Abundance, Diversity, and Structure of Geobacteraceae Community in Paddy Soil Under Long-Term Fertilization Practices

Applied Soil Ecology 153 (2020) 103577

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Applied Soil Ecology

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Abundance, diversity, and structure of Geobacteraceae community in paddy soil under long-term fertilization practices T ⁎ Xiaomin Lia,b, Longjun Dinga, , Xiaoming Lia, Yongguan Zhua,b,c a State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China b University of Chinese Academy of Sciences, Beijing 100049, China c Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China

ARTICLE INFO ABSTRACT

Keywords: Geobacteraceae is an important dissimilatory Fe(III) reducer that affects the cycles of multiple elements. Geobacteraceae However, the way in which different long-term fertilization regimes influence the Geobacteraceae community in Paddy soil paddy soils remains unknown. Therefore, the objective of this study was to explore the responses of Fertilization Geobacteraceae community in paddy soil to long-term chemical (nitrogen, phosphorus, and potassium) and/or Soil properties organic (manure) fertilization practices. Illumina sequencing results showed that the species richness and di- Iron reduction versity of Geobacteraceae community were not significantly changed by fertilizer treatments. Geobacteraceae in the treatments consisted of Geobacter (accounting for 90%–95% of total reads) and Geothermobacter genera (5%–10%), and all fertilizer treatments induced a significant (P < 0.05) decline in Geobacter and a marked enrichment of Geothermobacter. The taxonomic (based on Bray-Curtis distance) and phylogenetic structures (weighted-UniFrac distance) of the Geobacteraceae communities in all fertilizer treatments were clearly different from those in the non-fertilizer treatment; however, there were no significant changes among the different fertilization treatments. The variations in the Geobacteraceae community induced by long-term fertilization were mainly determined by changes in soil pH, total carbon, and total nitrogen. These findings provide an insight into the biogeochemistry of paddy soils and pave the way for harnessing the microbiome to improve soil fertility and environmental quality.

1. Introduction marine and lake sediments as well as paddy soils (Lovley et al., 2004; Bond et al., 2002; Fan et al., 2018; Yuan et al., 2016) and play a con- Dissimilatory iron reduction is a process used by microorganisms to siderable role in the biogeochemistry of elements. For example, Geo- reduce extracellular insoluble ferric iron oxides [Fe(III)] to ferrous iron bacter sulfurreducens PCA strain isolated from the surface sediment is [Fe(II)], accompanied by the oxidation of organic matter (e.g., acetate), able to oxidize acetate, coupling with elemental sulfur reduction hydrogen, or ammonium under anaerobic conditions. This microbe- (Caccavo et al., 1994). Phosphorus has a good affinity to Fe(III) oxide mediated process is central to several other biogeochemical cycles in surfaces (Norton et al., 2008) and could be released when Fe(III) mi- various anoxic environments, which, for instance, significantly influ- nerals are reduced by Geobacter genus (Wang et al., 2016). In addition, ence the cycles of carbon (C), nitrogen (N), and phosphorus (P) (Li Geothermobacter, Geoalkalibacter, and Geopsychrobacter also reduce Fe et al., 2012). The most well-known dissimilatory Fe(III) reducers belong (III) oxides, with acetate and amino acids acting as electron donors to in the Geobacteraceae family within the phylum Proteobacteria (Kashefi et al., 2003; Zavarzina et al., 2006; Holmes et al., 2004). (Lovley et al., 2004). This family has thus far been reported to en- Paddy soil is a transitional ecosystem between terrestrial and compass four genera, including Geobacter, Geothermobacter, Geoalk- aquatic ecosystems that is rich in Fe(III) oxides. The redox potential alibacter, and Geopsychrobacter (https://www.arb-silva.de/), with all gradient resulted from frequent alternate cycles of drying and wetting, being involved in dissimilatory Fe(III) reduction (Lovley et al., 2011; as well as an abundance of Fe(III), making paddy soils a potential Tully et al., 2017; Zavarzina et al., 2006; Holmes et al., 2004). These hotspot for dissimilatory iron reduction (Ding et al., 2014). It has been genera are widely distributed in many anaerobic environments, such as previously found that the Geobacter species play a major role in Fe(III)

⁎ Corresponding author at: State Key Lab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road, No.18, Haidian District, Beijing 100085, China. E-mail address: [email protected] (L. Ding). https://doi.org/10.1016/j.apsoil.2020.103577 Received 10 September 2019; Received in revised form 23 February 2020; Accepted 2 March 2020 Available online 08 March 2020 0929-1393/ © 2020 Elsevier B.V. All rights reserved. X. Li, et al. Applied Soil Ecology 153 (2020) 103577 reduction coupled to acetate or ammonium oxidation in paddy soils plot in April (early rice) and July (late rice) every year. The rice (Ding et al., 2015; Nercessian et al., 2012; Li et al., 2019). Thus, it seedlings were planted at a spacing of 20 cm × 20 cm (675 plants) per seems reasonable to expect that dissimilatory iron reduction mediated plot. Then, the field was flooded with 3–5 cm height of water for about by the Geobacteraceae family may influence the rice yields by affecting 90 days, and subjected to two-week drainage before harvest at July the biogeochemical cycles of nutrient elements. Therefore, in- (early rice) and October (late rice). During each rice growth period, vestigating the diversity and composition of the Geobacteraceae com- weeding and pest control were performed only once in June (early rice) munities found in paddy soils could provide valuable information for or September (late rice) every year. improving the soil fertility and the productivity of paddy soils. Fertilization is a universal and efficient way to improve rice yields. 2.3. Soil sampling and analyses Chemical and organic fertilization could elevate the soil nutrient levels and may affect the microbial communities found in the soil. For in- In late Aug 2017 (at elongation stage of rice), all plots were sampled stance, Zhong et al. (2010) found that increased levels of total nitrogen independently. At that time point, the field had not been subjected to and available phosphorus under long-term chemical fertilization sig- agronomic interventions such as weeding and pest control for nearly a nificantly influenced the microbial functional diversity in paddy soils month prior to sampling. For each plot five soil cores were collected (Zhong et al., 2010). The continuous input of organic and/or inorganic from the surface layer (0 to 15 cm in depth) by soil auger (diameter fertilizers could affect the overall bacterial community compositions in 5 cm) and completely mixed. Soil samples were then sealed in sterile paddy soils, which is attributed to changes in the levels of organic C, plastic bags and transported to the lab on ice within 24 h. alkali-hydrolyzable nitrogen, and available phosphorus in the soil (Cui Each soil sample was divided into three portions: one was air-dried et al., 2018). The community of dissimilatory iron-reducing bacteria and grounded through a 2-mm sieve for analyses of soil physicochem- was previously found to be significantly altered by long-term nitrogen ical properties in 2 weeks; the second samples were stored at 4 °C for fertilization, possibly due to the alterations in the total C, N, and about 1 month until soil iron speciation analysis; the remaining samples amorphous Fe(III) oxide in paddy soils (Ding et al., 2015). Nevertheless, were stored at −80 °C for about 1 month until molecular analysis. Soil the effects of long-term organic and/or chemical fertilization on the pH was analyzed at a dry soil to ultrapure water ratio of 1:2.5 (w/v) composition and diversity of Geobacteraceae community in paddy soils using a pH meter (FE20; Mettler Toledo, Zurich, Switzerland). Soil total have yet to be fully elucidated. Here, we hypothesize that the Geo- carbon (Total C) and total nitrogen (Total N) were measured using an bacteraceae community shifts due to long-term input of different fer- element analyzer (Vario EL III, Elementar, Germany). Soil organic + tilizers in paddy soils. matter (OM) and ammonium (NH4 -N) were determined by colori- The objectives of the present study were to investigate the changes metric methods of absorbance at 590 nm (A590) and 660 nm (A660), of diversity and composition of Geobacteraceae family in paddy soils respectively (Lu, 1999). Soil dissolved organic C (DOC) and dissolved under long-term chemical and/or organic fertilization practices from total N (DTN) were extracted at a soil to water ratio of 1:5 (w/v) and Southern China, and reveal the main factors influencing the structures determined by a TOC analyzer (LiquicTOC, Elementar, Germany). Soil of Geobacteraceae family in paddy soils. electrical conductivity was determined by a portable conductivity meter (MP521, China). The 2-mm sieved soil sample (0.25 g) was di-

2. Materials and methods gested with aqua regia (3HCl:HNO3, v/v) and condensed by perchloric acid (HClO4). The detailed digestion procedure was described in sup- 2.1. Site description plementary methods. Then the digested soils were analyzed using an inductively coupled plasma optical emission spectrometer (ICP-OES, The site of long-term different fertilization experiments was located Optimal 2000 DV; Perkin-Elmer, USA) for soil total iron (Total Fe), at the Qiyang Experimental Station for the Red Soil Eco-environment of manganese (Mn), aluminum (Al), total phosphorus (Total P), and total Chinese Academy of Agriculture Sciences, Yongzhou city, Hunan potassium (Total K) (Naozuka et al., 2011). Soil ferrous iron [Fe(II)] Province in China (26°45′N and 111°52′E, 120 m above sea level). This and microbial reducible ferric iron [Fe(III)] were analyzed using fresh region has a humid subtropical monsoon moist climate with an annual soil based on the methods proposed by Lovley and Phillips (1987). The mean temperature of 18 °C and annual mean precipitation of 1255 mm. basic properties of soil samples are shown in Table 1. In addition, it has 300 frost-free days and 1610 cumulative sunshine hours per year. The paddy soil in this station is developed from qua- 2.4. DNA extraction, quantitative PCR and Illumina sequencing ternary red clay and classified as a red soil (Ferralic Cambisol) (FAO, 1985). Genomic DNA was extracted from 0.5 g of fresh soil sample using the FastDNA Spin Kit (MP Biomedicals, USA). The quality and con- 2.2. Experimental design centration of the DNA were assessed using a NanoDrop ND-2000 spectrophotometer (NanoDrop Co., USA). The long-term fertilization experiment was started since 1982. The For quantitative PCR (qPCR), a primer set of Geo494F (5′-AGG AAG cropping system was double-cropped rice (Oryza sativa subsp. xian) and CAC CGG CTA ACT CC-3′) and Geo825R (5′-TAC CCG CRA CAC CTA winter fallow. Four treatments were selected in this study: (i) a non- GT-3′) was used to quantify the copy numbers of the 16S rRNA genes of fertilizer treatment (CK); (ii) application of composted cattle manure the Geobacteraceae family (denoted as “absolute abundance” of (M); (iii) application of chemical nitrogen, phosphorus, and potassium Geobacteraceae) (Holmes et al., 2002; Yuan et al., 2016). The universal fertilizers (NPK); and (iv) application of M combined with NPK primer set of BACT 1369F (5′-GGG GTG CGG TCY TTN ARY TC-3′) and (MNPK). The chemical fertilizer (NPK) was added in the form of urea PROK 1492R (5′-ACG GCT ACC TTG TTA CGA CTT-3′) and a probe TM (N, 46%), calcium superphosphate (P, 1.9%), and kalium chloratum (K, 1389F (5′-CTT GTA CAC ACC GCC CGT C-3′) were used to evaluate the 50%). The concentrations of total organic C, total N, P and K in com- bacterial 16S rRNA gene copy number in paddy soils (Zhang et al., − − − posted cattle manure are 397 g kg 1, 3.2 g kg 1, 1.1 g kg 1, and 2015). The ratio of the copy number of Geobacteraceae to that of the − 1.2 g kg 1, respectively. Each treatment had triplicate plots which were bacteria was denoted as the “normalized abundance” of Geobacter- randomly arranged in the field. Each plot (15 m × 1.8 m) was sepa- aceae. qPCR tests for Geobacteraceae and bacterial 16S rRNA genes rated by a 0.6 m deep cement block. The seasonal amounts of different were conducted using a real-time PCR instrument (LightCycler 480II; fertilization treatments are given in the Table S1. All fertilizers were Roche Company, Switzerland). The detailed amplification and thermal added as basal application in each plot before rice seedling trans- cycling profiles are shown in Table S2. Meanwhile, negative control planting. About 30 days-old rice seedlings were transplanted in each (template DNA-free) was performed in the same way during the qPCR

2 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

Table 1 structural diﬀerences among treatments was calculated using the ape † Soil characteristics in the four long-term fertilization treatments . package in R (Legendre and Gallagher, 2001). Pairwise comparisons of

Treatments CK M NPK MNPK the Spearman correlation between the soil properties and Geobacter- aceae absolute/normalized abundances were calculated and visualized ‡ pH 5.73c 5.53b 5.55b 5.43a using the corrplot package in R (Wei et al., 2017). Mantel tests were − Total C (g kg 1) 15.5a 20.3b 16.7a 20.8b − performed to determine the relationship between the soil properties Total N (g kg 1) 1.77a 2.19b 1.89a 2.23b − Organic matter (g kg 1) 29.6a 40.2a 33.3a 38.0a and Geobacteraceae taxonomic/phylogenetic dissimilarity using the − Dissolved organic C (mg kg 1) 48.1a 100b 69.3a 154c vegan package in R (Oksanen et al., 2013). The relative contribution of − Dissolved total N (mg kg 1) 29.9a 27.3a 30.7a 33.6a each significant soil factor on the structural variations of the Geo- + −1 NH4 -N (mg kg ) 28.0a 30.1a 35.8a 75.7b − bacteraceae community was calculated based on variance partition Electrical conductivity (μScm 1) 86.5a 100.9b 94.0ab 128.6c − analysis (VPA) using the capscale function of the vegan package in R Total Fe (g kg 1) 41.8a 41.2a 40.7a 41.8a − Fe(II) (g kg 1) 2.82a 2.63a 2.91a 5.88a (McArdle and Anderson, 2001; Anderson and Willis, 2003). The sig- − Fe(III) (g kg 1) 1.48a 2.64a 1.33a 7.93b nificantly changed OTUs between the CK and fertilizer treatments were − Mn (g kg 1) 0.61a 0.55a 0.47a 0.52a analyzed using t-test in Microsoft Excel 2007. The obtained sequences − Al (g kg 1) 66.5ab 65.3a 68.2b 68.1b − of Geobacteraceae were employed to establish a phylogenic tree in Total K (g kg 1) 12.1a 12.4a 12.6a 12.8a − Total P (g kg 1) 0.60a 0.70a 0.93b 1.15c MEGA X using a neighbor-joining method (Kumar et al., 2018; Saitou and Nei, 1987). Multiple comparison analysis among different treat- † CK, a non-fertilizer treatment; M, application of cattle manure; NPK, ap- ments was conducted by the Duncan method in SPSS (version 25). plication of chemical nitrogen, phosphorus, and potassium fertilizers; MNPK, M combined with NPK. The same in Table 2. 2.6. Accession number of nucleotide sequences ‡ Values within a row followed by the same letter are not significantly different at P < 0.05. The Geobacteraceae sequences have been deposited at the NCBI Sequence Read Archive (SRA) under accession number PRJNA561459. tests. All qPCR reactions were run in 3 replications. Melting curve fi fi analysis was performed to check for the speci city of ampli cation at 3. Results the end of qPCR runs. Additionally, the standard template DNA of Geobacteraceae and bacteria was prepared by the PCR amplicons using 3.1. Soil properties in different fertilization treatments the primer sets of Geo494F/Geo825R and BACT 1369F/PROK 1492R, fi respectively. The PCR amplicons were pooled, puri ed, and ligated to After long-term fertilization, signifi cantly lower pH values pGEM-T Easy vector (Promega, USA) and then transformed into Es- (P < 0.05) were measured in the M, NPK, and MNPK treatments cherichia coli JM109 cells. The most abundant positive clone with the (5.53 ± 0.06, 5.55 ± 0.03, and 5.43 ± 0.03, respectively) compared targeted gene was selected and sequenced for plasmid DNA extraction. with the CK treatment (5.73 ± 0.05), while the concentrations of total The sequences of plasmids have been deposited in GenBank under ac- C (TC), total N (TN), and dissolved organic C (DOC), as well as the cession numbers MT102380 (bacteria) and MT093379 (Geobacter- electrical conductivity (EC) value, were significantly higher (P < 0.05) 2 9 aceae). Standard curves ranging from 4.6 × 10 to 4.6 × 10 (for than those in CK (Table 1). The concentrations of soil total phosphorus 2 9 Geobacteraceae) and from 3.9 × 10 to 3.9 × 10 (for bacteria) copies + (TP) and ammonium (NH4 -N) in the MNPK treatment were sig- per reaction were generated from the plasmids (Fig. S1). The standard nificantly higher (P < 0.05) than those in the CK, M, and NPK treat- curves are shown in Fig. S1. ments. Overall, the soil physiochemical characteristics were dramati- fi For Illumina sequencing, the extracted DNA was ampli ed to target cally altered in the MNPK treatment, in which the levels of soil C, N, P, the Geobacteraceae 16S rRNA genes using the primer pairs Geo494F and salinity (represented by EC) increased markedly. In addition, the and Geo825R with sample-identifying barcodes for sample demulti- concentration of microbial reducible iron [Fe(III)] in the MNPK treat- fi − plexing (Yuan et al., 2016). The amplicons were puri ed using a uni- ment (7.93 ± 3.18 mg kg 1) was much greater (P < 0.01) than that fi − versal DNA puri cation kit (Tiangen, China) with equal amounts of the in the CK treatment (1.48 ± 0.32 mg kg 1)(Table 1). PCR products, and then submitted to Majorbio Corporation (Shanghai, China) for Illumina Miseq sequencing. Quantitative Insights into Mi- 3.2. Absolute and normalized abundance of Geobacteraceae in different crobial Ecology (QIIME) (Caporaso et al., 2010) was used to analyze the fertilization treatments sequencing data. The raw reads were filtered to eliminate adapter and low-quality sequences (e.g., containing > 20 low-quality bases) to ob- The absolute abundances of Geobacteraceae in all treatments tain high quality reads. Subsequently, any paired-end reads with ranged from 3.76 × 109 to 1.15 × 1010 copies per gram of dry soil overlaps were merged to the tags. The resultant tags were clustered into weight (Fig. 1, Table S3), with that in the MNPK treatment operational taxonomic units (OTUs) at a 97% similarity level. A re- (1.15 × 1010) being significantly higher (P < 0.05) than those in the presentative sequence for each OTU was selected for alignment and CK, M, and NPK treatments (3.76–4.27 × 109). This was also the case taxonomic assignment using the SILVA database version 128 (Quast regarding the bacterial abundance, which was determined by bacterial fi et al., 2013). Representative sequences classi ed as chloroplasts and 16S rRNA gene copy number. The normalized abundances of Geo- fi mitochondria were removed, and singleton OTUs were also ltered bacteraceae were approximately 7.6–11.3% in all fertilizer treatments before diversity analysis. (i.e., M, NPK, and MNPK) without significant differences (P > 0.05) when compared with the CK treatment (9.8%) (Fig. 1, Table S3). 2.5. Statistical analysis 3.3. The community structure of Geobacteraceae in different fertilization Taxonomic alpha-diversity indices, including the Chao1 estimator treatments and Shannon and Simpson indices, were calculated using the vegan R package (Oksanen et al., 2013). Bray-Curtis and weighted-UniFrac The Miseq sequencing results, based on Geo primers, showed that a distance matrices, which represent the taxonomic and phylogenetic total of 167,965 non-singleton reads of Geobacteraceae were detected dissimilarities, respectively, were calculated using the vegan and GU- in all treatments, with a count of reads that ranged from 8052 to 17,491 niFrac packages in R, respectively (Oksanen et al., 2013; Chen et al., per sample. All of these sequences were assigned to 27 operational 2012). Principal coordinate analysis (PCoA) of the Geobacteraceae taxonomic units (OTUs) at a 97% similarity level. The community

3 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

Bacteria OTU1 Normalized abundance Geobacteraceae a OTU2 3.00E+011 14 OTU3 100 OTU4 2.40E+011 12 OTU5 OTU6 1.80E+011 10 OTU7 80 OTU8 1.20E+011 8 OTU9

OTUs (%) OTU10 6.00E+010 6 60 OTU11 1.50E+010 OTU12 4 OTU13 1.00E+010 OTU14 (per gram dry soil)

Geobacteraceae (%) 40 Absolute abundance OTU15 5.00E+009 2 Normalized abundance of OTU16

Relative abundance of OTU17 0.00E+000 0 CK M NPK MNPK Geobacteraceae 20 OTU18 OTU19 Treatment OTU20 OTU21 Fig. 1. The absolute and normalized abundances of Geobacteraceae by qPCR in 0 CK NPK M MNPK OTU22 CK, M, NPK, and MNPK treatments. CK, non-fertilizer treatment; M, application OTU23 of cattle manure; NPK, application of chemical nitrogen, phosphorus, and po- Treatment OTU24 tassium fertilizers; MNPK, M combined with NPK. The same in Figs. 2 and 4. OTU25 b OTU26 35 OTU27 Table 2 OTU23 The average relative abundances of Geobacter and Geothermobacter genera in all 30 * * OTU22 treatments. ** OTU13 * * OTU11 Treatment Average relative abundances (%) 25 OTU1 ** * * Geobacter Geothermobacter 20 † CK 95b 5a M 90a 10b 15 NPK 92a 8b MNPK 91a 9b 10 ** † ** ** Values within a column followed by the same letter are not significantly Relative abundance (%) different at P < 0.05. 5 structure of Geobacteraceae in all treatments consisted of the genera 0 Geobacter (accounting for 90–95% of the total reads) and CK M NPK MNPK Geothermobacter (5.5–10%), where fertilization significantly decreased Treatment (P < 0.05) the relative abundance of Geobacter but increased that of c Geothermobacter (Table 2). The richness index (denoted by the Chao1 OTU25 20 estimator) and abundance index (denoted by the Simpson index) were OTU19 similar among all treatments, however, the Shannon diversity (denoting OTU18 fi OTU14 richness and abundance) in the MNPK treatment was signi cantly OTU7 lower (P < 0.05) than in the CK treatment (Table S3). 15 Among the 27 Geobacteraceae-related OTUs detected in all treat- – * ments, OTU16 was the most abundant, occupying 23% 26% of the total * reads in all treatments (Fig. 2a, Table S4). The relative abundances of 10 * ** OTU1 and OTU11 significantly increased (P < 0.05) in all fertilizer ** treatments compared with the CK treatment. Furthermore, OTU13 and * ** OTU23 were enriched (P < 0.05) in the M and MNPK treatments, Relative abundance (%) 5 ** while OTU22 was only enriched (P < 0.05) in the MNPK treatment ** ** (Fig. 2b). In contrast, the relative abundances of OTU7, OTU14, OTU18, ** and OTU25 were significantly decreased (P < 0.05) in all fertilizer 0 ** ** ** treatments compared with the CK treatment. Moreover, a significant CK M NPK MNPK (P < 0.05) decline in the relative abundance of OTU19 was observed Treatment in the M and MNPK treatments (Fig. 2c). Fig. 2. The composition of Geobacteraceae-related OTUs in CK, M, NPK, and MNPK treatments (a). Increased OTUs (b) and decreased OTUs (c) (relative 3.4. Taxonomic and phylogenetic analyses of Geobacteraceae abundance > 1%) in M, NPK, and MNPK treatments compared with CK treatment were calculated using the t-test method. *P < 0.05, **P < 0.01. The sequences of 27 Geobacteraceae-related OTUs were used to construct a phylogenetic tree using the neighbor-joining method “Clade 1”, “Clade 2”, and “Clade GM” (Geobacter metallireducens) (Fig. 3). The detected 27 OTUs all belonged to the Geobacteraceae fa- (Fig. 3). Clade 1 included OTU 22 and OTU 23, as well as Geobacter ffi mily, with OTU6, OTU13, OTU21, and OTU24 being a liated with the bremensis, G. bemidjiensis, and G. uraniireducens, which are commonly ffi Geothermobacter genus, and the remaining OTUs being a liated with found in petroleum- or uranium-contaminated sites. Clade 2 accounted the Geobacter genus. The majority of the sequences belonging to the for 63% of the obtained sequences, including 17 OTUs closely related to Geobacter genus fell into three phylogenetic clusters, designated as

4 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

Denitrifying

Uncult

red Geobac Uncultured delta proteobacterium JN03884

bacterium enrichment Geoalkalibac

Geobac

Geoalkalibac

teraceae bacterium EF668606.1

Geothermoba ter argillaceus G12 DQ1455 ter ferri

2.1 Uncultur 2 t er subterran 6117.1 96918 hydriticus Z-

ter sp. HQ875540.1 cter ehrlic culture FJ802228.1 AB65 ed proteo 6.1 9 093.1 Geothermoba Uncultu e er sp. JN0916 us Red1 b hii SS01 0531 DQ30 Geopsychrobact acterium EU298434.1

red bact red Geobac er luticola strain OSK6 NR 114303.1 er sp. JN0915 u t cter sp. strain EPR OTU21 erium AB656 red bacterium er pelophilus Dfr2 U 5 AY15E u U1822 OTU6 e 34 red Geobact rium u e 9326 8.1

OTU2 r electrodiphilus A1 Uncult Geobacter luticola OSK6 AB682759 Geobact red bact AB658004.1 5 OTU18 599 4 Uncult 32 EU660516 OTU1 7 Geobact -M Uncult 682968.1 MK OTU27 OTU24 Uncultu 135 3 Uncultured Geobac A95 KC OTU19 Geobact AY187303800.1 Uncultured bacterium AB658538.1rium clone er sp. R4-NIT Geobacter terpickeringii toluenoxydans G13 DQ145535 TMJ1 EU711072 Geobacter reddaltonii bacte FRC- -2015 LC076693.1 Clade Geobacu Uncult 0d-112 HQ875547.1 Geobacter bremensis Dfr1 U96917 GM OTU16 OTU23 OTU4 Geobacter bemidjiensis Bem AY187307 Uncultured Geobacter sp. clone HZ-3 OTU3 OTU17 OTU22 OTU14 27427 4 EF5 Uncultured Geobact Geobacter uraniireducens Rf 7.1 Clade 2 Uncultured Geobact er sp. clone D03 JX Uncultured Geobac OTU5 545158.1 ter sp. clone ZJ-30d-55 JN09162 Uncultu er sp. clone CH360 MH5395 ured Geobac 1561 Unculturedred bacte bacterium clone HWB5257- Uncult Geobac ter sp. clone CH356 hilus P35 AY653549541.1 OTU10 13.1 Uncult ter hephaestius AY73 MH539 ter psychrop AB660OTU2514.1 OTU20 rium clone AB660994.1 ter chapelleirium 172 U4 Geobact 509.1 Geobac 0830 505.1 ured proteo Uncultu 3-57 HM487998.1 Geobac OTU7 Uncultured bact red bacte Geobac Geobact OTU8 er sp. 2 MH539 Uncultu 7 Uncultu OTU15 OTU26 red Geobacter sp. cloneb 3Da 507.1 Uncultu OTU12 Uncultu acterium clone JF988221.1 4527.1 1 AM941463.1 10.1 1.1 8 1 LT62626 ter lovleyi SZ A er thiogenes OTU1 ucnU

OTU9 988.1 4 JX94 OTU 189.1 red Geobact cterium clone ARIC12 KM 1 tl sp. clone CH35 red Geobac erium LK 1.1 red Geobac

JN09160 eru

eraceae ba a 024815.2 AF223382Y914

eobact erium clone AB656087.1 er sp. MH5394

retc t 177 ui er sp. MH539 Uncultured Geobacter sp. ter sp. clone HZ- -4 Uncultured Geobacter JN098

rium cloneAB660

Uncultured G red bact er sp. cloneAY607

t 6BA m 418.1 6

erium clone F07 WMSP2 DQ450 Uncultu 65 88.1

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HQ875505.1

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Fig. 3. The neighbor-joining phylogenetic trees of the Geobacteraceae species and their close relatives derived from the RDP and NCBI databases. The unlabled branches are the cluster of Geothermobacter, Geoalkalibacter and Geopsychrobacter.

Geobacter psychrophilus, G. chapellei, G. thiogenes, G. lovleyi, and G. he- by non-fertilizer and fertilizer grouping (Adonis R2 > 0.5). In addition, phaestius. Clade GM contained 4 OTUs closely related to Geobacter ar- there was no significant difference between any two fertilizer treat- gillaceus, G. luticola, and G. pelophilus. ments (Adonis R2 ≤ 0.35, P ≥ 0.2). The PCoA of the taxonomic (based on Bray-Curtis distance) and phylogenetic (based on weighted-UniFrac distance) structures of the 3.5. Effects of soil variables on Geobacteraceae community Geobacteraceae communities both revealed remarkable differences between the CK and all the fertilizer treatments, with samples from the Spearman's correlation analysis was performed to explore the re- CK treatment being distinctly separated from the fertilizer treatments lationships between the soil properties and the absolute abundance or fi along PC1 (Fig. 4a and b). Moreover, there seemed to be no signi cant normalized abundance of Geobacteraceae in all treatments (Table S6). ff di erences between the taxonomic nor phylogenetic structures of the The absolute abundance of Geobacteraceae showed a significantly ne- Geobacteraceae communities among the M, NPK, and MNPK treat- gative correlation (P < 0.05) with soil pH, and positive correlations ments, as samples from these treatments were grouped together along with soil electrical conductivity (EC), Fe(II), microbial reducible Fe(III), ff PC1 (Fig. 4). Furthermore, to quantitatively compare the di erences and the concentrations of nutrients, including total carbon (TC), total between the taxonomic and phylogenetic structures of the Geobacter- nitrogen (TN), organic matter (OM), dissolved organic carbon (DOC), aceae community between any two treatments, Adonis analyses were + NH4 -N, total potassium (TK), and total phosphorus (TP). performed with the OTU-level data, calculated using the Bray-Curtis To determine the relationship between the soil properties and the and weighted-UniFrac methods, respectively (Table S5). The results structure of the Geobacteraceae community, we compared the distance- showed that the Geobacteraceae communities in all the fertilizer based taxonomic and phylogenetic dissimilarities in community com- fi ff treatments were marginally but signi cantly di erent (P = 0.1) from position with the soil variables using Mantel analysis (Fig. 5). Soil pH that in the CK treatment, and over 50% of the variations were explained showed the strongest correlations between both the phylogenetic and

5 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

a Bray-Curtis b weighted-UniFrac

Fig. 4. Ordination plots of taxonomic (a, based on Bray-Curtis distance matrix) and phylogenetic (b, based on weighted-UniFrac distance matrix) dissimilarities between the Geobacteraceae community in different treatments. taxonomic dissimilarities of Geobacteraceae community composition respectively (Fig. 6b). Furthermore, the interactions of these four soil (Mantel's r = 0.66 or 0.54 for taxonomic and phylogenetic dissim- factors accounted for 12% of the phylogenetic variations (Fig. 6b). ilatory, respectively; P < 0.01) in all treatments (Table S7). Soil TC Taken together, soil pH, TC, and TN were the top three factors sig- (P < 0.05) and TN (P < 0.01) were also significantly correlated with nificantly affecting the Geobacteraceae community. However, DOC, TP, two-distance-based Geobacteraceae community composition (Fig. 5, and TK were also found to play important roles in the shaping of the Table S7). On the other hand, soil DOC and TP were only significantly Geobacteraceae community structure (Fig. 6a and b). correlated (P < 0.05) with the taxonomic dissimilarity of Geobacter- aceae community structure, and TK was solely associated (P < 0.05) 4. Discussion with the phylogenetic dissimilarity of the Geobacteraceae community structure in all treatments (Fig. 5, Table S7). 4.1. Effects of long-term fertilization on the abundance and diversity of Variance partition analysis (VPA) was further performed to identify Geobacteraceae in paddy soils the relative importance of significant soil properties selected by the Mantel tests (Fig. 6). Overall, the soil pH, TC, TN, TP, and DOC ac- In this study, the absolute abundance of Geobacteraceae only counted for 4%, 9%, 13%, 4%, and 3% of the Geobacteraceae taxo- markedly increased in a paddy soil administered with organic and nomic dissimilarities based on the Bray-Curtis distance, respectively, chemical fertilizers (i.e., MNPK). A similar phenomenon was also ob- where the interactions between these soil properties account for 17% of served in previous studies (Ding et al., 2018)(Wang et al., 2019). the taxonomic variations (Fig. 6a). Moreover, the Geobacteraceae However, no significant difference was detected in the normalized phylogenetic dissimilarities based on the weighted-UniFrac distance abundance of Geobacteraceae between all fertilizer treatments (i.e., M, were accounted by soil pH (9%), TC (9%), TN (12%), and TK (5%), NPK, and MNPK) and CK treatment, indicating that long-term organic

Fig. 5. (a) The pairwise comparisons be- Mantel’s r a tween the soil properties are shown with > 0.5 Spearman's correlation (P < 0.05), the 0.25 - 0.5 black and white circles represent positive < 0.25 and negative correlations, respectively. The insigniﬁcant data are not shown. (b) -N

P-value +

4 The relationship between the taxonomic 0.001-0.01

NH (based on Bray-Curtis distance) or phylo- 0.01-0.05 genetic (based on weighted-UniFrac dis- > 0.05 tance) dissimilarities of the Geobacteraceae community and the soil properties were calculated using the Mantel test, shown at Phylogenetic the lower left. Line width in (b) was pro- dissimilarity portional to the Mantel's r value of the corresponding correlation, and line style represented the statistical signiﬁcance (P

+ value) based on 999 permutations. NH4 -N

b Taxonomic dissimilarity

6 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

a Bray-Curtis b weighted-UniFrac Fig. 6. Variance partition analysis (VPA) of the taxonomic (a, based on TC TN Bray-Curtis distance matrix) and phy- TC TN logenetic (b, based on weighted- 9% 13% UniFrac distance matrix) dissimilarities of the Geobacteraceae community in all 9% 12% TK pH treatments accounted for a selection of 9% 1% signiﬁcant soil variables based on 9% 8% 5% Mantel tests. Values < 0 were not shown in VPA. 17% 13% pH 4% 8% 4% TP 1% 12% 3% 6% 1% 1%

3% 1%

DOC Unexplained = 27% Unexplained = 24%

and/or chemical fertilization did not alter the normalized abundance of paddy soils and freshwater lakes were primarily clustered into Clade 2 Geobacteraceae. For M and NPK treatments, it is not surprising that (Yi et al., 2013)(Fan et al., 2018). These findings were similar to the there were no significant differences in terms of the normalized abun- results found in this study, which showed that the Geobacter species in dances of Geobacteraceae between these two treatments and CK, since Clade 2 were the dominant Geobacteraceae context in both the CK and the absolute abundances of Geobacteraceae and bacteria in these two fertilizer treatments (Fig. 3). Moreover, long-term chemical and/or treatments were not significantly different (P > 0.05) from those in organic fertilization significantly enriched OTU1 and OTU11, which are the CK treatment. For the MNPK treatment, a similar increase in the both affiliated with Clade 2 with closer genetic distance (Figs. 2 and 3). absolute abundances of Geobacteraceae and bacteria resulted in no This suggests the similar physiological and metabolic characteristics in significant differences between the normalized abundances of Geo- OTU 1 and OTU 11 which were the dominant phylotypes of Geo- bacteraceae compared with CK treatment. Moreover, no significant bacteraceae family in fertilized treatments. Numerous studies have re- changes in the alpha diversity indices of Geobacteraceae were induced ported that Clade 2 of the Geobacter species (e.g., G. lovleyi, G. thiogenes) by long-term organic and/or chemical fertilization regimes (M, NPK, isolated from paddy soils are capable of reducing Fe(III) via the oxi- and MNPK) (Table S3). This was consistent with a previous study, in dation of acetate (Lovley et al., 2011) and other macromolecular or- which the microbial richness and diversity in an upland soil were found ganic matter, including toluene and tetrachloroethene (De Wever et al., to be unaltered by the long-term input of chemical fertilizers (NPK) or 2000; Sung et al., 2006; Daprato et al., 2007). However, the enriched swine slurry, suggesting that the alpha diversity of microbes may be OTU1 and OTU11 could not be related to any known cultured Geobacter resilient to the original state (similar to non-fertilizer CK treatment) but were found to be closely related to uncultured Geobacter sequences after long-term fertilization (Suleiman et al., 2016). This may also ex- obtained from diverse environments. This may limit our investigation plain our finding that the absolute abundances of Geobacteraceae were to the effects of long-term fertilization on the Geobacteraceae com- not changed after the long-term application of organic or chemical munity structure, given the predominance of these unclassified OTUs fertilizers alone (Fig. 1, Table S3). That is, the absolute abundances of within the Geobacteraceae community in both the non-fertilized and Geobacteraceae may be temporarily increased by the application of fertilized paddy soils. Further research based on metagenomic se- organic or chemical fertilizers due to “priming effect” (Lourenco et al., quencing should be performed to determine the metabolic properties 2018), before subsequently returning to the original state in the paddy and environmental functions of uncultured Geobacteraceae species, soils. Furthermore, the numbers of Geobacteraceae-related OTUs (27) which may improve our understanding of the biodiversity and roles of targeted by Geo494F/Geo825R in present study were remarkably lower the Geobacteraceae family in the biogeochemistry of paddy soils. than those (about 70) targeted by the same primer set in flooded paddy soils based on clone library (Yi et al., 2013), but much higher than those 4.3. Relationship between soil variables and Geobacteraceae in paddy soils (from 13 to 19) targeted by Geo564F/Geo840R in freshwater lake sediments (Fan et al., 2018). This phenomenon may be explained by the Changes in the physiochemical characteristics of the soil could drive potential influences of sample characteristics and sequencing techni- shifts in the Geobacteraceae communities found in paddy soils (Ding ques. et al., 2015; Yuan et al., 2016). For example, Yuan et al. reported that soil properties, including amorphous iron, pH, and DOC, accounted for 4.2. Effects of long-term fertilization on the structure of Geobacteraceae in 18.2% of the variations in the Geobacteraceae community of 16 various + paddy soils paddy soils across China (Yuan et al., 2016). In addition, NH4 -N and different Fe(III) oxides affected the dissimilatory Fe(III) reducing mi- Geobacter spp. was the main constituent of the Geobacteraceae crobial community compositions, including the Geobacter species in community in present study (Fig. 3) and has been previously found in paddy soils (Ding et al., 2015). In the present study, the PCoA and various environments, including marine sediment, paddy soils, and Adonis results demonstrated that the structures of the Geobacteraceae freshwater lakes (Holmes et al., 2007; Yi et al., 2013; Fan et al., 2018). family in all fertilizer treatments were only marginally different from The obtained sequences of the Geobacter genus fell into three different CK (Adonis R2 > 0.50, P = 0.1) (Fig. 4, Table S5). The variations in phylogenetic clades: “Clade 1,”“Clade 2,” and “Clade GM” (Holmes the Geobacteraceae communities in fertilizer treatments may have been et al., 2007; Yi et al., 2013; Fan et al., 2018). Marine sediments have caused by changes in the soil properties induced by long-term fertili- been previously reported to harbor mainly Clade 1 of the Geobacter zation. sequences (Holmes et al., 2007), while the Geobacter sequences in Mantel tests showed that soil pH, TC, and TN were the key factors

7 X. Li, et al. Applied Soil Ecology 153 (2020) 103577

(P < 0.01) affecting the taxonomic and phylogenetic structures of soils and could be used to better harness the microbiome via agri- Geobacteraceae communities in all treatments (Fig. 5). Soil pH has also cultural management practice to improve the fertility and environ- been reported to be an important factor in determining the community mental quality of the soil. structure of Geobacteraceae in diverse paddy soils at a continental scale (Yuan et al., 2016). Spearman's correlation analysis showed that soil pH Acknowledgements was significantly correlated with many soil properties, such as being negatively correlated with microbial reducible Fe(III) (Fig. 5a). Micro- We would like to acknowledge Dr. Guoxin Sun for assistance in the bial reducible Fe(III) oxides, usually serving as electron acceptors of the collection of the soil samples. This work was conducted with financial Geobacteraceae family, tend to release with decreasing soil pH (Weber support from National Natural Science Foundation of China (grant et al., 2006) and thus affect the Geobacteraeae communities (Yuan et al., numbers 41601242 and 41430858), National Key Research and 2016). Here, the continuous input of organic and/or chemical fertilizers Development Program (grant number 2017YFD0801502), and Youth (M, NPK, and MNPK) was found to result in increased nutrient levels in Innovation Promotion Association, Chinese Academy of Sciences. the soil, including TC and TN concentrations (Table 1), which may then influence the Geobacteraceae communities in fertilizer treatments Declaration of competing interest (Figs. 5 and 6). The Geobacteraceae family can utilize various organic compounds as electron donors (Röling, 2014), where the donors vary The authors declare that they have no known competing financial for the different members of the Geobacteraceae community. For ex- interests or personal relationships that could have appeared to influ- ample, Geobacter bremensis uses C1-C5 organic acids and C2-C4 alcohols ence the work reported in this paper. as electron donors (Straub and Buchholz-Cleven, 2001), while Geobacter bemidjiensis has been found to use glucose as one of its electron donors Appendix A. Supplementary data (Nevin et al., 2005). Moreover, these species were found to be closely related to OTU22 and OTU23, which were significantly enriched in the Supplementary data to this article can be found online at https:// M and MNPK treatments (Fig. 2, Table S4). Additionally, Geobacter doi.org/10.1016/j.apsoil.2020.103577. lovleyi could only oxidize acetate and pyruvate within C1-C5 organic acids, and was found to be unable to use any C1-C5 alcohols as electron References donors in previous studies (Sung et al., 2006). This suggests that different types of electron donors may enrich various Geobacter species. Anderson, M.J., Willis, T.J., 2003. Canonical analysis of principal coordinates: a useful The application of manure (M and MNPK treatments) could result in a method of constrained ordination for ecology. Ecology 84, 511–525. Bond, D.R., Holmes, D.E., Tender, L.M., Lovley, D.R., 2002. Electrode-reducing micro- large number and a greater diversity of electron donors (e.g., DOC) for organisms that harvest energy from marine sediments. Science 295, 483–485. the Geobacteraceae species, which could then result in a shift of the Caccavo, F., Lonergan, D.J., Lovley, D.R., Davis, M., Stolz, J.F., McInerney, M.J., 1994. Geobacteraceae communities compared with the CK treatment. More- Geobacter sulfurreducens sp. nov., a hydrogen-and acetate-oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 60, 3752–3759. over, the composition of the Geobacteraceae community in the NPK Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., treatment also marginally significantly shifted compared with CK, Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., which may be caused by increases in the levels of nitrogen, phosphorus, Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., and potassium concentration (Figs. 5 and 6), since soil TN, TP, or TK Pirrung, M., Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., 2010. 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