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Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica
Acta Ecologica Sinica xxx (xxxx) xxx
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Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica Saleh Shahabivand a, *, Azar Parvaneh a, Ali Asghar Aliloo b a b
Department of Biology, Faculty of Sciences, University of Maragheh, Maragheh, Iran Department of Agronomy, Faculty of Agriculture, University of Maragheh, Maragheh, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2019 Received in revised form 9 July 2019 Accepted 29 September 2019 Available online xxx
Environmental pollution by heavy metals is a severe issue worldwide. Microbe-assisted phytoremediation is a safe, inexpensive, and promising strategy in refinement of metal-polluted regions. Current in vitro work was installed to study effects of the endophytic fungus Serendipita indica on some physiological traits and cadmium (Cd) bioaccumulation of Alyssum montanum and Helianthus annuus seedlings grown in MS medium, under varying levels of Cd (0, 20, 40 and 60 mg Cd/l medium). Even though Cd stress induced phyto-toxicity in both tested species, but a significant improvement was found in biomass accumulation, photosynthetic pigments content, and chlorophyll fluorescence indicators in inoculated seedlings by S. indica under different doses of Cd in media. The non-infected A. montanum plantlets accumulated more Cd in shoot than root, and illustrated the properties of an accumulator species as evidenced by translocation factor (TF) and bioaccumulation factor of shoot (BFS) higher than 1. Contrary to this, un-colonized H. annuus seedlings had higher amount of Cd in root than shoot and showed a phyto-stabilizer feature, as evidenced by TF˂1 and bioaccumulation factor of root (BFR) higher than 1. Presence of S. indica significantly enhanced Cd accumulation in root, while it noticeably diminished Cd amounts of shoot in both A. montanum and H. annuus seedlings, so that inoculated plants had higher values for BFR against lower values for BFS and TF, in compare to non-inoculated ones. These findings indicated that S. indica can be considered as a bio-fertilizer to improve the physiological characteristics of tested species under Cd stress, as well as a bio-stabilizer of Cd in the roots of A. montanum and H. annuus in the regions exposed to toxic levels of Cd. © 2019 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
Keywords: Alyssum Sunflower Cd toxicity Phyto-accumulator Phyto-stabilizer Serendipita indica
1. Introduction One of the important environmental problems worldwide is contamination of various ecosystems by heavy metal cadmium (Cd) due to its toxic nature [1]. The toxicity of Cd is linked to its unbiodegradable feature, long persistence in the environment and accumulation in the body of different living organisms including human and plant [2]. The phosphate fertilizers, sewage sludge, mining wastes and disposal of industrial effluents are the main sources of Cd pollution in soil and water by anthropogenic activities [3]. According to high mobility and solubility of Cd, it is easily taken up by plant roots and is subsequently transferred to human body
* Corresponding author. University of Maragheh, Madar Square, Golshahr, Maragheh, 55181-83111, Iran. Tel: þ98 41 3727 6068, Fax: þ98 41 3727 6060. E-mail addresses: [email protected], [email protected] (S. Shahabivand).
(as a carcinogenic element) via consuming Cd-contaminated foods [4]. In plants, Cd deleteriously affects normal physiological and biochemical processes, leading to a reduction/inhibition in growth and biomass accumulation, nutrient imbalances, leaf chlorosis and even death at its higher doses [5,6]. In the recent years, phyto-decontamination as a sustainable, promising and practicable method has been raised for the cleanup of metal-polluted soils and waters, regarding to its eco-friendly and cost effective features. Accordingly, phyto-extraction and phytostabilization are the two main kinds of phytoremediation to refining heavy metal polluted sites. Phyto-extraction or phytoaccumulation refers to the uptake of pollutants from environment by the roots and the accumulation of those in the above-ground parts of plants. Whereas, phyto-stabilization is the method to accumulate pollutants including heavy metals in plant roots or precipitation in the rhizosphere for preventing them to entry the food chains or underground water. It has been demonstrated that
https://doi.org/10.1016/j.chnaes.2019.09.002 1872-2032/© 2019 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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some plant-associated fungi can effectively elevate the efficiency of phytoremediation, as well as heavy metal tolerance of host plants. Therefore, study on the plant-associated fungi is a considerable factor to exploring the possible uses of these fungi for fungalassisted phytoremediation of heavy metal. Also, according to previous researches, the beneficial plant-microbe relationship yielded in an encouraging result on phytoremediation of metals both economically and environmentally than the use of plant alone [7,8]. Root endophytic fungus Serendipita indica (formerly known as Piriformospora indica), that belongs to the class basidiomycete and the order Sebacinales, has symbiotic interaction with various plants similar to that of mycorrhizal symbiosis [9,10]. S. indica attributes multiple beneficial property to the host such as promotion of growth and yield, elevation of macro- and micronutrient uptake, and tolerance to wide range of biotic and abiotic stresses including metals toxicity [10e12]. The selection of appropriate species is one of the significant features to improving phytoremediation efficacy. Alyssum montanum is a perennial herbaceous plant from family Brassicaceae, with a relatively small size. There is little information about A. montanum responses to metal-polluted environment, unlike other species of Alyssum genus such as Alyssum bertolonii as a ska et al. hyper-accumulator for Ni [13]. In a recent study by Muszyn [14], it was found that metallicolous ecotype of A. montanum showed higher tolerance to multi-metal stress in compare to nonmetallicolous one. However, they found that non-metallicolous ecotype accumulated more Cd in shoot than that of root. Helianthus annuus L., as a fast-growing crop plant, has a great biomass, extensive root system, and potent compatibility and strong tolerance to different stress conditions including heavy metal stress [15]. It is a non-hyper-accumulating species and suitable tool for Cd extraction in Cd-polluted sites because it merges great biomass yield with a high Cd bioaccumulation [16,17]. Regarding to the possible role of H. annuus (with reasonable biomass yields) and A. montanum (with low biomass yields) in the phytoremediation of Cd-contaminated environment, as well as S. indica beneficial impact in increasing tolerance to the hosts under the metals stress, in this paper, we tested the effect of S. indica on respond to Cd in the two above-mentioned species. For this goal, Cd bioaccumulation in root and shoot, photosynthetic pigments content, Fv/Fm and ETR values, and growth attributes in H. annuus and A. montanum under S. indica symbiosis and at increasing Cd doses were discussed. Our research was carried out under in vitro culture conditions due to the entirely controlled status and least environmental interaction in this medium. 2. Materials and methods 2.1. Plant and fungal materials Seeds of Alyssum montanum L. (collected from the farming land in University of Maragheh, Iran) and Helianthus annuus L. cv. Azargol (received from the Dryland Agricultural Research Institute, Maragheh, Iran) were surface sterilized for 2 min in ethanol followed by 10 min in a 1% NaClO solution (0.75% Cl), then rinsed with distilled water three times and germinated on wet filter paper at 4 C for 48 h. The endophytic fungus S. indica was cultured in Petri dishes on a Hill & K€ afer medium [18], then placed in a temperaturecontrolled growth chamber at 29 ± 1 C in dark for 10 days. 2.2. Experiment design In-vitro culture experiment was established to determine the effects of S. indica on A. montanum and H. annuus growing under different doses of Cd, and consisted of a completely randomized 2 4 factorial arrangements with the following factors for each
plant species: two S. indica treatments (with or without fungus inoculation), each having four Cd concentrations (0, 20, 40 and 60 mg Cd/l medium), in four replicates. Four germinated seeds (from A. montanum or H. annuus) were transplanted into the glass jars (8 20 cm) containing 30 g of autoclaved MS solid medium (as the culture substrate with the macro- and micronutrients and the MS vitamins without phytohormones; Murashige and Skoog) supplemented with 3% (w:v) sucrose. The solidification of MS medium was done by 0.75% Difco agar, and pH of medium was adjusted to 5.6 prior to solidification. Four Cd concentrations (0, 20, 40 and 60 mg Cd/l medium) were added to the MS medium prior to autoclaving, as cadmium (II) chloride salt. For co-cultivation of the two symbionts, two fungal plug (5 mm in diameter) was placed at a distance of 1 cm from the roots of 10-days-old seedlings. Non-S. indica treatments received the same quantity of autoclaved S. indica inoculum. Experimental jars, sealed with sterile AeraSeal film covers, were located in a temperature/moisture-controlled growth room under conditions of 14 h of light and 10 h darkness, an irradiance of 100 mmol m2 s1 by white fluorescent lamps, 26/20 C day/night temperature and relative humidity of 65%. The seedlings were harvested after 28 d for growth and other analyses. Roots and shoots of the samples were carefully washed with deionized water and dried by filter paper for measurement of root and shoot lengths, and then were frozen in the liquid nitrogen for pigments analysis. Some fresh root samples were stored in water for 1 h to study root colonization. To determine the dry weights and Cd amounts of root and shoot, the plant samples were dried in an oven at 75 C for 48 h. 2.3. Measurement of root colonization by S. indica The root colonization percent by S. indica was found by the method of Oelmüller et al. [19] after root cleaning in 10% KOH and staining with 0.1% Trypan blue [20]. In this method, S. indicachlamydospores distribution within the roots of colonized plants was estimated as an index for root colonization. 2.4. Cd measurement in plant materials The oven-dried root and shoot samples were separately ground and then digested in a mixture of concentrated HNO3 and HClO4 acids (7:1 ratio, v/v). The content of Cd in plant materials was determined using an atomic absorption spectrophotometry (Shimadzu, Japan) method. According to Yoon et al. [21], the translocation factor (TF) of Cd was determined as the ratio of Cd content in shoot to Cd content in root (Cd amount in shoot/Cd amount in root). Ha et al. [22] defined the bioaccumulation factor (BCF) as the ratio of metal content in shoot to that in the soil, whereas Yoon et al. [21] assumed BCF as ratio of metal content in root to soil concentration of metal. In this work, we defined the bioaccumulation factor of shoot (BFS) and the bioaccumulation factor of root (BFR) as following formulae: BFR ¼ Cd amount in root/Cd concentration in media, and BFS ¼ Cd amount in shoot/Cd concentration in media. 2.5. Photosynthetic pigments measurement in leaf For measurement of chlorophyll (Chl) content in the second fully expanded leaf, 0.1 g of fresh sample was homogenized with acetone 80% (v/v) and then filtrated through filter paper. The absorbance of filtrate was read at 663 and 645 nm for Chl a and Chl b respectively, and the pigments contents were estimated according to Arnon [23]. 2.6. Measurement of chlorophyll fluorescence After darkening the leaf for 30 min, the chlorophyll fluorescence
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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parameters including Fv/Fm (maximum quantum efficiency of PSII photochemistry) and ETR (the relative PSII electron transport rate), as physiological indicators, were measured on the second fully expanded leaf using a portable chlorophyll fluorometer (model Hansatech, Instruments LTD, UK). 2.7. Statistical analysis The experimental data were analyzed by SAS 9.4 software version 2013. Duncan’s Multiple Range Test at a 0.05 probability level was used to study the differences between treatments for each species separately. Finally, the results were presented as the means of four replicates ± standard deviation (SD). 3. Results and discussion 3.1. Root infection and growth attributes There are significant individual and interactive effects of Cd addition and S. indica infection on root colonization of A. montanum and H. annuus at p 0.001 (Table 1). The results of Table 2 illustrated that fungal symbiosis in A. montanum and H. annuus could easily be installed under Cd exposure. However, the roots of noninfected plants did not establish colonization with S. indica. The maximum colonization intensity of S. indica in the roots of A. montanum and H. annuus were 72.2- and 75.7%, respectively, at the control (0 mg Cd/l medium) treatment (Table 2). According to our previous researches [12,24], Cd stress reduced root colonization by S. indica in both A. montanum and H. annuus seedlings, and lowest level of colonization was found at highest level of Cd in substrate culture (60 mg Cd/l; Table 2). The lower percent of colonization in the roots of tested seedlings may be because of a delay/ diminish/eliminate on germination of S. indica chlamydospores and/or on its hyphal growth at higher doses of Cd. Also, van keulen et al. [25] showed that heavy metal toxicity caused an injury in fungal cell wall integrity via elevation in expression of the enzyme chitinase. Data of ANOVA (Table 1) showed that main effects of S. indica and Cd stress had significant influence on growth traits (p 0.001) in both A. montanum and H. annuus seedlings, however, their interaction did not significantly affect the growth indicators (except for shoot dry weight of H. annuus at p 0.05). Therefore, we presented here only the main effects of the factors on the growth and biomass. Increasing severity of Cd stress significantly reduced the plant length, and root and shoot dry weights in tested plants (Table 3). Compared with control (0 mg Cd), plant length, root dry weight and shoot dry weight were decreased by 31.2-, 27.7- and 27.2% in A. montanum, and by 25.1-, 37.4- and 33.2% in H. annuus under severe Cd stress (60 mg Cd/l culture substrate; Table 3), respectively. Regarding to the lower reduction in biomass accumulation (root and shoot dry matter) of A. montanum in compare to
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H. annuus, it can be considered that A. montanum suffered less from Cd toxicity than H. annuus at the higher doses of Cd. Taking into account fact that auxin level (as an important growth promotion phyto-hormone) is reduced under Cd stress (via elevated activity of auxin oxidase) [26], the reduction in the biomass of treated seedlings may be attributed to this phenomenon. On the other hand, inoculation by S. indica resulted in a significant increase on plant length, and root and shoot dry matters in A. montanum and H. annuus, in comparison to non-inoculated ones (Table 4). These increases in plant length, root dry weight and shoot dry weight were 15.6-, 24.3- and 9.2% in A. montanum, and were 8.7-, 18.6- and 19.3%, in H. annuus respectively (Table 4). These results are in accordance with our previous studies in wheat and sunflower under exposure to Cd in soil [12,24], and findings of Mohd et al. [11] in rice plants exposed to arsenic toxicity under in vitro conditions. Plant growth promotion activity of fungal endophytes can be done by producing phytohormones such as auxin and gibberllin, ACC deaminase (a key enzyme in consuming ethylene precursor, thus lowering ethylene level), and siderophores to the better uptake of iron by plant roots [27,28]. In our earlier work, symbiosis by S. indica significantly elevated Zn (by 44%) and Mn (by 34%) contents of lettuce leaves compared with control [29]. In the presence of S. indica, the amounts of N, P and K were markedly higher in mung bean plants, in compare to absence of the fungus [30]. Thus, one of the mechanisms involved in S. indica-mediated growth improvement may be increasing uptake of macro- and micronutrients from culture substrate. Furthermore, an enhanced growth under S. indica symbiosis, can be attributed to the elevation in photosynthetic pigment content and chlorophyll fluorescence indicators in A. montanum and H. annuus plantlets (Table 4). 3.2. Chlorophyll (Chl) contents and chlorophyll fluorescence parameters Results of ANOVA (Table 1) illustrated that interactive effect of S. indica and Cd treatments had no significant differences on Chl a and b contents, and Fv/Fm and ETR values, whereas main effect of the two factors had significant influence on above-mentioned traits at p 0.001 in A. montanum and H. annuus (except for Fv/Fm in A. montanum at p 0.05). By increasing Cd concentrations in the culture substrate, Chl a and b amounts in leaf, and Fv/Fm and ETR indicators were significantly decreased in both species seedlings, except for Chl b in A. montanum and ETR in H. annuus at highest level of Cd (60 mg; Table 3). The maximum level of Cd in medium induced minimum amount of the pigments and fluorescence parameters, so that the reductions in Chl a, Chl b, Fv/Fm and ETR were 37.3-, 29.6-, 13.5- and 33.2% in A. montanum, and 50.3-, 54-, 17.3and 20.1% in H. annuus, respectively, under 60 mg Cd as compared to control (Table 3). Several researches have described the reduction in Chl content of various plants such as rapeseed [31], wheat [32] and sunflower [12] under Cd stress which were consistent with
Table 1 Analysis of variance (ANOVA) of S. indica (S) treatment and Cd treatment on root colonization (RC), plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm), electron transport rate of PSII (ETR), root Cd (RCd), shoot Cd (SCd), translocation factor (TF), bioaccumulation factor of root (BFR) and bioaccumulation factor of shoot (BFS) in A. montanum and H. annuus seedlings. Source of variation A. montanum S Cd S Cd H. annuus S Cd S Cd
df
RC
PL
RDW
SDW
Chl a
Chl b
Fv/Fm
ETR
RCd
SCd
TF
BFR
BFS
1 3 3
*** *** ***
*** *** ns
*** *** ns
*** *** ns
*** *** ns
*** *** ns
* *** ns
*** *** ns
*** *** **
*** *** ***
*** *** ***
*** *** ***
*** *** ***
1 3 3
*** *** ***
*** *** ns
*** *** ns
*** *** *
*** *** ns
*** *** ns
*** *** ns
*** *** ns
*** *** ***
*** *** ***
*** *** ***
*** *** ***
*** *** ***
***, **, *, ns stand for significant at P 0.001, 0.01 and 0.05 probability levels and not significant, respectively.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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Table 2 Interaction effect of S. indica and Cd treatments on root colonization (RC) of A. montanum and H. annuus. S. indica treatment
Cd treatment (mg/l)
RC (%) A. montanum
RC (%) H. annuus
Without S. indica
0 20 40 60 0 20 40 60
n.d. n.d. n.d. n.d. 72.2 ± 3.7 a 63.2 ± 4.1 b 54.2 ± 2.6 c 48.7 ± 3.3 d
n.d. n.d. n.d. n.d. 75.7 ± 5.1 a 66.5 ± 3.8 b 58.2 ± 1.8 c 48.5 ± 2.0 d
With S. indica
The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at. p 0.001. n.d.: not determined.
Table 3 Main effect of Cd treatment on plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm) and electron transport rate of PSII (ETR) in A. montanum and H. annuus seedlings. Cd treatment A. montanum 0 20 40 60 H. annuus 0 20 40 60
PL (mm)
RDW (mg/jar)
SDW (mg/jar)
Chl. a (mg/g FW)
Chl. b (mg/g FW)
Fv/Fm
ETR
67.2 ± 5.8 a 58.9 ± 5.0 b 51.0 ± 4.3 c 46.2 ± 3.9 d
93.9 ± 13.0 a 84.7 ± 10.6 b 78.3 ± 9.9 c 67.9 ± 8.3 d
326.4 ± 19.6 a 293.3 ± 18.1 b 255.6 ± 13.4 c 237.7 ± 14.1 d
1.26 ± 0.10 a 1.13 ± 0.10 b 1.04 ± 0.17 c 0.79 ± 0.09 d
0.44 ± 0.06 a 0.36 ± 0.06 b 0.32 ± 0.04 c 0.31 ± 0.04 c
0.827 ± 0.01 a 0.783 ± 0.02 b 0.740 ± 0.01 c 0.715 ± 0.01 d
130.3 ± 9.6 a 117.7 ± 8.6 b 102.5 ± 8.0 c 87.1 ± 3.9 d
196.0 ± 7.8 a 170.9 ± 6.7 b 159.7 ± 9.2 c 146.8 ± 7.8 d
219.7 ± 21.3 a 184.4 ± 19.4 b 161.2 ± 15.3 c 137.5 ± 15.2 d
535.6 ± 56.7 a 467.4 ± 38.7 b 417.7 ± 42.9 c 357.7 ± 35.4 d
1.51 ± 0.13 a 1.25 ± 0.09 b 1.05 ± 0.13 c 0.75 ± 0.08 d
0.66 ± 0.11 a 0.49 ± 0.07 b 0.40 ± 0.06 c 0.31 ± 0.05 d
0.812 ± 0.01 a 0.778 ± 0.01 b 0.740 ± 0.02 c 0.688 ± 0.02 d
137.1 ± 6.2 a 126.1 ± 5.4 b 111.5 ± 3.5 c 109.5 ± 6.3 c
Values are mean ± SD, n ¼ 4. The different letter within each column (for each species) indicates significant difference among treatments using Duncan’s Multiple Range Test.
Table 4 Main effect of S. indica (S) on plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm) and electron transport rate of PSII (ETR) in A. montanum and H. annuus seedlings. S. indica treatment A. montanum -S þS H. annuus -S þS
PL (mm)
RDW (mg/jar)
SDW (mg/jar)
Chl a (mg/g FW)
Chl b (mg/g FW)
Fv/Fm
ETR
51.8 ± 7.9 b 59.9 ± 9.0 a
72.4 ± 9.5 b 90.0 ± 12 a
265.9 ± 34.7 b 290.5 ± 38.5 a
0.97 ± 0.18 b 1.14 ± 0.20 a
0.31 ± 0.05 b 0.40 ± 0.06 a
0.760 ± 0.04 b 0.773 ± 0.04 a
103.8 ± 14.7 b 115.0 ± 19.8 a
161.3 ± 19.1 b 175.4 ± 18.6 a
160.7 ± 29.7 b 190.7 ± 34.6 a
405.3 ± 62.1 b 483.8 ± 74.8 a
1.05 ± 0.28 b 1.24 ± 0.30 a
0.40 ± 0.12 b 0.53 ± 0.15 a
0.74 ± 0.05 b 0.77 ± 0.04 a
118.3 ± 10.4 b 123.7 ± 14.2 a
Values are mean ± SD, n ¼ 4. The different letter within each column (separately for each species) indicates significant difference among treatments using Duncan’s Multiple Range Test.
A. montanum and H. annuus in our work. The decreased amounts of the Chl a and Chl b in Cd-treated plants may be attributed to overproduction of reactive oxygen species (ROS) by oxidative stress, reduction of Chl biosynthesis, and Chl degradation via elevated chlorophylase enzyme activity at toxicity doses of Cd in medium [33]. The Chlorophyll fluorescence indicators (such as Fv/Fm and ETR) evaluation is a useful approach to determine the photosynthetic performance of plants under heavy metal stress. According to fluorescence responses in our study, a decline in Fv/Fm value of maize and wheat seedlings, as well as the reduction in ETR index of Alpine pennycress under Cd exposure were observed [34e36]. A reduction in the values of Fv/Fm and ETR, associated with decreased content of Chl in this study, suggests the role Cd in reducing PSII activity and its structure. The significant difference was resulted among the inoculated and non-inoculated seedlings for pigments amounts and fluorescence parameters, and inoculated plants of both species had better photosynthetic status in terms of Chl contents and fluorescence indicators than their respective controls (Table 4). Presence of S. indica produced higher amounts of Chl a (17.5%), Chl b (29%), Fv/ Fm (1.7%) and ETR (10.8%) in A. montanum, in comparison to absence of the endophyte (Table 4). Also, colonized H. annuus
seedlings with S. indica had higher values of Chl a (18.1%), Chl b (32.5%), Fv/Fm (4%) and ETR (4.5%) than those control ones (Table 4). Under heavy metal stress, Chl level and Fv/Fm value were significantly greater in the endophyte-infected rice seedlings [37]. Moreira et al. [38] in pineapple and Ghaffari et al. [39] in barley reported that S. indica symbiosis elevated Mg content, as a pivotal point in the chlorophyll structure. A remarkably elevation on ETR and a slight increase on Fv/Fm were resulted under inoculation by endophyte fungus in Chinese white poplar [40]. Considering these results, it is concluded that Cd toxicity results in functional disorder of photosynthetic machinery and disadvantage to electron transfer of PSII, however these disturbances can be attenuated by S. indica symbiosis in tested seedlings. 3.3. Cd uptake, transport and bioaccumulation Based on the results from Table 1, the main effect and interactive effect of the two factors S. indica colonization and Cd treatment had significant influence (p 0.001) on Cd content in root and shoot, translocation factor (TF), bioaccumulation factor of root (BFR) and bioaccumulation factor of shoot (BFS) in both A. monatum and H. annuus seedlings under in vitro conditions. Therefore, we
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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focused on interactive effect of mentioned parameters. No Cd was found in the seedlings grown on un-contaminated medium by Cd (0 mg Cd, date not shown). Based on Fig. 1, in infected and nuinfected A. monatum and H. annuus plantlets, by increasing Cd doses in the MS medium, Cd content in root and shoot were significantly increased (except shoot Cd of A. montanum from 20 to 60 mg Cd in un-infected, and shoot Cd of H. annuus from 40 to 60 mg Cd in infected ones). Cd amounts of roots in the both tested seedlings were higher than those Cd concentrations in MS medium, indicating the bioavailability of Cd in medium to plant roots, as well as capability of these species to the uptake of Cd via an active process along with energy consumption. The root cells of H. annuus seedlings bioaccumulated more Cd than A. montanum root under all levels of Cd in media, so that in un-infected seedlings, the maximum content of root Cd reached 240.1 mg Cd in H. annuus in compare to 118.2 mg Cd in A. montanum under 60 mg/l Cd in growth media (Fig. 1). In some species, the Casparian strip of endodermis cell as well as existence of pericycle layer in root tissue may be considered as the important barrier in transport of water and heavy metals from root to shoot, resulting in metal accumulation in root. In A. montanum and H. annuus, and in both colonized and non-colonized plants, TF values were approximately constant under various levels of Cd in media, except in A. montanum and in colonized ones at 60 mg Cd that TF value showed a significant increase in compare to 40 mg Cd in medium (Fig. 2). In response to the increase in Cd doses in substrate culture, a reduction in BFR and BFS were observed in the two species, and in both inoculated and un-inoculated seedlings (Fig. 3). Reduced values of BFR and BFS, when Cd level of media had elevating trend, indicated that these species were more sensitive to Cd toxicity at higher Cd amounts in growth media, and had a constraint to Cd accumulation under these levels of the metal. On the other hand, the two tested species (in un-colonized ones), illustrated a distinct pattern in TF, BFR and BFS responses to the addition of Cd in media. In non-inoculated
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H. annuus seedlings, TF values ranged between 0.33 and 0.36, BFR ranged from 4 to 5.4, and BFS ranged between 1.35 and 1.96, at various levels of Cd in media (Figs. 2 and 3). Plants with a TF value lower than 1, and a BFR value higher than 1 are benefit for phytostabilization process, whereas plants with TF, BFR and BFS higher than 1 have capability to be used in phyto-extraction process [21,41]. According to Hesami et al. [41], BFS or EF (extraction factor in their study), reflects both TF and BFR and can be considered as a useful index in phyto-extraction ability of plants. Regarding to our results, H. annuus illustrated a stand with TF lower than 1, and BFR higher than 1 (4e5.4), and may be appropriate in immobilization of Cd in its root and can be used in Cd phyto-stabilization. In the case of A. montanum, TF and BFR values were greater than 1 (1.03e1.11 for TF, and 1.97e3.03 for BFR) as well as BFS amounts were from 2.03 to 3.36, under different concentrations of Cd in nutrient media (Figs. 2 and 3). Furthermore, Cd amounts of shoot in non-infected A. montanum plants (at 40 and 60 mg Cd in growth media, Fig. 1) were greater than critical amount of Cd i.e. 100 mg/kg bioaccumulated in extractor/hyper-accumulator plants. Even though a small biomass is the constraint for metal extraction purpose, but the less biomass produced by A. montanum (in compare to H. annuus; Table 3), was compensated by its high TF and BFS values, leading to increased accumulation of Cd in shoot. Therefore, A. montanum can be seen as an appropriate option for Cd extraction from Cd polluted areas, because it tolerated more than 100 mg Cd in shoots along with a relatively low decline in the biomass of shoot (28.2%) under highest level of Cd (60 mg) in medium, in compare to control (Table 3). It is well known that plant-associated microbes have a direct influence on metal absorption from media by their different activities, as well as an indirect effect on host withstand under metal stress by changing morphological, biochemical and physiological features of host plant [42]. Therefore, microbe-assisted phytoremediation of toxic metals can be a practicable, safe and
Fig. 1. Interaction effect of S. indica and Cd treatments on Cd content in roots (up) and shoots (down) of A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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Fig. 2. Interaction effect of S. indica and Cd treatments on translocation factor (TF) of Cd in A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
Fig. 3. Interaction effect of S. indica and Cd treatments on bioaccumulation factor of root (BFR; up) and shoot (BFS; dowm) in A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
sustainable strategy in reclaiming metal polluted regions. In this research, presence of endophytic microbe S. indica noticeably enhanced Cd content in root, whereas markedly reduced Cd content in shoot in both treated species, in compare to absence of the endophyte (Fig. 1). In inoculated A. montanum plants, these enhancements in root Cd amounts were from 16.7% (under 60 mg Cd) to 35.6% (under 20 mg Cd in media), while the reductions in shoot Cd were from 19.9% (at 60 mg Cd) to 31.1% (at 40 mg Cd in growth medium). In the case of H. annuus, presence of S. indica increased root Cd between 20.6% (under 60 mg Cd) and 45.9% (under 20 mg Cd), but decreased shoot Cd between 37% (at 40 mg Cd) and 48.7% (at 20 mg Cd in growth substrate; Fig. 1). These findings are in accordance with our earlier studies in wheat cv. Sardari and sunflower cv. Zaria in contaminated soil by toxic levels of Cd [12,24]. Also, Sabra et al. [43] found that S. indica-infected sweet basal
plants, in comparison to uninfected ones, had higher lead and cupper content in roots, but lower Pb and Cu content in shoots when both mentioned heavy metals were present in media. It has been described that fungal endophytes could be expressed some genes related to metal-mobilize and metal-tolerance to their survival and function under metal stress, resulting in higher metal absorption, and confer resistance to their host. For instance, the EpNramp gene (as a bivalent transporter) from the endophyte Exophiala pisciphila, enhanced Cd uptake and conferred resistance to Cd toxicity [44]. Some fungi can participate in arising trace metal solubility in media through producing organic acids such as citrate and oxalate and forming their complexes with metals, leading to an increase in metal uptake as well as the un-contamination of polluted areas [45]. Inoculation of solanum nigrum and Populus canescens plants by fungal strains increased Cd content in the roots
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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via enhancing the availability and uptake of Cd [46,47]. Based on Figs. 2 and 3, presence of S. indica significantly reduced (p 0.001) TF values in A. montanum (32.1e46%) and H. annuus (49.4e65%), as well as BFS in A. montanum (20.2e31%) and H. annuus (37.1e48.5%), while significantly boosted (p 0.001) BFR values in A. montanum (16.7e35.6%) and H. annuus (20e44.4%), under various doses of Cd in media, in compare to absence of the fungus. Accordingly, a significant reduction in TF of Cd in mycorrhizal garlic chives and sunflower plants was observed under various metals toxicity [48]. It is likely that (1) a desirable larger surface area provided by extraradical hyphae of S. indica in A. montanum and H. annuus roots, (2) the maintain of Cd by fungal mycelia as an important sink for heavy metal via entering Cd ions inside the fungus cells (especially vacuolar compartmentalization), and (3) the adsorption of Cd to chitin or carboxyl groups of glucuronic and mannuronic acids in the fungal cell wall, could be involved in higher BFR quantity, and lower TF and BFS quantities of the infested species. Also, Wang et al. [49] reported that inoculation of maize plants by endophyte fungus regulated the expression of three maize genes that involved in chelation, uptake and transport of Cd, resulting in Cd sequestration in root, less transfer to shoot, and conferring tolerance to metal toxicity. Regarding to enhanced values for BFR and decreased values for TF and BFS in S. indica-inoculated seedlings, it seems that a possible detoxification mechanism exhibited by this fungus is the retention of Cd in root and the reduction in Cd translocation to above-ground parts, especially leaf as a physiologically more sensitive and active organ than root. Therefore, considering these findings, S. indica is a convenient and suitable agent for purpose of phyto-immobilization of Cd in A. montanum and H. annuus roots, which causes the species to act as an Cd phyto-stabilizer. 4. Conclusion The colonization of roots by S. indica had positive effects on improving Cd tolerance in A. montanum and H. annuus through enhancing growth and biomass accumulation, and the elevation on Chl a and Chl b contents, and Fv/Fm and ETR values. In non-infected seedlings, Cd exposure induced A. montanum to exhibit as an accumulator species, whereas H. annuus was suitable for immobilization purpose of Cd. However, in the presence of S. indica both species exhibited the characteristics of a Cd phyto-stabilizer via increasing BFR and a reduction in TF. Regarding to these results, S. indica by enhancing Cd accumulation in the root could be applied as an appropriate bio-tool for immobilization of Cd in A. montanum and H. annuus roots, as a result un-contamination of polluted zones with this metal for phytoremediation goal. References [1] S.A. Anjum, M. Tanveer, S. Hussain, M. Bao, L. Wang, I. Khan, E. Ullah, S.A. Tung, R.A. Samad, B. Shahzad, Cadmium toxicity in Maize (Zea mays L.): consequences on antioxidative systems, reactive oxygen species and cadmium accumulation, Environ. Sci. Pollut. Res. 22 (21) (2015) 17022e17030. [2] D. Mani, C. Kumar, N.K. Patel, Integrated micro-biochemical approach for phytoremediation of cadmium and lead contaminated soils using Gladiolus grandiflorus L cut flower, Ecotoxicol. Environ. Saf. 124 (2016) 435e446. [3] S.M. Gallego, L.B. Pena, R.A. Barcia, C.E. Azpilicueta, M.F. Iannone, E.P. Rosales, M.S. Zawoznik, M.D. Groppa, M.P. Benavides, Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms, Environ. Exp. Bot. 83 (2012) 33e46. [4] Q. Zhou, J. Guo, C. He, C. Shen, Y. Huang, J. Chen, J. Guo, J. Yuan, Z. Yang, Comparative transcriptome analysis between low- and high cadmiumaccumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress, Environ. Sci. Technol. 50 (2016) 6485e6494. [5] S.A. Anjum, M. Tanveer, S. Hussain, B. Shahzad, U. Ashraf, S. Fahad, W. Hassan, S. Jan, I. Khan, M.F. Saleem, A.A. Bajwa, L. Wang, A. Mahmood, R.A. Samad, S.A. Tung, Osmoregulation and antioxidant production in maize under combined cadmium and arsenic stress, Environ. Sci. Pollut. Res. 23 (12) (2016a) 11864e11875. [6] S.A. Anjum, et al., Morpho-physiological growth and yield responses of two contrasting maize cultivars to cadmium exposure, Clean. - Soil, Air, Water 44
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Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica Saleh Shahabivand a, *, Azar Parvaneh a, Ali Asghar Aliloo b a b
Department of Biology, Faculty of Sciences, University of Maragheh, Maragheh, Iran Department of Agronomy, Faculty of Agriculture, University of Maragheh, Maragheh, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2019 Received in revised form 9 July 2019 Accepted 29 September 2019 Available online xxx
Environmental pollution by heavy metals is a severe issue worldwide. Microbe-assisted phytoremediation is a safe, inexpensive, and promising strategy in refinement of metal-polluted regions. Current in vitro work was installed to study effects of the endophytic fungus Serendipita indica on some physiological traits and cadmium (Cd) bioaccumulation of Alyssum montanum and Helianthus annuus seedlings grown in MS medium, under varying levels of Cd (0, 20, 40 and 60 mg Cd/l medium). Even though Cd stress induced phyto-toxicity in both tested species, but a significant improvement was found in biomass accumulation, photosynthetic pigments content, and chlorophyll fluorescence indicators in inoculated seedlings by S. indica under different doses of Cd in media. The non-infected A. montanum plantlets accumulated more Cd in shoot than root, and illustrated the properties of an accumulator species as evidenced by translocation factor (TF) and bioaccumulation factor of shoot (BFS) higher than 1. Contrary to this, un-colonized H. annuus seedlings had higher amount of Cd in root than shoot and showed a phyto-stabilizer feature, as evidenced by TF˂1 and bioaccumulation factor of root (BFR) higher than 1. Presence of S. indica significantly enhanced Cd accumulation in root, while it noticeably diminished Cd amounts of shoot in both A. montanum and H. annuus seedlings, so that inoculated plants had higher values for BFR against lower values for BFS and TF, in compare to non-inoculated ones. These findings indicated that S. indica can be considered as a bio-fertilizer to improve the physiological characteristics of tested species under Cd stress, as well as a bio-stabilizer of Cd in the roots of A. montanum and H. annuus in the regions exposed to toxic levels of Cd. © 2019 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
Keywords: Alyssum Sunflower Cd toxicity Phyto-accumulator Phyto-stabilizer Serendipita indica
1. Introduction One of the important environmental problems worldwide is contamination of various ecosystems by heavy metal cadmium (Cd) due to its toxic nature [1]. The toxicity of Cd is linked to its unbiodegradable feature, long persistence in the environment and accumulation in the body of different living organisms including human and plant [2]. The phosphate fertilizers, sewage sludge, mining wastes and disposal of industrial effluents are the main sources of Cd pollution in soil and water by anthropogenic activities [3]. According to high mobility and solubility of Cd, it is easily taken up by plant roots and is subsequently transferred to human body
* Corresponding author. University of Maragheh, Madar Square, Golshahr, Maragheh, 55181-83111, Iran. Tel: þ98 41 3727 6068, Fax: þ98 41 3727 6060. E-mail addresses: [email protected], [email protected] (S. Shahabivand).
(as a carcinogenic element) via consuming Cd-contaminated foods [4]. In plants, Cd deleteriously affects normal physiological and biochemical processes, leading to a reduction/inhibition in growth and biomass accumulation, nutrient imbalances, leaf chlorosis and even death at its higher doses [5,6]. In the recent years, phyto-decontamination as a sustainable, promising and practicable method has been raised for the cleanup of metal-polluted soils and waters, regarding to its eco-friendly and cost effective features. Accordingly, phyto-extraction and phytostabilization are the two main kinds of phytoremediation to refining heavy metal polluted sites. Phyto-extraction or phytoaccumulation refers to the uptake of pollutants from environment by the roots and the accumulation of those in the above-ground parts of plants. Whereas, phyto-stabilization is the method to accumulate pollutants including heavy metals in plant roots or precipitation in the rhizosphere for preventing them to entry the food chains or underground water. It has been demonstrated that
https://doi.org/10.1016/j.chnaes.2019.09.002 1872-2032/© 2019 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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some plant-associated fungi can effectively elevate the efficiency of phytoremediation, as well as heavy metal tolerance of host plants. Therefore, study on the plant-associated fungi is a considerable factor to exploring the possible uses of these fungi for fungalassisted phytoremediation of heavy metal. Also, according to previous researches, the beneficial plant-microbe relationship yielded in an encouraging result on phytoremediation of metals both economically and environmentally than the use of plant alone [7,8]. Root endophytic fungus Serendipita indica (formerly known as Piriformospora indica), that belongs to the class basidiomycete and the order Sebacinales, has symbiotic interaction with various plants similar to that of mycorrhizal symbiosis [9,10]. S. indica attributes multiple beneficial property to the host such as promotion of growth and yield, elevation of macro- and micronutrient uptake, and tolerance to wide range of biotic and abiotic stresses including metals toxicity [10e12]. The selection of appropriate species is one of the significant features to improving phytoremediation efficacy. Alyssum montanum is a perennial herbaceous plant from family Brassicaceae, with a relatively small size. There is little information about A. montanum responses to metal-polluted environment, unlike other species of Alyssum genus such as Alyssum bertolonii as a ska et al. hyper-accumulator for Ni [13]. In a recent study by Muszyn [14], it was found that metallicolous ecotype of A. montanum showed higher tolerance to multi-metal stress in compare to nonmetallicolous one. However, they found that non-metallicolous ecotype accumulated more Cd in shoot than that of root. Helianthus annuus L., as a fast-growing crop plant, has a great biomass, extensive root system, and potent compatibility and strong tolerance to different stress conditions including heavy metal stress [15]. It is a non-hyper-accumulating species and suitable tool for Cd extraction in Cd-polluted sites because it merges great biomass yield with a high Cd bioaccumulation [16,17]. Regarding to the possible role of H. annuus (with reasonable biomass yields) and A. montanum (with low biomass yields) in the phytoremediation of Cd-contaminated environment, as well as S. indica beneficial impact in increasing tolerance to the hosts under the metals stress, in this paper, we tested the effect of S. indica on respond to Cd in the two above-mentioned species. For this goal, Cd bioaccumulation in root and shoot, photosynthetic pigments content, Fv/Fm and ETR values, and growth attributes in H. annuus and A. montanum under S. indica symbiosis and at increasing Cd doses were discussed. Our research was carried out under in vitro culture conditions due to the entirely controlled status and least environmental interaction in this medium. 2. Materials and methods 2.1. Plant and fungal materials Seeds of Alyssum montanum L. (collected from the farming land in University of Maragheh, Iran) and Helianthus annuus L. cv. Azargol (received from the Dryland Agricultural Research Institute, Maragheh, Iran) were surface sterilized for 2 min in ethanol followed by 10 min in a 1% NaClO solution (0.75% Cl), then rinsed with distilled water three times and germinated on wet filter paper at 4 C for 48 h. The endophytic fungus S. indica was cultured in Petri dishes on a Hill & K€ afer medium [18], then placed in a temperaturecontrolled growth chamber at 29 ± 1 C in dark for 10 days. 2.2. Experiment design In-vitro culture experiment was established to determine the effects of S. indica on A. montanum and H. annuus growing under different doses of Cd, and consisted of a completely randomized 2 4 factorial arrangements with the following factors for each
plant species: two S. indica treatments (with or without fungus inoculation), each having four Cd concentrations (0, 20, 40 and 60 mg Cd/l medium), in four replicates. Four germinated seeds (from A. montanum or H. annuus) were transplanted into the glass jars (8 20 cm) containing 30 g of autoclaved MS solid medium (as the culture substrate with the macro- and micronutrients and the MS vitamins without phytohormones; Murashige and Skoog) supplemented with 3% (w:v) sucrose. The solidification of MS medium was done by 0.75% Difco agar, and pH of medium was adjusted to 5.6 prior to solidification. Four Cd concentrations (0, 20, 40 and 60 mg Cd/l medium) were added to the MS medium prior to autoclaving, as cadmium (II) chloride salt. For co-cultivation of the two symbionts, two fungal plug (5 mm in diameter) was placed at a distance of 1 cm from the roots of 10-days-old seedlings. Non-S. indica treatments received the same quantity of autoclaved S. indica inoculum. Experimental jars, sealed with sterile AeraSeal film covers, were located in a temperature/moisture-controlled growth room under conditions of 14 h of light and 10 h darkness, an irradiance of 100 mmol m2 s1 by white fluorescent lamps, 26/20 C day/night temperature and relative humidity of 65%. The seedlings were harvested after 28 d for growth and other analyses. Roots and shoots of the samples were carefully washed with deionized water and dried by filter paper for measurement of root and shoot lengths, and then were frozen in the liquid nitrogen for pigments analysis. Some fresh root samples were stored in water for 1 h to study root colonization. To determine the dry weights and Cd amounts of root and shoot, the plant samples were dried in an oven at 75 C for 48 h. 2.3. Measurement of root colonization by S. indica The root colonization percent by S. indica was found by the method of Oelmüller et al. [19] after root cleaning in 10% KOH and staining with 0.1% Trypan blue [20]. In this method, S. indicachlamydospores distribution within the roots of colonized plants was estimated as an index for root colonization. 2.4. Cd measurement in plant materials The oven-dried root and shoot samples were separately ground and then digested in a mixture of concentrated HNO3 and HClO4 acids (7:1 ratio, v/v). The content of Cd in plant materials was determined using an atomic absorption spectrophotometry (Shimadzu, Japan) method. According to Yoon et al. [21], the translocation factor (TF) of Cd was determined as the ratio of Cd content in shoot to Cd content in root (Cd amount in shoot/Cd amount in root). Ha et al. [22] defined the bioaccumulation factor (BCF) as the ratio of metal content in shoot to that in the soil, whereas Yoon et al. [21] assumed BCF as ratio of metal content in root to soil concentration of metal. In this work, we defined the bioaccumulation factor of shoot (BFS) and the bioaccumulation factor of root (BFR) as following formulae: BFR ¼ Cd amount in root/Cd concentration in media, and BFS ¼ Cd amount in shoot/Cd concentration in media. 2.5. Photosynthetic pigments measurement in leaf For measurement of chlorophyll (Chl) content in the second fully expanded leaf, 0.1 g of fresh sample was homogenized with acetone 80% (v/v) and then filtrated through filter paper. The absorbance of filtrate was read at 663 and 645 nm for Chl a and Chl b respectively, and the pigments contents were estimated according to Arnon [23]. 2.6. Measurement of chlorophyll fluorescence After darkening the leaf for 30 min, the chlorophyll fluorescence
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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parameters including Fv/Fm (maximum quantum efficiency of PSII photochemistry) and ETR (the relative PSII electron transport rate), as physiological indicators, were measured on the second fully expanded leaf using a portable chlorophyll fluorometer (model Hansatech, Instruments LTD, UK). 2.7. Statistical analysis The experimental data were analyzed by SAS 9.4 software version 2013. Duncan’s Multiple Range Test at a 0.05 probability level was used to study the differences between treatments for each species separately. Finally, the results were presented as the means of four replicates ± standard deviation (SD). 3. Results and discussion 3.1. Root infection and growth attributes There are significant individual and interactive effects of Cd addition and S. indica infection on root colonization of A. montanum and H. annuus at p 0.001 (Table 1). The results of Table 2 illustrated that fungal symbiosis in A. montanum and H. annuus could easily be installed under Cd exposure. However, the roots of noninfected plants did not establish colonization with S. indica. The maximum colonization intensity of S. indica in the roots of A. montanum and H. annuus were 72.2- and 75.7%, respectively, at the control (0 mg Cd/l medium) treatment (Table 2). According to our previous researches [12,24], Cd stress reduced root colonization by S. indica in both A. montanum and H. annuus seedlings, and lowest level of colonization was found at highest level of Cd in substrate culture (60 mg Cd/l; Table 2). The lower percent of colonization in the roots of tested seedlings may be because of a delay/ diminish/eliminate on germination of S. indica chlamydospores and/or on its hyphal growth at higher doses of Cd. Also, van keulen et al. [25] showed that heavy metal toxicity caused an injury in fungal cell wall integrity via elevation in expression of the enzyme chitinase. Data of ANOVA (Table 1) showed that main effects of S. indica and Cd stress had significant influence on growth traits (p 0.001) in both A. montanum and H. annuus seedlings, however, their interaction did not significantly affect the growth indicators (except for shoot dry weight of H. annuus at p 0.05). Therefore, we presented here only the main effects of the factors on the growth and biomass. Increasing severity of Cd stress significantly reduced the plant length, and root and shoot dry weights in tested plants (Table 3). Compared with control (0 mg Cd), plant length, root dry weight and shoot dry weight were decreased by 31.2-, 27.7- and 27.2% in A. montanum, and by 25.1-, 37.4- and 33.2% in H. annuus under severe Cd stress (60 mg Cd/l culture substrate; Table 3), respectively. Regarding to the lower reduction in biomass accumulation (root and shoot dry matter) of A. montanum in compare to
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H. annuus, it can be considered that A. montanum suffered less from Cd toxicity than H. annuus at the higher doses of Cd. Taking into account fact that auxin level (as an important growth promotion phyto-hormone) is reduced under Cd stress (via elevated activity of auxin oxidase) [26], the reduction in the biomass of treated seedlings may be attributed to this phenomenon. On the other hand, inoculation by S. indica resulted in a significant increase on plant length, and root and shoot dry matters in A. montanum and H. annuus, in comparison to non-inoculated ones (Table 4). These increases in plant length, root dry weight and shoot dry weight were 15.6-, 24.3- and 9.2% in A. montanum, and were 8.7-, 18.6- and 19.3%, in H. annuus respectively (Table 4). These results are in accordance with our previous studies in wheat and sunflower under exposure to Cd in soil [12,24], and findings of Mohd et al. [11] in rice plants exposed to arsenic toxicity under in vitro conditions. Plant growth promotion activity of fungal endophytes can be done by producing phytohormones such as auxin and gibberllin, ACC deaminase (a key enzyme in consuming ethylene precursor, thus lowering ethylene level), and siderophores to the better uptake of iron by plant roots [27,28]. In our earlier work, symbiosis by S. indica significantly elevated Zn (by 44%) and Mn (by 34%) contents of lettuce leaves compared with control [29]. In the presence of S. indica, the amounts of N, P and K were markedly higher in mung bean plants, in compare to absence of the fungus [30]. Thus, one of the mechanisms involved in S. indica-mediated growth improvement may be increasing uptake of macro- and micronutrients from culture substrate. Furthermore, an enhanced growth under S. indica symbiosis, can be attributed to the elevation in photosynthetic pigment content and chlorophyll fluorescence indicators in A. montanum and H. annuus plantlets (Table 4). 3.2. Chlorophyll (Chl) contents and chlorophyll fluorescence parameters Results of ANOVA (Table 1) illustrated that interactive effect of S. indica and Cd treatments had no significant differences on Chl a and b contents, and Fv/Fm and ETR values, whereas main effect of the two factors had significant influence on above-mentioned traits at p 0.001 in A. montanum and H. annuus (except for Fv/Fm in A. montanum at p 0.05). By increasing Cd concentrations in the culture substrate, Chl a and b amounts in leaf, and Fv/Fm and ETR indicators were significantly decreased in both species seedlings, except for Chl b in A. montanum and ETR in H. annuus at highest level of Cd (60 mg; Table 3). The maximum level of Cd in medium induced minimum amount of the pigments and fluorescence parameters, so that the reductions in Chl a, Chl b, Fv/Fm and ETR were 37.3-, 29.6-, 13.5- and 33.2% in A. montanum, and 50.3-, 54-, 17.3and 20.1% in H. annuus, respectively, under 60 mg Cd as compared to control (Table 3). Several researches have described the reduction in Chl content of various plants such as rapeseed [31], wheat [32] and sunflower [12] under Cd stress which were consistent with
Table 1 Analysis of variance (ANOVA) of S. indica (S) treatment and Cd treatment on root colonization (RC), plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm), electron transport rate of PSII (ETR), root Cd (RCd), shoot Cd (SCd), translocation factor (TF), bioaccumulation factor of root (BFR) and bioaccumulation factor of shoot (BFS) in A. montanum and H. annuus seedlings. Source of variation A. montanum S Cd S Cd H. annuus S Cd S Cd
df
RC
PL
RDW
SDW
Chl a
Chl b
Fv/Fm
ETR
RCd
SCd
TF
BFR
BFS
1 3 3
*** *** ***
*** *** ns
*** *** ns
*** *** ns
*** *** ns
*** *** ns
* *** ns
*** *** ns
*** *** **
*** *** ***
*** *** ***
*** *** ***
*** *** ***
1 3 3
*** *** ***
*** *** ns
*** *** ns
*** *** *
*** *** ns
*** *** ns
*** *** ns
*** *** ns
*** *** ***
*** *** ***
*** *** ***
*** *** ***
*** *** ***
***, **, *, ns stand for significant at P 0.001, 0.01 and 0.05 probability levels and not significant, respectively.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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Table 2 Interaction effect of S. indica and Cd treatments on root colonization (RC) of A. montanum and H. annuus. S. indica treatment
Cd treatment (mg/l)
RC (%) A. montanum
RC (%) H. annuus
Without S. indica
0 20 40 60 0 20 40 60
n.d. n.d. n.d. n.d. 72.2 ± 3.7 a 63.2 ± 4.1 b 54.2 ± 2.6 c 48.7 ± 3.3 d
n.d. n.d. n.d. n.d. 75.7 ± 5.1 a 66.5 ± 3.8 b 58.2 ± 1.8 c 48.5 ± 2.0 d
With S. indica
The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at. p 0.001. n.d.: not determined.
Table 3 Main effect of Cd treatment on plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm) and electron transport rate of PSII (ETR) in A. montanum and H. annuus seedlings. Cd treatment A. montanum 0 20 40 60 H. annuus 0 20 40 60
PL (mm)
RDW (mg/jar)
SDW (mg/jar)
Chl. a (mg/g FW)
Chl. b (mg/g FW)
Fv/Fm
ETR
67.2 ± 5.8 a 58.9 ± 5.0 b 51.0 ± 4.3 c 46.2 ± 3.9 d
93.9 ± 13.0 a 84.7 ± 10.6 b 78.3 ± 9.9 c 67.9 ± 8.3 d
326.4 ± 19.6 a 293.3 ± 18.1 b 255.6 ± 13.4 c 237.7 ± 14.1 d
1.26 ± 0.10 a 1.13 ± 0.10 b 1.04 ± 0.17 c 0.79 ± 0.09 d
0.44 ± 0.06 a 0.36 ± 0.06 b 0.32 ± 0.04 c 0.31 ± 0.04 c
0.827 ± 0.01 a 0.783 ± 0.02 b 0.740 ± 0.01 c 0.715 ± 0.01 d
130.3 ± 9.6 a 117.7 ± 8.6 b 102.5 ± 8.0 c 87.1 ± 3.9 d
196.0 ± 7.8 a 170.9 ± 6.7 b 159.7 ± 9.2 c 146.8 ± 7.8 d
219.7 ± 21.3 a 184.4 ± 19.4 b 161.2 ± 15.3 c 137.5 ± 15.2 d
535.6 ± 56.7 a 467.4 ± 38.7 b 417.7 ± 42.9 c 357.7 ± 35.4 d
1.51 ± 0.13 a 1.25 ± 0.09 b 1.05 ± 0.13 c 0.75 ± 0.08 d
0.66 ± 0.11 a 0.49 ± 0.07 b 0.40 ± 0.06 c 0.31 ± 0.05 d
0.812 ± 0.01 a 0.778 ± 0.01 b 0.740 ± 0.02 c 0.688 ± 0.02 d
137.1 ± 6.2 a 126.1 ± 5.4 b 111.5 ± 3.5 c 109.5 ± 6.3 c
Values are mean ± SD, n ¼ 4. The different letter within each column (for each species) indicates significant difference among treatments using Duncan’s Multiple Range Test.
Table 4 Main effect of S. indica (S) on plant length (PL), root dry weight (RDW), shoot dry weight (SDW), chlorophyll a (Chl a), chlorophyll b (Chl b), maximum quantum efficiency of PSII (Fv/Fm) and electron transport rate of PSII (ETR) in A. montanum and H. annuus seedlings. S. indica treatment A. montanum -S þS H. annuus -S þS
PL (mm)
RDW (mg/jar)
SDW (mg/jar)
Chl a (mg/g FW)
Chl b (mg/g FW)
Fv/Fm
ETR
51.8 ± 7.9 b 59.9 ± 9.0 a
72.4 ± 9.5 b 90.0 ± 12 a
265.9 ± 34.7 b 290.5 ± 38.5 a
0.97 ± 0.18 b 1.14 ± 0.20 a
0.31 ± 0.05 b 0.40 ± 0.06 a
0.760 ± 0.04 b 0.773 ± 0.04 a
103.8 ± 14.7 b 115.0 ± 19.8 a
161.3 ± 19.1 b 175.4 ± 18.6 a
160.7 ± 29.7 b 190.7 ± 34.6 a
405.3 ± 62.1 b 483.8 ± 74.8 a
1.05 ± 0.28 b 1.24 ± 0.30 a
0.40 ± 0.12 b 0.53 ± 0.15 a
0.74 ± 0.05 b 0.77 ± 0.04 a
118.3 ± 10.4 b 123.7 ± 14.2 a
Values are mean ± SD, n ¼ 4. The different letter within each column (separately for each species) indicates significant difference among treatments using Duncan’s Multiple Range Test.
A. montanum and H. annuus in our work. The decreased amounts of the Chl a and Chl b in Cd-treated plants may be attributed to overproduction of reactive oxygen species (ROS) by oxidative stress, reduction of Chl biosynthesis, and Chl degradation via elevated chlorophylase enzyme activity at toxicity doses of Cd in medium [33]. The Chlorophyll fluorescence indicators (such as Fv/Fm and ETR) evaluation is a useful approach to determine the photosynthetic performance of plants under heavy metal stress. According to fluorescence responses in our study, a decline in Fv/Fm value of maize and wheat seedlings, as well as the reduction in ETR index of Alpine pennycress under Cd exposure were observed [34e36]. A reduction in the values of Fv/Fm and ETR, associated with decreased content of Chl in this study, suggests the role Cd in reducing PSII activity and its structure. The significant difference was resulted among the inoculated and non-inoculated seedlings for pigments amounts and fluorescence parameters, and inoculated plants of both species had better photosynthetic status in terms of Chl contents and fluorescence indicators than their respective controls (Table 4). Presence of S. indica produced higher amounts of Chl a (17.5%), Chl b (29%), Fv/ Fm (1.7%) and ETR (10.8%) in A. montanum, in comparison to absence of the endophyte (Table 4). Also, colonized H. annuus
seedlings with S. indica had higher values of Chl a (18.1%), Chl b (32.5%), Fv/Fm (4%) and ETR (4.5%) than those control ones (Table 4). Under heavy metal stress, Chl level and Fv/Fm value were significantly greater in the endophyte-infected rice seedlings [37]. Moreira et al. [38] in pineapple and Ghaffari et al. [39] in barley reported that S. indica symbiosis elevated Mg content, as a pivotal point in the chlorophyll structure. A remarkably elevation on ETR and a slight increase on Fv/Fm were resulted under inoculation by endophyte fungus in Chinese white poplar [40]. Considering these results, it is concluded that Cd toxicity results in functional disorder of photosynthetic machinery and disadvantage to electron transfer of PSII, however these disturbances can be attenuated by S. indica symbiosis in tested seedlings. 3.3. Cd uptake, transport and bioaccumulation Based on the results from Table 1, the main effect and interactive effect of the two factors S. indica colonization and Cd treatment had significant influence (p 0.001) on Cd content in root and shoot, translocation factor (TF), bioaccumulation factor of root (BFR) and bioaccumulation factor of shoot (BFS) in both A. monatum and H. annuus seedlings under in vitro conditions. Therefore, we
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focused on interactive effect of mentioned parameters. No Cd was found in the seedlings grown on un-contaminated medium by Cd (0 mg Cd, date not shown). Based on Fig. 1, in infected and nuinfected A. monatum and H. annuus plantlets, by increasing Cd doses in the MS medium, Cd content in root and shoot were significantly increased (except shoot Cd of A. montanum from 20 to 60 mg Cd in un-infected, and shoot Cd of H. annuus from 40 to 60 mg Cd in infected ones). Cd amounts of roots in the both tested seedlings were higher than those Cd concentrations in MS medium, indicating the bioavailability of Cd in medium to plant roots, as well as capability of these species to the uptake of Cd via an active process along with energy consumption. The root cells of H. annuus seedlings bioaccumulated more Cd than A. montanum root under all levels of Cd in media, so that in un-infected seedlings, the maximum content of root Cd reached 240.1 mg Cd in H. annuus in compare to 118.2 mg Cd in A. montanum under 60 mg/l Cd in growth media (Fig. 1). In some species, the Casparian strip of endodermis cell as well as existence of pericycle layer in root tissue may be considered as the important barrier in transport of water and heavy metals from root to shoot, resulting in metal accumulation in root. In A. montanum and H. annuus, and in both colonized and non-colonized plants, TF values were approximately constant under various levels of Cd in media, except in A. montanum and in colonized ones at 60 mg Cd that TF value showed a significant increase in compare to 40 mg Cd in medium (Fig. 2). In response to the increase in Cd doses in substrate culture, a reduction in BFR and BFS were observed in the two species, and in both inoculated and un-inoculated seedlings (Fig. 3). Reduced values of BFR and BFS, when Cd level of media had elevating trend, indicated that these species were more sensitive to Cd toxicity at higher Cd amounts in growth media, and had a constraint to Cd accumulation under these levels of the metal. On the other hand, the two tested species (in un-colonized ones), illustrated a distinct pattern in TF, BFR and BFS responses to the addition of Cd in media. In non-inoculated
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H. annuus seedlings, TF values ranged between 0.33 and 0.36, BFR ranged from 4 to 5.4, and BFS ranged between 1.35 and 1.96, at various levels of Cd in media (Figs. 2 and 3). Plants with a TF value lower than 1, and a BFR value higher than 1 are benefit for phytostabilization process, whereas plants with TF, BFR and BFS higher than 1 have capability to be used in phyto-extraction process [21,41]. According to Hesami et al. [41], BFS or EF (extraction factor in their study), reflects both TF and BFR and can be considered as a useful index in phyto-extraction ability of plants. Regarding to our results, H. annuus illustrated a stand with TF lower than 1, and BFR higher than 1 (4e5.4), and may be appropriate in immobilization of Cd in its root and can be used in Cd phyto-stabilization. In the case of A. montanum, TF and BFR values were greater than 1 (1.03e1.11 for TF, and 1.97e3.03 for BFR) as well as BFS amounts were from 2.03 to 3.36, under different concentrations of Cd in nutrient media (Figs. 2 and 3). Furthermore, Cd amounts of shoot in non-infected A. montanum plants (at 40 and 60 mg Cd in growth media, Fig. 1) were greater than critical amount of Cd i.e. 100 mg/kg bioaccumulated in extractor/hyper-accumulator plants. Even though a small biomass is the constraint for metal extraction purpose, but the less biomass produced by A. montanum (in compare to H. annuus; Table 3), was compensated by its high TF and BFS values, leading to increased accumulation of Cd in shoot. Therefore, A. montanum can be seen as an appropriate option for Cd extraction from Cd polluted areas, because it tolerated more than 100 mg Cd in shoots along with a relatively low decline in the biomass of shoot (28.2%) under highest level of Cd (60 mg) in medium, in compare to control (Table 3). It is well known that plant-associated microbes have a direct influence on metal absorption from media by their different activities, as well as an indirect effect on host withstand under metal stress by changing morphological, biochemical and physiological features of host plant [42]. Therefore, microbe-assisted phytoremediation of toxic metals can be a practicable, safe and
Fig. 1. Interaction effect of S. indica and Cd treatments on Cd content in roots (up) and shoots (down) of A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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Fig. 2. Interaction effect of S. indica and Cd treatments on translocation factor (TF) of Cd in A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
Fig. 3. Interaction effect of S. indica and Cd treatments on bioaccumulation factor of root (BFR; up) and shoot (BFS; dowm) in A. montanum and H. annuus seedlings. The mean values (±SD, n ¼ 4) for each species followed by the same letters are not significantly different at p 0.001.
sustainable strategy in reclaiming metal polluted regions. In this research, presence of endophytic microbe S. indica noticeably enhanced Cd content in root, whereas markedly reduced Cd content in shoot in both treated species, in compare to absence of the endophyte (Fig. 1). In inoculated A. montanum plants, these enhancements in root Cd amounts were from 16.7% (under 60 mg Cd) to 35.6% (under 20 mg Cd in media), while the reductions in shoot Cd were from 19.9% (at 60 mg Cd) to 31.1% (at 40 mg Cd in growth medium). In the case of H. annuus, presence of S. indica increased root Cd between 20.6% (under 60 mg Cd) and 45.9% (under 20 mg Cd), but decreased shoot Cd between 37% (at 40 mg Cd) and 48.7% (at 20 mg Cd in growth substrate; Fig. 1). These findings are in accordance with our earlier studies in wheat cv. Sardari and sunflower cv. Zaria in contaminated soil by toxic levels of Cd [12,24]. Also, Sabra et al. [43] found that S. indica-infected sweet basal
plants, in comparison to uninfected ones, had higher lead and cupper content in roots, but lower Pb and Cu content in shoots when both mentioned heavy metals were present in media. It has been described that fungal endophytes could be expressed some genes related to metal-mobilize and metal-tolerance to their survival and function under metal stress, resulting in higher metal absorption, and confer resistance to their host. For instance, the EpNramp gene (as a bivalent transporter) from the endophyte Exophiala pisciphila, enhanced Cd uptake and conferred resistance to Cd toxicity [44]. Some fungi can participate in arising trace metal solubility in media through producing organic acids such as citrate and oxalate and forming their complexes with metals, leading to an increase in metal uptake as well as the un-contamination of polluted areas [45]. Inoculation of solanum nigrum and Populus canescens plants by fungal strains increased Cd content in the roots
Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002
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via enhancing the availability and uptake of Cd [46,47]. Based on Figs. 2 and 3, presence of S. indica significantly reduced (p 0.001) TF values in A. montanum (32.1e46%) and H. annuus (49.4e65%), as well as BFS in A. montanum (20.2e31%) and H. annuus (37.1e48.5%), while significantly boosted (p 0.001) BFR values in A. montanum (16.7e35.6%) and H. annuus (20e44.4%), under various doses of Cd in media, in compare to absence of the fungus. Accordingly, a significant reduction in TF of Cd in mycorrhizal garlic chives and sunflower plants was observed under various metals toxicity [48]. It is likely that (1) a desirable larger surface area provided by extraradical hyphae of S. indica in A. montanum and H. annuus roots, (2) the maintain of Cd by fungal mycelia as an important sink for heavy metal via entering Cd ions inside the fungus cells (especially vacuolar compartmentalization), and (3) the adsorption of Cd to chitin or carboxyl groups of glucuronic and mannuronic acids in the fungal cell wall, could be involved in higher BFR quantity, and lower TF and BFS quantities of the infested species. Also, Wang et al. [49] reported that inoculation of maize plants by endophyte fungus regulated the expression of three maize genes that involved in chelation, uptake and transport of Cd, resulting in Cd sequestration in root, less transfer to shoot, and conferring tolerance to metal toxicity. Regarding to enhanced values for BFR and decreased values for TF and BFS in S. indica-inoculated seedlings, it seems that a possible detoxification mechanism exhibited by this fungus is the retention of Cd in root and the reduction in Cd translocation to above-ground parts, especially leaf as a physiologically more sensitive and active organ than root. Therefore, considering these findings, S. indica is a convenient and suitable agent for purpose of phyto-immobilization of Cd in A. montanum and H. annuus roots, which causes the species to act as an Cd phyto-stabilizer. 4. Conclusion The colonization of roots by S. indica had positive effects on improving Cd tolerance in A. montanum and H. annuus through enhancing growth and biomass accumulation, and the elevation on Chl a and Chl b contents, and Fv/Fm and ETR values. In non-infected seedlings, Cd exposure induced A. montanum to exhibit as an accumulator species, whereas H. annuus was suitable for immobilization purpose of Cd. However, in the presence of S. indica both species exhibited the characteristics of a Cd phyto-stabilizer via increasing BFR and a reduction in TF. Regarding to these results, S. indica by enhancing Cd accumulation in the root could be applied as an appropriate bio-tool for immobilization of Cd in A. montanum and H. annuus roots, as a result un-contamination of polluted zones with this metal for phytoremediation goal. References [1] S.A. Anjum, M. Tanveer, S. Hussain, M. Bao, L. Wang, I. Khan, E. Ullah, S.A. Tung, R.A. Samad, B. Shahzad, Cadmium toxicity in Maize (Zea mays L.): consequences on antioxidative systems, reactive oxygen species and cadmium accumulation, Environ. Sci. Pollut. Res. 22 (21) (2015) 17022e17030. [2] D. Mani, C. Kumar, N.K. Patel, Integrated micro-biochemical approach for phytoremediation of cadmium and lead contaminated soils using Gladiolus grandiflorus L cut flower, Ecotoxicol. Environ. Saf. 124 (2016) 435e446. [3] S.M. Gallego, L.B. Pena, R.A. Barcia, C.E. Azpilicueta, M.F. Iannone, E.P. Rosales, M.S. Zawoznik, M.D. Groppa, M.P. Benavides, Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms, Environ. Exp. Bot. 83 (2012) 33e46. [4] Q. Zhou, J. Guo, C. He, C. Shen, Y. Huang, J. Chen, J. Guo, J. Yuan, Z. Yang, Comparative transcriptome analysis between low- and high cadmiumaccumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress, Environ. Sci. Technol. 50 (2016) 6485e6494. [5] S.A. Anjum, M. Tanveer, S. Hussain, B. Shahzad, U. Ashraf, S. Fahad, W. Hassan, S. Jan, I. Khan, M.F. Saleem, A.A. Bajwa, L. Wang, A. Mahmood, R.A. Samad, S.A. Tung, Osmoregulation and antioxidant production in maize under combined cadmium and arsenic stress, Environ. Sci. Pollut. Res. 23 (12) (2016a) 11864e11875. [6] S.A. Anjum, et al., Morpho-physiological growth and yield responses of two contrasting maize cultivars to cadmium exposure, Clean. - Soil, Air, Water 44
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Please cite this article as: S. Shahabivand et al., Different response of Alyssum montanum and Helianthus annuus to cadmium bioaccumulation mediated by the endophyte fungus Serendipita indica, Acta Ecologica Sinica, https://doi.org/10.1016/j.chnaes.2019.09.002