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Healthy Response from ChromiumSurvived Pteridophytic Plant-Ampelopteris prolifera with theInteraction of Mycorrhizal Fungus-Glomus deserticolaJoginder Singh a , Manoj Kumar a & Anil Vyas ba Department of Biotechnology , Lovely Professional University ,Punjab , Indiab Department of Botany , Jai Narain Vyas University , Jodhpur ,Rajasthan , IndiaAccepted author version posted online: 22 May 2013.Publishedonline: 18 Oct 2013.

To cite this article: Joginder Singh , Manoj Kumar & Anil Vyas (2014) Healthy Response fromChromium Survived Pteridophytic Plant-Ampelopteris prolifera with the Interaction of MycorrhizalFungus-Glomus deserticola , International Journal of Phytoremediation, 16:5, 524-535, DOI:10.1080/15226514.2013.798619

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International Journal of Phytoremediation, 16:524–535, 2014Copyright C© Taylor & Francis Group, LLCISSN: 1522-6514 print / 1549-7879 onlineDOI: 10.1080/15226514.2013.798619

HEALTHY RESPONSE FROM CHROMIUM SURVIVEDPTERIDOPHYTIC PLANT-AMPELOPTERIS PROLIFERAWITH THE INTERACTION OF MYCORRHIZALFUNGUS-GLOMUS DESERTICOLA

Joginder Singh,1 Manoj Kumar,1 and Anil Vyas2

1Department of Biotechnology, Lovely Professional University, Punjab, India2Department of Botany, Jai Narain Vyas University, Jodhpur, Rajasthan, India

Interaction between arbuscular mycorrhizal fungus Glomus deserticola and pteridophyticmember Ampelopteris prolifera was found abundant on entire growth level based on elemen-tal composition and gaseous exchange as a potential remediation system for phytoextractionof chromium. Inoculated A. prolifera (AM) and non-inoculated A. prolifera (Non-AM) weresupplied with two Cr species: 12 mmol of trivalent cation (Cr+3) [Cr(III)] and 0.1 mmol ofdivalent dichromate anion (Cr2O7

−2) [Cr(VI)]. Both Cr species were found to be depressedin overall growth and inefficient stomatal conductance (gs) and net photosynthesis (NP).Mycorrhizal association was found to be natural scavenger of Cr toxicity as indicated bygreater growth in plants exposed to Cr species, and increased gas exchange of Cr(III) treatedplants. Though, chromium reduction resulted lower level of nitrogen (N), phosphorus (P),and potassium (K) but interestingly elevated the level of aluminum (Al), iron (Fe), and zinc(Zn) uptake in many folds which is the significance of sustainable growth of plant.

KEY WORDS: mycorrhization, mineral nutrition, phytoextraction, phytoremediation

INTRODUCTION

Chromium (Cr) is an abundant metal in earth crust, the stable forms are the trivalentCr(III) and the hexavalent Cr(VI) species, although there are various others valence stateswhich are unstable and short lived in biological systems. In addition, Cr(VI) is consideredthe most toxic form, and Cr(III) is less mobile, less toxic and is mainly found bound toorganic matter in soil and aquatic environments (Shanker et al. 2005).

Phytoremediation utilizes biological organisms such as Ampelopteris prolifera (Retz.)Copel that is a spreading pteridophyte with the objective of phytoextraction of heavy met-als taken up from polluted soils, and it represents an alternative to available chemical andphysical methods of treating contaminated soils. Normally heavy metals are not decom-posed biologically in soil, and they occur as free or exchangeable metal ions, soluble metalcomplexes, organically bound metals, precipitated or insoluble compounds such as oxides,carbonates and hydroxides.

Address correspondence to Joginder Singh, Department of Biotechnology, Lovely Professional University,Punjab, India 144 402. E-mail: joginder.15005@gmail.com

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Bioremediation is the globally recognized eco-friendly approach to address the en-vironmental issues wherein researchers have tried to understand the positive symbioticinteraction in nature. Mycorrhizal fungi such as Glomus deserticola (Trappe, Bloss, &Menge) present the direct link between soil and root, and consequently are involved duringphytoremediation process, because it enhances heavy metal availability into tissues andempowers plants to be tolerant under the inconsiderate conditions. However, the ability ofmycorrhizal fungi to enhance the tolerance capacity in plants such as phytoaccumulation ofCr was completely unknown. Previous studies already demonstrate different mechanismsfor mycorrhizal association with plant which are responsible for uptake of heavy metalscopper (Cu) (Carvalho et al. 2006), zinc (Zn) (Andrade et al. 2009), nickel (Ni) (Orłowskaet al. 2011), cadmium (Cd) (Carvalho et al. 2006), lead (Pb) (Ruyi et al. 2008), and othermetals (Gohre and Paszkowski 2006) in decimal concentration and marginal improvementat growth level.

The present study highlights the significance of chromium tolerant plants underpositive symbiotic interaction which could be subjected at different stretch where normalvegetation is in quest due to heavy metal contamination in soil.

MATERIALS AND METHODS

Plant Material And Mycorrhizal Fungus

The Ampelopteris prolifera (Retz.) Copel were completely uprooted from Abu region,Mount Abu, Rajasthan for collection of samples. Samples were placed in the polyethylenebags, labelled and then transported to the laboratory. Rhizomes of potted juvenile A.prolifera (Retz.) from greenhouse were subjected for mycorrhizal association. Cultures ofG. deserticola were initiated, multiplied and established with its best host Cenchrus ciliarisas standardized methodology (Mathur and Vyas 1995) without any Cr contamination.Further it has become the source of experimentation for positive culture.

Soil samples from the miniatures cultures, along with mycorrhizal inoculated rootsegments of C. ciliaris were used as inoculum. Twenty grams of inoculum containing≥2000 spores and inoculated root segments were inoculated in earthen pots having 700 gof sterilized soil. Three replicates were maintained to raise the inoculum. The inoculum wasapplied in a banding pattern at three fourth regions from bottom of the soil then rhizome ofthe A. prolifera were allowed to grow into the band to colonize.

Experimental Conditions

This experiment was conducted under greenhouse conditions in the department ofBotany, Jai Narain Vyas University, Jodhpur, India (geographic coordinate 26.28◦ N 73.02◦

E). Internal climatic conditions were followed as temperatures: 26.2 − 32.7◦C, Photosyn-thetic Active Radiation (PAR): measured by LI-COR 6200. At mid day PAR reading was652 mmol m−2s−1 and relative humidity was variable in a range of 54.1% to 76.7%. Exter-nal local climatic conditions were recorded as 20.5–39.0◦C (temperature) 12.8% to 17.5%(relative humidity); the variations between internal and external were high due to localconditions.

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Pot Medium

Pot medium containing coarse sand and sandy loam soil mixture in a defined ratio(1:1 v/v) with a texture composition reported and analyzed in soil laboratory as follows:80% sand, 15% clay, and 5% silt, pH 7.6, CEC 48.9 meq / 100 g, low organic matter(1.00%), and nutrient levels (1.2 μg N g−1, 2.9 μg P g−1, 27.7 μg Kg−1, 547.1 μg Ca g−1,42.3 μg Mg g−1, 3.0 μg Zn g−1, 3.7 μg Mn g−1, 0.08 μg Cu g−1, 3.4 μg Na g−1, and 5.4 μgS g−1). The pot medium was steamed pasteurized for two consecutive days by 5 hours perday at 80◦C.

Plant Conduction And Inoculation

Irrigation of pots with deionized water was done on alternate days, and 200 mlper pot modified Long Ashton nutrient solution [LANS] having 10 μg P ml−1 of P wassupplied twice in a week, after emerging of shoots. Pots (pot medium) were categorized asAM (Arbuscular Mycorrhiza) and Non-AM (without AM). AM pots contain mycorrhizalfungus Glomus deserticola Trappe, Bloss, & Menge, around 2000 spores per plant whereas Non-AM has been kept in the same conditions for comparative analysis.

Experimental Design And Treatments

The experiment was designed as factorial scheme (2 × 3) with randomized approach.Five sets of each treatment were contaminated with two species of chromium Cr (III) andCr (VI) whereas a negative control was maintained to check the Cr effect in Non-AM. Outof five sets, three which had healthy growth were taken into consideration and rest of thesets those had died plants were eliminated from the factorial model. Each pot having oneplant, served as replicates for the experimentation.

Chromium Application

100 g Soil, infused with 12 mmol of CrCl3 (Cr+3) [Cr(III)] and 0.1 mmol of Na2Cr2O7,(Cr2O7

−2) [Cr(VI)] were added to the potted plants, in a week for five weeks. The Cr(III)is less harmful compared to Cr(VI), hence its higher quantity was used.

Growth of Plants Versus Mycorrhizal Association

Measurement parameters associated with growth and development of plant such asheight, leaf area, dry matter, shoot dry matter, root dry matter, and total plant dry matterwere performed after 50 days of transplanting. Ten plants were taken for each treatment.The samples were dried in hot air oven at 70◦C for 48 h. Mycorrhizal inoculation effect(MIE), with minor adaptations, was estimated by the formula (Bagyaraj et al. 1988):

MIE(%) = PDMI − PDMC/PDMC × 100

Where: PDMI is plant dry matter of inoculated treatment, and PDMC is plant dry matterof control treatment.

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Gas Exchange

For gas exchange, net photosynthesis (NP) and stomatal conductance (gs), were mea-sured and completed with ambient CO2 concentration at about 360 μl l−1 under 1000 μmolm−2 s−1 of photosynthetic photon fluxes provided from a 1000W high pressure sodiumvapor lamp filtered through 5 cm of water enclosed in a Plexiglas box. Data were recordedbetween 9 and 11 am in 20 and 50 days intervals after transplantation. After 5 and 36 days ofCr treatment, gas exchange measurement was carried on four Cr treated plants. Two freshmatured leaves per plant (n = 8) were subjected for gaseous exchange experimentationusing Portable Photosynthesis System (LI-COR, 6200).

Study Of Inorganic Ingredients –Macro And Micro In Leaf

Leaves of treated and control plants were analyzed for macro and micro nutrients(nitrogen, phosphorus, potassium, aluminum, iron and zinc) with an inductively coupledatomic emission spectrophotometer. From 10 plants per treatment, newly matured leaveswere pooled (plants 1 to 3; 4 to 6, and 7to 9) into three combined samples for tissue analysis(n = 3).

Chromium Analysis

Potted plants were uprooted and roots were washed with running tap water, followedby rinsing in distilled de-ionized water. Roots, shoots and leaves were further taken apart andoven dried at 65◦C for three days. Grounded powder of A. prolifera tissues were digestedas per modified method of Cary and Olsen (1975). Digested samples were analyzed for Crconcentration by Atomic Absorption Spectrophotometer (Perkin-Elmer model 3110). Themean number of triplicate reading was calculated for each collected sample and furtherused for Cr content calculations.

Development of Mycorrhizal Association

To analyze inoculated plant associated with mycorrhiza, roots were taken for the ob-servation of arbuscules, vesicles and hyphae respectively. Short segment of root (1 cm) wassampled, harvested and collected to assess the colonization percentage after treating withKOH and followed by staining with typan blue (Phillips and Hayman 1970). Microscopic

Figure 1 Effect of chromium and mycorrhizal inoculation on height of A. prolifera.

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Figure 2 Effect of chromium and mycorrhizal inoculation on leaf area of A. prolifera.

study (40X) of each stained root sample was done, 10 slides per treatment which round up100 observations (Giovannetti and Mosse 1980) for statistical analysis.

Statistical Analysis

The data were analyzed and plotted with GraphPad prism 6.0 statistical software.Means were plotted as histograms with standard deviation error bar.

RESULTS

Application of Cr species affected the overall plant growth (plant height, leaf area,leaf dry matter, shoot dry matter, root dry matter, and plant dry matter) in AM and Non-AM. The concentration (0.1 mmol) of Cr(VI) was found to be fatal to the both AMand Non-AM, which caused the greatest decline in growth rate (Figures 1–3). Though amarginal sustainability against Cr accumulation in terms of growth and development hasbeen observed in the case of mycorrhizal association (AM), where Non-AM is decliningthe growth rate gradually in presence of Cr (Figures 1–3). The AM has greater scavengingeffect on Cr(III) level accumulation which is very much consistent in all healthy parameters,

Figure 3 Effect of chromium and mycorrhizal inoculation on dry matter of A. prolifera.

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Figure 4 Effect of chromium and mycorrhizal inoculation on mycorrhizal inoculation effect of A. prolifera.

although Cr (VI) was found to be toxic in both AM and Non-AM plants wherein colonizationwas intensive up to 71%. This observation suggesting that association needs to be moreimperative in case of Cr (VI) accumulation (Figure 4).

Within five days after the first Cr application (total 20 days after the experimentinitiation), 10% reduction in gs in the case of Cr(VI) treated plants (Figure 5) was noticed,whereas no significant changes were observed for NP (Figure 6) during this period. Sub-sequently after 5th day mycorrhizal inoculated plants treated with Cr(III) showed higherstomatal conductance as compared to non-inoculated plants, however gas exchange was notsignificantly affected by Cr(III) treatments compared to control. On 36th day (total 50 daysafter Cr treatment), both NP and gs of Cr(III) and Cr(VI)-treated plants were found to belower as compared to the initial 5th day treatment (Figures 5 and 6). Overall inoculatedCr(III) plants were found to possess higher gas exchange (both gs and NP) as compare tonon-inoculated plants, while in AM plants with Cr(VI), no significant affect in gas exchangewas noticed.

Both Cr(III) and Cr(VI) species significantly decreased N, P, and K values andalleviated level of Al., Fe, and Zn (Figure 7) in leaf tissue. The value of K was found to be

Figure 5 Effect of chromium and mycorrhizal inoculation on Net Photosynthesis (NP) of A. prolifera, 5 and36 days after initiation of Cr Treatment.

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Figure 6 Effect of chromium and mycorrhizal inoculation on Stomatal Conductance (gs) of A. prolifera, 5 and36 days after initiation of Cr Treatment.

greater in Non-AM and AM plants in non treated conditions while value of Fe and Zn waslow. Also Cr(III) inoculated plants possess high tissue P compared to Cr(VI).

AAS analysis has revealed the Cr concentrations in root, shoot and leaf tissueswere higher in Cr(III) treated plant as compared to Cr(VI) (Figure 8). The maximumaccumulation of Cr was reported in leaves, whereas other parts were having relatively lowaccumulation. Mycorrhizal inoculated plants with Cr(III) have more Cr accumulation ascompare to Cr(VI). Therefore a value based comparative report was prepared in order toexplain the bioaccumulation in Cr(III) and Cr(VI) treated plants in presence of mycorrhiza(Figure 9).

Though Cr uptake has not been found negative in mycorrhizal association in plants;hyphae, vesicle and root colonization were taken into consideration. Further colonizationof 83.9% and 73.2% was observed in Cr(III) and Cr(VI) inoculated plants, respectively(Figure 10). Visual observation of poor arbuscule formation was significance in Cr(VI)treated A. prolifera plants, compared to Cr(III) treated plants and control plants. Thisobservation might have a reason of toxicity at Cr uptake level which was very distinguishedat species level Cr(III) and Cr(VI).

Figure 7 Effect of chromium and mycorrhizal inoculation on leaf mineral concentration of A. prolifera.

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Figure 8 Effect of chromium (Cr) and mycorrhiza (AM) on Cr concentration and total Cr uptake in plant tissuesof A. prolifera.

DISCUSSION

Present study reporting mycorrhizal impact on Cr accumulation and changes at phyto-chemistry level checked in A. prolifera- a pteridophyte. In recent past several pteridophytes(with and without association of mycorrhiza) have been tested as model specifically forarsenic removal from contaminated soil (Khan et al. 2000; Liu et al. 2005; Khan 2005).

Increase in Cr phytoextraction was propped due to low Phosphorus fertility level(9 μg P ml−1), which inhibited CrO4

−2 adsorption in equilibrated soil suspensions (Yanget al. 2008), and due to competitive species for surface sites. Plant stress caused by excessiveconcentration of heavy metals is alleviated by mycorrhizal fungi (Shen et al. 2006; Rivera-Becerril et al. 2002). Mycorrhizal fungi may increase the tolerance of plants to heavy metalsthrough the following mechanisms: immobilization of heavy metals by compounds veiledby the fungus, precipitation in polyphosphate granules in the soil, adsorption in fungalcell walls, and chelating of metals inside the fungus (Gohre and Paszkowski 2006). Theutilization of G. deserticola to colonize A. prolifera plants presents advantages, because

Figure 9 Effect of chromium (Cr) and mycorrhiza (AM) on Cr concentration and total Cr accumulation in planttissues of A. prolifera.

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Figure 10 Effect of chromium on% arbuscules, hyphae, and vesicles in root cortical cells of mycorrhizal A.prolifera.

inoculated plants can augment uptake of less soluble metals, and alleviates nutritional stress,including P-stress, when compare to non-inoculated. Hence, can leads to enhance diseaseresistance, drought resistance, and stresses due to other abiotic factors come across in Crphytoremediation sites.

Inclusion of Cr to A. prolifera, dropped growth and eventually reduced both stomatalconductance (gs) as well as net photosynthesis (NP). Cr(VI) was noxious over Cr(III), andsharp drop in gs was observed after five days of Cr application. Inoculation of mycorrhizalfungi served well to the plants treated with Cr(III), this can be seen in terms of better growthand accrued gs and NP. Further, control inoculated A. prolifera plants flourished much betterthan non-inoculated ones, though showing very low MIE. The value of MIE was foundmore for Cr(III) and Cr(IV) treated plants as they became very much mycorrhizal reliant,MIE was increased 0.5 fold to 1.8 fold of in Cr treated plants than control.

P level was lower in leaf tissues of plants as these tissues possess elevated levels of Cr.It has already been evinced by other plant system (Davies et al. 2001, 2002 and Shahandehand Hossner 2000). The amount of tissue P was found to be nearly similar in non-inoculatedplants and control-inoculated i.e. (4.0 ± 0.3% vs. 2.7 ± 0.2%) and also involved in growthincrement. Many a times the P concentration remains conserved in leaf tissue, and incrementin P content resulted enhancement in leaf area, so as to maintain its concentration equalin tissue become a compulsive job to buffer the Cr uptake (Aguilera-Gomez et al. 1999).In addition to this there was more P acquirement on total plant basis, as seen by increasedbiomass of root and total plant in inoculated plants compare to non-inoculated ones. Therange of P in between 2.5 to 2.8 g P kg−1 was found to be critical and concentrationsranging from 1.4 to 1.8 g P kg−1 were considered to be marginal. Therefore, control A.prolifera plants where P was measured, 2.5 to 3.0 g P kg−1 were considered to be adequateand Cr species was supposed to reduce concentration of P to marginal i.e. 1.6 to 0.8 g Pkg−1. Tissue P levels in Cr(III) plants was increased by AM, which is reflected in terms ofincrease in growth and gas exchange, still tissue P levels was undersupplied, indicated bylower values of plant growth and gas exchange when compared to control plants. Additionof Cr(III) and Cr(VI) resulted in reduced amount of N and K in the leaf tissue, whereasalleviates values of Al, Fe, and Zn. Cr supply reduced N levels. The amount of N criticalto A. prolifera was around 45 g N kg−1. Of the total plants considered only Cr(III) were

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found to deficient as dilution effect might have played here major role, because they havelarger biomass compared to Cr(VI) plants (Davies et al. 2002). Addition of AM did notenhance tissue N level. The level of K was reduced by Cr treatment but all it remain abovethe critical level, 25 g K kg−1) for A. prolifera. Whereas the Zn level in all the treatmentswere in the range that considered to adequate (48 to 96 μg g−1). For Fe non-inoculatedplants have higher level than inoculated A. prolifera plants. The experiments conducted byShanker et al. (2005) are in agreement that P and Fe deficient plants can transport Cr betterfrom roots to shoots. As non-inoculated Cr(III) plants have marginal P concentrations, nodifference in tissue Cr concentration was noticed and lower total Cr accumulation in roots,leaves and total plant was found compared to AM inoculated one. The P uptake can beenhanced in A. prolifera by use of mycorrhiza.

Values of High oxidative potential of Cr(VI) seems to damage the membrane, sig-nifying its initial toxic effect. Also Cr is retained in the roots generally in vacuoles andcell wall and when moving to leaves Cr(III) may be adsorbed by cell wall, still mechanismof hyperaccretion is not clear (Schiavon et al. 2008). Higher concentration of Cr in soilhas direct effect on roots and indirect effect on leaves (Ahmad et al. 2011; Schiavon et al.2008), of which Cr(VI) mainly inhibit growth of the root. Stomatal conductance showedhigh sensitivity under the influence of Cr, which was around 5 to 6 fold reduction whilearound 2 to 3 fold reduction in values of NP at the end of the experiment. Moreover,leaf chlorosis was also noticed in A. prolifera that resulted in lower gas exchange, similarchlorosis in leaf under chromium toxicity was observed by Malmir (2011).

Both sub and supra-optimal P nutrition also reduces process of photosynthesis in A.prolifera. Control-inoculated and non-inoculated A. prolifera plants, taken in our study, hadsimilar P, gs and NP. However, A. prolifera plants treated with Cr(III) and Cr(VI) speciesresulted in lower tissue P and which resulted in less gs and NP activity. The value of tissueP was much higher in Mycorrhizal inoculated Cr(III) treated plants along with higher gasexchange efficacy as compared to non-inoculated plants. Whereas in pants treated withCr(VI) have showed low gas exchange values suggesting AM were not effective in themshown by the values of lower P, level in between inoculated and non-inoculated plants. Inall plants irrespective of Cr treatments hyphal development, root colonization and vesiclesformation was noticed in greater amount. Only in Cr(VI) AM inoculated plants showedcomparably less arbuscule formation, reduction in arbuscule formation signifies that theseare the important sites in cortical region where ion exchange between mycorrhizal hyphaeand host occurs (Rahmaty and Khara 2011). This reduced formation of arbuscules can beassociated to less active symbiosis of AM.

The concentration of the chromium was found maximum in leaves, moderate inshoots and least in roots. The entire sample analyzed for Cr distribution followed the trends:leaves > shoots > roots. This compartment indicated that chromium could be easily trans-ported from roots and accumulated in shoots, demonstrating great tolerance and accumula-tion to chromium. However, in A. prolifera significant amounts of Cr accumulated in shootsand leaves. Greater phytoextraction would occur if leaves were harvested in field sites.

The Cr level in elemental form present in the tissues of Cr(III) was more as compare toCr(VI) plants, this might be due to higher level of applied Cr(III), around 120-fold. Cr(VI)was used in lower amount, being soluble resulted in low plant growth and greater stressas compared to Cr(III). The value of gs within first five days of the treatment reduced byaround 55% also contributed overall reduced Cr uptake. Cr(III) species are less soluble andlargely retained on soil surfaces reduces its mobility and bioavailability in soil and water(Shahandeh and Hossner 2000). Non-mycorrhizal phytoextraction of Cr(VI) was found

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comparatively greater than Cr(III) in Plantago lanceolata (Estaun et al. 2010); Brassicajuncea and Thlaspi caerulescens (Salt et al. 1995).

Mycorrhizal inoculation in A. prolifera resulted greater accumulation of Cr comparedto non-inoculated ones. Not only Cr accumulation the biomass of mycorrhizal inoculatedplant was more than non-mycorrhizal on whole plant basis was noted. Cr(VI) being strongoxidant, will get reduced to Cr(III) and intermediate state. The plants used for Cr accumu-lation must hyperaccumulate Cr(III) from the soil, efficient enough to solubilize Cr presentin soil, capable to absorb soluble Cr and translocate absorbed Cr within itself to diminishits toxicity.

AM fungi can be found in polluted sites contaminated with Cd, Zn, Pb etc. whichsuggests them to be metal tolerant. Mycorrhizal extraredical hyphae present in soil canmobilize Cd from soil to the plants but from fungus to plant is not possible as becauseof fungal immobilization (Gaur and Adholeya 2004). Plant occurring in polluted sitesassociated with mycorrhiza which are tolerant to metals, mycorrhiza helps to accumulateheavy metals in root translocation to shoot is still doubtful. Different metal uptake efficaciesof AM species are associated with plants found in localities having varying concentrationof Zn, Mn, P etc. Still the mechanism of tolerance and alleviation of metal stress by theAM have to be separated from the tolerance efficacy of plant associated with mycorrhiza.

Our experiments can be employed for the efficient phytoremediation of Cr by dualimpact of A. prolifera along with Glomus deserticola. Involvement of arbuscular mycorrhizanot only provides tolerance against Cr accumulation can also impart A. prolifera as apotential candidate for phytoextraction.

ACKNOWLEDGMENT

This study was funded by a grant provided by Department of Science & Technology,Under Fast Track Research SERC Scheme, New Delhi.

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