Embothrium coccineum (Proteaceae) increases the survival of the cushion plant Acaena integerrima (Rosaceae) and modifies bacterial communities in their rhizosphere

Embothrium coccineum (Proteaceae) increases the survival of the cushion plant Acaena integerrima (Rosaceae) and modifies bacterial communities in their rhizosphere | 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 Embothrium coccineum (Proteaceae) increases the survival of the cushion plant Acaena integerrima (Rosaceae) and modifies bacterial communities in their rhizosphere Angela Sanchez-Salazar, Oscar Martínez, Susana Valle, Julieta Orlando, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5989779/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Background and Aims: Acaena integerrima and Embothrium coccineum are native plants colonizing volcanic deposits in southern South America. Both species can grow in poor nutrient availability, increasing their leaf nutrient status when growing together. Enhanced leaf P concentrations could be explained by cluster-root carboxylate exudation of E. coccineum; however higher leaf N in E. coccineum has an unknown origin and free-living N 2 -fixing microbes could be involved. Methods : To test this possible mechanism, we conducted an experiment using volcanic material (pumice) and seedlings. We studied bacterial communities (16S rRNA and nif H) in the rhizosphere of E. coccineum and A. integerrima , both grown alone and together. To evaluate microbial communities, we used Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis. We measured carboxylate-exudation, leaf nutrient concentrations (N, P) and leaf δ 15 N isotopic fractionation. Results : The survival and shoot growth of A. integerrima was increased when grown combined. E. coccineum showed higher N and P concentrations when grown together than alone. Carboxylate-exudation showed faster rates of citrate from both species, being malate detected only from E. coccineum. Isotopic fractionation showed values close to zero in E. coccineum , being indicative of a biologically fixed N. The rhizosphere of E. coccineum alone exhibited the most distinct presence of 16S rRNA and nifH. Conclusions : Our results show that the N 2 -fixing bacterial communities and δ 15 N indicate reliance on diazotrophic activity in E. coccineum rhizosphere. Additionally, A. integerrima appears to benefit from this N fixation and shows the importance of carboxylate exudation as strategy on colonizing plant species. Carboxylate exudation Microbial communities Diazotrophic Recent volcanic depositions Colonizing plants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Colonizing plants on volcanic deposits can grow with a low availability of soil nutrients, and in some cases, can act as nurse plants (Muñoz et al. 2021 ). Several environmental improvements, such as wind protection, increase in soil nutrient availability, avoidance of extreme temperatures, and increase in water provision below nurse plants, have been reported (Cavieres et al. 2014 ; McIntire and Fajardo 2014 ). However, belowground mechanisms that might involve microorganisms in the rhizosphere of these nurse plants, remains less studied than aboveground processes (Hernández et al. 2020 ; Leff et al. 2018 ). Microbial communities carry out fundamental processes that contribute to nutrient cycling, plant growth, root health (Marschner et al. 2007) and influence plant productivity and diversity (Hortal et al. 2017 ). These ecological roles are especially important in nutrient-poor ecosystems, where microorganisms are responsible for the acquisition of limited nutrients during early soil development stages (Hernández et al. 2020 ). As an example, the association with N 2 -fixing bacteria (in nodules or free-living) by plants is a fundamental strategy for their growth and establishment (Muñoz et al. 2021 ). In addition, the phosphorus (P) supply plays an equally important role in plant growth, but this nutrient is often poorly available in soils, especially in young volcanic material in southern South America (Gallardo et al. 2012 ; Hernández et al. 2020 ). However, in soils with low P availability or in recent volcanic deposits, inorganic P can be mobilized by carboxylate exudation from plant roots (Richardson and Simpson, 2011 ; Zhou et al. 2020 ). In this context, plant’s carboxylate exudation additionally it might also have an important role in the “recruitment” or “selection” of beneficial microorganisms that could contribute the acquisition of these essential nutrients (Rudrappa et al. 2008 ), that use carboxylates as carbon source (Wu et al. 2018 ). Additionally, in recent volcanic deposits and/or volcanic-originated soils from southern South America, certain plants exhibit root adaptations in response to P deficiency, known as cluster roots (CR) (Zúñiga-Feest et al. 2010 ; Avila-Valdés et al. 2019). These CR improve the efficiency of plants to cope with low P availability, by releasing large amounts of phosphatases and carboxylates into the rhizosphere (Delgado et al. 2014 ; Avila-Valdés et al. 2019; Zúñiga-Feest et al. 2020 ), producing chemical and biological effects in their rhizozpheric soil (Delgado et al. 2015 ; Renderos et al. 2022 ). Cluster-roots functioning has been reported in many Proteaceae species from South-western Australia (Shane et al., 2004 ; Lambers et al. 2012 ), and South America (de Britto Costa et al. 2015; Delgado et al. 2014 ). Carboxylate exudates such as malate and citrate are among the most frequently detected compounds (Neumann and Martinoia 2002 ; Shane and Lambers 2005 ), along with oxalate and aconitate (de Britto Costa et al. 2015; Denton et al. 2006). More recently, Zúñiga-Feest et al. ( 2020 ) and Delgado et al. ( 2021 ) reported several colonizing Proteaceae species growing in volcanic deposits in southern South America, which also exude oxalate, malate, citrate, and succinate. The released carboxylates may also serve as a source of carbon for rhizosphere microorganisms (Rudrappa et al. 2008 ; Weisskopf et al. 2006 ), suggesting that the composition of carboxylate in the exudates may determine the microbiota associated with specific root systems (Philippot et al. 2013 ; Rudrappa et al. 2008 ). However, other studies that include microorganisms associated with CR, conducted by Weisskopf et al. ( 2006 ), specifically on Lupinus albus L. (Fabaceae), suggest that abundant carboxylate production is associated with release of exudates that reduce the growth and viability of microorganisms, thereby limiting the microbial consumption of organic acids in the rhizosphere. Stafford et al. ( 2005 ) studied the microbial diversity associated with two endemic Proteaceae in South Africa ( Leucospermum truncatulum and Leucadendron xanthoconus ) using PCR-DGGE and sequencing of the 16S r DNA gene as fingerprinting techniques. In this study, the authors found that the rhizosphere soil was more bacterially diverse than non-rhizosphere soil, suggesting that these plant species select specific microorganisms in their rhizosphere, including some diazotrophic bacteria, such as Azospirillum and Frankia (Stafford et al. 2005 ). In addition, Zúñiga-Feest et al. (2019) reported that E. coccineum increases the number of colony-forming units (UFC) of cultivable bacteria from their rhizosphere when grown in combination with Sophora cassioides (Fabaceae), suggesting that this might be related to the root exudates of both species growing together. Similarly, Renderos et al. ( 2022 ) based on TRFLP and the profile of carbon (C) source consumption (Biolog) suggested that CR in the rhizosphere of E. coccineum may select specific microbial groups, especially during the early CR-development stage. Despite the association of beneficial microbial communities with plants, their role and mechanisms of plant-microbial improvement have not yet evaluated in plants growing on volcanic substrates. On recent volcanic deposits in southern South America, Piper et al. ( 2019 ) reported higher leaf P and N concentrations when E. coccineum grows alongside A. integerrima , compared with seedlings growing alone. These increased leaf nutrient concentrations have been detected in plants growing on recent volcanic depositions of the Hudson volcano, where the nutrient availability is low and A. integerrima acts as a nurse plant, as described by Badano and Cavieres ( 2006 ). Based on the proposed mechanism of CR functioning (Li et al. 2003 , Lambers et al. 2019), the P enrichment in the leaves of A. integerrima can be explained by the effect of CR activity that solubilizes sparkly-available P forms. We propose that the higher N concentration in the leaves of E. coccineum growing with A. integerrima , might be due to the recruitment of free-living N 2 fixing bacteria. These functional bacterial groups could be related to a specific carboxylate exudate composition in the rhizosphere of E. coccineum. Our main goal was to determine the rhizosphere bacterial communities associated with E. coccineum and A. integerrima when growing alone or together. In addition, we explored the possible relation of carboxylate exudation rates with the presence of specific beneficial functional microbial groups such as N 2 -fixation rhizosphere bacteria. Furthermore, we measured the survival, growth rate, biomass distribution, leaf N and P concentrations in both plant species and conditions. We expected differences in the bacterial community composition and a greater presence of N 2 -fixing functional microbial groups in the rhizosphere, when both species are growing together rather than alone. Additionally, we expected the carboxylate-exudation rate of E. coccineum to be faster when both plant species are grown together, compared with E. coccineum seedlings growing alone. Survival, growth rate, and leaf nutrient concentrations (N and P) were also expected to be higher when both species grow together. This controlled experiment could aid us to understand the mechanisms and dynamics of higher leaf nutrient concentrations of colonizing plant species and the possible role that bacterial communities play in natural environments. Material and methods Soil sampling and chemical properties We used a recent volcanic substrate (pumice) collected from the vicinity of Mocho-Choshuenco volcano (39° 54’ S, 72° 2’W, 200 m above sea level), Región de Los Ríos, Chile, which last erupted in 1864 (Rawson et al. 2015 ) and transported the sample immediately to the laboratory. Pumice was chosen because it is a substrate with low P availability, which has been extensively used as a model of recent volcanic substrate by our research group (Avila-Valdés et al. 2019; Zúñiga-Feest et al. 2019). We made chemical analyses prior to the experiment starting; briefly, the chemical features were assessed as follows: inorganic N was extracted with 2 M KCl and NO 3 -N was determined by the Devarda’s alloy distillation method (Radojević and Bashkin, 1999 ). Available P (P Olsen) was extracted using 0.5 M Na-bicarbonate and analysed using the molybdate method (Murphy and Riley, 1962 ). Organic matter content was determined by wet digestion (Walkley and Black, 1934 ). Soil pH was measured in a 1:2.5 soil/deionized water or CaCl 2 suspensions; these chemical features are shown in Table 1 . Table 1 Chemical properties of the volcanic substrate used in the assay (Pumice) at the beginning of this experiment Chemical properties Organic matter (%) 0.53 pH (H 2 O) 6.6 pH (CaCl 2 ) 5.8 N (mg kg − 1 ) 8.4 Olsen P (mg kg − 1 ) 2.2 Total P (mg kg − 1 ) 279 Extractable Al (mg kg − 1 ) 19.7 Al (cmol + kg − 1 ) 0.008 Ca (cmol + kg − 1 ) 2.49 Mg (cmol + kg − 1 ) 0.83 K (cmol + kg − 1 ) 0.38 Na (cmol + kg − 1 ) 0.67 Sum of cations (cmol + kg − 1 ) 4.36 Experimental design and seedling production The seeds were collected in Cerro Castillo, Patagonia (46°06’ S and 72°13’ W; 451 m above sea level), and stored at 4°C. The seeds were sterilized with sodium hypochlorite 0.01% (v/v) and then washed with tap water, to be later germinated in sterile Petri dishes at 20°C in a growth chamber. When the radicles emerged, seeds were transferred to pots, which contained a recent volcanic substrate (pumice), to be watered with tap water twice a week and maintained in a greenhouse at Universidad Austral de Chile, Valdivia. Then plants were randomly chosen for different pot treatments, as follows: a) one seedling of Acaena integerrima per pot; b) one seedling of Embothrium coccineum growing per pot c) one seedling of Acaena integerrima plus one seedling of E. coccineum , growing per pot. Each pot had a volume of 2 L, each seedling had around 4 cm of height. All plant treatments were cultivated in triplicate using a randomized block design, and the pots were maintained in the greenhouse for two years. To avoid water limitation, each pot was watered with tap water twice a week. Survival rate was registered during the experiment and compared among treatments at harvest time. Determination of biomass, leaf nitrogen and phosphorus concentration After two years, plants were harvested and separated into: green leaves, senesced leaves, stems, roots, and CR (only in the case of E. coccineum ). Subsequently, the plant material was washed with distilled water and then dried in an oven at 60°C for 48 h (MMM Medcenter, Einrichtungen GmbH Venticell, Germany). The dry weight was recorded, and shoot/root biomass calculated; then, samples were ground for chemical analyses (N, P). The plant material was subsequently heated in a muffle oven at 500ºC for 4–8 h. Determination of leaf and root N concentration was carried out using the digestion and steam Kjeldahl distillation with manual titration. Leaf and root P concentrations were spectrophotometrically assayed, using the molybdate-blue method at a wavelength of 420 nm (Sadzawka et al. 2004). Samples remained dry and stored until isotopic determinations. Determination of isotopic δ 15 N composition The δ 15 N analyses were carried out on ground whole-plant oven-dried material at the Archeometry Department of the University of Cape Town (Cape Town, South Africa). The isotopic ratio of δ 15 N was calculated as δ = 1000‰ (R sample/R standard), where R is the molar ratio of the heavier to the lighter isotope of the samples and standards are as described by Farquhar et al. (1989). Between 2.100 and 2.200 mg of each milled sample was weighed in 8 mm x 5 mm tin capsules (Elemental Micro-analysis Ltd., Devon, UK) on a Sartorius microbalance (Göttingen, Germany). The samples were then combusted in a Fisons NA 1500 (Series 2), CHN analyser (Fisons instruments SpA, Milan, Italy). The δ 15 N values for the N gas released were determined on a Finnigan Matt 252 mass spectrometer (Finnigan MAT GmbH, Bremen, Germany), which was connected to a CHN analyzer by a Finnigan MAT Conflo control unit. Three standards were used to correct the samples for machine drift: two in-house standards (Merck Gel and Nasturtium ) and the IAEA (International Atomic Energy Agency) standard (NH 4 ) 2 SO 4 . Determination of carboxylate exudation rates The exudates were collected from the entire root system, according to Delgado et al. (2013). Briefly, entire root systems were gently washed to remove particles of pumice on their surface and then submersed in a container with 50 mL of 0.25 mM CaSO 4 (pH = 5.5) and shaken gently for 2 h; the solution was then collected, filtered by a 0.2 µm membrane filter, and stored at − 20°C. Then, the samples were lyophilized and dissolved in 600–800 µL of deionized-sterile water and filtered again (0.22 µm) for HPLC injection. The chromatographic separation was performed in an HPLC system (Jasco LC-Net II/ADC, Maryland, USA) equipped with a photodiode array detector (DAD) (Jasco MD 2015 Plus, Maryland, USA) and a Sphere Column Heater, according to Meier et al. ( 2012 ). The flow rate was 1 mL min − 1 and injected samples were detected at a wavelength of 210 nm. Identification of organic anions was determined by comparing retention times and by comparison with standards for each organic acid (oxalate, malate, citrate, iso-citrate, fumarate and succinate). The exudation rate was calculated using the dry biomass of each root system, measuring carboxylate concentration and the time of collection. Soil DNA extraction During plant harvest, the soil was extracted from the rhizosheath of each plant, and DNA was then extracted from each sample. Each rhizosheath sample (≈ 1 g) was extracted using MoBio Power Soil® DNA Isolation Kit (Qiagen, Los Angeles, USA) according to the manufacturer’s instructions. DNA quality and quantity were determined by spectrophotometry (Nanoquandt 200Tecan, Grodig, Austria). The DNA purity was assessed at A260/A280 absorbance ratio ∼1.8. Analysis of rhizobacterial community composition The composition of rhizosphere bacterial communities was evaluated by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR–DGGE) as described by Jorquera et al. ( 2010 ), using a specific primer set for the bacterial 16 S rRNA gene (Table 2 ). PCR amplifications were carried out with reagents supplied with GoTaq Flexi DNA Polymerase (Promega Co., Madison, USA), as follows: a hot-start was performed at 95°C for 10 min, the annealing was initially set at 65°C, and was then decreased by 0.5°C every cycle until 55°C for 1 min, followed by extension at 72°C for 3 min. Ten additional cycles were then carried out at 55°C (annealing), followed by denaturation at 94°C for 1 min, and primer extension at 72°C for 3 min. The final extension step was 7 min at 72°C. Table 2 Primers used in this study Gen Primer Sequence Reference 16S rRNA 358F a 5′-CCT ACG GGA GGC AGC AG-3′ Muyzer et al. (1993) 907R 5′-CCG TCA ATT CMT TTG AGT TT-3’ nifH PolF 5′-TGC GAY CCS AAR GCB GAC TC-3’ Poly et al. (2001) PolR 5′-ATS GCC ATC ATY TCR CCG GA-3′ PolFI 5′-TGC GAI CCS AAI GCI GAC TC-3′ AQER-GC b 5′-GAC GAT GTA GAT YTC CTG-3′ phoD ALPS-F730 5′-CAG TGG GAC GAC CAC GAG GT-3 Sakurai et al., (2008) ALPS-R1101 c 5´-GAG GCC GAT CGG CAT GTC G-3´ *Base-modified primers: I = Inosine, Y = C/T, S = C/G, R = A/G, B = G/C/T. a GC-clamp 5´-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GG-3´ b GC-clamp 5´-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCC-3´ c GC clamp 5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G -3’ PCR conditions to amplify nif H gene consisted of 15 min denaturation at 95° C, followed by 30 cycles at 94° C for 1 min, alignment at 55° C for 1 min, and a 72° C extension per 1 min with a final extension of 10 min at 72° C. The DGGE analysis was performed using a DCode system (Bio-Rad Laboratories, Inc., California, USA). Twenty-microliter aliquots of PCR products were loaded onto 6% (w/v) polyacrylamide gel with a 35–75% denaturing gradient for 16S rRNA, nif H and pho D, the electrophoresis was then run for 16 h at 70 V. The gel was stained with SYBR Gold (Molecular Probes, Invitrogen Co., Massachusetts, USA), for 30 min and photographed on a UV trans-illuminator. Clustering of DGGE banding profiles using a dendrogram was carried out using Phoretix 1D analysis software (Total Lab Ltd, Newcastle, UK). Based on the matrix obtained from Phoretix 1D analysis, the changes in the presence (or absence) and abundance of bacterial groups were analyzed by non-metric multidimensional scaling (NMDS) using PAST freeware ( http://folk.uio.no/ohammer/past/ ), with the Bray–Curtis similarity index and used to estimate the bacterial diversity by the Shannon–Wiener index as described by Yang et al. ( 2003 ). Statistical analyses The data of growth rate, shoot-to-root ratio, N and P concentration, carboxylate-exudation rate, and isotopic fractionation values were analyzed by a one-way ANOVA, using treatment (alone or combined) as a factor, then comparisons were carried out with a post-hoc Tukey multiple range test. All experiments were carried out in triplicate, and the values are given as means ± standard errors. Differences were considered significant when the P value was < 0.05. The statistical differences between treatments with the Graph Pad Prism v5.01 program (Graph Pad Software), were evaluated using ANOVA. Based on the matrix obtained from Phoretix 1D analysis, the changes in the presence (or absence) and abundance of bacterial groups were analysed by non-metric multidimensional scaling (NMDS) using PRIMER6 + statistical package (Quest Research Limited, Auckland, New Zealand) (Anderson et al. 2008). To determine similarity in band presence, we used UPGMA, following Bray-Curtis. Results Survival and growth rate of Embothrium coccineum and Acaena integerrima combined Survival rate of E. coccineum was similar among treatments, being 89% when grown alone and 100% when grown combined. However, A. integerrima showed higher survival rate when grown combined compared when growing alone (100% vs 33%). Similar values of total dry biomass at the end of our experiment, without significant differences ( P < 0.05), were observed when both species were grown alone or combined (Fig. 1 ). A higher shoot-to-root ratio was observed in A. integerrima in all treatments, than in E. coccineum , being significantly higher among treatments when A. integerrima was grown together with E. coccineum (Fig. 1 ). Leaf nitrogen and phosphorus concentrations in Embothrium coccineum and Acaena integerrima growing together We observed significant differences comparing species and treatments. Leaf P concentration was significantly higher in E. coccineum when these plants were grown combined with A. integerrima , exhibiting its highest value (1.5 mg g − 1 ). However, leaf P concentration of A. integerrima was similar among treatments(≈ 0.6 mg g − 1 ) (Fig. 2 ). Also, we observed a higher root P concentration in A. integerrima than in E. coccineum root, without significant differences among treatments. Considering N leaf concentration, E. coccineum leaves showed the highest value (9 mg g − 1 ) when grown with A. integerrima ; however root N concentration showed no significant differences among treatments or species (Fig. 2 ). N/P leaf ratio did not show significant differences among species or treatments, with values lower than 14 in all cases. These values are indicative of N limitation, based on Koerselman et al. (1996), with the lowest values in leaves of E. coccineum growing combined with A. integerrima (6), and the highest in leaves of A. integerrima , growing combined with E. coccineum (13) (Fig. 3 ) Carboxylate exudation rate and composition Oxalate and citrate were detected in the exudates collected from the entire root system of E. coccineum and A. integerrima , but malate was only detected in the exudates collected of E. coccineum . Significant differences in exudation rate were observed, being generally faster when plants were grown together, compared with plants grown separately. Acaena integerrima showed a faster exudation rate of oxalate than E. coccineum did in all cases, the fastest rate detected being when grown together with E. coccineum (317 µmol g − 1 h − 1 ) (Fig. 4 ). Both species showed similar citrate exudation rates, without significant differences and with the highest values when both were grown together (Fig. 4 ). Embothrium coccineum showed similar malate exudation rates in all treatments (45 µmol g − 1 h − 1 ) (Fig. 4 ). The total contribution of carboxylate exudation by each seedling, when grown alone or combined, was calculated based on the mean root biomass per plant in each treatment and multiplied by the carboxylate exudation rate. Oxalate, citrate and malate were considered for E. coccineum , and oxalate and citrate for A. integerrima . In the case of E. coccineum growing alone, oxalate represented the main component of its carboxylate composition (63%), and this composition changed when grown combined, when citrate was the main carboxylate exuded (60%). The composition of exudates of A. integerrima did not show differences among treatments, being oxalate the main carboxylate exuded in both cases (Fig. 4 ). Isotopic N discrimination ( δ 15 N ) Values of δ 15 N (N 15 /N 14 ) showed significant differences between species, but not among treatments, being close to zero when E. coccineum was grown alone (+ 0.30). This value is indicative of N in leaves being dependent on biological N fixation. The lowest value was observed in leaves of A. integerrima grown alone (-2.72) (Fig. 5 ). Microbial communities Analyses of the 16 S rRNA gene by DGGE profiles and MNDS show clustering between two treatments, A. integerrima grown alone and A. integerrima + E. coccineum grown combined (Fig. 6 A). The UPGMA cluster and NMDS analyses revealed that only the treatment with E. coccineum growing alone was grouped separately from the other two treatments. UPGMA cluster and NMDS analyses based on the DGGE profiles of the nif H gene revealed the presence of one cluster with similarity over 60% from samples collected from the rhizosphere of E. coccineum growing alone, indicating N 2 -fixing guilds (Fig. 6 B). Cluster formed below 60% of similarity were also observed in the NMDS grouping the other treatments; however, they did not represent a strong sign of similarity. Principal component analysis (PCA) of the 16S rRNA gene and carboxylates exudates showed three clustered groups, being the cluster of E. coccineum the most distant along coordinate 2 (Fig. 7 ). The examined carboxylates exudates oxalate and citrate mostly explain the variation in the rhizosphere bacterial community structure, associated to the treatment combined of E. coccineum and A. integerrima growing together. On the other hand, the soil physicochemical parameters (N mineral and P total), was the main factor determining the clustering of A. integerrima growing alone. None of the factors associated evaluated explained the grouping of the treatment E. coccineum , however it is important to mention that the malate was only detected in the E. coccineum root exudates (Fig. 7 ). Discussion As we expected, the survival rate of A. integerrima increased when grown together with E. coccineum . Also A. integerrima shoot biomass increases, when grown combined with E. coccineum , in relation to their root. These results show that co-cultivation had positive effects on A. integerrima but not on E. coccineum . In this sense, the present results partly confirm those reported by Piper et al. ( 2019 ), who showed that when both species grow together under natural conditions, produce a net positive interaction. Considering nutrient concentration, in our experiment E. coccineum leaf showed higher P but not significant difference in N concentration, when grown combined with A. integerrima . This result differed from what we expected, considering the previous studies reported by Piper et al. ( 2019 ), where N concentration was higher in E. coccineum leaves growing in the cushion of A. integerrima . However, if we consider N leaves content (biomass x concentration), both species showed higher values when grown together, being eight times higher in E. coccineum compared when grown alone (1.26 mg to 0.15 mg, respectively) and A. integerrima showed the highest N content (2.13 mg), in this case explain by a significant higher shoot growth, probably stimulate by N availability from E. coccineum root system in the pot. Additionally, it is possible that the plant size of both species in our experiment was different from that observed under natural conditions, where small seedlings of E. coccineum were growing inside the cushion of A. integerrima formed by several individuals of several years, which could influence nutritional responses. In our experiment, we used a pair of seedlings of similar sizes at the beginning of this experiment and shoot, growth was probably positively influenced by higher temperatures occurring in the greenhouse, compared with natural conditions in Patagonia. As is well known greenhouse conditions promote higher aerial growth specially for A.integerrima compared with natural conditions, as we observed before on several experiments (personal observation). Additionally, Piper et al. ( 2019 ) results had influenced by several other plant species growing in the cushion, e.g., Trifolium repens , Gaulteria mucronata , Nothofagus pumilio ; these species might also influence nutrient dynamics and N availability for E. coccineum (Piper et al. 2019 ), considering that T. repens is an N 2 -fixing species. In any case, our initial predictions were not entirely confirmed and differ from the field results reported by Piper et al. ( 2019 ), because probably some competition by N occurred in the pots when both plants were growing together, as suggest values of N/P reported here, becaues E. coccineum showed the lowest values (6) and A. integerrima showed the highest values when grown combined (13). Further research with a long-term experiment that includes seedlings of more species, growing at a larger pot volume resembling natural conditions, may help in better understanding of nutrient benefits among plants. Our results confirm that both species described as colonizing; on recent volcanic substrates have the capacity to grow under low nutrient availability, in agreement with previous reports by Muñoz et al. ( 2021 ) and Delgado et al. (2019). Additionally, the carboxylate exudation rate was faster in the entire root system of both species when growing together rather than growing alone, confirming the importance of carboxylate exudation by roots as a mechanism of nutrient acquisition on early successional stages (Zhou et al 2020 , Yu et al 2020 ). As far as we know, this is the first report of A. integerrima carboxylate exudation rate measured on the entire root system of plants growing on volcanic substrates. Recently, other Rosaceae species ( Potentilla tanacetifolia ) with arbuscular mycorrhiza have been reported as P mobilising species, through carboxylate exudation (Yu et al. 2020 ). These authors used Mn as a proxy of this carboxylate exudation, because in natural environments carboxylate exudation measurements are unreliable (Abrahao et al. 2019). Probably in our experiment, where two individuals were growing in a pot had to compete for nutrients in this limited space, and exuded carboxylates at a higher rate. In our research group we also have found faster rates of exudation when plants were grown in groups of seedlings (Garcia, personal communication), as in the case of Orites myrtoidea (Proteaceae with CR) growing together with Gaulteria poepiggi (Ericaceae with ericoid mycorrhiza) seedlings on recent volcanic substrates. Also, carboxylate exudation shows variation depending on each neighbour as reported Fajardo and Piper ( 2019 ), when pears of conspecific and heterospecific species grow together on tephra showed different foliar Mn concentrations. The carboxylate exudation reported here promotes mobilization of P in the pots, where rhizosphere Olsen P at the end of this experiment was 30% higher when compared with the initial value on pumice (C. Reyes, personal communication). A similar effect was reported by Avila-Valdés et al. (2019) referring to Orites myrtoidea and E. coccineum (both Proteaceae species with CR), growing separately on similar substrate and greenhouse conditions, where P Olsen increased 120% and 50%, respectively, when compared with initial P Olsen values. These authors suggest that the greater availability was explained by citrate exudation. Based on Koerselman and Meuleman ( 1996 ), who proposed that the N/P ratio can be used to study nutritional limitations on plant communities, all the values in our experiment showed seedlings with N limitation, without significant differences among treatments. Under natural conditions similar limitations due to N have been observed in this species, with values around 5, with the exception of E. coccineum when growing together with A. integerrima (N/P = 14). Embothrium coccineum plants have a greater plasticity in their nutritional requirements, because they can grow at a faster rate when supplemented with nutrients and can maintain part of this growth rate when grown on low nutrient availability conditions (Zúñiga-Feest et al. 2010 ). All of this information suggests that the presence of N 2 -fixing microorganisms may be a key aspect of their colonizing capacity in harsh volcanic environments at the initial stages of pedogenesis. Considering the source of N, E. coccineum growing alone had an N-isotopic signature showing that some of the N came from N 2 -fixing microorganisms with the nif H gene in their rhizosphere, likely supported by exuded carboxylates (malate). All these results suggest that when E. coccineum grows alone, it could support the establishment of specialist bacterial groups, such as diazotrophic bacteria, perhaps due to specific interaction with their cluster roots (Stafford et al., 2005 ; Renderos et al., 2022 ). It is widely known that the rhizosphere harbors a wide diversity of microbial communities that are highly dynamic, and their structure is determined by specific characteristics of the plant species (Marschner et al., 2007; Zhang et al., 2020 ; Pathan et al., 2020 ). In our study, we show that nif H-harboring bacterial populations were associated with the rhizosphere of E. coccineum growing alone, comparing of the rhizosphere of plants growing together with A. integerrima (Fig. 6 ). Interestingly, the carboxylate exudation showed that the malate was exudated exclusively by E. coccineum , which might create an environmental condition that determines the establishment and colonization of some specific bacterial groups, such as N-fixing bacteria, however further research is need it to establish this mechanism. As far we know, in the rhizosphere of E. coccineum growing on recent volcanic depositions had shown bacterial consumption of several carboxylates and C sources as: maleate, oxalate, fumarate, succinate, lactate (personal communication A. Zúñiga-Feest) and lactose, glutamic acid, malate, asparagine from E. coccineum CR growing on rich volcanic soils (Renderos et al 2020). According to Meyer et al. ( 2011 ) malate is the major exudate among carboxylates, considered the second most preferred carbon source after glucose for microorganisms like the rhizobacterium Bacillus subtilis . In the same way, the malate and its derivatives have been reported in well-known nitrogen-fixing bacteria (such as Azospirillum brasilense and Paenibacillus polymyxa ) to act as a strong chemoattractant (Ling et al., 2011 ; Zhulin et al., 1993). Similarly, a study conducted by Rekha et al. ( 2018 ), demonstrated an increase in the expression of malate synthase in rice roots treated with Bacillus subtilis , suggesting a significant influence on malate biosynthesis, thereby enhancing plant metabolism and potentially promoting plant growth. This mechanism was elucidated by Rudrappa et al. ( 2008 ), who found that malate triggers the operon for Bacillus strains, attracting the bacteria to the rhizosphere and promoting growth and systemic resistance against potential pathogens. We surmise that a similar mechanism also could occur when E. coccineum is growing alone due to the high amount of malate, which represents a change and selection of the diazotrophic bacterial community as determined here by the nif H gene. However, further research is needed to dissect these possible mechanisms in the rhizosphere of these colonizing species. Oxalate and citrate exuded by plants explain the differentiation observed in the 16S representing the bacterial community in the rhizosphere of two groups: A. integerrima + E. coccineum and A. integerrima alone. Therefore, in substrates such as pumice, which have low bioavailability of nutrients like N, the high rates of plant exudation reported in this study would be a determining factor in the presence of certain diazotrophic populations. The results presented here constitute the first report on these mechanisms at the rhizospheric level, which could partially explain the nutritional enhancements observed in both species when grown together, as reported in field conditions by Piper et al. ( 2019 ), where plants are cultivated on volcanic ash from the Hudson volcano eruption. We have reported that when E. coccineum is grown in combination with Sophora cassioides (Fabaceae, N 2 fixing) it promotes an increase of total bacterial abundance (CFU) in their rhizosphere compared with plants growing alone (Zúñiga-Feest et al., 2018 ). These results suggest that Proteaceae may favor beneficial microbes in their rhizosphere and foster positive interactions with their neighboring plants. Furthermore, these results are consistent with the selection of bacterial genetic groups observed in the rhizosphere of various cluster-root stages by Renderos et al. ( 2022 ). These authors propose that new bacterial groups colonize the rhizosphere of young CR in adult E. coccineum plants growing under natural conditions. Conclusions Based on survival rate and shoot growth, E. coccineum had a positive effect on A. integerrima . However, our hypothesis was only partially supported because only E. coccineum showed a higher P concentration when grown together. Both species increased their carboxylate exudation rate through their root systems when grown together, but only E. coccineum exuded malate. The beneficial microbial group ( nif H) shows a strong correlation with E. coccineum rhizosphere when grown alone, and isotopic N discrimination on leaves shows bacterial N enrichments, suggesting some selective effect by its root exudate composition of diazotrophic microorganisms. These results demonstrate that plants growing together can modify their rhizosphere through carboxylate exudation, influencing vegetation nutrition and microbial dynamics of recent volcanic substrates on early successional stages. Declarations Acknowledgements : The authors thank Fondecyt 1180699 for financial support, Corporación Nacional Forestal (CONAF) and Dra. Frida Piper for seed collection. Also Dr. Mauricio Pereira for his assistance, Dr. Stefano Cesco and Dr. Hans Lambers for their revision with several helpful comments that improved this manuscript. Funding: T his work was supported by FONDECYT 1180699 grant (AZF), from Chilean Government Research Agency Competing interests: The authors have no relevant financial or non-financial interests to disclose Author contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Angela Sánchez-Salazar, Cecilia Reyes, Susana Valle, Alejandra Zúñiga-Feest. Julieta Orlando, Alexander Valentine. The first draft of the manuscript was written by Angela Sánchez-Salazar, Oscar Martínez and Alejandra Zúñiga Feest, all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Abrahão A, De Britto Costa P, Lambers H, et al. (2019) Soil types select for plants with matching nutrient-acquisition and -use traits in hyperdiverse and severely nutrient-impoverished Campos rupestres and Cerrado in Central Brazil. J Ecol 107:1302–16. Ávila A, Piper F, Zúñiga-Feest A (2019) Cluster root formation and function vary in two species with contrasting 2 geographic ranges. 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Plant Soil https://doi.org/10.1007/s11104-020-04640-y Zúñiga-Feest A, Muñoz G, Bustos-Salazar A, Ramírez F, Delgado M, Valle S, Díaz L (2018) The nitrogen fixing specie Sophora cassioides (Fabaceae), is nutritionally favored and their rhizosphere bacteria modified when is cocultivated with the cluster root forming Embothrium coccineum (Proteaceae). J Soil Sci Plant Nutr 18:597–616 Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2025 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 22 May, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Editor invited by journal 03 Mar, 2025 Editor assigned by journal 02 Mar, 2025 First submitted to journal 01 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5989779","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431786173,"identity":"b793a715-3c58-4d9a-9163-c16f1b598f53","order_by":0,"name":"Angela Sanchez-Salazar","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Sanchez-Salazar","suffix":""},{"id":431786174,"identity":"24d50d50-fed0-4afc-b14c-59d367499a30","order_by":1,"name":"Oscar Martínez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Martínez","suffix":""},{"id":431786175,"identity":"65c33f21-7fff-4f5b-8ece-7969dcf45c87","order_by":2,"name":"Susana Valle","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Valle","suffix":""},{"id":431786176,"identity":"d55f433e-1343-48e2-89e2-9b5f98d473f7","order_by":3,"name":"Julieta Orlando","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Julieta","middleName":"","lastName":"Orlando","suffix":""},{"id":431786177,"identity":"7ff7e6f6-8b78-4aeb-8ddd-db842625840a","order_by":4,"name":"Cecilia Reyes","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cecilia","middleName":"","lastName":"Reyes","suffix":""},{"id":431786178,"identity":"52cdd58f-7d39-46a6-bf33-2ab8668ac448","order_by":5,"name":"Alex Valentine","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Valentine","suffix":""},{"id":431786179,"identity":"77d24352-bc70-4beb-9613-8bcc433480aa","order_by":6,"name":"Alejandra Zúñiga-Feest","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABPklEQVRIie2RMUvDQBTH33GQLqlZL4PtV3hHoVKq9qs0dOjSopOLoJGDdEnr2k5+i+DYcmCXgKuQwUjBTkK6xVLBpEIlJtkd7gd3/O/xfjweB6BQ/EcqAAhAfx5dAGMfouRolRKF/lFMG4DcTVOFliuHO2WvCD1bzGAIOr/8fGxfGPd+Mwy358yojNar061XP6JAos0gpzCpdfnY77emc/8ErUmPma7PxXAScIcCNWdefozUEauORFi4TWa59AZfBkQM3YAkikareaUujYh/JUpd6qlyyzqv6zfRcoNOmYJSh0Y6BZ8SpRtLhgy4gDiwyhQuNWwcO33kvnbFLHvJmD/gs7Ed9BxKRNEutaV45x9OG2vP0jPj3TUzRsswinfB2cNILKJNwfrJl+EhEicTiF3Un0DD37zLBYVCoVDAN2l5a9CbFV6DAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3524-9813","institution":"Universidad Austral de Chile","correspondingAuthor":true,"prefix":"","firstName":"Alejandra","middleName":"","lastName":"Zúñiga-Feest","suffix":""}],"badges":[],"createdAt":"2025-02-08 21:23:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5989779/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5989779/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07985-4","type":"published","date":"2025-10-21T16:16:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79560056,"identity":"0b6638ec-1282-427d-a986-1174a2a74f8e","added_by":"auto","created_at":"2025-03-31 08:33:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":164892,"visible":true,"origin":"","legend":"\u003cp\u003ea) Total dry biomass and b) shoot/root biomass of \u003cem\u003eEmbothrium coccineum\u003c/em\u003e (E.c) and \u003cem\u003eAcaena integerrima\u003c/em\u003e (A.i) grown on pumice for two years either alone or together (E.c+A.i). Each value corresponds to the mean of 10 samples ± standard error (SE), different lowercase letters show significant differences (p \u0026lt;0.05). ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/46b05061f6a5733b62fbc407.png"},{"id":79560628,"identity":"6cfce780-a2ad-4de3-b9a5-281ee5a2549e","added_by":"auto","created_at":"2025-03-31 08:41:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":280616,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf and root phosphorus (P) and nitrogen (N) concentration of \u003cem\u003eEmbothrium coccineum\u003c/em\u003e(E.c) and \u003cem\u003eAcaena integerrima\u003c/em\u003e (A.i) grown on pumice for two years, either alone or together (E.c+A.i). Each value corresponds to the mean of three samples ± standard error (SE); different lowercase letters show significant differences (p\u0026lt;0.05). ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/78c5af94eaa34ad559b9fdf6.png"},{"id":79560058,"identity":"9f5bd512-e032-4ce6-80fd-b7106374008c","added_by":"auto","created_at":"2025-03-31 08:33:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102966,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf N/P ratio of\u003cem\u003eEmbothrium coccineum\u003c/em\u003e (E.c) and \u003cem\u003eAcaena integerrima\u003c/em\u003e (A.i) leaves from plants grown on pumice for two years either alone or together (E.c+A.i). Each value corresponds to the mean of three samples ± standard error (SE), different lowercase letters show significant differences (p \u0026gt;0.05). ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/7b4c670f700fb457af0e1c54.png"},{"id":79560627,"identity":"777e5680-21d3-4b45-a1bf-127ec63dea72","added_by":"auto","created_at":"2025-03-31 08:41:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176718,"visible":true,"origin":"","legend":"\u003cp\u003eCarboxylate-exudation rate for entire root systems of \u003cem\u003eEmbothrium coccineum\u003c/em\u003e (E.c.) and \u003cem\u003eAcaena integerrima \u003c/em\u003e(A.i.) grown alone or together. Each value corresponds to the mean of five samples ± standard error. Different lowercase letters show significant differences between treatments for each carboxylate (Tukey test, p\u0026lt;0.05). Different capital letters show significant differences for total carboxylate exudation detected between treatments (Tukey test, p\u0026lt;0.05). Malate was detected only in root exudates of \u003cem\u003eE. coccineum\u003c/em\u003e entire root system.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/ac44ba2bc52aebe4a3d97017.png"},{"id":79560625,"identity":"e083a7da-2d78-4fb5-8c25-63fac2e6c996","added_by":"auto","created_at":"2025-03-31 08:41:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":129415,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN natural abundance in leaves of \u003cem\u003eEmbothrium coccineum\u003c/em\u003e (E.c.) and \u003cem\u003eAcaena integerrima \u003c/em\u003e(A.i.) grown alone or together for two years under greenhouse conditions. Different lowercase letters show significant differences between species (Tukey test, p\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/8d1649854f64dd3cba5f71a4.png"},{"id":79561046,"identity":"c35290e3-1034-4936-9637-355f1a067fd0","added_by":"auto","created_at":"2025-03-31 08:49:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":404962,"visible":true,"origin":"","legend":"\u003cp\u003eNon-metric multidimensional scaling (NMDS) plots, using the similarity of Bray-Curtis based of DGGE profiles generated by Phoretix 1D Pro Gel Analysis Software (http://totallab.com/), and Dendrogram of DGGE profiles based on Unweighted Pair Group Method with Arithmetic Mean (UPGMA) algorithm, from top to down: of the 16S rRNA (a) and \u003cem\u003enif\u003c/em\u003eH gene (b). Samples from the rhizosphere of \u003cem\u003eAcaena integerrima\u0026nbsp;\u003c/em\u003e(A.c); \u003cem\u003eEmbothrium coccineum\u0026nbsp;\u003c/em\u003e(E.c)\u003cem\u003e \u003c/em\u003egrowing alone or\u003cem\u003e\u0026nbsp;Acaena integerrima\u003c/em\u003e and \u003cem\u003eEmbothrium coccineum\u003c/em\u003e growing together (A.c+E.c). The analysis was performed with the program PRIMERe v7 program (http://www.primer-e.com/).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/ac7780696f6a8c2a24800907.png"},{"id":79560067,"identity":"6365cf48-10cb-4004-b12a-9f4295f5132e","added_by":"auto","created_at":"2025-03-31 08:33:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":223121,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of 16S rRNA gene and carboxylates exuded in the rhizosphere of \u003cem\u003eAcaena integerrima \u003c/em\u003e(A.i); \u003cem\u003eEmbothrium coccineum \u003c/em\u003e(E.c)\u003cem\u003e \u003c/em\u003egrowing alone or\u003cem\u003e Acaena integerrima\u003c/em\u003e and \u003cem\u003eEmbothrium coccineum\u003c/em\u003e growing together (A.i+E.c). The analysis was performed with PAST software.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/99df1ad97c334a85875ea03b.png"},{"id":94490268,"identity":"c266f51e-a78c-497d-a476-acc508860b8c","added_by":"auto","created_at":"2025-10-27 17:08:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2456987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989779/v1/715dcfa1-a0ee-4cac-87ae-8f817c971fa0.pdf"}],"financialInterests":"","formattedTitle":"Embothrium coccineum (Proteaceae) increases the survival of the cushion plant Acaena integerrima (Rosaceae) and modifies bacterial communities in their rhizosphere","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColonizing plants on volcanic deposits can grow with a low availability of soil nutrients, and in some cases, can act as nurse plants (Mu\u0026ntilde;oz et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Several environmental improvements, such as wind protection, increase in soil nutrient availability, avoidance of extreme temperatures, and increase in water provision below nurse plants, have been reported (Cavieres et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; McIntire and Fajardo \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, belowground mechanisms that might involve microorganisms in the rhizosphere of these nurse plants, remains less studied than aboveground processes (Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Leff et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Microbial communities carry out fundamental processes that contribute to nutrient cycling, plant growth, root health (Marschner et al. 2007) and influence plant productivity and diversity (Hortal et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These ecological roles are especially important in nutrient-poor ecosystems, where microorganisms are responsible for the acquisition of limited nutrients during early soil development stages (Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As an example, the association with N\u003csub\u003e2\u003c/sub\u003e-fixing bacteria (in nodules or free-living) by plants is a fundamental strategy for their growth and establishment (Mu\u0026ntilde;oz et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, the phosphorus (P) supply plays an equally important role in plant growth, but this nutrient is often poorly available in soils, especially in young volcanic material in southern South America (Gallardo et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, in soils with low P availability or in recent volcanic deposits, inorganic P can be mobilized by carboxylate exudation from plant roots (Richardson and Simpson, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this context, plant\u0026rsquo;s carboxylate exudation additionally it might also have an important role in the \u0026ldquo;recruitment\u0026rdquo; or \u0026ldquo;selection\u0026rdquo; of beneficial microorganisms that could contribute the acquisition of these essential nutrients (Rudrappa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), that use carboxylates as carbon source (Wu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, in recent volcanic deposits and/or volcanic-originated soils from southern South America, certain plants exhibit root adaptations in response to P deficiency, known as cluster roots (CR) (Z\u0026uacute;\u0026ntilde;iga-Feest et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Avila-Vald\u0026eacute;s et al. 2019). These CR improve the efficiency of plants to cope with low P availability, by releasing large amounts of phosphatases and carboxylates into the rhizosphere (Delgado et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Avila-Vald\u0026eacute;s et al. 2019; Z\u0026uacute;\u0026ntilde;iga-Feest et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), producing chemical and biological effects in their rhizozpheric soil (Delgado et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Renderos et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cluster-roots functioning has been reported in many Proteaceae species from South-western Australia (Shane et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Lambers et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and South America (de Britto Costa et al. 2015; Delgado et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Carboxylate exudates such as malate and citrate are among the most frequently detected compounds (Neumann and Martinoia \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Shane and Lambers \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), along with oxalate and aconitate (de Britto Costa et al. 2015; Denton et al. 2006). More recently, Z\u0026uacute;\u0026ntilde;iga-Feest et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Delgado et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported several colonizing Proteaceae species growing in volcanic deposits in southern South America, which also exude oxalate, malate, citrate, and succinate.\u003c/p\u003e \u003cp\u003eThe released carboxylates may also serve as a source of carbon for rhizosphere microorganisms (Rudrappa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Weisskopf et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), suggesting that the composition of carboxylate in the exudates may determine the microbiota associated with specific root systems (Philippot et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rudrappa et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, other studies that include microorganisms associated with CR, conducted by Weisskopf et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), specifically on \u003cem\u003eLupinus albus\u003c/em\u003e L. (Fabaceae), suggest that abundant carboxylate production is associated with release of exudates that reduce the growth and viability of microorganisms, thereby limiting the microbial consumption of organic acids in the rhizosphere.\u003c/p\u003e \u003cp\u003eStafford et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) studied the microbial diversity associated with two endemic Proteaceae in South Africa (\u003cem\u003eLeucospermum truncatulum\u003c/em\u003e and \u003cem\u003eLeucadendron xanthoconus\u003c/em\u003e) using PCR-DGGE and sequencing of the \u003cem\u003e16S r\u003c/em\u003eDNA gene as fingerprinting techniques. In this study, the authors found that the rhizosphere soil was more bacterially diverse than non-rhizosphere soil, suggesting that these plant species select specific microorganisms in their rhizosphere, including some diazotrophic bacteria, such as \u003cem\u003eAzospirillum\u003c/em\u003e and \u003cem\u003eFrankia\u003c/em\u003e (Stafford et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In addition, Z\u0026uacute;\u0026ntilde;iga-Feest et al. (2019) reported that \u003cem\u003eE. coccineum\u003c/em\u003e increases the number of colony-forming units (UFC) of cultivable bacteria from their rhizosphere when grown in combination with \u003cem\u003eSophora cassioides\u003c/em\u003e (Fabaceae), suggesting that this might be related to the root exudates of both species growing together. Similarly, Renderos et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) based on TRFLP and the profile of carbon (C) source consumption (Biolog) suggested that CR in the rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e may select specific microbial groups, especially during the early CR-development stage. Despite the association of beneficial microbial communities with plants, their role and mechanisms of plant-microbial improvement have not yet evaluated in plants growing on volcanic substrates.\u003c/p\u003e \u003cp\u003eOn recent volcanic deposits in southern South America, Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported higher leaf P and N concentrations when \u003cem\u003eE. coccineum\u003c/em\u003e grows alongside \u003cem\u003eA. integerrima\u003c/em\u003e, compared with seedlings growing alone. These increased leaf nutrient concentrations have been detected in plants growing on recent volcanic depositions of the Hudson volcano, where the nutrient availability is low and \u003cem\u003eA. integerrima\u003c/em\u003e acts as a nurse plant, as described by Badano and Cavieres (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Based on the proposed mechanism of CR functioning (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, Lambers et al. 2019), the P enrichment in the leaves of \u003cem\u003eA. integerrima\u003c/em\u003e can be explained by the effect of CR activity that solubilizes sparkly-available P forms. We propose that the higher N concentration in the leaves of \u003cem\u003eE. coccineum\u003c/em\u003e growing with \u003cem\u003eA. integerrima\u003c/em\u003e, might be due to the recruitment of free-living N\u003csub\u003e2\u003c/sub\u003e fixing bacteria. These functional bacterial groups could be related to a specific carboxylate exudate composition in the rhizosphere of \u003cem\u003eE. coccineum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eOur main goal was to determine the rhizosphere bacterial communities associated with \u003cem\u003eE. coccineum\u003c/em\u003e and \u003cem\u003eA. integerrima\u003c/em\u003e when growing alone or together. In addition, we explored the possible relation of carboxylate exudation rates with the presence of specific beneficial functional microbial groups such as N\u003csub\u003e2\u003c/sub\u003e-fixation rhizosphere bacteria. Furthermore, we measured the survival, growth rate, biomass distribution, leaf N and P concentrations in both plant species and conditions. We expected differences in the bacterial community composition and a greater presence of N\u003csub\u003e2\u003c/sub\u003e-fixing functional microbial groups in the rhizosphere, when both species are growing together rather than alone. Additionally, we expected the carboxylate-exudation rate of \u003cem\u003eE. coccineum\u003c/em\u003e to be faster when both plant species are grown together, compared with \u003cem\u003eE. coccineum\u003c/em\u003e seedlings growing alone. Survival, growth rate, and leaf nutrient concentrations (N and P) were also expected to be higher when both species grow together. This controlled experiment could aid us to understand the mechanisms and dynamics of higher leaf nutrient concentrations of colonizing plant species and the possible role that bacterial communities play in natural environments.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSoil sampling and chemical properties\u003c/h2\u003e \u003cp\u003eWe used a recent volcanic substrate (pumice) collected from the vicinity of Mocho-Choshuenco volcano (39\u0026deg; 54\u0026rsquo; S, 72\u0026deg; 2\u0026rsquo;W, 200 m above sea level), Regi\u0026oacute;n de Los R\u0026iacute;os, Chile, which last erupted in 1864 (Rawson et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and transported the sample immediately to the laboratory. Pumice was chosen because it is a substrate with low P availability, which has been extensively used as a model of recent volcanic substrate by our research group (Avila-Vald\u0026eacute;s et al. 2019; Z\u0026uacute;\u0026ntilde;iga-Feest et al. 2019). We made chemical analyses prior to the experiment starting; briefly, the chemical features were assessed as follows: inorganic N was extracted with 2 M KCl and NO\u003csub\u003e3\u003c/sub\u003e-N was determined by the Devarda\u0026rsquo;s alloy distillation method (Radojević and Bashkin, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Available P (P Olsen) was extracted using 0.5 M Na-bicarbonate and analysed using the molybdate method (Murphy and Riley, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Organic matter content was determined by wet digestion (Walkley and Black, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1934\u003c/span\u003e). Soil pH was measured in a 1:2.5 soil/deionized water or CaCl\u003csub\u003e2\u003c/sub\u003e suspensions; these chemical features are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eChemical properties of the volcanic substrate used in the assay (Pumice) at the beginning of this experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical properties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic matter (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH (H\u003csub\u003e2\u003c/sub\u003eO)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH (CaCl\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlsen P (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal P (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e279\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtractable Al (mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAl (cmol\u003csub\u003e+\u003c/sub\u003e kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa (cmol\u0026thinsp;+\u0026thinsp;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSum of cations (cmol\u003csub\u003e+\u003c/sub\u003e kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.36\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\n\u003ch3\u003eExperimental design and seedling production\u003c/h3\u003e\n\u003cp\u003eThe seeds were collected in Cerro Castillo, Patagonia (46\u0026deg;06\u0026rsquo; S and 72\u0026deg;13\u0026rsquo; W; 451 m above sea level), and stored at 4\u0026deg;C. The seeds were sterilized with sodium hypochlorite 0.01% (v/v) and then washed with tap water, to be later germinated in sterile Petri dishes at 20\u0026deg;C in a growth chamber. When the radicles emerged, seeds were transferred to pots, which contained a recent volcanic substrate (pumice), to be watered with tap water twice a week and maintained in a greenhouse at Universidad Austral de Chile, Valdivia. Then plants were randomly chosen for different pot treatments, as follows: a) one seedling of \u003cem\u003eAcaena integerrima\u003c/em\u003e per pot; b) one seedling of \u003cem\u003eEmbothrium coccineum\u003c/em\u003e growing per pot c) one seedling of \u003cem\u003eAcaena integerrima\u003c/em\u003e plus one seedling of \u003cem\u003eE. coccineum\u003c/em\u003e, growing per pot. Each pot had a volume of 2 L, each seedling had around 4 cm of height. All plant treatments were cultivated in triplicate using a randomized block design, and the pots were maintained in the greenhouse for two years. To avoid water limitation, each pot was watered with tap water twice a week. Survival rate was registered during the experiment and compared among treatments at harvest time.\u003c/p\u003e\n\u003ch3\u003eDetermination of biomass, leaf nitrogen and phosphorus concentration\u003c/h3\u003e\n\u003cp\u003eAfter two years, plants were harvested and separated into: green leaves, senesced leaves, stems, roots, and CR (only in the case of \u003cem\u003eE. coccineum\u003c/em\u003e). Subsequently, the plant material was washed with distilled water and then dried in an oven at 60\u0026deg;C for 48 h (MMM Medcenter, Einrichtungen GmbH Venticell, Germany). The dry weight was recorded, and shoot/root biomass calculated; then, samples were ground for chemical analyses (N, P). The plant material was subsequently heated in a muffle oven at 500\u0026ordm;C for 4\u0026ndash;8 h. Determination of leaf and root N concentration was carried out using the digestion and steam Kjeldahl distillation with manual titration. Leaf and root P concentrations were spectrophotometrically assayed, using the molybdate-blue method at a wavelength of 420 nm (Sadzawka et al. 2004). Samples remained dry and stored until isotopic determinations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of isotopic\u003c/b\u003e \u003cem\u003eδ\u003c/em\u003e\u003csup\u003e\u003cem\u003e15\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eN\u003c/em\u003e \u003cb\u003ecomposition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe δ\u003csup\u003e15\u003c/sup\u003eN analyses were carried out on ground whole-plant oven-dried material at the Archeometry Department of the University of Cape Town (Cape Town, South Africa). The isotopic ratio of δ\u003csup\u003e15\u003c/sup\u003eN was calculated as δ\u0026thinsp;=\u0026thinsp;1000\u0026permil; (R sample/R standard), where R is the molar ratio of the heavier to the lighter isotope of the samples and standards are as described by Farquhar et al. (1989). Between 2.100 and 2.200 mg of each milled sample was weighed in 8 mm x 5 mm tin capsules (Elemental Micro-analysis Ltd., Devon, UK) on a Sartorius microbalance (G\u0026ouml;ttingen, Germany). The samples were then combusted in a Fisons NA 1500 (Series 2), CHN analyser (Fisons instruments SpA, Milan, Italy). The δ\u003csup\u003e15\u003c/sup\u003eN values for the N gas released were determined on a Finnigan Matt 252 mass spectrometer (Finnigan MAT GmbH, Bremen, Germany), which was connected to a CHN analyzer by a Finnigan MAT Conflo control unit. Three standards were used to correct the samples for machine drift: two in-house standards (Merck Gel and \u003cem\u003eNasturtium\u003c/em\u003e) and the IAEA (International Atomic Energy Agency) standard (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003eDetermination of carboxylate exudation rates\u003c/h3\u003e\n\u003cp\u003e The exudates were collected from the entire root system, according to Delgado et al. (2013). Briefly, entire root systems were gently washed to remove particles of pumice on their surface and then submersed in a container with 50 mL of 0.25 mM CaSO\u003csub\u003e4\u003c/sub\u003e (pH\u0026thinsp;=\u0026thinsp;5.5) and shaken gently for 2 h; the solution was then collected, filtered by a 0.2 \u0026micro;m membrane filter, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Then, the samples were lyophilized and dissolved in 600\u0026ndash;800 \u0026micro;L of deionized-sterile water and filtered again (0.22 \u0026micro;m) for HPLC injection. The chromatographic separation was performed in an HPLC system (Jasco LC-Net II/ADC, Maryland, USA) equipped with a photodiode array detector (DAD) (Jasco MD 2015 Plus, Maryland, USA) and a Sphere Column Heater, according to Meier et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The flow rate was 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and injected samples were detected at a wavelength of 210 nm. Identification of organic anions was determined by comparing retention times and by comparison with standards for each organic acid (oxalate, malate, citrate, iso-citrate, fumarate and succinate). The exudation rate was calculated using the dry biomass of each root system, measuring carboxylate concentration and the time of collection.\u003c/p\u003e\n\u003ch3\u003eSoil DNA extraction\u003c/h3\u003e\n\u003cp\u003eDuring plant harvest, the soil was extracted from the rhizosheath of each plant, and DNA was then extracted from each sample. Each rhizosheath sample (\u0026asymp;\u0026thinsp;1 g) was extracted using MoBio Power Soil\u0026reg; DNA Isolation Kit (Qiagen, Los Angeles, USA) according to the manufacturer\u0026rsquo;s instructions. DNA quality and quantity were determined by spectrophotometry (Nanoquandt 200Tecan, Grodig, Austria). The DNA purity was assessed at A260/A280 absorbance ratio \u0026sim;1.8.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of rhizobacterial community composition\u003c/h2\u003e \u003cp\u003eThe composition of rhizosphere bacterial communities was evaluated by Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR\u0026ndash;DGGE) as described by Jorquera et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), using a specific primer set for the bacterial \u003cem\u003e16\u003c/em\u003eS rRNA gene (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). PCR amplifications were carried out with reagents supplied with GoTaq Flexi DNA Polymerase (Promega Co., Madison, USA), as follows: a hot-start was performed at 95\u0026deg;C for 10 min, the annealing was initially set at 65\u0026deg;C, and was then decreased by 0.5\u0026deg;C every cycle until 55\u0026deg;C for 1 min, followed by extension at 72\u0026deg;C for 3 min. Ten additional cycles were then carried out at 55\u0026deg;C (annealing), followed by denaturation at 94\u0026deg;C for 1 min, and primer extension at 72\u0026deg;C for 3 min. The final extension step was 7 min at 72\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e16S rRNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e358F\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CCT ACG GGA GGC AGC AG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMuyzer et al. (1993)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e907R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CCG TCA ATT CMT TTG AGT TT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003enifH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TGC GAY CCS AAR GCB GAC TC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003ePoly et al. (2001)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-ATS GCC ATC ATY TCR CCG GA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePolFI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TGC GAI CCS AAI GCI GAC TC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAQER-GC\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GAC GAT GTA GAT YTC CTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ephoD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eALPS-F730\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-CAG TGG GAC GAC CAC GAG GT-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSakurai et al., (2008)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eALPS-R1101\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026acute;-GAG GCC GAT CGG CAT GTC G-3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Base-modified primers: I\u0026thinsp;=\u0026thinsp;Inosine, Y\u0026thinsp;=\u0026thinsp;C/T, S\u0026thinsp;=\u0026thinsp;C/G, R\u0026thinsp;=\u0026thinsp;A/G, B\u0026thinsp;=\u0026thinsp;G/C/T.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e GC-clamp 5\u0026acute;-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GG-3\u0026acute;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003eb\u003c/sup\u003e GC-clamp 5\u0026acute;-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCC-3\u0026acute;\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ec\u003c/sup\u003e GC clamp 5\u0026prime;-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G -3\u0026rsquo;\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ePCR conditions to amplify \u003cem\u003enif\u003c/em\u003eH gene consisted of 15 min denaturation at 95\u0026deg; C, followed by 30 cycles at 94\u0026deg; C for 1 min, alignment at 55\u0026deg; C for 1 min, and a 72\u0026deg; C extension per 1 min with a final extension of 10 min at 72\u0026deg; C. The DGGE analysis was performed using a DCode system (Bio-Rad Laboratories, Inc., California, USA). Twenty-microliter aliquots of PCR products were loaded onto 6% (w/v) polyacrylamide gel with a 35\u0026ndash;75% denaturing gradient for \u003cem\u003e16S\u003c/em\u003e rRNA, \u003cem\u003enif\u003c/em\u003eH and \u003cem\u003epho\u003c/em\u003eD, the electrophoresis was then run for 16 h at 70 V. The gel was stained with SYBR Gold (Molecular Probes, Invitrogen Co., Massachusetts, USA), for 30 min and photographed on a UV trans-illuminator.\u003c/p\u003e \u003cp\u003eClustering of DGGE banding profiles using a dendrogram was carried out using Phoretix 1D analysis software (Total Lab Ltd, Newcastle, UK). Based on the matrix obtained from Phoretix 1D analysis, the changes in the presence (or absence) and abundance of bacterial groups were analyzed by non-metric multidimensional scaling (NMDS) using PAST freeware (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://folk.uio.no/ohammer/past/\u003c/span\u003e\u003cspan address=\"http://folk.uio.no/ohammer/past/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with the Bray\u0026ndash;Curtis similarity index and used to estimate the bacterial diversity by the Shannon\u0026ndash;Wiener index as described by Yang et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe data of growth rate, shoot-to-root ratio, N and P concentration, carboxylate-exudation rate, and isotopic fractionation values were analyzed by a one-way ANOVA, using treatment (alone or combined) as a factor, then comparisons were carried out with a \u003cem\u003epost-hoc\u003c/em\u003e Tukey multiple range test. All experiments were carried out in triplicate, and the values are given as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors. Differences were considered significant when the P value was \u0026lt;\u0026thinsp;0.05. The statistical differences between treatments with the Graph Pad Prism v5.01 program (Graph Pad Software), were evaluated using ANOVA.\u003c/p\u003e \u003cp\u003eBased on the matrix obtained from Phoretix 1D analysis, the changes in the presence (or absence) and abundance of bacterial groups were analysed by non-metric multidimensional scaling (NMDS) using PRIMER6\u0026thinsp;+\u0026thinsp;statistical package (Quest Research Limited, Auckland, New Zealand) (Anderson et al. 2008). To determine similarity in band presence, we used UPGMA, following Bray-Curtis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSurvival and growth rate of\u003c/b\u003e \u003cb\u003eEmbothrium coccineum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eAcaena integerrima\u003c/b\u003e \u003cb\u003ecombined\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSurvival rate of \u003cem\u003eE. coccineum\u003c/em\u003e was similar among treatments, being 89% when grown alone and 100% when grown combined. However, \u003cem\u003eA. integerrima\u003c/em\u003e showed higher survival rate when grown combined compared when growing alone (100% vs 33%). Similar values of total dry biomass at the end of our experiment, without significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), were observed when both species were grown alone or combined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A higher shoot-to-root ratio was observed in \u003cem\u003eA. integerrima\u003c/em\u003e in all treatments, than in \u003cem\u003eE. coccineum\u003c/em\u003e, being significantly higher among treatments when \u003cem\u003eA. integerrima\u003c/em\u003e was grown together with \u003cem\u003eE. coccineum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eLeaf nitrogen and phosphorus concentrations in\u003c/b\u003e \u003cb\u003eEmbothrium coccineum\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eAcaena integerrima\u003c/b\u003e \u003cb\u003egrowing together\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe observed significant differences comparing species and treatments. Leaf P concentration was significantly higher in \u003cem\u003eE. coccineum\u003c/em\u003e when these plants were grown combined with \u003cem\u003eA. integerrima\u003c/em\u003e, exhibiting its highest value (1.5 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). However, leaf P concentration of \u003cem\u003eA. integerrima\u003c/em\u003e was similar among treatments(\u0026asymp;\u0026thinsp;0.6 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Also, we observed a higher root P concentration in \u003cem\u003eA. integerrima\u003c/em\u003e than in \u003cem\u003eE. coccineum\u003c/em\u003e root, without significant differences among treatments. Considering N leaf concentration, \u003cem\u003eE. coccineum\u003c/em\u003e leaves showed the highest value (9 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) when grown with \u003cem\u003eA. integerrima\u003c/em\u003e; however root N concentration showed no significant differences among treatments or species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eN/P leaf ratio did not show significant differences among species or treatments, with values lower than 14 in all cases. These values are indicative of N limitation, based on Koerselman et al. (1996), with the lowest values in leaves of \u003cem\u003eE. coccineum\u003c/em\u003e growing combined with \u003cem\u003eA. integerrima\u003c/em\u003e (6), and the highest in leaves of \u003cem\u003eA. integerrima\u003c/em\u003e, growing combined with \u003cem\u003eE. coccineum\u003c/em\u003e (13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCarboxylate exudation rate and composition\u003c/h2\u003e \u003cp\u003eOxalate and citrate were detected in the exudates collected from the entire root system of \u003cem\u003eE. coccineum\u003c/em\u003e and \u003cem\u003eA. integerrima\u003c/em\u003e, but malate was only detected in the exudates collected of \u003cem\u003eE. coccineum\u003c/em\u003e. Significant differences in exudation rate were observed, being generally faster when plants were grown together, compared with plants grown separately. \u003cem\u003eAcaena integerrima\u003c/em\u003e showed a faster exudation rate of oxalate than \u003cem\u003eE. coccineum\u003c/em\u003e did in all cases, the fastest rate detected being when grown together with \u003cem\u003eE. coccineum\u003c/em\u003e (317 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both species showed similar citrate exudation rates, without significant differences and with the highest values when both were grown together (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). \u003cem\u003eEmbothrium coccineum\u003c/em\u003e showed similar malate exudation rates in all treatments (45 \u0026micro;mol g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe total contribution of carboxylate exudation by each seedling, when grown alone or combined, was calculated based on the mean root biomass per plant in each treatment and multiplied by the carboxylate exudation rate. Oxalate, citrate and malate were considered for \u003cem\u003eE. coccineum\u003c/em\u003e, and oxalate and citrate for \u003cem\u003eA. integerrima\u003c/em\u003e. In the case of \u003cem\u003eE. coccineum\u003c/em\u003e growing alone, oxalate represented the main component of its carboxylate composition (63%), and this composition changed when grown combined, when citrate was the main carboxylate exuded (60%). The composition of exudates of \u003cem\u003eA. integerrima\u003c/em\u003e did not show differences among treatments, being oxalate the main carboxylate exuded in both cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsotopic N discrimination (\u003c/b\u003eδ\u003csup\u003e15\u003c/sup\u003eN\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eValues of δ\u003csup\u003e15\u003c/sup\u003eN (N\u003csup\u003e15\u003c/sup\u003e/N\u003csup\u003e14\u003c/sup\u003e) showed significant differences between species, but not among treatments, being close to zero when \u003cem\u003eE. coccineum\u003c/em\u003e was grown alone (+\u0026thinsp;0.30). This value is indicative of N in leaves being dependent on biological N fixation. The lowest value was observed in leaves of \u003cem\u003eA. integerrima\u003c/em\u003e grown alone (-2.72) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicrobial communities\u003c/h2\u003e \u003cp\u003eAnalyses of the \u003cem\u003e16\u003c/em\u003eS rRNA gene by DGGE profiles and MNDS show clustering between two treatments, \u003cem\u003eA. integerrima\u003c/em\u003e grown alone and \u003cem\u003eA. integerrima\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eE. coccineum\u003c/em\u003e grown combined (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The UPGMA cluster and NMDS analyses revealed that only the treatment with \u003cem\u003eE. coccineum\u003c/em\u003e growing alone was grouped separately from the other two treatments. UPGMA cluster and NMDS analyses based on the DGGE profiles of the \u003cem\u003enif\u003c/em\u003eH gene revealed the presence of one cluster with similarity over 60% from samples collected from the rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e growing alone, indicating N\u003csub\u003e2\u003c/sub\u003e-fixing guilds (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Cluster formed below 60% of similarity were also observed in the NMDS grouping the other treatments; however, they did not represent a strong sign of similarity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrincipal component analysis (PCA) of the 16S rRNA gene and carboxylates exudates showed three clustered groups, being the cluster of \u003cem\u003eE. coccineum\u003c/em\u003e the most distant along coordinate 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The examined carboxylates exudates oxalate and citrate mostly explain the variation in the rhizosphere bacterial community structure, associated to the treatment combined of \u003cem\u003eE. coccineum\u003c/em\u003e and \u003cem\u003eA. integerrima\u003c/em\u003e growing together. On the other hand, the soil physicochemical parameters (N mineral and P total), was the main factor determining the clustering of \u003cem\u003eA. integerrima\u003c/em\u003e growing alone. None of the factors associated evaluated explained the grouping of the treatment \u003cem\u003eE. coccineum\u003c/em\u003e, however it is important to mention that the malate was only detected in the \u003cem\u003eE. coccineum\u003c/em\u003e root exudates (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs we expected, the survival rate of \u003cem\u003eA. integerrima\u003c/em\u003e increased when grown together with \u003cem\u003eE. coccineum\u003c/em\u003e. Also \u003cem\u003eA. integerrima\u003c/em\u003e shoot biomass increases, when grown combined with \u003cem\u003eE. coccineum\u003c/em\u003e, in relation to their root. These results show that co-cultivation had positive effects on \u003cem\u003eA. integerrima\u003c/em\u003e but not on \u003cem\u003eE. coccineum\u003c/em\u003e. In this sense, the present results partly confirm those reported by Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who showed that when both species grow together under natural conditions, produce a net positive interaction.\u003c/p\u003e \u003cp\u003eConsidering nutrient concentration, in our experiment \u003cem\u003eE. coccineum\u003c/em\u003e leaf showed higher P but not significant difference in N concentration, when grown combined with \u003cem\u003eA. integerrima\u003c/em\u003e. This result differed from what we expected, considering the previous studies reported by Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where N concentration was higher in \u003cem\u003eE. coccineum\u003c/em\u003e leaves growing in the cushion of \u003cem\u003eA. integerrima\u003c/em\u003e. However, if we consider N leaves content (biomass x concentration), both species showed higher values when grown together, being eight times higher in \u003cem\u003eE. coccineum\u003c/em\u003e compared when grown alone (1.26 mg to 0.15 mg, respectively) and \u003cem\u003eA. integerrima\u003c/em\u003e showed the highest N content (2.13 mg), in this case explain by a significant higher shoot growth, probably stimulate by N availability from \u003cem\u003eE. coccineum\u003c/em\u003e root system in the pot. Additionally, it is possible that the plant size of both species in our experiment was different from that observed under natural conditions, where small seedlings of \u003cem\u003eE. coccineum\u003c/em\u003e were growing inside the cushion of \u003cem\u003eA. integerrima\u003c/em\u003e formed by several individuals of several years, which could influence nutritional responses. In our experiment, we used a pair of seedlings of similar sizes at the beginning of this experiment and shoot, growth was probably positively influenced by higher temperatures occurring in the greenhouse, compared with natural conditions in Patagonia. As is well known greenhouse conditions promote higher aerial growth specially for \u003cem\u003eA.integerrima\u003c/em\u003e compared with natural conditions, as we observed before on several experiments (personal observation). Additionally, Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) results had influenced by several other plant species growing in the cushion, e.g., \u003cem\u003eTrifolium repens\u003c/em\u003e, \u003cem\u003eGaulteria mucronata\u003c/em\u003e, \u003cem\u003eNothofagus pumilio\u003c/em\u003e; these species might also influence nutrient dynamics and N availability for \u003cem\u003eE. coccineum\u003c/em\u003e (Piper et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), considering that \u003cem\u003eT. repens\u003c/em\u003e is an N\u003csub\u003e2\u003c/sub\u003e-fixing species. In any case, our initial predictions were not entirely confirmed and differ from the field results reported by Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), because probably some competition by N occurred in the pots when both plants were growing together, as suggest values of N/P reported here, becaues \u003cem\u003eE. coccineum\u003c/em\u003e showed the lowest values (6) and \u003cem\u003eA. integerrima\u003c/em\u003e showed the highest values when grown combined (13). Further research with a long-term experiment that includes seedlings of more species, growing at a larger pot volume resembling natural conditions, may help in better understanding of nutrient benefits among plants.\u003c/p\u003e \u003cp\u003eOur results confirm that both species described as colonizing; on recent volcanic substrates have the capacity to grow under low nutrient availability, in agreement with previous reports by Mu\u0026ntilde;oz et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Delgado et al. (2019). Additionally, the carboxylate exudation rate was faster in the entire root system of both species when growing together rather than growing alone, confirming the importance of carboxylate exudation by roots as a mechanism of nutrient acquisition on early successional stages (Zhou et al \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Yu et al \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As far as we know, this is the first report of \u003cem\u003eA. integerrima\u003c/em\u003e carboxylate exudation rate measured on the entire root system of plants growing on volcanic substrates. Recently, other Rosaceae species (\u003cem\u003ePotentilla tanacetifolia\u003c/em\u003e) with arbuscular mycorrhiza have been reported as P mobilising species, through carboxylate exudation (Yu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These authors used Mn as a \u003cem\u003eproxy\u003c/em\u003e of this carboxylate exudation, because in natural environments carboxylate exudation measurements are unreliable (Abrahao et al. 2019). Probably in our experiment, where two individuals were growing in a pot had to compete for nutrients in this limited space, and exuded carboxylates at a higher rate. In our research group we also have found faster rates of exudation when plants were grown in groups of seedlings (Garcia, personal communication), as in the case of \u003cem\u003eOrites myrtoidea\u003c/em\u003e (Proteaceae with CR) growing together with \u003cem\u003eGaulteria poepiggi\u003c/em\u003e (Ericaceae with ericoid mycorrhiza) seedlings on recent volcanic substrates. Also, carboxylate exudation shows variation depending on each neighbour as reported Fajardo and Piper (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), when pears of conspecific and heterospecific species grow together on tephra showed different foliar Mn concentrations. The carboxylate exudation reported here promotes mobilization of P in the pots, where rhizosphere Olsen P at the end of this experiment was 30% higher when compared with the initial value on pumice (C. Reyes, personal communication). A similar effect was reported by Avila-Vald\u0026eacute;s et al. (2019) referring to \u003cem\u003eOrites myrtoidea\u003c/em\u003e and \u003cem\u003eE. coccineum\u003c/em\u003e (both Proteaceae species with CR), growing separately on similar substrate and greenhouse conditions, where P Olsen increased 120% and 50%, respectively, when compared with initial P Olsen values. These authors suggest that the greater availability was explained by citrate exudation.\u003c/p\u003e \u003cp\u003eBased on Koerselman and Meuleman (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), who proposed that the N/P ratio can be used to study nutritional limitations on plant communities, all the values in our experiment showed seedlings with N limitation, without significant differences among treatments. Under natural conditions similar limitations due to N have been observed in this species, with values around 5, with the exception of \u003cem\u003eE. coccineum\u003c/em\u003e when growing together with \u003cem\u003eA. integerrima\u003c/em\u003e (N/P\u0026thinsp;=\u0026thinsp;14). \u003cem\u003eEmbothrium coccineum\u003c/em\u003e plants have a greater plasticity in their nutritional requirements, because they can grow at a faster rate when supplemented with nutrients and can maintain part of this growth rate when grown on low nutrient availability conditions (Z\u0026uacute;\u0026ntilde;iga-Feest et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). All of this information suggests that the presence of N\u003csub\u003e2\u003c/sub\u003e-fixing microorganisms may be a key aspect of their colonizing capacity in harsh volcanic environments at the initial stages of pedogenesis. Considering the source of N, \u003cem\u003eE. coccineum\u003c/em\u003e growing alone had an N-isotopic signature showing that some of the N came from N\u003csub\u003e2\u003c/sub\u003e-fixing microorganisms with the \u003cem\u003enif\u003c/em\u003eH gene in their rhizosphere, likely supported by exuded carboxylates (malate). All these results suggest that when \u003cem\u003eE. coccineum\u003c/em\u003e grows alone, it could support the establishment of specialist bacterial groups, such as diazotrophic bacteria, perhaps due to specific interaction with their cluster roots (Stafford et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Renderos et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is widely known that the rhizosphere harbors a wide diversity of microbial communities that are highly dynamic, and their structure is determined by specific characteristics of the plant species (Marschner et al., 2007; Zhang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pathan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, we show that \u003cem\u003enif\u003c/em\u003eH-harboring bacterial populations were associated with the rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e growing alone, comparing of the rhizosphere of plants growing together with \u003cem\u003eA. integerrima\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, the carboxylate exudation showed that the malate was exudated exclusively by \u003cem\u003eE. coccineum\u003c/em\u003e, which might create an environmental condition that determines the establishment and colonization of some specific bacterial groups, such as N-fixing bacteria, however further research is need it to establish this mechanism. As far we know, in the rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e growing on recent volcanic depositions had shown bacterial consumption of several carboxylates and C sources as: maleate, oxalate, fumarate, succinate, lactate (personal communication A. Z\u0026uacute;\u0026ntilde;iga-Feest) and lactose, glutamic acid, malate, asparagine from \u003cem\u003eE. coccineum\u003c/em\u003e CR growing on rich volcanic soils (Renderos et al 2020). According to Meyer et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) malate is the major exudate among carboxylates, considered the second most preferred carbon source after glucose for microorganisms like the rhizobacterium \u003cem\u003eBacillus subtilis\u003c/em\u003e. In the same way, the malate and its derivatives have been reported in well-known nitrogen-fixing bacteria (such as \u003cem\u003eAzospirillum brasilense\u003c/em\u003e and \u003cem\u003ePaenibacillus polymyxa\u003c/em\u003e) to act as a strong chemoattractant (Ling et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zhulin et al., 1993). Similarly, a study conducted by Rekha et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), demonstrated an increase in the expression of malate synthase in rice roots treated with \u003cem\u003eBacillus subtilis\u003c/em\u003e, suggesting a significant influence on malate biosynthesis, thereby enhancing plant metabolism and potentially promoting plant growth. This mechanism was elucidated by Rudrappa et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), who found that malate triggers the operon for \u003cem\u003eBacillus\u003c/em\u003e strains, attracting the bacteria to the rhizosphere and promoting growth and systemic resistance against potential pathogens. We surmise that a similar mechanism also could occur when \u003cem\u003eE. coccineum\u003c/em\u003e is growing alone due to the high amount of malate, which represents a change and selection of the diazotrophic bacterial community as determined here by the \u003cem\u003enif\u003c/em\u003eH gene. However, further research is needed to dissect these possible mechanisms in the rhizosphere of these colonizing species.\u003c/p\u003e \u003cp\u003eOxalate and citrate exuded by plants explain the differentiation observed in the 16S representing the bacterial community in the rhizosphere of two groups: \u003cem\u003eA. integerrima\u0026thinsp;+\u0026thinsp;E. coccineum\u003c/em\u003e and \u003cem\u003eA. integerrima\u003c/em\u003e alone. Therefore, in substrates such as pumice, which have low bioavailability of nutrients like N, the high rates of plant exudation reported in this study would be a determining factor in the presence of certain diazotrophic populations. The results presented here constitute the first report on these mechanisms at the rhizospheric level, which could partially explain the nutritional enhancements observed in both species when grown together, as reported in field conditions by Piper et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where plants are cultivated on volcanic ash from the Hudson volcano eruption.\u003c/p\u003e \u003cp\u003eWe have reported that when \u003cem\u003eE. coccineum\u003c/em\u003e is grown in combination with \u003cem\u003eSophora cassioides\u003c/em\u003e (Fabaceae, N\u003csub\u003e2\u003c/sub\u003e fixing) it promotes an increase of total bacterial abundance (CFU) in their rhizosphere compared with plants growing alone (Z\u0026uacute;\u0026ntilde;iga-Feest et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These results suggest that Proteaceae may favor beneficial microbes in their rhizosphere and foster positive interactions with their neighboring plants. Furthermore, these results are consistent with the selection of bacterial genetic groups observed in the rhizosphere of various cluster-root stages by Renderos et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These authors propose that new bacterial groups colonize the rhizosphere of young CR in adult \u003cem\u003eE. coccineum\u003c/em\u003e plants growing under natural conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBased on survival rate and shoot growth, \u003cem\u003eE. coccineum\u003c/em\u003e had a positive effect on \u003cem\u003eA. integerrima\u003c/em\u003e. However, our hypothesis was only partially supported because only \u003cem\u003eE. coccineum\u003c/em\u003e showed a higher P concentration when grown together. Both species increased their carboxylate exudation rate through their root systems when grown together, but only \u003cem\u003eE. coccineum\u003c/em\u003e exuded malate. The beneficial microbial group (\u003cem\u003enif\u003c/em\u003eH) shows a strong correlation with \u003cem\u003eE. coccineum\u003c/em\u003e rhizosphere when grown alone, and isotopic N discrimination on leaves shows bacterial N enrichments, suggesting some selective effect by its root exudate composition of diazotrophic microorganisms. These results demonstrate that plants growing together can modify their rhizosphere through carboxylate exudation, influencing vegetation nutrition and microbial dynamics of recent volcanic substrates on early successional stages.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors thank Fondecyt 1180699 for financial support, Corporaci\u0026oacute;n Nacional Forestal (CONAF) and Dra. Frida Piper for seed collection. Also Dr. Mauricio Pereira for his assistance, Dr. Stefano Cesco and Dr. Hans Lambers for their revision with several helpful comments that improved this manuscript.\u003c/p\u003e\u003cp\u003eFunding: T\u003cem\u003ehis work was supported by FONDECYT 1180699 grant (AZF), from Chilean Government Research Agency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eCompeting interests: The authors have no relevant financial or non-financial interests to disclose\u003c/p\u003e\n\u003cp\u003eAuthor contribution: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Angela S\u0026aacute;nchez-Salazar, Cecilia Reyes, Susana Valle, Alejandra Z\u0026uacute;\u0026ntilde;iga-Feest. Julieta Orlando, Alexander Valentine. The first draft of the manuscript was written by Angela S\u0026aacute;nchez-Salazar, Oscar Mart\u0026iacute;nez and Alejandra Z\u0026uacute;\u0026ntilde;iga Feest, all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eData availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbrah\u0026atilde;o A, De Britto Costa P, Lambers H, et al. (2019) Soil types select for plants with matching nutrient-acquisition and -use traits in hyperdiverse and severely nutrient-impoverished Campos rupestres and Cerrado in Central Brazil. 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Plant Soil 334:113\u0026ndash;121\u003c/li\u003e\n\u003cli\u003eZ\u0026uacute;\u0026ntilde;iga-Feest A, Sep\u0026uacute;lveda F, Delgado M, Valle S, Mu\u0026ntilde;oz G, Pereira M, Reyes-Diaz M (2020) \u003cem\u003eGevuina avellana\u003c/em\u003e and \u003cem\u003eLomatia dentata\u003c/em\u003e, two Proteaceae species from evergreen temperate forests of South America exhibit contrasting physiological responses under nutrient deprivation. Plant Soil https://doi.org/10.1007/s11104-020-04640-y\u003c/li\u003e\n\u003cli\u003eZ\u0026uacute;\u0026ntilde;iga-Feest A, Mu\u0026ntilde;oz G, Bustos-Salazar A, Ram\u0026iacute;rez F, Delgado M, Valle S, D\u0026iacute;az L (2018) The nitrogen fixing specie\u003cem\u003e Sophora cassioides\u003c/em\u003e (Fabaceae), is nutritionally favored and their rhizosphere bacteria modified when is cocultivated with the cluster root forming \u003cem\u003eEmbothrium coccineum\u003c/em\u003e (Proteaceae). J Soil Sci Plant Nutr 18:597\u0026ndash;616\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Carboxylate exudation, Microbial communities, Diazotrophic, Recent volcanic depositions, Colonizing plants","lastPublishedDoi":"10.21203/rs.3.rs-5989779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5989779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Aims:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAcaena integerrima and \u003cem\u003eEmbothrium coccineum\u003c/em\u003e are native plants colonizing volcanic deposits in southern South America. Both species can grow in poor nutrient availability, increasing their leaf nutrient status when growing together. Enhanced leaf P concentrations could be explained by cluster-root carboxylate exudation of \u003cem\u003eE. coccineum;\u003c/em\u003e however higher leaf N in \u003cem\u003eE. coccineum\u003c/em\u003e has an unknown origin and free-living N\u003csub\u003e2\u003c/sub\u003e-fixing microbes could be involved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: To test this possible mechanism, we conducted an experiment using volcanic material (pumice) and seedlings. We studied bacterial communities (16S rRNA and \u003cem\u003enif\u003c/em\u003eH) in the rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e and \u003cem\u003eA. integerrima\u003c/em\u003e, both grown alone and together. To evaluate microbial communities, we used Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis. We measured carboxylate-exudation, leaf nutrient concentrations (N, P) and leaf δ\u003csup\u003e15\u003c/sup\u003eN isotopic fractionation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The survival and shoot growth of \u003cem\u003eA. integerrima\u003c/em\u003e was increased when grown combined. \u003cem\u003eE. coccineum\u003c/em\u003e showed higher N and P concentrations when grown together than alone. Carboxylate-exudation showed faster rates of citrate from both species, being malate detected only from \u003cem\u003eE. coccineum.\u003c/em\u003e Isotopic fractionation showed values close to zero in \u003cem\u003eE. coccineum\u003c/em\u003e, being indicative of a biologically fixed N. The rhizosphere of \u003cem\u003eE. coccineum\u003c/em\u003e alone exhibited the most distinct presence of \u003cem\u003e16S\u003c/em\u003e rRNA and \u003cem\u003enifH.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Our results show that the N\u003csub\u003e2\u003c/sub\u003e-fixing bacterial communities and δ\u003csup\u003e15\u003c/sup\u003eN indicate reliance on diazotrophic activity in \u003cem\u003eE. coccineum\u003c/em\u003e rhizosphere. Additionally, \u003cem\u003eA. integerrima\u003c/em\u003e appears to benefit from this N fixation and shows the importance of carboxylate exudation as strategy on colonizing plant species.\u003c/p\u003e","manuscriptTitle":"Embothrium coccineum (Proteaceae) increases the survival of the cushion plant Acaena integerrima (Rosaceae) and modifies bacterial communities in their rhizosphere","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 08:33:51","doi":"10.21203/rs.3.rs-5989779/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-05-22T17:17:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-31T12:24:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T22:09:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-03-03T07:46:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-03T03:09:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-03-01T06:26:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bf028d25-297d-485d-9282-4e4472610549","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:23:19+00:00","versionOfRecord":{"articleIdentity":"rs-5989779","link":"https://doi.org/10.1007/s11104-025-07985-4","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-10-21 16:16:44","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-03-31 08:33:51","video":"","vorDoi":"10.1007/s11104-025-07985-4","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07985-4","workflowStages":[]},"version":"v1","identity":"rs-5989779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5989779","identity":"rs-5989779","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}