Changes in microbiome assembly of the pioneer Andean tree Alnus acuminata in response to land degradation

Changes in microbiome assembly of the pioneer Andean tree Alnus acuminata in response to land degradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Changes in microbiome assembly of the pioneer Andean tree Alnus acuminata in response to land degradation Nubia Guzmán, Lara A. Quaglini, Raffaella Ansaloni, Mayra Jiménez, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8660278/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Land degradation in the Andes threatens ecosystems and biodiversity. The recovery of these areas often depends on pioneer species such as Alnus acuminata , which relies on symbiotic microorganisms for nutrient acquisition and stress tolerance. Understanding how degradation affects its microbiome is crucial for effective restoration. This study investigated how a land-use trajectory involving deforestation and abandonment impacts the diversity and structure of bacterial communities associated with Alnus acuminata . Next-generation sequencing of 16S rRNA gene amplicons was used to compare bacterial communities in bulk soil, roots, and root nodules between a native forest and degraded forest in the Ecuadorian Andes. Land degradation significantly altered bulk soil bacterial diversity and community structure, with pH and carbon content identified as key environmental drivers. Degraded soils were dominated by Actinomycetota, whereas native forest soils harbored more diverse communities, including Acidobacteriota and Pseudomonadota. Root endophytic and nodule-associated communities showed reduced diversity under degradation, although not statistically significant, suggesting that these niches are buffered from environmental changes. Nevertheless, their community structures differed significantly, indicating that Alnus acuminata may actively assemble a beneficial bacterial consortia to cope with degraded conditions. Alnus acuminata appears to respond to soil degradation by recruiting a more diverse and functionally beneficial endophytic microbiome, including stress-resilient, pathogen-defending, and plant growth-promoting genera such as Micromonospora , Rahnella , Rhodanobacter , Mycobacterium , and Deinococcus . This adaptive strategy likely supports its survival and establishment in nutrient-poor, degraded environments, highlighting the critical role of plant-microbe interactions in ecosystem recovery and suggesting that harnessing these interactions could improve restoration outcomes. Land degradation Soil restoration Andean alder Bacterial communities Endophytes NGS of 16S rRNA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Land degradation, generally defined as the long-term reduction or loss of biological productivity, ecological integrity, or human value (Olsson et al. 2022), is a pressing global challenge. In Ecuador, especially in the Andean mountains, forest ecosystems are under constant threat from human activities, primarily due to extensive livestock grazing, agriculture, mining, and logging. Additionally, large areas have been converted into plantations of exotic tree species such as Eucalyptus spp. and Pinus spp. (Hofstede et al. 2002; Günter et al. 2009; Tapia-Armijos et al. 2015). This land-use change has resulted in irreversible biodiversity loss and severely disrupted functioning ecosystems, affecting a significant proportion of native vegetation in an area of exceptionally high biological diversity. Nearly half (46%) of the original forest cover in southern Ecuador has been replaced by anthropogenic land cover types (Tapia-Armijos et al. 2015). These large-scale transformations not only threaten plant and animal biodiversity but can also have profound implications for the often-overlooked microbial communities that drive essential ecosystem functions. Microorganisms support almost all ecosystem functions, and ecosystem-scale responses fundamentally depend on their resident microbial communities (Osburn et al. 2023; Rawat et al. 2023). These communities, primarily bacteria and fungi, regulate a wide range of essential processes both below- and aboveground, including the transformation of organic matter, carbon storage, nutrient cycling, and plant productivity (Rawat et al. 2023; Dixit et al. 2024). Plant-associated microorganisms have gained increasing attention due to their central role in affecting plant establishment and adaptation, disease resistance, and resilience to environmental stress (Fitzpatrick et al. 2018; Muhammad et al. 2024; Zieschank et al. 2025). Despite their central role in these processes, microbial communities have long remained underexplored. This is partly due to the exceptional diversity of microorganisms, the challenges of studying them directly, their spatial heterogeneity, and their historical exclusion from most biodiversity surveys (Osburn et al. 2023). As a result, the link between land degradation and soil-plant-microbes remains insufficiently explored, representing a fundamental challenge for understanding ecosystem responses to land-use change (Zhou et al. 2018; Rawat et al. 2023). Land degradation affects key soil properties such as pH, organic matter content, nutrients availability, moisture, and temperature, which directly influence microbial activity, diversity, community composition, and abundance (Zhou et al. 2018; Peng et al. 2022; Mészárošová et al. 2024). For instance, soil pH is known to exert strong selective pressure on specific bacterial taxa, and its alteration can lead to shifts in microbial communities (Zhou et al. 2018; Pereira et al. 2022). Similarly, organic carbon content serves as a key driver of microbial dynamics by providing essential energy and nutrient sources that support community growth and metabolic activity (Li et al. 2024; Jiang et al. 2025). Furthermore, plant species significantly influence the soil microbiome through root architecture, exudate release, and rhizodeposition patterns, creating niches that favour specific microbial populations (Oppenheimer-Shaanan et al. 2022; Domeignoz-Horta et al. 2024). Given the ecological consequences of land degradation, especially for soil characteristics and microbial communities, ecological restoration becomes a key priority. Successful restoration often relies on the re-establishment of vegetation, particularly pioneer species that are capable of establishing in nutrient-poor, compacted, or contaminated soils where other plants may fail. These early colonizers can initiate positive feedback loops by improving soil structure, enhancing nutrient availability, and fostering microbial activity through root exudates and litter inputs (Wang et al. 2024; Xiuyu et al. 2024). Importantly, the ability of certain plant species to recruit beneficial microbial partners, including mycorrhizal fungi and nitrogen-fixing bacteria, can significantly accelerate the recovery of soil functions in degraded environments (Sun et al. 2018). Given this, selecting plant species that form effective mutualisms and can tolerate abiotic stress is essential for restoring both plant cover and the microbial networks that sustain a functioning ecosystem. Among the pioneer species with high potential for ecological restoration, Alnus (alder) species stand out for their remarkable ability to colonize adverse conditions, including high salinity, drought, extreme pH, and soils contaminated with heavy metals and organic pollutants (Lefrançois et al. 2010; Diagne et al. 2013; Thiem et al. 2018; Bhattacharyya et al. 2024). This success is attributed to a tripartite symbiosis with ectomycorrhizal and arbuscular mycorrhizal fungi, as well as nitrogen-fixing bacteria; particularly Frankia spp. (Becerra et al. 2005; Chen et al. 2020). However, current research on Alnus species has identified diverse bacterial taxa beyond Frankia that are associated with alder root nodules, uncovering even greater complexity in these symbiotic relationships (Carro et al. 2013; Aslani et al. 2020; Garneau et al. 2023a). Beyond plant–microbe interactions, microbe–microbe dynamics are also critical. For example, Pseudomonas has been shown to promote nodulation in Alnus rubra Bong. (Knowlton and Dawson 1982), while other bacteria genera such as Microvirga and Streptomyces can stimulate Frankia growth (Garneau et al. 2023a). While there are many studies that explored the microbial community associated with different Alnus species around the world (e.g. Aslani et al. 2020; Garneau et al. 2023a; Thiem et al. 2023; Dove et al. 2024), little information is available on Alnus acuminata Kunth, the only alder species native to the mountains of South America (Lægaard and Balslev 2014). In Ecuador, where extensive deforestation and land-use conversion threaten the integrity of Andean forest ecosystems, A. acuminata has been used for reforestation due to its pioneering character and soil-enriching capabilities (Günter et al. 2009). Yet, its belowground microbial associations remain largely unexplored, especially concerning non- Frankia bacteria and the broader structure of its root nodules and root associated bacterial communities. With the goal of improving ecosystem recovery in degraded Andean forests, this study sought to provide the necessary knowledge to exploit pioneer plant-microbe interactions. To this end, the diversity and structure of bacterial communities associated with the roots and nodules of Alnus acuminata , was investigated using metagenomic DNA sequencing comparing individuals and soils from a native and a degraded forest. Materials and Methods Study area and sample collection The study was conducted in the Andean mountains of southern Ecuador in the province of Azuay, across two distinct land-use types: (1) native intact forest (NF; 2°55’ S, 78°50’ W), a patch of high montane evergreen forest located within the Aguarongo Protected Vegetation Area; and (2) degraded forest (DF; 2°57’ S, 78°53’ W), corresponding to a high montane evergreen forest that has been completely deforested, which is located in close proximity to the Aguarongo Reserve (~ 6 km away) (Fig. 1 ). The native forest represents a well-preserved remnant of natural vegetation, showing no evidence of fire, logging, or other anthropogenic disturbances. The list of dominant species characterizing the NF is shown in Table S1 of the supplementary information. The degraded site, on the other hand, has undergone a complete land-use change. It was deforested over 50 years ago, mainly for livestock grazing and road construction, and has since been abandoned for about 10 years. It is now dominated by the grass Cenchrus clandestinus (Hochst. ex Chiov.) Morrone, commonly used as a forage crop, and by other non-native species such as the tree Eucalyptus globulus Labill., along with a few native pioneer species, including Alnus acuminata , the focal specie of this study. Both sites are located at elevations ranging from 2,800 to 3,140 m a.s.l. and share similar climatic conditions. The climate is cold temperate, with annual temperatures ranging from 9 to 12°C and an annual precipitation of 820 mm. Rainfall varies throughout the year, with a dry season from May to September, during which monthly precipitation ranges from 25 to 60 mm. From October to April, rainfall is more intense, reaching 75 to 110 mm per month (Minga 2002). Bulk soil, root, and nodule samples were collected from different individuals of A. acuminata growing in both the native and degraded forest sites. Four trees were randomly selected in each area, maintaining a maximum distance of about 150 m between individuals. The sampled trees had heights ranging from 4 to 7.5 m and diameters at breast height (DBH) between 6 and 15 cm. Around each focal tree, a 5 × 5 m plot was established with the tree in the center. Within each plot, all vascular plant species were identified according to the Angiosperm Phylogeny Group classification system (APG 2016). For bulk soil sampling, approximately 100 g of soil was randomly collected from a depth of 20–40 cm, at a distance of 10–20 cm from the trunk of each selected tree. Bulk soil samples were collected from three of the four selected trees per site, of which ~ 50 g was used for the analysis of soil chemical properties. Root and nodule samples were obtained from the four trees selected per site, including those used for soil sampling and one additional tree. In total, six bulk soil samples (three per land-use type) and eight root–nodule samples (four per land-use type) were collected for the analysis. All samples (bulk soil, roots, and nodules) were stored at -20°C until analysis. To compare root nodulation among A. acuminata individuals growing in native and degraded forests, the morphological characteristics of Frankia nodules were assessed (Fig. S1 ). For this, three replicates per individual (i.e. root subsamples of approximately 100 cm each) were randomly selected from the same individual trees sampled for bacterial analysis. Within each replicate, all nodule lobes were measured for size (length and width, mm) and weight (mg). Soil analysis Prior to analysis, soil samples were sieved through a 2 mm mesh and stored at room temperature. Soil pH was measured in water, while total nitrogen (%) and total carbon (%) contents were determined using a CN elemental analyzer (Flash EA 1112 NC Soil, Thermo Fisher Scientific, Pittsburgh, USA). Sample processing Prior to DNA extraction, roots and nodules of A. acuminata were first washed with tap water and then surface-sterilized by immersion in 70% ethanol for 1 min, followed by 3% sodium hypochlorite for 3 min, a second immersion in 70% ethanol for 30 s, and four rinses with sterile distilled water. To verify the effectiveness of sterilization, 100 µL from the final rinse was plated on trypticase soy agar and incubated to check for microbial contamination (Cheng et al. 2019). Sterilized roots and nodules were then ground in liquid nitrogen. Bacterial community diversity associated with bulk soil, A. acuminata roots and nodules (endophytes) in native and degraded forests was defined using 16S rRNA gene amplicons. DNA from all samples were extracted using the FastDNA® Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer’s protocol. An initial PCR amplification was performed using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCGCGGCKGCTG-3′) (Frank et al. 2008) on undiluted DNA extracts as well as on 1:10, 1:100, and 1:1,000 dilutions to assess the presence of potential PCR inhibitors. The thermal cycling conditions were as follows: initial denaturation at 95°C for 4 min; 29 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s; followed by a final extension at 72°C for 5 min. Because root and nodule samples also contain plant plastid-derived 16S rRNA sequences, these samples were processed differently from bulk soil, with a few additional steps required. Briefly, the full-length 16S rRNA gene was amplified from the DNA of roots and nodules using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). PCR conditions were as follows: initial denaturation at 95°C for 4 min; 29 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 2 min; followed by a final extension at 72°C for 5 min. PCR products were then purified using the Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. A second PCR amplification was performed on all samples (bulk soil, roots, and nodules), targeting the V5–V6 hypervariable regions of the bacterial 16S rRNA gene. Two different primer sets were used depending on the sample type: primers 783F and 1027R were used for bulk soil samples (Gandolfi et al. 2024), while primers 799F and 1107R, specifically designed for endophytic communities, were used for root and nodule samples (Chen et al. 2022). All primers were tagged with custom 6 bp oligonucleotide barcodes (sequences listed in Table S2). PCR conditions were identical for both primer sets and included an initial denaturation at 94°C for 4 min, followed by 28 cycles of denaturation at 94°C for 50 s, annealing at 47°C for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. PCR products were purified, and DNA concentrations were quantified using a Qubit® 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Amplicon libraries were prepared in batches of nine samples, each distinguished by the unique barcode pair (Table S2). Library preparation included the addition of standard Nextera indexes (Illumina, Inc., San Diego, CA, USA), and sequencing was carried out on an Illumina MiSeq platform using a 2 x 300 bp paired-end protocol. Amplicon Sequence Variants (ASVs) were inferred using the DADA2 algorithm (Callahan et al. 2016). Data analysis Due to the use of different primer sets for bulk soil and endophytes (roots and nodules) samples, these compartments were analyzed separately. All statistical analyses were performed within the R environment v. 4.3.2 (R Core Team 2023). First, different α-diversity metrics on the rarified bacterial data were calculated using the vegan package. Specifically, bacterial richness was estimated as the number of ASV using the specnumber() function. Bacterial evenness was assessed using the Berger-Parker index, which measures the dominance of the most abundant taxa in a community (Berger and Parker 1970). Additionally, the Shannon diversity index was calculated using the diversity() function. Next, differences were analyzed for bacterial α-diversity between land-use types (native and degraded forests), compartments (bulk soil, roots, and nodules), and their interaction. ANOVA was used for bacterial evenness and Shannon diversity index, while generalized linear models (GLMs) with a quasi-Poisson distribution were applied to ASV richness. To visualize differences in bacterial community structure between land-use types, Non-metric Multidimensional Scaling (NMDS) was conducted based on Bray-Curtis dissimilarities using the metaMDS() function from the vegan package. Additionally, significant differences were tested with PERMANOVA using the adonis2() function. To evaluate the influence of soil variables and plant richness on bacterial community composition, a Redundancy Analysis (RDA) was conducted on bulk soil and endophytes (roots and nodules) ASVs, constrained by environmental variables including soil pH, total nitrogen, total carbon, and plant richness. However, because total carbon and soil pH were highly correlated, total carbon was excluded from the analysis, and soil pH was retained. Before these multivariate analyses, the bacterial ASV abundance matrix was transformed using the Hellinger method to downweigh the influence of highly abundant ASVs, emphasize their presence or absence, and address the double-zero problem commonly encountered when comparing community compositions across samples (Borcard et al. 2018). The 10 most abundant phyla were identified across all land-used types and compartments and compared to their relative abundances between native and degraded forests with one-way ANOVA. Next, the core endophytic bacterial communities present in the roots and nodules of A. acuminata were identified by selecting ASVs detected in at least 80% of samples, regardless of land-used type (Cheng et al. 2019; Neu et al. 2021). The core microbiome refers to microbial taxa that are consistently associated with a specific host or environment, typically identified by their presence across multiple microbial communities (Neu et al. 2021). These taxa are thought to play key ecological and functional roles within their host or environment under prevailing conditions (Risely 2020). Lastly, A. acuminata root nodulation (nodule lobe length, width, and weight) was analyzed using linear mixed models (LMMs) with land-use type (native and degraded forests) as a fixed factor, root length as a covariate, and replicate (i.e. root subsample) nested into individual tree identity as a random factor. To do this, the lme4 and lmerTest packages were implemented. Results Differences in soil properties and plant composition between native and degraded forests Soil properties were altered in the degraded forest (DF) compared to the native forest (NF). The DF exhibited a markedly higher soil pH (5.4 ± 0.04 vs. 4.3 ± 0.05) and a lower total carbon content (4.22 ± 0.06% vs. 5.20 ± 0.02%). However, total nitrogen content did not substantially differ between the two sites (0.35 ± 0.20% in the DF and 0.34 ± 0.06% in the NF). A total of 19 vascular plant species, belonging to 14 families were recorded in the plots surrounding A. acuminata individuals. In the degraded site, the vegetation was almost completely dominated by two introduced herbaceous species: Cenchrus clandestinus (Hochst. ex Chiov.) Morrone and Trifolium repens L, along with a few native shrubs such as Baccharis latifolia (Ruiz & Pav.) Pers., Cestrum tomentosum L.f., and Rubus floribundus Kunth. In contrast, the native forest was characterized by a dominance of native tree species typical of high mountain forest ecosystems, including Axinaea macrophylla Triana, Lomatia hirsuta (Lam.) Diels, Myrica parvifolia Benth., Polylepis lanuginosa Kunth, and Vallea stipularis L.f. The understory featured species such as Valeriana hirtella Kunth, Verbesina latisquama S.F. Blake, and the fern Lophosoria quadripinnata (J.F. Gmel.) C. Chr. Effect of land degradation on bacterial alpha-diversity When analyzing the differences of bacterial α-diversity between DF and NF, no effect of degradation was found on ASV richness of either bulk soil (GLMs: p = 0.519; Table S3), or endophytes (root and nodule samples) of A. acuminata (GLMs: p = 0.918; Fig. 2 a, Table S4). Conversely, bacterial evenness of bulk soil samples was significantly lower in the degraded forest (ANOVA: F = 32.93, p = 0.00457; Fig. 2 b); a similar trend was observed for Shannon index, although the difference was marginally significant (ANOVA: F = 7.164, p = 0.0554; Fig. 2 c, Table S3). Bacterial diversity in endophytic samples did not differ significantly between root and nodule compartments, between land-use types, or due to their interaction (Table S4). Root samples showed a pattern similar to that of bulk soil, with lower evenness and Shannon indices in DF compared to NF; however, these differences were not statistically significant, likely due to high variability in index values. Degradation also appeared not to affect the bacterial diversity of A. acuminata nodules (Fig. 2 a, b, c). Effect of land degradation on the structure and composition of bacterial communities The results indicate that bacterial community structure across different compartments (bulk soil, roots, and nodules of A. acuminata ) was influenced by land-use type, as revealed by NMDS analysis (Fig. 3 ). In bulk soil, PERMANOVA showed that land use explained a substantial portion (86.4%) of the variation in community composition, although the effect was not statistically significant, likely due to the small sample size ( F = 25.38, R ² = 0.864, p = 0.1; Table S5). In contrast, for A. acuminata endophytes (roots and nodules), the effect of land use was statistically significant, accounting for 10.6% of the variation ( F = 1.761, R ² = 0.106, p = 0.037; Table S6). These findings suggest that land degradation can alter bacterial community structure, with a marked effect in the bulk soil compared to the endosphere. Additionally, differences between root and nodule compartments explained 8.9% of the variation ( F = 1.472, R ² = 0.089, p = 0.088; Table S6), indicating that compartmentalization within the plant also contributes to shaping the endophytic bacterial community, albeit to a lesser extent than land use. Among the environmental variables examined, soil pH and plant richness were the main drivers of bacterial community composition, as revealed by the RDA analysis (Fig. 4 ). Together, these factors explained 91.3% of the variation in bulk soil bacterial communities (RDA: p 0.05). Soil pH emerged as a key determinant of bacterial community structure in bulk soil, especially under land degradation. In contrast, total carbon, excluded from the plot due to multicollinearity with pH, and plant richness were more strongly associated with bacterial communities in the native forest (Fig. 4 ). The relative abundance of the dominant bacterial phyla shifted in response to land degradation, with more pronounced effects in bulk soil bacterial communities compared to the endophytic communities of A. acuminata (Fig. 5 , Table S7). In bulk soil, degradation significantly decreased the abundance of Pseudomonadota, Acidobacteriota, Bacteroidota, Chloroflexota, and Verrucomicrobiota, while increasing the abundance of Actinomycetota compared to native forest (ANOVA: p < 0.05; Fig. 5 , Table S8). In the endosphere, bacterial communities associated with the roots and nodules of A. acuminata were largely dominated by Pseudomonadota in both DF and NF. Root endophytes in DF showed a reduction in the abundance of the phyla Bacillota, Bacteroidota, and Acidobacteriota, while Bacteroidota, Chloroflexota, and Cyanobacteriota declined in nodules. Conversely, degradation led to an increase in the abundance of Planctomycetota, Actinomycetota, and Deinococcota in nodule endophytes compared with NF; however, these changes were not statistically significant, likely due to high variability among samples (Fig. 5 , Tables S9, S10). Unique phyla were detected exclusively in bulk soil samples, including candidate division WPS-2 (DF), Chlamydiota (NF and DF), Chlorobiota (DF), Elusimicrobiota (NF and DF), Latescibacteria (NF and DF), and Parcubacteria (DF). In contrast, the only unique phylum detected in endophytic samples (roots and nodules) was Abditibacteriota (DF). At lower taxonomic ranks, the dominance of specific bacterial taxa became more apparent not only within each land-use type, but also across different compartments. For example, in bulk soil from degraded forest, the most abundant genera were Streptomyces (relative abundance of 4.7%), Reyranella (2.6%), and Mycobacterium (2.2%). In contrast, in the native forest, Paraburkholderia (5.2%), GP2 (5%), Subdivision3_genera_incertae_sedis (2%), and Caballeronia (1.8%) were the most abundant (Fig. 6 ). Regarding the endophytic samples, Buttiauxella , Rahnella , Variovorax , and Rhizobium dominated the root endosphere of A. acuminata in the degraded site, with relative abundances of 8.1%, 5%, 4.4%, and 3.5%, respectively. Conversely, in native forest, the most abundant genera were Paraburkholderia (8.3%), Bradyrhizobium (7.4%), Pseudomonas (2.8%), and Caballeronia (1.7%) (Fig. 6 ). Notably, genera classified within the order Enterobacterales (belonging to the phylum Pseudomonadota) were by far the most abundant under both land-use types, accounting for 56% and 30% of the total root endophytic community in DF and NF, respectively. With regard to nodule endophytes, a remarkably higher abundance of Paraburkholderia was found in NF compared to DF (42.4% vs. 1.1%, respectively). In DF, Rhodanobacter (14.1%), Rahnella (12%), and Caballeronia (5.7%) were the most abundant genera (Fig. 6 ). Interestingly, among nodule endophytes, Pseudomonas and Bradyrhizobium appeared to be unaffected by land degradation. Pseudomonas , a dominant genus in the nodule endosphere, showed similar abundances in DF and NF (21% and 17.6%, respectively) and was detected only in NF bulk soil. Similarly, Bradyrhizobium displayed comparable abundances, 1.1% in NF and 2.2% in DF, but showed a consistent dominance in bulk soil samples across both land-use types (approximately 3%). Analysis of the core endophytic bacterial communities of roots and nodules of Alnus acuminata Among the 3,225 ASVs detected in the root and nodule endosphere of A. acuminata , 10 ASVs were identified consistently present in root samples at a frequency greater than 80%, and 7 ASVs met the same criterion in nodules, independently of land-use type (Table 1 ). Notably, all core ASVs identified in nodules were also part of the root core community. These shared ASVs (i.e. ASV146, 207, 261, 298, 353, 775, and 826) were taxonomically assigned to Paraburkholderia , Caballeronia , Rahnella , Pseudomonas , and two unclassified Enterobacterales . Among root core endophytes, the most abundant ASV was ASV826, assigned to the order Enterobacterales , which accounted for 26.85% of the total relative abundance. In nodules, the most abundant core ASV was ASV775 (5.92%), assigned to the genus Pseudomonas . Table 1 Core endophytic bacterial community of A. acuminata samples. The table displays taxonomic classification, relative abundance (Ab), and frequency (Frq) of bacterial Amplicon Sequence Variants (ASVs) in root and nodule samples Phylum Class Order Family Genus ASV Roots Nodules Ab% Frq% Ab% Frq % Pseudomonadota Alphaproteobacteria Hyphomicrobiales Bradyrhizobiaceae Bradyrhizobium 702 0.89 87.5 / / Betaproteobacteria Burkholderiales Burkholderiaceae Paraburkholderia 146 0.38 87.5 0.98 100 797 1.82 87.5 / / Caballeronia 207 0.61 87.5 3.2 87.5 Gammaproteobacteria Enterobacterales Yersiniaceae Rahnella 261 0.63 87.5 1.13 100 / / 231 3.88 87.5 / / / / 298 5.53 100 0.94 87.5 / / 826 26.85 100 4.5 87.5 Pseudomonadales Pseudomonadaceae Pseudomonas 775 0.3 87.5 5.92 87.5 Xanthomonadales Rhodanobacteraceae Rhodanobacter 353 0.14 87.5 1.07 100 Effect of land degradation on the morphology of Alnus acuminata nodules Land degradation did not appear to affect the root nodule morphology of A. acuminata . Linear mixed models showed no significant differences in nodule length, width, or weight between degraded and native forests (LMMs: p length = 0.372, p width = 0.8574, p weight = 0.9215; Fig. 7 , Table S11). DISCUSSION By comparing bacterial communities between a native forest and a degraded forest in Ecuador, this study investigated how a land-use trajectory (deforestation for pasture followed by abandonment) affects their diversity and structure in the soil, roots, and root nodules of the pioneer tree Alnus acuminata . Pioneer trees are key engineer species in the restoration of degraded landscapes. As a nitrogen-fixing pioneer species, A. acuminata is critical for facilitating soil recovery and ecological succession in these abandoned pastures. Therefore, understanding the bacterial communities that support its establishment and growth is pivotal. This knowledge can inform effective, long-term recovery strategies that enhance ecosystem resilience. The study report here demonstrated that, compared with native forest, degraded forest shows a marked alteration of soil physicochemical properties, plant community composition and the structure of bacterial communities associated with both bulk soil and the endosphere of A. acuminata . Specifically, soil pH increased and total carbon decreased in degraded forest soils compared to native forest, while total nitrogen content remained relatively unchanged. These findings agree with previous research showing that forest disturbance often leads to soil alkalization (Pedrinho et al. 2020; Pereira et al. 2022) and carbon loss due to reduced organic matter inputs and altered nutrient cycling (Zhou et al. 2018; Peng et al. 2022). The results of this study showed that bacterial richness was not significantly affected by land degradation in either bulk soil or root and nodule endophytes of A. acuminata . However, bacterial evenness and Shannon diversity indices were significantly lower in degraded bulk soils but not in both roots and nodules, indicating that degradation affected principally bulk soil community structure by reducing the evenness of bacterial taxa rather than richness. This pattern agrees with findings from other studies where disturbance often leads to dominance by a few opportunistic taxa at the expense of a more balanced community (Sigler and Zeyer 2004). For example, similar reductions in soil bacterial diversity have been reported in degraded semiarid tropical regions (Pereira et al. 2022; Silva et al. 2024). In contrast, other studies have documented increases (Rodrigues et al. 2013; Zhou et al. 2018; Pedrinho et al. 2020) or no significant changes (Lee-Cruz et al. 2013; Lozano et al. 2014; Li et al. 2024) following land degradation, highlighting the context dependency of microbial responses. Such differences likely reflect variation in spatial scale, disturbance type and intensity, soil physicochemical properties, and ecosystem characteristics (Zhou et al. 2018). With regard to endophytic bacterial diversity between land-use types, the lack of significant differences (evenness was lower but not significant) suggests that, compared with bulk soil bacterial communities, plant-associated microbial communities are more buffered against external environmental changes due to host-mediated selection (Lundberg et al. 2012; Hardoim et al. 2015). Plants usually select beneficial microorganisms to face unfavorable environmental conditions, but in some cases, that selection can lead to a reduced root-associated bacterial diversity as reported for Alnus glutinosa under high-salinity stress (Thiem et al. 2018, 2023). In agreement with evenness reduction, the multivariate analyses in this study revealed that land-use type strongly influenced bacterial community structure in both bulk soil and the plant endosphere. Land degradation explained a substantial portion of variance in soil bacterial communities (PERMANOVA: R ² = 0.864) and a smaller but significant fraction in root and nodule endophytes ( R ² = 0.106). While the effect on soil communities was not statistically significant, likely due to limited sample size, the magnitude of the effect is ecologically relevant and consistent with previous findings that land-use change drives pronounced shifts in soil microbial assemblages (Rodrigues et al. 2013; Lozano et al. 2014; Zhou et al. 2018). The observed compartment effect within A. acuminata (roots vs. nodules) also highlights the role of plant microhabitats in shaping endophytic communities, though this effect was less pronounced than land use. Soil pH along with total carbon emerged as the dominant environmental drivers shaping bulk soil bacterial communities, consistent with numerous studies identifying them as principal determinants of soil microbial diversity and composition worldwide. These factors act as critical environmental filters, determining which bacterial groups can thrive under specific soil conditions (Zhou et al. 2018; Lopes et al. 2021; Mészárošová et al. 2024; Zhou et al. 2024). In the present study, degraded soils were dominated by stress-tolerant Actinomycetota, whereas native forest soils supported more diverse communities rich in Acidobacteriota and Pseudomonadota, typical of resource-rich, acidic environments. Actinomycetota, known for their adaptability, tolerate stressors such as pH fluctuations, salinity, and nutrient scarcity, enabling them to dominate in degraded soils (Mohammadipanah and Wink 2016; Lopes et al. 2021). Their abundance often decreases as ecosystems recover, suggesting a preference for degraded conditions (Li et al. 2024). In contrast, Acidobacteriota, which thrive in acidic soils and typically decline as soil pH increases (Bai et al. 2023; Silva et al. 2024; Jiang et al. 2025) was dominant in NF along with Pseudomonadota, which prosper in high-resource environments with labile carbon inputs (Peng et al. 2022; Bai et al. 2023; Li et al. 2024). Both these taxa declined with degradation. Verrucomicrobiota, another group sensitive to soil fertility changes, also declined with degradation, consistent with previous findings (Navarrete et al. 2015; Lopes et al. 2021). Similar but less pronounced changes were detected in endophytic communities, with Pseudomonadota dominating both roots and nodules irrespective of land use, suggesting that a core microbiome was maintained by the host plant (Knowlton and Dawsonm 1982; Dove et al. 2024). It is important to underline that although Actinomycetota, particularly Frankia spp., was typically dominant in Alnus nodules (Diagne et al. 2013; Chen et al. 2020), a low relative abundance of Frankia was detected. This is likely attributable to the specific physio-morphological state of these endophytes, which may have hindered efficient DNA extraction relative to other genera (Akkermans et al. 1991). Additionally, the universal primers used for endophytic community detection, while designed to capture a broad spectrum, are known to introduce amplification biases, which likely resulted in the underrepresentation of these groups (Thiem et al. 2018). Nevertheless, anatomical observations confirmed its presence in both land-use types (Fig. S1 ). At lower taxonomic rank, Paraburkholderia , Caballeronia , Rahnella , Pseudomonas , and two unclassified Enterobacterales genera were identified as core members of the root and nodule microbiomes, consistent with their known roles as nitrogen-fixing and plant growth-promoting bacteria in symbiotic associations (Garneau et al. 2023a; Dove et al. 2024; Thompson et al. 2025). Although endosphere communities are primarily shaped by host plant identity (Brown et al. 2020; Mészárošová et al. 2024) significant differences in the endospheric bacterial communities from A. acuminata plants grown in degraded and native forests was found. Specifically, changes in the abundance of these core symbionts between degraded and native forests and their association with other bacterial genera suggest that plants can form specific symbiotic bacteria consortia to function better in their specific environments. The marked increase of Rahnella in A. acuminata roots grown in degraded soil accompanied by other genera, such as Micromonospora , shows the capacity of the plant to select and enter into symbiosis with the most useful endophytes for those environmental conditions. Both Rahnella and Micromonospora are plant endophytes. Rahnella is a plant growth-promoting bacterium (PGPB) which enhances nutrient acquisition, such as phosphate solubilization, and Micromonospora has been shown to contribute to plant health and pathogen defense, through the synthesis of many metabolites (Carro et al. 2013; Garneau et al. 2023a, b). Similarly, there was a marked decline of Paraburkholderia in nodules from degraded forests, accompanied by an increased abundance of other species such as Rhodanobacter , primarily recognized for its role in biogeochemical cycling, Mycobacterium and Deinococcus , recognized as plant associated resilience enhancers. These results, suggests that forest degradation may alter symbiotic partnerships to maintain or even improve nitrogen fixation efficiency in that environment (Ghodhbane-Gtari et al. 2021; Tariq et al. 2025; Thompson et al. 2025). Consistently, nodule morphology remained unaffected by degradation, suggesting that nodulation capacity in A. acuminata is resilient to changes in land use at least within the studied context. This resilience highlights the potential for physiological plasticity or redundancy among nodule- and root-associated bacteria of A. acuminata to maintain or improve symbiotic functions (Gyaneshwar et al. 2011; Tong et al. 2025). Overall, the results reported in this study highlight the complex interactions between land degradation and microbial assemblages in soil and in the root of the pioneer plant A. acuminata . The strong influence of soil pH and total carbon on soil bacterial communities underscores the need to consider abiotic and biotic factors in restoration strategies. Furthermore, the A. acuminata endophytic microbiome exhibits both relative stability and significant plasticity, allowing the plant to modulate its community structure to ensure efficient function under different environmental conditions. By maintaining and improving these beneficial microbial partners, A. acuminata may sustain its own growth and contribute to soil recovery, creating a positive feedback loop that facilitates the re-establishment of diverse plant and microbial assemblages over time. This adaptive capacity strongly suggests that exploiting plant-microbe interactions is a promising avenue for the recovery of degraded Andean ecosystems. Future work integrating bacteria selection, functional analyses and experimental inoculations will be needed to determine the appropriate bacteria consortia useful to accelerate spontaneous restoration. Declarations Competing interests The authors have no competing interests to declare. Supplementary information Supplementary data associated with this article Fig. S1 and tables Tab S1- Tab S11 can be found online at doi: XXXX Funding This work was funded by the Italian Ministry of University and Research. The authors declare they have no financial interests Author Contribution N.G.: design of the research, sample collection, sample processing and laboratory analyses, data analysis and interpretation, preparation of the original draft.L.A.Q.: data analysis and interpretation, preparation of the original draft.R.A.: design of the research, revision of the manuscript.M.J.: sample collection and measurement of nodules, revision of the manuscript.R.G.: design of the research, revision of the manuscript.S.C.: design of the research, supervision and guidance, revision and editing of the manuscript. Acknowledgement NG acknowledges the support of the Department of Earth and Environmental Sciences, University of Milano-Bicocca, through a fellowship. The authors sincerely thank Sarah Caronni for laboratory assistance, Miguel Ángel Vizcho for his help with field sampling, and Dr. Wayne Hanson for reviewing the English of the manuscript. 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Quaglini","email":"","orcid":"","institution":"Museo di Storia Naturale di Milano, Sezione di Botanica","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"A.","lastName":"Quaglini","suffix":""},{"id":578422358,"identity":"a0623f65-17de-4d84-9c47-7474a569086f","order_by":2,"name":"Raffaella Ansaloni","email":"","orcid":"","institution":"University of Azuay","correspondingAuthor":false,"prefix":"","firstName":"Raffaella","middleName":"","lastName":"Ansaloni","suffix":""},{"id":578422359,"identity":"426ede0a-efb6-4362-b38c-b88e5c19c90c","order_by":3,"name":"Mayra Jiménez","email":"","orcid":"","institution":"University of Azuay","correspondingAuthor":false,"prefix":"","firstName":"Mayra","middleName":"","lastName":"Jiménez","suffix":""},{"id":578422360,"identity":"34912750-e45c-4ad7-a632-953462bbdcd0","order_by":4,"name":"Rodolfo Gentili","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Rodolfo","middleName":"","lastName":"Gentili","suffix":""},{"id":578422363,"identity":"c4ce67f6-34b0-4bd1-bdaa-c2acfb21717b","order_by":5,"name":"Sandra Citterio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIie3PsQrCMBCA4SuFuBTnFsS8QkpBKBR8FUvBLiqODg4pYt109i06drQITnFP0UUEJxcXQRAxRnCLdXTIP4RQ+nEXAJ3uT7NfB5JXAk0Ag0JIwaomyJTE+5BvRhKQBF4/g1TKMXi2LXajMeA6XhTH0TCIs91kmh1yaLQVhLBB5LMNuCkyTZeRbj/bFykPmXoxAr2WQxF0xFuQQ8m6n/FQkFRN8OIsyEOS2k2QmFQR4GJKkr6nGIJ0KgnhZ89P5rZ4S+SJxbruUhJmW9ZKtVjPLek1wHhSHC70HuA6j0/lLQ/aNaraTGb/8EWn0+l0v/cEC5tTuM3HLSwAAAAASUVORK5CYII=","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":true,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Citterio","suffix":""}],"badges":[],"createdAt":"2026-01-21 13:23:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8660278/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8660278/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101415334,"identity":"d5ad0851-7e40-4b82-ab7e-f8087862025c","added_by":"auto","created_at":"2026-01-29 12:28:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1042413,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the sampling locations in the province of Azuay across two distinct land-use types: native forest and degraded forest\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/b771e878b7d03efe50f46988.png"},{"id":101751477,"identity":"ac21b434-2532-4f6f-abcd-9626b01a5c29","added_by":"auto","created_at":"2026-02-03 10:20:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129159,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of \u003cstrong\u003e(a)\u003c/strong\u003e ASV richness, \u003cstrong\u003e(b)\u003c/strong\u003e evenness, and \u003cstrong\u003e(c)\u003c/strong\u003e Shannon diversity in bulk soil, root, and nodule samples of \u003cem\u003eAlnus acuminata\u003c/em\u003e from degraded (orange boxes) and native (light green boxes) forest sites\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/12b2ba95ac536d6de685fd30.png"},{"id":101415333,"identity":"e0e4e7a2-706c-46c2-9271-0336253b73be","added_by":"auto","created_at":"2026-01-29 12:28:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":235769,"visible":true,"origin":"","legend":"\u003cp\u003eNon-metric multidimensional scaling (NMDS) of bacterial community associated with \u003cem\u003eAlnus acuminata\u003c/em\u003e in two land-use types: degraded forest (orange polygons) and native forest (light green polygons). The left plot shows bulk soil; the right plot shows endophytic samples (roots and nodules). The diagram also displays the main bacterial phyla associated with each land-use type and compartment\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/82a98b4fc2eb56e152c1a7db.png"},{"id":101751415,"identity":"17dc641d-6007-4e99-a192-7ff4d71a7035","added_by":"auto","created_at":"2026-02-03 10:20:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90801,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis (RDA) showing the influence of soil pH, total nitrogen, and plant richness on bacterial communities in bulk soil (left) and endophytes (right) of \u003cem\u003eAlnus acuminata\u003c/em\u003efrom degraded forest (orange) and native forest (light green). Significant variables are indicated with red arrows\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/05b0fc8aab14a1c286de29d8.jpeg"},{"id":101415336,"identity":"40db14a2-00a8-485f-a0de-9cfe20445c53","added_by":"auto","created_at":"2026-01-29 12:28:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169472,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of relative abundance (%) of main bacterial phyla across compartments (bulk soil, roots, nodules) in degraded forest (orange bars) and native forest (light green bars). Significant differences between land-use types are indicated by asterisks\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/f4ac392acf398e0237e7a45e.png"},{"id":101751962,"identity":"75b32317-d279-4a09-b224-942281dce89e","added_by":"auto","created_at":"2026-02-03 10:24:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":534412,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of relative abundances (%) of bacterial genera across compartments (bulk soil, roots and nodules) in degraded forest (DF) and native forest (NF)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/edb99edda64e6558ae0e262d.png"},{"id":101751698,"identity":"eb126a19-e25b-4379-964d-a61ccc3938a6","added_by":"auto","created_at":"2026-02-03 10:22:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22046,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of nodule length (mm), width (mm), and weight (mg) of \u003cem\u003eAlnus acuminata \u003c/em\u003eindividuals growing in degraded forest (orange boxes) and native forest (light green boxes)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/6af3e7393035bde7cf91c5ce.png"},{"id":101942768,"identity":"7e18be6b-fea5-46ff-94fb-2648ac00b0bd","added_by":"auto","created_at":"2026-02-05 09:37:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3200238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/436ecb1f-bb8d-4748-82b1-7b3db9d328eb.pdf"},{"id":101415338,"identity":"7222de72-87cd-44d2-ae77-76c071ccb29a","added_by":"auto","created_at":"2026-01-29 12:28:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3991841,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8660278/v1/aff6c85c0ea8c78b6f4424bd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in microbiome assembly of the pioneer Andean tree Alnus acuminata in response to land degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLand degradation, generally defined as the long-term reduction or loss of biological productivity, ecological integrity, or human value (Olsson et al. 2022), is a pressing global challenge. In Ecuador, especially in the Andean mountains, forest ecosystems are under constant threat from human activities, primarily due to extensive livestock grazing, agriculture, mining, and logging. Additionally, large areas have been converted into plantations of exotic tree species such as \u003cem\u003eEucalyptus\u003c/em\u003e spp. and \u003cem\u003ePinus\u003c/em\u003e spp. (Hofstede et al. 2002; G\u0026uuml;nter et al. 2009; Tapia-Armijos et al. 2015). This land-use change has resulted in irreversible biodiversity loss and severely disrupted functioning ecosystems, affecting a significant proportion of native vegetation in an area of exceptionally high biological diversity. Nearly half (46%) of the original forest cover in southern Ecuador has been replaced by anthropogenic land cover types (Tapia-Armijos et al. 2015).\u003c/p\u003e \u003cp\u003eThese large-scale transformations not only threaten plant and animal biodiversity but can also have profound implications for the often-overlooked microbial communities that drive essential ecosystem functions. Microorganisms support almost all ecosystem functions, and ecosystem-scale responses fundamentally depend on their resident microbial communities (Osburn et al. 2023; Rawat et al. 2023). These communities, primarily bacteria and fungi, regulate a wide range of essential processes both below- and aboveground, including the transformation of organic matter, carbon storage, nutrient cycling, and plant productivity (Rawat et al. 2023; Dixit et al. 2024). Plant-associated microorganisms have gained increasing attention due to their central role in affecting plant establishment and adaptation, disease resistance, and resilience to environmental stress (Fitzpatrick et al. 2018; Muhammad et al. 2024; Zieschank et al. 2025). Despite their central role in these processes, microbial communities have long remained underexplored. This is partly due to the exceptional diversity of microorganisms, the challenges of studying them directly, their spatial heterogeneity, and their historical exclusion from most biodiversity surveys (Osburn et al. 2023).\u003c/p\u003e \u003cp\u003eAs a result, the link between land degradation and soil-plant-microbes remains insufficiently explored, representing a fundamental challenge for understanding ecosystem responses to land-use change (Zhou et al. 2018; Rawat et al. 2023). Land degradation affects key soil properties such as pH, organic matter content, nutrients availability, moisture, and temperature, which directly influence microbial activity, diversity, community composition, and abundance (Zhou et al. 2018; Peng et al. 2022; M\u0026eacute;sz\u0026aacute;rošov\u0026aacute; et al. 2024). For instance, soil pH is known to exert strong selective pressure on specific bacterial taxa, and its alteration can lead to shifts in microbial communities (Zhou et al. 2018; Pereira et al. 2022). Similarly, organic carbon content serves as a key driver of microbial dynamics by providing essential energy and nutrient sources that support community growth and metabolic activity (Li et al. 2024; Jiang et al. 2025). Furthermore, plant species significantly influence the soil microbiome through root architecture, exudate release, and rhizodeposition patterns, creating niches that favour specific microbial populations (Oppenheimer-Shaanan et al. 2022; Domeignoz-Horta et al. 2024).\u003c/p\u003e \u003cp\u003eGiven the ecological consequences of land degradation, especially for soil characteristics and microbial communities, ecological restoration becomes a key priority. Successful restoration often relies on the re-establishment of vegetation, particularly pioneer species that are capable of establishing in nutrient-poor, compacted, or contaminated soils where other plants may fail. These early colonizers can initiate positive feedback loops by improving soil structure, enhancing nutrient availability, and fostering microbial activity through root exudates and litter inputs (Wang et al. 2024; Xiuyu et al. 2024). Importantly, the ability of certain plant species to recruit beneficial microbial partners, including mycorrhizal fungi and nitrogen-fixing bacteria, can significantly accelerate the recovery of soil functions in degraded environments (Sun et al. 2018). Given this, selecting plant species that form effective mutualisms and can tolerate abiotic stress is essential for restoring both plant cover and the microbial networks that sustain a functioning ecosystem.\u003c/p\u003e \u003cp\u003eAmong the pioneer species with high potential for ecological restoration, \u003cem\u003eAlnus\u003c/em\u003e (alder) species stand out for their remarkable ability to colonize adverse conditions, including high salinity, drought, extreme pH, and soils contaminated with heavy metals and organic pollutants (Lefran\u0026ccedil;ois et al. 2010; Diagne et al. 2013; Thiem et al. 2018; Bhattacharyya et al. 2024). This success is attributed to a tripartite symbiosis with ectomycorrhizal and arbuscular mycorrhizal fungi, as well as nitrogen-fixing bacteria; particularly \u003cem\u003eFrankia\u003c/em\u003e spp. (Becerra et al. 2005; Chen et al. 2020). However, current research on \u003cem\u003eAlnus\u003c/em\u003e species has identified diverse bacterial taxa beyond \u003cem\u003eFrankia\u003c/em\u003e that are associated with alder root nodules, uncovering even greater complexity in these symbiotic relationships (Carro et al. 2013; Aslani et al. 2020; Garneau et al. 2023a). Beyond plant\u0026ndash;microbe interactions, microbe\u0026ndash;microbe dynamics are also critical. For example, \u003cem\u003ePseudomonas\u003c/em\u003e has been shown to promote nodulation in \u003cem\u003eAlnus rubra\u003c/em\u003e Bong. (Knowlton and Dawson 1982), while other bacteria genera such as \u003cem\u003eMicrovirga\u003c/em\u003e and \u003cem\u003eStreptomyces\u003c/em\u003e can stimulate \u003cem\u003eFrankia\u003c/em\u003e growth (Garneau et al. 2023a).\u003c/p\u003e \u003cp\u003eWhile there are many studies that explored the microbial community associated with different \u003cem\u003eAlnus\u003c/em\u003e species around the world (e.g. Aslani et al. 2020; Garneau et al. 2023a; Thiem et al. 2023; Dove et al. 2024), little information is available on \u003cem\u003eAlnus acuminata\u003c/em\u003e Kunth, the only alder species native to the mountains of South America (L\u0026aelig;gaard and Balslev 2014). In Ecuador, where extensive deforestation and land-use conversion threaten the integrity of Andean forest ecosystems, \u003cem\u003eA. acuminata\u003c/em\u003e has been used for reforestation due to its pioneering character and soil-enriching capabilities (G\u0026uuml;nter et al. 2009). Yet, its belowground microbial associations remain largely unexplored, especially concerning non-\u003cem\u003eFrankia\u003c/em\u003e bacteria and the broader structure of its root nodules and root associated bacterial communities.\u003c/p\u003e \u003cp\u003eWith the goal of improving ecosystem recovery in degraded Andean forests, this study sought to provide the necessary knowledge to exploit pioneer plant-microbe interactions. To this end, the diversity and structure of bacterial communities associated with the roots and nodules of \u003cem\u003eAlnus acuminata\u003c/em\u003e, was investigated using metagenomic DNA sequencing comparing individuals and soils from a native and a degraded forest.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area and sample collection\u003c/h2\u003e \u003cp\u003eThe study was conducted in the Andean mountains of southern Ecuador in the province of Azuay, across two distinct land-use types: (1) native intact forest (NF; 2\u0026deg;55\u0026rsquo; S, 78\u0026deg;50\u0026rsquo; W), a patch of high montane evergreen forest located within the Aguarongo Protected Vegetation Area; and (2) degraded forest (DF; 2\u0026deg;57\u0026rsquo; S, 78\u0026deg;53\u0026rsquo; W), corresponding to a high montane evergreen forest that has been completely deforested, which is located in close proximity to the Aguarongo Reserve (~\u0026thinsp;6 km away) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe native forest represents a well-preserved remnant of natural vegetation, showing no evidence of fire, logging, or other anthropogenic disturbances. The list of dominant species characterizing the NF is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e of the supplementary information. The degraded site, on the other hand, has undergone a complete land-use change. It was deforested over 50 years ago, mainly for livestock grazing and road construction, and has since been abandoned for about 10 years. It is now dominated by the grass \u003cem\u003eCenchrus clandestinus\u003c/em\u003e (Hochst. ex Chiov.) Morrone, commonly used as a forage crop, and by other non-native species such as the tree \u003cem\u003eEucalyptus globulus\u003c/em\u003e Labill., along with a few native pioneer species, including \u003cem\u003eAlnus acuminata\u003c/em\u003e, the focal specie of this study. Both sites are located at elevations ranging from 2,800 to 3,140 m a.s.l. and share similar climatic conditions. The climate is cold temperate, with annual temperatures ranging from 9 to 12\u0026deg;C and an annual precipitation of 820 mm. Rainfall varies throughout the year, with a dry season from May to September, during which monthly precipitation ranges from 25 to 60 mm. From October to April, rainfall is more intense, reaching 75 to 110 mm per month (Minga 2002).\u003c/p\u003e \u003cp\u003eBulk soil, root, and nodule samples were collected from different individuals of \u003cem\u003eA. acuminata\u003c/em\u003e growing in both the native and degraded forest sites. Four trees were randomly selected in each area, maintaining a maximum distance of about 150 m between individuals. The sampled trees had heights ranging from 4 to 7.5 m and diameters at breast height (DBH) between 6 and 15 cm. Around each focal tree, a 5 \u0026times; 5 m plot was established with the tree in the center. Within each plot, all vascular plant species were identified according to the Angiosperm Phylogeny Group classification system (APG 2016).\u003c/p\u003e \u003cp\u003eFor bulk soil sampling, approximately 100 g of soil was randomly collected from a depth of 20\u0026ndash;40 cm, at a distance of 10\u0026ndash;20 cm from the trunk of each selected tree. Bulk soil samples were collected from three of the four selected trees per site, of which\u0026thinsp;~\u0026thinsp;50 g was used for the analysis of soil chemical properties. Root and nodule samples were obtained from the four trees selected per site, including those used for soil sampling and one additional tree. In total, six bulk soil samples (three per land-use type) and eight root\u0026ndash;nodule samples (four per land-use type) were collected for the analysis. All samples (bulk soil, roots, and nodules) were stored at -20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eTo compare root nodulation among \u003cem\u003eA. acuminata\u003c/em\u003e individuals growing in native and degraded forests, the morphological characteristics of \u003cem\u003eFrankia\u003c/em\u003e nodules were assessed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For this, three replicates per individual (i.e. root subsamples of approximately 100 cm each) were randomly selected from the same individual trees sampled for bacterial analysis. Within each replicate, all nodule lobes were measured for size (length and width, mm) and weight (mg).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSoil analysis\u003c/h3\u003e\n\u003cp\u003ePrior to analysis, soil samples were sieved through a 2 mm mesh and stored at room temperature. Soil pH was measured in water, while total nitrogen (%) and total carbon (%) contents were determined using a CN elemental analyzer (Flash EA 1112 NC Soil, Thermo Fisher Scientific, Pittsburgh, USA).\u003c/p\u003e\n\u003ch3\u003eSample processing\u003c/h3\u003e\n\u003cp\u003ePrior to DNA extraction, roots and nodules of \u003cem\u003eA. acuminata\u003c/em\u003e were first washed with tap water and then surface-sterilized by immersion in 70% ethanol for 1 min, followed by 3% sodium hypochlorite for 3 min, a second immersion in 70% ethanol for 30 s, and four rinses with sterile distilled water. To verify the effectiveness of sterilization, 100 \u0026micro;L from the final rinse was plated on trypticase soy agar and incubated to check for microbial contamination (Cheng et al. 2019). Sterilized roots and nodules were then ground in liquid nitrogen.\u003c/p\u003e \u003cp\u003eBacterial community diversity associated with bulk soil, \u003cem\u003eA. acuminata\u003c/em\u003e roots and nodules (endophytes) in native and degraded forests was defined using 16S rRNA gene amplicons. DNA from all samples were extracted using the FastDNA\u0026reg; Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) following the manufacturer\u0026rsquo;s protocol. An initial PCR amplification was performed using primers 27F (5\u0026prime;-AGAGTTTGATCMTGGCTCAG-3\u0026prime;) and 519R (5\u0026prime;-GWATTACCGCGGCKGCTG-3\u0026prime;) (Frank et al. 2008) on undiluted DNA extracts as well as on 1:10, 1:100, and 1:1,000 dilutions to assess the presence of potential PCR inhibitors. The thermal cycling conditions were as follows: initial denaturation at 95\u0026deg;C for 4 min; 29 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 45 s, and extension at 72\u0026deg;C for 45 s; followed by a final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e \u003cp\u003eBecause root and nodule samples also contain plant plastid-derived 16S rRNA sequences, these samples were processed differently from bulk soil, with a few additional steps required. Briefly, the full-length 16S rRNA gene was amplified from the DNA of roots and nodules using primers 27F (5\u0026prime;-AGAGTTTGATCCTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-GGTTACCTTGTTACGACTT-3\u0026prime;). PCR conditions were as follows: initial denaturation at 95\u0026deg;C for 4 min; 29 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 45 s, and extension at 72\u0026deg;C for 2 min; followed by a final extension at 72\u0026deg;C for 5 min. PCR products were then purified using the Wizard\u0026reg; SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA), according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eA second PCR amplification was performed on all samples (bulk soil, roots, and nodules), targeting the V5\u0026ndash;V6 hypervariable regions of the bacterial 16S rRNA gene. Two different primer sets were used depending on the sample type: primers 783F and 1027R were used for bulk soil samples (Gandolfi et al. 2024), while primers 799F and 1107R, specifically designed for endophytic communities, were used for root and nodule samples (Chen et al. 2022). All primers were tagged with custom 6 bp oligonucleotide barcodes (sequences listed in Table S2). PCR conditions were identical for both primer sets and included an initial denaturation at 94\u0026deg;C for 4 min, followed by 28 cycles of denaturation at 94\u0026deg;C for 50 s, annealing at 47\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s, with a final extension at 72\u0026deg;C for 5 min. PCR products were purified, and DNA concentrations were quantified using a Qubit\u0026reg; 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA).\u003c/p\u003e \u003cp\u003eAmplicon libraries were prepared in batches of nine samples, each distinguished by the unique barcode pair (Table S2). Library preparation included the addition of standard Nextera indexes (Illumina, Inc., San Diego, CA, USA), and sequencing was carried out on an Illumina MiSeq platform using a 2 x 300 bp paired-end protocol. Amplicon Sequence Variants (ASVs) were inferred using the DADA2 algorithm (Callahan et al. 2016).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eDue to the use of different primer sets for bulk soil and endophytes (roots and nodules) samples, these compartments were analyzed separately. All statistical analyses were performed within the R environment v. 4.3.2 (R Core Team 2023).\u003c/p\u003e \u003cp\u003eFirst, different α-diversity metrics on the rarified bacterial data were calculated using the \u003cem\u003evegan\u003c/em\u003e package. Specifically, bacterial richness was estimated as the number of ASV using the specnumber() function. Bacterial evenness was assessed using the Berger-Parker index, which measures the dominance of the most abundant taxa in a community (Berger and Parker 1970). Additionally, the Shannon diversity index was calculated using the diversity() function. Next, differences were analyzed for bacterial α-diversity between land-use types (native and degraded forests), compartments (bulk soil, roots, and nodules), and their interaction. ANOVA was used for bacterial evenness and Shannon diversity index, while generalized linear models (GLMs) with a quasi-Poisson distribution were applied to ASV richness.\u003c/p\u003e \u003cp\u003eTo visualize differences in bacterial community structure between land-use types, Non-metric Multidimensional Scaling (NMDS) was conducted based on Bray-Curtis dissimilarities using the metaMDS() function from the \u003cem\u003evegan\u003c/em\u003e package. Additionally, significant differences were tested with PERMANOVA using the adonis2() function. To evaluate the influence of soil variables and plant richness on bacterial community composition, a Redundancy Analysis (RDA) was conducted on bulk soil and endophytes (roots and nodules) ASVs, constrained by environmental variables including soil pH, total nitrogen, total carbon, and plant richness. However, because total carbon and soil pH were highly correlated, total carbon was excluded from the analysis, and soil pH was retained. Before these multivariate analyses, the bacterial ASV abundance matrix was transformed using the Hellinger method to downweigh the influence of highly abundant ASVs, emphasize their presence or absence, and address the double-zero problem commonly encountered when comparing community compositions across samples (Borcard et al. 2018).\u003c/p\u003e \u003cp\u003eThe 10 most abundant phyla were identified across all land-used types and compartments and compared to their relative abundances between native and degraded forests with one-way ANOVA.\u003c/p\u003e \u003cp\u003eNext, the core endophytic bacterial communities present in the roots and nodules of \u003cem\u003eA. acuminata\u003c/em\u003e were identified by selecting ASVs detected in at least 80% of samples, regardless of land-used type (Cheng et al. 2019; Neu et al. 2021). The core microbiome refers to microbial taxa that are consistently associated with a specific host or environment, typically identified by their presence across multiple microbial communities (Neu et al. 2021). These taxa are thought to play key ecological and functional roles within their host or environment under prevailing conditions (Risely 2020).\u003c/p\u003e \u003cp\u003eLastly, \u003cem\u003eA. acuminata\u003c/em\u003e root nodulation (nodule lobe length, width, and weight) was analyzed using linear mixed models (LMMs) with land-use type (native and degraded forests) as a fixed factor, root length as a covariate, and replicate (i.e. root subsample) nested into individual tree identity as a random factor. To do this, the \u003cem\u003elme4\u003c/em\u003e and \u003cem\u003elmerTest\u003c/em\u003e packages were implemented.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in soil properties and plant composition between native and degraded forests\u003c/h2\u003e \u003cp\u003eSoil properties were altered in the degraded forest (DF) compared to the native forest (NF). The DF exhibited a markedly higher soil pH (5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs. 4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) and a lower total carbon content (4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% vs. 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%). However, total nitrogen content did not substantially differ between the two sites (0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% in the DF and 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% in the NF).\u003c/p\u003e \u003cp\u003eA total of 19 vascular plant species, belonging to 14 families were recorded in the plots surrounding \u003cem\u003eA. acuminata\u003c/em\u003e individuals. In the degraded site, the vegetation was almost completely dominated by two introduced herbaceous species: \u003cem\u003eCenchrus clandestinus\u003c/em\u003e (Hochst. ex Chiov.) Morrone and \u003cem\u003eTrifolium repens\u003c/em\u003e L, along with a few native shrubs such as \u003cem\u003eBaccharis latifolia\u003c/em\u003e (Ruiz \u0026amp; Pav.) Pers., \u003cem\u003eCestrum tomentosum\u003c/em\u003e L.f., and \u003cem\u003eRubus floribundus\u003c/em\u003e Kunth. In contrast, the native forest was characterized by a dominance of native tree species typical of high mountain forest ecosystems, including \u003cem\u003eAxinaea macrophylla\u003c/em\u003e Triana, \u003cem\u003eLomatia hirsuta\u003c/em\u003e (Lam.) Diels, \u003cem\u003eMyrica parvifolia\u003c/em\u003e Benth., \u003cem\u003ePolylepis lanuginosa\u003c/em\u003e Kunth, and \u003cem\u003eVallea stipularis\u003c/em\u003e L.f. The understory featured species such as \u003cem\u003eValeriana hirtella\u003c/em\u003e Kunth, \u003cem\u003eVerbesina latisquama\u003c/em\u003e S.F. Blake, and the fern \u003cem\u003eLophosoria quadripinnata\u003c/em\u003e (J.F. Gmel.) C. Chr.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of land degradation on bacterial alpha-diversity\u003c/h3\u003e\n\u003cp\u003eWhen analyzing the differences of bacterial α-diversity between DF and NF, no effect of degradation was found on ASV richness of either bulk soil (GLMs: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.519; Table S3), or endophytes (root and nodule samples) of \u003cem\u003eA. acuminata\u003c/em\u003e (GLMs: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.918; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Table S4). Conversely, bacterial evenness of bulk soil samples was significantly lower in the degraded forest (ANOVA: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;32.93, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00457; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb); a similar trend was observed for Shannon index, although the difference was marginally significant (ANOVA: \u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.164, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0554; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, Table S3). Bacterial diversity in endophytic samples did not differ significantly between root and nodule compartments, between land-use types, or due to their interaction (Table S4). Root samples showed a pattern similar to that of bulk soil, with lower evenness and Shannon indices in DF compared to NF; however, these differences were not statistically significant, likely due to high variability in index values. Degradation also appeared not to affect the bacterial diversity of \u003cem\u003eA. acuminata\u003c/em\u003e nodules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEffect of land degradation on the structure and composition of bacterial communities\u003c/h3\u003e\n\u003cp\u003eThe results indicate that bacterial community structure across different compartments (bulk soil, roots, and nodules of \u003cem\u003eA. acuminata\u003c/em\u003e) was influenced by land-use type, as revealed by NMDS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In bulk soil, PERMANOVA showed that land use explained a substantial portion (86.4%) of the variation in community composition, although the effect was not statistically significant, likely due to the small sample size (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25.38, \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.864, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.1; Table S5). In contrast, for \u003cem\u003eA. acuminata\u003c/em\u003e endophytes (roots and nodules), the effect of land use was statistically significant, accounting for 10.6% of the variation (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.761, \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.106, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.037; Table S6). These findings suggest that land degradation can alter bacterial community structure, with a marked effect in the bulk soil compared to the endosphere. Additionally, differences between root and nodule compartments explained 8.9% of the variation (\u003cem\u003eF\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.472, \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.089, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.088; Table S6), indicating that compartmentalization within the plant also contributes to shaping the endophytic bacterial community, albeit to a lesser extent than land use.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the environmental variables examined, soil pH and plant richness were the main drivers of bacterial community composition, as revealed by the RDA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Together, these factors explained 91.3% of the variation in bulk soil bacterial communities (RDA: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but only 19.5% of the variation in endophytic communities, which was not statistically significant (RDA: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Soil pH emerged as a key determinant of bacterial community structure in bulk soil, especially under land degradation. In contrast, total carbon, excluded from the plot due to multicollinearity with pH, and plant richness were more strongly associated with bacterial communities in the native forest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative abundance of the dominant bacterial phyla shifted in response to land degradation, with more pronounced effects in bulk soil bacterial communities compared to the endophytic communities of \u003cem\u003eA. acuminata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table S7). In bulk soil, degradation significantly decreased the abundance of Pseudomonadota, Acidobacteriota, Bacteroidota, Chloroflexota, and Verrucomicrobiota, while increasing the abundance of Actinomycetota compared to native forest (ANOVA: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table S8).\u003c/p\u003e \u003cp\u003eIn the endosphere, bacterial communities associated with the roots and nodules of \u003cem\u003eA. acuminata\u003c/em\u003e were largely dominated by Pseudomonadota in both DF and NF. Root endophytes in DF showed a reduction in the abundance of the phyla Bacillota, Bacteroidota, and Acidobacteriota, while Bacteroidota, Chloroflexota, and Cyanobacteriota declined in nodules. Conversely, degradation led to an increase in the abundance of Planctomycetota, Actinomycetota, and Deinococcota in nodule endophytes compared with NF; however, these changes were not statistically significant, likely due to high variability among samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Tables S9, S10).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnique phyla were detected exclusively in bulk soil samples, including candidate division WPS-2 (DF), Chlamydiota (NF and DF), Chlorobiota (DF), Elusimicrobiota (NF and DF), Latescibacteria (NF and DF), and Parcubacteria (DF). In contrast, the only unique phylum detected in endophytic samples (roots and nodules) was Abditibacteriota (DF).\u003c/p\u003e \u003cp\u003eAt lower taxonomic ranks, the dominance of specific bacterial taxa became more apparent not only within each land-use type, but also across different compartments. For example, in bulk soil from degraded forest, the most abundant genera were \u003cem\u003eStreptomyces\u003c/em\u003e (relative abundance of 4.7%), \u003cem\u003eReyranella\u003c/em\u003e (2.6%), and \u003cem\u003eMycobacterium\u003c/em\u003e (2.2%). In contrast, in the native forest, \u003cem\u003eParaburkholderia\u003c/em\u003e (5.2%), \u003cem\u003eGP2\u003c/em\u003e (5%), \u003cem\u003eSubdivision3_genera_incertae_sedis\u003c/em\u003e (2%), and \u003cem\u003eCaballeronia\u003c/em\u003e (1.8%) were the most abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the endophytic samples, \u003cem\u003eButtiauxella\u003c/em\u003e, \u003cem\u003eRahnella\u003c/em\u003e, \u003cem\u003eVariovorax\u003c/em\u003e, and \u003cem\u003eRhizobium\u003c/em\u003e dominated the root endosphere of \u003cem\u003eA. acuminata\u003c/em\u003e in the degraded site, with relative abundances of 8.1%, 5%, 4.4%, and 3.5%, respectively. Conversely, in native forest, the most abundant genera were \u003cem\u003eParaburkholderia\u003c/em\u003e (8.3%), \u003cem\u003eBradyrhizobium\u003c/em\u003e (7.4%), \u003cem\u003ePseudomonas\u003c/em\u003e (2.8%), and \u003cem\u003eCaballeronia\u003c/em\u003e (1.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, genera classified within the order \u003cem\u003eEnterobacterales\u003c/em\u003e (belonging to the phylum Pseudomonadota) were by far the most abundant under both land-use types, accounting for 56% and 30% of the total root endophytic community in DF and NF, respectively. With regard to nodule endophytes, a remarkably higher abundance of \u003cem\u003eParaburkholderia\u003c/em\u003e was found in NF compared to DF (42.4% vs. 1.1%, respectively). In DF, \u003cem\u003eRhodanobacter\u003c/em\u003e (14.1%), \u003cem\u003eRahnella\u003c/em\u003e (12%), and \u003cem\u003eCaballeronia\u003c/em\u003e (5.7%) were the most abundant genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, among nodule endophytes, \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eBradyrhizobium\u003c/em\u003e appeared to be unaffected by land degradation. \u003cem\u003ePseudomonas\u003c/em\u003e, a dominant genus in the nodule endosphere, showed similar abundances in DF and NF (21% and 17.6%, respectively) and was detected only in NF bulk soil. Similarly, \u003cem\u003eBradyrhizobium\u003c/em\u003e displayed comparable abundances, 1.1% in NF and 2.2% in DF, but showed a consistent dominance in bulk soil samples across both land-use types (approximately 3%).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the core endophytic bacterial communities of roots and nodules of Alnus acuminata\u003c/h2\u003e \u003cp\u003eAmong the 3,225 ASVs detected in the root and nodule endosphere of \u003cem\u003eA. acuminata\u003c/em\u003e, 10 ASVs were identified consistently present in root samples at a frequency greater than 80%, and 7 ASVs met the same criterion in nodules, independently of land-use type (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, all core ASVs identified in nodules were also part of the root core community. These shared ASVs (i.e. ASV146, 207, 261, 298, 353, 775, and 826) were taxonomically assigned to \u003cem\u003eParaburkholderia\u003c/em\u003e, \u003cem\u003eCaballeronia\u003c/em\u003e, \u003cem\u003eRahnella\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and two unclassified \u003cem\u003eEnterobacterales\u003c/em\u003e. Among root core endophytes, the most abundant ASV was ASV826, assigned to the order \u003cem\u003eEnterobacterales\u003c/em\u003e, which accounted for 26.85% of the total relative abundance. In nodules, the most abundant core ASV was ASV775 (5.92%), assigned to the genus \u003cem\u003ePseudomonas\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCore endophytic bacterial community of \u003cem\u003eA. acuminata\u003c/em\u003e samples. The table displays taxonomic classification, relative abundance (Ab), and frequency (Frq) of bacterial Amplicon Sequence Variants (ASVs) in root and nodule samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePhylum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eASV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eRoots\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eNodules\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAb%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFrq%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAb%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFrq %\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003ePseudomonadota\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlphaproteobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHyphomicrobiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eBradyrhizobiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eBradyrhizobium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBetaproteobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eBurkholderiales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eBurkholderiaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eParaburkholderia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCaballeronia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eGammaproteobacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cem\u003eEnterobacterales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eYersiniaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRahnella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e/\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e26.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePseudomonadales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ePseudomonadaceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eXanthomonadales\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eRhodanobacteraceae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eRhodanobacter\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffect of land degradation on the morphology of Alnus acuminata nodules\u003c/h2\u003e \u003cp\u003eLand degradation did not appear to affect the root nodule morphology of \u003cem\u003eA. acuminata\u003c/em\u003e. Linear mixed models showed no significant differences in nodule length, width, or weight between degraded and native forests (LMMs: \u003cem\u003ep\u003c/em\u003e\u003csub\u003elength\u003c/sub\u003e = 0.372, \u003cem\u003ep\u003c/em\u003e\u003csub\u003ewidth\u003c/sub\u003e = 0.8574, \u003cem\u003ep\u003c/em\u003e\u003csub\u003eweight\u003c/sub\u003e = 0.9215; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Table S11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBy comparing bacterial communities between a native forest and a degraded forest in Ecuador, this study investigated how a land-use trajectory (deforestation for pasture followed by abandonment) affects their diversity and structure in the soil, roots, and root nodules of the pioneer tree \u003cem\u003eAlnus acuminata\u003c/em\u003e. Pioneer trees are key engineer species in the restoration of degraded landscapes. As a nitrogen-fixing pioneer species, \u003cem\u003eA. acuminata\u003c/em\u003e is critical for facilitating soil recovery and ecological succession in these abandoned pastures. Therefore, understanding the bacterial communities that support its establishment and growth is pivotal. This knowledge can inform effective, long-term recovery strategies that enhance ecosystem resilience.\u003c/p\u003e \u003cp\u003eThe study report here demonstrated that, compared with native forest, degraded forest shows a marked alteration of soil physicochemical properties, plant community composition and the structure of bacterial communities associated with both bulk soil and the endosphere of \u003cem\u003eA. acuminata\u003c/em\u003e. Specifically, soil pH increased and total carbon decreased in degraded forest soils compared to native forest, while total nitrogen content remained relatively unchanged. These findings agree with previous research showing that forest disturbance often leads to soil alkalization (Pedrinho et al. 2020; Pereira et al. 2022) and carbon loss due to reduced organic matter inputs and altered nutrient cycling (Zhou et al. 2018; Peng et al. 2022).\u003c/p\u003e \u003cp\u003eThe results of this study showed that bacterial richness was not significantly affected by land degradation in either bulk soil or root and nodule endophytes of \u003cem\u003eA. acuminata\u003c/em\u003e. However, bacterial evenness and Shannon diversity indices were significantly lower in degraded bulk soils but not in both roots and nodules, indicating that degradation affected principally bulk soil community structure by reducing the evenness of bacterial taxa rather than richness. This pattern agrees with findings from other studies where disturbance often leads to dominance by a few opportunistic taxa at the expense of a more balanced community (Sigler and Zeyer 2004). For example, similar reductions in soil bacterial diversity have been reported in degraded semiarid tropical regions (Pereira et al. 2022; Silva et al. 2024). In contrast, other studies have documented increases (Rodrigues et al. 2013; Zhou et al. 2018; Pedrinho et al. 2020) or no significant changes (Lee-Cruz et al. 2013; Lozano et al. 2014; Li et al. 2024) following land degradation, highlighting the context dependency of microbial responses. Such differences likely reflect variation in spatial scale, disturbance type and intensity, soil physicochemical properties, and ecosystem characteristics (Zhou et al. 2018).\u003c/p\u003e \u003cp\u003eWith regard to endophytic bacterial diversity between land-use types, the lack of significant differences (evenness was lower but not significant) suggests that, compared with bulk soil bacterial communities, plant-associated microbial communities are more buffered against external environmental changes due to host-mediated selection (Lundberg et al. 2012; Hardoim et al. 2015). Plants usually select beneficial microorganisms to face unfavorable environmental conditions, but in some cases, that selection can lead to a reduced root-associated bacterial diversity as reported for \u003cem\u003eAlnus glutinosa\u003c/em\u003e under high-salinity stress (Thiem et al. 2018, 2023).\u003c/p\u003e \u003cp\u003eIn agreement with evenness reduction, the multivariate analyses in this study revealed that land-use type strongly influenced bacterial community structure in both bulk soil and the plant endosphere. Land degradation explained a substantial portion of variance in soil bacterial communities (PERMANOVA: \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.864) and a smaller but significant fraction in root and nodule endophytes (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.106). While the effect on soil communities was not statistically significant, likely due to limited sample size, the magnitude of the effect is ecologically relevant and consistent with previous findings that land-use change drives pronounced shifts in soil microbial assemblages (Rodrigues et al. 2013; Lozano et al. 2014; Zhou et al. 2018). The observed compartment effect within \u003cem\u003eA. acuminata\u003c/em\u003e (roots vs. nodules) also highlights the role of plant microhabitats in shaping endophytic communities, though this effect was less pronounced than land use.\u003c/p\u003e \u003cp\u003eSoil pH along with total carbon emerged as the dominant environmental drivers shaping bulk soil bacterial communities, consistent with numerous studies identifying them as principal determinants of soil microbial diversity and composition worldwide. These factors act as critical environmental filters, determining which bacterial groups can thrive under specific soil conditions (Zhou et al. 2018; Lopes et al. 2021; M\u0026eacute;sz\u0026aacute;rošov\u0026aacute; et al. 2024; Zhou et al. 2024).\u003c/p\u003e \u003cp\u003eIn the present study, degraded soils were dominated by stress-tolerant Actinomycetota, whereas native forest soils supported more diverse communities rich in Acidobacteriota and Pseudomonadota, typical of resource-rich, acidic environments. Actinomycetota, known for their adaptability, tolerate stressors such as pH fluctuations, salinity, and nutrient scarcity, enabling them to dominate in degraded soils (Mohammadipanah and Wink 2016; Lopes et al. 2021). Their abundance often decreases as ecosystems recover, suggesting a preference for degraded conditions (Li et al. 2024). In contrast, Acidobacteriota, which thrive in acidic soils and typically decline as soil pH increases (Bai et al. 2023; Silva et al. 2024; Jiang et al. 2025) was dominant in NF along with Pseudomonadota, which prosper in high-resource environments with labile carbon inputs (Peng et al. 2022; Bai et al. 2023; Li et al. 2024). Both these taxa declined with degradation. Verrucomicrobiota, another group sensitive to soil fertility changes, also declined with degradation, consistent with previous findings (Navarrete et al. 2015; Lopes et al. 2021).\u003c/p\u003e \u003cp\u003eSimilar but less pronounced changes were detected in endophytic communities, with Pseudomonadota dominating both roots and nodules irrespective of land use, suggesting that a core microbiome was maintained by the host plant (Knowlton and Dawsonm 1982; Dove et al. 2024). It is important to underline that although Actinomycetota, particularly \u003cem\u003eFrankia\u003c/em\u003e spp., was typically dominant in \u003cem\u003eAlnus\u003c/em\u003e nodules (Diagne et al. 2013; Chen et al. 2020), a low relative abundance of \u003cem\u003eFrankia\u003c/em\u003e was detected. This is likely attributable to the specific physio-morphological state of these endophytes, which may have hindered efficient DNA extraction relative to other genera (Akkermans et al. 1991). Additionally, the universal primers used for endophytic community detection, while designed to capture a broad spectrum, are known to introduce amplification biases, which likely resulted in the underrepresentation of these groups (Thiem et al. 2018). Nevertheless, anatomical observations confirmed its presence in both land-use types (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt lower taxonomic rank, \u003cem\u003eParaburkholderia\u003c/em\u003e, \u003cem\u003eCaballeronia\u003c/em\u003e, \u003cem\u003eRahnella\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and two unclassified Enterobacterales genera were identified as core members of the root and nodule microbiomes, consistent with their known roles as nitrogen-fixing and plant growth-promoting bacteria in symbiotic associations (Garneau et al. 2023a; Dove et al. 2024; Thompson et al. 2025).\u003c/p\u003e \u003cp\u003eAlthough endosphere communities are primarily shaped by host plant identity (Brown et al. 2020; M\u0026eacute;sz\u0026aacute;rošov\u0026aacute; et al. 2024) significant differences in the endospheric bacterial communities from \u003cem\u003eA. acuminata\u003c/em\u003e plants grown in degraded and native forests was found. Specifically, changes in the abundance of these core symbionts between degraded and native forests and their association with other bacterial genera suggest that plants can form specific symbiotic bacteria consortia to function better in their specific environments. The marked increase of \u003cem\u003eRahnella\u003c/em\u003e in \u003cem\u003eA. acuminata\u003c/em\u003e roots grown in degraded soil accompanied by other genera, such as \u003cem\u003eMicromonospora\u003c/em\u003e, shows the capacity of the plant to select and enter into symbiosis with the most useful endophytes for those environmental conditions. Both \u003cem\u003eRahnella\u003c/em\u003e and \u003cem\u003eMicromonospora\u003c/em\u003e are plant endophytes. \u003cem\u003eRahnella\u003c/em\u003e is a plant growth-promoting bacterium (PGPB) which enhances nutrient acquisition, such as phosphate solubilization, and \u003cem\u003eMicromonospora\u003c/em\u003e has been shown to contribute to plant health and pathogen defense, through the synthesis of many metabolites (Carro et al. 2013; Garneau et al. 2023a, b).\u003c/p\u003e \u003cp\u003eSimilarly, there was a marked decline of \u003cem\u003eParaburkholderia\u003c/em\u003e in nodules from degraded forests, accompanied by an increased abundance of other species such as \u003cem\u003eRhodanobacter\u003c/em\u003e, primarily recognized for its role in biogeochemical cycling, \u003cem\u003eMycobacterium and Deinococcus\u003c/em\u003e, recognized as plant associated resilience enhancers. These results, suggests that forest degradation may alter symbiotic partnerships to maintain or even improve nitrogen fixation efficiency in that environment (Ghodhbane-Gtari et al. 2021; Tariq et al. 2025; Thompson et al. 2025). Consistently, nodule morphology remained unaffected by degradation, suggesting that nodulation capacity in \u003cem\u003eA. acuminata\u003c/em\u003e is resilient to changes in land use at least within the studied context. This resilience highlights the potential for physiological plasticity or redundancy among nodule- and root-associated bacteria of \u003cem\u003eA. acuminata\u003c/em\u003e to maintain or improve symbiotic functions (Gyaneshwar et al. 2011; Tong et al. 2025).\u003c/p\u003e \u003cp\u003eOverall, the results reported in this study highlight the complex interactions between land degradation and microbial assemblages in soil and in the root of the pioneer plant \u003cem\u003eA. acuminata\u003c/em\u003e. The strong influence of soil pH and total carbon on soil bacterial communities underscores the need to consider abiotic and biotic factors in restoration strategies. Furthermore, the \u003cem\u003eA. acuminata\u003c/em\u003e endophytic microbiome exhibits both relative stability and significant plasticity, allowing the plant to modulate its community structure to ensure efficient function under different environmental conditions. By maintaining and improving these beneficial microbial partners, \u003cem\u003eA. acuminata\u003c/em\u003e may sustain its own growth and contribute to soil recovery, creating a positive feedback loop that facilitates the re-establishment of diverse plant and microbial assemblages over time. This adaptive capacity strongly suggests that exploiting plant-microbe interactions is a promising avenue for the recovery of degraded Andean ecosystems.\u003c/p\u003e \u003cp\u003eFuture work integrating bacteria selection, functional analyses and experimental inoculations will be needed to determine the appropriate bacteria consortia useful to accelerate spontaneous restoration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary information\u003c/h2\u003e \u003cp\u003eSupplementary data associated with this article Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and tables Tab S1- Tab S11 can be found online at doi: XXXX\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by the Italian Ministry of University and Research.\u003c/p\u003e \u003cp\u003eThe authors declare they have no financial interests\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.G.: design of the research, sample collection, sample processing and laboratory analyses, data analysis and interpretation, preparation of the original draft.L.A.Q.: data analysis and interpretation, preparation of the original draft.R.A.: design of the research, revision of the manuscript.M.J.: sample collection and measurement of nodules, revision of the manuscript.R.G.: design of the research, revision of the manuscript.S.C.: design of the research, supervision and guidance, revision and editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNG acknowledges the support of the Department of Earth and Environmental Sciences, University of Milano-Bicocca, through a fellowship. The authors sincerely thank Sarah Caronni for laboratory assistance, Miguel \u0026Aacute;ngel Vizcho for his help with field sampling, and Dr. Wayne Hanson for reviewing the English of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkkermans A, Hahn D, Mirza S (1991) Molecular ecology of \u003cem\u003eFrankia\u003c/em\u003e: Advantages and disadvantages of the use of DNA probes. Plant and Soil 137:49\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAslani F, Tedersoo L, P\u0026otilde;lme S, Knox O, Bahram M (2020) Global patterns and determinants of bacterial communities associated with ectomycorrhizal root tips of \u003cem\u003eAlnus\u003c/em\u003e species. 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PLoS ONE 10(9). https://doi.org/10.1371/journal.pone.0133701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTariq A, Tanvir A, Barasarathi J, Alsohim A, Mastinu A, Sayyed R, Nazir A (2025) Endophytes: key role players for sustainable agriculture: mechanisms, omics insights and future prospects. Plant Growth Regulation. https://doi.org/10.1007/s10725-025-01370-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiem D, Gołebiewski M, Hulisz P, Piernik A, Hrynkiewicz K (2018) How does salinity shape bacterial and fungal microbiomes of \u003cem\u003eAlnus glutinosa\u003c/em\u003e roots? Front Microbiol 9. https://doi.org/10.3389/fmicb.2018.00651\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThiem D, Goebel M, Gołębiewski M, Baum C, Koczorski P, Szymańska S, Hrynkiewicz K (2023) Endophytic microbiota and ectomycorrhizal structure of \u003cem\u003eAlnus glutinosa\u003c/em\u003e Gaertn. at saline and nonsaline forest sites. 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Science of The Total Environment, 940:173584.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiuyu X, Fangke C, Liyong F, Peng F, Minhuai W, Zhiquan C (2024) A pioneer tree species rapidly facilitating ecosystem restoration in coastal regions depends on soil traits. Catena, 238:107825.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Z, Wang C, Luo Y (2018) Effects of forest degradation on microbial communities and soil carbon cycling: A global meta-analysis. Global Ecology and Biogeography 27:110\u0026ndash;124. https://doi.org/10.1111/geb.12663\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X, Tahvanainen T, Malard L, Chen L, P\u0026eacute;rez-P\u0026eacute;rez J, Berninger F (2024) Global analysis of soil bacterial genera and diversity in response to pH. Soil Biology and Biochemistry, 198:109552. https://doi.org/10.1016/j.soilbio.2024.109552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZieschank V, Muola A, Janssen S, Lach A, Junker R (2025) Tolerance to land-use changes through natural modulations of the plant microbiome. The ISME Journal 19(1). https://doi.org/10.1093/ismejo/wraf010\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"symbiosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Symbiosis](https://link.springer.com/journal/13199)","snPcode":"13199","submissionUrl":"https://submission.springernature.com/new-submission/13199/3","title":"Symbiosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Land degradation, Soil restoration, Andean alder, Bacterial communities, Endophytes, NGS of 16S rRNA","lastPublishedDoi":"10.21203/rs.3.rs-8660278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8660278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLand degradation in the Andes threatens ecosystems and biodiversity. The recovery of these areas often depends on pioneer species such as \u003cem\u003eAlnus acuminata\u003c/em\u003e, which relies on symbiotic microorganisms for nutrient acquisition and stress tolerance. Understanding how degradation affects its microbiome is crucial for effective restoration. This study investigated how a land-use trajectory involving deforestation and abandonment impacts the diversity and structure of bacterial communities associated with \u003cem\u003eAlnus acuminata\u003c/em\u003e. Next-generation sequencing of 16S rRNA gene amplicons was used to compare bacterial communities in bulk soil, roots, and root nodules between a native forest and degraded forest in the Ecuadorian Andes. Land degradation significantly altered bulk soil bacterial diversity and community structure, with pH and carbon content identified as key environmental drivers. Degraded soils were dominated by Actinomycetota, whereas native forest soils harbored more diverse communities, including Acidobacteriota and Pseudomonadota. Root endophytic and nodule-associated communities showed reduced diversity under degradation, although not statistically significant, suggesting that these niches are buffered from environmental changes. Nevertheless, their community structures differed significantly, indicating that \u003cem\u003eAlnus acuminata\u003c/em\u003e may actively assemble a beneficial bacterial consortia to cope with degraded conditions. \u003cem\u003eAlnus acuminata\u003c/em\u003e appears to respond to soil degradation by recruiting a more diverse and functionally beneficial endophytic microbiome, including stress-resilient, pathogen-defending, and plant growth-promoting genera such as \u003cem\u003eMicromonospora\u003c/em\u003e, \u003cem\u003eRahnella\u003c/em\u003e, \u003cem\u003eRhodanobacter\u003c/em\u003e, \u003cem\u003eMycobacterium\u003c/em\u003e, and \u003cem\u003eDeinococcus\u003c/em\u003e. This adaptive strategy likely supports its survival and establishment in nutrient-poor, degraded environments, highlighting the critical role of plant-microbe interactions in ecosystem recovery and suggesting that harnessing these interactions could improve restoration outcomes.\u003c/p\u003e","manuscriptTitle":"Changes in microbiome assembly of the pioneer Andean tree Alnus acuminata in response to land degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 12:28:12","doi":"10.21203/rs.3.rs-8660278/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-20T04:02:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T23:55:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-08T22:05:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73819830630187778838376486815395949230","date":"2026-02-03T19:19:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173485268667940392107004251175605306952","date":"2026-02-03T14:14:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T10:30:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-02T02:50:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T01:27:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Symbiosis","date":"2026-01-21T13:14:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"symbiosis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Symbiosis](https://link.springer.com/journal/13199)","snPcode":"13199","submissionUrl":"https://submission.springernature.com/new-submission/13199/3","title":"Symbiosis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6b0d420f-9f68-4e07-b3c8-1bf65ffc8596","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T07:27:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 12:28:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8660278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8660278","identity":"rs-8660278","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}