Introduction

Photosynthesis is directly or indirectly with many processes finely regulated in the cells and variations in them are reflected in changes in plant physiology and finally in their growth and survival. Several studies have reported that exposure to suboptimal conditions or stress conditions (water stress, extreme temperatures, excessive solar radiation, mineral stress, etc.) result in a decrease of photosynthesis as a result of the suppression or activation of certain metabolic pathways (^{Rakic, 2015}). For example, the decrease in the amount of solar radiation, and generally under any kind of stress photosynthetic activity reduces by decreasing chlorophyll content (^{Cambron-Sandoval et al., 2011}).

The secondary metabolites are essential phytochemicals that are affected by the light spectrum and act as defense compounds, as well as protection against ultraviolet radiation and oxidizing agents. In addition to this, certain flavonoids and phenolic compounds, show microbial, antioxidant, antifungal and free radical trapping activity (^{Ouzounis et al., 2015}).

Phenolic compounds are widely distributed in plants, produced in shikimate pathways, pentose phosphate and phenylpropanoid; its importance, besides their role they play in growth, reproduction and protection, lies on contributing to color and sensory characteristics of fruits (^{Balasundram et al., 2006}). In peel of avocado fruits have been identified various pigments, primarily carotenoids, chlorophylls and cyanides (^{Donetti and Terry, 2012}). In avocados with green peel, the color is primarily due to chlorophyll, whereas in the case of 'Hass', whose peel turns black as it matures, its color is caused in part by the content of anthocyanins (^{Ashton et al., 2006}). The content of phytochemicals in avocado peel has been reported for different crops, highlighting 'Hass' (Guatemalan x Mexican race) for its high concentration of total phenolic compounds and lower chlorophyll A and B, opposite behavior to cv. Tonnage (Guatemalan x Antillean race) (^{Wang et al., 2010}).

The variation in agronomic conditions (species, crop, development stage, plant organ, competition, fertilization, etc.), the season, weather conditions, water availability and light (intensity, quality and duration) have significant effects on the content and phytochemical profile in a crop (^{Björkman et al., 2011}). Avocado crop thrives from temperate to warm climates with a temperature range for photosynthesis from 10 to 35 °C and rainfall from 800 to 1 500 mm per year (^{Ruiz et al., 2013}), though with irrigation can be grown under rain absence.

For “Hass” producing areas in the semi-warm climate from the state of Nayarit was developed a prediction model for floral development of winter vegetative f lush shoot which was associated with temperatures ≤ 21 °C and summer f lush shoot at temperatures ≤ 19 °C (^{Salazar-García et al., 2007}). These models were tested on three types of climate from the state of Michoacan (warm sub humid, semi warm sub humid and temperate sub humid) and the model only ran for summer vegetative f lush shoots (temperatures ≤ 19°C. ^{Salazar-García et al., 2009}). In another study, ^{Alvarez-Bravo and Salazar-Garcia (2015)} evaluated a prediction model of floral development developed for Michoacan (based on chilling days accumulated with temperatures ≤ 16°C), which showed good predictive capability of winter vegetative flush shoots in 'Hass' grown under six types of climate from the avocado region of Michoacan. This indicates that there are different temperature thresholds related with floral development of 'Hass' avocado. On the other hand, in warm climates, 'Hass' peel often has greater roughness and thickness, which is usually taken as an argument to decrease fruit price, compared to that from cooler regions in which predominates fruit with thinner and less wrinkled peel. However, these characteristics do not seem to affect the physical, chemical or organoleptic quality of the fruit (Salazar-García et al., 2016).

The changes in the components of the climate system obey to a complex relationship between forcing agents (such as the production of greenhouse gases, changes in solar irradiation and insolation and changes in land cover), natural processes and human activities. This leads to certain climatic responses as the variation in precipitation and temperature (^{Pielke et al., 2007}). With climate change, the variations in agro-climatic parameters will affect cell function, and therefore, crop physiology, besides affecting fruit quality in terms of taste, appearance, flavor and nutrient content, phytochemicals and other dietary components. This may be due to a high photo- oxidation, to an increase in the synthesis of reactive oxygen species (ROS) and secondary metabolites (^{Singh et al., 2015}). The aim of this study was to evaluate the effect of soil moisture condition and incident solar radiation on the content of phytochemicals in peel of 'Hass' avocado.

Materials and methods

Orchards characteristics. Two commercial orchards of 'Hass' avocado were selected in the Ejido El Rodeo, municipality of Tepic, Nayarit, one located at an altitude of 950 m (without irrigation) and the other at 1 200 m (irrigated) with semi-warm sub-humid climate (A)C(w2) and soil type Andosol.

The orchard with irrigation has a micro sprinkler of 34 L h^-1 per tree and frequency and irrigation depth is determined by tensiometers, trying to keep the soil moisture tension between 10 and 30 centibars, giving irrigations of 3 to 4 hours at intervals of 14 to 21 days. Rainfall distribution in the orchard without irrigation is from June to September, with precipitation during these months over 1 000 mm which represents about 90% annually.

Climate characterization. With data from normal weather stations of the National Weather Service, a monthly climograph was built with time series 1981-2010 from the weather station 18 038 in Tepic, located in 21° 30' 00" north latitude and 104° 53' 00" west longitude with an altitude of 935 masl.

Meteorology of the study area. During the study period from May to September 2014, in each orchard were recorded hourly ambient temperature with automated loggers HOBO H8 (Onset Computer, Witzprod, Englewood Cliffs, NJ, USA). Using the database engine Microsoft Access 2010 temperature data from the temperature sensors were integrated from the beginning of the warm and dry period until harvest (May- September), calculating the number of days that exceeded the monthly historical average of maximum (Tmax) and minimum (Tmin) temperature; also quantified the monthly maximum maximorum (Mmax) and minimum minimorum (Mmin) temperature. To calculate accumulated heat units (UCA) per month the following expression was used:

Fruit collection. In each of the orchards, ten trees were selected and in each tree eight fruits were marked, four for "covered" treatment and four for "uncovered" treatment. The fruits from "covered" treatment were covered with a brown paper cone with the base open, once it reached olive size (2-3 cm in diameter). The fruits analyzed were originated by the main f lowering (winter 2013) and harvested in October 2014, with dry matter content in the pulp ≥ 21.5%. After harvest, the fruits were taken to the Fruit-physiology Laboratory from the Experimental Santiago Ixcuintla, where these were processed. The fruits were washed with distilled water with sodium hypochlorite (200 mg L^-1) and weighed on a precision balance (Ohaus model P2001, Florham, NJ, USA.). Length and diameter were obtained with a digital vernier (MTC500-196, Mitutoyo Co., Japan). Subsequently, the peel was separated from the pulp with a vegetable peeler. The peel was frozen at -20 °C until the time of preparing extracts in which the concentration of phytochemicals was determined.

Concentration of phytochemicals in peel. The concentration of total compounds and total chlorophy ll A, B, on ‘Hass' peel was determined spectrophotometrically in four replicates, each of which consisted of three fruits, each from different tree. Absorbance readings were made in triplicate. Total phenolic compounds (CFT) were quantified with the methodology proposed by ^{Rodríguez-Carpena et al. (2011)}: the extraction was performed in 10 g of peel with 30 mL of a mixture acetone: water (70:10). Once homogenized, the mixture was centrifuged at 2 500 rpm for 3 minutes at 4 °C. The supernatant was collected and the residue was subjected to the same extraction process again. Both extracts were combined. Total phenolic content of each extract was determined using the Folin-Ciocalteu method proposed by ^{Soong and Barlow (2004)} and modified by ^{Rodriguez-Carpena et al. (2011)}. Absorbance was measured at 765 nm and the total phenolic content was calculated from a standard curve of gallic acid and reported in mg equivalents gallic acid per gram of peel (mg EAG g^-1 peel). A portion of the extract obtained for the determination of total phenolic compounds was incubated in dark for 48 h at 4 °C for chlorophylls A, B, and totals (CA, CB and CT, respectively) analysis. The sample was centrifuged at 4 000 rpm and the supernatant absorbance was measured at 647 and 664 nm. The concentration of chlorophyll A, B and Total (mg L^-1) was determined with the following equations (^{Gandolfo-Wiederhold, 2008}) and reported in mg g-1 peel:

CA= 12.64 Abs_{664 nm}-2.99 Abs_{647 nm}

CB= -5.60 Abs_{664 nm}+23.26 Abs_{647 nm}

CT= 7.04 Abs_{664 nm}+20.27 Abs_{647 nm}

Statistical analysis. A factorial experiment 2 x 2, water management (irrigated and non-irrigated) and fruit cover (covered and uncovered) was used. The size of the fruit, according to weight, length and diameter, thus concentration of phyto chemicals were subjected to analysis of variance and mean test (Duncan, p< 0.05) with SAS.

Results

Climatology

From the climatological analysis of the period 1981-2010, it was observed that the monthly average temperature is above 15 °C; the warmest period occurs between May and September with an average Tmax above 26 °C; the cold period (December to March) has an average Tmin lower than 10 °C. From June to September 86% of the rain is present (1 100 mm) and the wettest month exceeds 400 mm (July). The annual average maximum and minimum temperature are 27.1 and 13.6 °C, respectively, while the annual rainfall is 1 286 mm (Figure 1).

Figure 1 Climograph for average monthly precipitation, maximum and minimum temperature in the study area from 1981 to 2010. 

Meteorology of the study period (May-September 2014)

The days above the historical average temperature (Tmax) and days below the historical average (Tmin) temperature were quantified. Orchards with irrigation accumulated 37 days with temperatures above average, while in orchards without irrigation were 85 days. In contrast, accumulated days in which temperature was below average temperature were 47 and 17 days for orchards with and without irrigation, respectively. The orchard without irrigation was hotter (greater amount of warm days and less cool days) than those without irrigation. The comparison showed that orchards with irrigation accumulated monthly fewer days with Tmax above average and a higher number of days with Tmin below average; while Mmax was higher in orchards without irrigation, ranging between 0.7 and 1.8 °C; Mmin in orchard with irrigation was the coolest, with differences between -0.2 and -1.5 °C. In orchard without irrigation accumulated 2 141.6 heat units, being higher than orchard with irrigation (1990.7) Table 1.

Table 1 Days with temperature higher than monthly average, extreme values and accumulated heat units. 

Fruit size

In orchard with irrigation, weight, length and diameter, were higher in uncovered than in covered fruits. For the orchard without irrigation, fruit size was not affected by fruit covered with paper (Table 2).

Table 2 Fruit size of ‘Hass’ avocado with different degrees of exposure to light and water management. 

^zMedias con la misma letra en una columna no presentan diferencia significativa, de acuerdo con la prueba de Duncan, p≤ 0.05.

Water management affected the size of uncovered fruits. The fruits of the orchard with irrigation had higher weight and length than orchard without irrigation. However, fruit diameter was not affected by water management (Table 3).

Table 3 Fruit size of uncovered ‘Hass’ avocado according to water management. 

^zMedias con la misma letra en una columna no presentan diferencia significativa, de acuerdo con la prueba de Duncan, p≤ 0.05.

Phytochemicals

In uncovered fruits both irrigated and not irrigated, showed a higher concentration of total phenolic compounds as well as chlorophyll A, B and total in peel (Table 4); moreover, in uncovered fruits, water management did not affect the concentration of phytochemical compounds analyzed (Table 5).

Table 4 Concentration of phytochemicals in fruit peel of ‘Hass’ avocado with different degrees of exposure to light and water management. 

^zCFT= compuestos fenólicos totales, mg equivalentes de ácido gálico (EAG) g^-1 piel. ^yCA= clorofila A, mg g-1 piel; ^xCB= clorofila B, mg g-1 piel; ^wC_T= clorofila total, mg g^-1 piel; ^vMedias con la misma letra en una columna no presentan diferencia significativa, de acuerdo con la prueba de Duncan, p≤ 0.05.

Table 5.Concentration of phytochemicals in fruit peel of’ Hass’ avocado exposed to light and with different water management. 

^zCFT= compuestos fenólicos totales, mg equivalentes de ácido gálico (EAG) g^-1 piel. ^yCA= clorofila A, mg g-1 piel; ^xCB= clorofila B, mg g-1 piel; ^wC_T= clorofila total, mg g^-1 piel; ^vMedias con la misma letra en una columna no presentan diferencia significativa, de acuerdo con la prueba de Duncan, p≤ 0.05.

According to the factorial analysis, the degree of exposure to light was the factor with high statistical significance (p< 0.01) for all the analyzed variables in the model. The interaction between both factors, exposure to light and water management, was significant only for fruit weight and fruit length (Table 6).

Table 6 Mean squares of the factorial model for water management and fruit coverage. 

** Significativo a p< 0.01; * Significativo a p< 0.05. FV= fuente de variación; GL= grados de libertad; MA= manejo del agua; CF= cobertura del fruto.

Discussion

Climatic conditions of the places where this work was developed match the description of a semi-warm climate described by Garcia (1964), where the annual average and the warmest month exceeds the thermal threshold of 18 °C and 22 °C, respectively. These climatic conditions are consistent with previous work in the study region, where it was determined the presence of favorable chilling for reproductive development of 'Hass' avocado (^{Salazar-García et al., 2007}; ^{Salazar-García et al., 2009}).

Water management affected fruit size as the orchard with irrigation produced heavier and longer fruits than the orchard without irrigation. This differs to that found by ^{Salazar-García et al. (2011)} in Michoacan where there was no difference in fruit weight of 'Hass' from orchards with irrigation and without irrigation. This divergence of results could be attributed that in Michoacan fruits grow in climates with higher annual accumulated rainfall and a wider distribution of rainfall during the year, which could result in lower water stress in the crop.

The concentration of total phenolic compounds in peel of uncovered fruit was lower than that reported by ^{Wang et al. (2010)}, who conducted the phytochemical extraction with a mixture of acetone: water: acetic acid (70: 29.7: 0.3, v/v/v), which could explain the difference between their results and those in this research as the extractant was acetone: water (70:10v/v). Certain phenolic groups such as flavonoids, act as a protective mechanism against ultraviolet radiation (^{Cheynier et al., 2013}), hence the fruits exposed to light have been those who had a higher concentration of total phenolic compounds. In grape (Vitis vinifera), uncovered clusters had higher contents of total phenolic compounds, compared with fruits that were covered with nets blocking part of the light (^{Leguizamón-M et al., 2008}).

The lowest chlorophyll content was in covered fruits, showed the stress condition to which fruits were subjected. In uncovered fruits chlorophyll content A, B and total, was higher than that found by ^{Wang et al. (2010)}, in 'Hass', who reported pigments of 0.019, 0.010 and 0.029 mg g-1 peel, respectively; this difference can be explained due to Wang et al. (2010) analyzed mature fruit ready for consumption, while in the present study the analysis was performed at physiological maturity. ^{Cox et al. (2004)} reported for 'Hass' a reduction in chlorophyll content between fruits with forest green and dark color.

The content of secondary metabolites usually has seasonal variations (^{Scogings et al., 2015}), which could explain the differences between the results of this study and the previously mentioned. By obtaining chlorophyll A / B ratio in uncovered fruits (1.73 and 2.08, orchard with and without irrigation, respectively) and in covered fruits (1.80 and 1.54, orchard with and without irrigation, respectively) was found that it was lower in the latter which was similar to that found in pine (Pinus pseudostrobus) and associated with a decrease in the amount of light (^{Cambron-Sandoval et al., 2011}).

Conclusions

Water management in 'Hass' avocado orchards did not modify the content of phytochemicals in peel. Regarding to the degree of exposure to solar radiation, uncovered fruits had a higher concentration of total phenolic compounds and chlorophyll, compared with those subjected to lack of light stress. As variation in solar radiation is a forcing agent of climate change, this factor should be considered when analyzing the production of phytochemicals in crops.