1 Introduction

Lentil (Lens culinaris Medik.) is an important legume crop as it is the world’s third important cool season grain after chickpea and pea, accounting for 6% of global dry pulse output with an average yield of 926 kg ha−1 between 2010 and 2015 [57]. Lentil is an ancient and widely consumed pulse worldwide. Lentil is rich in complex form of carbohydrates, proteins, amino acids, minerals, vitamins and dietary fibers [47]. Lentil plants get most of their nitrogen (N2) via N2 fixation by forming symbiotic association with Rhizobium in root nodules. Rhizobium transforms atmospheric N2 to ammonia (NH3), which is then taken up by plants in exchange for carbon-containing molecules [17]. Biological N2 fixation alone accounts for about 64% of the total N2 fixed and the rest by abiotic means [22]. Rhizobium also plays an important role in metal phytoremediation, allowing plants to grow and improve soil health in the presence of inhibitory levels of heavy metals [33]. Lentils also form symbiotic association with arbuscular mycorrhizal fungi (AMF) in addition to Rhizobium [25]. Most agricultural crops, shrubs and trees preferentially associate with AMF [12]. AMF increase uptake of diffusion limited nutrients and water, and tolerance to biotic and abiotic stresses [76]. Because of their complementary functions in delivering primary nutrients, AMF and Rhizobium are considered to be the major drivers of legume growth and productivity [75]. Plants associated with more than one symbiont result in higher growth when compared to the individual symbiont because of synergistic benefits. Many studies on legumes, inoculated with AMF and rhizobia have shown significant increase in plant growth as compared to single inoculation with either of them [2, 11, 28,29,30].

Fly ash (FA) is a major solid waste bi-product of thermal power plants and its disposal is a serious management issue. FA consists of several macro- and micronutrients essential for plant growth [6]. It can be utilized in agriculture as a source of nutrients or as a supplement to improve the quality of soils [66]. Therefore, FA supplication in cropland reduces the cost spent on chemical and organic fertilizers. Besides, presence of several beneficial nutrients, FA is deficient in N2 as it is burnt with coal and released in the air. The association of N2 fixing Rhizobium with leguminous crops overcomes this deficiency [58]. Several experiments have been conducted to determine the optimum beneficial quantity of FA to be applied in the croplands as nutrient supplements [4, 35, 69]. Application of 25% FA levels significantly increased plant length, plant biomass, number of leaves and yield of many crops and higher amounts reduced growth performance of plants [35]. In addition to being rich in macro- and micronutrients, FA is also a rich source of heavy metals such as Cd, Pb, Ni and Zn [39]. Several studies [24, 60, 69] have focused on the roles of microorganisms (AMF and Rhizobium) on reducing the uptake of these toxic heavy metals in plants as well as improving plant growth.

Thus, the goal of the present study was to elucidate how a 25% FA addition to soil combined with Rhizobium and AMF (F. caledonius or G. bagyarajii) inoculation affects plant length, fresh and dry weight, photosynthetic pigments, nitrate reductase activity and protein content of lentil crop as well as nodulation, mycorrhizal population along with heavy metal uptake in roots and shoots of lentil.

2 Materials and methods

2.1 Soil preparation and FA application

FA was collected from the Kasimpur Thermal Power Plant in Aligarh (India), air dried, finely powdered, and sieved through a 2 mm mesh before being utilized for soil amendment. The soil for this investigation was taken from the Department of Botany of Aligarh Muslim University, air dried and divided into two parts. One part of the soil was used without fly ash and other half part of the soil was mixed with fly ash in the ratio of 3:1 v/v. Half of each part (FA amended and unamended soil) was autoclaved twice at 121 °C for 20 min each time, with 1-day interval and used as the sterilized substrates for the experiment while the other half part of amended and unamended soil was used without sterilization.

2.2 Preparation of the inoculum

For Rhizobium treatments, 50 g authorized and active culture of the Rhizobium bacteria specific for lentil (Rhizobium leguminosarum) was purchased from the “Krishi Vigyan Kendra” (Agriculture Science Centre, Aligarh, India). 100 ml of distilled water, 10 g of jaggery, and the obtained Rhizobium culture were thoroughly mixed. Seeds were surface sterilized with 0.5% sodium hypochlorite (NaClO) for 15 min and washed thoroughly. Ten such surface-sterilized seeds were then inoculated with 1 g of Rhizobium culture, dried in the shade before being sown in the pots as needed.

Pure cultures of AMF [Funneliformis caledonius (Nicolson & Gerd.) and Glomus bagyarajii Mehrotra] were purchased from Centre for Natural Biological Resources and Community Development (CNBRCD), Bengaluru, Karnataka, India. Before the start of experiment, the purchased AMF cultures were individually multiplied in the Department of Botany. To do this, a 1:1 w/w sand/soil mixture was made in clay pots and autoclaved twice for 20 min at 121 °C, separated by 1 day. Each pot received a layer of the corresponding AMF cultures, which were subsequently covered with a layer of sand/soil mixture. For the purpose of the experiment, the Lolium perenne seeds in these pots were planted, kept watered, and allowed to grow for around 2 months. Following multiplication, addition of 10 g of the corresponding inoculum was made to the potting hole (as per treatments) along with a soil/sand combination containing infected roots and spores and the lentil seeds were sown in each pot to a depth of 0.5 cm.

2.3 Pot experiment

The soil alone and soil supplemented with FA were filled in pots holding 4 kg soil and placed in a randomized block design. Lentil (Lens culinaris Medik. var. KLS-218) as test plant were selected and purchased from Chola Beej Bhandar (Agro company), Aligarh (UP) India. The 12 treatments in sterilized soil are given below. The same 12 treatments were maintained with unsterilized soil totaling to 24 treatments. Each treatment had 3 replications.

Details of the treatments:

(i) Soil (S)

(vii) S with fly ash (FA)

(ii) S + Rhizobium (R) (S + R)

(viii) FA + Rhizobium (R) (FA + R)

(iii) S + Funneliformis caledonius (Fc) (S + Fc)

(ix) FA + Funneliformis caledonius (Fc) (FA + Fc)

(iv) S + Glomus bagyarajii (Gb) (S + Gb)

(x) FA + Glomus bagyarajii (Gb) (FA + Gb)

(v) S + R + Fc

(xi) FA + R + Fc

(vi) S + R + Gb

(xii) FA + R + Gb

The pots were then maintained in a glasshouse of Botany Department, Aligarh Muslim University and watered as and when necessary. Thinning was done when the seedlings had five to six leaves and two plants were maintained in each pot. To assess the effects of FA, AMF and Rhizobium application singly and in combination on plant length, fresh and dry weight, physiology, biochemical content and microbial population at 90 days after sowing (DAS). Heavy metal analysis was performed at the time of harvesting i.e. 120 DAS.

2.4 Growth parameters

One plant from each replicate of each set of treatment was uprooted and washed carefully. A metric scale was used to determine the entire plant length. Plants were weighed individually to determine their fresh weight. Plant’s root nodules were also counted and weighed using electronic balance (AH220A, AHN Germany). The dry weight of plants and the nodules were determined after the samples were dried in an oven at 70 °C for 72 h and dry weight was recorded using electronic balance.

2.5 Photosynthetic pigments

The photosynthetic pigments (chlorophyll-a, chlorophyll-b, total chlorophyll and carotenoid contents) were determined using the standard procedures by Mackinney [44]. 100 mg fresh leaves were crushed with a pestle and mortar in 10 ml of 80% acetone. The extract was filtered through a Whatman No.1 filter paper and final volume of extract was made to 10 ml by adding 80% acetone. A UV–VIS spectrophotometer (T70 UK) was used to determine the O.D. of the supernatant at 663, 645, 510, and 480 nm.

2.6 Estimation of biochemical content

Nitrate reductase (NR) activity and protein content of the plants were determined using the standard procedures Jaworski [32], Lowry et al. [43], respectively. For NR activity, fresh leaves weighing 200 mg were cut into bits and 2.5 ml phosphate buffer (pH 7.5), 0.5 ml potassium nitrate solution, and 2.5 ml of 5% isopropanol were introduced to it. The samples were incubated in a BOD incubator for 2 h at 30 ± 2 °C in the dark. 0.4 ml of the mixture was then transferred into a test tube and 0.3 ml of 1% sulphanilamide solution and 0.02% NED-HCl were included. The combination was left in the test tube for 20 min for maximum color development and was diluted to 5 ml with double distilled water (DDW). Absorbance was read at 540 nm on a UV–VIS spectrophotometer (T70 UK) against a blank reagent.

For protein analysis, 300 mg of leaf tissues were blended in 10 ml of 20% trichloroacetic acid in a pre-cooled mortar and pestle. The leaf samples were then centrifuged for 15 min at 600 rpm. The supernatant was removed. 5.0 ml of 0.1 N NaOH was added to the pellet, agitated, and again centrifuged for 15 min. The protein segments was remove from the supernatant and 5.0 ml alkaline copper solution was given to 0.5 ml protein extract and left to wait for 10 min. The Folin–Ciocalteau reagent was then incorporated into it and mixed. The blue color appeared and the absorbance of solution was read at 660 nm on a UV–VIS spectrophotometer (T70 UK).

2.7 Mycorrhizal parameters

AMF spore numbers from root zone soil samples were determined by wet sieving and decantation procedure [23]. The roots were sliced into 1 cm bits, treated with trypan blue and observed with the help of the microscope to calculate the percentage of mycorrhizal colonization by the method described by Phillips and Hayman [50].

2.8 Heavy metal analysis

Plants were also analyzed for heavy metal content after 120 DAS following the methods described by Allen et al. [8]. The roots and shoots were set ready by washing them with distilled water and drying them in an oven at 75 °C for 3 days. The dried sections were blended into a powdered form. Sample weighing 300 mg were digested in a tri-acid mixture (HNO3/H2SO4/HClO4; 5:1:1 v/v) at a temperature of 80 °C. The treated solution was cooled, diluted with distilled water, and filtered using a Whatman 42 filter paper, before being evaluated for Cd, Cr, Pb, Ni and Zn concentration using atomic absorption spectrophotometer (AAS) (GBC, 932 plus; GBC Scientific Instruments, Braeside, Australia).

2.9 Statistical analysis

The statistical analysis was carried separately for sterilized and unsterilized set of treatments using one-way ANOVA by SPSS software version 17 (SPSS, Chicago, IL, USA). Duncan’s multiple range test (DMRT) was used to determine the significance of differences between the treatments at p < 0.05.

3 Results

3.1 Impact of symbiosis on growth parameters of lentil in FA amended soil

In the present study, the results clearly brought out the positive effect of FA and microbial inoculation on the growth parameters of lentil particularly in sterilized pots as compared to unsterilized ones. Plants modified with 25% FA showed comparatively higher growth parameters (total length, total fresh and dry weight) of lentil than in soil alone. The amendment of only FA showed a notable surge in total length and weight of lentil plants in both autoclaved and unautoclaved sets, as compared to their treatments in soil alone. Additionally, plants when inoculated with both Rhizobium + AMF mainly F. caledonius alongside with FA recorded the maximum growth parameters in lentil plants when compared to their respective single inoculations (Figs. 1, 2 and 3).

Fig. 1
figure 1

Effect of fly ash and microbial inoculation on the total length of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (Duncan’s multiple range test; analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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Fig. 2
figure 2

Effect of fly ash and microbial inoculation on the fresh weight of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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Fig. 3
figure 3

Effect of fly ash and microbial inoculation on the dry weight of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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3.2 Impact of symbiosis on physiological attributes of lentil in FA amended soil

Lentil plants amended with 25% FA showed relatively higher physiological parameters (chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents) as compared to the soil alone. Amendment with FA was more noticeable when inoculated alongside with Rhizobium + AMF compared to their inoculation in soil alone. The dual inoculation of microbes showed greater photosynthetic contents in FA amended soil when compared to their single inoculation. The higher chlorophyll and carotenoid content in lentil was obtained in plants inoculated with a combination of FA as well as Rhizobium and F. caledonius as compared to the rest of the treatments (Fig. 4a–d).

Fig. 4
figure 4

a–d Effect of fly ash and microbial inoculation on the photosynthetic pigments (chlorophyll ‘a’, chlorophyll ‘b’, total chlorophyll and carotenoid contents of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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3.3 Impact of symbiosis on biochemical content of lentil in FA amended soil

Application of FA alone in soil significantly enhanced the NR activity and protein content in lentil plants in both sterilized and unsterilized sets, as compared to their respective control (soil alone). In FA amended soil, dual inoculation exhibited higher NR activity and protein content as compared to the single inoculation in amended soil (Figs. 5 and 6).

Fig. 5
figure 5

Effect of fly ash and microbial inoculation on the nitrate reductase activity (NRA) of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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Fig. 6
figure 6

Effect of fly ash and microbial inoculation on the protein content of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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3.4 Impact of symbiosis on nodulation and AMF population of lentil in FA amended soil

Effects of varying treatments on nodule counts in lentil are summarized in (Table 1). FA amelioration with AMF and Rhizobium significantly increased nodule count in lentil as compared to soil. Lentil inoculated with Rhizobium and/or AMF increased nodules number in the following order: [(FA + Rhizobium + F. caledonius ≥ FA + Rhizobium + G. bagyarajii) > FA + Rhizobium] > [(Soil + Rhizobium + F. caledonius ≥ Soil + Rhizobium + G. bagyarajii) > Soil + Rhizobium] indicating that tripartite symbiosis of lentil and FA amendment led to highest increase in nodulation than when only Rhizobium was inoculated. Similarly, fresh and dry weight of nodules in lentil increased significantly in tripartite symbiosis with Rhizobium + F. caledonius and Rhizobium + G. bagyarajii in FA amended soil both in sterilized and unsterilized soil. Fresh and dry weight of nodules in 90 DAS old lentil at varying treatments were in the order: [(FA + Rhizobium + F. caledonius ≥ FA + Rhizobium + G. bagyarajii) > FA + Rhizobium] > [(Soil + Rhizobium + F. caledonius ≥ Soil + Rhizobium + G. bagyarajii) > Soil + Rhizobium]. Highest nodule count and nodule fresh and dry weight in lentil were recorded on treatment with FA + Rhizobium + F. caledonius as compared to rest of the treatments.

Table 1 Effect of fly ash and microbial inoculation on the nodule number, nodule fresh and dry weight in lentil (Lens culinaris Medik.)
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Spore density in root zone and mycorrhizal root colonization in varying treatments of lentil was in the order: [(FA + Rhizobium + F. caledonius ≥ FA + Rhizobium + G. bagyarajii) > (FA + F. caledonius ≥ FA + G. bagyarajii)] > [(Soil + Rhizobium + F. caledonius ≥ Soil + Rhizobium + G. bagyarajii) > (Soil + F. caledonius ≥ Soil + G. bagyarajii)]. Both the mycorrhizal parameters were significantly higher in the treatment FA + R + Fc but not differing statistically from the treatment FA + R + Gb in both sterilized and unsterilized soil. Also, the mycorrhizal population in sterilized sets was relatively higher as compared to the respective treatments in unsterilized sets (Table 2).

Table 2 Effect of fly ash and microbial inoculation on percent root colonization and spore count of AMF in lentil (Lens culinaris Medik.)
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3.5 Impact of symbiosis on heavy metal uptake by lentil roots and shoots in FA amended soil

The result of the concentration of heavy metals (Cd, Cr, Pb, Ni and Zn) present in roots and shoots of lentil are shown in Fig. 7a–c. Use of 25% FA in soil resulted in a significant increase of Cd, Cr, Pb, Ni and Zn in roots and shoots of lentil plants. Inoculation with Rhizobium and either of the selected AMF in FA treated or untreated soil caused higher concentration of heavy metals in roots in comparison to shoots. Single inoculation of microbes also decreased the concentration of heavy metals in roots and shoots of lentil to certain level but the dual inoculation displayed superior tolerance level to these metals in roots and also prevented their translocation to shoots. Maximum response was noted in Rhizobium and F. caledonius treatment in FA amended soil with reference to lower amount of heavy metals in roots and shoots when compared to the other treatments.

Fig. 7
figure 7

Effect of fly ash and microbial inoculation on the heavy metal (Cd, Pb, Zn) accumulation in roots and shoots of lentil (Lens culinaris Medik.) (mean ± SE; n = 3). Bars with different small letters show statistically significant variation at p < 0.05 as per DMRT (analyzed separately for sterilized and unsterilized sets). S: soil; FA: fly ash; R: Rhizobium; Fc: Funneliformis caledonius; Gb: Glomus bagyarajii

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4 Discussion

4.1 Impact of symbiosis on growth and photosynthetic attributes of lentil in FA amended soil

In the present study, overall growth performance of lentil is relatively higher in sterilized FA amended soil than in unsterilized sets (Figs. 1, 2 and 3). Significant increases in growth parameters of some plants have been reported in a pathogen free environment carried out in autoclaved sets as reported by earlier study [3]. Higher growth promoting effects in sterilized soil signifies that presence of some unknown biotic stress partially inhibited symbiotic benefits [28]. Furthermore, implementation of nutrient rich FA in soil promoted the growth parameters (total plant length, fresh and dry weight) of lentil. Similar findings have been reported for other crops due to ample availability of nutrients [4, 16, 26]. Increases in biomass of a legume on application of FA in lower amounts have also been documented [52]. Single application of Rhizobium increased the overall growth attributes of lentil crop. Ahmad [5] reported the similar effects of Rhizobium inoculation on the plant growth treated with FA. Furthermore, AMF increasing the uptake of limited nutrients resulting in plant growth stimulation is well documented [12]. Extra-radical mycelium of AMF has been shown to boost nutrient uptake and thereby plant growth and development [41]. Inoculation with Rhizobium + AMF further improved plant growth parameters studied. Similar effects of synergistic interaction between Rhizobium + AMF have been reported in other leguminous crops [28,29,30, 40].

Photosynthetic pigments (chlorophyll a, b, total chlorophyll, and carotenoid) were significantly more in lentil grown in both sterilized and unsterilized soil amended with 25% FA compared to unamended soil (Fig. 4a–d). Pigment content in beetroots significantly increased on application of FA in soil [59]. Application of 10 to 30% FA enhanced photosynthetic activity in plants [37, 68] and these beneficial impacts were attributed to improved nutrients availability in FA amended soil. The increase in chlorophyll content was because of high concentration of Mg in FA [59]. Mg, an essential and nuclear component of the chlorophyll molecule occurs adequately in FA and its uptake enriched the leaves with chlorophyll content [27]. Carotenoids are the auxiliary pigments and functions as non-enzymatic antioxidant to chlorophyll pigment [36]. In the present study, Rhizobium applications influenced photosynthetic pigments along with 25% FA in soil. Chaudhary et al. [15] reported significant enhancement in total chlorophyll and carotenoid content in Rhizobium inoculated Vigna species. Pea (Pisum sativum) inoculated with Rhizobium and grown in soil amended with FA also showed increased chlorophyll and carotenoid [34]. Similarly, the association of plants with AMF significantly enhanced the concentrations of chlorophyll in host plants [13]. AMF inoculated plants had considerably more leaf pigment than non-mycorrhizal plants [7]. Wheat plants inoculated with Glomus mosseae and Glomus fasciculatum also showed increased photosynthetic pigment [9]. Krishna et al. [38] reported increased concentrations of carotenoids in leaves of Vitis vinifera associated with AMF. Baslam et al. [14] also reported that Lactuca sativa L. inoculated with Glomus fasciculatum had a higher carotenoid content. Moreover, co-inoculation of lentil plants with Rhizobium + AMF further improved the photosynthetic activity as documented earlier [28,29,30].

4.2 Impact of symbiosis on the biochemical attributes of lentil in FA amended soil

The results of the present study brought out that addition of FA to soil significantly increased the NR activity and protein content of the plant (Figs. 5 and 6). FA in lower amounts increases NR activity and protein content but higher FA application can reduce these attributes in plants [26]. Shakeel et al. [59] reported that FA application in soil enhanced NR activity and protein contents in beetroots. Ashfaque and Inam [10] also attributed the increase in NR activity and protein synthesis in FA amended soil. Similarly, Mushtaq et al. [45] reported that NR activity in chickpea increased on Rhizobium inoculation under stressed condition. The increase in NR activity resulted from higher uptake of nitrite is due to microbial association [1]. Higher protein content in plants inoculated with Rhizobium has also been reported by earlier [28, 30, 45]. Inoculation of AMF further increased NR activity and protein content. Similar findings were also reported in Talaat and Shawky [65] in Triticum aestivum, Tisserant et al. [67] in Medicago truncatula and Stancheva et al. [64] in Vigna unguiculata. However, dual inoculation with Rhizobium and AMF in Vigna unguiculata and white clover resulted in higher NR activity and protein content than their single applications [64, 73].

4.3 Impact of symbiosis on microbial population of lentil in FA amended soil

Applications of 25% FA with Rhizobium inoculation increased nodulation in lentil grown in sterilized and unsterilized soil (Table 1). In agreement to present finding, Ahmad [5] reported that nodulation in chickpea increased on application of 10% FA in soil. Faizan and Kausar [19] also reported that application of 25% coal ash in soil significantly increased number and dry weight of nodules in Lens culinaris. In addition, dual inoculation of Rhizobium and AMF showed higher nodulation, and the similar results were also observed by Xie et al. [74]. The positive outcome was possible might be because Rhizobium and AMF do not fight for each other’s colonized sites, thereby improving nodulation by adding vigor for nodule development and nitrogen fixation [48].

Applications of either of the species of AM fungi in FA amended soil increased mycorrhizal colonization in lentil as compared to application of AMF in soil without FA (Table 2). Parab et al. [49] reported positive effects of lower levels of FA applications on root colonization and spore counts of inoculated mycorrhizal fungi in onion. Ultra [70] reported that applying compost and FA with mycorrhiza enhanced AMF root colonization in Delonix regia. Ultra and Manyiwa [71] also reported that AM colonization rates were significantly higher in FA-amended mine tailings in three Acacia species. Moreover, dual inoculation enhances AMF colonization as compared to application of AMF alone was also reported earlier [30, 74].

4.4 Impact of symbiosis on heavy metal uptake of lentil in FA amended soil

Uptake of heavy metals in lentil roots and shoots was higher in FA amended soil than soil without FA (Fig. 7a–c). Heavy metals uptake in mung bean also increased on application of 5 to 25% FA [61]. Singh et al. [63] reported that uptake of heavy metals increased significantly with increasing concentrations of FA. Increasing doses of FA led to corresponding increase heavy in metals uptake in plants [46]. On contrary Upadhyay et al. [72] reported that 40% FA supplementation with manure reduced the translocation of toxic metals in edible parts of chickpea. In our study, the order of accumulation of all three heavy metals (Cd, Pb and Zn) in roots and shoots of lentil was Zn > Pb > Cd in respective treatments of microbes and FA (Fig. 7). Similar levels of uptake of heavy metalshas been reported in plants grown in amended soil [31, 46, 62]. On uptake, relatively larger proportion of heavy metals was retained in the roots of lentil than the amounts translocate in shoot. Higher accumulation of heavy metals in roots than shoots might be because roots are the first organs to encounter heavy metals and consequently absorb and retain larger proportions of absorbed heavy metals in roots than partitioning to shoots [21]. In contrast, root to shoot translocation of heavy metals was higher in Typha latifolia [56] grown in FA amended soil. Similarly, Rai et al. [51] reported that Rhizobium inoculated plants of Prosopis juliflora accumulated relatively higher amounts of heavy metals in roots than shoots. Nodule bacteria have very high heavy metal tolerance, accumulation and detoxifying mechanisms [54]. Furthermore, Garg and Chandel [20] reported that Cd concentration in pigeon pea inoculated with Glomus mosseae was remarkably reduced as compared to uninoculated plants. Liu et al. [42] reported that amendment with mycorrhizal fungi significantly reduced Cd accumulation in maize tissues. In line with effects of Rhizobium + AMF, various microbes have been found to reduce heavy metal accumulation in legume plants grown in metal polluted soils as in Lens culinaris [28]; Cicer arietinum [30], Medicago ciliaris [53], Pisum sativum [55] and Vigna radiata [18]. These results suggest that dual inoculation with Rhizobium + AMF would enable lentil to be grown in heavy metal polluted soil.

5 Conclusions

Finally, we may conclude that soil amendments with 25% FA, Rhizobium and selected AM fungi (G. bagyarajii or F. caledonius) either applied singly or in dual or triple combination remarkably enhanced plant growth, photosynthetic pigments, biochemical attributes as well as nodulation and mycorrhizal population in lentil as compared to the soil without any of these amendments. Dual inoculation with Rhizobium and AMF not only further enhanced plant growth but also reduced the uptake of heavy metals, hence assisting lentil plants to effectively cope with heavy metal stress. The improvement in plant growth, biochemical attributes as well as in microbial population was highest in the combined application of FA along Rhizobium and F. caledonius. Among the two selected species of mycorrhizae, F. caledonius was better in stimulating plant development and nutrient uptake as compared to G. bagyarajii. Future prospect of using fly ash in soil as a nutrient enhancer and using these symbionts to control the heavy metals uptake in aerial parts of the plants and improving productivity of the lentil crop for agricultural use can be utilized. In future, a long term research vision should include the use of fly ash that builds on a thorough understanding of fly ash properties and its appropriate doses along with their interactions with soil microorganisms when utilized in agriculture.