Inhibitory effect of TiO₂ NPs on symbiotic arbuscular mycorrhizal fungi in plant roots

Abstract

While nanoparticles (NPs) are known to exhibit antimicrobial properties, their effects on symbiotic arbuscular mycorrhizal fungi (AMF) in plant roots has to be carefully examined as NPs particularly of titanium dioxide (TiO₂) reach plant roots through varied sources such as fertilisers, plant protection products and other nanoproducts. The objective of the present study is to assess the effect of TiO₂ NPs on the symbiotic behaviour of AMF colonising rice (Oryza sativa L.) plants. Using sol–gel method, TiO₂ NPs with three different sizes were successfully synthesised employing doping. Characterisation of the prepared material was done by X‐ray powder diffraction and scanning electron microscopy. The synthesised materials were applied at 0, 25, 50 and 100 mg plant–1 to the rhizosphere of mycorrhizal rice plants maintained in pots. The study revealed that the prepared NPs had an inhibitory effect on arbuscular mycorrhizal symbiosis in plant roots. Development of AMF structures such as vesicles and arbuscules was significantly reduced in TiO₂ ‐doped NPs with a relatively more inhibition in 2% TiO₂ ‐doped NPs. Among the concentrations of TiO₂ NPs applied to different treatments, %F was significantly (P < 0.001) affected at medium to higher levels of application.

1 Introduction

In the area of heterogeneous photocatalysis, titanium dioxide (TiO₂) is particularly employed to enhance the efficiency of the environmental remediation process involving reduction–oxidation reaction. TiO₂ is one of the most important transition metal oxides that has applications in various fields such as material in fuel cell, solar energy conversion, photocatalysts, white pigment in paints and paper, ultraviolet (UV) absorber in sunscreen cream, antimicrobial agent and additive in food products [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. Semiconductor TiO₂ absorbs UV radiation (λ  < 400 nm) and photo‐stimulate redox reactions on its surface producing reactive oxygen species (ROS) such as hydroxyl radical (·OH), superoxide radical (·O2⁻) and singlet oxygen (¹ O₂) [3, 4]. The production of ROS contributes the biocidal activity with strong oxidative activity and destroys organic compounds and pollutants [6]. TiO₂ shows photoactivity only under UV light excitation because of its wide band‐gap (∼3.2 eV). Several researchers proposed to extend the photoactivity response of TiO₂ to the visible range (400–500 nm) through metal ion doping [12, 13, 14, 15, 16, 17, 18, 19, 20].

Many works have been reported for the photocatalytic disinfection studies in which TiO₂ was used against bacteria, viruses and fungi [5, 6, 7, 8, 9, 10, 11]. However, very little information is available on its effect on beneficial microorganisms which play a crucial role in ecosystem services such as driving carbon and nitrogen cycling [21] and mineral weathering [22] as well as serving as plant growth promoting agents.

Arbuscular mycorrhizal fungi (AMF) belonging to the phylum Glomeromycota form obligate symbioses with more than 80% of vascular plants [23]. AMF structures include intra‐ and extra‐matrical hyphae and two specialised structures: vesicles and arbuscules. Vesicles are produced by the internal mycelium mostly intercellularly. They are regarded as the structures of storage and are absent in certain forms. The internal mycelium also penetrates root cell walls and form dendritic structures called arbuscules within the host plant root cells [23, 24]. The fungi take up inorganic ions, particularly from the soil using their hyphal network, or mycelium, and transfer them to the host plant in exchange for carbohydrates via the arbuscules [23, 24]. The symbiosis is also important in enhancing plant drought resistance [25], disease resistance [26] and heavy metal tolerance. Since an intimate interaction exists between mycorrhizae and soil, mycorrhizae maybe particularly sensitive to an increased availability of TiO₂ in soil by way of fertiliser or as plant protection products. This may result in secondary consequences to the terrestrial plants that depend on mycorrhizae as well as potential impacts to nutrient and carbon cycling.

In the present work, nanophase TiO₂ samples with three different sizes were prepared by sol–gel method by loading with ceria (Ce) ions at various molar concentrations, thereby extending the photoactive response to visible regime. As synthesised nanoparticles (NPs) samples were characterised by X‐ray powder diffraction (XRD) and scanning electron microscopy (SEM). The effect of prepared TiO₂ NPs on the symbiotic behaviour of AMF colonising the roots of rice (Oryza sativa L.) was also evaluated.

2 Materials and methods

2.1 Materials

Ti (IV)‐n ‐butoxide (98%, Alfa Aesar), Ce(NO₃)₃. 6H₂ O (99.5%, Alfa Aesar), isopropyl alcohol (99%, Fisher Scientific) and nitric acid (99.9%, Merck) were used for the synthesis of Ce‐doped nanocrystalline TiO₂. Distilled water was used in all the synthesis process.

2.2 Synthesis

Nanophase TiO₂ powder samples loaded with Ce at various molar percentages (0, 2 and 3 mol %) were synthesised by sol–gel process, as described elsewhere [13]. The powder samples with different molar percentages were then calcined at 400°C for 3 h. S1, S2 and S3 are Ce loaded TiO₂ samples with 0, 2 and 3 mol%, respectively. The scheme for preparing the Ce loaded TiO₂ NPs is shown in Fig. 1.

Fig. 1.

Scheme of preparation of Ce loaded TiO₂ NPs

2.3 Characterisation

The XRD patterns of the synthesised TiO₂ NPs were studied using Bruker D8 Advance X‐ray diffractometer with Cu‐Kα radiation (λ  = 1.5406 Å, X‐ray tube voltage = 40 kV and current = 35 mA) in the 2θ range from 0° to 90°. The average crystallite size was estimated from Scherrer's equation [27], given by

$$D=\frac{0.9\lambda }{\beta \cos \theta },$$ (1)

where λ is the X‐ray wavelength, β is the full width at half maximum of diffraction peak and θ is Bragg's angle. The surface morphology of the sample was analysed with a scanning electron microscope JEOL MODEL JSM‐6390LV operating at 20 kV.

2.4 Evaluation of the effect of TiO₂ NPs on AM fungal colonisation in plants

Seeds of rice (O. sativa L. var. Jyothi) was sown in plastic pots (15 cm diameter) filled with 3 kg sandy (entisol) soil (pH 5.5, organic carbon 12.5 g kg⁻¹ soil and available phosphorus 5.5 µg g⁻¹ soil). The soil was steam sterilised using an autoclave for 20 min at 121°C prior to filling the pots. At potting, inocula consisting of a consortium of indigenous AM species [Diversispora versiformis (P. Karst.) Oehl, G.A. Silva and Sieverd., Funneliformis dimorphicus (Boyetchko and J.P. Tewari) Oehl, G.A. Silva and Sieverd. and Glomus indicum Blazk, Wubet and Harikumar] procured from rice fields were multiplied using sterilised sand soil mix (1:1 v/v) as substrate and Sorghum as the host. After six weeks of growth, shoots of the host plants were severed and the substrate containing the hyphae, spores and root bits was diluted to provide equal inoculum densities (∼1.5 × 10⁵ propagules pot⁻¹) and placed uniformly 5 cm below the surface of the soil in all the pots. The pots were arranged on the green house bench and watered at 3 days interval with distilled water. After emergence of seedlings, the number of plants was thinned to one per pot and treatments applied to selected pots. Pots of rice plants designated as non‐treated controls received 100 ml of distilled water, while treated pots received 25, 50 and 100 mg of TiO₂ ‐anatase, TiO₂ ‐2 mol% doped or TiO₂ 3 mol%‐doped NP dissolved in 100 ml of distilled water. Shallow (2 cm) plastic plates were placed under each planted pot to prevent loss of NP during watering. Our experiment allowed for a 4 × 3 × 3 factorial design that tested for the effect of NP (non‐treated, TiO₂ ‐treated, TiO₂ ‐2 mol% doped treated and TiO₂ ‐3 mol% doped treated), three concentrations (25, 50 and 100 mg) and three stages of growth [30, 60 and 90 days after application (DAA)] on root symbiosis by AMF. Three plants from each treatment were pulled out with the roots intact at 30 days interval for monitoring the AM fungal colonisation behaviour. Final sampling of plants from each treatment was done at the termination of the experiment at 90 DAA.

The undamaged fine roots of test plants were washed thoroughly in running tap water to remove the adherent debris and cut into 1 cm root segments. About 30 root segments selected at random were subjected to clearing and staining procedure as described by Koske and Gemma [28]. The stained root samples were mounted on microscopic slides in a mixture of glycerol and lactic acid (v/v), gently squashed and covered by a glass cover slip and observed under a compound microscope (Olympus CX31). Simultaneous evaluation of frequency of colonisation (%F), intensity of colonisation (%M) and the proportion of vesicles (%V) and arbuscules (%A) present in the roots was performed following the method of Trouvelot et al. [29]. The results are subjected to two‐way ANOVA suitable for RD for the test of significance and the means were separated using Tukey's honestly significant difference (HSD) test using SYSTAT 9.

3 Results and discussion

3.1 XRD analysis

The powder X‐ray diffraction patterns of Ce loaded TiO₂ NPs are shown in Fig. 2. All the peaks correspond to a tetragonal structure (space group: I4₁ amd⁻¹) containing 12 atoms per unit cell with lattice parameters a  = 3.776 Å and c  = 9.486 Å, which is in agreement with the JCPDS file no. 21‐1272 for TiO₂. The strong diffraction peak at 25.7 corresponds to the TiO₂ ‐anatase phase. Other peaks observed are 38.2, 48.1, 54.9, 63.1, 69.9, 75.7 and 82.9 which ensures the presence of crystalline anatase TiO₂. Only a very small content of rutile is present at 30.9 position. However, no peaks for Ce is observed due to very low amount of doping concentration. There is no phase change after Ce doping as no other polymorph of titania is observed in the XRD spectra.

Fig. 2.

XRD patterns of TiO₂ samples

The average crystallite sizes of TiO₂ NPs were calculated from XRD patterns using (1). The calculated values are 7.42, 5.25 and 4.56 nm, respectively, for 0, 2 and 3 mol%‐doped TiO₂ NP samples. It is interesting that size of the NPs decreases with doping. This may occur due to the inclusion of Ce into the TiO₂ lattice.

3.2 Scanning electron microscopy

Fig. 3 shows the scanning electron micrographs of pure and Ce loaded TiO₂ NPs. The photographs of the samples contain both homogeneous and irregular crystallised NP clusters. Also, it can be seen from Fig. 3 that doping changes the morphology of the sample slightly. SEM images also demonstrate that average particle size decreases with doping concentration. From the energy dispersive X‐ray spectroscopy, the amount of Ti, O and Ce in percentage are found to be 79.53, 20.06, 0.41 and 79.21, 20.02, 0.77, respectively for 2 and 3 mol% Ce loaded TiO₂.

Fig. 3.

SEM images of undoped TiO₂ and Ce loaded TiO₂ samples

3.3 Effect of TiO₂ NPs on AM fungal colonisation in plants

There was a significant (P  < 0.001) difference in the development of fungal structures within plant roots between TiO₂ NP treatments and untreated control (Table 1). Development of AMF structures such as vesicles and arbuscules were significantly reduced in all sizes of TiO₂ samples. However, relatively more inhibition is found in 2% TiO₂ ‐doped NPs. However, only a weak effect was observed for TiO₂ ‐2 mol%‐doped NP on fungal characters such as %F and %M. Since vesicles are the structures of storage and arbuscles meant for the transference of nutrients between fungus and the plant, the inhibition of these structures is likely to affect the functional symbiosis within the host roots. The extent of inhibition however varied with different NP treatments (Fig. 4). Earlier studies on AM fungal symbiosis with plant roots suggest that mechanical or chemical disturbances of soil can substantially reduce AM fungal propagules, vigour and functioning [30] which in turn could influence the nutrient exchange process [31]. In the present paper, the treatment with TiO₂ NPs of all sizes altered to a great extent the AM fungal characters in plant roots as compared with untreated control indicating the deleterious effects of NPs on AM symbiosis possibly due to the binding of TiO₂ NPs to plant roots [32] or increases in internal concentration of Ti in root tissue. However, offering such comments need more detailed investigations. Though previous studies witnessed the antimicrobial properties of NPs [33, 34], the effect of TiO₂ NPs on AM fungal colonisation and development of fungal structures in plant roots is meager, except for some stray reports on its effect on composition of AMF in soil [35] and mycorrhizal effects on plant growth [36].

Table 1.

Effects of TiO₂ NPs on the development of AM fungal structures in rice plants

Treatment	F %	M %	%V	%A
control	59.00 ± 0.42a	12.04 ± 0.14a	15.48 ± 0.09a	16.42 ± 0.05a
TiO2 ‐anatase	41.41 ± 0.41c	6.49 ± 0.14c	1.94 ± 0.09b	3.02 ± 0.05b
TiO2 ‐2 mol% doped	50.57 ± 0.41b	9.49 ± 0.14b	0.00 ± 0.09c	0.00 ± 0.05c
TiO2 ‐3 mol% doped	41.22 ± 0.41c	6.90 ± 0.14c	1.06 ± 0.09bc	1.01 ± 0.05b

Means in a column followed by same letter do not differ significantly (P  < 0.05) by Tukey's HSD.

Fig. 4.

Inhibition (%) of AM fungal structures in rice roots as influenced by the application of TiO₂ NPs

(a) Frequency of colonisation, (b) Intensity of colonisation, (c) Proportion of vesicles, (d) Arbuscules

Among the concentrations of TiO₂ NPs applied to different treatments, %F was found to be significantly (P  < 0.001) affected at medium to higher levels of application. Whereas, the fungal characters such as %M, %V and %A had no influence on the concentration of TiO₂ NPs (Table 2). An explanation to microbial inhibition vis‐à‐vis concentration of NPs is rather difficult as varied dose‐dependent response is seen for the inhibition of different microorganism. For example, at a far low concentration of 0.5 mg l⁻¹ silver, the growth of Escherichia coli PHL 628 gfp was inhibited to the tune of 55 ± 8% [37]. In another study along with a concentration gradient of ferrous oxide NPs, Feng et al. [36] have observed a significant reduction of glomalin glycoprotein produced by hyphae and spores of AMF in soil and in roots at 3.2 mg kg h⁻¹ level of application.

Table 2.

Effect of concentration of TiO₂ ‐2 mol%‐doped NPs on the development of AM fungal structures in rice plants

Concentration, mg plant−1	F %	M %	%V	%A
25	52.75 ± 0.35a	8.75 ± 0.12a	4.17 ± 0.08b	4.12 ± 0.04b
50	49.22 ± 0.35a	8.86 ± 0.12a	5.47 ± 0.08a	5.59 ± 0.04a
100	42.16 ± 0.35b	8.57 ± 0.12a	4.22 ± 0.08b	5.60 ± 0.04a

Means in a column followed by same letter do not differ significantly (P  < 0.05) by Tukey's HSD.

The effect of application of TiO₂ NPs on the inhibition of AM fungal colonisation in plant roots was more pronounced during early stages of application but later on found to recover during late stages of application (Table 3). This is possibly due to the high mobility of TiO₂ through the porous substratum, i.e. sandy soil [38]. Production of more mycorrhizal roots in the absence of a high concentration of NPs could be assigned as a second possibility for a higher scoring of mycorrhizal variables at late stages of application.

Table 3.

Effect of time of application of TiO₂ ‐2 mol%‐doped NPs on the development of AM fungal structures in rice roots

DAA	F %	M %	%V	%A
30	45.37 ± 0.35b	8.04 ± 0.12b	3.29 ± 0.08b	4.66 ± 0.04b
60	50.16 ± 0.35b	9.57 ± 0.12a	5.30 ± 0.08a	4.29 ± 0.04b
90	48.60 ± 0.35ab	8.57 ± 0.12ab	5.27 ± 0.08a	6.35 ± 0.04a

Means in a column followed by same letter do not differ significantly (P  < 0.05) by Tukey's HSD.

4 Conclusions

Ce loaded TiO₂ NPs were successfully synthesised by sol–gel method and characterised by XRD and SEM. The study on effect of TiO₂ NPs on the symbiotic behaviour of AMF colonising the roots of rice brought meaningful insights into the fact that TiO₂ NPs could be a possible candidate in inhibiting AM colonisation in plant roots at medium to higher levels of NP application, particularly, during the early stages of its application. Therefore, the deleterious effects of TiO₂ NPs on beneficial microorganisms such as AMF are to be seriously looked into while applying NPs in terrestrial ecosystems.