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Article

Bacterial Communities in the Rhizosphere of Common Bean Plants (Phaseolus vulgaris L.) Grown in an Arable Soil Amended with TiO2 Nanoparticles

by
Gabriela Medina-Pérez
1,2,
Laura Afanador-Barajas
1,
Sergio Pérez-Ríos
2,
Yendi E. Navarro-Noya
3,
Marco Luna-Guido
1,
Fabián Fernández-Luqueño
4 and
Luc Dendooven
2,*
1
Laboratory of Soil Ecology, Cinvestav, Mexico City 07360, Mexico
2
Instituto de Ciencias Agropecuarias, Universidad Autónoma del Estado de Hidalgo, Tulancingo de Bravo 43600, Mexico
3
Laboratory of Biotic Interactions, Centro de Investigación en Ciencias Biológicas, Universidad Autónoma de Tlaxcala, Tlaxcala 90120, Mexico
4
Programa en Sustentabilidad de los Recursos Naturales y Energía, Cinvestav Saltillo Industrial, Saltillo 25900, Mexico
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(1), 74; https://doi.org/10.3390/agronomy14010074
Submission received: 6 December 2023 / Revised: 23 December 2023 / Accepted: 23 December 2023 / Published: 28 December 2023
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

:
The use of nanoparticles, such as titanium dioxide (TiO2-NPs), has increased substantially over the years. Some of them will end up in the soil, where their effect on plants and the soil bacterial community needs to be studied to determine their possible environmental risks. In this paper, beans (Phaseolus vulgaris L.) were cultivated in soil with 0, 150, or 300 mg TiO2-NPs kg−1. Plant development, nodule formation, chlorophyl content, and the bacterial community were monitored in uncultivated, non-rhizosphere, and rhizosphere soils. TiO2-NPs did not affect the beans’ growth and their chlorophyl content, but they did increase bacterial diversity and had a significant effect on the bacterial community structure in the rhizosphere, but not in the bulk and non-rhizosphere soil. Although the relative abundance of most bacterial groups varied with the TiO2-NP application rate, the cultivation of the bean plants, or the exposure time, that of Acidobacteria decreased, while that of Planctomycetes increased in the TiO2-NP-amended soil. Many bacterial groups were affected by the cultivation of the bean plants, i.e., the relative abundance of Acidobacteria, Gemmatimonadetes, Deltaproteobacteria, and Firmicutes mostly decreased in the rhizosphere independent of the application of TiO2-NPs or the time of exposure, while most groups belonging to Actinobacteria, Bacteroidetes, Rhizobiaceae, Phyllobacteriaceae, and Sphingomonadaceae were enriched.

1. Introduction

Nanotechnology is a fast-growing research field with many applications in medicine, energy production, agriculture, electronics, drug administration, and medical diagnostics [1,2,3,4,5]. The global production of engineered nanosized particles, i.e., <100 nm, reached 260,000–309,000 kg in 2010, and large amounts of these nanoparticles (≤ 28%) could end up in the soil, water (≤7%), and the atmosphere (1.5%) [6]. Titanium dioxide nanoparticles (TiO2-NPs) are one of the most used nanomaterials. They are turbid with a whitish appearance, and its photocatalytic activity has been applied for products such as paints, colorants, plastics, cosmetics, cleaning and personal care products, toothpaste, and sunscreen [7]. In the United States, the production of TiO2-NPs could reach 2.5 million tons by 2025, and a large amount could end up in the environment [8].
Titanium dioxide nanoparticles could affect plant development and alter the bacterial community structure in the soil [9]. Plants naturally interact with titanium as it is the second most abundant transition metal in the soil (on average 6.3 mg kg−1) [10,11]. Titanium can change the content of some essential elements in plant tissues and affects the activity of some enzymes (peroxidase, catalase, and nitrate reductase) [12,13]. Engineered TiO2-NPs could negatively or positively affect plant growth. The reactivity of nanoparticles depends on their composition, form, weight, size, aggregation, sedimentation or diffusion into cells, soil conditions, the dose and method of application, and plant conditions [14,15,16,17,18,19,20]. At specific concentrations, Fe3O4, ZnO, nTiO2, Ag, and SiO2-NPs improve plant development, such as germination, antioxidant, and enzymatic activity; the synthesis of chlorophyll; and growth [11,21,22,23,24,25,26,27,28,29]. Bao-Shan et al. [22] applied nano-SiO2 (nSiO2) to Larix olgensis H. var. changpaiensis seedlings and found that SiO2-NPs improved root length, mean height, root collar diameter, and the number of lateral roots. TiO2-NPs, however, did not affect the root elongation of oilseed rape (Brassica napus L.), wheat (Triticum aestivum L.), Arabidopsis (Arabidopsis thaliana L.), cucumber (Cucumis sativus L.), lettuce (Lactuca sativa L.), rice (Oryza sativa L), and radish (Raphanus sativus L.) [30,31,32]. Asli and Neuman [33] found that TiO2-NPs inhibited leaf growth and transpiration as the root water transport was inhibited in maize seedlings.
Titanium dioxide (TiO2) applied in agricultural practices at low concentrations to leaves or roots of crops has been shown to improve plant development by increasing photosynthesis and chlorophyll content and stimulating the activity of certain enzymes, thereby improving stress tolerance, increasing nutrient uptake, and in turn, crop yield and quality [34,35]. As such, it is important to understand the possible effect of nanoparticles on plant roots [36]. For instance, Fan et al. [37] found that the exposure of garden peas (Pisum sativum L.) to TiO2-NP decreased the number of secondary lateral roots. They also analyzed cultured R. leguminosarum Viciae 3841 as its common rhizobial partner and found alterations in the morphology of cultivated bacterial cells. The interaction between plants and bacteria was also affected by TiO2-NP as root nodule formation and N2 fixation were reduced, while the polysaccharide composition of the nodules’ walls changed.
The accumulation of specific nanoparticles alters the soil microorganisms’ activity, diversity, abundance, and growth [38,39,40]. Ge et al. [41] reported that TiO2-NPs changed the abundance and diversity of bacterial communities in grassland soils. The relative abundance of bacteria involved in refractory organic compound mineralization increased, and those potentially involved in methane oxidation and N2 fixation decreased. Beans have a symbiotic relationship with N2-fixing bacteria, but it is unknown if TiO2-NP affects this symbiosis and thus the development of the plants. Therefore, in this paper, common bean plants (Phaseolus vulgaris L.) are cultivated in an arable soil applied with TiO2-NPs at 0, 150, and 300 mg TiO2 kg−1 soil, while plant development, soil characteristics, and the bacterial community are determined 45 and 90 days after planting. This work aims to study the effect of TiO2-NPs on (i) bean plant development, (ii) soil characteristics, and (iii) the bacterial community in uncultivated soil, non-rhizosphere soil, and the rhizosphere of common bean plants (Phaseolus vulgaris L.). It was hypothesized that the TiO2-NPs and bean plants alter the bacterial community, but TiO2-NPs do not affect bean plant development.

2. Materials and Methods

2.1. Common Bean, Soil, and Nanoparticles

Common bean (Phaseolus vulgaris L.) var. Pinto Saltillo-certified seeds were purchased from “El Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias” (INIFAP, Celaya, México). The seeds were stored in the dark at 4 °C until further use. Uncoated TiO2-NPs were obtained from “Materiales Nanoestructurados S.A de CV” (San Luis Potosí, México). The physicochemical characteristics of TiO2-NPs were provided by the manufacturer and are presented in Table S1.
The arable soil was sampled at the Institute of Agricultural Sciences of the Autonomous University of Hidalgo (Pachuca, Hidalgo State, Mexico), latitude 20°04′53″ N, latitude 98°22′07″ W. The 0–20 cm top-soil layer was sampled ten times from three 400 m2 areas. The 10 soil samples taken in each area were pooled, passed separately (n = 3) through a 2 mm sieve, characterized, and extracted for DNA as described below. The different treatments were applied to each of the soil samples (n = 3) to avoid pseudo-replication [42]. The agricultural soil was a haplic phaeozem (FAO/UNESCO soil classification system) with pH of 7.5, electrolytic conductivity (EC) of 5.3 dS m−1, and a water holding capacity (WHC) of 625 g kg−1. The organic carbon content was 3.6 g C kg−1 and the total N content was 0.21 g N kg−1. All soil characteristics provided in the manuscript are on a dried soil base.

2.2. Experimental Design and Greenhouse Experiment

More details of the experimental design schematized in Figure S1 can be found in Medina-Pérez [43]. The experimental design is. Six different treatments were applied to the arable soil combining the application of 0, 150, and 300 mg TiO2-NPs kg−1 and soil left uncultivated or cultivated with common beans (Phaseolus vulgaris L.). Plant development and the bacterial community in the uncultivated, non-rhizosphere, and rhizosphere soils were determined after 45 and 90 days. The experiment was done in a greenhouse at Cinvestav-Zacatenco (Mexico). A completely randomized block design was used.
Thirty-six PVC columns (17 cm ∅ × 60 cm heigh) closed at the bottom with a perforated PVC tap were amended with red tezontle, which is a porous, highly oxidized, and volcanic rock (0.5 kg). Twice-washed sand was placed on top of the tezontle (5 cm) to avoid washing out of the soil. A 7 kg sub-sample of soil was added on top of the sand.
Titanium dioxide NPs were suspended in 200 mL deionized water and sonicated for 30 min. The soil of one third of the columns (n = 12) was mixed with 150 mg TiO2-NPs kg−1 dry soil, a third (n = 12) with 300 mg TiO2-NPs kg−1 dry soil, and a third (n = 12) was mixed but left unamended. As such, three different amounts of TiO2-NPs were applied to the soil, i.e., 0, 150, and 300 mg TiO2-NPs kg−1. Three bean seeds were planted at 2 cm of depth in half of the columns amended with the three different concentrations of TiO2-NPs kg−1 (n = 6). As such, six treatments were applied to the three soil samples, i.e., three different application rates of TiO2-NPs kg−1 left uncultivated or cultivated with the bean plants. The PVC columns were placed in the greenhouse for 90 days. Two of the three emerging plantlets were taken from the soil columns one week after emergence, so that only one plantlet, i.e., the most vigorous, was kept per column. Plant chlorophyll content was determined on four aleatory leaves from three plants of each treatment using a Minolta SPAD-502 chlorophyll meter [44] every two days, starting 12 days after sowing. The plants were irrigated every third day for 90 days so that the water content remained at 50 ± 5% WHC during the entire experiment. The water content in the uncultivated soil was also adjusted to 50 ± 5% WHC when required. The irrigation was such that no leaching occurred during the entire experiment. The average daily temperature in the greenhouse ranged between 20 and 25 °C and the moisture content was in the range of 35–48%.
Three PVC columns were randomly selected from each treatment (n = 6) after 45 and 90 days. The soil and plants were removed from the columns. The plants were gently shaken by hand for 5 min and the detached soil was considered as the non-rhizosphere soil. The soil was brushed from the roots, collected, and considered as the rhizosphere soil. At the same time, the soil was removed from the uncultivated soil columns and considered as the uncultivated or bulk soil. The soil was characterized from the bulk, non-rhizosphere, and rhizosphere and extracted for DNA as described below. The root and shoot lengths and fresh weight of the bean plants were determined. The roots and shoots were dried at 60 °C and weighed.

2.3. Soil and Nanoparticle Characterization

The gravimetric soil moisture content was determined by drying the soil at 120 °C for 48 h and the measuring the weight loss. The WHC was measured by saturating a 20 g soil sub-sample with distilled water and leaving it to drain freely for 24 h. The water retained in the soil sample was considered the WHC and expressed as the amount of water (g) per kg soil [45]. The EC was determined in a 1:5 soil/H2O suspension in water paste with a HI 933300 microprocessor (HANNA Instruments, Smithfield, RI, USA) [46]. The pH was determined in a 1:2.5 soil/H2O suspension soil–water paste using a calibrated potentiometer (Denver Instrument, Bohemia, NY, USA) [47] with a glass electrode (3007281 pH/ATC Thermo Fisher Scientific, Waltham, MA, USA). The soil particle size distribution was determined with the hydrometer method [48]. A 10 g soil sub-sample was extracted for mineral N (nitrate (NO3−), nitrite (NO2−), and ammonium (NH4+) with 100 ml 0.5 M K2SO4. The mineral N content in the extract was measured on a San Plus System-SKALAR automatic analyzer (Skalar, Breda, The Netherlands) [49]. A JEOL 2000 transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 80 kV was used to determine the size and morphology of the NPs. The software ImageJ was used to measure the particles.

2.4. DNA Extraction and PCR Amplification

A 0.5 g sub-sample of soil was used for each extraction. The samples were washed with 0.15 mol L−1 sodium pyrophosphate until the samples were clear [50]. Excess pyrophosphate was removed with 0.15 mol L−1 phosphate buffer with pH 8. The DNA was extracted using three different methods to lyse the cells [51,52,53]. A washed 0.5 g soil sub-sample was used for each extraction method applied. The metagenomic DNA obtained with the three techniques was pooled. The PCR amplified the hypervariable regions V3 and V4 of the bacterial 16S rRNA gene. The PCR products were pooled and purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and quantified with a Nanodrop 3300 (TermoFisher, USA) with PicoGreen dsDNA. The samples were mixed in equimolar amounts and sequenced with an Ilumina MiSeq by Macrogen Inc. (Seoul, Republic of Korea).

2.5. rDNA Sequence Analysis

The raw sequences were analyzed with the “Quantitative insights into microbial ecology pipeline” (QIIME) version 1.9.0 software pipeline [54]. First, poor quality reads were excluded from further analyses, i.e., no ambiguous base calls and quality values less than 25 Phred Q score. Paired-end sequences were assembled with the fastq-join method within QIIME. Operational taxonomic units (OTUs) were clustered at a 97% similarity level using the UCLUST algorithm [55]. Chimeras detected were removed from the data using the Chimera Slayer [56]. Sequence alignments were conducted against the Greengenes core set using representative sequences of each OTU using PyNAST and filtered at a threshold of 75% [57]. Taxonomic assignment was conducted using the naïve Bayesian rRNA classifier from the Ribosomal Data Project [58] at a confidence threshold of 80%. The obtained 16S dataset was filtered, all operational taxonomic units (OTUs) assigned to Archaea were discarded, and the dataset was normalized.

2.6. Data Accessibility

Sequence reads generated in this study were submitted to the NCBI Sequence Read Archive under accession number SUB13480672.

2.7. Phylogenetic and Statistical Analyses

All statistical analyses were performed in R (v. 4.2.2) [59]. An ANOVA test (aov function) was used to determine the effect of TiO2-NPs on plant development. The alpha diversity of the soil bacterial community using the assigned groups, assigned up to the taxonomic level of genus, was determined based on the Hill numbers at different q orders (at q = 0, 1, and 2) [60]. The Hill number at q = 0 reflects genus richness, q = 1 is the Shannon entropy and denotes frequently occurring genera, and q = 2 is the inverse Simpson and characterizes dominant genera. Hill numbers were calculated as proposed by Chao et al. [61] using the Package hillR (v. 0.5.1) [62]. A non-parametric test was used to determine the effects of the TiO2-NP application and the cultivation of the bean plants on the Hill numbers. The t1way test of the WRS2 package (A collection of robust statistical methods, v. 1.1-0) was used [63]. The phylogenetic beta diversity was determined with the betapart R package [64].
Ordination (principal component analysis (PCA)) and multivariate comparison (perMANOVA) were conducted with converted sequence data using the centered log-ratio transformation test returned by the aldex.clr argument [65]. The ALDEx2 package (v. 1.21.1, Analysis of differential abundance taking sample variation into account [66]) was used for this centered log-ratio transformation. The effects of the application of TiO2-NPs (0, 150, and 300 mg TiO2-NPs kg−1) and the cultivation of the bean plants (bulk, non-rhizosphere, and rhizosphere soil) on the bacterial community structure were visualized with a PCA analysis using the FactoMineR package (v. 2.3, Multivariate exploratory data analysis and data mining [67]). A perMANOVA test was used to determine the effects of TiO2-NPs, soil, and time (days 0, 45, and 90) and their interactions on the bacterial community structure. The perMANOVA test was conducted with the vegan package (v. 2.5-6, Community ecology package [68]).
A non-parametric Kruskal–Wallis test was used to determine the effects of the application of TiO2-NPs and the cultivation of bean plants on the different bacterial groups with the ALDEx2 package (v. 1.21.1 [64]). The “effect size” [69] was calculated with the ALDEx2 package (v. 1.21.1, [66]) comparing the relative abundance of the bacterial groups in the different soils, i.e., left unamended or amended with different amounts of TiO2-NPs and left uncultivated or cultivated with bean plants. The effect size was plotted versus the expected p-value of the Kruskal–Wallis test in a volcano plot generated in Microsoft® Excel for Mac v.16.62.

3. Results

3.1. Plant Soil Characteristics

None of the bean plant characteristics were affected significantly by the application of TiO2-NPs, although some of them increased significantly over time (p ≤  0.05) (Table 1). Chlorophyll values increased until approximately day 45, remained constant until day 70, and then decreased (Figure S2). The application of TiO2-NPs had no significant effect on the chlorophyll content of the plants.
The application of TiO2-NPs had no significant effect on the soil characteristics after 45 days (Table 2 and Table S2). The cultivation of the bean plants decreased the EC significantly after 45 days (p ≤ 0.05). After 90 days, the application of TiO2-NPs increased the WHC and the concentration of NO3−, as well as the interaction with the cultivation of the bean plants (p ≤ 0.01).

3.2. Bacterial Community

The number of reads was filtered and rarefied at 4494 to include all 72 samples in the analysis, generating a total of 323,568 reads, whose sequences resulted in 63,075 OTUs. The rarefication curve of the number of sequences versus the number of OTUs was asymptotic; therefore, analyzing more sequences would yield only a limited number of new OTUs.
The Hill numbers at q = 0 and q = 1 showed some variation over time in some treatments (Figure S3). The dominant bacteria or the Hill number at q = 2 was similarly independent of the application TiO2-NPs, the cultivation of common bean plants, or time. The application of TiO2-NPs significantly increased the Hill numbers at q = 0 or the bacterial richness and at q = 1 or frequent genera in the rhizosphere compared to the unamended soil, but not in the bulk and non-rhizosphere soils (Table 3). The Hill number at q = 1 was significantly higher in the rhizosphere than in the bulk and non-rhizosphere soils amended with 150 mg TiO2-NPs kg−1.
The Jaccard pair-wise dissimilarity (beta.jac, Table S3) was similar in the TiO2-NP-applied soils compared to the unamended soil. The dissimilarity was mostly explained by a 1-to-1 substitution or turnover of bacterial genera (beta.jtu, Table S3). However, the loss (nestedness) of genera, i.e., not being detected (beta.jne, Table S3), was sometimes higher than a 1-to-1 substitution, e.g., when comparing the unamended soil with the soil to which 300 mg TiO2-NPs kg−1 was applied. The loss of bacterial genera was also often larger after 90 days than after 45 days. A similar pattern emerged when comparing the bulk soil with the rhizosphere and non-rhizosphere soils.
Proteobacteria dominated in the unamended and uncultivated soil (mean relative abundance of 52.5%), followed by Acidobacteria (21.4%) and Firmicutes (7.3%) (Figure 1). Phylotypes belonging to iii1-15 (Acidobacteria, 8.2%), other Rhodospirillaceae (5.5%), and SC-I-84 (Betaproteobacteria, 5.4%) were the most abundant bacterial groups assigned up to the taxonomic level of genus, while Bacillus (3.8%) and Halomonas (3.5%) were the most dominant genera. The application of nanoparticles had only a small effect on the relative abundance of the bacterial phyla, but many bacterial genera were strongly and significantly affected by their application in the bulk, non-rhizosphere, and rhizosphere soils (p ≤ 0.05, Table 4, Figure S4a). The PCA separated the unamended rhizosphere soil from the rhizosphere soil to which 300 kg TiO2-NPs kg−1 was applied considering all bacterial groups assigned up to the taxonomic level of genus, but not in the bulk and non-rhizosphere soils (Figure 2).
The application of nanoparticles had a significant effect on the bacterial genera or OTU community structure in the rhizosphere (p ≤ 0.05), but not in the bulk and non-rhizosphere soils (Table 4 and Table S4). Many bacterial groups assigned up to the taxonomic level of genus were affected by the application of TiO2-NPs (Table S5). More bacterial groups were affected when 300 mg TiO2-NPs kg−1, rather than 150 mg TiO2-NPs kg−1, was applied (considering days 45 and 90) and more were affected in the non-rhizosphere and rhizosphere soils than in the uncultivated soil amended with 300 mg TiO2-NPs kg−1 (Table 5).
The cultivation of bean plants had a strong effect on a wide range of bacterial groups considering all TiO2-NP application rates (Table S6, Figure S4b). The PCA separated the bacterial community structure in the uncultivated soil from that in the rhizosphere, and was the most accentuated when 300 mg TiO2-NPs kg−1 was applied to the soil (Figure 3). The perMANOVA analysis showed that the cultivation of the bean plants had a significant effect on the bacterial genera and OTU community structure (p ≤ 0.016) (Table 4 and Table S4).

4. Discussion

4.1. Soil and Plant Characteristics

Nanoparticles might affect plant development, but not always [70]. Cox et al. [71], in a review of the effect of silver and TiO2-NP toxicity in plants, reported that exposure to NPs produces reactive oxygen species in the cells, which may have positive and negative effects on plant growth. They also stated that the NP characteristics, such as size, shape, surface coating, and concentration, determine a possible effect and that different plants respond differently to NP exposure, with some showing positive, but others negative effects. Burke et al. [70] found no significant effect of TiO2-NPs on soybean (Glycine max L.) and maize (Zea mays L.) growth and their nutrient content. In this study, the application of TiO2-NPs had no significant effect on the development and chlorophyll content of the bean plants, which confirms the above-mentioned factors reported by Cox et al. [71], and soil characteristics [72], time of exposure [73], and experimental conditions determine whether NPs affect plant growth.
Kaur et al. [74] reported an increase in the nutrient content, i.e., available N, P, Cu, Fe, Mn, nitrate-N, and ammoniacal-N, in the rhizosphere of mung beans (Vigna radiata (L.) R. Wilczek) when TiO2-NPs were applied to the soil. In this study, the amount of NO3− also increased when TiO2-NPs were applied, but not that of NH4+. The effect of the TiO2-NPs on the amount of NO3− in the soil might be a combination of abiotic and biotic processes. The application of TiO2-NPs might increase the availability of NH4+, i.e., increased mineralization, which is then oxidized to NO2− and further to NO2−. This increase in NH4+ availability will increase the nitrifier activity, but might also enrich them. In this study, members of the Nitrosomonadaceae, mostly Nitrosovibrio, ammonia-oxidizing bacteria [75] were not enriched, but the relative abundance of Nitrospirales, chemolithoautotrophic nitrite-oxidizing bacteria [76], increased with the increase in the application of TiO2-NPs from 0.71% in the unamended soil to 0.77% in the soil amended with 300 mg TiO2-NPs kg−1. Simonin et al. [77] reported a negative effect on nitrification enzyme activity even at an exposure of 1 mg TiO2-NPs kg−1 and changes in the bacterial community structure after 90 days. This suggest that not only amount of TiO2-NPs applied and the time of exposure, but also the soil characteristics, e.g., texture of the soil and organic matter, determine the effect of the application of TiO2-NPs on soil processes and microbial activity [73]. In another study, Simonin et al. [72] applied 1 or 500 mg TiO2-NPs kg−1 to six arable soils with different textures and organic matter contents to study their effect on C mineralization and bacterial abundance. They found that, after 90 days, TiO2-NPs did not affect C mineralization and the bacterial community, except in one soil, and suggested that the toxicity of the TiO2-NPs most likely depended on pH and soil organic material and less on soil texture.
Komendová et al. [78] stated that the strong hydration of nanoparticles, i.e., platinum nanoparticles in their study, affects the water structural network in the soil, but the interaction with soil organic matter partially reduces this effect. In this study, the application of TiO2-NPs increased the soil WHC after 90 days presumably due to their hydration, but the cultivation of bean plants reduced these effects as dying roots and exudates increased the soil organic matter content. However, Suazo-Hernández et al. [79] stated that the possible effects of metal- or metallic oxide-engineered NPs on the soil physical and chemical characteristics depend on the soil properties, time of exposure, amounts of NP applied, and the type of NPs.

4.2. Bacterial Community

Simonin et al. [72] studied the effect of 1 or 500 mg TiO2-NPs kg−1 on the bacterial community and reported that some taxa were enriched, but the relative abundance of more taxa decreased, indicating that the NPs mostly reduced diversity. Ge et al. [80] also reported that TiO2-NPs reduced bacterial diversity and suggested that it was the result of direct toxicity rather than TiO2-NPs indirectly affecting soil water and organic matter pools. In this study, however, TiO2-NPs increased the bacterial diversity in the rhizosphere, but had no effect in the bulk and non-rhizosphere soils. The Hill number at q = 0 or the bacterial diversity increased from 182 in the unamended soil to 259 when 150 mg TiO2 kg−1 and to 325 when 300 mg TiO2 kg−1 were applied. This indicates that the application of TiO2-NPs increased the availability of root exudates, i.e., providing more C substrate, thereby increasing bacterial diversity, or the dying roots and exudates reduced the direct toxicity of TiO2-NPs, i.e., chelate formation [81].
The application of TiO2-NPs often alters the bacterial community structure (e.g., [82,83]; as reviewed by Sun et al. [84]), but not always. For instance, Asadishad et al. [85] reported that the microbial community in agricultural soil amended with biosolids and spiked with TiO2-NPs at 1, 10, or 100 mg kg−1 soil showed negligible changes after 30 days. Gorczyca et al. [86], however, found that TiO2NPs applied at 100 mg L−1 significantly increased the number of bacteria in the rhizoplane of 21-day old flax plants (Linum usitatissimum L.). In this study, TiO2-NPs also had a highly significant effect on the bacterial community structure in the rhizosphere, but not in the uncultivated and non-rhizosphere soils.
Gorczyca et al. [86] found that Pseudomonas and Bacillus were enriched by TiO2NPs in the rhizoplane of 21-day-old wheat plants and Bacillus in the flax rhizoplane. They also found that TiO2NPs reduced the relative abundance of Clostridium in the rhizoplanes of wheat and flax, but less so than AgNPs. In this study, members of Bacillus were also enriched when TiO2NPs was applied to soil after 45 and 90 days (Table S7). The relative abundance of Pseudomonas, however, was strongly reduced in the rhizosphere of the bean plants by the application TiO2NPs after 90 days, while that of Clostridium increased after 90 days when 300 mg TiO2NPs kg−1 was applied. Ge et al. [80] found that TiO2-NPs reduced the relative abundance of taxa known to be associated with nitrogen fixation (Rhizobiales, Bradyrhizobiaceae, and Bradyrhizobium) and methane oxidation (Methylobacteriaceae), enriched taxa involved in the decomposition of recalcitrant organic pollutants (Sphingomonadaceae) and biopolymer production (Streptomycetaceae and Streptomyces). Considering the effect of TiO2-NPs on these bacterial groups and on Pseudomonas, Bacillus, and Clostridium [87], we confirmed in this study that the possible effect of the NPs depends on the cultivation of the bean plants, the concentration of the NPs applied, and the residence time (Table S7). For instance, the relative abundance of Rhizobium mostly increased when TiO2-NPs were applied to the soil, but not in the uncultivated soil and the non-rhizosphere soil amended with 150 mg TiO2-NPs kg−1 after 45 days. Additionally, the application of TiO2-NPs had no significant effect on the relative abundance of these bacterial groups and the effect size was lower than 0.8 and larger than −0.8 (Table S6). Interestingly, the relative abundance of Pseudoxanthomonas, whose species were detected recently in the nodule of Phaseolus vulgaris plants [88] and some members have N2-fixing and indoleacetic acid (IAA) production capacity (P. mexicana) [89], was strongly reduced by the application of 150 and 300 mg TiO2-NPs kg−1. Additionally, the relative abundance of members of Pseudomonas, a genus that includes species that have a N2-fixing capacity [90] and others that were detected inside healthy Lotus burttii nodules [91], increased, while that of Devosia, N2-fixing bacteria capable of forming nodules [92], was strongly reduced by the application of TiO2-NPs in the rhizosphere after 90 days.
Although the TiO2-NPs had different effects on the relative abundance of N2-fixing bacteria, some bacterial groups showed a more consistent response to TiO2-NPs in this study. For instance, of all the acidobacterial groups that were affected strongly by TiO2-NPs, i.e., an effect size of ≤−0.8 and ≥0.8 [69], only one showed a decrease, i.e., CCU21 (Acidobacteria-6), in the rhizosphere applied with 300 mg TiO2-NPs kg−1 after 45 days (Table S5). All the others showed an increase independent of the cultivation of the bean plants, the application rate of TiO2-NPs, or the time of exposure. A similar pattern emerged considering members of Planctomycetes. Only twice was the relative abundance of a group of Planctomycetes reduced when TiO2-NPs were applied to the soil; all others showed an increase independent of the cultivation of the bean plant, the application rate of TiO2-NPs, or the time of exposure.
The development of a rhizosphere when plant roots penetrate the soil is known to have a strong effect on the bacterial community structure (e.g., [93]). In this study, the bacterial community and a wide range of bacterial groups were significantly affected by the cultivation of the bean plants. The relative abundance of all groups belonging to Acidobacteria, Gemmatimonadetes, and Deltaproteobacteria and most Firmicutes, when comparing the uncultivated soil with the rhizosphere soil, was reduced (large effect size of ≤−0.8 and ≥0.8 [69]) by the cultivation of the bean plants, independent of the application of TiO2-NPs or the time of exposure. As such, these groups belonging to Acidobacteria, Gemmatimonadetes, and Deltaproteobacteria were characterized by an oligotrophic behavior with a K-strategy lifestyle, i.e., enriched in nutrient-poor environments [94]. Most groups belonging to Actinobacteria, except for the Rubrobacteraceae, and all Bacteroidetes, Rhizobiaceae (e.g., Agrobacterium and Rhizobium), Phyllobacteriaceae, and Sphingomonadaceae were enriched in the bean-plant-cultivated soil independent of the application of the TiO2-NPs or the time of exposure. The microorganisms that were enriched in the nutrient-rich rhizosphere participate in the degradation of that organic material, which often is mostly easily degradable carbohydrates and (hemi)cellulose, and are considered copiotrophic [95] or R-strategists [96].

5. Conclusions

It was found that the development of common bean plants was not affected by the application of 150 or 300 mg TiO2-NPs kg−1. The application of TiO2-NPs significantly increased the bacterial richness and frequent genera in the rhizosphere compared to the unamended soil, but not in the bulk and non-rhizosphere soils. The application of TiO2-NPs had little effect on the Jaccard pair-wise dissimilarity compared to the unamended soil, and the majority of the dissimilarity was explained by a 1-to-1 substitution or turnover of bacterial genera, although some loss of genera, i.e., not being detected, also occurred when comparing the unamended soil with the soil to which 300 mg TiO2-NPs kg−1 was applied. Proteobacteria dominated in the unamended and uncultivated soils followed by Acidobacteria, while Bacillus and Halomonas were the most dominant genera. The application of TiO2-NPs had a highly significant effect on the bacterial community structure in the rhizosphere, but not in the bulk and non-rhizosphere soils. Many bacterial groups assigned up to the taxonomic level of genus were affected by the application of TiO2-NPs, and more were affected when 300 mg TiO2-NPs kg−1, rather than 150 mg TiO2-NPs kg−1, was applied. Although the effect of TiO2-NPs on the relative abundance of most bacterial groups was not consistent and depended on the application rate, the cultivation of the bean plants, and the exposure time, the relative abundance of Acidobacteria mostly decreased, while that of Planctomycetes increased. The relative abundance of most bacterial groups belonging to Acidobacteria, Gemmatimonadetes, Deltaproteobacteria, and Firmicutes was reduced by the cultivation of the bean plants independent of the application of TiO2-NPs or the exposure time, while most groups belonging to Actinobacteria, except for the Rubrobacteraceae, and all Bacteroidetes, Rhizobiaceae, Phyllobacteriaceae, and Sphingomonadaceae were enriched in the bean-plant-cultivated soil.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14010074/s1.

Author Contributions

Conceptualization, G.M.-P., L.A.-B., F.F.-L. and L.D.; Methodology, M.L.-G. and Y.E.N.-N.; Data analysis, G.M.-P., Y.E.N.-N. and L.D.; Writing and correction of the manuscript, G.M.-P., L.A.-B., S.P.-R., M.L.-G. and L.D.; Funding acquisition, F.F.-L. and L.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Cinvestav. G.M.-P. (Grant number 426133) and L.A.-B. (Grant number 464393) received grant-aided support from the “Consejo Nacional de Humanidades, Ciencias y Tecnologías” (CONAHCYT). Part of the data analysis and part of the writing of the manuscript was conducted while L.D. was on sabbatical leave from Cinvestav at the “Centro de Investigación en Ciencias Biológicas” of the “Universidad Autónoma de Tlaxcala” (Mexico).

Data Availability Statement

The sequence reads generated in this study were submitted to the NCBI Sequence Read Archive under accession number SUB13480672.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 00074 g001
Figure 1. Bar plots of the relative abundance (%) of the 15 most abundant (a) bacterial phyla and (b) bacterial groups assigned up to the taxonomic level of genus in the uncultivated (bulk), non-rhizosphere (NoR), and rhizosphere (Rhi) soils left unamended or to which 150 mg TiO2-NPs kg−1 dry soil or 300 mg TiO2-NPs kg−1 dry soil was applied at the onset of the experiment, i.e., day 0, and after 45 and 90 days.
Figure 1. Bar plots of the relative abundance (%) of the 15 most abundant (a) bacterial phyla and (b) bacterial groups assigned up to the taxonomic level of genus in the uncultivated (bulk), non-rhizosphere (NoR), and rhizosphere (Rhi) soils left unamended or to which 150 mg TiO2-NPs kg−1 dry soil or 300 mg TiO2-NPs kg−1 dry soil was applied at the onset of the experiment, i.e., day 0, and after 45 and 90 days.
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Figure 2. A principal component analysis (PCA) with the relative abundance of bacterial groups assigned up to the taxonomic level of genus in the uncultivated, non-rhizosphere, and rhizosphere soils of the bean plants (Phaseolus vulgaris L.). Unamended soil (◯) and soil amended with 150 mg TiO2-NPs kg−1 dry soil (●) and 300 mg TiO2-NPs kg−1 dry soil (●).
Figure 2. A principal component analysis (PCA) with the relative abundance of bacterial groups assigned up to the taxonomic level of genus in the uncultivated, non-rhizosphere, and rhizosphere soils of the bean plants (Phaseolus vulgaris L.). Unamended soil (◯) and soil amended with 150 mg TiO2-NPs kg−1 dry soil (●) and 300 mg TiO2-NPs kg−1 dry soil (●).
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Figure 3. A principal component analysis (PCA) with the relative abundance of bacterial groups assigned up to the taxonomic level of genus in unamended soil, soil amended with 150 mg TiO2-NPs kg−1 dry soil or 300 mg TiO2-NPs kg−1 dry soil left uncultivated (◯), and non-rhizosphere (●) and rhizosphere soils of the bean plants (Phaseolus vulgaris L.) (●).
Figure 3. A principal component analysis (PCA) with the relative abundance of bacterial groups assigned up to the taxonomic level of genus in unamended soil, soil amended with 150 mg TiO2-NPs kg−1 dry soil or 300 mg TiO2-NPs kg−1 dry soil left uncultivated (◯), and non-rhizosphere (●) and rhizosphere soils of the bean plants (Phaseolus vulgaris L.) (●).
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Table 1. Characteristics of common bean plants (Phaseolus vulgaris L.) cultivated in soil amended with titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) after 45 and 90 days [43].
Table 1. Characteristics of common bean plants (Phaseolus vulgaris L.) cultivated in soil amended with titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) after 45 and 90 days [43].
TiO2 Length (cm)ShootFresh Weight (g)Dry Weight (g)
Time-NPsNodulesDiameter
(Days)(mg kg−1)(Number)ShootRoot(cm)ShootRootShootRoot
45010 a118 c60 b0.5 a41 b30 a6.4 b3.7 a
15015 a139 bc81 a0.4 a47 b29 a6.7 b3.6 a
30020 a144 bc62 b0.5 a44 b34 a6.5 b3.7 a
90023 a221 a77 ab0.5 a78 a38 a17.1 a5.6 a
15035 a203 ab67 ab0.5 a77 a38 a17.0 a5.8 a
30015 a255 a81 a0.5 a87 a39 a19.0 a5.9 a
Values with the same letter are not significantly different at p ≤ 0.05 within the columns.
Table 2. Characteristics of soil left uncultivated (Beans: No) or cultivated with common bean plants (Phaseolus vulgaris L.) (Beans: Yes) in soil amended with titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) after 45 and 90 days [43].
Table 2. Characteristics of soil left uncultivated (Beans: No) or cultivated with common bean plants (Phaseolus vulgaris L.) (Beans: Yes) in soil amended with titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) after 45 and 90 days [43].
Time TiO2-NPs ECWHCNH4+NO2−NO3−
(days)Beans(mg kg−1)pH(dS m−1)(g kg−1)(g kg−1)
45No07.9 ab2.23 ab625 bcd1.3 ab0.24 a12.5 ab
1507.8 b3.09 a643 bc1.5 ab0.18 a10.0 ab
3007.9 ab2.48 ab629 bcd1.4 ab0.19 a12.6 a
Yes07.9 ab1.54 b659 b1.4 ab0.25 a6.6 ab
1508.0 ab1.62 b677 cd1.5 ab0.17 a3.5 ab
3007.9 ab1.76 b840 a2.1 a0.30 a9.5 ab
90No08.2 abND504 e1.1 b0.18 a4.8 ab
1508.1 abND616 bcde1.3 ab0.16 a5.9 ab
3008.1 abND715 b1.4 ab0.20 a10.6 ab
Yes08.3 aND598 cde1.6 ab0.23 a4.6 ab
1508.0 abND517 ed1.7 ab0.26 a1.7 b
3008.1 abND594 cde2.1 a0.18 a1.8 b
Values with the same letter are similar over time in soil left uncultivated or cultivated with bean plants and in soil applied with 0, 150 or 300 mg TiO2-NPs kg−1 dry soil, ND: Not determined.
Table 3. The effect of application of titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) and cultivation of common bean plants (Phaseolus vulgaris L.) (bulk, non-rhizosphere or rhizosphere soil) on the Hill numbers at q = 0, q = 1 and q = 2 of the bacterial genera.
Table 3. The effect of application of titanium dioxide nanoparticles (0, 150 or 300 mg TiO2-NPs kg−1 dry soil) and cultivation of common bean plants (Phaseolus vulgaris L.) (bulk, non-rhizosphere or rhizosphere soil) on the Hill numbers at q = 0, q = 1 and q = 2 of the bacterial genera.
q = 0q = 1q = 2
SoilF Valuep ValueF valuep ValueF Valuep Value
Effect of nanoparticles
Bulk soil 1.260.3323.850.0570.001.000
Non rhizosphere soil1.220.3380.350.7130.001.000
Rhizosphere soil6.230.045 *7.430.034 *0.001.000
Effect of cultivation of bean plants
Unamended 0.500.6340.650.5610.001.000
Amended with 150 mg TiO2-NPs kg−10.730.5120.630.5630.001.000
Amended with 300 mg nTiO2-NPs kg−13.530.10719.670.003 **0.001.000
* p value ≤ 0.05 and ≥ 0.01, ** p value < 0.01 and ≥ 0.001.
Table 4. Permutational multivariate analysis of variance (perMANOVA) to determine the effect of nanoparticles (0, 150 or 300 TiO2-NPs kg−1 dry soil), common bean plants cultivation (Phaseolus vulgaris L.) (uncultivated, non-rhizosphere and rhizosphere soil), time (45 and 90 days) and their interactions on the bacterial groups assigned up to the taxonomic level of genus.
Table 4. Permutational multivariate analysis of variance (perMANOVA) to determine the effect of nanoparticles (0, 150 or 300 TiO2-NPs kg−1 dry soil), common bean plants cultivation (Phaseolus vulgaris L.) (uncultivated, non-rhizosphere and rhizosphere soil), time (45 and 90 days) and their interactions on the bacterial groups assigned up to the taxonomic level of genus.
perMANOVA
R2F Valuep Value
Nanoparticles (0, 150, 300 mg TiO2-NPs kg−1)0.0221.530.005 **
Bean plant (bulk, non-rhizosphere, rhizosphere)0.0381.310.016 *
Time (day, 0, 45, 90)0.0201.350.800
Interaction nanoparticles and bean plant 0.0321.110.127
Interaction nanoparticles and time 0.0171.190.091
Interaction bean plant and time0.0341.170.065
Interaction nanoparticles, bean plant and time0.0311.050.240
Effect of nanoparticles
Bulk soil0.0380.960.602
Non-rhizosphere soil0.0420.970.391
Rhizosphere soil0.1051.750.005 **
Effect of bean plant
Unamended soil 0.1021.130.124
Soil with 150 mg TiO2 kg−10.0891.020.300
Soil with 300 mg TiO2 kg−10.1251.210.056
* p value ≤ 0.05 and ≥0.01, ** p value < 0.01 and ≥0.001.
Table 5. Effect of the application of titanium dioxide nanoparticles (TiO2-NPs) on the relative abundance of bacterial genera in uncultivated (bulk) and non-rhizosphere soil, and rhizosphere of common bean plant (Phaseolus vulgaris L.). A negative effect size means that the relative abundance of the bacterial group was higher in the unamended soil than in the soil amended with 150 or 300 mg TiO2-NPs kg−1 on days 45 and 90, while a positive value means the opposite.
Table 5. Effect of the application of titanium dioxide nanoparticles (TiO2-NPs) on the relative abundance of bacterial genera in uncultivated (bulk) and non-rhizosphere soil, and rhizosphere of common bean plant (Phaseolus vulgaris L.). A negative effect size means that the relative abundance of the bacterial group was higher in the unamended soil than in the soil amended with 150 or 300 mg TiO2-NPs kg−1 on days 45 and 90, while a positive value means the opposite.
Unamended soil versus soil amended with 150 mg TiO2 kg−1 soil.
Bulk soil: other Enterobacteriaceae (−0.8 a, b * c)
Non-rhizosphere soil: other Gemmataceae (−1.0*), Afifella (−0.8)
Rhizosphere soil: Pseudoxanthomonas (−0.8)
Unamended soil versus soil amended with 300 mg TiO2-NPs kg−1 soil.
Bulk soil: other Piscirickettsiaceae (−1.2 *), Steroidobacter (−1.1 *), other Solibacterales (−0.8), Thermomonas (−0.8), other Alcaligenaceae (−0.8)
Non-rhizosphere soil: other iii1-15 (Acidobacteria-6, −1.2 *), other [Entotheonellaceae] (−1.1 *), mb2424 (Acidobacteria, −1.0 *), PK29 (Acidobacteria, −1.0 *), other Oxalobacteraceae (−0.9 *), WD2101 (Planctomycetes, −0.9 *), S0208 (Chlorofle S0208xi, −0.9 *) Ellin6075 (Acidobacteria, −0.8 *), Bacillus (−0.8), A4b (Chloroflexi, −0.8), other Syntrophobacteraceae (−0.8), other Gemmatimonadetes (−0.8)
Rhizosphere soil: other Pseudomonadaceae (−1.0), Pseudoxanthomonas (−0.9), Devosia (−0.9), Pseudomonas (−0.8), other iii1-15 (0.8), mb2424 (0.8), PK29 (0.8), Candidatus Solibacter (0.8), Ellin6067 (0.8), Halomonas (0.9), RB41 (0.9 *), RB25 (0.9), Acidobacteria-5 (1.4 *), Ellin6075 (1.5 *)
a Only bacterial genera with a large effect size ≤ −0.8 and ≥0.8 are given [69], b The effect size, which is defined as the difference between groups divided by the maximum dispersion within group A or B, was calculated with the aldex.ttest argument (ALDEx2 (version, 1.18), [66]), c *: significant at p ≤ 0.05 as determined with the non-parametric Kruskal Wallis calculated with the aldex.kw argument (ALDEx2 (version, 1.18), [66]).
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Medina-Pérez, G.; Afanador-Barajas, L.; Pérez-Ríos, S.; Navarro-Noya, Y.E.; Luna-Guido, M.; Fernández-Luqueño, F.; Dendooven, L. Bacterial Communities in the Rhizosphere of Common Bean Plants (Phaseolus vulgaris L.) Grown in an Arable Soil Amended with TiO2 Nanoparticles. Agronomy 2024, 14, 74. https://doi.org/10.3390/agronomy14010074

AMA Style

Medina-Pérez G, Afanador-Barajas L, Pérez-Ríos S, Navarro-Noya YE, Luna-Guido M, Fernández-Luqueño F, Dendooven L. Bacterial Communities in the Rhizosphere of Common Bean Plants (Phaseolus vulgaris L.) Grown in an Arable Soil Amended with TiO2 Nanoparticles. Agronomy. 2024; 14(1):74. https://doi.org/10.3390/agronomy14010074

Chicago/Turabian Style

Medina-Pérez, Gabriela, Laura Afanador-Barajas, Sergio Pérez-Ríos, Yendi E. Navarro-Noya, Marco Luna-Guido, Fabián Fernández-Luqueño, and Luc Dendooven. 2024. "Bacterial Communities in the Rhizosphere of Common Bean Plants (Phaseolus vulgaris L.) Grown in an Arable Soil Amended with TiO2 Nanoparticles" Agronomy 14, no. 1: 74. https://doi.org/10.3390/agronomy14010074

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