Effect of cadmium contamination on the rhizosphere bacterial diversity of Echinocactus platyacanthus

Rhizosphere 13 (2020) 100187

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Effect of cadmium contamination on the rhizosphere bacterial diversity of Echinocactus platyacanthus

T

Margarita María Sarria Carabalía,b, Felipe García-Olivac, Luis Enrique Cortés Páezd, Nguyen E. López-Lozanob,∗ a

Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia Sede Palmira, Carrera 32 No 12-00, Chapinero, Palmira, Valle del Cauca, A.A 237, Colombia CONACyT-División de Ciencias Ambientales, Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), Camino a laPresa San José 2055, Lomas 4ta. Sección, San Luis Potosí, S.L.P., 78216, Mexico c Instituto de Investigaciones en Ecosistemas y Sustentabilidad, Universidad Nacional Autónoma de Mexico, 58090, Morelia, Mexico d Departamento de Ciencias Básicas, Facultad de Ingeniería y Administración, Universidad Nacional de Colombia Sede Palmira. Carrera 32 No 12-00, Chapinero, Palmira, Valle del Cauca, A.A 237, Colombia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial community Cacti Heavy metals Diazotrophs Microbial biomass Photosynthetic efficiency

To better understand the mechanism of tolerance to heavy metals, it is relevant to analyze the effects of Cd contamination on the rhizospheric microbiota of plants with high tolerance to environmental stress such as cacti species. In this work, the effects of soil contamination with Cd on bacterial diversity, microbial carbon, abundance of diazotrophs in the soil, and on the photosynthetic efficiency and absorption of Cd of the candy barrel cactus (Echinocactus platyacanthus) were evaluated. Experimental pots sown with E. platyacanthus were contaminated with different concentrations of Cd+2. Changes in rhizospheric soil community were evaluated after 30 days of contamination. In general, soil contamination with Cd significantly changes the structure of the rizospheric bacterial community which could have serious effects on the functioning of this community, especially in N mobilization. Despite the plant not showing signs of physiological stress reflected in the photosynthetic yield, revealing its high resistance to contamination with Cd, a slight increase in the electron transport rate at low Cd concentration was observed. This increase correlates with an increase in abundance of diazotrophs and microbial C, suggesting an interesting response by the entire system to the presence of low Cd concentration in the soil. In this work, we have demonstrated that the factors analyzed here can be indicators of the quality and health of the soil, being a complement to explaining the behavior of metals in the soil matrix, and thus be able to understand the mechanism of tolerance to Cd contamination in soil.

1. Introduction One of the most worrisome soil degradation factors is the presence of heavy metals, which can come from industrial processes, mining, as well as the inadequate disposal of domestic and agricultural waste that increase heavy metals concentration in soils to risk levels (Park et al., 2011). This kind of human activities increase Cadmium (Cd) contamination, a highly toxic metal that decreases soil biodiversity, and whose negative effects can be transferred to vegetation (Shentu et al., 2014; Thavamani et al., 2012; Hu et al., 2007) and eventually reach food chains (Rahman et al., 2017). In this context, the study of plantassociated microbes is gaining importance, not only for the ecosystem services it offers, such as energy flow, nutrient cycling, maintenance of soil structure, among others (Chodak et al., 2013; Bordez et al., 2016), but also due to microorganisms actively participate in soil remediation ∗

of heavy metals and stress tolerance of plants (Tiwari and Lata, 2018). Besides, microorganisms are recognized as perturbation sensors, being much more sensitive than macro-organisms to environmental stress (Giller et al., 2009; Khan et al., 2018). Due to any decline in microbial populations resulting from pollutants entering to soils have proportionately harmful effects on the function of natural ecosystems, it is possible to use essential functions such as those involved in the nitrogen (N) cycle as indicators to evaluate the effect of heavy metal pollution. A detrimental effect of heavy metal additions on different steps of the nitrogen cycle has been reported (Smolders et al., 2001; Chen et al., 2003; Gremion et al., 2004; Sobolev and Begonia, 2008; Niemeyer et al., 2012). The entry of N to the soil can be compromised with the increase in the concentrations of Cd by a direct effect over the N fixers populations and activity (Brookes et al., 1986; Chen et al., 2003). However, the N and Cd availability in the soil matrix are closely

Corresponding author. E-mail address: [email protected] (N.E. López-Lozano).

https://doi.org/10.1016/j.rhisph.2020.100187 Received 26 June 2019; Received in revised form 14 January 2020; Accepted 14 January 2020 Available online 23 January 2020 2452-2198/ © 2020 Elsevier B.V. All rights reserved.

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2. Materials and methods

related. The ammonium ions compete with Cd for exchange sites; its displacement could favor the losses of available N in soil (Mitchell et al., 2000; Gray et al., 2002). In this way, soil factors such as pH and ionic strength, interact with microbial community activities, plant biomass growth and root exudation to increase the concentration of Cd in the soil solution (Mitchell et al., 2000). Although great efforts have been made to evaluate the impacts of heavy metals such as Cd on the biomass and structure of the soil microbial community (Shentu et al., 2014; Zhang et al., 2015), some studies do not show such an obvious relationship between these factors (Gillan et al., 2005; Grandlic et al., 2006; Zhu et al., 2013). This could be due to the effect of toxicity of heavy metals on the structure of the microbial community also being influenced by the physicochemical properties of the soil (Boivin et al., 2006; Kenarova et al., 2014; Stemmer et al., 2007) and the plant species present in the contaminated sites (Sharma et al., 2015; Muehe et al., 2015). Given the mixed effects of various factors present in nature, a possible solution to dissect all these factors is carry out experimental studies that allow us to understand heavy metals tolerance mechanisms in plants, as well as when and how this pollutants could modify essential soil processes by changes in microbial communities. For a more realistic scenario is necessary use model organisms that lives in the specific conditions of the soil at risk of contamination. In this sense, the family Cactaceae is a very interesting group of plants because in general they are highly resistant to different type of stress (Levresse et al., 2012), however few studies have explored the microbiota associated with them (Fonseca-García et al., 2016; Kavamura et al., 2018). We hypothesize that, in addition to the intrinsic adaptation strategies of cacti, the rhizospheric microbiota is involved in modulating their response to Cd contamination. The definition of reliable risk levels of heavy metals in soil is controversial. In Mexico, through official Mexican Standard (NOM147-SEMARNAT/SS1-2004), it was established that the concentration of Cd in agricultural soils should not exceed 37 mg kg−1 (SEMARNAT, 2007); a value that depended mainly on the study of the total content of this metal in the soils. In this regard, Epelde et al. (2016) evaluated the effect of different concentrations of this metal on the enzymatic activity of the soil (β-Glucosidase, arylsulphatase and alkaline and acid phosphatase), biomass and diversity of soil microbial communities, finding that values around 44 mg kg−1 presented strong negative effects; a value similar to that defined by the legislation of several European countries. However, the United States Environmental Protection Agency (US EPA) considered that the data on the toxicity of metals in soil microorganisms and microbial processes are insufficient and therefore the interpretation was too uncertain to establish thresholds based on risk using these parameters (Giller et al., 2009). Faced with this, in our experiments we used Cd concentrations below to the Mexican and European official standards (a maximum ~40 mg kg−1) with the intention of testing if harmful effects are being underestimated at lower concentrations. In this context, this research aimed at experimentally determining the short-term effects of Cd contamination on the rhizospheric microbial communities of Echinocactus platyacanthus (candy barrel cactus), an endemic cactus from Mexico of rural use (Aragón et al., 2017) and which naturally inhabits sites at risk of contamination by heavy metals (Levresse et al., 2012). For this, the effect of the chemical properties of the soil and three levels of contamination by Cd on multiple indicators were evaluated, such as: 1) the diversity and structure of the bacterial community (by sequencing the V1–V3 region of the 16 S rRNA gene) in Illumina Miseq, 2) Microbial carbon (as an indirect measure of biomass), 3) abundance of N-fixing bacteria (based on quantification by qPCR of the nifH gene), 4) photosynthetic efficiency and Cd absorption both in the stem and in the root of Echinocactus platyacanthus.

2.1. Experimental soil The soil used in the experiments comes from the municipality of Guadalcázar, San Luis Potosí, Mexico (22° 37′ 15.95″ N; 100° 24′ 20.62″ W); altitude between 1090 and 2500 m.a.s.l., sandy loam texture, defined as Leptosols. The soil was sieved in 2 mm mesh (sieve # 10), mixed homogeneously and physicochemically characterized (Table S1). The experimental units were made with plastic pots containing 600 g of soil and three-year-old transplanted Echinocactus platyacanthus plants. After two months of acclimatization subsequent to the transplant, these were contaminated with Cd solutions prepared from a standard solution of Cd (Cd Standard for AAS 1000 ppm in 2% of HNO3, SIGMA - ALDRICH), which were added to the soil with a water content percentage of < 1% until saturation to obtain concentrations to 0, 20, 30 and 40 mg kg−1 (control, CdL, CdM and CdH, respectively); each treatment was prepared in triplicate. Subsequently, they were kept contaminated for 30 days in greenhouse conditions in order to allow an absorption and interaction of the metal with the soil matrix and the rhizosphere. Temperature and irrigation conditions controlled, 150 ml of sterilized water were added every week to prevent desiccation of the plants. After mixing all the soil that will be used as substrate, a composite sample was taken before contamination to perform the general soil characterization. Then, a new characterization of the soil attached to the roots for each replicate was carried out at the end of the experiment in order to determine any differences between treatments in the chemical properties of the soil after contamination. Water content soil was determined using the gravimetric method, samples were oven-dried at 60 °C. Soil pH (pHW) was measured in deionized water (soil: solution ratio, 1:2 w/v). For Organic C (OC) determination, the calcination method was used (Schulte and Hopkins, 1996). To measure macronutrients (Na, K, Ca, Mg), micronutrients (Zn, B, Fe, Mo, Mn, Cu) and soluble Phosphorous, an inductively coupled plasma optical emission spectroscopy was used (ICP-OES) (730-ES, Varian, Palo Alto, CA). Amonium (NH4) and nitrate (NO3) were measured using colorimetric methods (Forster, 1995; Miranda et al., 2001; Doane and Horwath, 2003). Total carbon and total nitrogen were determined with an elemental combustion system (ECS) (4010, Costech Analytical Technologies, Valencia, CA). Microbial C was measured by the fumigation/extraction method of Vance et al. (1987). The total Cd (CdT) was determined by acid digestion (HNO3+HCl); soluble Cd (CdW) by extraction using a soil:water ratio of 1:10 w/v and acidified with concentrated HNO3 (Sposito et al. 1982); and fractionation of Cd forms (bioavailable), extractions were made in a suspension of soil: EDTA 0.05 M pH 7.0 (1:10 ratio) (CdE); in all three cases the concentration was measured by atomic absorption spectrophotometry using a graphite furnace, the practical limit of detection is 0.05 mg/L. 2.2. Rizosphere sampling for DNA analysis Four (4) equidistant points were located around the root of Echinocactus platyacantus plants for a better representation of the rhizosphere. Sub-samples of approximately 1 g of soil were taken at a depth of 5 cm from the fraction that remained attached to the roots with a sterile spatula. The sample was pooled and homogenized in 2 ml Eppendorf tubes and subsequently stored at −80 °C. This sampling was done to the treatments before contaminating (US) and treatments contaminated with Cd after 30 days (CS). 2.3. DNA extraction and sequencing The metagenomic DNA was extracted from 0.25 g of each sample of contaminated and uncontaminated soil. We worked with the ZR soil microbe DNA MiniPrep™ extraction kit (Epigenetics Company™) following the manufacturer's instructions. 2

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Echinocactus platyacanthus by using a portable pulse amplitude modulation fluorometer (Mini-PAM; H. Walz, Effeltrich, Germany) in treatments before contamination, and then in treatments before 30 days’ contamination with Cd. The first round of photosynthetic efficiency measures was conducted on dark-adapted cacti at predawn (between 05:00 and 06:00 h) in order to assess the maximum quantum efficiency of photosystem II (Fv/Fm). The second round of photosynthetic efficiency measurements was conducted at noon (between 13:00 and 14:00 h), when plants faced the maximum daily temperature. These data were used to estimate the effective quantum yield of photosystem II (ΦPSII). We also calculated the electron transport rate (ETR) across the electron chain of chloroplasts, which is directly and positively related to the generation of chemical energy (ATP and NADPH/ H+) that will be later used in the Calvin cycle (Aragón et al., 2014). The Cd absorbed in stem and roots was recovered by acid digestion (HNO3+H2O2) using 4 g of dry tissue and measured by atomic absorption spectrophotometry using a graphite furnace. The accumulation and adsorption of Cd contamination in E. platyacanthus were analyzed using the bioconcentration factor (BCF) and translocation factor (TF) (Wang et al., 2019):

The DNA samples were sent to the Molecular Research laboratory (Mr.DNA) of Shallowater, Texas, USA, for sequencing in Illumina Miseq 2 × 300 bp, 20 K, using the 16 S rRNA gene V1–V3 variable regions PCR primers 27Fmod-AGRGTTTGATCMTGGCTCAG and 519Rmodbio-GWATTACCGCGGCKGCTG. 2.4. Sequence processing The obtained sequences were processed using the Mothur software (version 1.35.1). Reads were assembled and filtered according to their quality (Q value = 25, length = 300 bp, elimination of primers, barcodes, chimeras and homopolymers ≥ 8). The assembled sequences were aligned with the gene database of the 16 S rRNA SILVA v. 128. A distance matrix was calculated through the set of non-redundant sequences and operational taxonomic units (OTUs) were assigned with a similarity threshold of 97%. The sequences and OTUs were categorized taxonomically using a Bayesian classifier from program Mothur and the set of bacterial references from SILVA v.128 (https://www.arb-silva. de/). A representative sequence of each OTU was selected and taxonomic assignments were made with a confidence threshold greater than 80% of the bootstrap value. The sample sets were normalized to the same size (34,763 sequences) based on the library with the lowest number of reads. The richness was estimated with non-parametric statistics from Chao1 and Ace, and the diversity Shannon indexes were calculated using the same program. Based on a literature review, in order to identify possible functional changes between treatments using the relative abundance of the 16 S rRNA gene sequences, the genera that were identified taxonomically with a degree of certainty greater than or equal to 80% of bootstrap according to the Naïve Bayesian Classifier (Wang et al., 2007) were grouped into categories according to their ability to adapt to the environment, tolerate stress or present metabolic pathways that participate in the cycling of nutrients (Table S2), considering the possession of genes involved in the trait or detected activity under laboratory conditions reported in the literature. The relative abundance of each functional category was calculated by adding the number of sequences of the genera within that category and a comparison between the treatments was made using ANOVA. Divisions classified as candidates, or for which there is no information available in the literature, constituted only 3% of the total community. The reads obtained from sequencing have been deposited in the GenBank Sequence Read Archive under the Bioproject accession PRJNA516923.

BCF = CMIP / CMIS CMIP is the total concentration of Cd in the plant; CMIS is the concentration of Cd in the soil.

TF = CMIL/ CMIR CMIL is the concentration of Cd in stem and CMIR is the concentration of Cd in root of the cacti. 2.7. Statistical analysis The normality of the data was verified using the Kolmogorov Smirnov test. The effect of Cd concentration over soil chemical properties for the samples taken after 30 days of contamination was assessed using one-way ANOVA for normal data and Kruskal Wallis for nonnormal data. A post hoc test was used to determine group differences for significance (Tukey test: for normal data, Mann Whitney: for nonnormal data). In order to compare bacterial diversity indexes, photosynthetic yield and electron transport rate of Echinocactus platyacanthus in treatments before and after 30 days of contamination, we carried out a two-way analysis of variance (ANOVA) (factor 1 levels: control, CdL, CdM, CdH; factor 2 levels: before and after contamination) and PERMANOVA for Beta Diversity Profiling with Principal Coordinates Analysis (PCoA). General linear models were used in order to find out which chemical properties of the soil (pHW, OC, NO3, NH4, CdT, CdW, CdE) significantly influenced the behavior of dependent variables after contamination: bacterial diversity (Shannon index), abundance of diazotrophs (in gene copies g−1 dw), microbial C (μg−1 dw) and Cd absorption in the stem and in the root (mg kg−1). For each dependent variable, the corresponding model that best fits data was obtained by stepwise forward regression. Later, the significant variables were plotted according to simple regression models to illustrate their behavior. The statistical package SPSS v. 23.0 (SPSS Inc., Chicago, USA) was used for all these analyses. Furthermore, a correlation analysis was performed (Pearson: for normal data, Spearman: non-normal data) between the parameters considered as indicators and the chemical properties of the soil, and clustering analyses based in Bray -Curtis distance and heatmaps of relative abundance of bacterial genera (> 1%) were generated using R software v 3.4.0.

2.5. Quantification of nitrogen-fixing microbial communities The abundance of N fixers bacteria (diazotrophs) was quantified by qPCR targeting the nifH gene, using the primers FPGH19 (Simonet et al., 1991) and PolR (Poly et al., 2001). Absolute quantification of each soil sample was used for the PikoReal 96 Real-Time PCR System (TCR0096, Thermo Fisher Scientific Inc.). with amplification regime of 40 PCR cycles. Three reactions per replicate were performed using SYBR Green PCR Master Mix (Applied Biosystems). The specificity of the amplification products was confirmed by melting curve analysis. The standard curves were obtained using serial dilutions of the plasmid vector, containing a cloned fragment of the Geobacter sulfurreducens nifH gene (in pGEM-T Vector Systems, Promega, Madison, WI). The standard curve was optimized close to 100% efficiency. Inhibition in the PCR reactions was tested by mixing serial dilutions of DNA extracted from soil against a known amount of standard DNA before qPCR. The Ct values of the standard DNA did not change in the presence of diluted soil DNA, indicating the absence of severe inhibition.

3. Results 3.1. Chemical properties of Cd contaminated soils

2.6. Photosynthetic efficiency measures and determination of Cd absorption in plant tissues

The chemical properties of the soil collected for experimental procedures before contamination are shown in Table S1. Total and soluble

Two rounds of photosynthetic efficiency measures were taken on 3

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Table 1 Chemicals properties of soil treatments in the Cd contamination experiment after 30 days (mean ± SD, n = 3). pHW: solution soil pH; CEW: solution electrical conductivity; OC: organic carbon; NH4: ammonium; NO3: nitrate; CdT: total cadmium; CdW: soluble cadmium; CdE: cadmium extractable with EDTA (bioavailable). Control: without Cd; CdL: Low Cd concentration; CdM: Medium Cd concentration; CdH: High Cd concentration. P value: significance level. Treatment

pHW (1:10)

OC (%)

Control CdL CdM CdH p value

7.0 ± 0.1a 6.4 ± 0.04b 6.1 ± 0.07c 5.8 ± 0.28c < 0.001

1.33 ± 1.24 ± 1.26 ± 1.25 ± 0.215

∗

0.04a 0.05a 0.02a .0.07a

NH4 (mg kg−1)

NO3 (mg kg−1)

CdT (mg kg−1)

CdW (mg kg−1)

CdE (mg kg−1)

CdE/CdT

4.69 ± 4.96 ± 5.25 ± 2.79 ± 0.929

16.64 ± 6.75b 7.86 ± 1.63b 26.83 ± 0.02b 78.49 ± 6.53a 0.005

0.00 ± 0.00∗ 18.12 ± 1.44b 24.95 ± 1.58 ab 32.47 ± 8.21a < 0.001

0.00 ± 0.63 ± 0.72 ± 2.58 ± 0.002

0.00 ± 0.00∗ 18.40 ± 2.01b 17.64 ± 0.62b 29.43 ± 2.89a < 0.001

– 1.00 ± 0.11a 0.71 ± 0.04a 0.93 ± 10a 0.12

0.25a 0.40a 0.52a 0.27a

0.00∗ 0.04b 0.08b 0.25a

these values were excluded from the analysis.

simple correlation analysis using the data after 30 days of contamination. Using the same dataset, a positive correlation was also observed between the Shannon index and the pHW (rho value = 0,837**) (**p < 0.01). The general linear model carried out to determine the effect of the set of chemical variables of the soil (pHW, NH4, NO3, organic C, CdT, CdW and CdE) on the bacterial diversity (Shannon index) showed that the variable which has the greatest influence on the behavior of bacterial diversity was the Cd extractable with EDTA (CdE) (Table 3). In this regard, in Fig. 2a the relationship between the CdE concentration and the Shannon index is illustrated by a simple regression model. The results of the clustering analysis based on relative abundance of the bacterial genera present in each experimental sample showed a clear separation between the treatments according to their categorization of uncontaminated (US, all treatments before contamination with Cd) and contaminated (CS, treatments contaminated with Cd after 30 days), which evidences a change in the microbial composition product of the Cd contamination. Among the most notable differences in the uncontaminated treatments, a greater relative abundance of the groups RB41 (Acidobacteria) and Rubrobacter (Actinobacteria) is observed. In contrast, in contaminated soils Tumebacillus (Firmicutes), Bacillus (Firmicutes) and Massilia (Proteobacteria) increased their relative abundance (Fig. 3). The analysis based on the literature review of the functional capacities of the taxa identified, considering the possession of genes involved in the trait or detected activity under laboratory conditions, showed significant differences in the abundance of the functional categories between treatments. Mainly, the genera with the ability to form spores that exhibit tolerance to desiccation, temperature and metals, which have reported participation during bioremediation processes and nitrate reducers, increased their relative abundance in treatments contaminated with Cd, while the genera that have some route for carbon fixation decreased their abundance (*p < 0.05, **p < 0.01) (Fig. 4).

content Cd and Cd extractable with EDTA (CdT, CdW, and CdE respectively) were non-detectable before contamination, which means that the concentrations determined by our methods was negligible. The contamination with Cd was done by adding three concentrations of this metal (20, 30 and 40 mg kg−1) to the soil. Despite the high variability in the final Cd concentrations, significant differences were observed between treatments (Table 1) where most of the Cd was bioavailable or extractable with EDTA (CdE/CdT), due to recent time of contamination with Cd. The pHW of CdL treatment was neutral, while for contaminated treatments (CdM and CdH) it was slightly and moderately acidic, respectively, due to increased contamination. The organic carbon (OC) and NH4+ content had no significant differences. While the NO3 content was significantly higher in CdH treatment due to higher concentration of nitric acid of the Cd standard solution.

3.2. Effect of Cd concentration on the bacterial diversity of the soil After processing by quality, a total of 403,748 reads were analyzed. To avoid the bias of different depths of sequencing in the alpha diversity measures, each library was normalized to a size of 34,763 sequences, which corresponds to the size of the library with the lowest number of sequences obtained during sequencing and after processing by quality. The number of OTUs observed in the treatments contaminated with Cd after 30 days (CS) was lower compared to the treatments before contamination (US) (Table 2). The expected OTU richness was estimated by Chao1 and Ace, which did not show significant differences between periods or treatments. However, the contamination with Cd in the soil generated a decrease in the Shannon index (p < 0.05) (Table 2), as well as a change in beta diversity of the bacterial community, where treatments with soil contaminated with Cd form a individual group of uncontaminated soil and control treatments (Fig. 1). This behavior was also evidenced by the negative correlations that this index showed with the total and soluble concentrations of Cd and Cd extractable with EDTA (bioavailable) (rho values = −0,845**, −0,824** and −0,852**, respectively) (**p < 0.01), obtained by

Table 2 Bacterial diversity indexes based on 16 S rRNA gene sequencing (Mean ± SD; n = 3) of treatments before and after contamination with Cd. Control: without Cd; CdL: Low Cd concentration; CdM: Medium Cd concentration; CdH: High Cd concentration. Period

Treatment

Observed OTUs97

Chao

Uncontaminated

Control CdL CdM CdH Control CdL CdM CdH Period Treatment Period*Treatment

10,006 ± 346 10,125 ± 424 10,122 ± 273 10,387 ± 427 10,544 ± 83 8964 ± 54 8942 ± 506 7730 ± 1122 28.375*** 5.757** 9.742***

29,781 27,564 28,796 31,245 31,314 23,728 26,480 24,401 4.553* 2.249 1.683

Contaminated

F value and significance

p ≤ 0.001 = ***, p ≤ 0.01 = **, p ≤ 0.05 = *. 4

Ace ± ± ± ± ± ± ± ±

3197 1523 1616 4667 1657 821 2997 6102

54,440 47,590 51,397 56,157 58,204 39,293 48,336 45,208 2.104 2.722 1.030

Shannon ± ± ± ± ± ± ± ±

8082 2828 5283 10,210 4699 2766 7509 4099

7.47 ± 0.04 7.61 ± 0.11 7.54 ± 0.02 7.61 ± 0.03 7.60 ± 0.05 7.38 ± 0.11 7.14 ± 0.30 6.56 ± 0.36 28.645*** 8.125** 11.993***

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Fig. 1. Microbial Beta diversity profiling and significance testing (PCoA) for uncontaminated soil, control and contaminated soil with Cd samples. PERMANOVA: Fvalue: 10.951; R-squared: 0.51052; p-value < 0.001.

concentrations of CdT, while at high concentrations it decreased, which suggests that although the presence of CdT in low concentrations can stimulate the increase in microbial biomass, there is a point of inflection in which the effects start to become negative. However, the behavior of the microbial C is also influenced positively by the organic carbon (OC) of the soil and negatively by the soil NO3 concentration (rho values = −0.699*, −0.734**, respectively) (*p < 0.05, **p < 0.01).

Table 3 Summary of automatic linear modeling results of soil chemical properties (transformed variables) that predict the Shannon diversity, nifH abundance (gene copy numbers) and Cd absorption in stem and root of Echinocactus platyacanthus considering soil properties; only the significant variables in the final models are shown. %SS percentage of the total sum of squares explained by each variable.

Shannon CdE OC nifH CdW CdT CdE Microbial C CdT CdW NO3 ETR NH4 CdE pHW Cd Stem pHW Cd Root pHW NO3

%SS

F

p

Adjusted R2

56 18

13 4

0.011 0.87

0.60

76 17 3

422 96 19

< 0.001 0.002 0.022

0.99

58 36 6

38 24 4

0.002 0.004 0.099

0.88

44 40 7

29 27 4

0.004 0.004 0.010

1.0

91

99

< 0.001

0.89

89 7

46 101

0.001 < 0.001

0.94

3.4. Effect of Cd concentration on nitrogen-fixing soil bacteria The variables that significantly influenced the abundance of nitrogen-fixing bacteria (number of copies of the nifH gene), based on the general linear model, are the total Cd and soluble Cd (CdW); the latter being the most important (Table 3). Therefore, we observed that the abundance of nitrogen-fixing bacteria increased at low and medium concentrations of CdW, while at higher concentrations a negative effect on their abundance was observed (Fig. 2c).

3.5. Photosynthetic efficiency of Echinocactus platyacanthus The photosynthetic performance data (YIELD) and electron transport rate (ETR) of Echinocactus platyacanthus both in the measurement carried out at 6:00 h (Fv/Fm) and at 13:00 h (ΦPSII), did not show significant differences between Cd levels. There were only significant differences between the samples before and after the incubation period, being slightly higher in the plants after 30 days (Table 4). According to the general linear model, one of the factors with the greatest influence on the photosynthetic efficiency of the plant was the Cd extractable with EDTA (CdE) (Table 3). In the regression graph between these two factors (Fig. 2d), it was observed that ETR is only higher when CdE is lower than 20 mg kg−1; above this concentration ETR decreased.

3.3. Effect of Cd concentration on the carbon within microbial biomass in the soil According to the general linear model, we found that the total Cd (CdT) is the variable with the greatest influence on the behavior of the microbial C of the soil under study. In this regard, in Fig. 2b we observed that the microbial C increased in low and medium 5

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Fig. 2. Regression models between Cd concentration and (a) Shannon Index, (b) Microbial carbon (μg g−1 of soil), (c) Abundance of N fixers (nifH gene copies g−1 of soil), (d) Electron transport rate (ETR, μmol m−2 s−1), (e) Cd stem absorption (mg kg−1), (f) Cd root absorption (mg kg−1) in Echinocactus platyacanthus.

plant to these concentrations of metal in the soil. A similar behavior occurred for the translocation factor (TF) (Table 5), indicating that the plant regulates the entry of Cd to its tissues.

3.6. Cd absorption in Echinocactus platyacanthus The general linear model for Cd absorption in the stem and root revealed that the variable that best explains its behavior is the pHW. However, the pHW has a high negative correlation with the Cd forms (r value = −0.98***, −0.83***, −0.93***; CdT, CdW and CdE, respectively) (***p < 0.001), In this regard, by simple linear regression it was observed that the accumulation of Cd in the stem and the root of Echinocactus platyacanthus was directly proportional to the soil CdT concentration (p < 0.05) (Fig. 2e and f). In addition, the positive correlation between Cd absortion in both tissues and the CdT, CdW and CdE (Stem: r values = 0.93***, 0.81*** and 0.89***, respectively; Root: 0.93***, 0.88*** and 0.90***, respectively) (***p < 0.001) indicate that the plant has a high capacity for Cd accumulation. The bioconcentration factor (BCF) for Cd had significant differences between contaminated treatments and the control (p < 0.05). However there were not significant differences within contaminated treatments (CdL, CdM and CdH), indicating a similar response of the

4. Discussion In order to understand the response to Cd contamination of plants that are highly resistant to stress such as cacti in association with their rizospheric bacterial community, in this study the effect in short term of soil contamination by Cd was evaluated using several indicators: rhizospheric bacterial diversity, microbial carbon, abundance of nitrogenfixing bacteria (nifH gene), photosynthetic efficiency and Cd absorption in stem and root of Echinocactus platyacanthus. It was evidenced that Cd contamination significantly changes the structure of the rizospheric bacterial community, in addition to negatively affecting bacterial diversity, which could have serious effects on the functioning of this community, especially in N mobilization. These negative effects in the rizospheric bacterial communities have a clear relationship with the 6

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Fig. 3. Clustering analysis and Heatmap based on the relative abundance of the most abundant bacterial genera in soil before contamination (US) and soil after contamination (CS), treatments with low (CdL), medium (CdM) and high (CdH) Cd concentrations. Numbers indicate replicates of treatments (three replicates per treatment).

et al., 2007, 2015; Li et al., 2014; Chodak et al., 2013). The increase in abundance of Tumebacillus and Bacillus may be related to their ability to tolerance environmental stress (Wang et al., 2014; Radhakrishnan et al., 2017). For Massilia genus, it has been reported that several of its members produce siderophores, which chelate heavy metals to increase their bioavailability and facilitate their absorption through the roots of the plants to which they are associated (Wang et al. 2014; Radhakrishnan et al., 2017; Ofek et al., 2012). These three microbial groups have great importance in the nitrogen cycle in the soil, mainly in nitrate reduction (Rosenberg et al., 2014a, 2014b; Caiola and Billi, 2007); for this reason, an increase of nitrate reducers abundance in the contaminated treatments with respect to the soil prior to contamination with Cd was observed (Fig. 4). High-throughput sequencing such as that performed in this work allows a much deeper exploration of microbial diversity; however, the definition of the functional characteristics of the taxonomic groups is limited to the closest culturable representatives from each sequence. Despite this limitation, the allocation of functional traits of closely related bacteria is based on the degree of generalized phylogenetic conservation of many of these functional traits in prokaryotes (Martiny et al., 2013). Approaches based on the allocation of functional features of close relatives have been proposed for the successful prediction of gene content (Langille et al. 2013; Goberna and Verdú, 2016), therefore, it is considered that the effects observed in this work reflect the possible effects of Cd contamination on the functional groups of the rhizosphere microbial community. The chemical properties of soil can enhance the negative effect of Cd on bacterial diversity since the behavior of this metal (adsorption-

increase in Cd absorption by the plant. Despite the plant not showing signs of physiological stress reflected in the photosynthetic yield, revealing its high resistance to contamination with Cd, a slight increase in the electron transport rate at low Cd concentration was observed. This increase correlates with an increase in the gene copy of diazotrophs and microbial C, suggesting an interesting response from the entire system to the presence of low Cd concentration in soil (Fig. 2b and d). Thus, soil contamination with Cd selected microorganisms that are tolerant or resistant to this metal (Valls and De Lorenzo, 2002). In the analysis of changes in the relative abundance of functional groups based on the literature review, we observed a significant increase of groups of bacteria with a tolerance to heavy metals, and which have been identified in bioremediation processes mainly due to the presence of genera such as Kribella (Actinobacteria), Nocardioides (Actinobacteria), Burkholderia-Paraburkholderia (Proteobacteria) and Massilia (Proteobacteria) (Table S2). In general, it has been reported that Actinobacteria exhibit tolerance to high concentrations of Cd in soils contaminated with heavy metals due to their ability to mobilize metals through various metabolic mechanisms (Hema et al., 2014; Sathya et al., 2017). Among the most relevant changes in the microbial composition, a negative effect of Cd was observed in groups of Acidobacteria (Uncultured RB41 and Sub-group 6) and Proteobacteria (Microvirga) that could fulfill functions of carbon and nitrogen fixation in the soil (Ai et al. 2018). In contrast, other groups such as Tumebacillus (Firmicutes), Bacillus (Firmicutes) and Massilia (Proteobacteria) thrived under the stress caused by the metal, a behavior similar to that found in other soils contaminated with heavy metals (Bourceret et al. 2016; Zhang 7

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Fig. 4. Changes in the relative abundance of functional groups (%) in contaminated soil after 30 days with respect to soil treatments before contamination with Cd. Asterisks indicate significant differences (*p < 0.05, **p < 0.01). Bars represent standard errors.

enter the cell membrane by diffusion to the cytoplasm leading to a decrease from bacterial growth (Bolan et al., 2014; Huang et al., 2008). Nevertheless, the NH4 content and Cd availability in the soil matrix studied are closely related because ammonium ions compete with Cd for exchange sites, the displacement of ammonium by Cd could favor

desorption in exchange sites of clay and soil organic matter) is strongly influenced by the cationic exchange capacity of the soil, organic carbon content and pH of the soil solution. The positive correlation shown between the pH of the soil and the Shannon index indicates that as the pH decreases the Cd is more bioavailable, which allows the Cd+2 to

Table 4 Photosynthetic yield and electron transport rate (ETR) (μmol m−2 s−1) (Mean ± SD; n = 3) of Echinocactus platyacanthus in treatments before contamination and after 30 days of contamination. Control: without Cd; CdL: Low Cd concentration; CdM: Medium Cd concentration; CdH: High Cd concentration. Period

Uncontaminated

Contaminated

F value and significance

Treatment

YIELD

Control CdL CdM CdH Control CdL CdM CdH Period Tratment Show Uncited Period*Treatment

p ≤ 0.001 = ***, p ≤ 0.01 = **, p ≤ 0.05 = *. 8

ETR

Fv/Fm 6:00 h

ΦPSII 13:00 h

13:00 h

0.85 ± 0.00 0.85 ± 0.01 0.84 ± 0.01 0.82 ± 0.03 0.85 ± 0.01 0.86 ± 0.01 0.86 ± 0.00 0.86 ± 0.00 19.187*** 1.656 3.040

0.77 ± 0.01 0.76 ± 0.02 0.7 ± 0.02 0.75 ± 0.02 0.82 ± 0.02 0.81 ± 0.00 0.77 ± 0.06 0.78 ± 0.02 5.575* 1.659 2.387

76.03 ± 9.12 72.00 ± 3.34 68.83 ± 3.53 64.47 ± 3.68 44.03 ± 11.13 63.17 ± 17.38 89.17 ± 11.57 57.80 ± 21.22 15.224** 2.199 1.275

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2012; Li et al., 2015); however, an interesting increase in the ETR was observed at low Cd concentration (Fig. 2d) suggesting a positive stimulation. According to previous findings there is some potentially positive influence of Cd on plant growth at low concentrations (Guo et al., 2004; Li et al., 2015), especially in some hyperaccumulators such as Arabis paniculata F. (Qiu et al., 2008), Sedum alfredii Hance (Yang et al., 2004), Picris divaricata (Ying et al., 2010), Thlaspi caerulescens (Liu et al., 2008), Potentilla griffithii (Hu et al., 2009) and Viola baoshanensis (Liu et al., 2004). The reason why the treatment by Cd can facilitate the growth of plants is unclear; however, there is some evidence as to their role as nutrient by substituting Zn to form the carbonic anhydrase, which is a Cd-containing enzyme involved in growth promotion (Lane et al., 2005); this is an interesting mechanism to be understood. The BCF and TF indicates that the ability of cacti to absorb and translocate Cd from the soil is not entirely dependent on the Cd concentration in the soil. It is also related to the plant physiology and shielding effect, so when Cd stresses the plant, it has the ability to actively regulate the concentration of this metal in their tissues (Wang et al., 2019). The high accumulation of Cd in E. platyacanthus, which is a wild plant of rural use as food and for ornamental proposals, indicates that it has a great capacity for the phytoacummulation of heavy metals, but by continuing its consumption this plant could represent a risk to animal and human health. As we have seen, the effect of pollutants such as cadmium on soil quality and plant health depends on multiple factors for which it does not necessarily follow a linear pattern; instead, it can show ecological thresholds or inflection points (Huggett, 2018; Groffman et al., 2006; Epelde et al., 2016). Establishing risk limits based solely on the concentration of Cd in the soil is not adequate since bioavailable fractions can be affected by both physicochemical properties and bacterial communities (Chen et al., 2019). This work also suggests a close relationship between the availability of Cd and the N mobilization in soil. Due to the complexity of factors that determine the Cd bioavailability in the soil and their negative effects, risk assessments and regulations need to consider several aspects of soil properties to establish ecological risk limits. Combining information from different properties including soil physicochemical parameters, microbial community diversity, microbial functions and plant stress signaling might be more indicative of overall soil health and less dependent on the response of individual properties. In this work, we have demonstrated that the factors analyzed here can be indicators of the quality and health of the soil, being a complement to explaining the behavior of metals in the soil matrix, and thus be able to understand the mechanism of tolerance to Cd contamination in soil.

Table 5 Bioconcentration factor (BCF) and translocation factor (TF) of Cd in Echinocactus platyacanthus. (mean ± SD, n = 3). Treatment

BCF

TF

Control CdL CdM CdH p value

0.00 ± 0.00a 0.55 ± 0.17b 0.58 ± 0.09b 0.60 ± 0.10b < 0.001

0.00 ± 0.00a 0.20 ± 0.03b 0.20 ± 0.02b 0.17 ± 0.01b < 0.001

the losses of available N (Mitchell et al., 2000; Gray et al., 2002); in this way when the Cd concentration increased there was a decrease of ammonium. Coupled with this, the increase of nitrate concentration and nitrate reducers abundance could stimulate denitrification, further enhancing the loss of N from soil. Contrary to expectations, the solubilization of Cd in the soil induced a significant increase in microbial carbon, but only in concentrations lower than 30 mg kg−1, indicating that low Cd concentrations promotes the microbial physiological adaptation induced by Cd stress relied on cellular mechanisms, which are energy demanding. In addition, this result could be due to the biological response to Cd at low concentrations may be more meaningful as it is closer to field conditions (Shentu et al., 2008). Thus, above 30 mg kg−1 cellular mechanisms would increase the maintenance energy and reduce the conversion of substrate into new microbial biomass. We found that microbial C decreased to concentrations higher than 40 mg kg−1, subsequent to the reduction of carbon mineralization and nitrogen fixation in soils (Shentu et al., 2014; Zhang et al., 2018) given by the negative correlation found between microbial C, organic carbon (CO) and NH4+. Similarly, CdW caused an increase in the abundance of diazotrophs at concentrations lower than 30 mg kg−1 (Fig. 2c). Similar behavior was reported by Moreira et al. (2008) and attributed to the fact that diazotrophs acquire an adaptation to excess of heavy metals, since they can detect signs of environmental stress that result from the stimulation of the entire population, which respond by forming biofilms on the surface of the roots of plants by “quorum sensing”. This increment in their population density in association with other regulatory systems, such as the production of chelating agents, siderophores and phytohormones, activate environmental signals and tolerance of the microbes and plants to heavy metals stress (Ullah et al., 2015) (Fig. 4). However, at higher concentrations a negative effect of Cd in diazotrophs is observed, represented by the decrease in their abundance. The high tolerance to environmental stress of Echinocactus platyacanthus was confirmed (Aragón et al., 2017). Large accumulation of Cd in its stem and root was observed, being proportional to the Cd content in the soil (Fig. 2e and f), which is verified by its positive correlations with the forms of Cd (CdT, CdW and CdE, p < 0.01); while its negative correlation with pH indicates that as the rhizosphere becomes acidic, a greater accumulation of metal in the plant is produced due to molecules that bind and immobilize it as amino acids, organic acids, phenolic compounds, among others (Benavides et al., 2005; Antoniadis et al., 2017). The greater accumulation of Cd in the root in comparison to its accumulation in the stem (p < 0.05) is possibly due to the limitation of the translocation of the metal in the plant that generates the exudates of the root by means of the formation of organo-metallic complexes on the surface of it; in addition to another series of internal tolerance mechanisms that plants can perform to maintain homeostasis (Antoniadis et al., 2017). Furthermore, when comparing the absorption of Cd in E. platyacanthus with other plant species of Echinocactus, Berberis, Opuntia and Larrea grown in soils contaminated with heavy metals (Levresse et al., 2012), it is observed that this plant has a wide absorption capacity. Despite a considerable accumulation of Cd in their tissues, the photosynthetic yield was not affected, which reflects its high resistance to this metal since this parameter has been reported as an indicator of the negative effect of Cd contamination in other plants (Bouzon et al.,

Funding This work was supported by the Consejo Nacional de Ciencia y Tecnología, Meé through the project SEP-CONACYT Basic Science 254,406. Acknowledgements We would like to thank to Rodrigo Velazquez-Duran for the microbial C analyses, Ma. del Carmen Rocha for measurements of heavy metal concentrations, the Department of Science, Technology and Innovation (COLCIENCIAS). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.rhisph.2020.100187. References Ai, C., Zhang, S., Zhang, X., Guo, D., Zhou, W., Huang, S., 2018. Distinct responses of soil bacterial and fungal communities to changes in fertilization regime and crop rotation.

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