant organ was recorded. Representative samples were obtained from each organ and dried in a forced air oven at 70 °C until constant weight was reached. Dry matter was determined for each sample by gravimetry. The dry matter was subjected to analysis to determine the nitrogen concentration, with the micro Kjeldahl method (Bremner, 1965); phosphorus, with the colorimetric method of Olsen et al. (1954); and potassium, by atomic emission spectroscopy (Chapman, 1973).
On each sampling date, the content of nutrients (Cn) in each plant organ was determined using the equation Likewise, the nutrient content per plant (Cnp) was calculated using the equation = + + + + (Maia et al., 2016).
The fruits were harvested at 551 DAP, when the fruits presented an external ripening of 50% (Soler, 1990).
The content of N, P and K in each organ and in the plant by sampling date are represented with the arithmetic mean and standard deviation. The influence of planting density and cultivar on nutrient content was determined with mixed models. These models considered each factor independently and the interaction between them. A null model was also included, under the assumption that none of the factors was a good predictor of nutrient content, with a total of five models used for each organ. The best model was selected considering the lowest value of the Akaike Information Criterion (AIC), which is a measure of the relative quality of a statistical model for a data set, which manages a trade-off between the goodness of fit and the complexity of the model (Vrieze, 2012). A posthoc test (Tukey with p<0.05) was performed on the selected model to identify differences between the levels of the fixed effects.

Nutrient content during plant growth
Of the total N content by the plant, independent of the planting density, cultivar, and phenological stage, the leaf is the one that accumulated the greatest amount, while the least amount is accumulated by the root (Figure 1). At densities of 30000 and 45000 plants ha -1 , in the three cultivars, the highest N content in the plant occurred at 441 DAP (Figure 1 A The leaf was the organ with the highest P content until before harvest (506 DAP), in the three planting densities and in the three cultivars ( Figure 2). In all planting densities, the highest P content in 'Champaka' and ' In the three planting densities, until before 551 DAP, the highest K content was detected in the leaf ( Figure  3). In all of them, the highest K content in 'MD-2' occurred at 441 DAP (Figure 3 B, E, H). The situation was different with 'Champaka' and 'Smooth Cayenne', where the highest K content changed as a function of planting density (Figure 3 A, C, D, F, G, I).

Nutrient content during fruit harvest
The N, P and K content in the different organs of the plant, in some cases, depending on the planting density, the cultivar or the interaction of both factors ( Table 1). The N and K content in the stem, as well as the P content in the stem and leaf, are because of planting density. The N, P and K content in the peduncle depends on the cultivar. The N and K content in the leaf, as well as the P and K content in the fruit, are explained by the effect of planting density + cultivar + planting density:cultivar (Table 1).  The density of 30000 plants ha -1 stimulated the highest N content in the stem compared to the density of 60000 plants ha -1 . The N content in the peduncle was similar in the three pineapple cultivars. The N content in the leaves was higher with 'Champaka' at the density of 30000 plants ha -1 compared to 'MD-2' at the densities of 30000 and 45000 plants ha -1 (  (Table 4).

DISCUSSION
Previous reports indicate that nutrient content changes as a function of the cultivar (Hanafi et al., 2009), plant organ (Pegoraro et al., 2014b) or planting density (Souza et al., 2019). This situation was corroborated in the peduncle and stem, since, the N, P and K content depended on the cultivar, while the stem depended on the planting density. However, none of the factors considered in this study were able to explain the behavior of N, P and K content in the root, indicating that other factors are involved in the accumulation of nutrients in this organ. The N, P and K content in leaf and fruit is not attributed to a single factor but is influenced by the interaction of planting density and cultivar, therefore, it is speculated that the factors that affect the accumulation of nutrients in the leaf will also affect the accumulation of nutrients in the fruit. Nutrient content depends on the amount of biomass produced, increased leaf biomass promotes increased plant biomass (Hanafi et al., 2009;Mota et al., 2016), which is reflected in larger fruits (Teixeira et al., 2011;Vilela et al., 2015) and consequently with higher N, P and K content. In this study, the order of nutrient demand was little influenced by the cultivar and planting density, since the order of nutrient content in the three cultivars and in the three densities was: K>N>P, somewhat similar was observed with cv. 'Vitória' that, at different planting densities, the content order was K>N>P (Souza et al., 2019).
Changes in nutrient content as a function of plant organ is consistent with that reported by Pegoraro et al. (2014b). The greater accumulation of N, P and K in the leaf is attributed to the presence of chlorophyll molecules, enzymes, proteins, and nucleic acids that are required for different metabolic processes of the plant. Although the stem and the root are lignified organs, a greater N, P and K content in the stem compared to root, is attributed to the fact that the stem is a reserve organ for photoassimilates in the form of vascular parenchyma tissues and starch (d'Eeckenbrugge and Leal, 2018). On the other hand, the lower N, P and K content in the root is attributed to the fact that it is an absorption organ with lower N content, mainly constituted by fibrous tissues, rich in lignin and with a lower concentration of proteins (López-Herrera et al., 2014). It is very possible that during the development of the fruit a phenomenon of translocation of nutrients from the leaf and stem to the fruit has occurred, since the proportion of nutrients extracted by the leaf and stem increased compared to the first phenological stages of the crop.
No other studies were found to compare the behavior of N, P and K content throughout the pineapple growth cycle; therefore, these results will serve as a point of comparison for other studies. The results indicate that the maximum N, P and K content is achieved after TFI like those reported by Pegoraro et al. (2014b). In this case, the highest total N and K content occurred at the beginning of fruit development, while the highest total P content was detected at the final stage of fruit development, mainly in 'Champaka' and 'Smooth cayenne'. This can be related to the amount of dry matter produced or to the nutrient concentration in the plant tissue (Ávila-Escobedo et al., 2022). In the cv. 'Red Spanish', total dry matter accumulation and linear N content were reported as plant age increased (José et al., 2007). However, other research has shown that pineapple can reach its maximum dry matter accumulation at the time when fruit development begins and after this stage this value decreases (Malézieux, 1993;Zhang and Barthomew, 1997;Souza et al., 2007). That reduction can be attributed to the loss of dry matter in the stem (Bartholomew, 2018) or by the disintegration of old leaves (Zhang and Barthomew, 1997;Pegoraro et al., 2014a), since, at the time of sampling only the green leaves present on the plant were considered. This explains the higher N and K content at the time when fruit development begins (441 DAP), since the highest accumulation of total dry matter in the plant was detected at this phenological stage in the three pineapple cultivars (Rebolledo-Martínez et al., 2005). Regarding P content, the highest values presented at the end of the fruit development stage (506 DAP) are attributed to the concentration of the nutrient in plant tissues and in the fruit. Previous studies indicate that the nutrient concentration in plant tissues can change depending on the phenological stage of pineapple, depending on the nutrient, the concentration can increase, decrease, or remain stable as plant age increases (Ramos et al., 2011). In this study, P concentration increased in the vegetative part, mainly with 'Champaka' and 'Smooth cayenne'. The average P concentration of these two cultivars in root, stem and leaf was 0.02, 0.12 and 0.07 mg kg -1 at 441 DAP, values that increased to 0.07, 0.14 and 0.09 mg kg -1 at 506 DAP, respectively. On the other hand, of all organs, the highest P concentrations were detected in the fruit at 506 DAP, specifically in the crown and peel with 0.23 and 0.17 mg kg -1 , respectively. This higher requirement of N, P and K at the beginning of fruit development coincides with the fertilization recommendation Rebolledo Martínez et al., 2016, which suggest concluding fertilization before the anthesis stage.   Greater nutrient content with higher planting density has also been detected in other producing regions of the world (Silva et al., 2009;Souza et al., 2019), this study confirms that the fertilization dose should increase as the number of plants per hectare increases.
In the fruit harvest, the lowest planting density presented the highest content of N, P and K per plant, while per unit area it was presented by the density of 60000 plants ha -1 . 'Smooth Cayenne' accumulated 9.725, 1.129 and 12.516 g plant -1 of N, P and K and 587, 68, and 750 kg ha -1 of the same elements. On the other hand, 'Champaka' accumulated 9.626, 0.972, and 9.207 g plant -1 of N, P, and K, which represents 577, 58, and 522 kg ha -1 of N, P, and K, respectively. It is important to point out that, at harvest time, 20-22, 39-54, and 29-47% of the total N, P, and K accumulated in the plant were accumulated in the fruit, values that changed to 19-24, 45-50, and 32-34% for 'MD-2', as well as 20-24, 50-54, and 36-42% for 'Smooth Cayenne', respectively. These percentages indicate that, when 'Champaka' fruits of 2.2 to 2.5 kg plant -1 are obtained, the amount of N, P and K removed from the production unit is around 84-123, 18-28 and 164-259 kg ha -1 , respectively. In the case of 'MD-2', with fruits from 1.8 to 2.1 kg plant -1 , 50-110, 18-25 and 141-203 kg ha -1 of N, P and K are removed, respectively. As for 'Smooth cayenne', with fruits from 2.3 to 2.7 kg plant -1 , about 84-123, 18-28 and 164-259 kg ha -1 of N, P and K, respectively, withdraw from the production unit. These results corroborate the recommendations of Rebolledo Martínez et al. (2016) and Uriza-Ávila et al. (2018), who, for pineapple plantations that will be kept in the field to obtain a second or third fruit harvest, indicate applying between 40% and 50% of the fertilization dose applied for the first fruit harvest. The results mainly support the dose of N and K, however, in the case of P, in the three Cultivars and in the three planting densities, the percentage accumulated in the fruit was less than 25%. Therefore, for plantations that will be maintained for a second or third fruit harvest, the P recommendation should be less than 30% of the dose applied at the beginning of the plantation.

CONCLUSIONS
The results indicate that, regardless of the Cultivar and planting density, the highest content of N and K occurs during fruit development, while the highest content of P occurs close to harvest. The greatest amount of N, P, and K accumulates in the leaves, mainly before the fruit develops. A higher content of N, P, and K per plant occurs at the lowest planting density, however, the highest content per hectare occurs at the highest planting density. 'Smooth Cayenne' is the cultivar that accumulates the greatest amount of N and K, while the least amount of N and P is accumulated from the production unit with 'MD-2' fruits. This information must be considered in the nutritional program of commercial pineapple plantations, especially if some of the cultivars studied are used.