Host plant growth promotion and cadmium detoxification in Solanum nigrum, mediated by endophytic fungi

Ecotoxicology and Environmental Safety 136 (2017) 180–188

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Host plant growth promotion and cadmium detoxification in Solanum nigrum, mediated by endophytic fungi Abdur Rahim Khan a, Ihsan Ullah a,b, Muhammad Waqas a, Gun-Seok Park a, Abdul Latif Khan c, Sung-Jun Hong a, Rehman Ullah d, Byung Kwon Jung a, Chang Eon Park a, Shafiq Ur-Rehman e, In-Jung Lee a, Jae-Ho Shin a,n a

School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Republic of Korea Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Pakistan c UoN Chair of Oman's Medicinal Plants & Marine Natural Products, University of Nizwa, 616 Nizwa, Oman d Department of Botany, University of Peshawar, Peshawar, Pakistan e Department of Plant Sciences, Kohat University of Science and Technology (KUST), Kohat 26000, Pakistan b

art ic l e i nf o

a b s t r a c t

Article history: Received 20 October 2015 Received in revised form 4 March 2016 Accepted 8 March 2016

Current investigation conducted to evaluate the associated fungal endophyte interactions of a Cd hyperaccumulator Solanum nigrum Korean ecotype under varying concentrations of Cd. Two indole-3-acetic acid (IAA) producing fungal strains, RSF-4L and RSF-6L, isolated from the leaves of S. nigrum, were initially screened for Cd tolerance and accumulation potential. In terms of dry biomass production, the strain RSF-6L showed higher tolerance and accumulation capacity for Cd toxicity in comparison to RSF4L. Therefore, RSF-6L was applied in vivo to S. nigrum and grown for six weeks under Cd concentrations of 0, 10, and 30 mg Kg
1 of dry sand. The effect of fungal inoculation assessed by plant physiological responses, endogenous biochemical regulations, and Cd profile in different tissues. Significant increase were observed in plant growth attributes such as shoot length, root length, dry biomass, leaf area, and chlorophyll contents in inoculated RSF-6L plants in comparison to non-inoculated plants with or without Cd contamination. RSF-6L inoculation decreased uptake of Cd in roots and above ground parts, as evidenced by a low bio-concentration factor (BCF) and improved tolerance index (TI). However, Cd concentration in the leaves remained the same for inoculated and non-inoculated plants under Cd spiking. Fungal inoculation protected the host plants, as evidenced by low peroxidase (POD) and polyphenol peroxidase (PPO) activities and high catalase (CAT) activity. Application of appropriate fungal inoculation that can improve tolerance mechanisms of hyper-accumulators and reduce Cd uptake can be recommended for phyto-stabilisation/immobilisation of heavy metals in crop fields. & 2016 Elsevier Inc. All rights reserved.

Keywords: Endophytic fungi Solanum nigrum Cd-hyperaccumulator Phytoremediation

1. Introduction To satisfy the needs of an exponentially growing human population, industrialisation, urbanisation, intensive agriculture, and extensive mining have accelerated, thereby devastating natural resources and creating widespread environmental contamination (Wan et al., 2012). The unprecedented accumulation and magnification of heavy metals in the environment has posed a lifethreatening dilemma for all living organisms including plants (Gerhardt et al., 2009). Rapid industrialisation has been one of the major contributing factors to soil contamination by heavy metals, and this poses a significant risk to the global ecosystem. Heavy n

Corresponding author. E-mail address: [email protected] (J.-H. Shin).

http://dx.doi.org/10.1016/j.ecoenv.2016.03.014 0147-6513/& 2016 Elsevier Inc. All rights reserved.

metals are non-biodegradable, can enter living beings, including humans, through the food chain, where they accumulate and cause lethal effects (Dong et al., 2001). Cadmium (Cd) is a nonessential, considerably toxic, widespread heavy metal, present in trace amounts in the soil; however, its concentration in the environment is increasing rapidly due to anthropogenic activities such as industrialisation, urbanisation, pesticide application in agricultural fields, and mine exploration (Nagajyoti et al., 2010; Prapagdee et al., 2013). The presence of Cd in the environment, particularly in the soil, poses several ecological problems and creates negative impacts on the environment, plants, and human health (Xu and Wang, 2014). Cd is relatively mobile in soil and is easily absorbed by plants, thus, leading to severe disturbances in the normal metabolic processes of plants, such as respiration, photosynthesis, and chlorophyll synthesis, and interference with

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enzyme activity. Moreover, Cd also produces reactive oxygen species (ROS), which lead to DNA destabilisation (Rivetta et al., 1997; Sanità et al., 1999). In addition, high concentrations of Cd retard the growth of roots and shoots, and cause leaf withering and chlorosis, nutrient imbalances, and biomass reduction (Zhang et al., 2010). Among the strategies used in the remediation of heavy metal contaminated soils, phytoremediation has been considered recently as an alternative, environmentally friendly, and cost effective strategy (Salt et al., 1998). Plant species used for phytoremediation, which have the ability to accumulate exceptionally high concentrations of heavy metals, are referred to as hyperaccumulators. The majority of hyperaccumulators belong to the non-mycorrhizal family of Brassicaceae and Solanaceae (Van de Mortel and Aarts, 2006). Plant species such as Arabidopsis halleri (Dahmani-Muller et al., 2001), Thlaspi caerulescens (Baker et al., 1994), and Solanum nigrum L. (Wei et al., 2005) can hyperaccumulate Cd in aerial parts without showing any adverse effects (Lasat, 2002). Although they possess numerous advantages, some limitations have also been reported for these hyperaccumulators, such as low tolerance levels, slow growth with low biomass production, and variations in bioavailability of heavy metals (Gerhardt et al., 2009; Glick, 2010). In addition, a high level of heavy metals in soil impairs metabolism and reduces growth of hyperaccumulators, consequently, highly restricting the phytoextraction potential of such plants (Rajkumar et al., 2010). The development of other phytoremediation strategies to detoxify contaminated soils is therefore necessary. Possible substitute solutions to improving the overall efficiency of hyperaccumulators include increasing the tolerance level of plants against contaminant toxicity by processes such as microbe-assisted phytoremediation (Rajkumar et al., 2012). Fungal endophytic association with host plants is a relatively new approach to enhancing multiple plant characteristics such as growth, biomass, and phytoremediation potential (Mei and Flinn, 2010). In addition, heavy metal resistant fungal endophytes capable of promoting plant growth show high potential for eco-friendly, costeffective strategies towards the remediation of heavy metal contaminated soils and the regulation of heavy metal induced toxicity in crops and other plants (Aishwarya et al., 2014; Khan and Lee, 2013). Endophytes play various important roles in host plant growth through different mechanisms. Through symbiotic association, host plant growth can be enhanced by morphological and chemical changes triggered by endophytes inside the plant tissues that affect the composition of nutrients and provide protection against biotic and abiotic stresses (Singh et al., 2011). This potential of endophytes are attributed to the production of various phytohormones such as gibberellins and auxins, the combined effects of which provide the arsenal to combat the adverse effects of extreme environmental conditions (Waqas et al., 2014). Such heavy metal resistant phytohormone-producing fungi have the ability to detoxify toxic metals by immobilising them through insoluble metal oxalate formation, biosorption, or chelation onto melanin-like polymers. Fungal isolates of various genera such as Trichoderma and Aspergillus, and arbuscular mycorrhizal fungi (AMF) have been studied for their phytoremediation potentials in contaminated soil (Firmin et al., 2015). In the present study, efforts were made to understand how the association of indole-3-acetic acid (IAA)-producing fungal endophytes could enhance the efficiency of the Cd hyperaccumulator, S. nigrum (Teixeira et al., 2011). Several studies have shown the phytoremediation potential of different ecotypes of S. nigrum in Australia, China, Portugal, and South Korea (Khan et al., 2014; Wei et al., 2013, 2005), focusing mainly on the associated bacterial endophytes, whereas other studies have extensively explored endophytic interactions under conditions of Cd

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contamination (Chen et al., 2014, 2010; Khan et al., 2015b). However, further research on S. nigrum-associated fungal endophytes and their interactions in the presence of Cd is needed. The isolation and characterisation of S. nigrum-associated fungal endophytes is a prerequisite for the development of effective phytoremediation techniques for Cd contaminated soils. Sufficient information about the potential interaction of endophytic fungi isolated from S. nigrum in Cd phytoremediation is unavailable. In the present study, we investigated the mutual interactions of endophytic fungi with the host plant S. nigrum under Cd contaminated conditions. Pure sand was used to avoid interference by other factors during the interaction between potential endophytic fungi and the host plant. Responses of fungal endophyte infected S. nigrum plants were evaluated in sand spiked with different Cd concentrations. In a previous study, we isolated and identified two endophytic fungal strains, i.e. Fusarium tricinctum RSF-4L and Alternaria alternata RSF-6L, capable of actively producing IAA from the leaves of the S. nigrum plant (Khan et al., 2015a). We hypothesised that such IAA producing fungal endophytes might be helpful in the mitigation of heavy metal stress, by improving the physiological status of plants. An important consideration of the present study was that such IAA producing endophytic fungi might restore endogenous IAA levels required for normal plant growth, which inhibited under Cd stress (Yuan and Huang, 2015). Initially, both isolates (RSF-4L and RSF-6L) were screened for Cd tolerance and bioaccumulation. RSF-6L was then selected based on high Cd tolerance and bioaccumulation. After the initial screening of Cd bioaccumulation ability, we assessed the potential of the phytohormone-producing endophytic fungus RSF-6L, to enhance plant physiological processes during bioaccumulation and uptake of Cd metal in hyperaccumulator S. nigrum plants and its subsequent effects on biochemical responses.

2. Materials and methods 2.1. Fungal endophyte isolation source, identification, and stock preservation Two fungal endophytes, F. tricinctum RSF-4L and A. alternata RSF-6L (GenBank accession numbers: KM100450 and KM100451, respectively) were previously isolated from the leaves of S. nigrum Korean ecotype plants (Khan et al., 2015a). Both isolates were capable of growth promotion in rice plants, which was attributed to innate IAA production. The isolates were identified by sequencing the ITS1-ITS4 region of the internal transcribed spacer (ITS) of extracted gDNA by using the universal primers ITS1-F (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS-4R (5′-TCC TCC GCT TAT TGA TAT GC-3′). Glycerol stock (50%) was made of each isolate and stored at
80 °C for future use. Both isolates were grown on potato dextrose agar (PDA) medium for 7 days at 25 °C, for further characterisation. 2.2. Determination of minimum inhibitory concentration under conditions of Cd contamination The minimum inhibitory concentrations (MIC) of Cd for the fungal endophytes were determined using potato dextrose agar (PDA) medium spiked with increasing concentrations of Cd (0, 1, and 2 mM). Three replicates of each treated PDA plate were inoculated with a 5 mm agar plug from the edge of seven-day-old culture plates of both isolates. PDA plates without Cd augmentation were inoculated with both isolates and used as controls. The PDA plates were then incubated at 25 °C for seven days. The diameter of growing mycelia was measured daily and the initial diameter was subtracted from the subsequent diameter. The

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highest concentration of Cd that completely inhibited fungal growth was considered the minimum inhibitory concentration (MIC). Furthermore, the tolerance index (TI) was calculated from the extent of growth in each isolate exposed to Cd, divided by the extent of growth in the control plates. RSF-6L was selected based on high Cd tolerance, and subjected to Cd bioaccumulation analysis. 2.3. Bioaccumulation of Cd by growing fungus in culture broth The capacity of fungal isolate RSF-6L for biosorption of Cd ions was measured by inoculating 100 mL Czapek broth spiked with 1 mM Cd. Endophyte RSF-6L was grown for 8 days at 28 °C at 150 rpm and pH values of the broth were measured at 24 h intervals. Mycelia and liquid medium were separated by vacuum filtration using 0.22 mm filter paper. The mycelia were dried in an oven at 70 °C for 48 h and culture filtrates were preserved at
20 °C until further processing. Filtrates and dried mycelia were used for Cd quantification, according to the method outlined by Deng et al. (2014b). All measurements were done in triplicate, and results were presented as mean values. 2.4. Host plant and endophytic fungi interaction under Cd stress Sand (particle size 1.0 mm, pH 7) purchased from was washed with autoclaved deionised water, sterilised twice (at 120 °C for 15 min, 15 psi) and dehydrated at 70 °C overnight (Ulsan MansaeDae Susuck Garden, Ulsan, South Korea). The sand was then spiked with CdCl2 aqueous solution to obtain the final concentrations of Cd of 0, 10, and 30 mg Kg
1. The investigation consisted of six treatments: (i) control plants without Cd or RSF-6L inoculation; (ii) plants inoculated with RSF-6L; (iii) Cd-10 mg Kg
1 treated plants; (iv) Cd-10 mg Kg
1 plants infected with RSF-6L; (v) Cd-30 mg Kg
1,; and (vi) Cd-30 mg Kg
1 infected with RSF-6L. The Cd spiked sand was stabilised under growth chamber conditions for a minimum of two weeks. In the meanwhile, S. nigrum seeds were surface sterilised using 2.5% sodium hypochlorite for 30 min, treated with 70% ethanol (EtOH) for 30 s, and washed with autoclaved distilled water (D.W.) twice. The seeds were germinated for 4–5 days on autoclaved filter papers in 9-cm petri plates moistened with autoclaved distilled water. Subsequently, the germinated seeds were grown for 15 days in multi well pots containing autoclaved sand and supplied with half strength Hoagland's solution in a growth chamber with a day/night regime of 25 °C, 16/8 h light/dark cycle, and 60% relative humidity. At the same time, plastic pots lined with polythene bags were filled with stabilised Cd spiked sand (500 g in each pot) with five replicates for each treatment, resulting in 30 pots. The strain RSF-6L cultured in 500 ml Czapek broth for 10 days at 25 °C using shaking incubator. Mycelia were collected by filtration using vacuum pump and re-suspended in sterile distilled water. Equal size seedling were selected from pre-germinated seeds and their roots were dipped in the fungal mycelial suspension for 1 h. Seedlings were then transplanted into plastic pots containing Cd modified sand and shifted to the aforementioned growth chamber programme. The plants were left to grow for 6 weeks after fungal inoculation. The investigation was performed in a completely randomised block design. Pots were daily irrigated as per requirement with D. W. and nutrients were supplemented by the application of half strength Hoagland's solution on a weekly basis. 2.5. Assessment of inoculated and non-inoculated plant growth attributes with and without Cd contamination After 6 weeks of growth, chlorophyll contents (measured by a chlorophyll metre, SPAD-502 Minolta, Japan) and leaf area of

samples from each treatment were determined. Plants were harvested by cutting the stem above the sand surface. Shoot length and fresh weight were measured and immediately stored in liquid nitrogen for biochemical analysis. Likewise, roots were carefully separated from sand and washed with D.W. to determine root length and fresh weight. For Cd analysis, randomly selected root and shoot samples were washed with D.W., blotted with tissue paper, and oven dried at 80 °C for 48 h. Dry weights of samples were determined, after which they were ground to powder and subjected to Cd content analysis. 2.6. Biochemical analyses of inoculated and non-inoculated plants with and without Cd contamination Antioxidant contents and enzymes in fresh leaf samples stored at
80 °C. For antioxidant enzymatic activities, leaf samples (0.5 g) from each treatment were ground in liquid nitrogen and the powder was suspended in extraction buffer consisting of 50 mM sodium phosphate (pH 7.0), 1% polyvinyl-polypyrrolidone (PVP; w/v), and 0.1 mM EDTA. The homogenate was centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant was used to determine total protein contents according to the Bradford (1976) assay method using bovine serum albumin (BSA, Sigma) as the standard. Furthermore, the activity of antioxidant enzymes including catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) were measured according to the methods previously outlined by Khan et al. (2014). The activity of POD and PPO enzymes were measured at a wavelength of 420 nm. One unit each of POD and PPO was indirectly measured by an increase of 0.1 unit of absorbance. 2.7. Microscopic analyses of endophyte infected plants with and without Cd contamination To determine the association of fungal endophyte with the roots of S. nigrum, microscopic analyses were carried out with a light microscope. Plants infected with RSF-6L were carefully uprooted from the sand, washed with deionised water, and treated with 2.5% sodium hypochlorite for 10 min for surface sterilisation. The sterilised roots were cut aseptically into pieces of about 1 cm in length under a laminar flow hood. The root pieces were subsequently kept in KOH (20%) solution for 24 h and then rinsed extensively with D.W. The roots were stained overnight in 0.5% acid fuchsin and 95% lactic acid. The roots were de-stained with lactic acid for 24 h and visualised under a light microscope. 2.8. Cadmium measurement and determination of related parameters Cadmium ions were analysed in dried plant tissues, fungal biomass, and culture broth throughout the experiment. Ovendried plant tissue (0.1 g) and fungal biomass (0.1 g) samples were ground with a mortar and pestle to a fine powder. The ground samples and culture broth were digested in a mixture of HNO3 and HClO4. Cd contents were determined by inductively coupled plasma spectroscopy (ICP) (Optima 79000DV, Perkin Elmer, USA). The Cd translocation efficiency from roots to shoots of plants was evaluated by calculating the translocation factor (TF) using the following formula:

TF =

Concentration of Cd in shoots mgKg−1 Concentration of Cd in roots mgKg−1

(1)

The bioconcentration factor (BCF) was calculated as the ratio of Cd concentration in plant tissues to the Cd concentration in the soil, according to the formula of Gonzalez-Mendoza et al. (2007) as

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follows:

BCF =

Concentration of Cd in plant mgKg

−1

Concentration of Cd in soil mg·Kg−1

(2)

The Cd tolerance index (TI) was determined using the following formula developed by Wilkins (1978)

TI(%)=

Root length in Cd treated plants × 100 Root length in control plants

(3)

2.9. Statistical analysis All experiments were performed in triplicate and values are presented as the means 7SD. The data were analysed using GraphPad Prism software (version 5.01, San Diego, CA, USA). Significant differences (p o0.05) between the mean values of different treatments were compared and evaluated using Duncan's multiple range test (DMRT) on a Statistical Analysis System (SAS, Cary, NC, USA).

3. Results 3.1. Endophytic fungi tolerance to Cd and its accumulation in body mass The two selected phytohormone producing fungal endophytes were tested for Cd tolerance at concentrations of 0, 1, and 2 mM (Fig. 1a). F. tricinctum RSF-4L showed sensitivity and intolerance towards Cd and didnot grow even on the lowest concentration of

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Cd; whereas, A. alternata RSF-6L grew well on 1 mM Cd supplemented medium over 8 days of incubation. In contrast, the growth of RSF-6L was strongly inhibited at 2 mM of Cd-spiked PDA plates. The RSF-6L strain showed a relatively higher (58%) TI at a concentration of 1 mM Cd in comparison to 2 mM Cd. Therefore, only RSF-6L was processed and used for further experiments. In addition, the Cd accumulation capability of the strain RSF-6L was also evaluated in 0.5 mM Cd spiked Czapek broth. The pH of the culture media increased slightly from 7.0 to 7.5 and remained minimally changed over one week of fungal incubation (Fig. 1b). The strain accumulated over 78% and 57% Cd when cultured under 0.5 and 1 mM Cd spiked medium, respectively (Fig. 1c). The dry biomass showed an inverse relationship with Cd concentration and was reduced by as much as 36% at 0.5 mM Cd and 81% at 1 mM Cd (Fig. 1d). 3.2. Active colonisation of RSF-6L improved Solanum nigrum growth attributes under Cd contamination Seedlings of the S. nigrum plant were inoculated with A. alternata RSF-6L, in the absence and presence of different concentrations of Cd (0, 10, and 30 mg/kg). After 6 weeks of endophyte association, the visible growth promoting effects based on plant morphological characteristics including shoots and roots were compared to non-inoculated controls (Fig. 2a, b). Endophytic fungi colonisation was confirmed in stained root fragments and microscopic images reflected the association with RSF-6L, whereas plants without inoculation showed no fungal association (Fig. 2c, d, e, and f). In addition, the effect of fungal inoculation on S. nigrum plants under Cd contamination were determined by measuring chlorophyll contents, leaf area, root and shoot length, and fresh

Fig. 1. Determination of minimum inhibitory concentration (MIC) of Cd for selected fungal endophytes; in vitro response of RSF-6L and RSF-4L grown on 0, 1, and 2 mM cadmium (Cd) contaminated PDA media (a). Effect of Cd concentrations on pH curve of RSF-6L grown in Czapek broth modified with 0 mM, 0.5 mM, and 1.0 mM Cd for 8 days (b). Dry biomass of RSF-6L (c). Uptake and accumulation of Cd in RSF-6L biomass (d). Pooled data of all experiments; each value represents the mean 7SE of three replicates per treatment from three independently conducted experiments. For each set of treatments, different letter(s) indicate significant differences between different treatments of Cd (0 mM, 0.5 mM, and 1.0 mM) at po 0.05, by Duncan's multiple range test (DMRT).

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Fig. 2. Rescue effect of RSF-6L inoculation on the growth attributes of Solanum nigrum grown with 0, 10, and 30 mg/kg Cd. Endophytic fungi inoculation has clearly promoted plant growth parameters as compared to their respective control plants in the absence and presence of Cd heavy metal stress (a). Effect of RSF-6L on growth and morphology of root growth in the absence and presence of Cd heavy metal stress (b). Microscopic observation of plant root colonisation by RSF-6L was found in control plants (c), 0 mg Cd treated plants (d), as well as in 10 mg (e), and 30 mg (f), Cd treated plants.

Fig. 3. Effects of RSF-6L inoculation on growth attributes of S. nigrum over six weeks: shoot and root length (a), dry biomass (b), leaf area (c), and chlorophyll contents (d) in the absence and presence of Cd at concentrations of 0, 10, and 30 mg/Kg of dry sand. Each value is the mean 7 SE of three replicates per treatment from three independently conducted experiments. For each treatment, different letter(s) indicate significant differences for growth characteristics noted on plants grown at different concentrations of Cd (0, 10, and 30 mg/Kg) by DMRT (po 0.05).

and dry weight (Fig. 3). Cd augmentation significantly retarded the shoot and root growth and total dry biomass production (Fig. 3a, b). In the absence of Cd, endophytic fungi inoculation significantly increased shoot and root length of S. nigrum plants by 30% and 24% respectively, as compared to the untreated control. On the other hand, without endophytic association, Cd contamination significantly retarded the shoot (63%) and root length (46%) of plants as compared to the untreated controls. Plants inoculated with endophytic fungi had significantly longer shoots (53%) and roots (37%) in the presence of Cd as compared to non-inoculated

counterparts. Similarly, in fungi-inoculation treatments, the plants had significantly greater total dry biomass production, leaf area, and chlorophyll contents as compared to non-infected Cd treatments (Fig. 3b, c, d). The total dry biomass of plants inoculated with endophytic fungi was 25%, 30%, and 15% higher than noninoculated untreated, 10, and 30 mg/Kg Cd treatments, respectively. The leaf area was significantly greater in RSF-6L inoculated plants than in non-inoculated plants (Fig. 3c). Cd contamination significantly reduced the leaf area in non-inoculated plants, whereas this parameter was significantly increased in plants

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Table 1 Cadmium uptake and concentration profile in different tissues of fungal endophyte RSF-6L infected and non-infected Solanum nigrum plants grown in 0, 10 and 30 mg Kg
1 concentrations of Cd. Cd treatment (mg Kg
1 sand DW)

0 10 30

Association

Control RSF-6L Control RSF-6L Control RSF-6L

Cd concentration (mg Kg
1 plant DW) Leaf

Stem

Root

ND ND 265 7 14ab 2617 21ab 293 7 21a 250 7 20b

ND ND 8477 25b 228 7 18d 1130 766a 5737 31c

ND ND 15007 50c 1021 798d 2322 7 96a 1820 7 50b

TF

BCF

TI%

– – 0.7 0.5 0.6 0.5

– – 1611 1070 2464 1902

– – 38 61 10 12

Control¼ fungal endophyte free plants, RSF-6L ¼ Solanum nigrum plants infected with fungal endophyte RSF-6L, TF ¼Translocation factor, BCF ¼ Bio-concentration factor, TI% ¼ Percentage of tolerance index. The different alphabets are representing significant differences among the samples. Cd concentration was calculated on the basis of dry weight (DW). Values in each column represent the mean 7 SE. Mean values of the control and RSF-6L inoculated plants grown under different Cd concentrations (mg Kg
1 dry sand) in each column denoted by the different letters are significantly different at p o 0.05 as analysed by Duncan's multiple range test.

inoculated with RSF-6L. Furthermore, chlorophyll contents were significantly higher only in RSF-6L inoculated plants under 10 mg/ kg Cd contamination in comparison to non-inoculated Cd treated plants (Fig. 3d). 3.3. RSF-6L inoculation decreased Cd uptake and accumulation in S. nigrum tissues The effects of RSF-6L inoculation on Cd accumulation/uptake and distribution in different tissues of S. nigrum plants are summarised in Table 1. Generally, the concentration of Cd in both inoculated and non-inoculated plants increased in different plant parts with increasing concentrations of Cd in sand, with the exception of the leaves where the concentration did not change significantly with Cd application (Fig. 4). Higher concentrations of Cd accumulated in roots in comparison to stems and leaves irrespective of treatment. The trend of Cd concentration in different tissues was root 4stem4leaf in both Cd treatments (10 and 30 mg/kg), with and without RSF-6L inoculation. On the other hand, fungal inoculation significantly reduced uptake and distribution of Cd in roots and stems for both concentrations of Cd in comparison to non-inoculated treatments. However, Cd contents in leaves were not significantly changed irrespective of treatment. Endophyte RSF-6L inoculation decreased Cd accumulation in roots by as much as 31% and 22% under 10 and 30 mg/kg Cd treatments, respectively. Whereas RSF-6L inoculation caused a reduction in Cd contents of stems by as much as 73% and 49% in 10 and 30 mg/Kg Cd treatments, respectively, in comparison to non-inoculated Cdtreated plants.

3.4. RSF-6L inoculation inhibited Cd to above ground tissues as revealed by decreased BCF and increased TI Cd uptake, distribution, and translocation were further evaluated by determining total Cd contents, TF, BCF, and TI (Table 1). The TF ranged from 0.5 to 0.7, which showed negligible difference irrespective of treatment. Moreover, the BCF increased in non-inoculated Cd-treated plants with increasing Cd contamination. However, the BCF was significantly reduced by fungal inoculation under Cd contamination. On the other hand, TI was significantly increased by RSF-6L inoculation with the 10 mg/kg Cd treatment and slightly increased with the 30 mg/kg treatment. 3.5. Antioxidant enzymes responses of S. nigrum The oxidative stress responsive enzymes are summarised in Table 2. The activity of antioxidative enzymes including CAT, POD, and PPO were significantly influenced by Cd contamination. Particularly, POD and PPO activities were significantly enhanced primarily in the leaves of non-inoculated plants grown in Cd contaminated soil. In addition, PPO enzyme activity was not influenced by RSF-6L inoculation without Cd contamination; whereas RSF-6L inoculation significantly modified POD and PPO activity in plants grown in Cd contaminated sand. RSF-6L inoculation clearly caused significant reductions of POD and PPO activity in plants grown in Cd spiked sand. CAT activity on the other hand was significantly higher in fungal inoculated plants with the 30 mg/Kg Cd treatment only. Table 2 Antioxidant enzymes response in fresh leaves of fungal endophyte RSF-6L infected and non-infected Solanum nigrum plants grown in 0, 10 and 30 (mg Kg
1 sand DW) concentrations of Cd. Cd treatment Association CAT (units/mg POD (units/mg PPO (units/mg protein) protein) protein) (mg Kg
1 sand DW) 0 10 30

Fig. 4. Accumulation of Cd in different parts of inoculated and non-inoculated S. nigrum. The asterisk (*) denotes significant differences among treatments (po 0.05); the error bar represents the standard error.

Control RSF-6L Control RSF-6L Control RSF-6L

2.058 7 0.11a 0.577 0.03c 0.2437 0.04e 0.3557 0.03d 0.3777 0.05d 1.253 7 0.04b

31.5 7 2.57f 44.5 7 3.42e 111.0 7 3.61c 71.7 7 3.16d 172.8 7 4.47a 124.87 2.95b

4.5 70.90c 3.6 70.92c 9.4 71.00b 5.3370.99c 13.9 70.70c 9.1 71.33b

Control¼ fungal endophyte free plants, RSF-6L ¼ Solanum nigrum plants infected with fungal endophyte RSF-6L. The different alphabets are representing significant differences among the samples. Values in each column represent the mean 7 SE. Mean values of the control and RSF-6L inoculated plants grown under different Cd concentrations (mg Kg
1 dry sand) in each column denoted by the different letters are significantly different at p o0.05 as analysed by Duncan's multiple range test.

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4. Discussion Many fungal and bacterial species such as Rhizopus arrhizus, Mucor rouxii, Cupriavidus taiwanensis TJ208, Aspergillus niger, Bacillus jeotgali, and Pseudomonas veronii 2E have been reported to have the potential for accumulation of Cd and other heavy metals (Vinichuk et al., 2013; Vullo et al., 2008; Xiao et al., 2010). However, maximum Cd accumulation capacity, up to 173 mg/g from culture media, was reported for Mucor sp. CBRF59 (Deng et al., 2011a). The strain RSF-6L, which is capable of IAA production (Khan et al., 2015a) was found to be more Cd resistant than RSF-4L, as evidenced by the MIC of Cd. This finding could be the reason for its tolerance of higher concentrations of Cd. Based on its Cd resistance, the fungal endophyte RSF-6L was assessed for Cd accumulation efficiency. The strain was more efficient in Cd accumulation at lower concentrations (probably because of higher biomass production), in comparison to higher concentrations, which significantly reduced fungal biomass production. In the present study, strain RSF-6L accumulated lower amounts of Cd in comparison to other reported fungal strains i.e. Microsphaeropsis sp. LSE10 isolated from S. nigrum (Xiao et al., 2010). This might be due to the difference in tolerance mechanisms and compositions of the cell wall in the different fungal species. Phytoremediation with the use of hyperaccumulator plants has emerged as a promising and cost effective eco-friendly technique for the rehabilitation of heavy metal contaminated soils. However, the lower biomass production and lower tolerance levels to higher concentrations of contaminants are some of the obstacles to the efficiency of hyperaccumulator plant-based remediation technology (Gerhardt et al., 2009; Glick 2010). To overcome these challenges, alternative strategies such as the use of endophytic bacteria and fungi have recently been employed to improve the phytoremediation potential of plants. For this purpose, the selected endophytes should possess the ability to promote the growth and biomass yield of the host plant, resist the effects of the contaminant, and should sequester the target contaminant to reduce phytotoxicity. Our results reveal that fungal inoculation enhanced host plant growth in the presence of Cd by improving all plant growth attributes. The results of the present study clearly demonstrate that RSF-6L inoculation enhances the fresh and dry biomass of S. nigrum plants under conditions of Cd contamination. The outcomes of the plant growth experiments reveal that the endophytic fungal strain RSF-6L showed stimulatory effects on both shoots and roots of plants under conditions of Cd contamination. Enhanced plant growth and a significant increase in dry biomass was observed on plants inoculated with RSF-6L in the presence of Cd. Root elongation and proliferation of lateral and adventitious roots were significantly promoted in RSF-6L inoculated, Cd-treated plants in comparison to non-inoculated Cdtreated plants. These findings confirm an integral plant-fungal relationship, which is considered a pivotal factor for plant development and growth. Endophytic fungi have been reported to be plant growth regulators and protectants both in normal and in stressful environmental conditions. Several studies have reported many endophytic fungal species like Penicillium citrinum, Piriformospora indica, Neotyphodium sp., Curvularia protuberata, and Colletotrichum sp. that have improved plant growth in the presence of heavy metals (Vinichuk et al., 2013; Vullo et al., 2008). The ability of endophytic fungi to produce phytohormones is beneficial to the host plant in its defence against the adverse effects of abiotic stressors (Khan and Lee, 2013; Waqas et al., 2014). RSF-6L inoculation in plants yielded comparatively higher leaf area, chlorophyll contents, root/shoot length, and increased biomass. Moreover, the strain also mitigated the adverse effects of metal contamination on these plant growth parameters. The increased growth rate and total dry biomass yield could be

attributed to the superior physiological responses of S. nigrum induced by increased endogenous phytohormone levels, supplied by IAA-producing RSF-6L (Khan et al., 2015b). The supplementation of endophytic fungi RSF-6L IAA in S. nigrum under heavy metal stress may therefore diminish the deleterious effects of Cd, such as the inhibition of primary root growth and key metabolic and physiological processes (Yuan and Huang, 2015). Increased IAA production is evidenced by longer roots in Cd stressed endophytic infected plants. On the other hand, non-inoculated plants were found to have mostly reduced growth attributes, including biomass. Our results are consistent with our previous findings in which endophytic bacterial inoculation enhanced both plant growth and biomass (Khan et al., 2015a). Higher concentrations of Cd were found in the roots of S. nigrum, which also suggests its application as a phytostabiliser. Results of the present study are consistent with previous reports in which higher levels of Cd accumulated in the roots (Alvarenga et al., 2008). However, the disparity in accumulation patterns could be attributed to the use of different types of substrate media for plant growth, which causes variation in the availability of Cd for plant uptake (Chen et al., 2010; Khan et al., 2014; Teixeira et al., 2011). In addition, higher Cd availability does not necessarily lead to higher levels of translocation to different tissues of the plant. The present results reveal that accumulation of Cd in plant roots and stems increased with increasing Cd concentration. However, the Cd contents of leaves were not significantly different among treatments and concentrations of Cd. This can be attributed to a certain capacity for metal transfer from stem to leaf that is regulated to minimise further translocation once a certain threshold is attained. However, the transfer of Cd from root to stem also seems to be affected by a concentration gradient. Because of its relatively high tolerance and higher accumulation capacity of the root to retain Cd, S. nigrum might also be applicable to the phytostabilisation of Cd contaminated soil. Plants used for phytostabilisation should have a dense extensive root system with a large biomass production capacity in the presence of relatively high contamination levels, to keep the TF as low as possible (Alvarenga et al., 2008). The BCF and TF are the important parameters to be considered in determining the phytostabilisation potential of certain plant species (Alvarenga et al., 2008). However, using different substrate media for plant growth within the same species, the TF and BCF vary greatly in-vitro. This finding is evidenced by a number of reports in which the same plant species have different BCF and TF due to a difference in substrate media during screening (Chen et al., 2010; Khan et al., 2014; Teixeira et al., 2011). In the present study, only Cd treated plants without fungal inoculation showed a higher BCF and lower TF ( o1), suggesting that large amounts of Cd accumulated in roots and relatively smaller portions were translocated to the aerial parts of the plant. S. nigrum exhibits higher BCF and lower TF values, suggesting its potential application as a candidate plant for phytostabilisation of Cd contaminated sites, to thus minimise the migration of Cd into surface- and groundwater and reduce the risk of entry into the food chain. However, fungal inoculation significantly reduced Cd accumulation in roots as well as its subsequent translocation to stems. The immobilisation of significant amounts of Cd in the roots is a characteristic of certain plants. Such a mechanism is regarded as being protective against excessive accumulation of contaminants, especially in leaves to support the vital process of photosynthesis (Thurman, 1981). However, in the present study the concentration of Cd in the inoculated and non-inoculated plant leaves remained unchanged. The differences were evident in fungal inoculated plants, which produced significantly higher root and shoot biomass than those without inoculation treated only with Cd. Reducing the toxic effects of Cd contamination in plants might entail

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the use of endophytic fungi and plant growth promoting (PGP) bacteria. In the present study, fungal inoculation reduced total Cd accumulation in plants, but increased plant growth and biomass. It is well documented that fungal association with plants provide a filter function against heavy metals that leads to increased plant tolerance (Schulz and Boyle, 2005). Our results showed a positive effect of RSF-6L inoculation on the protection of S. nigrum grown in Cd contaminated sand. Fungal inoculation was found to alleviate oxidative stress in leaves, although similar Cd levels were detected in the leaves of inoculated and non-inoculated plants. Production of reactive oxygen species rapidly increased in plant cells under a variety of abiotic and biotic stress conditions, and this poses various challenges to normal cellular function. Consequently, radical scavenging activities are deployed to support homoeostasis through antioxidants like CAT, POD, and PPO. Our findings demonstrate that Cd contamination significantly increases the activity of the antioxidant enzymes POD and PPO in leaves. These results are consistent with previous studies, which showed that Cd treatment increases the activity of antioxidant enzymes in the leaves of S. nigrum (Khan et al., 2015a; Khan et al., 2014). Fungal inoculation conferred protection and reduced the deleterious effects of oxidative stress caused by Cd, as evidenced by decreased activity of two important enzymatic antioxidants (POD and PPO). The down regulation of PPO and POD activities of in the present study also verify the results obtained in two major maize and sunflower crops infected with AMF Glomus intraradices and Glomus mosseae species. The AMF species not only downregulated PPO and POD activity, but also enhanced growth attributes and reduced uptake of Cd in shoots of maize and sunflower (Aghababaei and Raiesi, 2015). However, CAT activity showed dissimilar effects and was increased in inoculated plants. This finding is consistent with those of Babu et al. (2015), who demonstrated the protective effect of the endophytic bacteria Pseudomonas koreensis AGB-1 on host plant Miscanthus sinensis exposed to mine-tailing soil (containing Cd and other heavy metals), increased uptake of heavy metal, and significant increase in CAT activity. However, in the present study, the up-regulation of CAT activity in the leaves of RSF-6L inoculated S. nigrum, is the opposite to that found in the leaves of AMF infected maize and sunflower exposed to Cd, as presented by Aghababaei and Raiesi (2015). Furthermore, a more comprehensive explanation can be inferred from the experiments of Zhao et al. (2015), with the dark septate endophyte Exophiala pisciphila, which was grown in broth media, either with or without Cd. At the end of their investigation, the transcriptome of E. pisciphila exposed to Cd stress, revealed that 575 genes were differentially expressed, 40% of which were related to ten established heavy metal tolerant pathways. Among them, 12 loci were identified that were responsible for the generation of free radicle scavengers. Genes controlling CAT activity were up regulated, whereas those related to POD activity were down regulated. According to our findings, the protection conferred by fungi might not be limited to the stimulation of antioxidant enzyme activity, nor did it appear to be related to Cd uptake in leaves (Firmin et al., 2015).

5. Conclusion Solanum nigrum has been extensively reported as a promising Cd hyperaccumulator. However, further details are yet to be explored for its associated endophytic fungi and their role in phytotoxicity reduction. Furthermore, no previous study has reported on phytohormones, particularly IAA producing fungal endophytes and the mechanisms by which they facilitate Cd accumulation/ stress resistance in S. nigrum. The present study demonstrated that endophytes such as RSF-6L, isolated from S. nigrum could grow in

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Cd contaminated agar and broth media. RSF-6L rescued host plant growth attributes, by immobilising Cd in the roots, reducing its uptake, and by enhancing enzymatic activities. Finally, we can conclude that each of the endophytic fungi harboured by the host plant, perform specific functions and collectively shape the specific traits of the plant. In the present study, the selected endophyte produced IAA to balance the endogenous levels needed for proper physiological functions that are compromised by Cd stress. Other endophytes may enhance Cd uptake. This unique, delicately balanced relationship with endophytic fungi might make S. nigrum a perfect choice for Cd phytoremediation. Endophytes such as RSF6L, capable of IAA production and suppression of Cd uptake in host plants, can also be recommended for crops grown in Cd contaminated soil.

Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF2015R1D1A1A01057187).

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