Ecotoxicology and Environmental Safety 136 (2017) 180–188

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Ecotoxicology and Environmental Safety

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Host plant growth promotion and cadmium detoxification in Solanumnigrum, 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 Koreab Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Pakistanc UoN Chair of Oman's Medicinal Plants & Marine Natural Products, University of Nizwa, 616 Nizwa, Omand Department of Botany, University of Peshawar, Peshawar, Pakistane Department of Plant Sciences, Kohat University of Science and Technology (KUST), Kohat 26000, Pakistan

a r t i c l e i n f o

Article history:Received 20 October 2015Received in revised form4 March 2016Accepted 8 March 2016

Keywords:Endophytic fungiSolanum nigrumCd-hyperaccumulatorPhytoremediation

x.doi.org/10.1016/j.ecoenv.2016.03.01413/& 2016 Elsevier Inc. All rights reserved.

esponding author.ail address: jhshin@knu.ac.kr (J.-H. Shin).

a b s t r a c t

Current investigation conducted to evaluate the associated fungal endophyte interactions of a Cd hyper-accumulator Solanum nigrum Korean ecotype under varying concentrations of Cd. Two indole-3-aceticacid (IAA) producing fungal strains, RSF-4L and RSF-6L, isolated from the leaves of S. nigrum, were in-itially screened for Cd tolerance and accumulation potential. In terms of dry biomass production, thestrain RSF-6L showed higher tolerance and accumulation capacity for Cd toxicity in comparison to RSF-4L. Therefore, RSF-6L was applied in vivo to S. nigrum and grown for six weeks under Cd concentrations of0, 10, and 30 mg Kg�1 of dry sand. The effect of fungal inoculation assessed by plant physiological re-sponses, endogenous biochemical regulations, and Cd profile in different tissues. Significant increasewere observed in plant growth attributes such as shoot length, root length, dry biomass, leaf area, andchlorophyll contents in inoculated RSF-6L plants in comparison to non-inoculated plants with or withoutCd contamination. RSF-6L inoculation decreased uptake of Cd in roots and above ground parts, as evi-denced by a low bio-concentration factor (BCF) and improved tolerance index (TI). However, Cd con-centration 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 polyphenolperoxidase (PPO) activities and high catalase (CAT) activity. Application of appropriate fungal inoculationthat can improve tolerance mechanisms of hyper-accumulators and reduce Cd uptake can be re-commended for phyto-stabilisation/immobilisation of heavy metals in crop fields.

& 2016 Elsevier Inc. All rights reserved.

1. Introduction

To satisfy the needs of an exponentially growing human po-pulation, industrialisation, urbanisation, intensive agriculture, andextensive mining have accelerated, thereby devastating naturalresources and creating widespread environmental contamination(Wan et al., 2012). The unprecedented accumulation and magni-fication of heavy metals in the environment has posed a life-threatening dilemma for all living organisms including plants(Gerhardt et al., 2009). Rapid industrialisation has been one of themajor contributing factors to soil contamination by heavy metals,and this poses a significant risk to the global ecosystem. Heavy

metals are non-biodegradable, can enter living beings, includinghumans, through the food chain, where they accumulate andcause lethal effects (Dong et al., 2001). Cadmium (Cd) is a non-essential, considerably toxic, widespread heavy metal, present intrace amounts in the soil; however, its concentration in the en-vironment is increasing rapidly due to anthropogenic activitiessuch as industrialisation, urbanisation, pesticide application inagricultural 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 andcreates negative impacts on the environment, plants, and humanhealth (Xu and Wang, 2014). Cd is relatively mobile in soil and iseasily absorbed by plants, thus, leading to severe disturbances inthe normal metabolic processes of plants, such as respiration,photosynthesis, and chlorophyll synthesis, and interference with

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188 181

enzyme activity. Moreover, Cd also produces reactive oxygenspecies (ROS), which lead to DNA destabilisation (Rivetta et al.,1997; Sanità et al., 1999). In addition, high concentrations of Cdretard the growth of roots and shoots, and cause leaf witheringand chlorosis, nutrient imbalances, and biomass reduction (Zhanget al., 2010).

Among the strategies used in the remediation of heavy metalcontaminated soils, phytoremediation has been considered re-cently as an alternative, environmentally friendly, and cost effec-tive strategy (Salt et al., 1998). Plant species used for phytor-emediation, which have the ability to accumulate exceptionallyhigh concentrations of heavy metals, are referred to as hyper-accumulators. The majority of hyperaccumulators belong to thenon-mycorrhizal family of Brassicaceae and Solanaceae (Van deMortel 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 hyper-accumulate Cd in aerial parts without showing any adverse effects(Lasat, 2002). Although they possess numerous advantages, somelimitations have also been reported for these hyperaccumulators,such as low tolerance levels, slow growth with low biomass pro-duction, and variations in bioavailability of heavy metals (Gerhardtet al., 2009; Glick, 2010).

In addition, a high level of heavy metals in soil impairs meta-bolism and reduces growth of hyperaccumulators, consequently,highly restricting the phytoextraction potential of such plants(Rajkumar et al., 2010). The development of other phytoremedia-tion strategies to detoxify contaminated soils is therefore neces-sary. Possible substitute solutions to improving the overall effi-ciency of hyperaccumulators include increasing the tolerance levelof plants against contaminant toxicity by processes such as mi-crobe-assisted phytoremediation (Rajkumar et al., 2012). Fungalendophytic association with host plants is a relatively new ap-proach to enhancing multiple plant characteristics such as growth,biomass, and phytoremediation potential (Mei and Flinn, 2010). Inaddition, heavy metal resistant fungal endophytes capable ofpromoting plant growth show high potential for eco-friendly, cost-effective strategies towards the remediation of heavy metal con-taminated soils and the regulation of heavy metal induced toxicityin crops and other plants (Aishwarya et al., 2014; Khan and Lee,2013). Endophytes play various important roles in host plantgrowth through different mechanisms. Through symbiotic asso-ciation, host plant growth can be enhanced by morphological andchemical changes triggered by endophytes inside the plant tissuesthat affect the composition of nutrients and provide protectionagainst biotic and abiotic stresses (Singh et al., 2011). This poten-tial of endophytes are attributed to the production of variousphytohormones such as gibberellins and auxins, the combinedeffects of which provide the arsenal to combat the adverse effectsof extreme environmental conditions (Waqas et al., 2014). Suchheavy metal resistant phytohormone-producing fungi have theability to detoxify toxic metals by immobilising them through in-soluble metal oxalate formation, biosorption, or chelation ontomelanin-like polymers. Fungal isolates of various genera such asTrichoderma and Aspergillus, and arbuscular mycorrhizal fungi(AMF) have been studied for their phytoremediation potentials incontaminated soil (Firmin et al., 2015).

In the present study, efforts were made to understand how theassociation of indole-3-acetic acid (IAA)-producing fungal en-dophytes could enhance the efficiency of the Cd hyper-accumulator, S. nigrum (Teixeira et al., 2011). Several studies haveshown 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 associatedbacterial endophytes, whereas other studies have extensively ex-plored endophytic interactions under conditions of Cd

contamination (Chen et al., 2014, 2010; Khan et al., 2015b). How-ever, further research on S. nigrum-associated fungal endophytesand their interactions in the presence of Cd is needed. The isola-tion and characterisation of S. nigrum-associated fungal en-dophytes is a prerequisite for the development of effective phy-toremediation techniques for Cd contaminated soils. Sufficientinformation about the potential interaction of endophytic fungiisolated from S. nigrum in Cd phytoremediation is unavailable. Inthe present study, we investigated the mutual interactions of en-dophytic fungi with the host plant S. nigrum under Cd con-taminated conditions. Pure sand was used to avoid interference byother factors during the interaction between potential endophyticfungi and the host plant. Responses of fungal endophyte infected S.nigrum plants were evaluated in sand spiked with different Cdconcentrations. In a previous study, we isolated and identified twoendophytic fungal strains, i.e. Fusarium tricinctum RSF-4L and Al-ternaria alternata RSF-6L, capable of actively producing IAA fromthe leaves of the S. nigrum plant (Khan et al., 2015a). We hy-pothesised that such IAA producing fungal endophytes might behelpful in the mitigation of heavy metal stress, by improving thephysiological status of plants. An important consideration of thepresent study was that such IAA producing endophytic fungimight restore endogenous IAA levels required for normal plantgrowth, which inhibited under Cd stress (Yuan and Huang, 2015).Initially, both isolates (RSF-4L and RSF-6L) were screened for Cdtolerance and bioaccumulation. RSF-6L was then selected based onhigh Cd tolerance and bioaccumulation. After the initial screeningof Cd bioaccumulation ability, we assessed the potential of thephytohormone-producing endophytic fungus RSF-6L, to enhanceplant physiological processes during bioaccumulation and uptakeof Cd metal in hyperaccumulator S. nigrum plants and its sub-sequent effects on biochemical responses.

2. Materials and methods

2.1. Fungal endophyte isolation source, identification, and stockpreservation

Two fungal endophytes, F. tricinctum RSF-4L and A. alternataRSF-6L (GenBank accession numbers: KM100450 and KM100451,respectively) were previously isolated from the leaves of S. nigrumKorean ecotype plants (Khan et al., 2015a). Both isolates werecapable of growth promotion in rice plants, which was attributedto innate IAA production. The isolates were identified by sequen-cing the ITS1-ITS4 region of the internal transcribed spacer (ITS) ofextracted gDNA by using the universal primers ITS1-F (5′-TCC GTAGGT GAA CCT GCG G-3′) and ITS-4R (5′-TCC TCC GCT TAT TGA TATGC-3′). Glycerol stock (50%) was made of each isolate and stored at�80 °C for future use. Both isolates were grown on potato dex-trose agar (PDA) medium for 7 days at 25 °C, for furthercharacterisation.

2.2. Determination of minimum inhibitory concentration underconditions of Cd contamination

The minimum inhibitory concentrations (MIC) of Cd for thefungal 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 in-oculated with a 5 mm agar plug from the edge of seven-day-oldculture plates of both isolates. PDA plates without Cd augmenta-tion were inoculated with both isolates and used as controls. ThePDA plates were then incubated at 25 °C for seven days. The dia-meter of growing mycelia was measured daily and the initialdiameter was subtracted from the subsequent diameter. The

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188182

highest concentration of Cd that completely inhibited fungalgrowth was considered the minimum inhibitory concentration(MIC). Furthermore, the tolerance index (TI) was calculated fromthe extent of growth in each isolate exposed to Cd, divided by theextent of growth in the control plates. RSF-6L was selected basedon high Cd tolerance, and subjected to Cd bioaccumulationanalysis.

2.3. Bioaccumulation of Cd by growing fungus in culture broth

The capacity of fungal isolate RSF-6L for biosorption of Cd ionswas measured by inoculating 100 mL Czapek broth spiked with1 mM Cd. Endophyte RSF-6L was grown for 8 days at 28 °C at150 rpm and pH values of the broth were measured at 24 h in-tervals. Mycelia and liquid medium were separated by vacuumfiltration using 0.22 mm filter paper. The mycelia were dried in anoven at 70 °C for 48 h and culture filtrates were preserved at�20 °C until further processing. Filtrates and dried mycelia wereused for Cd quantification, according to the method outlined byDeng et al. (2014b). All measurements were done in triplicate, andresults 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 washedwith autoclaved deionised water, sterilised twice (at 120 °C for15 min, 15 psi) and dehydrated at 70 °C overnight (Ulsan Man-saeDae Susuck Garden, Ulsan, South Korea). The sand was thenspiked with CdCl2 aqueous solution to obtain the final con-centrations of Cd of 0, 10, and 30 mg Kg�1. The investigationconsisted of six treatments: (i) control plants without Cd or RSF-6Linoculation; (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 condi-tions for a minimum of two weeks. In the meanwhile, S. nigrumseeds were surface sterilised using 2.5% sodium hypochlorite for30 min, treated with 70% ethanol (EtOH) for 30 s, and washed withautoclaved distilled water (D.W.) twice. The seeds were germi-nated for 4–5 days on autoclaved filter papers in 9-cm petri platesmoistened with autoclaved distilled water. Subsequently, the ger-minated seeds were grown for 15 days in multi well pots con-taining autoclaved sand and supplied with half strength Hoa-gland's solution in a growth chamber with a day/night regime of25 °C, 16/8 h light/dark cycle, and 60% relative humidity. At thesame time, plastic pots lined with polythene bags were filled withstabilised Cd spiked sand (500 g in each pot) with five replicatesfor each treatment, resulting in 30 pots. The strain RSF-6L culturedin 500 ml Czapek broth for 10 days at 25 °C using shaking in-cubator. Mycelia were collected by filtration using vacuum pumpand re-suspended in sterile distilled water. Equal size seedlingwere selected from pre-germinated seeds and their roots weredipped in the fungal mycelial suspension for 1 h. Seedlings werethen transplanted into plastic pots containing Cd modified sandand 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 randomisedblock design. Pots were daily irrigated as per requirement with D.W. and nutrients were supplemented by the application of halfstrength Hoagland's solution on a weekly basis.

2.5. Assessment of inoculated and non-inoculated plant growth at-tributes with and without Cd contamination

After 6 weeks of growth, chlorophyll contents (measured by achlorophyll metre, SPAD-502 Minolta, Japan) and leaf area of

samples from each treatment were determined. Plants were har-vested by cutting the stem above the sand surface. Shoot lengthand fresh weight were measured and immediately stored in liquidnitrogen for biochemical analysis. Likewise, roots were carefullyseparated from sand and washed with D.W. to determine rootlength and fresh weight. For Cd analysis, randomly selected rootand shoot samples were washed with D.W., blotted with tissuepaper, and oven dried at 80 °C for 48 h. Dry weights of sampleswere determined, after which they were ground to powder andsubjected to Cd content analysis.

2.6. Biochemical analyses of inoculated and non-inoculated plantswith and without Cd contamination

Antioxidant contents and enzymes in fresh leaf samples storedat �80 °C. For antioxidant enzymatic activities, leaf samples(0.5 g) from each treatment were ground in liquid nitrogen andthe powder was suspended in extraction buffer consisting of50 mM sodium phosphate (pH 7.0), 1% polyvinyl-polypyrrolidone(PVP; w/v), and 0.1 mM EDTA. The homogenate was centrifuged at13,000 rpm for 10 min at 4 °C and the supernatant was used todetermine total protein contents according to the Bradford (1976)assay method using bovine serum albumin (BSA, Sigma) as thestandard. Furthermore, the activity of antioxidant enzymes in-cluding catalase (CAT), peroxidase (POD), and polyphenol oxidase(PPO) were measured according to the methods previously out-lined by Khan et al. (2014). The activity of POD and PPO enzymeswere measured at a wavelength of 420 nm. One unit each of PODand PPO was indirectly measured by an increase of 0.1 unit ofabsorbance.

2.7. Microscopic analyses of endophyte infected plants with andwithout Cd contamination

To determine the association of fungal endophyte with theroots of S. nigrum, microscopic analyses were carried out with alight microscope. Plants infected with RSF-6L were carefully up-rooted from the sand, washed with deionised water, and treatedwith 2.5% sodium hypochlorite for 10 min for surface sterilisation.The sterilised roots were cut aseptically into pieces of about 1 cmin length under a laminar flow hood. The root pieces were sub-sequently kept in KOH (20%) solution for 24 h and then rinsedextensively with D.W. The roots were stained overnight in 0.5%acid fuchsin and 95% lactic acid. The roots were de-stained withlactic acid for 24 h and visualised under a light microscope.

2.8. Cadmium measurement and determination of relatedparameters

Cadmium ions were analysed in dried plant tissues, fungalbiomass, and culture broth throughout the experiment. Oven-dried plant tissue (0.1 g) and fungal biomass (0.1 g) samples wereground with a mortar and pestle to a fine powder. The groundsamples and culture broth were digested in a mixture of HNO3 andHClO4. Cd contents were determined by inductively coupledplasma spectroscopy (ICP) (Optima 79000DV, Perkin Elmer, USA).The Cd translocation efficiency from roots to shoots of plants wasevaluated by calculating the translocation factor (TF) using thefollowing formula:

=( )

−

−TFConcentration of Cd in shoots mgKgConcentration of Cd in roots mgKg 1

1

1

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

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188 183

follows:

=· ( )

−

−BCFConcentration of Cd in plant mgKgConcentration of Cd in soil mg Kg 2

1

1

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

( )= ×( )

TI %Root length in Cd treated plants

Root length in control plants100

3

2.9. Statistical analysis

All experiments were performed in triplicate and values arepresented as the means7SD. The data were analysed usingGraphPad Prism software (version 5.01, San Diego, CA, USA). Sig-nificant differences (po0.05) between the mean values of differ-ent treatments were compared and evaluated using Duncan'smultiple 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 bodymass

The two selected phytohormone producing fungal endophyteswere tested for Cd tolerance at concentrations of 0, 1, and 2 mM(Fig. 1a). F. tricinctum RSF-4L showed sensitivity and intolerancetowards Cd and didnot grow even on the lowest concentration of

Fig. 1. Determination of minimum inhibitory concentration (MIC) of Cd for selected funcadmium (Cd) contaminated PDA media (a). Effect of Cd concentrations on pH curve of RS(b). Dry biomass of RSF-6L (c). Uptake and accumulation of Cd in RSF-6L biomass (d). Pooper treatment from three independently conducted experiments. For each set of treatmeof Cd (0 mM, 0.5 mM, and 1.0 mM) at po0.05, by Duncan's multiple range test (DMRT)

Cd; whereas, A. alternata RSF-6L grew well on 1 mM Cd supple-mented medium over 8 days of incubation. In contrast, the growthof 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 con-centration of 1 mM Cd in comparison to 2 mM Cd. Therefore, onlyRSF-6L was processed and used for further experiments. In addi-tion, the Cd accumulation capability of the strain RSF-6L was alsoevaluated in 0.5 mM Cd spiked Czapek broth. The pH of the culturemedia increased slightly from 7.0 to 7.5 and remained minimallychanged over one week of fungal incubation (Fig. 1b). The strainaccumulated over 78% and 57% Cd when cultured under 0.5 and1 mM Cd spiked medium, respectively (Fig. 1c). The dry biomassshowed an inverse relationship with Cd concentration and wasreduced 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 growthattributes under Cd contamination

Seedlings of the S. nigrum plant were inoculated with A. alter-nata RSF-6L, in the absence and presence of different concentra-tions of Cd (0, 10, and 30 mg/kg). After 6 weeks of endophyte as-sociation, the visible growth promoting effects based on plantmorphological characteristics including shoots and roots werecompared to non-inoculated controls (Fig. 2a, b). Endophytic fungicolonisation was confirmed in stained root fragments and micro-scopic images reflected the association with RSF-6L, whereasplants without inoculation showed no fungal association (Fig. 2c,d, e, and f). In addition, the effect of fungal inoculation on S. nigrumplants under Cd contamination were determined by measuringchlorophyll contents, leaf area, root and shoot length, and fresh

gal endophytes; in vitro response of RSF-6L and RSF-4L grown on 0, 1, and 2 mMF-6L grown in Czapek broth modified with 0 mM, 0.5 mM, and 1.0 mM Cd for 8 daysled data of all experiments; each value represents the mean7SE of three replicatesnts, different letter(s) indicate significant differences between different treatments.

Fig. 2. Rescue effect of RSF-6L inoculation on the growth attributes of Solanum nigrum grownwith 0, 10, and 30 mg/kg Cd. Endophytic fungi inoculation has clearly promotedplant 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 morphologyof 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 Cdtreated 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) inthe 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 independentlyconducted experiments. For each treatment, different letter(s) indicate significant differences for growth characteristics noted on plants grown at different concentrations ofCd (0, 10, and 30 mg/Kg) by DMRT (po0.05).

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188184

and dry weight (Fig. 3). Cd augmentation significantly retarded theshoot and root growth and total dry biomass production (Fig. 3a,b). In the absence of Cd, endophytic fungi inoculation significantlyincreased shoot and root length of S. nigrum plants by 30% and 24%respectively, as compared to the untreated control. On the otherhand, without endophytic association, Cd contamination sig-nificantly retarded the shoot (63%) and root length (46%) of plantsas compared to the untreated controls. Plants inoculated withendophytic 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 plantshad significantly greater total dry biomass production, leaf area,and chlorophyll contents as compared to non-infected Cd treat-ments (Fig. 3b, c, d). The total dry biomass of plants inoculatedwith endophytic fungi was 25%, 30%, and 15% higher than non-inoculated untreated, 10, and 30 mg/Kg Cd treatments, respec-tively. The leaf area was significantly greater in RSF-6L inoculatedplants than in non-inoculated plants (Fig. 3c). Cd contaminationsignificantly reduced the leaf area in non-inoculated plants,whereas this parameter was significantly increased in plants

Table 1Cadmium 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) Association Cd concentration (mg Kg�1 plant DW) TF BCF TI%

Leaf Stem Root

0 Control ND ND ND – – –

RSF-6L ND ND ND – – –

10 Control 265714ab 847725b 1500750c 0.7 1611 38RSF-6L 261721ab 228718d 1021798d 0.5 1070 61

30 Control 293721a 1130766a 2322796a 0.6 2464 10RSF-6L 250720b 573731c 1820750b 0.5 1902 12

Control¼ fungal endophyte free plants, RSF-6L¼Solanum nigrum plants infected with fungal endophyte RSF-6L, TF¼Translocation factor, BCF¼ 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 plantsgrown under different Cd concentrations (mg Kg�1 dry sand) in each column denoted by the different letters are significantly different at po0.05 as analysed by Duncan'smultiple range test.

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188 185

inoculated with RSF-6L. Furthermore, chlorophyll contents weresignificantly higher only in RSF-6L inoculated plants under 10 mg/kg Cd contamination in comparison to non-inoculated Cd treatedplants (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/uptakeand distribution in different tissues of S. nigrum plants are sum-marised in Table 1. Generally, the concentration of Cd in both in-oculated and non-inoculated plants increased in different plantparts with increasing concentrations of Cd in sand, with the ex-ception of the leaves where the concentration did not changesignificantly with Cd application (Fig. 4). Higher concentrations ofCd accumulated in roots in comparison to stems and leaves irre-spective of treatment. The trend of Cd concentration in differenttissues was root4stem4 leaf in both Cd treatments (10 and30 mg/kg), with and without RSF-6L inoculation. On the otherhand, fungal inoculation significantly reduced uptake and dis-tribution of Cd in roots and stems for both concentrations of Cd incomparison to non-inoculated treatments. However, Cd contentsin leaves were not significantly changed irrespective of treatment.Endophyte RSF-6L inoculation decreased Cd accumulation in rootsby as much as 31% and 22% under 10 and 30 mg/kg Cd treatments,respectively. Whereas RSF-6L inoculation caused a reduction in Cdcontents of stems by as much as 73% and 49% in 10 and 30 mg/KgCd treatments, respectively, in comparison to non-inoculated Cd-treated plants.

Fig. 4. Accumulation of Cd in different parts of inoculated and non-inoculated S.nigrum. The asterisk (*) denotes significant differences among treatments(po0.05); the error bar represents the standard error.

3.4. RSF-6L inoculation inhibited Cd to above ground tissues as re-vealed by decreased BCF and increased TI

Cd uptake, distribution, and translocation were further eval-uated by determining total Cd contents, TF, BCF, and TI (Table 1).The TF ranged from 0.5 to 0.7, which showed negligible differenceirrespective of treatment. Moreover, the BCF increased in non-in-oculated Cd-treated plants with increasing Cd contamination.However, the BCF was significantly reduced by fungal inoculationunder Cd contamination. On the other hand, TI was significantlyincreased by RSF-6L inoculation with the 10 mg/kg Cd treatmentand slightly increased with the 30 mg/kg treatment.

3.5. Antioxidant enzymes responses of S. nigrum

The oxidative stress responsive enzymes are summarised inTable 2. The activity of antioxidative enzymes including CAT, POD,and PPO were significantly influenced by Cd contamination. Par-ticularly, POD and PPO activities were significantly enhanced pri-marily in the leaves of non-inoculated plants grown in Cd con-taminated soil. In addition, PPO enzyme activity was not influ-enced by RSF-6L inoculation without Cd contamination; whereasRSF-6L inoculation significantly modified POD and PPO activity inplants grown in Cd contaminated sand. RSF-6L inoculation clearlycaused significant reductions of POD and PPO activity in plantsgrown in Cd spiked sand. CAT activity on the other hand wassignificantly higher in fungal inoculated plants with the 30 mg/KgCd treatment only.

Table 2Antioxidant enzymes response in fresh leaves of fungal endophyte RSF-6L infectedand non-infected Solanum nigrum plants grown in 0, 10 and 30 (mg Kg�1 sand DW)concentrations of Cd.

Cd treatment(mg Kg�1

sand DW)

Association CAT (units/mgprotein)

POD (units/mgprotein)

PPO (units/mgprotein)

0 Control 2.05870.11a 31.572.57f 4.570.90c

RSF-6L 0.5770.03c 44.573.42e 3.670.92c

10 Control 0.24370.04e 111.073.61c 9.471.00b

RSF-6L 0.35570.03d 71.773.16d 5.3370.99c

30 Control 0.37770.05d 172.874.47a 13.970.70c

RSF-6L 1.25370.04b 124.872.95b 9.171.33b

Control¼ fungal endophyte free plants, RSF-6L¼Solanum nigrum plants infectedwith fungal endophyte RSF-6L. The different alphabets are representing significantdifferences among the samples.Values in each column represent the mean 7 SE. Mean values of the control andRSF-6L inoculated plants grown under different Cd concentrations (mg Kg�1 drysand) in each column denoted by the different letters are significantly different atpo0.05 as analysed by Duncan's multiple range test.

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188186

4. Discussion

Many fungal and bacterial species such as Rhizopus arrhizus,Mucor rouxii, Cupriavidus taiwanensis TJ208, Aspergillus niger, Ba-cillus jeotgali, and Pseudomonas veronii 2E have been reported tohave the potential for accumulation of Cd and other heavy metals(Vinichuk et al., 2013; Vullo et al., 2008; Xiao et al., 2010). How-ever, maximum Cd accumulation capacity, up to 173 mg/g fromculture 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 forits tolerance of higher concentrations of Cd. Based on its Cd re-sistance, the fungal endophyte RSF-6L was assessed for Cd accu-mulation efficiency. The strain was more efficient in Cd accumu-lation at lower concentrations (probably because of higher bio-mass production), in comparison to higher concentrations, whichsignificantly reduced fungal biomass production. In the presentstudy, strain RSF-6L accumulated lower amounts of Cd in com-parison to other reported fungal strains i.e. Microsphaeropsis sp.LSE10 isolated from S. nigrum (Xiao et al., 2010). This might be dueto the difference in tolerance mechanisms and compositions of thecell wall in the different fungal species.

Phytoremediation with the use of hyperaccumulator plants hasemerged as a promising and cost effective eco-friendly techniquefor the rehabilitation of heavy metal contaminated soils. However,the lower biomass production and lower tolerance levels to higherconcentrations of contaminants are some of the obstacles to theefficiency of hyperaccumulator plant-based remediation technol-ogy (Gerhardt et al., 2009; Glick 2010). To overcome these chal-lenges, alternative strategies such as the use of endophytic bac-teria and fungi have recently been employed to improve thephytoremediation potential of plants. For this purpose, the se-lected endophytes should possess the ability to promote thegrowth and biomass yield of the host plant, resist the effects of thecontaminant, and should sequester the target contaminant to re-duce phytotoxicity. Our results reveal that fungal inoculation en-hanced host plant growth in the presence of Cd by improving allplant growth attributes. The results of the present study clearlydemonstrate that RSF-6L inoculation enhances the fresh and drybiomass of S. nigrum plants under conditions of Cd contamination.The outcomes of the plant growth experiments reveal that theendophytic fungal strain RSF-6L showed stimulatory effects onboth shoots and roots of plants under conditions of Cd con-tamination. Enhanced plant growth and a significant increase indry biomass was observed on plants inoculated with RSF-6L in thepresence of Cd. Root elongation and proliferation of lateral andadventitious roots were significantly promoted in RSF-6L in-oculated, Cd-treated plants in comparison to non-inoculated Cd-treated plants. These findings confirm an integral plant-fungalrelationship, which is considered a pivotal factor for plant devel-opment and growth. Endophytic fungi have been reported to beplant growth regulators and protectants both in normal and instressful environmental conditions. Several studies have reportedmany endophytic fungal species like Penicillium citrinum, Pir-iformospora indica, Neotyphodium sp., Curvularia protuberata, andColletotrichum sp. that have improved plant growth in the pre-sence of heavy metals (Vinichuk et al., 2013; Vullo et al., 2008).The ability of endophytic fungi to produce phytohormones isbeneficial to the host plant in its defence against the adverse ef-fects of abiotic stressors (Khan and Lee, 2013; Waqas et al., 2014).RSF-6L inoculation in plants yielded comparatively higher leafarea, chlorophyll contents, root/shoot length, and increased bio-mass. Moreover, the strain also mitigated the adverse effects ofmetal contamination on these plant growth parameters. The in-creased growth rate and total dry biomass yield could be

attributed to the superior physiological responses of S. nigruminduced by increased endogenous phytohormone levels, suppliedby IAA-producing RSF-6L (Khan et al., 2015b). The supplementa-tion of endophytic fungi RSF-6L IAA in S. nigrum under heavymetal stress may therefore diminish the deleterious effects of Cd,such as the inhibition of primary root growth and key metabolicand physiological processes (Yuan and Huang, 2015). IncreasedIAA production is evidenced by longer roots in Cd stressed en-dophytic infected plants. On the other hand, non-inoculated plantswere found to have mostly reduced growth attributes, includingbiomass. Our results are consistent with our previous findings inwhich endophytic bacterial inoculation enhanced both plantgrowth and biomass (Khan et al., 2015a).

Higher concentrations of Cd were found in the roots of S. ni-grum, which also suggests its application as a phytostabiliser. Re-sults of the present study are consistent with previous reports inwhich higher levels of Cd accumulated in the roots (Alvarengaet al., 2008). However, the disparity in accumulation patternscould be attributed to the use of different types of substrate mediafor plant growth, which causes variation in the availability of Cdfor plant uptake (Chen et al., 2010; Khan et al., 2014; Teixeira et al.,2011). In addition, higher Cd availability does not necessarily leadto higher levels of translocation to different tissues of the plant.The present results reveal that accumulation of Cd in plant rootsand stems increased with increasing Cd concentration. However,the Cd contents of leaves were not significantly different amongtreatments and concentrations of Cd. This can be attributed to acertain capacity for metal transfer from stem to leaf that is regu-lated to minimise further translocation once a certain threshold isattained. However, the transfer of Cd from root to stem also seemsto be affected by a concentration gradient. Because of its relativelyhigh tolerance and higher accumulation capacity of the root toretain Cd, S. nigrum might also be applicable to the phytostabil-isation of Cd contaminated soil. Plants used for phytostabilisationshould have a dense extensive root system with a large biomassproduction capacity in the presence of relatively high con-tamination levels, to keep the TF as low as possible (Alvarengaet al., 2008).

The BCF and TF are the important parameters to be consideredin determining the phytostabilisation potential of certain plantspecies (Alvarenga et al., 2008). However, using different substratemedia for plant growth within the same species, the TF and BCFvary greatly in-vitro. This finding is evidenced by a number ofreports in which the same plant species have different BCF and TFdue to a difference in substrate media during screening (Chenet al., 2010; Khan et al., 2014; Teixeira et al., 2011). In the presentstudy, only Cd treated plants without fungal inoculation showed ahigher BCF and lower TF (o1), suggesting that large amounts ofCd accumulated in roots and relatively smaller portions weretranslocated to the aerial parts of the plant. S. nigrum exhibitshigher BCF and lower TF values, suggesting its potential applica-tion as a candidate plant for phytostabilisation of Cd contaminatedsites, to thus minimise the migration of Cd into surface- andgroundwater and reduce the risk of entry into the food chain.However, fungal inoculation significantly reduced Cd accumula-tion in roots as well as its subsequent translocation to stems. Theimmobilisation of significant amounts of Cd in the roots is acharacteristic of certain plants. Such a mechanism is regarded asbeing protective against excessive accumulation of contaminants,especially in leaves to support the vital process of photosynthesis(Thurman, 1981). However, in the present study the concentrationof Cd in the inoculated and non-inoculated plant leaves remainedunchanged. The differences were evident in fungal inoculatedplants, which produced significantly higher root and shoot bio-mass than those without inoculation treated only with Cd. Redu-cing the toxic effects of Cd contamination in plants might entail

A.R. Khan et al. / Ecotoxicology and Environmental Safety 136 (2017) 180–188 187

the use of endophytic fungi and plant growth promoting (PGP)bacteria. In the present study, fungal inoculation reduced total Cdaccumulation in plants, but increased plant growth and biomass.

It is well documented that fungal association with plants pro-vide a filter function against heavy metals that leads to increasedplant tolerance (Schulz and Boyle, 2005). Our results showed apositive effect of RSF-6L inoculation on the protection of S. nigrumgrown in Cd contaminated sand. Fungal inoculation was found toalleviate oxidative stress in leaves, although similar Cd levels weredetected in the leaves of inoculated and non-inoculated plants.Production of reactive oxygen species rapidly increased in plantcells under a variety of abiotic and biotic stress conditions, and thisposes various challenges to normal cellular function. Conse-quently, radical scavenging activities are deployed to supporthomoeostasis through antioxidants like CAT, POD, and PPO. Ourfindings demonstrate that Cd contamination significantly in-creases the activity of the antioxidant enzymes POD and PPO inleaves. These results are consistent with previous studies, whichshowed that Cd treatment increases the activity of antioxidantenzymes in the leaves of S. nigrum (Khan et al., 2015a; Khan et al.,2014). Fungal inoculation conferred protection and reduced thedeleterious effects of oxidative stress caused by Cd, as evidencedby decreased activity of two important enzymatic antioxidants(POD and PPO). The down regulation of PPO and POD activities ofin the present study also verify the results obtained in two majormaize and sunflower crops infected with AMF Glomus intraradicesand Glomus mosseae species. The AMF species not only down-regulated PPO and POD activity, but also enhanced growth attri-butes and reduced uptake of Cd in shoots of maize and sunflower(Aghababaei and Raiesi, 2015). However, CAT activity showeddissimilar effects and was increased in inoculated plants. Thisfinding is consistent with those of Babu et al. (2015), who de-monstrated the protective effect of the endophytic bacteria Pseu-domonas koreensis AGB-1 on host plant Miscanthus sinensis ex-posed to mine-tailing soil (containing Cd and other heavy metals),increased uptake of heavy metal, and significant increase in CATactivity. However, in the present study, the up-regulation of CATactivity in the leaves of RSF-6L inoculated S. nigrum, is the oppositeto that found in the leaves of AMF infected maize and sunflowerexposed to Cd, as presented by Aghababaei and Raiesi (2015).Furthermore, a more comprehensive explanation can be inferredfrom the experiments of Zhao et al. (2015), with the dark septateendophyte Exophiala pisciphila, which was grown in broth media,either with or without Cd. At the end of their investigation, thetranscriptome of E. pisciphila exposed to Cd stress, revealed that575 genes were differentially expressed, 40% of which were re-lated to ten established heavy metal tolerant pathways. Amongthem, 12 loci were identified that were responsible for the gen-eration of free radicle scavengers. Genes controlling CAT activitywere up regulated, whereas those related to POD activity weredown regulated. According to our findings, the protection con-ferred by fungi might not be limited to the stimulation of anti-oxidant enzyme activity, nor did it appear to be related to Cduptake in leaves (Firmin et al., 2015).

5. Conclusion

Solanum nigrum has been extensively reported as a promisingCd hyperaccumulator. However, further details are yet to be ex-plored for its associated endophytic fungi and their role in phy-totoxicity reduction. Furthermore, no previous study has reportedon phytohormones, particularly IAA producing fungal endophytesand the mechanisms by which they facilitate Cd accumulation/stress resistance in S. nigrum. The present study demonstrated thatendophytes such as RSF-6L, isolated from S. nigrum could grow in

Cd contaminated agar and broth media. RSF-6L rescued host plantgrowth attributes, by immobilising Cd in the roots, reducing itsuptake, and by enhancing enzymatic activities. Finally, we canconclude that each of the endophytic fungi harboured by the hostplant, perform specific functions and collectively shape the specifictraits of the plant. In the present study, the selected endophyteproduced IAA to balance the endogenous levels needed for properphysiological functions that are compromised by Cd stress. Otherendophytes may enhance Cd uptake. This unique, delicately ba-lanced relationship with endophytic fungi might make S. nigrum aperfect choice for Cd phytoremediation. Endophytes such as RSF-6L, capable of IAA production and suppression of Cd uptake in hostplants, can also be recommended for crops grown in Cd con-taminated soil.

Acknowledgement

This research was supported by the Basic Science ResearchProgram through the National Research Foundation of Korea(NRF), funded by the Ministry of Education (NRF-2015R1D1A1A01057187).

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