Certain Physiological and Chemical Indicators Drive the Yield and Quality of Cladode Mucilage in Three Fodder Nopal Morphotypes (Opuntia spp.) Under Different Soil Water Content Conditions

Abstract

Nopal cladode mucilage is a product of great importance in fodder, agri-food, industry, and health areas. This study aimed to evaluate the effect of three soil moisture contents on some physiological and chemical variables associated with the mucilage yield and quality of three morphotypes of fodder nopal (Opuntia spp.). A randomized block experimental design in a split–split plot arrangement with three replicates was used. The large plots represented the following soil moisture contents (SMC): optimum soil moisture content (OSMC) from 22 to 27%; suboptimum soil moisture content (SSMC) from 16 to 21%; and deficient soil moisture content (DSMC) from 10 to 15%. The subplots consisted of three cactus pear genotypes identified by the following IDs: C-CH, -C-NA, and C-HE. The relative water content (RWC) was significantly higher in the C-HE morphotype across each soil moisture content, and the lowest value was recorded in C-NA with OSMC; -C-CH had the lowest values in SSMC and DSMC, with 71.3% and 44.3%, respectively. There were slight variations in chlorophyll with SSMC; the C-NA and C-CH morphotypes had significantly higher chlorophyll contents, with values of 10.3 mg g⁻¹ 100 FW of chlorophyll a. and 5.87 mg 100 g⁻¹ FW of chlorophyll b. The C-CH morphotype had the best mucilage yield, with 800 mL kg⁻¹ FW and 712.6 mL kg⁻¹ FW in OSMC and SSMC, respectively; DSMC showed the lowest yield at 552.3 mL kg⁻¹ FW. The quality of cladode mucilage by treatment did not vary by soil moisture content or among nopal morphotypes. Additionally, there was a positive correlation among the relative water content and the chlorophyll a, b, and total chlorophyll contents with the yield of the nopal cladode mucilage, but not with the ash content or total solids as variables of mucilage quality vs. yield.

1. Introduction

Droughts in arid ecosystems have negative environmental effects, such as soil loss, low vegetation cover, and decreased biological productivity [1]. Droughts have intensified due to extreme weather events caused by climate change; they are changing high-rainfall areas into flood-prone areas and low-rainfall areas into drier ones, leading to desertification problems [2].

Nopal (Opuntia spp.) is an endemic plant genetic resource in arid ecosystems with an important role in biodiversity and productivity [3]. The Opuntia genus includes succulent plants that perform CAM photosynthetic metabolism, which, through the optimization of photosynthesis, allows for high water use efficiency and, therefore, adaptation to water scarcity [4]. These plants retain adequate water content and avoid hydric stress by maintaining high water potential in their tissues in dry environments as a mechanism to tolerate water stress [5,6]. In drought seasons, the mucilage in succulent plants, such as nopal, is the main structural component that increases the water storage capacity of the apoplast [7], which not only becomes a source of survival for native fauna during droughts but also for extensive livestock that feeds on succulent cladodes with high water and fiber content [8]. Nopal cladode mucilage is a compound of parenchymal and collenchyma tissue, and it is the cellular site with the highest concentration of fresh matter, which is the main fodder for livestock during dry seasonal times in arid zones [9]. Indeed, the nopal cladode mucilage is part of the tissue that has the ability to store water in the apoplast [10] in times of extreme drought [8,11].

Nopal mucilage is recognized as a long-chain polyelectrolyte [12] composed of negatively charged functional groups throughout the molecule, which gives it a viscous property [13,14]. It is a hydrocolloid compound based on arabinose (10.1–44.0%), xylose (5.1–22.1%), galactose (20.4–33.0%), galacturonic acid (0.18–18.5%), and rhamnose (4.5–15.70%) [10,14]. Because of all these characteristics, nopal mucilage is a product that is used on the market in the fodder, medical, cosmetic, agri-food, and industrial areas [10,11]. The mucilage of this plant contains not only water and fiber but also different active chemical elements with nutritional, pharmaceutical, and industrial value [11,13,14].

From the point of view of the industry, nopal mucilage is a water-soluble compound that stands out for its benefits as a thickening agent, gelling agent, stabilizer, antioxidant, and coating film on fresh fruits and vegetables to improve shelf life [15,16]. Rebah and Siddeeg [17] reported that nopal mucilage is used for the treatment of water contaminated with heavy metals, for the control of fecal coliforms, and for reducing foul odors; it is also used as an additive in paints, an adhesive in construction, and as a natural waterproofing material for roofs [18].

Sáenz et al. [19] reported that nopal mucilage is beneficial for glycemic control; Alarcón-Aguilar [20] identified a polysaccharide with hypoglycemic properties in the mucilage; and Basurto and Magos [21] found that it is associated with lowering cholesterol, protecting the gastric mucosa from ulcers, and providing analgesic and anti-inflammatory properties.

Nopal is widely consumed as a fresh vegetable when the cladode is in the early stages of development and as a fresh fruit when the plant reaches the production stage of the fruit, which is called tuna [22]. As fresh vegetables, the quantity and quality of the mucilage are decisive factors in the selection of the cultivar for culinary use [23]. Still, it is also essential as a fodder crop for extensive livestock farming during the dry season, which is characterized by low rainfall and a scarcity of grasses due to the low water content in the soil [24].

Although the nopal is a crop with wide distribution and use in dry lands, research continues into technologies to improve its management and obtain advantages such as higher cladode quality and quantity, mainly in terms of mucilage yield, which is the most important indicator of fodder and agri-food in nopal production [25,26]. This plant species is used as an emergency fodder source for livestock in arid regions due to its high water content and fiber-rich mucilage. However, little is known about how soil moisture content influences mucilage yield and its nutritional composition. Additionally, differences among nopal genotypes in mucilage production and their suitability as livestock fodder remain unclear. Understanding these variations is essential for improving nopal’s efficiency as a drought-resilient fodder crop. This study aimed to evaluate three nopal genotypes under different soil moisture contents and their response to various physiological and chemical characteristics associated with the yield and quality of the mucilage in northern Mexico.

2. Materials and Methods

2.1. Location of the Study Area

The experiment was carried out from 2019 to 2020 at the Unidad Regional Universitaria de Zonas Aridas (URUZA) of the Universidad Autónoma Chapingo (UACh) in Bermejillo, Durango, Mexico. The experimental site is located at 23°54′ NL and 103°37′ WL, at an average elevation of 1100 m. The area has a dry, desert-type climate, with an average annual rainfall of 258 mm, mainly in summer and autumn, and the average temperature is 21 °C, with a maximum of 33.7 °C and a minimum of 7.5 °C [27].

2.2. Genetic Materials of the Nopal Used in the Experiment

The genetic materials of the nopal (Opuntia ssp.) used in this study are part of the Nopal Germplasm Bank of the URUZA-UACh in Bermejillo, Durango, Mexico, which consists of 55 collections of nopal from different ecological regions of the country, mainly from the states of Mexico, San Luis Potosí, Coahuila, Aguascalientes, and Zacatecas [28]. They are the product of natural crosses that share characteristics similar to those of the species Opuntia megacantha and Opuntia ficus-indica. The three genetic materials used in this study have not been taxonomically identified to the variety level yet; they have been grouped by morphometric similarities [29] and by using isoenzymatic technics [30]. Based on these studies, the three nopal genetic materials correspond to groups with similar characteristics among themselves but differ from each other and have been conventionally classified as morphotypes with the following ID keys: C-CH, C-N, and C-EH. The asexual reproduction of the nopal is the most common cultivation method, using cladodes that are planted in commercial nopal orchards. The rainfall was recorded using a Davis Instruments model 6162 microclimatic station (Hayward, CA, USA) located 600 m from the experimental area.

2.3. Experimental Design and Treatment Arrangements

A randomized block design in a split-plot arrangement with three replicates was used. The large plots represented the following soil moisture contents (SMC): optimum soil moisture content (OSMC) from 22–27%; suboptimum soil moisture content (SSMC) from 16–21%; and deficient soil moisture content (DSMC) from 10–15%, corresponding to SMC averages of 24.5%, 18.5%, and 12.5%, respectively. The subplots consisted of three nopal morphotypes identified as C-CH, C-NA, and C-HE. They were named with these codes since these genetic materials were collected from three different nopal ecological areas in Zacatecas and San Luis Potosi States, Mexico; they are not yet genetically characterized, but phenotypic differences have been recognized. The experimental unit (treatment) consisted of four rows of nopal, each 8 m long, in double rows with 0.75 m between rows and 50 cm between plants in each row. In each treatment, 16 plants were planted, and there were 27 treatments across three replicates; thus, there was a total of 648 plants in the whole experimental area. Four plants were randomly selected from the two middle rows of each treatment to measure the variables. (Figure 1 and Figure 2). In addition, the experimental area was 24 m long and 30.6 m wide, with a total surface area of 734.4 m², which corresponds to a relatively small area, which was selected for its similar soil characteristics. A random block design was used based on the assumption that the area could be heterogeneous in its physical and chemical characteristics. Analysis of variance performed with the database confirmed that the soil was homogeneous since no significant statistical differences were found among the blocks.

2.4. Irrigation System Set Up

Irrigation regime performance was evaluated on the basis of the physicochemical characteristics of the soil, which were determined beforehand; the apparent density was 1.2 g cm⁻³, the capillarity exhibited values of water sheet = 2.3669 cm and t = 0.4215 min, the infiltration rate was 1.3 cm h⁻¹, the average daily evapotranspiration was 11 mm, and an experimental evaporation coefficient of 70% was obtained. The field capacity (FC) was 27%, and the permanent wilting point (PWP) was 13%, according to a soil moisture drawdown curve (Figure 3) determined using the membrane pot method [31]. In the 10–15% soil moisture content range, the lower limit (10%) was lower than the PWP (13%), which induced water stress. This is because the capacity to tolerate water deficit is very high in succulent plants such as nopal, which uses the Crassulaceae Acid Metabolism (CAM) photosynthetic pathway [5].

For the watering of the experiment, according to Figure 1, a pressurized irrigation system was used. A main PVC sprinkler with lateral connections was installed for each treatment, controlling the irrigation time using a stopcock. An irrigation hose was used with drippers spaced 0.5 m apart. Irrigation times were calculated according to the method proposed by Jiménez-Galindo and Acosta-Gallegos [32]. During the first two weeks, the experimental area was irrigated at CC (27%). After this time, the differentiation of the three soil moisture regimes was established: 22–27%, 16–21%, and 10–15%. Each soil moisture regime was allowed to reach its upper limit and then allowed to reach its lower limit: 22%, 16%, and 10%, respectively. After that, each moisture range was restored to its upper limit, with 6% soil moisture in each range, corresponding to 27%, 21%, and 15%, respectively. This recovery was achieved in approximately 2.5 h between each irrigation time across the three soil moisture regimes.

The soil moisture percentage was monitored daily using a Lutron brand digital meter, model PMS-714 (Lutron Electronic Enterprise Co., Taipei, Taiwan). With this sensor, real-time readings of soil moisture were obtained, generating precise information for irrigation control and adjustment in the experiment. The average annual rainfall is extremely low in the study area, with an average of 258 mm per year [27]. The rainfall recorded in 2024 was 192.6 mm, and the main pluvial precipitation occurred from July to September, with values of 93.2 mm, 39.8 mm, and 44.2 mm in July, August, and September, respectively. To reduce the influence of this rainy period on the soil moisture contents evaluated in this study, after each rainfall event, drip irrigation was suspended until each soil moisture content reached its lower limit, and then irrigation was restarted until the upper limit of each soil moisture content was reached.

2.5. Variables

Measurements of variables started six months after the experiment was established.

2.5.1. Physiological Variables Measured

The relative water content (RWC) (%) was calculated using the following equation proposed by Kramer [33]:

$$\mathrm{R}\mathrm{W}\mathrm{C}=\left[{\displaystyle \frac{\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}-\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}{\mathrm{T}\mathrm{u}\mathrm{r}\mathrm{g}\mathrm{i}\mathrm{d} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}}\right]\times 100$$

The mucilage moisture content (MMC) (%) was determined using 2 g of fresh biomass sample, which was weighed and subsequently placed in a crucible to be dried in a HAFO^® brand oven, model 1600 (Radnor, PA, USA), at 135 °C ± 2 °C for 2 h. The MMC was calculated using the following formula:

$$\mathrm{M}\mathrm{M}\mathrm{C}=\left[{\displaystyle \frac{\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}{\mathrm{F}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}}}\right]\times 100$$

Photosynthetic pigments refer to chlorophyll a content (mg 100 g⁻¹ FW), chlorophyll b content (mg 100 g⁻¹ FW), and total chlorophyll content (mg 100 g⁻¹ FW), which were determined according to the methodology described by Wellburn [34]. Fresh nopal cladodes were sliced into 5 mm circles. The cladode samples were placed in a test tube; then, 10 mL of methanol (CH₃OH) was added and incubated in the dark at room temperature for 24 h. Absorbance (A) was measured with a spectrophotometer at 653 nm (chlorophyll b = Chl b) and 666 nm (chlorophyll a = Chl a). The calculation of the pigment concentration was performed using the following formulas [4]:

Chlor a = [15.65·(A₆₆₆) − 7.34·(A₆₅₃)] [V/1000 × FW]

Chlor b = [27.05·(A₆₅₃) − 11.21·(A₆₆₆)] [V/1000 × FW]

TChlor = [Chlor a + Chlor b]

where V = extraction volume, and FW = fresh weight of plant tissue. The concentrations of Chl a, Chl b, and TChlor = total chlorophyll. All variables are expressed in mg per 100 g of fresh weight (FW).

2.5.2. Quality and Quantity of Mucilage Production

The pH was determined in a mucilage suspension using a Conductronic brand potentiometer, model PH140 (Puebla, Mexico).

Using the method described by Wang and Strong [35], the ash percentage was calculated according to the official Mexican standard: NMX-F-066-S-1978 (1978). After two hours at 540 °C in a muffle furnace, a porcelain crucible was taken out and filled with two milliliters of pure nitric acid. Before its ultimate weight (W1) was determined using an analytical balance AY220 model (Shimadzu Corporation, Kyoto, Japan), the crucible was allowed to cool for an hour in a desiccator after being dried on an electric heating plate Thermo Scientific SP131635Q model (Clarkson Laboratory and Supply Inc., San Diego, CA, USA) to evaporate the acid. After adding around 2 g of mucilage (W2), the crucible was put back in the muffle furnace and heated to 540 °C for 2 h. The crucible was then taken out, allowed to cool for 1 h, and weighed once again using the dried sample (W3). The ash percentage was calculated using the following equation:

$$\mathrm{A}\mathrm{s}\mathrm{h} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\left(\mathrm{\%}\right)=\left[{\displaystyle \frac{\mathrm{W}3-\mathrm{W}1}{\mathrm{W}4}}\right]\times 100$$

where W1 is the weight of the empty crucible, W2 is the weight of the crucible with more fresh or partially dried sample, W3 is the weight of the crucible with more ash, and W4 is the weight of the fresh or partially dried sample (W2 − W1).

The total solids content was determined according to the methodology cited by Wang and Strong [35] using a refractometer PAL-1 model (ATAGO, Saitama, Japan). The determination was made at room temperature using 5 g of mucilage in a porcelain crucible. Mucilage samples were oven-dried at 105 °C for 24 h. The methanol-precipitated solids were derived from the weight of total solids. Then, to each dry gel sample, 25 mL of 99% methanol was added and placed in 50 mL Eppendorf tubes, and the tubes were shaken at medium speed Thermo Scientific 2346 model (Capitol Scientific, Inc., Austin, TX, USA) for 30 min at room temperature. Therefore, tubes were allowed to stand for 12 h and then centrifuged. The precipitate was washed three times with 25 mL of methanol. The samples were placed in test tubes and oven-dried for 24 h at 45–60 °C. Finally, the test tubes were placed in a desiccator to cool at room temperature.

Mucilage production (mL kg⁻¹ FW) was obtained using the methodology proposed by Pérez et al. [36], which consists of cutting 1 kg of the nopal leaf cladode into small pieces and leaving them to macerate in distilled water for 24 h. Subsequently, using a 40-mesh sieve, the mucilaginous suspension was filtered to remove fiber and cuticle residues, and using a test tube, the amount of mucilage produced in mL kg⁻¹ FW was measured. The mucilage yield, in this case of the nopal cladode, was determined by the weight–volume relationship [24].

2.6. Data Analysis

A variance analysis and Tukey’s multiple range test (p < 0.05) were performed on the database to identify the effects among treatments; additionally, a simple Pearson correlation analysis was conducted. All analyses were performed using Minitab 16 and SAS Version 9.0 software. Moreover, the Excel V. 6.0 program was used to perform regression analysis, which allowed us to identify the relationships between cause variables and effect variables.

3. Results

3.1. Description of Some Morphological Characteristics of the Nopal Morphotypes

As part of this study, some morphological characteristics of the nopal genetic materials were recorded. The C-CH morphotype is a plant with large, thick, dark green cladodes that are circular or racket-shaped and without thorns. It has separate areoles, with a low presence of glochidia (small thorns) grouped in averages of 10 to 15; when the plant bears fruit, the flowers and fruits are red. The C-N morphotype is a plant with large, light-dark green cladodes, which are elongated and slightly thin, with a low presence of thorns measuring 1 to 1.5 cm in length and white in color, along with small sharp lateral thorns and separate areoles with glochidia (small thorns) grouped in averages of four to seven; when the plant bears fruit, the flowers and fruits are yellow. The C-EH morphotype is a plant with large, elongated, and flat pale green cladodes, with a regular presence of thorns measuring 1 to 1.5 cm in length and white in color, along with small sharp lateral thorns, regularly separated areoles, and glochids (small thorns) grouped in the areoles in averages of four to seven; when the plant bears fruit, the flowers and fruits are yellow.

3.2. Measured, Chemical, and Physiological Variables

Relative water content (RWC) is the percentage of water that can be stored in plant tissues [5]. Physiologically, it is related to the water potential of the plant (⨚p) since this and its components, referred to as the turgor potential (⨚t) and the osmotic potential (⨚o), are a function of the volume of water in the protoplasm [37]. In this study, the RWC decreased when the soil moisture content was reduced. The highest values (p < 0.05, n = 6, g.l. = 5) corresponded with RWC values of 82.5%, 78.5%, and 67.2% in the C-HE morphotype under OSMC, SSMC, and DSMC conditions, respectively, compared to the other two nopal genotypes; the lowest values were observed in the DSMC in the C-HE and C-NA morphotypes, with 67.2% and 63.9%, respectively. No statistical difference among them was found, compared to 44.3%, which was the lowest RWC value in C-CH (Figure 4).

Chlorophyll is a pigment that enables photosynthesis [38]. Maintaining consistent pigment levels during water stress is crucial for photosynthesis, stress tolerance, growth, and survival in multiple species; however, we are focused on Opuntia spp., which thrives in challenging environments [39]. Pigment content was not affected by extreme soil moisture contents (OSMC and DSMC) and only showed statistical differences in SSMC (16–21%); the C-CH and C-NA morphotypes showed higher values of chlorophyll a, chlorophyll b, and total chlorophyll. The C-HE genotype recorded lower values in the median soil moisture content (

Table 1. Average value of chlorophyll a, b, and total chlorophyll content (mg g⁻¹ FW) in three genotypes of nopal (Opuntia spp.) under different soil moisture content conditions.

Nopal Morphotypes	Soil Moisture Content (%)
	OSMC (22–27)			SSMC (16–21)			DSMC (10–15)
	Chl a	Chl b	TChl	Chl a	Chl b	TChl	Chl a	Chl b	TChl
C-CH	9.44 a ±0.08	5.3 a ±0.56	14.74 a ±0.47	10.2 ab ±0.07	5.87 a ±0.02	16.11 a ±0.10	5.31 a ±0.29	4.80 a ±0.65	10.11 a ±0.94
C-HE	9.34 a ±0.05	4.7 a ±0.0	14.03 a ±0.05	10.1 b ±0.05	4.72 b ±0.00	14.86 b ±0.05	5.20 a ±0.18	4.44 a ±0.07	9.64 a ±0.24
C-NA	9.52 a ±0.09	5.0 a ±0.57	14.55 a ±0.53	10.3 a ±0.13	4.74 b ±0.01	15.10 ab ±0.14	5.27 a ±0.04	4.40 a ±0.07	9.67 a ±0.06

Tukey test (p ≤ 0.05). Figures with the same letters in the same column are not significantly different. OSMC = optimum soil moisture content; SSMC = suboptimum soil moisture content; DSMC = deficient soil moisture content; Chl a = chlorophyll a; Chl b = chlorophyll b; TChl = total chlorophyll; ± = standard deviation.

).

3.3. Quality and Mucilage Yield

The physicochemical characteristics of mucilage and its yield mainly depend on the physiological response of the plant to the environment [40]. In this study, the pH of the mucilage was lower than the neutral value, with a statistical effect (p ≤ 0.05, n = 6, g.l.= 5) at the optimum soil moisture content (24.5% ± 2.5) between the C-NA and C-CH morphotypes, with values of 5.02 and 4.76, respectively; the C-HE morphotype exhibited intermediate values between the other two previously mentioned genotypes. In addition, the mucilage moisture content, associated with water content in the plant tissue, did not vary significantly among nopal genotypes at the different soil moisture contents (

Table 2. Average acidity, mucilage moisture content, and mucilage yield in three morphotypes of nopal (Opuntia spp.) under different soil moisture content conditions.

Nopal Morphotypes	Soil Moisture Content (%)
	OSMC (22–27)			SSMC (16–21)			DSMC (10–15)
	pH	MMC (%)	MY (mL kg−1 FW)	pH	MMC (%)	MY (mL kg−1 FW)	pH	MMC (%)	MY (mL kg−1 FW)
C-CH	4.76 b ±0.03	98.2 a ±0.18	800.0 a ±0.0	4.55 a ±0.03	97.6 a ±0.03	712.6 a ±2.5	4.45 a ±0.03	97.1 a ±0.07	552.3 b ±7.5
C-HE	4.81 ab ±0.08	98.2 a ±0.01	789.3 c ±1.1	4.57 a ±0.05	97.5 a ±0.02	701.3 b ±4.1	4.49 a ±0.07	96.9 b ±0.11	571.0 a ±7.9
C-NA	5.02 a ±0.08	98.2 a ±0.06	795.6 b ±1.1	4.57 a ±0.03	97.5 a ±0.03	706.0 b ±5.2	4.48 a ±0.02	97.0 ab ±0.02	573.3 a ±5.7

Tukey test (p ≤ 0.05). Figures with the same letters in the same column are not significantly different. pH = hydrogen potential as an indicator of mucilage acidity; OSMC = optimum soil moisture content; SSMC = suboptimum soil moisture content; DSMC = deficient soil moisture content; MMC = mucilage moisture content; MY = mucilage yield; ± = standard deviation.

).

Nopal mucilage yield was significantly higher in the C-CH genotype under optimum and suboptimum soil moisture content conditions, with values of 800 kg⁻¹ FW and 712.6 mL kg⁻¹ FW, respectively. However, this same genotype showed the lowest mucilage yield value, with only 552.3 mL kg⁻¹ FW. In contrast, the C-NA and C-HE genotypes showed a high yield of mucilage under deficient soil moisture content conditions (Table 2).

The different genotypes and soil moisture contents examined did not affect the ash content and total solids content of the nopal mucilage in this study, which means that these variables are independent of the quality of the nopal cladode mucilage. The values were similar among treatments, varying only from 2.36% to 4.14 in ash content and from 5.6% to 6.3% in total solids (

Table 3. Average values of ash content and total solids of the mucilage of three morphotypes of nopal (Opuntia ssp.) under different soil moisture content conditions.

Nopal Morphotypes	Soil Moisture Content (%)
	OSMC (22–27)		SSMC (16–21)		DSMC (10–15)
	Ash Content (%)	Total Solids (%)	Ash Content (%)	Total Solids (%)	Ash Content (%)	Total Solids (%)
C-CH	2.44 a ±0.00	6.0 a ±0.00	2.56 a ±0.02	6.0 a ±0.00	4.14 a ±0.05	6.0 a ±0.00
C-HE	2.42 a ±0.07	6.3 a ±0.57	2.52 a ±0.02	6.3 a ±0.57	4.11 a ±0.02	6.0 a ±0.00
C-NA	2.36 a ±0.07	5.6 a ±0.57	2.51 a ±0.02	6.0 a ±0.00	4.08 a ±0.00	6.0 a ±0.00

Tukey test (p ≤ 0.05). Figures with the same letters in the same column are not significantly different. OCMC = optimum soil moisture content; SSMC = suboptimum soil moisture content; DSMC = deficient soil moisture content; ± = standard deviation.

).

3.4. Correlation Analysis

A positive correlation between nopal mucilage yield and physiological variables was found, except for the ash content and total solids. The ash content exhibited a significant negative correlation, and the total solids exhibited no significant correlation. Nopal mucilage yield is directly associated with the contents of chlorophyll a, b, and total chlorophyll since these were highly statistically significant (p < 0.01) for chlorophyll a and total chlorophyll and statistically significant (p < 0.05) for chlorophyll b content (

Table 4. Correlation analysis between nopal mucilage production and physiological variables.

	MMC	AC	MY	TS	Chl a	Chl b	TChl
MY	0.95721 <0.0001	−0.94340 ** <0.0001	1.00000	0.00227 0.9910	0.85278 ** <0.0001	0.42214 * 0.0283	0.84003 ** <0.0001

Pearson correlation analysis (p ≤ 0.05). MMC = mucilage moisture content; AC = ash content; MY = mucilage yield; TS = total solids; Chl a = chlorophyll a; Chl b = chlorophyll b; TChl = total chlorophyll. * p < 0.05, ** p < 0.01.

).

4. Discussion

First, the morphological description of the three genetic materials of nopal used in this study suggests that they are genetically distinct materials that could possibly be recognized as varieties descended from Opuntia megacantha and O. ficus-indica. This could be determined through phylogenetic studies that are carried out in the future within this same line of research.

This study examined key physiological and chemical factors affecting mucilage yield and quality in fodder nopal, which is crucial for the plant’s drought tolerance. The results demonstrated the genotypes’ resilience in water-limited environments by revealing notable variations in their capacity to tolerate soil moisture fluctuations while retaining production. Because of its versatility, Opuntia spp. is a crucial crop for dry and semi-arid areas, providing a long-term solution to problems with water shortages in agricultural systems. The findings highlight how these genotypes might promote resource efficiency and food security in regions that are vulnerable to drought.

The response of nopal genotypes to varying soil moisture contents demonstrates significant adaptability, particularly in the recovery of cladode turgidity. The flaccid state of the cladode in C-CH, caused by a low RWC, is reversed when moving from DSMC to OSSM, with results comparable to the best response observed in the C-HE morphotype. This suggests that the C-CH morphotype has the flexibility to adapt under environmental stress conditions by tolerating low water potential at the cellular level under conditions of a water deficit in the soil and is capable of recovering its water status, as reflected in the relative water content, under favorable soil moisture content conditions [41,42], which corresponds to a capability reported in various plant species [43]. The optimum or deficient availability of water in the soil directly affects the water content of the plants, even though they have specific mechanisms for tolerating water deficits [44] since declining water availability triggers different adaptive processes to cope with water deficit stress [45]. This response is related to the mechanisms of escape, evasion, and tolerance, which suggests that the Opuntia genus evades dry environments by maintaining high water potential in its tissues as a plant in the photosynthetic pathway group of Crassulaceae Acid Metabolism (CAM) [46,47]. This tolerance exists within certain limits to maintain adequate development, growth, and production of fresh biomass [5,48].

The relative water content (RWC) of the three nopal genotypes showed variations of 5% to 10% under optimum and suboptimum soil moisture content conditions, while under deficient moisture conditions, the differences increased to 30%. These results partially align with those of Pimienta-Barrios et al. [46], who reported RWC values of 82–87% in the wet season and 60–82% in the dry season. However, their study was conducted in a temperate climate with higher soil moisture retention, which explains the discrepancies observed. These physiological processes require further elucidation [49].

The response of nopal plants to pigment content reveals notable stability under varying environmental conditions. This suggests that even with fluctuating soil moisture and other external factors, pigmentation remains stable due to an interaction between genotype and crop conditions. Such stability in pigmentation also raises the question of how other physiological responses, like chlorophyll content, react to water stress, which is explored in the next section [50,51].

While the pigments in the cladodes show tolerance to soil moisture changes, chlorophyll content appears more sensitive, revealing distinct physiological responses. This contrast suggests that, whereas pigmentation remains stable, chlorophyll dynamics may be influenced by gene expression and environmental interactions, allowing some genotypes to regulate photosynthesis more efficiently under water stress. The acid metabolism of Crassulaceae (CAM) in Opuntia spp. allows for high efficiency in the use of water by fixing CO₂ during the night, reducing daytime perspiration, and maximizing water retention in the tissues. This metabolic strategy is essential in arid and semi-arid environments, as it provides a physiological advantage in terms of maintaining cell turgor and biomass production under conditions of water deficit. However, it is important to note that other studies have shown variations in chlorophyll content under different water stress conditions [44], as was shown in this study, in which the median soil moisture content (16–21%) between the field capacity (FC) and permanent wilting point (PWP) resulted in the nopal genotypes showing significant differences. Nevertheless, this was not observed when the soil moisture content regimes was extremely close to FC or PWP. This effect on the soil moisture content could be associated with the response of pigments, which may be complex and influenced by various genetic and environmental factors [48,52].

However, the chlorophyll content in the evaluated nopal morphotypes was lower, ranging from 4.4 to 16.1 mg g⁻¹ FW. These values align with those reported by Maki-Díaz et al. [53] in nopal destined for national consumption and export. This lower chlorophyll content might indicate a trade-off between water conservation and photosynthetic efficiency in these genotypes, which is crucial for their adaptation to arid environments [54]. Notably, cladode age and season, irradiance, temperature, water availability, and fertilizer delivery may all affect chlorophyll content. Also, in these plant species, the chlorophyll content is higher, as reported by González-Espíndola et al. [50] in fodder clover, Lotus corniculatus, with values ranging from 61.4 to 194.6 mg g⁻¹ FW. This is congruent since the nopal is a succulent plant with low photosynthetic activity and slow biomass production, in comparison to Lotus, which is a fodder species with high photosynthetic activity, a high growth rate, and high biomass production [55].

From the perspective of chemical characteristics, the acidity reported in this study coincides with that reported by authors such as Betancourt-Domínguez et al. [48] and Aguilar-Sánchez et al. [56]. pH can fluctuate depending on several factors, such as crop variety, growth conditions, and mucilage processing [9]. In some cases, the required acidity level can serve as a good physiological indicator, depending on the focus. The mucilage yield results obtained are similar to those reported by Galicia-Villanueva et al. [24], who showed values of 816.4 mL kg⁻¹ FW. This implies that the nopal mucilage output fluctuates in response to the interaction between the plant’s genetic makeup and the soil’s moisture content [14]. Since nopal mucilage is primarily composed of water, its yield is related to the availability of water in the soil, further emphasizing the critical role of soil moisture in determining mucilage content and overall yield. [57]. According to Xiong et al. [58], the soil moisture content influences C₄ and CAM metabolism, stomatal closure, and changes in gene expression, including those encoding potentially protective proteins, key enzymes in the osmolyte synthesis pathway, antioxidant enzymes, and transcription factors. These factors regulate stress-induced gene expression through the signaling pathways of the hormonal action of abscisic acid that lead to plant tolerance to water stress, which could explain the differential behavior among the nopal morphotypes evaluated in this study.

However, the moisture content of the mucilage did not show significant differences in the percentages of soil moisture among the nopal genotypes evaluated in this study. However, Rodríguez-Félix and Cantwell [57] obtained values ranging from 92 to 95% for this variable, suggesting that this difference in moisture in the cladodes is mainly due to their size. Additionally, RWC is closely related to mucilage production capacity under median soil moisture (18.5% ± 2.5). The mucilage effect was shown at a genetic level since the C-CH morphotypes had higher mucilage content under OSMC and SSMC conditions; however, the same genotype’s mucilage production was more affected under DSMC conditions (12.5% ± 2.5). The C-NA and C-HE morphotypes showed a moderate effect with this variable. Thus, mucilage yield is more influenced by genetic effects than by soil moisture content [12], at least for the regimes evaluated in this study. This suggests the need for molecular studies of genetic expression, as reported by Tomkowiak et al. [59], to identify the different responses to water deficits and performance among different genetic materials, such as the nopal morphotypes evaluated in this study.

The findings regarding mucilage quality show that at least in the soil water contents evaluated in this study, water shortage had no discernible effect on nopal mucilage quality. This means that the cactus plant has a high tolerance to stress, and the lowest level of soil moisture included in this study may not have been low enough to influence mucilage quality in response to hydric stress. Reports on other succulent crops, such as aloe, have linked water deficiency in the soil to better mucilage quality [43,60], which means that this plant could be more sensitive to water deficits. Studies on other plant species that have been subjected to environmental stress, however, have shown detrimental effects on the amounts of ash and total solids [61,62]. The ash content and total solids values linked to water deficit show that these parameters are affected by several variables, such as the genotype, growth conditions, and the duration and intensity of the water deficit [40]. These findings highlight how crucial it is to take species-specific and environmental interactions into account when evaluating the impact of water stress on mucilage quality. This study observed no significant changes in mucilage quality under water deficit conditions. However, research on other succulent species, such as aloe, has shown an increase in viscosity and polysaccharide concentration under stress [43]. These differences may stem from variations in mucilage polysaccharides’ structure and gene regulation across species. In Opuntia spp., mucilage production appears to be more influenced by genetics than by water availability, showing its functional stability in the genotypes. However, it is important to evaluate other physicochemical parameters, such as the molecular composition of the mucilage and its water-holding capacity, to better understand its response to water stress.

The positive correlation between nopal mucilage yield and physiological variables, such as tissue water content, chlorophyll a, and total chlorophyll, highlights the critical role of these indicators in mucilage production, as previously reported by Hernández and Briones [63]. Chlorophyll is a green pigment in plants that absorbs light energy and transforms it into chemical energy through photosynthesis, which is then used to synthesize organic compounds [64] such as nopal mucilage. The nopal morphotypes differ in the intensity of the green color of the cladode, which is stronger in the case of the C-CH morphotype, which may explain the different responses among the nopal genetic materials evaluated in this study. These findings demonstrate the nopal’s remarkable tolerance and adaptive capacity to water deficits, influenced by the genetic material used, with variations in mucilage yield but consistent quality. This relationship between key physiological traits and mucilage production underscores the importance of nopal as a resilient crop, particularly for use as animal fodder in arid and semi-arid livestock regions, as emphasized by Maniçoba et al. [65]. In addition, Parent et al. [66] showed that ABA affects plant hydraulic properties through aquaporin activity, which contributes to the maintenance of a favorable plant water status. The findings of this study suggest that the genetic materials evaluated in this study may serve as a basis for selecting and improving tolerance to water stress in livestock regions in arid zones. This would allow for the availability of genetic materials that exhibit greater efficiency in the use of water and improve the quality of fresh matter for animal feed in dry areas.

Finally, this study shows how different nopal morphotypes tolerate water stress and its effect on mucilage production; however, some limitations should be noted. Future studies should incorporate transcriptomic and metabolomic analyses to further understand the molecular mechanisms underlying these responses. In addition, evaluating the stability of mucilage under different storage conditions and its potential application in the agri-food industry could provide valuable information for its use in sustainable production systems in arid and semi-arid regions.

5. Conclusions

This study highlights the potential of Opuntia ssp. morphotypes for sustainable agricultural practices by demonstrating their resilience and adaptation to changing soil moisture content in arid and semi-arid locations. The findings indicate that certain physiological and chemical markers related to mucilage yield are significantly influenced by water availability. The C-CH morphotype produced the most mucilage under both optimal soil water and near-wilting conditions. The C-NA and C-HE morphotypes, on the other hand, showed a low level of resilience in the face of deficient watering. Water availability boosts productivity, as shown by the direct correlation between mucilage yield and physiological traits like relative water content (RWC) and chlorophyll content. Mucilage quality measures, such as pH, ash content, and total solids, were stable among genotypes despite differences in soil moisture content, suggesting that genetic variables have a more significant influence on mucilage quality than environmental factors. The results highlight the suitability of Opuntia ssp. as a crop for agricultural systems that are resource-efficient and climate-resilient, promoting food security and the production of animal feed with high mucilage yields while preserving quality in water-limited environments.