Plants and their root-associated microorganisms have evolved intricate symbiotic relationships that facilitate adaptation to environmental fluctuations. The rhizosphere, the narrow zone surrounding plant roots, is a complex environment where plant-microbe interactions are shaped by both abiotic (environmental) and biotic (biological) factors. The plant microbiome plays a crucial role in enhancing stress tolerance, disease resistance, and nutrient uptake. Consequently, understanding the factors that govern the assembly of rhizosphere microbial communities is fundamental to comprehending plant evolution and improving agricultural yields. Most microbiome assembly studies have focused on cultivated crops and the model plant Arabidopsis thaliana. These studies have revealed that abiotic factors, primarily soil properties like nutrient levels and pH, directly influence microbial communities by affecting their growth and indirectly by altering plant physiology and root exudation patterns. Plant genotype has also been identified as a major biotic factor, influencing microbiome composition through the production of diverse hormones and exudates, thereby exerting selective pressures on bacterial and fungal communities. Different genotypes of the same species may exhibit distinct traits, root architectures, growth rates, and physiological processes that shape rhizosphere microbiome assembly. However, research on rhizosphere communities and the factors governing microbiome assembly in wild plant species within their native habitats remains limited, particularly in high-altitude ecosystems such as the Andes. This study aimed to investigate the impact of soil (abiotic) and plant genotype (biotic) factors on the composition and diversity of bacterial and fungal communities in the rhizosphere microbiome of the Andean blueberry (Vaccinium floribundum Kunth) in its natural environment. V. floribundum was selected due to its endemic status in the Andean region, its unique habitat in the páramo ecosystem (characterized by acidic soils, high UV radiation, low temperatures, and high humidity), its nutritional value, its cultural significance in Ecuador, and its lack of domestication, making it an ideal subject for studying plant-microbe interactions in a largely undisturbed setting. The genetic structure of V. floribundum in the Ecuadorian Highlands is well-documented, enabling the investigation of plant genotype effects on the associated microbiome.

Numerous studies have examined the factors shaping plant microbiomes, largely focusing on cultivated species and model organisms. These studies highlight the significant influence of both abiotic and biotic factors. Abiotic factors such as soil pH, nutrient availability (particularly phosphorus), and the presence of heavy metals have been shown to strongly influence microbial community structure and function. The oligotrophic-copiotrophic theory suggests that copiotrophic bacteria thrive in nutrient-rich environments, while oligotrophic bacteria are better adapted to nutrient-poor conditions. This theory is often applied to explain the distribution of bacterial phyla across diverse soil types. Biotic factors, particularly plant genotype, have also been demonstrated to shape microbiome assembly. Different plant genotypes produce different root exudates that selectively support the growth of specific microbial taxa. Studies have shown that root architecture, growth rates, and physiological characteristics of the plant also play a role. Despite these advancements, research on wild plant species in their native environments remains relatively scarce, particularly in high-altitude ecosystems like the Andean páramo. Understanding the factors that structure microbiomes in these unique environments is essential for understanding plant adaptation and conservation efforts.

This study analyzed 39 rhizosphere soil samples collected from V. floribundum individuals across two distinct soil regions (northern and southern) in the Ecuadorian Highlands. The samples were selected based on previously identified genetic clusters within the V. floribundum population. Northern soils were classified as Andisols (higher nutrient content), while southern soils were classified as Paleosols (poorer soils due to erosion). Rhizosphere soil was collected, and DNA was extracted using the PowerSoil DNA Isolation Kit. Amplicon sequencing of the V3-V4 region of the 16S rRNA gene was performed to characterize bacterial communities, while the internal transcribed spacer (ITS1) region was sequenced for fungal community analysis. Sequencing was conducted using Illumina MiSeq v3. Sequence data processing was carried out using MT-Toolbox and DADA2 software for bacteria and DADA2 and MOTHUR for fungi. Amplicon sequence variants (ASVs) were identified, and taxonomic assignments were made using the SILVA 132 and UNITE databases. Alpha diversity (Shannon diversity index) was calculated to assess microbial diversity within samples. Beta diversity (Bray-Curtis dissimilarity) and principal coordinate analysis (PCoA) were used to compare community composition between samples. Linear mixed models (ANOVAs and stepwise regression) were employed to investigate the effects of soil region, plant genotype, altitude, and edaphic factors (pH, organic carbon, conductivity, total nitrogen, and various metal concentrations) on bacterial and fungal alpha diversity. Physicochemical properties of the bulk soil were also measured to investigate the relationship between soil properties and microbial communities.

Analysis of the V. floribundum rhizosphere microbiome revealed that Proteobacteria and Acidobacteria were the most abundant bacterial phyla across all samples. However, the relative abundance of Actinobacteria, Chloroflexi, and Gemmatimonadetes differed significantly between the two soil regions. Fungal communities were more evenly distributed, with no single dominant taxa. Soil region was identified as the primary predictor of bacterial alpha diversity. Stepwise regression analysis showed that phosphorus (P) and lead (Pb) concentrations in the soil were the most important edaphic factors explaining this diversity. Higher phosphorus levels were associated with higher bacterial diversity. In contrast, the interaction between plant genotype and altitude was the most significant factor influencing fungal alpha diversity. Plant genetic clusters 3 and 4 exhibited higher fungal diversity compared to clusters 1 and 2, and this difference was more pronounced at higher altitudes. Beta diversity analysis showed that soil region explained more variance in bacterial community composition compared to fungal composition, while plant genotype had similar effects on both bacterial and fungal beta diversity. Overall, the results indicated that abiotic factors (soil properties, specifically P and Pb) were more influential in shaping bacterial communities, whereas biotic factors (plant genotype and altitude) were more critical in determining fungal community assembly.

This study demonstrates that the assembly of bacterial and fungal communities in the Vaccinium floribundum rhizosphere is influenced by distinct factors. Bacterial communities are primarily structured by soil region and its associated edaphic characteristics, particularly phosphorus and lead concentrations. The abundance of Proteobacteria, generally considered copiotrophic, was higher in the nutrient-rich northern region, while Acidobacteria, associated with nutrient-poor environments, were more abundant in the southern region. Phosphorus's role in shaping bacterial communities aligns with previous findings indicating that plant genes involved in the phosphate starvation response coordinate microbiome assembly and immunity. Lead, while less studied, likely contributes to microbiome diversity by influencing the distribution of specific taxa. In contrast, fungal community assembly is primarily driven by the interaction between plant genotype and altitude. This suggests a strong host-specific influence on fungal communities, likely reflecting co-evolutionary processes between the plant and its fungal symbionts. The results support the notion that fungal communities are more strongly influenced by biotic factors than bacterial communities, which are more susceptible to edaphic factors. This finding aligns with previous research illustrating the stronger relationship between plant community composition and fungal diversity compared to bacterial diversity.

This study provides novel insights into the factors influencing rhizosphere microbiome assembly in a wild Andean blueberry species. Abiotic factors, particularly soil phosphorus and lead levels, are major determinants of bacterial community structure, while the interaction of plant genotype and altitude is the most significant factor shaping fungal communities. This highlights the complex interplay between soil properties and plant genetics in structuring the rhizosphere microbiome. Further research should expand beyond this single species and region, investigating additional wild plant species across diverse environments to obtain a more comprehensive understanding of plant-microbe interactions in natural settings.

This study was conducted in a specific geographic region and focused on a single plant species. The findings might not be generalizable to all wild blueberry species or other plant species in different ecosystems. The sampling design, while based on previously established genetic clusters, could still be improved to provide greater statistical power. Further studies could incorporate more detailed analyses of root exudates and other plant-microbe interactions to enhance our understanding of the underlying mechanisms.