Altitudinal Gradient Drives Rhizosphere Microbial Structure and Functional Potential in Prickly Pear Cactus (Opuntia ficus-indica L.)

Abstract

The prickly pear cactus (Opuntia ficus-indica L.) is an emblematic crop for Mexico’s economy, gastronomy, and culture. Microbial communities play an important role in the health, development, and productivity of crops. This study used 16S rRNA high-throughput sequencing and bioinformatic analyses to evaluate the rhizosphere microbiome of prickly pear cactus across an altitudinal gradient in Milpa Alta (Mexico). A microbial core consisting of Bacillus, Acidibacter, and Sphingomonas was detected, reflecting strong co-adaptation between plants and soil microorganisms under different agroecosystems. However, in the lower-altitude zones, Conexibacter, Agromyces, Domibacillus, Pedomicrobium, and Rokubacteriales predominated, which are associated with humid environments and high organic matter content. In contrast, in the middle-altitude zones, Acidothermus, Gemmatimonas, Mesorhizobium, and Pseudoxanthomonas were enriched, which are involved in carbon and nitrogen cycles. Higher-altitude zones exhibited greater bacterial specialization, with genera adapted to more extreme conditions such as Halocella, Solirubrobacter, Rhodomicrobium, Phenylobacterium, Roseomonas, Pseudarthrobacter, Crossiella, Aquicella, and others. Overall, our data show that altitude acts as an ecological filter structuring soil microbial communities associated with prickly pear cactus, influencing the diversity and functional potential. This study on microbial diversity not only provides insights into the health of the agroecosystem but also represents a valuable source of microorganisms with functional potential for sustainable agriculture.

1. Introduction

The genus Opuntia of the Cactaceae family contains almost 300 species, although only a dozen are cultivated for agro-industrial purposes, such as food production, fodder, and breeding of the carmine cochineal insect (Dactylopius coccus) [1]. This genus is distributed throughout the American continent from Canada to Patagonia [2]. Among the most commonly consumed species, the prickly pear or prickly pear cactus (Opuntia ficus-indica L.) has historical and cultural significance in Mexico [3]; its cladodes (pads) and fruits (cactus pear) represent a fundamental pillar of national agriculture due to their nutritional, ecological, economic, and ethnobotanical value [4]. Mexico is the world’s leading producer of prickly pear, with more than 864,000 tons annually, accounting for 5.1% of its horticultural production [5,6].

Prickly pear producers face limitations associated with poor agronomic and input management, environmental variability, water scarcity, soil degradation and pest and disease pressure, all of which can negatively impact yield, crop quality, and long-term sustainability. In this context, one of the main diseases affecting cladodes from Opuntia is black spot, caused by Pseudocercospora opuntiae, which initially presents as transparent, oily spots that darken over time due to conidiophore and conidia formation [7,8]. Other pathogens such as Lasiodiplodia theobromae and Alternaria spp. are associated with cladode rot, though the latter, like Fusarium spp., is considered opportunistic [7,9]. Understanding the multiple factors that influence plant health and contribute to crop protection is essential for optimizing prickly pear production and ensuring its long-term sustainability.

Environmental factors such as altitude, temperature, water availability, and soil nutrient content play a critical role in the growth and development of Opuntia ficus-indica [10,11,12]. Prickly pear production occurs throughout the year. Optimal cultivation typically occurs under specific agroclimatic conditions, particularly average temperatures ranging from 18 to 23 °C and annual precipitation between 200- and 400-mm. Regarding altitude, the ideal range is considered to be between 150 and 1000 m above sea level. At higher elevations, plant growth tends to be slower; however, water requirements decrease due to cooler nighttime temperatures. Notably, under these conditions, the crop requires less rainfall than in warmer lowland regions. High levels of solar radiation further enhance productivity by promoting CAM photosynthesis, particularly under the combination of warm days and cool nights, which maximizes CO₂ fixation efficiency. However, persistent levels of relative humidity below 40% can adversely affect plant growth and yield [10,11,12].

Another important factor influencing crop development and health is the plant-associated microbiota, particularly that of the rhizosphere, the narrow zone of soil surrounding the roots where complex interactions between plants, microorganisms, and soil components take place, affecting nutrient availability, pathogen suppression, and overall plant resilience [13]. These microbial communities contribute to host performance through a variety of direct and indirect mechanisms [14]. For instance, certain microorganisms can enhance plant growth by producing phytohormones, volatile compounds, and by solubilizing nutrients. Additionally, the microbiota can inhibit phytopathogen development through the production of secondary metabolites and can alter the physicochemical properties of the soil, thereby improving plant resilience [15,16].

Recently, the study of microbiota has gained significant relevance in fields such as soil ecology and agronomy, enabling a deeper understanding of the dynamics and functions of microbial communities across diverse environments [17,18]. The characterization of these communities can be achieved through both culture-dependent and culture-independent approaches [19,20]. Culture-dependent methods rely on classical microbiological techniques, involving the isolation of microorganisms followed by their morphological, biochemical, and functional characterization. In contrast, culture-independent techniques, based on marker gene sequencing or whole-genome analysis, have revolutionized microbial ecology by providing a more comprehensive and representative view of microbial diversity, including taxa that are not cultivable under standard laboratory conditions [20,21]. Among these, 16S rRNA high-throughput sequencing has become a cornerstone for the taxonomic identification and profiling of bacterial communities. This method offers valuable insights into the structure, composition, and temporal dynamics of microbiomes, while also enabling the detection of microbial interactions and their implications for soil health and agricultural productivity [22,23,24].

Given the potential role of environmental gradients in modulating Opuntia spp. physiology, we hypothesize that altitude may influence the diversity and functionality of their microbiota. Therefore, the main objective of this study was to use 16S rRNA high-throughput sequencing and bioinformatics tools to describe the microbial communities associated with prickly pear cactus cultivated at different altitudes in the agroecosystems of Milpa Alta, one of Mexico’s main producing regions.

2. Materials and Methods

2.1. Study Area

The experimental sites were located in the Milpa Alta municipality, Mexico City (Mexico), a region with diverse topography and edaphoclimatic conditions highly suitable for Opuntia spp. cultivation. The local climate is classified as temperate semi-arid with predominantly summer rainfall. Average annual temperatures range from 13 to 16 °C (55–61 °F), with summer peaks reaching up to 27 °C (81 °F). Annual precipitation ranges from 600 to 800 mm, and relative humidity typically remains between 60% and 70%. Their soils are medium-textured Andosols with a loamy texture and variations between sites. Bulk density ranges from 1.03 to 1.16 g/cm³, field capacity ranges from 36% to 47%, and permanent wilting point (PWP) ranges from 19% to 24%. Clay content reaches up to 27% in some areas. The pH fluctuates from 6.0 to 8.0, and the organic matter content ranges from 2.5% to 4%. Inorganic nitrogen levels range from 45 to 130 mg kg⁻¹ and extractable phosphorus levels range from 186 to 405 mg kg⁻¹ [12].

Notably, Mexico City accounts for approximately 24.2% of national prickly pear production, with Milpa Alta municipality contributing over 90% of this output. Local producers have developed collective strategies to address the region’s climatic, social, and economic conditions [25]. These strategies consolidate agroecological practices aimed at sustainability. One notable practice is the use of local O. ficus-indica varieties that are well adapted to the climate and volcanic soils. Similarly, phytosanitary management relies on natural resources to prevent and control pests and diseases. This reduces dependence on agrochemicals and promotes ecological balance in agricultural systems.

2.2. Soil Sampling

In September 2024, rhizosphere samples were taken from the “Milpa Alta” variety of prickly pear cactus, which is characterized by its robust, erect growth and oval-shaped cladodes. This variety is highly productive in the summer but sensitive to low temperatures. Four sampling zones were selected, as illustrated in Figure 1: (1) High Zone 1 (HZ1) and High Zone 2 (HZ2), both located in the Tecuahupanco region (19°10′7.9″ N, 99°1′11.08″ W, ~2650 masl); (3) Mid Zone (MZ), in the Acxotla region (19°13′0.5″ N, 99°1′59.53″ W, ~2400 masl); and (4) Low Zone (LZ), in the Cuahuiltlcoch region (19°13′6.03″ N, 99°2′1.62″ W, ~2250 masl). These areas were determined by the “Comisión de Recursos Naturales y Desarrollo Rural” (CORENADR), as there is a long history of traditional cultivation of Opuntia spp. using similar agronomic practices, but at different altitudes. Prior to sampling, the soil surface at each site was manually cleared of organic debris and leaf litter. Zigzag transects were established within each plot, and soil samples (50–150 g) were collected using a sterile metal spatula at a depth of 0–30 cm. Samples were placed in sterile, sealable plastic bags and kept at 4 °C during transport to the laboratory for further processing.

2.3. DNA Extraction and 16S rRNA High-Throughput Sequencing

Metagenomic DNA was isolated from 0.2 g of each rhizosphere sample using the DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Three samples per plot were analyzed. DNA integrity was verified by 1% agarose gel electrophoresis, run for 50 min at 90 V. The hypervariable regions V2, V3, V4, and V6–V9 of the 16S rDNA gene were amplified in two independent PCR reactions, using the Ion 16S™ Metagenomics Kit (Thermo Fisher Scientific, Waltham, MA, USA) on a SelectCycler thermal cycler (Select BioProducts, Waltham, MA, USA). An equimolar mixture of PCR products was used to construct the 16S rDNA libraries with the Ion Plus Fragment Library Kit and Ion Xpress™ Barcode Adapters (Thermo Fisher Scientific). The libraries were purified using the Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA) and quantified with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on the Ion S5™ System (Thermo Fisher Scientific).

2.4. Bioinformatics and Statistical Analyses

A quality assessment of the raw sequencing data was conducted using FastQC v0.12.1, followed by trimming of low-quality reads and adapter sequences with TrimGalore [26,27]. The trimmed reads were then processed using the QIIME2 pipelines (Quantitative Insights Into Microbial Ecology) for microbiome analysis [28]. Quality filtering, chimera removal, demultiplexing, and identification of amplicon sequence variants (ASVs) were performed using the DADA2 plugin. ASVs were aligned with MAFFT, and a phylogenetic tree was constructed using FastTree v2.1 [29]. Taxonomic classification of ASVs was carried out using a scikit-learn-based Naive Bayes classifier trained on the SILVA 16S rRNA database (v138). Alpha and beta diversity metrics were computed to assess bacterial community composition and distribution. Alpha diversity indices included observed ASVs, Chao1, Shannon, and Simpson, followed by ANOVA with Tukey’s post hoc test to identify significant differences among altitudes. Beta diversity was analyzed through non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarities, with statistical significance evaluated using PERMANOVA and beta dispersion tests. Differential abundance analysis of ASVs was performed using Linear Discriminant Analysis Effect Size (LEfSe) to identify bacterial taxa that serve as bioindicators across the altitudinal gradient. A core microbiome associated with O. ficus-indica was defined independently of altitude, considering a relative abundance ≥ 0.1% and a prevalence ≥ 75% across samples. Finally, ecological functions of the microbiota (e.g., nitrogen fixation, sulfate respiration, hydrocarbon degradation) were inferred using FAPROTAX (Functional Annotation of Prokaryotic Taxa) [30].

3. Results and Discussion

The production of the prickly pear cactus is determined by environmental variables such as altitude, temperature, water availability, and soil nutrients. These variables can also influence the composition of its associated microbial communities. In light of the recognized role of microbiota in plant health and soil function, we compared the microbial diversity and functionality associated with this crop grown in four agroecosystems at different altitudes.

3.1. Microbial Diversity Associated with Prickly Pear Cactus

The 16S rRNA high-throughput sequencing of each soil sample produced a total of 18,000 reads after applying quality and rarefaction filters and removing sequences associated with mitochondria and chloroplasts (Figure S1). Alpha diversity indices were calculated to assess the richness and evenness of the microbial communities. The observed ASVs and the Chao1 estimator revealed that the LZ samples had significantly higher bacterial richness (ANOVA, p < 0.05), while the HZ1 samples had the lowest values (Figure 2a,b). The MZ samples showed intermediate richness. The HZ2 samples were outliers with respect to this trend. In terms of diversity, the Shannon index exhibited a similar pattern (Figure 2c), demonstrating a significant decrease in diversity as altitude increased (ANOVA, p < 0.05). However, HZ2 showed an increase in this metric. The Simpson index, which emphasizes evenness, indicated that bacterial communities were slightly less even in LZ and HZ1 compared to MZ and HZ2 (Figure 2d). These patterns suggest altitudinal changes in the structure of the prickly pear cactus microbiota, with a decrease in richness from low to medium altitudes and a partial recovery at higher altitudes. The distinctive diversity of the HZ2 samples may reflect unique selective pressures or microclimatic conditions in the upper high zone of Milpa Alta. Also, this area has much greater vegetation coverage and crop diversity. This promotes a more heterogeneous environment that favors the coexistence of a greater variety of microorganisms [31].

Differences in bacterial community composition further emerged in the NMDS analysis based on Bray-Curtis distances (PERMANOVA, p < 0.001). Four distinct groups corresponding to the sampling zones were identified: LZ, MZ, HZ1, and HZ2 (Figure 3).

Samples from LZ formed a tight cluster, suggesting a relatively homogeneous microbial composition in soils at lower elevations. In contrast, samples from the MZ showed moderate dispersion and were positioned closer to the LZ cluster, possibly reflecting transitional microclimatic conditions between low and high altitudes. Notably, samples from the high zones (HZ1 and HZ2) were more dispersed and clearly separated from the lower zones, indicating a greater heterogeneity in microbial communities at higher elevations. This distinct behavior may reflect microclimatic differences (e.g., slope orientation, solar radiation), changes in humidity, soil heterogeneity, or local management practices such as variation in organic amendments, which could have contributed to the observed divergence. Multiple studies show that altitudinal gradients modify soil microbial diversity by altering soil characteristics, plant traits, and temperature [32,33,34].

3.2. Rhizosphere Microbiota Structure Associated with Prickly Pear Cactus

In relation to the microbial composition associated with prickly pear cactus, the majority of the community was dominated by Pseudomonadota (30–50%) (Figure 4), which represented the highest relative abundance across all samples, regardless of the altitude at which the cactus was grown. Pseudomonadota constitute a phylum of Gram-negative bacteria that are widely distributed in various environments, including soil [35,36]. This group encompasses a vast diversity of species that play key roles in major biogeochemical cycles such as those of carbon, nitrogen, and sulfur [35,37]. These functional traits make Pseudomonadota essential components in plant nutrition and soil quality regulation. Their capacity to degrade organic matter and facilitate nutrient conversion positions them as central players in soil fertility and ecosystem functioning [38,39,40].

The second most abundant phylum was Actinobacteriota. This group consists mainly of Gram-positive, mostly aerobic bacteria that are ubiquitously distributed in both terrestrial and aquatic ecosystems. Notably, many Actinobacteria are capable of producing secondary metabolites and enzymes [41,42]. In soils, they contribute to the decomposition of plant residues, nutrient cycling, and the inhibition of microbial competitors through antimicrobial production [43,44,45].

Other prominent phyla identified were Acidobacteriota and Bacillota. Members of Acidobacteriota are Gram-negative, non-spore-forming, and typically oligotrophic, which enables them to thrive in nutrient-poor environments [46]. Despite being less studied, these bacteria are found in diverse terrestrial ecosystems from tundra to deserts and agricultural soils [47,48]. Their ability to degrade complex organic compounds and contribute to nutrient cycling, particularly nitrogen, highlights their potential importance in promoting plant growth [49]. Bacillota, on the other hand, are primarily composed of Gram-positive bacteria, many of which can form endospores. They are commonly found in agricultural soils and the gastrointestinal tracts of animals [50,51]. Bacillota members are key contributors to organic matter degradation, fermentation, and the production of short-chain fatty acid compounds crucial for soil health and plant nutrition [51,52]. In agricultural systems, species of Bacillota are also involved in mineral solubilization and nitrogen cycling, further supporting soil fertility and plant development [53,54].

3.3. The Core Microbiome Reveals Specific Recruitment of Bacterial Groups by Prickly Pear Cactus

A core microbiome composition was identified across different cultivation zones (Figure 5). Bacillus, Acidibacter, and Sphingomonas exhibited high prevalence in most samples even at high detection thresholds, suggesting that these bacterial genera are highly adapted to the rhizosphere of prickly pear cactus. The genus Bacillus is widely recognized for its ability to promote plant growth and suppress soil pathogens [55]. Sphingomonas species have been isolated from a variety of anthropogenically contaminated environments and have demonstrated the ability to degrade different types of pollutants [56,57]. Acidibacter are found in a variety of environments, including soil. They possess the ability to produce a wide range of enzymes that degrade soil litter [58]. Collectively, they are recognized for promoting plant growth and modulating the surrounding microbial community [59,60,61].

The prevalence of Vicinamibacteraceae, Acidibacter, Gaiella, and Nitrospira was consistent, although these taxa showed lower abundance. Regarding their ecological functions, Gaiella contributes to the degradation of organic matter [62], while Nitrospira plays a central role in nitrification as an aerobic chemolithoautotrophic nitrite-oxidizing bacterium [63]. Acidibacter belongs to the Gammaproteobacteria and has been characterized by colonizing many plant species, as well as being associated with soil nutrient and iron cycles [64]. Finally, the Vicinamibacteraceae family stands out for its ability to utilize a wide variety of substrates, including easily degradable carbon compounds, making it a functionally versatile group. This versatility enables it to play a crucial role in maintaining soil functions, particularly in processes related to the carbon cycle [65]. Overall, these core microbial groups are functionally linked to organic matter turnover and nutrient cycling processes within the soil ecosystem.

The consistent presence of these bacterial groups across altitudinal gradients suggests a strong selective influence exerted by prickly pear cactus roots. Through the secretion of root exudates comprising sugars, amino acids, organic acids, and species-specific compounds, plants shape their rhizosphere microbiome by recruiting specialized microorganisms capable of establishing close, often mutualistic, interactions with the host [66]. These compounds serve not only as nutrient sources but also as chemical signals, while some exhibit antimicrobial activity that selects for tolerant and adapted microbial taxa capable of colonizing the rhizosphere [67]. Additionally, in Milpa Alta municipality, farmers typically propagate prickly pear cactus using cladodes from a single “mother plant,” leading to clonal crop populations. This uniform genetic background, combined with repeated cultivation practices, may contribute to the consistent and selective recruitment of microbial communities associated with the prickly pear cactus “Milpa Alta” variety. From an ecological perspective, the identification of a stable and functionally relevant core microbiome in prickly pear cactus highlights the strong co-adaptation between host plants and soil microorganisms under environmental gradients.

3.4. Biomarkers Linked to Prickly Pear Cactus Microbiomes Along an Altitudinal Gradient

Among the main bacterial groups found in differential abundance across altitude levels, we identified several genera that could serve as microbiological markers potentially linked to plant quality and health. These biomarkers reflect not only the influence of environmental factors such as altitude, temperature, humidity, and soil nutrient content, but also the agroecological practices adopted by local producers. To detect these bacterial genera, we employed two complementary strategies: first, we estimated log fold changes (LogFC) based on variations in genus abundance across altitudinal gradients (Figure 6); second, we conducted a LEfSe analysis, which integrates statistical significance with biological consistency to identify taxa with discriminative potential (Figure 7).

In the low zone (LZ), the predominant taxa were Conexibacter, Agromyces, Domibacillus, Pedomicrobium, and Rokubacteriales. Conexibacter, a genus involved in the decomposition of organic matter, has been associated with more humid environments, which could facilitate the proliferation of pathogens in lowland areas [68,69]. Agromyces is a genus of Gram-positive Actinobacteria commonly isolated from soil and rhizosphere environments, where they play a role in nutrient cycling. Some species have been reported as potential plant growth-promoting rhizobacteria [70]. Domibacillus is commonly found in soils rich in organic matter and has been associated with nitrogen-fixing bacteria [71]. Pedomicrobium is recognized as an iron- and manganese-oxidizing and -accumulating bacterium; it is primarily distributed in aquatic environments [72,73]. Finally, Rokubacteriales has been detected in terrestrial environments such as forest soils, mines, jungles, and cultivated fields. Some members are considered non-culturable bacteria with methanotrophic potential and the ability to produce sulfite reductases [74].

In the middle zone (MZ), the genera Acidothermus, Gemmatimonas, Mesorhizobium, and Pseudoxanthomonas were identified as differentially enriched (Figure 6). Acidothermus, a member of the Actinobacteria, is Gram-negative, non-spore-forming, and non-flagellated [75]. It is known for its ability to produce cellulases capable of degrading plant debris [76]. Gemmatimonas includes species distributed across various environments [77] and is notable for its capacity to transform nitrous oxide (N₂O) [78]. Mesorhizobium, a genus that forms symbiotic associations with legume roots to fix atmospheric nitrogen, may benefit tender cactus cultivation by enhancing nutrient availability and stress tolerance [79]. Pseudoxanthomonas has been linked to the degradation of organic matter and nitrate denitrification, contributing nutrients to the surrounding soil [80]. Although these genera may contribute to plant development, the presence of disease and pests in the mid-zone suggests that they may not provide the same level of protection as those found in the high zone.

With regard to the elevated zone, HZ1 samples exhibited a preponderance of bacteria from the genera Halocella, Solirubrobacter, Rhodomicrobium, Phenylobacterium, Roseomonas, and Streptomyces. Halocella is a genus of moderately halophilic anaerobic bacteria that have been isolated from saline and anaerobic environments [81]. Its presence suggests that in HZ1, there may be microhabitats with saline conditions or organic content that favor their growth. In addition, some species possess the ability to degrade cellulose [82]. Solirubrobacter, a genus within the phylum Actinobacteria, is commonly associated with arid or semi-arid soils. These bacteria are typically aerobic, resistant to extreme conditions, and have been found in deserts, in UV-exposed environments, and in association with plants [83,84]. Some species have been linked to the degradation of complex organic matter and the production of bioactive compounds that could inhibit pathogenic microorganisms and promote plant growth [85,86,87]. Rhodomicrobium, belonging to the phylum Pseudomonadota, is a genus of purple non-sulfur bacteria that carry out anoxygenic photosynthesis. These bacteria are typically found in soils, in the plant microbiome, and in aquatic or humid environments with light, suggesting that the site may have temporary or permanent moist areas with light exposure that support their development [88,89]. Their metabolism suggests a role in nutrient cycles that may indirectly promote plant growth. Additionally, they have significant biotechnological potential due to their ability to produce bioplastics [90]. Phenylobacterium includes species known for degrading aromatic compounds, including pollutants such as herbicides and industrial products. Their detection may indicate the presence and transformation of complex organic compounds or contaminants [91,92,93]. Their presence could help maintain a chemically stress-free environment for plants, supporting their development and defense. Streptomyces is widely distributed in soils and plays a crucial role in the decomposition of organic residues [94].

On the other hand, the samples from HZ2 stood out due to the presence of Pseudarthrobacter, Crossiella, and Aquicella. Pseudarthrobacter is a group of Gram-positive endophytic bacteria that belongs to the family Micrococcaceae [95]. Species of this genus have been the focus of numerous studies due to their potential for promoting plant growth, bioremediation, and the production of antibiotics and enzymes. Thanks to their ability to use both organic and inorganic compounds as metabolic substrates, these bacteria have been employed as biofertilizers in agriculture [95,96,97]. Crossiella is another genus reported in diverse environments such as horses, caves, and soils [98,99]. It has demonstrated the ability to inhibit certain pathogens. Its presence in HZ2 could be contributing to the overall improvement of cactus crop health, and it is likely linked to the use of horse manure as fertilizer. Finally, Aquicella is a bacterial genus that thrives in humid and aquatic environments. Species of this genus require activated carbon for their growth [100,101], which could explain their presence in the system, as carbon is part of the fertilization practices used by the producer. Additionally, this genus has been reported as abundant in the rhizosphere and is known to increase plant resilience to contaminants by improving nutrient availability and stimulating root growth [102]. These findings underscore the potential role of microbiota not only in plant health and productivity but also in the resilience and ecological sustainability of traditional agroecosystems, particularly in arid and semi-arid regions where resource efficiency and microbial partnerships are key to long-term ecosystem function.

3.5. Altitude as a Determining Factor for Microbiota Diversity and Functionality

In our study, LZ exhibited markedly richer and more diverse bacterial communities than MZ and HZ, reflecting environmental conditions that are more stable and conducive to microbial coexistence (Figure 2). Factors such as warmer temperatures, higher water availability, and greater accumulation of organic matter likely drive these patterns, enhancing microbial activity and functional redundancy [103,104]. This is evident in the elevated abundance of microorganisms involved in cellulolysis, xylanolysis, fermentation, and nitrification in LZ soils, which contribute to the decomposition of plant residues and the release of nutrients (Figure 8). In contrast, MZ soils were characterized by a comparatively lower abundance of fermentative microorganisms but showed higher representation of nitrogen-fixing taxa, indicating a functional shift towards processes that directly support nitrogen availability. Together, these microbial functions play a central role in sustaining nutrient cycling and maintaining soil fertility across the gradient [105].

At higher altitudes, HZ1 samples showed the lowest richness and diversity, indicating that colder temperatures, reduced water availability, and higher UV radiation act as strong environmental filters. As a result, the microbiome becomes more specialized, dominated by stress-tolerant taxa and enriched in functions related to nitrogen cycling, particularly denitrification, nitrite respiration, and nitrogen respiration, reflecting a shift toward anaerobic or microaerophilic processes in nutrient-poor soils (Figure 8). This functional reconfiguration highlights the central role of nitrogen availability in sustaining cactus cultivation under harsh conditions. Interestingly, HZ2 emerged as an outlier. Despite its high elevation, it displayed partial recovery of microbial diversity, with higher abundance of groups involved in nitrogen fixation, ureolysis, cellulolysis, and chemoheterotrophy. This recovery is likely linked to distinct Mexican agricultural practices for Opuntia spp., such as compost incorporation, manure application, and maintenance of secondary vegetation, which buffer environmental stress and generate more heterogeneous microhabitats [3,106].

A limitation of this study is the absence of direct measurements of soil physicochemical and environmental parameters, which constrains our ability to attribute the observed patterns to specific drivers. Another weakness is that microbial functional profiles were inferred from 16S rRNA data. A more comprehensive understanding would require shotgun metagenomics or metatranscriptomics to accurately capture the functional potential and activity of microbial communities. However, our results provide valuable initial insights into how altitude and soil management influence the microbial functions in Opuntia spp. agroecosystems.

4. Conclusions

Our study reveals that altitude strongly shapes the rhizosphere microbiome of Opuntia ficus-indica, influencing both its taxonomic diversity and functional potential across Milpa Alta’s agroecosystems in Mexico. These findings underscore the importance of native soil microbiota in sustaining cactus pear health and productivity, while also providing a basis for site-specific management strategies. Future work should assess whether inoculation with site-adapted core microbiota or microbial biomarkers can enhance crop resilience and performance in the face of climate change.