Beneficial Effects of a Root-Endophytic Bacterium with Quorum-Sensing Traits on Growth and Drought Tolerance in the Vulnerable Conifer Araucaria araucana

Abstract

Climate change-induced drought threatens the persistence of Araucaria araucana, an endangered and endemic conifer of the Southern Andes. Beneficial plant–microbe interactions may contribute to drought resilience. Here, we evaluated the effects of a root-endophytic bacterium with the capacity to produce N-acyl homoserine lactones (AHLs) on the growth and drought tolerance of A. araucana. For this, a root endophytic bacterium was isolated from A. araucana and identified as Erwinia billingiae. It was characterized for plant growth-promoting traits, and inoculated into A. araucana seedlings under drought conditions). The bacteria produced N-butyryl-L-homoserine lactone (C4-HSL) under control conditions and C4-HSL and N-hexanoyl-L-homoserine lactone (C6-HSL) under drought stress. The strain also produces indoleacetic acid, ammonia, siderophores and solubilizes phosphate. Under drought stress, non-inoculated seedlings showed marked reductions in shoot and root biomass, chlorophyll content, relative water content (RWC), and soluble sugars. In contrast, inoculated seedlings under drought displayed significantly higher shoot and root biomass, reaching levels comparable to those of well-watered controls. Chlorophyll content increased from 5.42 to 9.35 mg L⁻¹, and RWC increased from 62% to 71% in inoculated plants under drought conditions. Soluble sugar content increased from 25.74 to 36.34 mg g⁻¹ fresh weight following inoculation. Drought-induced oxidative stress was significantly alleviated in inoculated seedlings, with lower malondialdehyde and proline accumulation compared to non-inoculated drought-stressed plants. Antioxidant responses were modulated, indicating improved redox balance under water limitation. These results demonstrate that a root-endophytic bacterium with AHL production can enhance drought tolerance in A. araucana seedlings. This study provides novel evidence supporting the role of beneficial endophytes in microbiome-based strategies for conserving native forest species under climate change.

1. Introduction

Global environmental changes, including rising temperatures and reduced rainfall, are affecting the performance of tree species, leading to reduced growth, forest decline, and widespread tree mortality worldwide [1]. Under current climate change scenarios, severe drought events represent one of the most pressing environmental threats to tree populations, with projections indicating an increase in both the frequency and intensity of droughts in the near future [2]. The negative effects of drought on tree performance and potential mortality are particularly severe for long-lived species with marginal distributions, as these species typically exhibit low genetic diversity and long generational intervals, which constrain adaptive responses to rapid climate change [3]. In this context, Araucaria araucana (Molina) K. Koch (Araucariaceae) is a long-lived, slow-growing tree species native to Chile and Argentina. Its distribution is highly restricted, in the Andean Range between 37°40′ and 40°30′ S latitude, and in the Nahuelbuta Coastal Range between 37°30′ and 38°40′ S [4,5]. Populations of A. araucana have significantly declined due to natural disturbances, such as volcanic activity and wildfires, as well as anthropogenic factors including extensive logging, habitat fragmentation, and climate change-induced alterations [6]. Additionally, the prevalent water scarcity in soils inhabited by A. araucana exacerbates these pressures, compromising the species growth, health and survival. As a result, A. araucana is currently classified as an endangered species by the International Union for Conservation of Nature (IUCN) [7].

In Chilean native forests, Araucaria commonly grows in Andisols, which are characterized by acidic conditions and low nutrient availability [8]. Under these conditions, this species likely benefits from colonization by plant-growth-promoting microorganisms, forming mutualistic associations that enhance its overall health [9]. Numerous studies recognize the importance of the plant-associated microbiome, including microorganisms inhabiting the soil near the roots and those residing within host plants [10,11]. In this context, the endophytic microorganisms associated with A. araucana remain largely unexplored, generating interest in their identification due to their potential beneficial effects on plant health and growth [12]. These microorganisms colonize the internal tissues of the plant for at least part of their life cycle without causing visible symptoms of infection or adverse effects on the host [13]. Recent studies have demonstrated that root-endophytic bacteria can promote plant growth and enhance adaptation to environmental stresses through the production of various metabolites and mechanisms such as exopolysaccharides (EPS), phosphate solubilization, ammonia production, and indoleacetic acid (IAA) [14,15,16].

Quorum sensing is the most extensively studied chemical communication mechanism among microorganisms, providing bacteria with significant competitive advantages by coordinating synchronized responses to environmental stimuli, thus enhancing survival in challenging habitats [17]. N-acyl-L-homoserine lactones (AHLs), characterized by short, medium, or long carbon chains, are the primary quorum-sensing signaling molecules produced by Gram-negative bacteria [18]. Recent studies indicate that plant roots can perceive and respond to these bacterial quorum-sensing molecules, suggesting intricate interkingdom communication [19,20,21]. This capability plays a crucial role in fostering positive interactions between plants and beneficial bacteria, ultimately resulting in growth promotion and enhanced pathogen defense [22].

Scientific reports have demonstrated that AHLs play essential roles in plant responses to abiotic stress, including drought stress [23]. In maize, AHL-producing bacteria enhance biochemical systems related to drought-stress tolerance, including chlorophyll, carotenoids, proline, soluble sugars, and antioxidant enzymes [24]. Similarly, inoculation of rice with Gluconacetobacter diazotrophicus PAL5 enhances biomass production, gas exchange, and the accumulation of osmoprotectants such as proline and glycine betaine, while reducing lipid peroxidation under drought conditions [25]. Despite growing evidence in crops, the role of AHL-producing endophytes in native trees remains unexplored. No studies have addressed their physiological impact on keystone species such as A. araucana, leaving a critical gap in understanding microbiome-based strategies for forest resilience under climate change. Exploring these microbial interactions could offer new, microbiome-based strategies for the conservation and resilience of A. araucana forests under increasing climate stress, particularly in the context of prolonged droughts affecting its native range.

The aim of this study is to characterize root-endophytic bacterium capable of producing AHLs and to evaluate their effects on drought tolerance and plant-growth promotion in A. araucana.

2. Results

2.1. Isolation, Detection of AHL-Producing Bacteria and Molecular Identification

A total of 28 endophytic bacterial strains were isolated from the roots of six adult A. araucana trees. Screening for AHL production using the CV026 reporter detected one AHL-producing isolate (Figure S1). Based on 16S rRNA gene sequencing, the strain was identified as Erwinia billingiae. The nucleotide sequence was submitted to the GenBank database under the code PX929575.

2.2. Drought Tolerance

E. billingiae growth under different concentrations of polyethylene glycol 6000 (PEG 6000) to simulate drought stress tolerance is presented in Figure 1. Optimal growth was observed under non-stressed conditions (0% PEG). At a moderate PEG concentration (15%), bacterial growth was sustained but reduced compared to the control. In contrast, higher PEG concentration (30% and 40%) significantly inhibited bacterial growth, resulting in minimal biomass accumulation.

2.3. Quantification of AHLs

Quantitative analysis using HPLC-MS confirmed the production of AHL molecules by E. billingiae under both control and drought-induced (20% PEG) conditions. The strain produced detectable levels of N-butyryl-L-homoserine lactone (C4-HSL) under both treatments, whereas N-hexanoyl-L-homoserine lactone (C6-HSL) was only detected under PEG-induced osmotic stress (

Table 1. Quantification of AHL production by E. billingiae under simulated drought stress conditions (20% PEG) by HPLC-MS. Data are expressed as means ± standard deviation (n = 4). Different letters denote statistically significant differences between treatments, according to Student’s t-test (p ≤ 0.05).

Strain	PEG Dose (%)	AHLs (ng L−1)
		C4-HSL	C6-HSL
Erwinia billingiae	0	2459 ± 13.75 b	Not detected
	20	1209 ± 22.41 a	319 ± 8.38 a

). Notably, C4-HSL concentrations varied significantly depending on osmotic conditions, with a marked reduction under stress.

2.4. Characterization of Plant Growth-Promotion Traits

The isolated strain exhibited multiple PGP traits. It produced 61.4 ± 2.57 µmol mL⁻¹ of ammonia and 0.39 ± 0.05 µmol mL⁻¹ of indoleacetic acid (IAA). Siderophore production was positive, with a production rate of 1.98 ± 0.26 mm day⁻¹. Phosphorus solubilization assay demonstrated selective activity. E. billingiae showed measurable solubilization of AlPO₄ (4.2 ± 0.35) and phytic acid (3.24 ± 0.47), expressed as phosphate solubilization index (PSI). In contrast, Ca₃(PO₄)₂ and FePO₄ showed no solubilization activity.

2.5. Inoculum Prevalence, Biomass and Physiological Variables

Concerning inoculum prevalence, bacterial colonies consistent with E. billingiae were recovered from inoculated plants, whereas no colonies with morphology similar to E. billingiae were detected in non-inoculated controls. In addition, 16S rRNA gene sequencing of re-isolated colonies confirmed species-level identification as E. billingiae.

The effects of drought stress and bacterial inoculation on shoot and root dry biomass in A. araucana are presented in Figure 2. Plants subjected to drought stress (40% field capacity) exhibited significantly lower shoot and root biomass compared to those under well-watered conditions (80% field capacity). Inoculation with E. billingiae significantly increased shoot and root biomass under drought conditions, reaching values statistically comparable to those observed under the 80% irrigation regime. Regarding total chlorophyll content (

Table 2. Total chlorophyll, relative water content and total sugar content from leaves of A. araucana growing under drought stress conditions. Values are expressed as means ± standard deviation (n = 6). Different letters indicate significant differences according to Tukey’s multiple range test (p ≤ 0.05).

Treatment	Total Chlorophyll Content (mg L−1)	Relative Water Content (%)	Total Sugars (mg g−1 Sample)
C 80%	7.60 ± 1.3 ab	75 ± 2.5 bc	34.94 ± 3.8 b
IN 80%	7.62 ± 1.2 ab	78 ± 2.1 c	39.27 ± 3.3 b
C 40%	5.42 ± 1.3 a	62 ± 3.4 a	25.74 ± 0.4 a
IN 40%	9.35 ± 1.0 b	71 ± 2.5 b	36.34 ± 1 b

), no differences were observed under optimal irrigation conditions, both inoculated and non-inoculated plants showed similar values (7.60 ± 1.3 and 7.62 ± 1.2 mg L⁻¹, respectively). Non-inoculated plants under drought stress exhibited the lowest chlorophyll content (5.42 ± 1.3 mg L⁻¹), while inoculated plants under the same stress condition showed the highest values (9.35 ± 1.0 mg L⁻¹). Similarly, the lowest relative water content (RWC) was recorded in non-inoculated drought-stressed plants (62 ± 3.4%), whereas the highest RWC was observed in inoculated, well-watered plants (78 ± 2.1%). Inoculated plants under drought stress exhibited significantly higher RWC (71 ± 2.5%) compared to their non-inoculated plants (Table 2). Regarding total soluble sugars, no significant differences were observed between inoculated and non-inoculated plants under well-watered conditions (39.27 ± 3.3 and 34.94 ± 3.8 mg g⁻¹, respectively). However, drought stress led to a marked reduction in sugar content in non-inoculated plants (25.74 ± 0.4 mg g⁻¹), while inoculated drought-stressed plants showed significantly higher sugar content (36.34 ± 0.4 mg g⁻¹).

2.6. Oxidative Damage and Proline Content

Lipid peroxidation and proline accumulation were significantly influenced by irrigation regime and bacterial inoculation (Figure 3a,b). Under well-watered conditions, non-inoculated plants exhibited the lowest malondialdehyde (MDA) levels, whereas inoculated plants showed a slight but statistically significant increase. In contrast, drought stress induced a marked increase in MDA content in non-inoculated plants, reaching the highest values among all treatments. Notably, inoculated plants under drought stress showed significantly lower MDA levels compared to their non-inoculated plants, although these values remained higher than those observed under optimal irrigation (Figure 3a). Similarly, drought stress led to a significant increase in proline accumulation in non-inoculated plants. However, inoculated plants under drought conditions accumulated significantly less proline. Under well-watered conditions, no significant differences in proline content were observed between inoculated and non-inoculated plants (Figure 3b).

2.7. Antioxidant Activity

Total phenolic content and antioxidant activity, assessed by TEAC, CUPRAC, and DPPH assays, varied significantly depending on irrigation regime and bacterial inoculation (Figure 4).

Under well-watered conditions (80% field capacity), inoculation with E. billingiae resulted in lower total phenolic content and reduced antioxidant activity compared to non-inoculated plants. This decrease was consistent across CUPRAC, DPPH, and TEAC assays and indicates a reduced requirement for antioxidant defenses in inoculated plants under non-stress conditions.

In contrast, drought stress (40% field capacity) induced a pronounced increase in total phenolic content and antioxidant activity in non-inoculated plants, reflecting the activation of oxidative stress responses. Inoculated plants subjected to drought exhibited a distinct antioxidant profile, characterized by lower total phenolic content and reduced CUPRAC activity (Figure 4a,b), while DPPH and TEAC activities were maintained at levels comparable to non-inoculated stressed plants (Figure 4c,d).

2.8. Principal Component Analysis (PCA)

The first two principal components explained 83.3% of the total experimental variance (PC1 = 67.6%, PC2 = 15.7%) (Figure 5). Along PC1, samples were clearly separated according to irrigation regime: plants under 80% field capacity clustered on the positive axis, associated with shoot and root biomass, RWC, and soluble sugar content, whereas those under 40% field capacity were positioned on the negative axis, associated with proline accumulation, lipid peroxidation, total phenolic content, CUPRAC, and TEAC activity. Treatment differences were mainly observed under the 40% irrigation condition along PC2: Control plants were associated with proline accumulation and phenolic-based antioxidant responses, while Inoculated plants shifted toward higher DPPH activity and chlorophyll content. Under well-watered conditions (80% field capacity), Control and Inoculated plants overlapped in the PCA space and were primarily defined by biomass and relative water content.

3. Discussion

This study provides the first evidence that an AHL-producing endophytic bacterium, Erwinia billingiae, enhances drought tolerance in Araucaria araucana, a keystone and endangered conifer native to the southern Andes. A total of 28 endophytic bacterial strains were isolated from root tissues, a relatively low number that likely reflects the specialized composition of endophytic communities in conifers adapted to nutrient-poor Andisols. The isolation of E. billingiae from the root tissues of A. araucana is consistent with previous high-throughput sequencing characterizing the endophytic bacterial communities of this ancient conifer [26], where reported that Proteobacteria (the phylum containing Erwinia) and specifically Erwiniaceae family are part of the core microbiome of Araucaria. Similar patterns have been reported in other conifer species, where endophyte communities exhibit low taxonomic diversity but functionally specialized, particularly in traits associated with host tolerance to abiotic stress [27]. Within this context, the ability of E. billingiae to tolerate osmotic stress induced by PEG is a key attribute supporting its functional role as a beneficial symbiont in drought-prone environments, a trait that has also been reported in other Erwinia species [28]. Although bacterial growth declined at higher PEG concentrations (30–40%) (Figure 1), this response is consistent with patterns observed in other plant-associated bacteria adapted to fluctuating soil water potentials, including Bacillus megaterium [29], Pantoea agglomerans [30], Rhizobium sp. and Pseudomonas indica [31]. The ability to survive and remain metabolically active under water stress is particularly relevant, as endophytic bacteria must persist within host tissues or in the rhizosphere even when water availability is limited.

In addition to drought tolerance, E. billingiae exhibited active production of AHLs, signaling molecules that may further support its survival and interaction with the host plant under stress conditions. Our results reveal that E. billingiae produced C4-HSL under non-stress conditions, while its synthesis decreased under drought stress. Conversely, C6-HSL was only detected under drought conditions (Table 1). This shift suggests a dynamic adjustment of quorum-sensing signals in response to environmental conditions, with C4-HSL and C6-HSL potentially functioning in a complementary and synergistic manner. This interaction could be relevant for plant responses, as C4-HLS has been associated with the activation of defense responses [32], whereas C6-HLS has been linked to plant growth promotion [33]. In this sense, studies using LC-MS/MS have confirmed that root-associated bacteria such as Paraburkholderia, and Pseudomonas are AHL producers. For example, Hoang et al. [34] quantified AHL production in an endophytic Paraburkholderia strain using supercritical fluid chromatography coupled to high-resolution mass spectrometry, reporting concentrations of 0.43–18.96 mg L⁻¹ in the culture supernatant for at least 9 different AHLs. Furthermore, Ortiz et al. [35] quantified N-heptanoyl-L-Homoserine lactone and N-nonanoyl-L-Homoserine lactone in Pseudomonas brassicacearum isolated from stressed soils, reporting concentrations ranging from 0.07 and 6.72 mg L⁻¹. Recent studies have shown that AHLs can function as interkingdom signaling molecules, modulating root development, stomatal regulation, and defense responses in host plants [35,36]. For example, von Rad et al. [37] demonstrated that inoculation of Arabidopsis thaliana roots with different types of AHLs, including C4-HSL and C6-HSL, promoted root elongation through alterations in transcriptional activity and the levels of cytokinins and auxins. Similarly, Gupta et al. [38] reported that a nanocomposite fertilizer incorporating Fe-carbon nanofibers with C4-HSL enhanced seed germination, plant growth, and chlorophyll content in Cicer arietinum, while also improving resistance to oxidative and salinity stresses. In another study, Babenko et al. [23] found that C6-HSL significantly increased total chlorophyll content under simulated acid rain conditions and mitigated stress-induced chlorophyll loss. These reports are consistent with our findings, extending evidence to a long-lived gymnosperm and suggesting that AHL-mediated signaling may contribute to drought adaptation.

The characterization of plant growth-promoting (PGP) traits revealed that E. billingiae possesses multiple mechanisms that may contribute to enhanced seedling performance under drought stress conditions (Table 2). The production of IAA and ammonia are consistent with its role in stimulating root development and nutrient mobilization [39]. IAA is well known for promoting lateral root formation and enhancing water and nutrient uptake, processes that are particularly critical under limited water availability [40]. Ammonia production, in turn, can improve nitrogen availability in nutrient-poor volcanic soils, aligning with previous studies reporting improved nitrogen nutrition in conifers following endophytic bacterial inoculation [27,41]. Another important trait was the selective solubilization of phosphate, particularly from AlPO₄ and phytic acid. This ability to mobilize recalcitrant phosphorus sources is especially relevant given that A. araucana grows in volcanic soils where phosphorus is bound to aluminum or organic complexes [42]. These PGP mechanisms likely contribute to the increased biomass observed in inoculated seedlings under both irrigation regimes (Figure 2a).

The greenhouse experiment demonstrated that inoculation with E. billingiae effectively mitigated the adverse effects of drought stress on biomass accumulation, chlorophyll content, relative water content, and soluble sugar content. Notably, inoculated seedlings subjected to severe drought achieved shoot and root biomass values statistically similar to those of well-watered control plants (Figure 2a,b), highlighting the protective role of the bacterial symbiont. In addition, inoculated plants under the same stress condition exhibited the highest total chlorophyll content, consistent with the reported effects of AHLs in different plants [23,38,43]. In addition, RWC was significantly improved in inoculated seedlings under drought conditions, indicating a more efficient water balance. In contrast, non-inoculated plants exhibited a pronounced decline in RWC (Table 2). The improved water balance status is likely associated with PGPR-mediated regulation of stomatal behavior, as several studies suggest that beneficial bacteria can induce partial stomatal closure under drought, thereby reducing transpiration and limiting water loss [44]. Furthermore, the higher accumulation of total soluble sugars in inoculated seedlings may contribute to drought tolerance through osmotic adjustment. These sugars act as osmoprotectants by stabilizing cellular membranes and proteins during dehydration and substituting for water molecules via hydrogen bonding, thereby preserving turgor and structural integrity [45]. Beyond their osmoprotective role, soluble sugars also serve as signaling molecules that activate drought-responsive gene expression and metabolic pathways, further enhancing plant resilience [46].

Another notable effect of inoculation was the significant reduction in MDA levels, a key marker of oxidative damage (Figure 3a). This reduction may be partially attributed to the siderophore production observed in E. billingiae (Table 2). In addition to enhancing iron bioavailability, siderophores can mitigate oxidative stress by limiting the formation of reactive oxygen species (ROS) through inhibition of Fenton reactions [47]. Furthermore, PGPR are known to activate plant antioxidant systems; consequently, the redox balance may have shifted towards a reduction in ROS through the activation of enzymatic defenses, such as, superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, as well as increased levels of non-enzymatic antioxidants like glutathione and ascorbate [48]. The induction of this antioxidant machinery likely contributed to the observed decrease in oxidative stress, which in turn may have explained the lower proline accumulation in inoculated seedlings (Figure 3b). Proline is widely recognized as a compatible solute and secondary ROS scavenger, commonly synthesized under drought conditions. However, its reduced levels in inoculated plants suggest that the bacteria alleviated drought stress intensity, thereby lessening reliance on this metabolite. Similar findings were reported by Santander et al. [49], where Lactuca sativa plants inoculated with different Bacillus spp. strains under drought conditions showed decreased proline accumulation, indicating a positive effect of inoculation against drought stress.

The modulation of phenolic compounds and antioxidant activity represents an additional mechanism by which E. billingiae contributes to drought stress tolerance in A. araucana. Non-inoculated plants under drought conditions accumulated higher total phenolic content and CUPRAC activity, reflecting a generalized stress response (Figure 4). In contrast, inoculated plants showed reduced total phenols but maintained or even enhanced radical-scavenging capacity in the DPPH and TEAC assays (Figure 4). This pattern suggests that E. billingiae may differentially modulate phenolic metabolism, shifting antioxidant responses toward more efficient or targeted pathways rather than inducing a broad increase in phenolic content. Accumulating evidence indicates that bacterial quorum-sensing molecules such as AHLs may play a key role in this regulation. AHLs have been shown to activate both enzymatic antioxidant defenses (e.g., superoxide dismutase, catalase, peroxidases) and non-enzymatic systems, thereby improving ROS detoxification under abiotic stress conditions [21]. For instance, exposure to C6-HSL has been associated with reduced ROS accumulation and improved tolerance to drought and salinity in A. thaliana and crop species. Moreover, C6-HSL has been shown to stimulate the accumulation of phenolic compounds, flavonoids, and proline under stress [23,50], and seed priming with C6-HSL enhanced biomass, chlorophyll, and carotenoid content in winter wheat under field conditions [33]. These findings support the hypothesis that the antioxidant response in E. billingiae-inoculated seedlings does not solely depend on phenolic biosynthesis but could also be mediated by AHL-induced signaling pathways that fine-tune redox regulation. Similarly, PCA analyses demonstrated that drought was the primary factor structuring plant responses, but inoculation induced distinct effects under severe water stress (Figure 5). Inoculated plants were aligned with chlorophyll maintenance and enhanced DPPH activity, suggesting improved ROS scavenging capacity and pigment protection.

In contrast, control plants relied more on proline accumulation and phenolic-based antioxidant systems, typically associated with higher oxidative stress. Together, these results indicate that E. billingiae promotes drought tolerance in A. araucana not only by improving water balance and osmotic regulation but also by fine-tuning oxidative stress responses and secondary metabolism. This multifaceted modulation likely explains the convergence of biomass and physiological traits in inoculated drought-stressed plants to levels comparable with well-watered controls. However, further research should focus on identifying specific phenolic compounds and elucidating their functional roles to clarify the mechanistic basis of these responses.

4. Materials and Methods

4.1. Root Samples

Six Araucarias trees growing in the surroundings of China Muerta National Reserve (38°42′24.4″ S 71°26′53.9″ W) were selected for the isolation and characterization of root endophytic bacteria. A portion of roots from these adult trees of Araucaria were carefully cut and placed in sterilized Falcon tubes and stored at 4 °C until transported to the laboratory.

4.2. Isolation of Root Endophytic Bacteria and Screening of AHLs Production

For the isolation of root endophytic bacteria associated with Araucaria, the roots were thoroughly washed with tap water to remove adhering soil and then surface-disinfected according to Herrera et al. [51]. The disinfected roots were thereafter ground in a mortar with sterile phosphate-buffered saline (PBS) in a laminar flow chamber. The solution was serially diluted from 10⁻¹ to 10⁻⁵ in sterile PBS and plated in triplicate onto Petri dishes containing full-strength and 1/10 strength Tryptic Soy Agar medium, full-strength and 1/10 strength Reasoner’s 2A and full-strength, and 1/10 strength Luria–Bertani (LB) agar. Plates were incubated at 26 ± 1 °C until individual bacterial strains were detected and subsequently purified. Purified colonies were stored on individual plates at 4 °C and periodically subcultured for further analysis. The ability of each strain to produce AHL molecules was assessed using the biosensor strain Chromobacterium violaceum (CV026). Each bacterial isolate was streaked onto an LB agar plate against CV026 and incubated at 26 ± 1 °C for 3 days [52]. The presence of AHL was confirmed by the appearance of a purple halo surrounding the bacterial colony.

4.3. Molecular Identification of AHL-Producing Isolates

AHL-producing root-endophytic bacterium was cultured in LB broth for 48 h. DNA was extracted using UltraClean^® Microbial DNA Isolation Kit (Mo Bio Laboratories. Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, polymerase chain reaction (PCR) was performed using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) [53]. PCR products were visualized in 1% agarose gel electrophoresis and sequenced (Macrogen, Inc.; Seoul, Republic of Korea). For identification, species assignment was made on EzBioCloud 16S database searches to find the closest match, accepting the genus and species classification according to Chun et al. [54].

4.4. Assessment of Drought Tolerance in Bacterial Isolates

To assess drought stress tolerance, the AHL-producing isolate was cultured in LB broth with increasing concentrations of PEG 6000 to achieve drought-induced osmotic stress (0, 15, 30 and 40%, corresponding to 0, −0.62, −2.80 and −6.00 MPa, respectively [55]). Isolate was incubated for 72 h (corresponding to the beginning of the stationary phase, as determined from the previously established bacterial growth curve) in a shaker at 26 °C ± 1 °C. Growth was assessed every 24 h by measuring the optical density at 600 nm using a spectrophotometer (BioTek Synergy H1, Agilent Technologies, Palo Alto, CA, USA) and compared with the 0% PEG control. The strain was categorized based on OD values to assess its tolerance to drought stress, following the criteria established by Susilowati et al. [56]. Each treatment was conducted with five replicates.

4.5. Quantification of AHLs by HPLC-MS

Quantification of AHLs was measured in HPLC-MS according to the method described by Ortiz et al. [35] with minor modifications. The strain was cultured in 500 mL of LB medium at 26 °C under stirring for 48 h. Additionally, to simulate osmotic stress conditions, the strain was cultured under identical conditions in LB medium supplemented with PEG 6000 20% w/v). Samples were collected at late exponential growth phase, as the concentration of AHLs typically reaches its maximum when bacterial growth transitions from exponential to stationary phase [57]. After incubation, the supernatant was extracted twice using ethyl acetate acidified with 0.1% formic acid (1:1 v/v). Finally, the extracts were concentrated in a rotary evaporator and subsequently dissolved in 1 mL of acetonitrile acidified with 0.1% formic acid until analysis. Each treatment was performed with four biological replicates. The HPLC-MS/MS analysis was carried out in UPLC-UV (Prominence LC-20AD, Shimadzu, Kyoto, Japan) coupled with a triple quadrupole/linear ion trap instrument (LIT) (QTRAP3200, AB Sciex, Foster, CA, USA) with an electrospray ionization (ESI) source. The chromatographic separation occurred with a C18 reverse-phase column (3.0 µm particle size, 100 mm × 4.6 mm, Supelco, Bellefonte, PA, USA), and mobile phases were 0.1% formic acid v/v in acetonitrile (solvent A) and 0.1% formic acid v/v in water (solvent B). AHLs concentrations were calculated using standard curves with synthetic AHLs (N-butyril homoserine lactone, N- hexanoyl homoserine lactone, N-heptanoyl homoserine lactone, N-nonanoyl homoserine lactone, N-dodecanoyl homoserine lactone and N-tetradecanoyl homoserine lactone) purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).

4.6. Evaluation of Plant Growth-Promoting Traits

Standard procedures were used to assess the PGP traits of AHL-producing root-endophytic bacterium. Ammonia production was determined according to the method of Bhattacharyya et al. [58]. P solubilization was evaluated following Nautiyal [59], using National Botanical Research Institute’s phosphate growth medium (NBRIP) supplemented with 0.1 g L⁻¹ bromophenol blue. The phosphate source assayed included Ca₃(PO₄)², AlO₄P, FeO₄P·2H₂O and phytic acid. Phosphate solubilization index (PSI) was calculated using the following formula: PSI: [(CD + HR)/CD], where CD is the colony diameter in millimeters (mm) and HR is the halo ratio in mm [60]. Indoleacetic acid (IAA) production was analyzed as described by Chiboub et al. [61], while siderophores production was assessed following the method of Milagres et al. [62] using chrome azurol S (CAS) agar. CAS reaction rate was determined based on Machuca and Milagres [63] and expressed in mm per day (mm day⁻¹). All measurements were conducted with five replicates.

4.7. Greenhouse Experiment

Seeds of Araucaria were soaked in a fungicide solution (Captan 13 WP^®, ANASAC, Santiago, Chile) for 3 days and sown in sterile sand and allowed to grow until seedlings reached 10 cm in the greenhouse of the Bioremediation Laboratory (Universidad de La Frontera, Temuco, Chile). Nine months after germination, the Araucaria seedlings were transplanted into 1 L pots filled with a sterile substrate mixture containing composted pine bark and perlite (3:1 v/v). Fifteen days after transplantation, seedlings were inoculated with a root endophytic bacteria previously selected for its ability to produce AHLs and its plant growth-promoting traits.

For seedling inoculation, root endophytic bacteria was grown in LB medium for 48 h at 26 ± 1 °C. After incubation, the culture was centrifuged at 6000 rpm for 5 min and resuspended in sterilized magnesium sulphate 0.01 M to reach an optical density of 0.8 at 600 nm (OD 600) [49]. Then, 10 mL of inoculum was directly applied to the root zone by irrigation. Inoculum application was repeated at 15 days, 1 month, and 4 months post-transplantation. Non-inoculated plants received 10 mL of sterile 0.01 M magnesium sulphate at all inoculation times.

The experiment was designed with two inoculation treatments (control without inoculation and inoculation with root endophytic bacteria), each subjected to two irrigation regimes (80% and 40% of field capacity). A total of four treatments with ten biological replicates were performed (n = 40). Fifteen days post-transplantation, the plants were subjected to different irrigation levels. Field capacity of the substrate was determined by weighing the dry substrate and subtracting this weight from that of the water-saturated substrate. The difference between wet and dry weights represents 100% of the field capacity. This value was then used to calculate the corresponding weights for the irrigation levels (80% and 40% of field capacity). Plants were grown for eight months in a greenhouse under controlled conditions with a 16/8 h day/night cycle at 24/16 °C and 50% relative humidity. Field capacity was adjusted twice a week until harvest. At the end of the experiment, root samples from non-inoculated and inoculated plants were collected to determine the prevalence of the inoculum. For this a re-isolation of the inoculated strain was performed according to the previously described procedure.

4.8. Dry Biomass

Each Araucaria seedling was carefully removed from substrate, and the entire root system was gently washed to remove adhering soil particles. Subsequently, the shoot (aboveground tissues) and the whole root system were separated, dried at 70 °C for 5 days in a forced-air oven and weighed to determine dry biomass.

4.9. Total Chlorophyll, Relative Water Content and Total Sugar Content

Chlorophyll a, b and total were quantified from 50 mg of fresh leaf tissue following extraction with dimethyl sulfoxide (DMSO) and concentrations were determined using a UV/Vis spectrophotometer by measuring the absorbance at 645 nm and 663 nm [64]. Chlorophyll concentrations were calculated using the following equations: Chl a (g L⁻¹) = 0.0127 × A663 − 0.00269 × A645; Chl b (g L⁻¹) = 0.0229 × A645 − 0.00468 × A663; Tot Chl (g L⁻¹) = 0.0202 × A645 + 0.00802 × A663.

Relative water content (RWC) was determined following Chávez et al. [12] using three leaves collected from the middle portion of the shoot of three independent experimental units. Fresh (FW), turgid (TW) and dry (DW) weight were used for RWC calculation following the formula: RWC (%) = [(FW − DW)/(TW − DW)] × 100.

Total soluble sugar was determined from 100 mg leaf tissue using the anthrone colorimetric method as described by Dubois et al. [65]. Sugar concentration was calculated using a sucrose standard calibration curve ranging from 0 to 100 µg mL⁻¹.

4.10. Lipid Peroxidation and Proline Content

Lipid peroxidation in leaves was determined following Du and Bramlage [66], based on the quantification of malondialdehyde (MDA) through its reaction with thiobarbituric acid (TBA). For this analysis, 100 mg of leaf tissue was used. Proline content was measured according to Zouari et al. [67]. For this, 250 mg of leaf tissue was homogenized in 3% (w/v) sulfosalicylic acid. Proline concentration was quantified spectrophotometrically using a standard L-proline calibration curve ranging from 0 to 0.2 mM, prepared by dissolving L-proline in 3% (w/v) sulfosalicylic acid.

4.11. Total Phenols and Antioxidant Activity

Phenolic compounds were extracted from 300 mg of leaf tissue following the method described by Santander et al. [49], using an extraction solvent consisting of methanol:formic acid (95:5, v/v). Total phenolic content was determined using the Folin–Ciocalteu method, with gallic acid as standard ranging from 100 to 500 mg L⁻¹ [68]. Absorbance was measured spectrophotometrically at 750 nm using HPLC (Shimadzu, Tokyo, Japan) equipped with a UV-visible diode-array detector (SPD-M20A), as described by Santander et al. [69]. Antioxidant activity was assessed using the same phenolic extract by the TEAC (Trolox equivalent antioxidant capacity), CUPRAC (cupric ion reducing antioxidant capacity), and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays, according to Parada et al. [70]. Trolox was used as the calibration standard, with concentration ranges from 0.01 to 0.7 mmol L⁻¹. Absorbance readings were collected at 734 nm for TEAC, 450 nm for CUPRAC, and 517 nm for DPPH. All antioxidant measurements were performed using a microplate spectrophotometer EPOCH (BioTek Instruments, Inc., Winooski, VT, USA).

4.12. Statistical Analysis

All statistical analyses were conducted using the R software (version 4.5.0) [71]. Data sets were tested for normality and equal variance (Shapiro–Wilk and Levene’s test). Quantitative data underwent one or two-way ANOVA analysis, with significant distinctions determined at p < 0.05. To assess variations among treatments, Tukey’s multiple range test was employed. Principal component analysis (PCA) was performed to summarize the variation in physiological and biochemical variables measured under two inoculation treatments (control and inoculation with E. billingiae) and two irrigation regimes (80% field capacity and 40% field capacity). The dataset was standardized to zero mean and unit variance prior to analysis, and the PCA was computed from the correlation matrix.

5. Conclusions

This study provides the first evidence that an AHL-producing endophytic bacterium can enhance drought resilience in the native conifer A. araucana. Inoculation with E. billingiae significantly improved biomass accumulation, chlorophyll content, water status, and osmotic adjustment under water-limited conditions. These beneficial effects are likely associated with a combination of bacterial functional traits, including AHL production and classical plant growth-promoting mechanisms such as IAA production, siderophore activity, and phosphate solubilization, rather than being attributable to a single factor. Notably, E. billingiae exhibited a shift in its AHL production profile under drought stress, producing C4-HSL under non-stress conditions and inducing C6-HSL synthesis under water limitation. This change suggests that quorum-sensing signals may be part of the bacterial physiological response to drought and could contribute to the modulation of plant–microbe interactions under stressful conditions.

In addition to improving plant water relations, inoculation was associated with reduced oxidative damage and altered secondary metabolism, highlighting the multifunctional nature of E. billingiae as a beneficial endophyte. Overall, our findings indicate that endophytic bacteria with quorum-sensing traits represent a promising component of microbiome-based approaches aimed at enhancing the resilience and conservation of endangered forest species such as A. araucana under climate change scenarios.