Oxalotrophic bacterial assemblages in the ectomycorrhizosphere of forest trees and their effects on oxalate degradation and carbon fixation potential

Chemical Geology 514 (2019) 54–64

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Oxalotrophic bacterial assemblages in the ectomycorrhizosphere of forest trees and their effects on oxalate degradation and carbon fixation potential Qibiao Suna, Jing Lia, Roger D. Finlayb, Bin Liana, a b

T

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Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of Agricultural Sciences, Uppsala SE 75 007, Sweden

ARTICLE INFO

ABSTRACT

Editor: Dong Hailiang

Ectomycorrhizal (ECM) fungi can promote soil mineral weathering through acidification and complexation by secreting oxalic acid. However, the metabolism of oxalate in the ectomycorrhizosphere and identity of the bacteria involved are still poorly understood. Here, we investigated the abundance and community structure of oxalotrophic bacteria in the ectomycorrhizospheres of Pinus massoniana and Quercus serrata trees using quantitative PCR and high-throughput sequencing. The secondary minerals formed in the degradation of oxalate were analyzed using SEM, XRD and HRTEM. Our results showed that oxalotrophic bacteria are abundant in the ectomycorrhizosphere (2.60–5.03 × 108/g soil) and that ECM fungi can influence oxalotrophic bacteria, resulting in communities that are compositionally distinct from those in non-mycorrhizosphere soils. The results also showed that approximately one third of these bacterial species were nitrogen-fixing, accounting for 43–60% of the total sequences of oxalotrophic bacteria. An oxalotrophic Streptomyces sp. NJ10 was isolated from the ectomycorrhizosphere of P. massoniana and shown to be able to degrade calcium oxalate and induce the formation of carbonate minerals (calcite or dolomite). This study provides novel evidence that ECM fungi can enrich specific oxalotrophic bacteria in the ectomycorrhizosphere, that degrade oxalate using the oxalate-carbonate pathway, representing a potential long-term sink for photosynthetically fixed carbon derived from the atmosphere. These findings improve our understanding of the possible ecological functioning and environmental effects of plant-fungal-bacterial interactions in forests.

Keywords: Formyl-CoA transferase Oxalatrophic bacteria Ectomycorrhizal fungi Carbon sequestration Quantitative PCR

1. Introduction Ectomycorrhizal (ECM) fungi form symbiotic associations with fine roots of woody plants, that are ubiquitous in temperate and boreal forests. ECM fungi can improve the uptake of water and mineral nutrients by their tree hosts, and improve their stress tolerance, receiving photosynthetically-fixed carbohydrates (Smith and Read, 2008; Finlay et al., 2009; Behie and Bidochka, 2014; Quirk et al., 2014; Sun et al., 2017). Essential plant macronutrients such as potassium (K) and phosphorus (P) are limited in bioavailability, and often immobilized in soil minerals or rocks. ECM fungi play a critical role in acquiring insoluble nutrients from soil primary minerals through weathering (Jongmans et al., 1997; Landeweert et al., 2001; Finlay et al., 2009). ECM fungi are able to weather minerals through local acidification around the hyphae and by exuding metal-complexing weathering agents such as organic acids and siderophores (van Hees et al., 2006; Winkelmann, 2007; Rosling et al., 2009; Bonneville et al., 2011). Oxalic acid is proven to be an important agent secreted by ECM fungi involved in ectomycorrhizal fungal mineral

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weathering (Arvieu et al., 2003; Casarin et al., 2003; van Hees et al., 2006; Smits et al., 2012; Schmalenberger et al., 2015). In vitro studies have shown that ECM fungi can secrete large amounts of oxalic acid to acquire structural K and P from minerals (Paris et al., 1996; Wallander, 2000). Griffiths et al. (1994) found that colonization by the ECM fungus Gautieria monticola notably increased the amount of oxalic acid in soil. Calcium oxalate (CaOx) can accumulate in forest soils (Graustein et al., 1977) and mats of hypogeous fungi (Cromack et al., 1979) and deposition of Ca from the weathering of apatite as CaOx crystals on the hyphal surfaces of Rhizopogon sp. mycorrhizal hyphae growing from Pinus muricata seedlings has been shown in microcosm studies (Wallander et al., 2002). CaOx is one of the most insoluble by-products (Ksp = 2.32 × 10−9) of mineral weathering mediated by oxalic acid, often in the form of whewellite or weddellite (Gadd et al., 2014). Glowa et al. (2004) detected no CaOx minerals in a study of mineral composition in the ectomycorrhizosphere (the volume of soil under the influence of ECM roots, normally around 2–3 mm in scale) of hybrid spruce in the sub-boreal forest, which may imply that no significant CaOx accumulates in the

Corresponding author at: College of Life Sciences, Nanjing Normal University, Nanjing 210023, China. E-mail address: [email protected] (B. Lian).

https://doi.org/10.1016/j.chemgeo.2019.03.023 Received 6 January 2019; Received in revised form 22 March 2019; Accepted 25 March 2019 Available online 28 March 2019 0009-2541/ © 2019 Elsevier B.V. All rights reserved.

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ectomycorrhizosphere in forests. In contrast, CaOx encrustation is often observed within cortical cells of plant fine-roots rather than on surfaces of mycelia and the mycorrhizal mantle (Gonzalez et al., 2009; Pylro et al., 2013). In nature, spontaneous oxidation of CaOx is thermodynamically unlikely due to the high activation energy required and microbiallymediated degradation is the main mechanism of CaOx removal from most soils, which could explain the absence of oxalate in paleosols as well as in the geological sedimentary record (Verrecchia et al., 2006). Bacteria belonging to diverse taxonomical groups, able to utilize oxalate as a sole carbon, electron and energy source, are described as oxalotrophic bacteria (OxB) (Sahin, 2003). According to the literature, OxB are restricted to three phyla, namely Actinobacteria, Firmicutes and Proteobacteria (Sahin, 2003; Hervé et al., 2016). ECM fungi represent the nutrient exchange link between plant and soil, and degradation of oxalate at this interface, not only helps to eliminate the barrier of insoluble oxalate on the surface of minerals and mycelia, but can also improve the re-uptake of metal nutrients immobilized in soil oxalate. Despite their potential importance, there are few reports about OxB in the ectomycorrhizosphere, except for that of Knutson et al. (1980), who isolated and identified OxB from ectomycorrhizas of Douglas-fir (Pseudotsuga menziesii) using a culture-dependent method. ECM fungal-bacterial interactions are widespread and crucial in ecosystem functioning (Johansson et al., 2004; Bonfante and Anca, 2009). For example, ECM fungi can select fluorescent pseudomonads, which function to promote hyphal growth and ectomycorrhization, as well as mobilizing nutrients from soil minerals (Frey et al., 1997; Uroz et al., 2007; Frey-Klett et al., 2010). To date, the community structure and ecological function of OxB in the ectomycorrhizosphere are still a black box. However, the finding of formyl-CoA transferase, encoded by the frc gene, involved in the oxidation of oxalate (Sidhu et al., 1997; Svedružić et al., 2005) and the development of specific primers targeting the frc gene, designed by Khammar et al. (2009), make studies of quantification and diversity of OxB possible. The biodegradation of oxalates is an important component of the biogeochemical cycles of different metals (e.g. calcium, iron, magnesium, zinc etc.) and of carbon (Gadd et al., 2014). Large amounts of oxalate produced by both plants and fungi can be used as the sole carbon source by OxB and converted into carbonate (such as calcite), via the oxalate-carbonate pathway (OCP, Eq. (1)) which is well-studied in the bulk soil of oxalogenic trees (Braissant et al., 2002; Cailleau et al., 2005; Verrecchia et al., 2006; Cailleau et al., 2011; Martin et al., 2012).

CaC2 O4 + 1/2O2

CaCO3 + CO2

(1)

In the OCP, the oxidation of oxalate is associated with a strong local alkalinization and accumulation of calcite in carbonate-poor soils, which may also improve soil properties. Many ECM fungi are prolific producers of oxalic acid (Lapeyrie, 1988; Wallander, 2000; Rineau et al., 2008), however, whether the OCP is functional in the ectomycorrhizosphere, it is still unknown. In the present study, we determined contents of oxalate and carbonate in the ectomycorrhizospheres of Pinus massoniana and Quercus serrata. Quantitative PCR (qPCR) and high-throughput sequencing were used to determine the abundance and community structure of OxB in the ectomycorrhizospheres of these two plants. In addition, we isolated an oxalotrophic bacterium, NJ10, from the ectomycorrhizosphere of Pinus massoniana and determined its characteristics of degradation of soluble and insoluble oxalate under solid and liquid culture. The central aim was to identify the diverse OxB taxa and their possible role in the OCP in the ectomycorrhizosphere, and to improve our understanding of their ecological functions and potential to immobilize carbon. 2. Materials and methods 2.1. Study area and sampling The study area was situated within mixed forests, mainly dominated by P. massoniana, Q. serrata, Quercus acutissima, Quercus fabri and Liquidambar formosana, and located on the campus of Nanjing Normal University in Nanjing, China. At all locations the soils are brown, acidic and on siliceous parent rock. We collected samples from the soil adhering to ECM roots of P. massoniana and Q. serrata, the most abundant ECM trees in the forest, and the corresponding bulk soils. In this study, soil adhering to ectomycorrhizas was identified as ectomycorrhizosphere soil. In total, five plants of each species were selected and each plant was at least 200 m apart. The selected trees form diverse types of ectomycorrhizas (Fig. 1). Three different sub-samples of mycorrhizosphere soil collected from each tree were homogenized and combined into one sample. The bulk soil samples were at least 20 cm distant from the plots of selected ECM roots, and the soil cores (5 cm × 5 cm) at depth 0–10 cm were collected after removing surficial debris of plants and animals. In total, 10 mycorrhizosphere soil samples and 10 bulk soil samples were collected. All soil samples were transported back to the laboratory on ice. Each soil sample

Fig. 1. Ectomycorrhizal roots of P. massoniana and Q. serrata visualized using a dissecting microscope. Morphotypes of P. massoniana ectomycorrhizas were dichotomous with ochre sheath (A, C), dichotomous with whitish hairy extramatrical hyphae (B), monopodial-pinnate with abundant whitish rhizomorphs (D) or irregularly dichotomous with yellowish tips (E). Morphotypes of Q. serrata ectomycorrhizas were monopodial-pyramidal with yellowish brown surface (F), irregularly and tortuously pinnate with golden mantle (G) or solitary unramified (H). Bar scales = 1 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 55

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was sieved through a 2 mm mesh and separated into two sub-samples. One was stored in a −80 °C freezer for DNA extraction, and the other was air dried for analysis of soil properties.

2.4. Isolation, identification and growth characteristics of OxB A plating method was used to isolate OxB. The isolation medium used was modified Schlegel solid medium supplemented with CaOx as the sole carbon source (Aragno and Schlegel, 1992). The medium was poured in two layers. The first layer (c. 20 mL) was prepared from solutions A and B (ratio 100:1) without the addition of the carbon source. The upper layer (c. 5 mL) was prepared using the same solutions supplemented with CaOx (25 mM, as the sole carbon source). The composition of solution A was: Na2HPO4·12H2O 25 mM; KH2PO4 11 mM; NH4Cl 19 mM; MgSO4·7H2O 0.8 mM; trace element solution 1 mL/L. The final pH of solution A was adjusted to 7.2. The composition of solution B was (in 250 mL): Fe(NH4) citrate 500 mM. Solid medium was prepared by adding 2% agar. Bacterial colonies showing dissolution halos were selected and transferred to fresh Schlegel medium for further purification. Finally, we selected the isolate (NJ10) with the largest dissolution halo for subsequent studies, isolated from the ectomycorrhizosphere of P. massoniana. The NJ10 bacteria were observed using an S-3400 N scanning electron microscope (Hitachi, Japan). The isolate NJ10 was incubated on solid Schlegel medium prior to DNA extraction using the Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech, China). PCR amplification of the 16S rRNA gene was carried out using the universal primers 27F (5′-AGAGTTTGATCCTGGCT CAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR mixtures used for bacterial DNA amplification contained (in 50 μL of final volume): 25 μL 2× Phanta Max Master Mix (Vazmye Biotech, China), 20 μM of each primer and 1 μL DNA template. PCR was performed with initial denaturation at 95 °C for 3 min followed by 30 cycles with denaturation for 15 s at 95 °C, annealing for 15 s at 58 °C, extension for 80 s at 72 °C, and complete extension at 72 °C for 5 min. The PCR product was sent for Sanger sequencing at Sangon Biotech, China. The search for similarity against sequences from the 16S rRNA gene was performed using BLASTn (Altschul et al., 1997) comparing the query sequence at NCBI. The 16S rRNA gene sequence from the isolate was deposited in GenBank (accession number: MH145452).

2.2. Assay of soil properties Soil pH was determined at a 1:2.5 (w/v) soil-to-solution ratio with 1 M KCl solution. The moisture content was determined gravimetrically, in an aliquot of moist soil dried at 105 °C for 24 h. The oxalate content of soil samples was determined using a colorimetric method. The pretreatment of soil samples were performed using the method of Cailleau et al. (2014). Briefly, 25 mL of 1 M HCl were added to 10 g of dried sample for an overnight reaction on a reciprocating shaker, after shaking, pH was adjusted to 2 with 6 M HCl. The samples were centrifuged at 6000 rpm for 10 min and the supernatant was analyzed using the method of Jin et al. (2007). Measurements were carried out using a microplate reader SpectraMax M2 (Molecular Devices, USA) at 660 nm. Final results are expressed in mg calcium oxalate per kg of air-dried soil. The carbonate content was evaluated using a back-titration method. Briefly, 0.5 M HCl was added to 5 g soil and 0.5 M NaOH was used to back-titrate the resulting solution using 1% phenolphthalein as an end indicator. Carbonate content is expressed in mg of calcium carbonate per g of air-dried soil. 2.3. High-throughput sequencing and analysis of the OxB community Genomic DNA was extracted using the FastDNA® SPIN Kit for soil (MP Biomedicals, USA) according to the manufacturer's instructions. The DNA extracts were sent to a commercial company (Sangon Biotech, China) for Illumina MiSeq sequencing. Specific steps were carried out as follows: the frc gene was amplified with a set of primers frc171-F (5′-CTSTAYTTCACSATGCTSAAC-3′) and frc627-R (5′-TGCTGRTCRCGYAGYTTSAC-3′) (Khammar et al., 2009). PCR amplification was performed in triplicate using a 30 μL reaction mixture containing 15 μL 2× KAPA HiFi Hot Start Ready Mix (Takara, Japan), 1 μL of each primer (10 μM), and 20 ng of template DNA. PCR was performed in an ABI 9700 thermocycler (Applied Biosystems, USA) using the following program: 1 cycle of denaturing at 95 °C for 3 min, first 5 cycles of denaturing at 95 °C for 30 s, annealing at 45 °C for 30 s, elongation at 72 °C for 30 s, then 20 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 30 s and a final extension at 72 °C for 5 min. The PCR products were checked using electrophoresis in 1% agarose gels (1% in TBE buffer). Free primers and primer dimer species in the amplicon products were removed using Agencourt® AMPure XP beads (QIAGEN, Germany). Library construction used universal Illumina adaptors and index sequences. Before sequencing, the DNA concentration of each PCR product was determined using a Qubit® 2.0 Fluorometer (Invitrogen, USA) and it was quality controlled using an Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). The amplicons from each reaction mixture were pooled in equimolar ratios based on their concentration and sequenced on an Illumina MiSeq PE300 platform. Sequencing raw data were separated by sample according to barcode sequences. Due to the Illumina MiSeq PE300 platform adopting a paired-end sequencing strategy, raw reads were merged into complete sequences using FLASH 1.2.11 software (Magoč and Salzberg, 2011). Barcode sequences were subsequently removed from each sample and sequences < 200 bp were discarded using Cutadapt v.1.14 software (http://cutadapt.readthedocs.io). The processed sequences were clustered into operational taxonomic units (OTUs) at 97% similarity using USEARCH (Edgar, 2013). A representative sequence with the highest abundance in each OTU was selected for taxonomic assignment. Taxonomic annotation of representative sequences was performed using BLASTn method (Altschul et al., 1997) in the National Center for Biotechnology Information (NCBI) non-redundant database and sequences with similarity < 90% and coverage < 90% were defined as unclassified. The sequencing raw data were deposited in the SRA (http://www.ncbi.nlm.nih.gov/sra) at NCBI (Accession number: SRR7091383).

2.5. Quantitative detection of bacteria in soil The qPCR was performed in triplicate on an ABI StepOne™ RealTime PCR system (Applied Biosystems, USA) using primer sets frc171F/frc306-R (5′-GDSAAGCCCATVCGRTC-3′) for the frc gene of OxB (Khammar et al., 2009) and 338F (5′-ACTCCTACGGGAGGCAGCAG-3′)/ 518R (5′-ATTACCGCGGCTGCTGG-3′) for the bacterial 16S rRNA gene. Each 20 μL of qPCR reaction contained 10 μL of AceQ® SYBR Green Master mix (Vazmye Biotech, China), 0.4 μL of each primer (10 μM), and 1 μL of diluted DNA template and adjusted with RNase-free ddH2O to the final volume. Amplification of the frc and 16S rRNA genes was conducted using an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of 10 s at 95 °C, 30 s at 60 °C. Melting curve analysis was carried out to verify amplicon specificity. A negative control without DNA template was used in all of the qPCR amplifications. Standard curves were generated based on decimally diluted standard frc fragments from the oxalatrophic bacterium NJ10 and 16S rRNA gene from Escherichia coli DH5α, respectively, cloned into pEASY®-Blunt Zero vector (TransGen Biotech, China). The amplification efficiencies of frc and 16S rRNA were 93.40% and 108.58%, and the R2 values of the standard curves were 0.9945 and 0.9902, respectively. 2.6. Characteristics of biodegradation of oxalate by OxB Since the isolate NJ10 was unable to grow in liquid Schlegel medium, we chose modified King's B medium to determine the ability of NJ10 to degrade oxalate. The composition of King's B medium is as follows: peptone 5.0 g/L, potassium or calcium oxalate 25 mM, K2HPO4 11 mM, MgSO4·7H2O 6 mM, pH 7.2. The isolate NJ10 was incubated on solid Schlegel medium at 30 °C for 5 days for sporation. The spores were collected using sterilized ddH2O, washed three times, and diluted. 56

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Erlenmeyer flasks (250 mL) containing 100 mL of medium with 108 spores were incubated at 30 °C and 150 rpm in an orbital shaker for 6 days. The pH and oxalate content in the medium were determined at intervals of 12 h and the by-products on the sixth day were collected for mineralogical analysis. Mineral particles formed on Schlegel medium were extracted from the agar after removal of the colony and gently melting the agar with 100 °C ddH2O in a beaker. Crystals were transferred to a clean beaker using a pipette and washed several times with boiled, deionized water to ensure the removal of all agar and soluble elements. A scanning electron microscope Zeiss-Supra55 (ZEISS, Germany) equipped with an energy dispersive X-ray spectrometer (EDS) and field emission transmission electron microscopy (JEOL, Japan) were used to analyze morphology and elemental composition of biominerals induced by the isolate NJ10. The structure and composition of biogenic minerals were determined using an X-ray diffractometer BTX-526 (Olympus, Japan) using Co-Kα radiation with a voltage of 30 kV and current of 300 μA (λ = 1.79 Å). In addition, soft X-ray microscope imaging and three-dimensional scanning were performed using the beamline BL07W station at the National Synchrotron Radiation Laboratory, Hefei, China.

3. Results 3.1. Soil properties The pHKCl of the mycorrhizospheric soil of P. massoniana and Q. serrata was significantly lower than that in the corresponding bulk soil (t = −0.4251, P = 0.003; t = −2.783, P = 0.024, respectively, Table 1). The oxalate content in the ectomycorrhizospheres of P. massoniana and Q. serrata was statistically significantly lower than that in the corresponding bulk soil (t = −3.493, P = 0.008; t = −3.878, P = 0.005, respectively). Although the pHKCl of the mycorrhizospheric soil was lower than that of the bulk soil, the average carbonate content was higher than that of the corresponding bulk soil. The mean contents of carbonate in the ectomycorrhizospheres of P. massoniana and Q. serrata were 19.78 ± 4.94 mg/g soil and 18.90 ± 4.87 mg/g soil, respectively. The respective mean contents of carbonate in the bulk soil of P. massoniana and Q. serrata were 12.52 ± 7.34 mg/g soil and 14.0 ± 15.04 mg/g soil, but the difference between the means was not statistically significantly with the degree of replication used in this experiment (t = 1.833, P = 0.104; t = 1.557, P = 0.158, respectively). However, the ratio of carbonate to oxalate in the ectomycorrhizospheres of P. massoniana and Q. serrata was significantly higher than that of the corresponding bulk soil (t = 2.732, P = 0.026 and t = 2.549, P = 0.034, respectively).

2.7. Statistical analysis Nonmetric multidimensional scaling (NMDS) and canonical correspondence analysis (CCA) were performed in R software, using the Vegan package. Permutational multivariate analysis of variance (PERMANOVA) tests were conducted to determine the significance of differences among groups. t-Tests and Kruskal-Wallis one-way ANOVA tests were used to determine the significance of differences in soil properties and bacterial communities and were performed in SPSS 20. Data are expressed as mean ± standard deviation.

3.2. Diversity of OxB in soil The high-throughput sequencing yielded 1,507,741 raw sequences. A total of 1,341,131 high-quality sequences was obtained and clustered into 23,972 OTUs using a 97% similarity threshold. The proportion of OTUs assigned to each taxonomic level was different: 65.17% of the OTUs were assigned to phylum level, and 58.54% to genus level, while only 26.10% of the OTUs could be classified at species level (Fig. 2A). In the ectomycorrhizospheres of P. massoniana and Q. serrata, 3762 OTUs

Table 1 pH and contents of oxalate and carbonate in ectomycorrhizosphere and bulk soil. Sample

pHKCl

Pm-EM Pm-BS Qs-EM Qs-BS

4.56 ± 0.19⁎⁎ 5.04 ± 0.16 4.46 ± 0.30⁎ 4.90 ± 0.17

Oxalate (mg/kg soil)

Carbonate (mg/g soil)

110.00 ± 7.18⁎ 127.24 ± 8.38 107.74 ± 7.55⁎ 132.22 ± 11.92

19.78 12.52 18.90 14.01

± ± ± ±

4.93 7.34 4.87 5.04

Carbonate/oxalate ratio 178.09 ± 35.84⁎ 96.48 ± 56.37 174.30 ± 35.88⁎ 108.40 ± 20.27

Pm-EM: ectomycorrhizosphere soil of P. massoniana; Pm-BS: bulk soil adjacent to P. massoniana ectomycorrhizas; Qs-EM: ectomycorrhizosphere soil of Q. serrata; QsBS: bulk soil adjacent to Q. serrata ectomycorrhizas. Statistically significant differences were identified using t-test. Data represent mean values ( ± SD) of five replicates. ⁎ P < 0.05. ⁎⁎ P < 0.01.

Fig. 2. (A) Percentage of sequences assigned to different taxa; (B) the distribution of oxalotrophic bacteria at phylum level; (C) the relative abundance of nitrogenfixing genera. Abbreviations are shown in Table 1. 57

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Table 2 α-Diversity indices of bacterial communities in ectomycorrhizosphere and bulk soil samples (n = 5). Sample

OTU

Pm-EM Pm-BS Qs-EM Qs-BS

3762 2935 2957 2962

Chao1 ± ± ± ±

397 700 492 951

4775 3703 3816 3561

± ± ± ±

513 966 609 1302

Shannon

Simpson

6.07 5.61 5.40 5.90

0.02 0.03 0.05 0.02

± ± ± ±

0.53 0.32 0.44 0.49

± ± ± ±

Coverage 0.02 0.02 0.05 0.01

0.98 0.99 0.98 0.99

± ± ± ±

0.01 0.00 0.01 0.01

Data represent mean values ( ± SD) of five replicates.

and 2957 OTUs were obtained, respectively, while 2935 OTUs and 2962 OTUs were found in the corresponding bulk soil (Table 2). Oneway Kruskal-Wallis analysis of variance showed that the Chao1, Shannon, and Simpson α-diversity indices did not differ significantly between ectomycorrhizosphere and bulk soil. The OTUs from all samples were annotated to 4 phyla and 35 genera. Acidobacteria and Proteobacteria were the dominant phyla in the tested samples (Fig. 2B). Moreover, 12 genera were nitrogen-fixing bacteria, accounting for

42.77% of the total sequences in ectomycorrhizosphere samples of P. massoniana and 60.35% in ectomycorrhizosphere sample of Q. serrata of total sequences (Fig. 2C). The heatmap (Fig. 3A) and NMDS (Fig. 3B) analyses suggested that the community composition of oxalotrophic bacteria in the ectomycorrhizosphere differed markedly from that in the bulk soil at the genus level. PERMANOVA test results based on BrayCurtis distance showed a statistically significant difference between bacterial community structure of the ectomycorrhizosphere of Q.

Fig. 3. Heatmap of the composition of OxB at genus level based on normalized abundance (A); Nonmetric multidimensional scaling (NMDS) analysis (B); CCA analysis of effects of environmental parameters on bacterial community structure (C); the ratio of environmental parameters explained on the variation of oxalotrophic bacteria community (D). Abbreviations are shown in Table 1. 58

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Fig. 4. Quantification of frc (A) and 16S rRNA (B) gene copies in soil and the proportion of OxB in total soil bacteria (C). Data represent mean values ( ± SD) of five replicates. Statistically significant differences were identified using t-test. Abbreviations are shown in Table 1. *P < 0.05. **P < 0.01.

serrata and the corresponding bulk soil (F = 1.632, P = 0.032), but there was no statistically significant difference between the ectomycorrhizosphere of P. massoniana and the corresponding bulk soil (F = 1.443, P = 0.104). CCA analysis was used to illustrate the effects of environmental parameters (soil pH, carbonate content, and oxalate content) on the community structure of OxB (Fig. 3C). Mantel tests indicated that soil pH was significantly related to changes in the bacterial community (r = 0.202, P = 0.047), but not content of oxalate (r = 0.132, P = 0.115) or carbonate (r = −0.033, P = 0.560). Soil parameters determined in this study only explained 18.02% of the total variance in bacterial community structure (Fig. 3D), suggesting that this was also affected by other factors.

3.5. Characteristics of oxalate degradation in solid and liquid media Isolate NJ10 was able to form clear dissolution halos and visible radial crystalline deposits around the colony on solid Schlegel medium during incubation for 15 days (Fig. 6). At the micrometer scale, these deposits consisted of long columnar crystals, composed of carbon, oxygen, calcium and phosphorus (Fig. 7A). XRD analysis showed that the deposits formed on the medium were mainly composed of phosphate and carbonate minerals, of which octacalcium phosphate pentahydrate, and brushite, accounted for 80.5%, and the proportion of calcite was 19.5% (Fig. 7B). Isolate NJ10 was unable to grow in liquid Schlegel medium, and other media were tried, including Gauze's, Luria-Bertani and King's B medium. Only King's B medium was suitable for the growth of NJ10 under oxalate-containing liquid conditions. King's B medium was therefore used to determine the degradation rate of soluble (potassium oxalate) and insoluble (CaOx) oxalate. The results showed the degradation rate of potassium oxalate by NJ10 was significantly higher than that of CaOx (Fig. 8). The degraded amount of potassium oxalate increased dramatically after 96 h, with complete degradation after 144 h culture (Fig. 8A). However, the degradation of CaOx was slightly slower and the amount of degradation was only 87.84 ± 0.15% after incubation for 144 h (Fig. 8B). The pH values of media in degrading potassium oxalate and CaOx both increased gradually, reaching 9.52 ± 0.01 and 9.15 ± 0.03 at 144 h, respectively. Isolate NJ10 formed micro-scale, cuboidal particles in the process of degrading CaOx, mainly composed of carbon, oxygen, magnesium, calcium and phosphorus, tested by EDS (Fig. 7C). The XRD results showed small amounts of dolomite (7.6 ± 3.6%, Fig. 7D) in the sediments of the CaOx-containing medium after a 144-h incubation, suggesting that NJ10 can convert CaOx into carbonate precipitates.

3.3. Gene copies of frc and 16S rRNA in soil The abundance of the frc gene in the ectomycorrhizosphere of P. massoniana, estimated using qPCR, was 2.60 × 108 ± 1.45 × 108 copies/ g oven-dried soil, significantly (t = 3.157, P = 0.013) higher than that in the corresponding bulk soil (5.34 × 107 ± 1.64 × 106 gene copies, Fig. 4A). The abundance of the frc gene in the ectomycorrhizosphere of Q. serrata was 5.03 × 108 ± 2.11 × 108 copies/g oven-dried soil, which was also significantly (t = 4.886, P = 0.001) higher than that of the corresponding bulk soil (3.97 × 107 ± 1.95 × 106 gene copies, Fig. 4A). The qPCR also showed that the abundance of the 16S rRNA gene in the ectomycorrhizospheres of P. massoniana (1.53 × 1010 ± 5.50 × 109 gene copies) and Q. serrata (1.87 × 1010 ± 8.12 × 109 gene copies) was significantly (t = 3.273, P = 0.011; t = 3.073, P = 0.015) higher than that in the corresponding bulk soils (8.19 × 109 ± 1.13 × 109 gene copies, 9.17 × 109 ± 1.99 × 109 gene copies, respectively, Fig. 4B). The relative abundance of OxB compared to total soil bacteria in the ectomycorrhizospheres of P. massoniana and Q. serrata was respectively 1.64 ± 0.34% and 2.86 ± 1.20%, significantly (t = 5.212, P = 0.001; t = 4.096, P = 0.003) higher than that in the corresponding bulk soil (Fig. 4C).

4. Discussion The production of oxalic acid by ECM fungi has a positive impact on mineral weathering, especially promoting the dissolution of phosphorus from apatite (Wallander, 2000; Landeweert et al., 2001; Arvieu et al., 2003). CaOx precipitates are frequently observed on the surfaces of minerals and hyphae in microcosm experiments (Wallander, 2000; Casarin et al., 2003) or on ectomycorrhizas in field (Cromack et al., 1979; Griffiths et al., 1994). However, some studies also documented that CaOx is rarely observed on the surfaces of mycelia or ECM roots in field (Glowa et al., 2004; Gonzalez et al., 2009; Pylro et al., 2013), indicating that oxalate may be degraded. A similar phenomenon was also found in this study. Our results showed the contents of oxalate in the ectomycorrhizospheres of P. massoniana and Q. serrata were significantly lower than those in the corresponding bulk soil (Table 1). ECM fungi not only significantly increase the total numbers of bacteria

3.4. Isolation and identification of OxB An isolate NJ10, with the highest efficiency of oxalate degradation among 15 isolates, was identified and selected for the subsequent experiments (Fig. 5A). Electron scanning microscopy showed the spiral spore filaments of the isolate NJ10 strain had a spore diameter of approximately 0.61 to 0.80 μm (Fig. 5B). The taxonomic category of NJ10 was identified by sequencing its 16S rRNA gene and closest match in GenBank was Streptomyces sp. HP1 (KX710212) using BLASTN at NCBI. Phylogenetic analysis showed NJ10 was significantly different from known Streptomyces species (Fig. 5C), suggesting that this isolate may be a new species within the genus Streptomyces. 59

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Fig. 5. (A) Colony of strain NJ10 formed on Schlegel medium at 30 °C for 5 days; (B) scanning electron micrograph of the isolate; (C) phylogenetic tree based on 16S rRNA gene sequences using Mega 7.0 software, Neighbor-joining method, showing the phylogenetic relationship of Streptomycetes sp. NJ10 with respect to related species. The 16S rRNA gene of Kitasatospora aureofaciens DM-1, a species in the family Streptomycetaceae, was used as an outgroup. The scale bar indicates 0.005 substitutions per nucleotide position.

in the ectomycorrhizosphere, but also increase the proportion of specific, functional bacteria. In the present study, qPCR analysis showed that the proportions of OxB in the ectomycorrhizospheres of these two plant species were significantly higher than those in the bulk soil (Fig. 4), indicating that OxB play a key role in the degradation of oxalate in the ectomycorrhizosphere. The high-throughput sequencing of the formyl-CoA transferase gene (frc) was conducted using specific primers and results signal an abundant community of OxB in the ectomycorrhizosphere soil. Reviewed literature suggests that OxB are restricted to Gram-negative bacteria in the Proteobacteria and Grampositive bacteria in the Actinobacteria and Firmicutes (Sahin, 2003; Hervé et al., 2016). Nonetheless, Candidatus Solibacter usitatus in the Acidobacteria and Gemmatirosa kalamazoonesis in the Gemmatimonadetes, detected in the bulk soil of Q. serrata, also contain frc gene found in this study (Fig. 2B), extending the observed taxonomic distribution of OxB. ECM fungi not only increase the abundance of OxB in the ectomycorrhizosphere, but can also enrich the specific community of OxB, such as the community structure of OxB in the ectomycorrhizosphere of

Q. serrata was distinct from that in the corresponding bulk soil (Fig. 3A, B). In this study, soil pH had a significant influence on oxalate decomposing bacterial community structure but not oxalate or carbonate content and these three factors together only explained a low proportion of the variation (18.02%, Fig. 3C, D), indicating that the OxB community was also affected by other factors, such as ECM fungi and host plants. The annotated rate of frc genes obtained by sequencing at the phylum level was only 65.1%, and 58.54% at the genus level (Fig. 2A), suggesting that there are still large numbers of unknown OxB taxa to be identified. The bacterially-mediated OCP in the biodegradation of oxalate has received extensive attention (Aragno and Schlegel, 1992; Verrecchia et al., 2006; Cailleau et al., 2011), and is considered to be a promising biological approach for improving underground sequestration of atmospheric CO2. Due to the limited number of studies focused on OxB in the ectomycorrhizosphere (Knutson et al., 1980), a number of questions regarding the OCP in this special niche remain to be addressed. Our results provide some support for the idea that the OCP is active in the

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Fig. 6. Light micrographs of crystalline precipitates formed on solid Schlegel medium after incubation for 15-days (A, B) and morphology of crystals extracted from agar (C, D).

Fig. 7. A) SEM-EDS and B) XRD profiles of crystals from Schlegel medium; C) SEM-EDS and D) XRD profiles of crystals from CaOx-containing liquid and King's B medium, respectively.

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environment. The results of high-resolution transmission electron microscope (HRTEM) showed that the biogenic mineral aggregates at the nanometer-scale were well-developed lamellar crystals (Fig. S1). Field and laboratory results (Table 1, Fig. 7) indicated that the OCP may also function in the ectomycorrhizosphere, suggesting that these taxa make a functional contribution to long-term carbon sequestration by transforming oxalate into carbonate minerals, such as calcite. The mycorrhizogenic calcite can ultimately form calcretes under certain conditions (Wright et al., 1995; Verboom and Pate, 2006), resulting in a much longer residence time in soils than organic substrates. Although ectomycorrhizosphere soils often are acidic, CaOx are still insoluble based on its property, and biogenic calcium carbonate can still accumulate duo to its stability in a wild range of pH values (Liu et al., 2018). Oxalic acid in soil also is a potential pathogenicity factor for phytopathogens, suppressing the oxidative burst of the host plant and weakening Ca2+-dependent defense responses by chelating plant cell wall Ca2+ (Cessna et al., 2000; Williams et al., 2011). Therefore, OxB in the ectomycorrhizosphere may also play a role as plant growth-promoting rhizobacteria, functioning in biological control. This suggests that ECM fungi can regulate multifunctional bacteria by secreting fungal-derived metabolites (Duponnois and Kisa, 2006; Uroz et al., 2007). In this study, our results showed that about one third of genera were nitrogen-fixing bacteria and the relative abundance of these genera was 42.77% to 60.35% (Fig. 2C), further implying that ECM can enrich bacteria with multiple ecological functions. For example, the Streptomyces sp. NJ10 isolated from the ectomycorrhizosphere may act as a mycorrhiza helper bacterium, possibly promoting mycelial growth of ECM fungi and the mycorrhization of plant roots by influencing the gene expression of the ECM fungi (Schrey et al., 2005). OxB can move and disperse on fungal hyphae (Bravo et al., 2013), which may function as “fungal highways” (Wong and Griffin, 1976; Wick et al., 2007). ECM fungi may recruit multifunctional bacteria from surrounding soil through fungal highways. However, the existence of these types of functional interaction in the case of the particular OxB investigated in the present study remains to be tested in further experiments. Based on the results above, interactions between plants, ECM fungi and OxB may be a key mechanism for maintaining ectomycorrhizosphere stability, and improving nitrogen supply as shown in Fig. 9. Moreover, the alkalinization and carbonate formation associated with oxalate biodegradation not only improves the soil microenvironment, but also plays an important role in the soil carbon cycle, through promoting long-term sequestration of carbon derived from the atmosphere and photosynthetically fixed by the plant hosts that supply it to their fungal symbionts.

Fig. 8. Degradation characteristics of potassium oxalate (A) and calcium oxalate (B) by Streptomycetes sp. NJ10. Data points represent mean values ( ± SD) of three biological replicates.

ectomycorrhizosphere since there was a significant increase in the ratio of carbonate to oxalate, compared to the bulk soil (Table 1). In order to obtain a deeper understanding of the role of OxB from the ectomycorrhizosphere in the OCP, we isolated an oxalotrophic bacterium NJ10, identified as Streptomyces by 16S rRNA sequencing (Fig. 5). When potassium oxalate or CaOx was used as the sole carbon source, the final pH of the culture medium was alkaline after incubating Streptomycetes NJ10 for 6 days (Fig. 8). This may be an important way to regulate the acidity of microenvironments (Cromack et al., 1977), especially in the strongly acidic ectomycorrhizosphere (Table 1). The carbonate mineral (dolomite) was observed in King's B liquid medium with CaOx as sole carbon source after a 6-day incubation (Fig. 7B) and the alkalinization of the medium may be a key factor in facilitating precipitation of carbonate (Fig. 8B). Braissant et al. (2002) found the OxB Ralstonia eutropha and Xanthobacter autotrophicus can form calcium phosphate carbonate on the solid Schlegel bilayer medium. We also found radial, long, cubic crystals induced by Streptomyces sp. NJ10 on the Schlegel bilayer medium (Fig. 6), composed of octacalcium phosphate hexahydrate (54.6 ± 1.2%), brushite (25.9 ± 3.7%) and calcite (19.5 ± 3.4%) as determined by EDS and XRD (Fig. 7A, B). This composition of crystals is not surprising because of the high level of phosphorus in Schlegel medium. The calcium ion released from CaOx easily forms secondary precipitates with phosphate in the alkaline

5. Conclusions This study demonstrates that the content of oxalate in the ectomycorrhizosphere of P. massoniana and Q. serrata was significantly lower than that of bulk soil. Using qPCR and high-throughput sequencing, we revealed, for the first time, the elevated abundance and altered community structure of OxB in ectomycorrhizosphere compared to bulk soil. Our results also clearly showed that this physiological group in ectomycorrhizosphere soil differed from that in bulk soil and suggested that ECM fungi can select special multifunctional bacteria that may benefit the mycorrhizal associations, by mechanisms such as fixing nitrogen, promoting mycorrhizal formation and resisting plant pathogens. Our results obtained in situ and in vitro, suggest that the bacterial oxalate-carbonate pathway demonstrated in the ectomycorrhizospheres of P. massoniana and Q. serrata, has a significant potential capacity for sequestering carbon ultimately derived from the atmosphere. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.chemgeo.2019.03.023.

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Fig. 9. Simplified schematic diagram of plant-ectomycorrhiza-oxalotrophic bacteria interactions. ECM fungi and trees uptake nutrients released from soil minerals by secreting oxalic acid; plants enable the allocation of photosynthetic products to ECM fungi, accelerating the growth and reproduction of these fungi, and in particular guaranteeing the large amount of carbon needed for fruit-body formation; oxalate accumulated in the ectomycorrhizosphere forms encrustations on the surface of minerals and mycelia that inhibit mineral weathering and nutrient uptake, and may also increase plant susceptibility; ECM fungi recruit multifunctional OxB to degrade oxalate and mitigate the abiotic stress caused by oxalic acid. CaOx, calcium oxalate; OxB, oxalotrophic bacteria.

Acknowledgements

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