Biopriming with Piriformospora indica ameliorates cadmium stress in rice by lowering oxidative stress and cell death in root cells

Ecotoxicology and Environmental Safety 186 (2019) 109741

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Biopriming with Piriformospora indica ameliorates cadmium stress in rice by lowering oxidative stress and cell death in root cells

T

Surbhi Dabrala, Yashasweea, Ajit Varmaa, Devendra Kumar Choudharya,∗∗, Rajeev Nayan Bahugunaa,b,∗∗∗, Manoj Natha,c,∗ a

Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Sector 125, Noida, 201313, India Center for Advance Studies on Climate Change, Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, Bihar, 848125, India c ICAR-Directorate of Mushroom Research, Chambaghat, Solan, Himachal Pradesh, 173213, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Rice Piriformospora indica Cadmium stress Root

Piriformospora indica is known for plant growth promotion and abiotic stress alleviation potential in several agricultural crops. However, a systemic analysis is warranted to explore potential application of this important fungus to augment heavy metal tolerance in rice. The present study explores potential of P. indica in ameliorating the effect of cadmium (Cd) stress in rice cultivars N22 and IR64. Seedlings inoculated with P. indica recorded significantly higher root-shoot length and biomass as compared to non-inoculated plants under control and Cd stress, respectively. Moreover, P. indica inoculated stressed roots accumulated more Cd as compared to noninoculated stressed roots in both the varieties. Interestingly, cell death and reactive oxygen species (ROS) accumulation were significantly lower in the inoculated plant roots as compare with non-inoculated roots under Cd stress. The results emphasized significantly higher accumulation of Cd in fungal spores could reduce ROS accumulation in root cells resulting in lower cell death.

1. Introduction Rice is major food crop for more than 3.5 billion people worldwide. People in major rice growing countries rely on rice as a chief source of their dietary requirement (Cheng et al., 2013; Zhang et al., 2019). Heavy metal stress arising due to various anthropogenic regions is one of major yield constrain for rice production in the heavy metal contaminated soils (Solidum et al., 2012; Xue et al., 2014). There are several anthropogenic routes including contaminated irrigation water, industrial and municipal wastes, mining and smelting, use of pesticides and fertilizers, waste discharge from leather industries and ecological disturbances by which heavy metals reach to the paddy fields (AlHobaib et al., 2012; Arunakumara et al., 2013; Gallego et al., 2012). Besides yield losses, there are severe health constrains associated with heavy metal accumulation in crop plants when enters in to the food chain (Li et al., 2016). Cadmium (Cd) is one of the most common heavy metal contaminant in water, which is reaching to the agricultural fields (Adriano, 2001). Cadmium stress primarily target the inhibition of the photosynthetic activities (Paunov et al., 2018; Bączek-Kwinta et al.,

2019; Song et al., 2019). Moreover, Cd toxicity is responsible for various deleterious effects on plants like decrease in growth, late germination of seeds, leaf chlorosis and necrosis (Hall, 2002; Xu et al., 2009; Dias et al., 2013). Conversely, Cd uptake and translocation from root to aerial part of the plant such as rice straw and grains, ultimately may cause deleterious effects to the consumers (Du et al., 2013; Aziz et al., 2015; Song et al., 2015). Rice is sensitive to Cd stress, which causes reduction of growth and biomass (Rahman et al., 2015; Zhou et al., 2015). Rice growing under Cd contaminated soil shows phytotoxic symptoms like leaf necrosis, chlorosis and decreased photosynthetic activity, stomatal conductance and transpiration rate (Liu et al., 2003a,b; Rascio et al., 2008; Wang et al., 2014). In addition, stunted root-shoot, reduced leaf area and number of leaves are also the consequences of Cd stress (Yu et al., 2006; Song et al., 2015). Moreover, cell death in plants due to heavy metal stress includes an elevation in reactive oxygen species (ROS). Notably, high levels of Cd tend to accumulate in root and shoot of rice leading to oxidative burst, which is responsible for high ROS accumulation and cell death in rice plant (Guo et al., 2009; Tripathi et al., 2012).

∗

Corresponding author. ICAR-Directorate of Mushroom Research, Chambaghat, Solan, Himachal Pradesh, 173213, India. Corresponding author. Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Sector 125, Noida, 201313, India. ∗∗∗ Corresponding author. Center for Advance Studies on Climate Change, Dr. Rajendra Prasad Central Agricultural University, Pusa, Samastipur, Bihar, 848125, India. E-mail addresses: [email protected] (D.K. Choudhary), [email protected] (R.N. Bahuguna), [email protected] (M. Nath). ∗∗

https://doi.org/10.1016/j.ecoenv.2019.109741 Received 24 June 2019; Received in revised form 25 September 2019; Accepted 28 September 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.

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2.2. In vitro growth of P. indica under Cd stress

Utilization of the beneficial microbe such as Piriformospora indica is one of the approach to combat heavy metal stress (Nanda and Agrawal, 2018). P. indica, a symbiotic mycorrhizal fungus, is a root endophyte and well documented for promoting the plant growth (Varma et al., 1999). Moreover, P. indica has unique abilities to thrive under harsh environments and has been reported to augment stress tolerance of the host plant under both biotic and abiotic stress environment (Waller et al., 2005; Deshmukh and Kogel, 2007; Serfling et al., 2007; Sarma et al., 2011; Dolatabadi et al., 2012; Varma et al., 2012; Jogawat et al., 2013; Das et al., 2014; Gill et al., 2016; Zhang et al., 2017). Interestingly, P. indica has been used for mitigating heavy metal stress in plants such as wheat and sunflower (Shahabivand et al., 2012, 2017), tobacco (Hui et al., 2015), Cassia angustifolia (Nanda and Agrawal, 2018) and rice (Mohd et al., 2017). Particularly, reports in tobacco (Hui et al., 2015) and sunflower (Shahabivand et al., 2017) illustrates that P. indica inoculation under Cd stress tend to accumulate more Cd in roots to restrict heavy metal movement to aerial parts of the plant. However, the mechanistic explanation of P. indica mediated heavy metal stress tolerance in rice is not well understood. Thus, this study was done to explore the effect of biopriming with P. indica on rice growth under control and Cd stressed soil. Additionally, impact of Cd stress on P. indica growth and Cd accumulation, ROS generation and cell death was determined in inoculated and non-inoculated Cd stressed rice roots to dissect the mechanistic basis of P. indica mediated heavy metal tolerance.

The study was done in terms of circularly extending fungal mycelia in Hill and Kaefer agar plates under Cd (CdCl2,Merck International, Germany) influence. Fungal disc of P. indica (about 4 mm) was inoculated on Hill and Kaefer agar medium made with different Cd concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.6 mM, 0.9 mM and 1.2 mM) in 90 mm petri plates. The plates were incubated in dark at 30 °C in incubator (Biosync Technology) for 15–20 days (Hill and Kafer, 2001). Growth of P. indica was assessed by measuring the radius of radially growing hyphae on 5th, 10th, 15th, 20th and 25thday of P. indica inoculation. 2.3. Cadmium detection in spores Spores were isolated from P. indica inoculated plates under different Cd concentrations as described in section 2.1. Leadmium Green AM dye (Molecular Probes, Life Technologies, California, USA) was used to detect the Cd accumulation as described by Zhao et al. (2013) with slight modifications. Isolated spores were treated with 20 mM disodium ethylenediamine tetra-acetic acid (Na2-EDTA) for 15 min followed by rinsing with deionized water for 3-4times. The spores were then treated with Leadmium Green AM dyefor 45 min in dark at room temperature followed by washing three times with deionized water. The Leadmium green AM dye (50 μg) was mixed with 50 μL of dimethyl sulfoxide (DMSO). The prepared stock solution was further diluted 1:10 with 0.85% NaCl. A drop of stained spores sample was put over glass slide followed by covering it with a coverslip carefully so that no air bubbles were formed. Confocal microscope (Model:Nikon A1, Confocal Microscopy Facility, Amity Institute Of Microbial Technology Amity University Uttar Pradesh, India) was used to visualize the sample. The confocal settings used in confocal microscope for Leadmium green AM dye are as follows; excitation- 488 nm, emission- 515 nm, 60× magnification. The mean fluorescence pixel intensity (MFPI) was determined using randomly selected five fluorescent confocal images by ImageJ program (Collins, 2007).

2. Material and methods A pot experiment was conducted in the walk-in growth chambers at Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, India to evaluate the potential of P. indica to improve rice growth and tolerance to cadmium stress at vegetative stage. The pure culture of P. indica was obtained from Amity Institute of Microbial Technology (AIMT), Amity University, Noida, UP, India. Seeds of rice cultivars IR64 and N22 were obtained from International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. The experiment was designed with two cultivars and four treatments. Treatments were categorized as 1. control (C), plants without P. indica inoculation and without cadmium treatment (non-inoculated and nonstressed), 2. plants inoculated with P. indica (Pi) only (inoculated and non-stressed), 3. plants treated with cadmium (Cd) only (non-inoculated and stressed) and 4. Plants treated with P. indica grown under Cd enriched soil (Cd + Pi); (inoculated and stressed).

2.4. Seedling growth, cadmium treatment and environmental conditions Seeds of rice cultivars N22 and IR64 were incubated at 50 °C for 24 h to break dormancy. Seeds were surface sterilized in 10% sodium hypochlorite for 10 min and then washed six times with sterile water as described earlier by Bagheri et al. (2013). Before germination, seeds were divided in two equal parts and half of the seeds were bioprimed with P. indica spores (5 × 105 spores/ml) while other half was kept untreated. Seeds of respective group were then placed separately on germination sheets in the petri plates and kept in dark for 3 day at 28 °C in incubator (Biosync Technology, India). Seeds moisture was maintained in the petri plates by adding distilled water as needed. Pre-germinated seeds of same appearance were sown in germination tray filled with autoclaved clay loam soil. Fourteen days after germination, healthy seedlings of both the cultivars were selected from each group and divided in two sets. Conversely, methodology for fortification of soil with heavy metal (Cd) was adopted from Shahabivand et al. (2012). In brief, autoclaved soil was fortified with 0.1 mM CdCl2 solution to reach 10 mg/kg Cd level in soil and mixed thoroughly in plastic trays followed by incubation at 20 °C for one month for proper distribution of heavy metal within the soil. One set was transplanted in pots (11 cm height and 9 cm diameter) filled with approximately 265 g autoclaved clay loam soil and the other set was transplanted in the pots filled with Cd enriched soil. One established seedling in a pot was considered as a biological replicate and each treatment had ten biological replicates. Five replicates were used for the morphological observations and measurement of growth parameters. Three replicates were used for confocal study while two replicates were taken to observe root colonization in the respective treatment. The growth conditions were

2.1. P. indica growth condition and spore isolation The P. indica culture was maintained by inoculating 4 mm fungal discs on Hill and Kaefer agar medium in 90 mm petri plates. The plates were incubated in dark at 30 °C in incubator (Biosync Technology, India) for 7–8 days (Hill and Kafer, 2001). The methodology for isolation of P. indica spores was adopted from Ghabooli et al. (2013) with slight modifications. In brief, distilled water mixed with 0.02% Tween 20 was autoclaved (Narang Scientific, India). The autoclaved solution was cooled at room temperature and then refrigerated at 4 °C. Further, 1 ml of the solution was added to freshly prepare P. indica culture plates followed by a gentle scraping of the fungal spores with the help of spreader. This step was repeated two to three times and the suspension was collected in 50 ml falcon tubes (Tarsons, India). The suspension was vortexed and then centrifuged at 5000 g for 10 min in tabletop centrifuge (Labogene, Denmark). The supernatant was discarded and the pellet obtained was mixed with 10 ml sterile distilled water. The spores were observed and counted with the help of hemocytometer. The final concentration was adjusted to 5 × 105spores/ml by distilled water. 2

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Pradesh). The confocal settings for Propidium iodide; excitation560nm, emission- 595 nm and for H2DCFDA and Leadmium green AM; excitation-488nm, emission- 515. Imaging of root tips stained with propidium iodide and H2DCFDA was done together while cadmium staining experiment was performed separately as excitation and emission of H2DCFDA and Leadmium green AM were similar. Each experiment was performed in triplicates. The images captured through Nikon NIS Elements software were used to calculate the MFPI of all the signals (propidium iodide, H2DCFDA and Leadmium green, AM) through Image J software and the graph was plotted for the same. The respective MFPI was calculated as described in section 2.3.

maintained throughout the experiment in the controlled environmental walk-in chambers with 32 °C/23 °C day/night temperature, 60–80% relative humidity and photoperiod of 16 h with a photon flux density of 700 μmol m−2s−1. The seedlings were supplemented with half strength Hoagland's solution thrice in a week. 2.5. Inoculation of P. indica in rice and root colonization P. indica spore suspension with a concentration of 5 × 105 spores ml−1 was inoculated again in the rhizoshpere soil of respective pots 5 days after transplanting. Two punctures were done in the soil near the vicinity of roots and 1 ml of spore suspension was inoculated in the rhizospheric zone. Three seedlings were chosen randomly and uprooted to check the root colonization after five days of inoculation. P. indica colonization in the roots was observed as described by Tripathi et al. (2015). In brief, rice roots were reaped followed by gentle washing with tap water for the elimination of adhered soil particles. The roots were then excised and cut in small pieces measuring up to 1–2 cm and were heated in 10% KOH for approximately 10–15 min. The root samples were washed with distilled water three-four times followed by the treatment with 1N HCl for 4 min. Root samples were kept in petri plate and stained with 0.02% trypan blue dye overnight. Destaining was done with 50% lactophenol treatment for 2 h. The root samples were then observed under Nikon light microscope for P. indica colonization under 10X magnification.

2.6. Statistical analysis The experiment was designed as completely randomized design (CRD) with treatments and cultivars as factors. Means were compared by Tukey's HSD test. The statistical analyses were performed using the Package for Social Sciences version 16 (SPSS16, USA). 3. Results 3.1. In vitro growth of P. indica under Cd stress In vitro studies on P. indica growth under different heavy metal (Cd) concentrations revealed that the fungus was able to survive in 0.01 mM, 0.05 mM and 0.1 mM Cd concentrations. Complete hyphal growth of P. indica was achieved within 15 days in control (3.98 ± 0.01 cm) and 0.01 mM (3.92 ± 0.03 cm). Moreover, 0.05 mM (3.83 ± 0.08 cm) and 0.1 mM (3.55 ± 0.07 cm) acquired 25 days to achieve full growth in petri plate. However, 0.2 mM and 0.3 mM Cd concentration restricted fungal growth to 1.5 ± 0.01 cm and 1 cm respectively even after 25 days. No growth of P. indica was observed in the media having cadmium concentrations above 0.6 mM (Fig. 1A and B).

2.5.1. Observations and sampling Plants of both the cultivars were uprooted from the pots across the treatments in three biological replicates at 30 days after transplanting to measure the different growth parameters. Uprooted plants were thoroughly washed with tap water for the removal of adhered soil particles. After washing, plants were placed over a black cloth to measure the shoot and root length with the help of measuring scale. Shoot length was measured from the root-shoot junction to the tip of longest leaf while root length was measured from root-shoot junction to the root tip. The weight of the samples was taken immediately after harvesting and fresh weight was recorded.

3.2. Cadmium accumulation in P. indica spores The P. indica spores were stained with Leadmium Green AM dye to investigate the Cd accumulation under different Cd stress condition. The Cd accumulation was observed in 0.01 mM, 0.05 mM and 0.1 mM Cd concentration only. However, P. indica spores were not isolated from remaining Cd concentrations due to restricted growth (0.2 mM and 0.3 mM Cd) or no fungal growth (0.6 mM, 0.9 mM and 1.2 mM Cd). Similar level of Cd accumulation was observed in the spores treated with 0.01 mM, 0.05 mM and 0.1 mM Cd concentrations (Fig. 2A and B) and further corroborated with mean fluorescence pixel intensity analysis (MFPI) (Fig. 2C).

2.5.2. Histochemical analysis for cell death, ROS and cadmium Three biological replicates from IR 64 and N22 were harvested and the roots were washed under tap water to remove all the soil particles. The experiment was performed with crown roots of each treatment to maintain uniformity. After washing the roots were spread neatly over a solid surface with the help of brush and distilled water. Further, tip portionwas excised with the help of scalpel and the excised root tip was transferred in 1 ml distilled water in multiwell plates. The root tips were further treated with propidium iodide (Hi-Media), 2′,7′-dichlorodihydrofluorescin diacetate (H2DCFDA; Sigma-Aldrich) and Leadmium green AM dyes for determining cell viability, ROS accumulation and cadmium respectively. The protocol followed for cell death and ROS staining was adopted from Nath et al.(2016), while for Cd detection the methodology was adopted as described by Zhao et al. (2013), with slight modifications. In brief, root samples were stained with 4 μl of 10 μM H2DCFDA for 40 min in dark at room temperature for determining ROS accumulation. To estimate cell death, 2 μl of 3 μg/ ml propidiumiodide (Sigma Aldrich, USA) was used for 10 min in dark at room temperature. For Cd detection in root tips methodology described in section 2.3 was followed. The multiwell plate with root samples was subjected to continuous shaking in dark in shaker (Lab Companion, India) at room temperature, after completion of the incubation period the root tip was picked with the help of forceps and carefully placed on the glass slide (Hi-Media) and was covered with the coverslip making sure that air bubbles not formed. The samples were observed under 10× magnification and the images were captured using the Confocal microscope (Nikon A1, Confocal Microscopy Facility, Amity Institute of Microbial Technology Amity University Uttar

3.3. P. indica colonization and Cd accumulation in inoculated and noninoculated rice roots Roots of rice varieties N22 and IR64 grown in Cd fortified soil with (inoculated) and without P. indica (non-inoculated) were stained with Leadmium Green AM dye along with respective control. Further, Leadmium Green AM stained roots were subjected to confocal analysis for detecting Cd accumulation. The results indicated that presence of P. indica resulted in high Cd buildup in roots of both the varieties. Confocal studies revealed that Cd levels were elevated in inoculated roots of both rice cultivars as compared with non-inoculated one (Fig. 3A–D). Though, there was a significant difference in Cd accumulation in both the rice cultivars with both the treatments (inoculated, stressed and non-inoculated stressed), higher Cd was accumulated in the inoculated and non-inoculated roots of IR64 as compared with N22. In addition, plants of both the cultivars (N22 and IR64) were randomly chosen across the treatments to observe P. indica root colonization. The finding of the Cd accumulation was further validated by mean fluorescence pixel intensity (MFPI) analysis. This MFPI analysis further 3

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Fig. 1. Effect of different cadmium (Cd) concentrations on P. indica growth under in vitro condition. (A) Hyphal growth of P. indica under various Cd concentrations (0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.6 mM, 0.9 mM and 1.2 mM), (B) Analysis of hyphal radius after 5, 10,15, 20 and 25 day of P. indicainoculation. DAI: Days after inoculation. Each data point represents mean ± standard error of three biological replicates.

inoculated). Stressed, non-inoculated plants and P. indica inoculated non-stressed plants displayed similar height. Fungus inoculated seedlings grown in Cd fortified soil displayed higher root length as compared to Cd alone. Combination of Cd with P. indica proved to be beneficial as higher growth traits were observed relative to control (Fig. 4A ad 4B). It was evident that P. indica significantly improved plant growth under control and Cd stress conditions. Exposure to Cd reduced plant shoot and root length of rice cultivar IR64. P. indica inoculation significantly reduced this effect in case of IR64. P. indica colonized nonstressed plants recorded highest shoot (13.54 ± 0.2 cm) and root length (6.78 ± 0.2 cm). However, P. indica inoculated stressed plants recorded significantly higher shoot (11.4 ± 0.3 cm) and root length (6.92 ± 0.5 cm) as compared to non-inoculated stressed (5.28 ± 0.4 cm) and non-inoculated and non-stressed plants (9.94 ± 0.4 cm). For N22 non-stressed and non-inoculated control seedlings exhibited 5.46 ± 0.2 cm and 9.14 ± 0.3 cm of root and shoot length respectively while P. indica inoculated non-stressed plants recorded (7.66 ± 0.4 cm) and (15.82 ± 0.2 cm) of root and shoot length respectively. Stressed and inoculated plants recorded (6.50 ± 0.5 cm) and (11.50 ± 0.4 cm) of root and shoot length

corroborated i.e. higher Cd level in inoculated roots of N22 (20.57) and IR64 (59.56) as compared with non-inoculated roots of N22 (9.66) and IR64 (42.06) (Fig. 3E). Trypan blue stained fungal spores colonizing the roots were witnessed in both the rice varieties under light microscope (Supplementary Fig. 1). 3.4. Effect of Cd stress on rice growth Rice seedlings of both the varieties N22 and IR64subjected to different treatments were observed regularly on the basis of morphological and physiological changes occurring in plants. In IR64, P. indica inoculated non-stressed plants displayed 36.21% and 22.82% increase in shoot and root length as compared to non-stressed and non-inoculated plants. Stressed and non-inoculated seedlings (Cd alone) exhibited 46.88% and 32.97% decreased shoot and root length respectively in comparison to non-stressed non-inoculated seedlings. An increase of 115.90% and 87.02% was observed in P. indica inoculated stressed plants in comparison to stressed and non-inoculated plants. In N22, percentage increase recorded in the shoot and root length of P. indica inoculated non-stressed seedlings was 67.17 and 40.29, respectively as compared to seedlings without any treatment (non-stressed and non4

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Fig. 2. Cadmium (Cd) accumulation in P. indica spores under Cd stress. (A) Bright-field and (B) Fluorescent confocal image of P. indicaspores under control, 0.01 mM, 0.05 mM and 0.1 mM Cd concentrations; (C) Respective mean of fluorescence pixel intensity (MFPI) for fungal spores under different Cd concentration. Each vertical column represents mean ± standard error of ten replicates.

IR64plants as compared with the other treatments under Cd stress. Though, higher ROS was generated in IR64 as compared with N22 roots under Cd stress (Fig. 6A). Stressed and non-inoculated roots of N22 and IR64 recorded MFPI value of25.57 and 61.83 respectively, while P. indica inoculated non-stressed roots represented the value of 2.91 (N22) and 3.68 (IR64). Moreover, P. indica inoculated stressed roots displayed MFPI values 5.04 (N22) and 1.00 (IR64) (Fig. 6 C). Roots without any treatment (non-stressed and non-inoculated) exhibited very less ROS generation.

respectively (Fig. 4C and D). In IR64 and N22, P. indica only inoculated non-stressed plants exhibited significantly high fresh weight in comparison to non-inoculated non-stressed control plants. IR64 stressed and inoculated seedlings displayed significantly high fresh weight with respect to stressed and non-inoculated seedlings while N22 showed a different pattern as stressed and non-inoculated seedlings and the ones which are stressed and inoculated exhibited almost same fresh weight (Fig. 4 and Supplementary Table 1). 3.5. P. indica treatment reduce ROS accumulation and cell death in root cells

4. Discussion 4.1. P. indica has potential to survive under Cd stress

Propidium iodide staining was performed in roots of rice cultivars (N22 and IR64) to investigate the cell viability under Cd stress. The results of cell viability analysis revealed augmented cell death in noninoculated stressed roots as compared to P. indica inoculated stressed roots (Fig. 5 A and C). Further, the mean fluorescence pixel intensity (MFPI) analysis also indicated higher MFPI value for non-inoculated stressed roots of N22 (48.31) and IR64 (59.75) than P. indica inoculated stressed N22 (8.95) and IR64 (7.85) roots. Moreover, P. indica only (inoculated and non-stressed) roots of N22 demonstrated higher MFPI value (19.93) as compared with IR64 (11.94) roots.(Fig. 5 B and D). Overall, MFPI analysis further substantiated the cell viability analysis. Similarly, H2DCFDA-based ROS localization study demonstrated significantly high levels of H2O2 generated in roots of N22 and

The findings of the present study suggest that P. indica was able to grow in 0.01 mM, 0.05 mM and 0.1 mM of Cd stress and growth was as at par with control (Fig. 1). Notably, survival ability in heavy metal stress was reported in various filamentous fungi belonging to the genera Aspergillus, Trichoderma, Mucor and Penicillium (Ezzouhri et al., 2009; Oladipo et al., 2017). Zuniga-Silva et al. (2016) studied the response of five soil-borne fungi towards heavy metal stress and reported declining pattern of the mycelial growth with increasing concentration of heavy metal concentration. In contrast, we could document that P. indica was able to grow well in Cd concentration (0.01–0.1 mM) at par to control. Growth at 0.1 mM Cd concentration acquired more days to attain 5

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Fig. 3. Cadmium (Cd) accumulation in P. indica inoculated and non-inoculated stressed rice roots. Confocal based detection of Cd accumulation by Leadmium Green AM stained non-inoculated (A) N22 and (B) IR64 roots and P. indica inoculated (C) N22 and (D) IR64 roots and their respective (E) Mean fluorescence pixel intensity (MFPI) analysis. Each vertical column represents mean ± standard error of ten replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

in Cassia angustifolia (Nanda and Agrawal, 2018); Cd in tobacco (Hui et al., 2015); wheat and sunflower (Shahabivand et al., 2012, 2017). Most plants are sensitive for Cd stress, which reduce root-shoot growth as a consequence of modification in photosynthesis rate as well as in nutrient uptake and translocation (Lozano-Rodríguez et al. 1997; Sandalio et al., 2001). Previously, studies have reported that Cd stressed pea plants recorded decreased root length (Rodriguez-Serrano et al., 2006), while Nicotiana tabacum recorded reduced root and shoot growth (Hui et al., 2015). In our study we have observed significantly reduced root and shoot growth in IR64 under Cd fortified soil (stressed and non-inoculated). The results for IR64 are in accordance with the study conducted on rice with arsenic stress, in which reduction was observed in the root and shoot length of stressed rice seedlings (Shri et al., 2009; Upadhyay et al., 2016). Moreover, fresh weight, shoot and root growth was higher in P. indica inoculated stressed plants as compared to non-inoculated stressed plants of IR64, but no significant difference was observed in N22. It has been reported that adverse impact of Cd on tobacco growth was successfully overcome by P. indica treatment (Hui et al., 2015). In yet another study, high Cd concentration in soil caused stunted growth and lower fresh weight in wheat and P. indica ameliorated the effect and enhanced root and shoot length under

similar growth as compared to control could be explained as the possible induction of adaptive response, which was noted earlier for Amanita muscaria under heavy metal stress (Crane et al., 2010). P. indica was also reported to show tolerance towards arsenic up to 1 mM concentration beyond which the growth was significantly reduced (Mohd et al., 2017). In this study, we have observed tolerance of fungus up to 0.1 mM Cd concentration, while reduction in growth up to 0.3 mM and negligible growth at higher concentrations (0.6 mM, 0.9 mM and 1.2 mM). 4.2. P. indica treatment improved rice growth under Cd stress The fungus P. indica is well known to stimulate plant growth and development (Varma et al., 2012; Jogawat et al., 2013; Gill et al., 2016; Zhang et al., 2017; Dehury et al., 2018). P. indica has been reported in antagonizing abiotic stress conditions like drought stress in Arabidopsis (Sherameti et al., 2008), barley (Ghaffari et al., 2018) and maize (Hosseini et al. 2018); and salinity stress in Cucumis melo L.(Hassani et al., 2019) and tomato (Ghorbani et al., 2019). Conversely, previous studies revealed that P. indica has been used for vindicating heavy metal stress in plants such as arsenic in rice (Mohd et al., 2017); copper 6

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Fig. 4. Effect of P. indica on rice growth under cadmium (Cd) stress. (A) N22 and (B) IR64 growth under different treatments: Control(C), P. indica (Pi), Cadmium (Cd), and both Cadmium and P. indica (Cd + Pi); and their root and shoot lengths of (C) N22 and (D) IR64. Each vertical column represents mean ± standard error of five replicates.

Fig. 5. Cell death analysis of P. indica inoculated and non-inoculated rice roots. Propidium iodide based cell viability analysis of(A) N22 and (C) IR64 and its respective comparative quantification of mean fluorescence pixel intensity (B) N22 and (D) IR64. Each vertical column represents mean ± standard error of ten replicates. 7

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Fig. 6. Reactive oxygen species (ROS) accumulation in P. indica inoculated and non-inoculated rice roots. H2DCFDA based analysis of the ROS accumulation in (A) N22 and (C) IR64 and its respective mean fluorescence pixel intensity (MFPI) (B) N22 and (D) IR64. Each vertical column represents mean ± standard error of five replicates.

are tolerant and sensitive cultivars, respectively (Shankar et al., 2016).

Cd stress (Shahabivand et al., 2012). P. indica could promote growth of host plant by harvesting more nutrients from the soil (Hartley and Gange, 2009), which in turn helps to run essential biochemical pathways required to achieve optimum growth (Sirrenberg et al., 2007; Vadassery et al., 2009). IR64 seedlings exhibited short shoot length under Cd stress which could be attributed to high Cd accumulation towards root tips, which is the most active part for root development and nutrient acquisition. High Cd accumulation in active root zone resulted in high ROS production and increased cell death. Interestingly, better growth of N22 seedlings under Cd stress could be attributed to low Cd accumulation towards root tips. However, P. indica inoculated plants under Cd stress showed improved growth with reduced ROS production and cell death in both N22 and IR64. Uptake of Cd, transport and accumulation by plants are strongly influenced by soil properties and vary with plant species (Zhang et al., 2009). Rice cultivars (N22 and IR64) exhibited different amount of Cd uptake in roots with and without P. indica treatment. Under similar growth conditions and Cd treatment, heavy metal distribution and accumulation may differ among different rice varieties (Li et al., 2012a, b; Yan et al., 2013; Cheng et al., 2014; Song et al., 2015; Yao et al., 2015). Notably, two different rice cultivars indica and japonica showed varied Cd accumulation in roots (Liu et al., 2005; Ye et al., 2012). In addition, Kurz et al. (1999) also reported that different cultivars of same species can have immense difference in metal uptake and translocation. Apart from rice, differential Cd tolerance was also observed among different brassica cultivars (Gill et al., 2011). The above studies are in agreement with our results as high Cd accumulation was observed in roots of IR64 as compared to N22. Moreover, differential response of these two cultivars towards Cd stress indicates different behavior similar to drought stress as N22 and IR64

4.3. P. indica inoculated plants show decreased cell death and ROS production Abiotic stress including heavy metal stress are well documented to induce ROS generation and oxidative stress in plants, which further elicits cell death (Apel and Hirt, 2004; Halliwell, 2006; RodriguezSerrano et al., 2006; Gill and Tuteja, 2010; Rasool et al., 2013). H2DCFDA dye is non-fluorescent dye, which can readily enter into the cells and can convert into fluorescent form, it has been routinely used to visualize the intracellular ROS level (Peng et al., 2014; Nie et al., 2015; Nath et al., 2016, Nath et al., 2019). On the other hand, propidium iodide is a good indicator to differentiate the viable and non-viable cells as it does not cross the membrane of the live cells (Truernit and Haseloff, 2008; Nguyen and McCurdy, 2015; Nath et al., 2016, Nath et al., 2019). Therefore, these indicators were used for the ROS profiling and cell viability of the stressed roots of the rice cultivars. Our results demonstrated that roots of both the rice varieties (N22 and IR64) grown in Cd fortified soil exhibited higher ROS production and cell death across the treatments. Fluorescence microscopy study conducted in pea plants with Cd stress revealed that Cd induced ROS accumulation and high cell death in stressed pea roots (Rodriguez-Serrano et al., 2006). Moreover, accumulation of Cd has been documented to cause ROS production and lipid peroxidation in rice, which in turn elevated oxidative stress (Tripathi et al., 2012). Our confocal analysis conducted on stressed rice roots (N22 and IR64) also revealed high levels of ROS generation and cell death. However, plants pre-inoculated with P. indica recorded significantly lower levels of ROS and cell death in both the rice varieties under Cd stress. These results are in agreement with the 8

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Fig. 7. Model for the P. indica mediated Cadmium (Cd) tolerance. The Cd accumulation in non-colonized roots may increase the ROS accumulation and further leads to the reduction in cell viability while this is just reverse in the case of P. indica colonized roots though higher Cd accumulated in the colonized roots which can be sequestered by P. indica and ultimately alleviate the negative impact of stress. Non-colonized roots: without P. indica (-Pi); Colonized roots: + Pi, Cd: Cadmium.

4.4. P. indica sequester excess cadmium

study of P. indica and rice interaction under arsenic stress (Mohd. et al., 2017). Similar results regarding P. indica role in copper sequestration and reduction of oxidative stress and cell death are also reported in Cassia angustifolia under heavy metal stress (Nanda and Agarwal, 2018). Interestingly, higher cell death in P. indica colonized plant roots was found as compared with non-colonized one, which indicates mutual association initially require cell death during root colonization. Similar findings were previously reported for P. indica colonized roots of barley (Deshmukh et al., 2006) and Arabidopsis (Qiang et al., 2012). P. indica colonizes the plant root system and induce a systemic signaling mediated by ROS generation and other defense pathways involving salicylic acid and jasmonic acid (Stein et al., 2008). This signaling results in the activation of a broad-spectrum defense pathway, which majorly includes a fortified ROS scavenging pathway with increased production of antioxidant enzymes that protects the plant against oxidative damage. The role of fungus mediated stress tolerance linked with ROS metabolism in various crops under stressful environment (Waller et al., 2005; Baltruschat et al., 2008; Kumar et al., 2009; Hashem et al., 2016). Recently, Saddique et al (2018) reported that P. indica association with rice alleviate the osmotic stress and further provide the drought tolerance. Similarly, in the current study, fungus mediated reduction in heavy metal stress as demonstrated by attenuated ROS generation and cell death despite the presence of high Cd level in the stressed colonized roots, clearly indicates as one of the major reason of heavy metal tolerance.

However, here P. indica treatment significantly enhanced Cd uptake in stressed roots of both the rice cultivars as compared to non-inoculated one, which further suggests P. indica mediated Cd sequestration and alleviation of stressful environment. Similar findings were also reported in tobacco (Hui et al., 2015) and sunflower (Shahabivand et al., 2017), where P. indica treated stressed plants showed high Cd accumulation in roots as compared to non-inoculated stressed roots. Our results with N22 and IR64 under Cd stress are also in agreement with the above reports as Cd stressed inoculated roots has more Cd accumulation as compared to only stressed roots. Endophytes have the ability to increase host tolerance under heavy metal stress by metal chelating mechanisms thus helping in alleviating stress conditions. Adsorption mechanism of fungus associated with Cd tolerance level could be due to the presence of different functional group in the cell walls, which binds with metal ions during the biosorption process (Xu et al., 2012). Accumulation of heavy metals by spores was observed in many AMF fungi as a way to sequester heavy metal and protect the plant from heavy metal stress. Electron microscopy analysis of spores and hyphae of Glomus intraradices has been reported to accumulate cadmium, zinc and copper (González-Guerrero et al., 2008). In another study, confocal microscopy results revealed the accumulation of aluminium (Al) in spores isolated from Al treated Glomus intraradices (Aguilera et al., 2011). However, Mohd et al (2017) also reported presence of arsenic in vacuoles and cell wall of P. indica spores under heavy metal stress. Similarly, our results of confocal microscopy 9

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revealed accumulation of Cd in spores isolated from 0.01 mM, 0.05 mM and 0.1 mM concentration which indicates sequestration ability of P. indica towards Cd. In a previous study, augmented level of heavy metal tolerance in colonized maize plants correlated with the high Cd accumulation in the metal tolerant fungus-Exophiala pisciphila, (H93) (Li et al., 2011). Similar findings of this study, suggests stress tolerance of the rice was linked with the increased level of Cd accumulation in the P. indica colonized roots under stress condition. Overall, the findings of this study indicate that Cd adsorption by fungus P. indica spores and hyphal mass within the root tissues could be a major mechanism that confined Cd accumulation in microbial cells within the root tissues and thus preventing its translocation to aerial parts of plants.

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5. Conclusion Our findings concluded that P. indica survived under very high concentration of Cd and alleviate the stressful environment in the rice cultivars (N22 and IR64). When grown in association with host plants, this fungus has remarkably sequestered Cd in ambient root tissues which were evident from better plant growth despite the presence of high Cd and attenuated ROS and cell death in stressed colonized roots. Further, a schematic model is also illustrated highlighting the P. indica mediated Cd sequestration to alleviate the stressful environment in rice (Fig. 7). Author contributions S.D. planned and conducted the experiments, data interpretation and helped in manuscript writing. Yashaswee helped in the different experiments and manuscript preparation. AV and DKC helped in finalizing the manuscript and contributed towards data interpretation. MN and RNB conceived and designed the experiment, contributed in planning, data analysis and manuscript writing. All authors read and approved the final manuscript. Acknowledgments We are thankful for partial financial support from the Science & Engineering Research Board, Department of Science & Technology (DST); (Grant: ECR/2016/000653), Govt. of India. RNB acknowledge the support from Science & Engineering Research Board, Young Scientist Grant (YSS/2015/000523). SD is thankful for the Confocal Microscopy Facility, AIMT, AUUP, Noida funded by DST-FIST. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2019.109741. References Adriano, D.C., 2001. Cadmium in Trace Elements in Terrestrial Environments: Biogeochemistry, Bioavailability, and Risks of Metals, second ed. Springer, NewYork, pp. 264–314. Aguilera, P., Borie, F., Seguel, A., Cornejo, P., 2011. Fluorescence detection of aluminum in arbuscular mycorrhizal fungal structures and glomalin using confocal laser scanning microscopy. Soil Biol Biochem 43 (12), e2427–e2431. https://doi.org/10.1016/ j.soilbio.2011.09.001. Al-Hobaib, A.S., Al-Jaseem, K.Q., Baioumy, H.M., Ahmed, A.H., 2012. Environmental impact assessment inside and around Mahd Adh Dhahab gold mine, Saudi Arabia. Arab J Geosci 4, 199–205. Apel, K., Hirt, H., 2004. Reactive oxygen species :metabolism,oxidative stress and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. https://doi.org/10.1146/annurev. arplant.55.031903.141701. Arunakumara, K.K.I.U., WalpolaBC, Yoon, M.H., 2013. Current status of heavy metal contamination in Asia's rice lands. Environ. Sci. Technol. https://doi.org/10.1007/ s11157-013-9323-1. Aziz, R., Rafiq, M.T., Li, T., Liu, D., He, Z., Stoffella, P.J., Sun, K.W., Xiaoe, Y., 2015. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/ toxicity in human cell lines (Caco-2/HL-7702). J. Agric. Food Chem. 63, 3599–3608.

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