3.1. AMF colonization under different treatments
As shown in Table 1, the frequency of mycorrhiza (F%) of reeds inoculated with F. mosseae were determined before those were transplanted into the VFCWs, being 50.00%, 53.30%, 50.00%, 53.30%, 50.00%, 46.70%, respectively. When the experiment ended at the 60th day, the F% values of the corresponding treatment increased by 40.00%, 25.14%, 20.00%, 18.76%, 13.40%, 7.07%, respectively, indicating that AMF could continue to infect the root system under flooded conditions. In particular, the F% values of the "IA+FM" treatment under the three concentrations of combined HMs (0, low, and high) were significantly higher than those of the "NA+FM" treatment (P<0.05), which increased by 10.58%, 17.64%, and 20.00%, respectively. Meanwhile, the intensity of mycorrhizal colonization (M%) and arbuscular abundance (A%) also showed a similar trend with the F%. These results suggested that AA had a positive effect on improving AMF colonization in VFCWs, especially under the high concentrations of combined HMs, which was consistent with the results of Xu et al. (2021).
Table 1
Root colonization of reeds in the six VFCWs inoculated with F. mosseae.
Treatment
|
HMs level
|
Days
|
F%
|
M%
|
A%
|
IA+FM
|
0
|
0
|
50.00±2.00fg
|
13.10±0.40d
|
0.80±0.10de
|
60
|
70.00±2.00a
|
24.30±0.80a
|
2.80±0.30a
|
low
|
0
|
53.30±1.60ef
|
14.80±0.30c
|
0.80±0.10de
|
60
|
66.70±2.60ab
|
23.70±0.60a
|
2.10±0.20b
|
high
|
0
|
50.00±1.70fg
|
8.50±0.50g
|
0.50±0.10f
|
60
|
60.00±2.00cd
|
15.00±0.50c
|
1.10±0.20c
|
NA+FM
|
0
|
0
|
53.30±2.30ef
|
9.30±0.30g
|
0.40±0.10f
|
60
|
63.30±2.60bc
|
17.40±0.40b
|
1.00±0.10cd
|
low
|
0
|
50.00±1.70fg
|
11.70±0.50e
|
0.60±0.10ef
|
60
|
56.70±2.20de
|
17.00±0.40b
|
0.90±0.10cd
|
high
|
0
|
46.70±1.90g
|
10.50±0.50f
|
0.50±0.10f
|
60
|
50.00±2.60fg
|
13.00±0.40d
|
0.60±0.10ef
|
F%: the frequency of mycorrhiza; M%: the intensity of mycorrhizal colonization; A%: the arbuscule abundance; IA: intermittent aeration; NA: no aeration; FM: inoculation with F. mosseae; 0: the day of the reeds were transplanted; 60: the day of the reeds were harvested. |
Data are presented as Means ± SD. Significant differences between different treatments are presented by different letters (Duncan’s test, P<0.05). |
Lower oxygen content in wetland habitats may be one of the most important factors leading to AMF not to colonize roots at a high level like in the terrestrial ecosystems (Huang et al. 2021, Wang et al. 2015). Similarly, Hu et al. (2020b) reported that AMF colonization under fluctuating water depth was higher than high water regimes, which was ascribed to the increasing oxygen content of the rhizosphere by constantly changing water regime. In addition to improving the availability of oxygen, the addition of aeration also promoted the growth of wetland plants, thus providing more surface areas for microorganisms growth, and also altered the diversity of microbial communities distributed in the rhizosphere (Feng et al. 2021). Ferreira et al. (2021) and Viollet et al. (2017) revealed that the diverse microbial communities (e.g. Plant growth-promoting rhizobacteria, nitrogen-fixing bacteria) had a significant influence on helping AMF acquire more nutrient and facilitating hyphal growth to produce spores, which played a positive role in promoting AMF colonization. We inferred here that it happened in wetlands environment as well. As the concentration of combined HMs increased, the F%, M%, and A% of the two inoculation treatments all showed a decreasing trend. A similar study by Hu et al. (2021) showed that the F% were 49.70% at 0 mg/ kg Cr concentration, but decreased to 40.90% and 23.90% at 5 and 25 mg/kg Cr concentration, respectively. This may be attributed to the negative influence of HMs on the germination and growth of hyphal (Göhre &Paszkowski 2006). What’s more, HMs had a great negative impact on the establishment of symbiotic relationship between AMF and plant roots, thereby decreasing root infectivity and colonization (Wu et al. 2019). Therefore, the AA provided progress for the lower AMF colonization in wetland plant roots.
3.2. Plant growth and physiological indexes under different treatments
3.2.1. Growth indexes, chlorophyll content and root vitality
Under the three concentrations of combined HMs, compared with the CK treatment ("NA+NM"), the reeds shoot height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, and chlorophyll content of AMF inoculation and/or AA treatments increased by 9.40-56.17%, 0.14-76.47%, 12.22-169.32%, 2.07-126.62%, 11.84-58.33%, 28.62-238.77%, and 36.52-519.67%, respectively (Table S1). Among them, those growth indexes and chlorophyll content of the "NA+FM" and "IA+FM" treatments were significantly higher than those in the CK treatment (P<0.05), and the growth promotion of the "IA+FM" treatment was the most significant. The root vitality of the AMF inoculation and/or AA treatments was significantly higher than the non-inoculated or non-aerated treatments (P<0.05). Compared with the reeds grown without combined HMs, the root vitality of reeds grown at low concentrations increased slightly, while that dropped significantly at high concentrations (P<0.05). Principal Component Analysis (PCA) results showed that these parameters were largely determined by Dim1 (67.90%), implying there was a strong correlation among them. Meanwhile, the results of scores of 12 treatments showed that AMF inoculation and AA had different effects on wetland plant physiological functions under the three concentrations of combined HMs (Fig. S2). The results of hierarchical clustering analysis indicated that AMF inoculation and AA showed obvious synergistic effects on the growth characteristics of wetland plants (Fig. 1).
For aquatic plants, HMs stress could inhibit the root vitality, decrease the content of chlorophyll and plant biomass by reducing the uptake of essential macro and micronutrients (Wu et al. 2019, Zhao et al. 2019). The plant biomass and chlorophyll content of wetland plant (Iris wilsonii) inoculated with AMF under the stress of Cr contaminated-water were higher than those of non-inoculated control, which was consistent with our results (Hu et al. 2020c). Schück andGreger (2020) conducted a 5-day HMs exposure experiment on 34 types of wetland plants and measured 20 traits of each plant, and finally found that high biomass was one of the most important characteristics of the plant's ability to remove HMs. Therefore, wetland plants inoculated with AMF could strengthen their ability to remove HMs by increasing plant biomass. Meanwhile, AMF-inoculated plants can also alleviate the damage of combined HMs to chloroplast in leaves by restricting the transport to the aboveground parts (Janeeshma &Puthur 2020).
Similar with our result, Ban et al. (2021) revealed that the root vitality of reeds increased significantly under the low concentrations of CuO-NPs-polluted wastewater (50 mg/L), but decreased significantly at high concentrations (500 mg/L). A likely explanation was that plants themselves had the potential to improve their physiological metabolism level by increasing the root vitality to resist combined HMs stress at low concentrations. The widespread underground network between the mycelium and plant roots was beneficial for facilitating nutrient flow between roots, and preventing combined HMs from entering root cells (Alam et al. 2019). However, excessive HMs could destroy the root cell structure, significantly reduce root vitality, and cause obvious physiological and metabolic disorders (Liu et al. 2014).
Meanwhile, the AA not only increased the dissolved oxygen in the rhizosphere microenvironment and improved the vent ability in reed tissues, but also promoted the absorption of nutrients by roots, thereby promoting the growth of the reeds. There was a very marked synergistic effect of AMF inoculation and AA on growth indexes, chlorophyll, and root vitality of wetland plants for treating combined HMs polluted wastewater. Three-way ANOVA analysis also showed that AMF, aeration, combined HMs pollution, and the interaction of AMF with aeration had significant effects on the growth indexes, chlorophyll content, and root vitality (P<0.05) (Table 2).
Table 2
Three-way analysis of variance of AMF inoculation, aeration and combined HMs stress on reeds.
Index
|
AMF
(F Value)
|
Aeration
(F Value)
|
C
(F Value)
|
AMF×Aeration
(F Value)
|
AMF×C
(F Value)
|
Aeration× C
(F Value)
|
AMF×Aeration× C
(F Value)
|
|
Shoot height
|
2928.66***
|
632.59***
|
1310.79***
|
10.35**
|
0.07 NS
|
27.83***
|
31.04*
|
|
Root length
|
2622.60***
|
126.29***
|
415.67***
|
40.96***
|
103.98***
|
18.86***
|
0.77NS
|
|
Aboveground fresh weight
|
300.55***
|
67.99***
|
153.61***
|
7.28**
|
1.15 NS
|
2.86 NS
|
2.81NS
|
|
Underground fresh weight
|
4509.45***
|
130.50***
|
337.32***
|
33.87***
|
61.31**
|
5.52*
|
2.17NS
|
|
Aboveground dry weight
|
601.27***
|
500.25***
|
1849.43***
|
89.10***
|
196.20***
|
94.55***
|
22.85***
|
|
Underground dry weight
|
57081.50***
|
8096.28***
|
920.36***
|
482.69***
|
256.66***
|
334.20***
|
2509.49***
|
|
Root vitality
|
39134704.50***
|
3325620.50***
|
8585079.50***
|
243602.00***
|
398605.50***
|
36639.50***
|
166784.00***
|
|
Chlorophyll
|
12745.40***
|
1674.39***
|
5347.21***
|
26.48***
|
236.40***
|
16.98***
|
102.30***
|
|
SOD
|
6316.69***
|
1271.17***
|
22993.76***
|
79.45***
|
626.93***
|
50.59***
|
106.24***
|
|
POD
|
62721.01***
|
2289.32***
|
175562.61***
|
12.90***
|
1574.04***
|
879.96***
|
124.05***
|
|
CAT
|
3254.36***
|
43.41***
|
21422.46***
|
241.29***
|
697.20***
|
41.62***
|
60.19***
|
|
MDA
|
6212.11***
|
239.08***
|
19795.75***
|
8.87**
|
1059.52***
|
74.87***
|
50.77***
|
|
ROS
|
352.80***
|
57.800***
|
491.79***
|
6.96**
|
59.89***
|
4.96*
|
1.36 NS
|
|
* means P<0.05, ** means P<0.01, *** means P<0.001, and NS means no significant difference. AMF represent inoculation of F. mosseae, C represent combined HMs stress. |
3.2.2. ROS levels, lipid peroxidation and antioxidant response
It can be seen from Table S2 that compared with the CK treatment ("NA+NM"), AMF inoculation and/or AA reduced the contents of MDA and ROS, and the "IA+FM" treatment showed the lowest values at different combined HMs concentrations. Conversely, the reeds SOD, CAT and POD activities of AMF inoculation and/or AA treatments were significantly higher than those of the "NA+NM" treatment under the three concentrations (P<0.05), with an increase of 7.32-176.78%, 3.70-74.88%, and 3.74-40.89%, respectively. Among them, the SOD, CAT and POD activities of reeds in the "IA+FM" treatment were the highest. PCA results showed the plant growth indexes had a strong negative correlation with ROS and MDA, which was consistent with Hu et al. (2020a) (Fig. S2). Those results of hierarchical clustering analysis indicated that inoculation with AMF and/or AA had a positive effect on alleviating oxidative stress and promoting the antioxidant enzyme activities of wetland plants under the stress of combined HMs (Fig. 1).
HMs pollution was one of the abiotic factors leading to oxidative stress in plants, which could induce excessive ROS and lipid peroxidation, resulting in cellular damage and disturbing cellular ionic homeostasis (Dubey et al. 2018). SOD, CAT and POD were the main antioxidant enzymes to remove ROS in plants, which could protect the structure and function of the cell membrane system and maintain the redox state of plant cells. Nafady andElgharably (2018) found that AMF inoculation significantly reduced MDA content when studying the effect of mycorrhizal symbiosis on Zea mays growth under combined HMs (Fe, Mn, Zn, Cu, Cd, and Pb) stress, which was consistent with our results. Similarly, whether in aquatic habitat or terrestrial habitat, mycorrhizal plants had better ROS scavenging ability and antioxidant ability than non-inoculated plants under HMs stress (Hu et al. 2020c, Zhan et al. 2018). In addition, AA had the potential to alleviate oxidative damage by enhancing plant antioxidant capacity and leaf photosynthesis, and upregulate the expression of the genes maintaining cell redox balance (Xiaochuang et al. 2020). Therefore, AMF inoculation and AA synergistically improved the physiological indexes of reeds and increased antioxidant enzyme activities (SOD, CAT, and POD) under the stress of combined HMs. Three-way ANOVA analysis also showed that the interaction of AMF with aeration had significant effects on antioxidant enzyme contents (P<0.05) (Table 2).
3.3. Distribution of Pb, Zn, Cu and Cd in VFCWs
3.3.1. Distribution of Pb, Zn, Cu and Cd in wetland plants
As shown in Fig. 2, the concentrations of Pb, Zn, Cu and Cd in the roots of the reeds were higher than those in the shoots under the low and high concentrations of combined HMs, indicating that HMs were mainly accumulated in the roots of wetland plants. Meanwhile, the concentrations of Pb, Zn, Cu and Cd in the reeds were all increased greatly as the concentration increased. Under the two concentrations of combined HMs, the concentrations of Pb, Zn, Cu and Cd in the roots of AMF inoculation and/or AA treatments were higher than those of the CK treatment ("NA+NM"), particularly, the "IA+FM" treatment showed the highest values, which increased by 28.07-168.98% and 42.83-486.31%, respectively (P<0.05). Conversely, the concentrations of Pb, Zn, Cu and Cd in the shoots of the "IA+FM" treatment at low and high levels were significantly lower than those of the CK treatment, which decreased by 48.80-89.82% and 54.56-76.49%, respectively (P<0.05). Interestingly, the "IA+NM" treatment showed the highest concentrations of HMs in the shoots at high level (P<0.05), which may be related to the improvement of water and nutrients delivery to the shoots with the help of AA owing to the inhibition of transpiration by HMs (Rucińska-Sobkowiak 2016). Meanwhile, the ranking of HMs concentrations in reeds was Cu > Pb > Zn >> Cd. Moreover, it could also be seen from the Table S3 that AMF inoculation and AA deeply increased the BCF and decreased the TF of the reeds.
Similar results were obtained by Han et al. (2021b) and de los Angeles Beltrán-Nambo et al. (2021), AMF played a positive role in promoting the accumulation of HMs in roots and inhibiting their transfer to the shoots. Moreover, Huang et al. (2017) reported that AMF symbiosis with higher BCF and lower TF promoted the tolerance of P. australis to Cd stress compared to the non-inoculated groups, which was consistent with our results. Meanwhile, previous studies also proved that the longer roots of mycorrhizal plants had the potential to increase the accumulation of combined HMs in roots, and combined HMs were preferentially accumulated in mycelium of fungi rather than root cells (Wu et al. 2016). This might be the underlying mechanism that AMF plants were able to increase HMs influx in roots. The differences in the ability to accumulate Pb, Zn, Cu and Cd between the results of the present study and Fritioff andGreger (2006) might be determined by the characteristics of plants and HMs. AA accelerated the absorption and transportation of water and nutrients, improving the growth of plants, which was helpful to strengthen the resistance to combined HMs stress and the transfer of HMs from roots to shoots. What’s more, AA improved the colonization of AMF in plant roots, and AMF colonization led to higher combined HMs accumulation in roots and improved plant physiological status, which could strengthen the ecological function of VFCWs. In summary, AMF inoculation and AA were beneficial for wetland plants to resist combined HMs stress.
3.3.2. Distribution of Pb, Zn, Cu, and Cd in the substrates
As shown in Fig. 3A and B, AMF inoculation and/or AA significantly promoted the adsorption of Pb, Zn, Cu, and Cd in the substrates than those in the CK treatment ("NA+NM") under the low and high concentrations (P<0.05), particularly, the "IA+FM" treatment showed the highest Pb, Zn, Cu, and Cd accumulation in the substrates. Compared with the CK treatment, the concentrations of Pb, Zn, Cu, and Cd in the substrates of the "IA+FM" treatment at low concentrations were 44.31, 32.34, 42.28 and 1.58 mg/kg, respectively, with an increase of 45.40%, 6.52%, 47.20%, and 17.10%, respectively, and the concentrations at high concentrations were 85.47, 105.97, 159.44, and 3.89 mg/kg, respectively, with an increase of 50.86%, 61.30%, 59.49%, and 48.80%, respectively. What’s more, the ranking of Pb, Zn, Cu, and Cd accumulation contents in the substrates was Cu > Pb ≥ Zn >> Cd.
In this study, it can be seen that inoculation with AMF enhanced the adsorption of combined HMs by the substrates. We speculated here that this was related to the fact that AMF could secrete some active matters (e.g. glomalin related soil protein (GRSP)) to chelate the combined HMs in substrates, absorb the HMs ions via their hyphae and change the rhizosphere microbial community (Ma et al. 2019, Wang &Feng 2021, Wang et al. 2020). GRSP released by AMF via mycelium and spore walls was water-insoluble glycoprotein, performing a remarkable role in chelating HMs (Wu et al. 2014). It was confirmed that AMF inoculation presented a noteworthy decrease of Cd, Pb, Zn, and Cu concentrations due to the release of GRSP by Nafady andElgharably (2018). Meanwhile, AA increased the oxygen content in the VFCWs, leading to the growth of microorganisms on the surface of the substrates and roots to form more biofilms, which can produce more extracellular polymeric substances (EPS) to remove combined HMs through chelation, surface precipitation, and ion exchange (Li &Yu 2014, More et al. 2014, Riaz et al. 2021). What’s more, AA could also increase the frequencies of mycorrhizal symbiosis, thereby promoting the HMs adsorption of the substrates through the potential mechanisms mentioned above. Under the two concentrations of combined HMs, the adsorption difference of Cu, Pb, Zn and Cd might be attributed to the pH, redox potential, different chemical composition, and structure of the substrate material. For example, Hernández-Montoya et al. (2013) reported that zeolitic showed a selectivity in the adsorption process of Pb, Zn and Cd, and the ranking was Pb > Zn > Cd.
3.3.3. Removal rates of Pb, Zn, Cu, and Cd under different treatments
It can be seen from Fig. 4A that the removal rates of Pb, Zn, Cu and Cd of the four treatments all exceeded 80% at low concentrations, and those of the CK treatment ("NA+NM") were 95.17%, 80.36%, 91.66% and 80.68%, respectively. Among the four treatments, the removal rates of the "IA+FM" treatment were the highest, being 98.49%, 92.64%, 97.72%, 92.06%, respectively, increased by 3.48%, 15.28%, 6.61%, 14.11%, respectively, comparing with the CK treatment. At high concentrations, the removal rates of Pb, Zn, Cu, and Cd were lower than those at low concentrations. Comparing with the CK treatment, the removal rates of the AMF inoculation and/or AA treatments increased by 7.84-22.73%, 16.34-30.31%, 5.62-12.63%, 27.88-50.22%, respectively (Fig. 4B). At high concentrations, AMF inoculation and/or AA could significantly improve the removal efficiencies of Pb, Zn, Cu and Cd (P<0.05). However, AMF inoculation and/or AA could significantly improve the removal rates of only Zn and Cd at low concentrations (P<0.05).
The Zn and Cd removal rates of the inoculated treatments ("IA+FM", "NA+FM") were significantly higher than that of the CK treatment under the low concentrations (P<0.05), but not Pb and Cu. This may be due to the fact that the removal rates of Pb and Cu had been at a very high level (more than 90%), which made the role of AMF not obvious in promoting the performance. However, with the increase of combined HMs concentration, the adsorption efficiency of the substrates would be lower, and the toxicity of HMs to plants would be greater. Therefore, compared with the CK treatment, the addition of AMF had a more obvious effect on the removal of Pb, Zn, Cu and Cd at high concentrations than that at low concentrations, with an increase of 11.34%, 20.03%, 5.62%, 32.62%, respectively. Gunathilakae et al. (2018) found that AMF inoculation showed a more obvious effect on the removal of the high Cd level (50 ppm) than the low Cd levels (0, 5, 10, and 20 ppm) when evaluating the role of AMF on the phytoremediation potential of water hyacinth in Cd uptake, which was consistent with our result.
3.3.4. Distribution of Pb, Zn, Cu, and Cd in subcellular components
As shown in Fig. S3, Pb, Zn, Cu and Cd were mainly distributed in the cell wall, especially for the underground part of the reeds. The proportions of Pb, Zn, Cu, and Cd in the cell wall in the aboveground part of the reeds under the two concentrations were 30.39-73.93%, 46.74-58.43%, 43.90-72.73% and 35.23-44.71%, respectively, and those of the underground part of the reeds were 71.85-92.44%, 70.27-88.56%, 72.42-90.11% and 48.40-69.07%, respectively. As the concentration of combined HMs increased, there was a decline in the proportions of Pb, Zn, Cu and Cd in the cell wall in the aboveground part of the reeds, while a contrasting tendency was found in the underground part, except for "IA+FM" treatment. For the aboveground and underground parts, the Pb, Zn, Cu, and Cd proportions in the reeds cell wall of the "IA+FM" treatment were higher than those of the CK treatment ("NA+NM") at low concentrations. The cumulative concentration of single Pb, Zn, Cu, Cd in the aboveground and underground parts of the reeds all was cell wall > organelle ≥ soluble component.
The different types and concentrations of HMs induced a distinct subcellular combined HMs distribution. The components of cell wall (e.g. polysaccharides and proteins) provided a large number of retention sites to bind HMs and restricted their transmembrane transport, leading to a lower toxicity than the free state of that in soluble component and organelle (Han et al. 2021a, Mwamba et al. 2016). Gao et al. (2021) revealed that AMF inoculation improved the ability of the plant cell wall in fixing Cd by increasing the cell wall components, resulting in more Cd fixed in the cell walls, which was consistent with our study. More importantly, the fixation of HMs by cell wall was considered as the first barrier to protect protoplasts from the toxic effects of HMs in plants, while the mycorrhizal plants could promote the deposition of HMs on the plant cell walls and reduce the distribution in organelles, thereby alleviating the damage of HMs to the host plant organelles (Huang et al. 2018). Additionally, Fritz (2007) reported that the HMs ions affinity in pectin (a component of cell wall) showed a difference depending on the characteristics of them (Al3+ > Pb2+ > Cu2+ > Co2+ > Ni2+ > Zn2+ > Cd2+ > Ca2+), which might account for the differences in the distribution of Pb, Zn, Cu and Cd in subcellular components in our study. According to the result of Considine andFoyer (2021), oxygen may be as a signal for plant growth, which affecting the root and shoot growth and structure, resulting in different distributions of combined HMs in plant subcellular. This study provides sufficient evidence on a subcellular level to illustrate how AMF inoculation and AA benefitted combined HMs tolerance.
3.3.5. Localization of Pb, Zn, Cu, Cd in ultrastructure of mycorrhiza
The normal root cells of reeds were presented in Fig. 5A (e.g. i: nucleolus, h: nucleus, g: cell membrane) applying transmission electron microscopy (TEM). However, under the stress of combined HMs, the root cell morphology of reeds not inoculated with AMF changed greatly, such as deformation of cell wall (k) and helical curl of cell membrane (l) (Fig. 5B). In addition, there were many thickened cell walls (j) and compartmentalized structures about 2-3 µm in diameter in reeds inoculated with AMF under the stress of high combined HMs concentrations (Fig. 5C, D, E, F). Meanwhile, there were many black deposits (a, b, c, d, e, and f) in cell walls and cell interiors, which were suspected to the Pb, Zn, Cu, and Cd. Interestingly, the mycelium of AMF penetrating the cell wall (red circle) of reed from one cell to another were observed (Fig. 5F), which showed the process of AMF infecting plant cells. Additionally, Energy Dispersive Spectrometer (EDS) was conducted for those selected black deposits (a, b, c, d, e, and f), and the results were shown in (Fig. S4). The presence of Pb, Zn, Cu and Cd peaks indicated that the added HMs ions entered reed cells (a, b, c, d) and AMF structures (e, f). Since the samples were organic matter and the sample holder was made of aluminum material, there was no doubt that the C, O and Al elements were also identified.
TEM can be used to observe the injuries to different organs of plants in response to combined HMs stress. In this part, we can clearly observe how AMF-inoculated plants resist HMs damage at the cellular level. Huang et al. (2018) found that the organelle integrity of plant cells became worse with the increase of Cd concentration applying TEM, while the root cell structure of plants inoculated with AMF were more complete. In addition, Abdel-Fattah et al. (2011) and Xu et al. (2018) found that increasing cell wall thickness and altering subcellular compartmentalization by AMF was a significant mechanism for plants detoxification to HMs, which was consistent with our observations. Meanwhile, a similar result reported by González-Guerrero et al. (2008) that Zn, Cu and Cd also could be accumulated in the cell walls of AMF when these HMs were added to the extra-root mycelium.
3.3.6. Combined HMs mass balance
As shown in Fig. 6, we found that substrate was the main component for HMs removal in these VFCWs, accounting for more than 50%, which was consistent with the results of Ban et al. (2021). Under the two concentrations, the proportions of combined HMs in the plants of the AMF inoculation and/or AA treatments were higher than those of the CK treatment ("NA+NM"), with the following ranking order: "IA+FM" > "NA+FM" > "IA+NM" > "NA+NM". Besides, the proportions of combined HMs in the roots of "NA+FM" treatment (more than 85%) were higher than those of the CK treatment at the low and high concentrations. Conversely, compared with the CK treatment, the proportions of combined HMs in the water of the AMF inoculation and/or AA treatments under the two concentrations decreased by 34.11-64.34% and 15.44-30.63%, respectively. The proportion of combined HMs in water of the "IA+FM" treatment under the two concentrations was the lowest among the four treatments at 4.60% and 27.40%, respectively.
The removal rates of combined HMs in VFCWs were closely related to the synergistic effects of the plants, microorganisms and substrates. Under the stress of combined HMs, AMF decreased the combined HMs concentration in water by improving the plants uptake, promoting that absorbed in substrates, which was consistent with Riaz et al. (2021). In this study, the combined HMs contents of the inoculated treatments in substrates were lower than that in the CK treatment at low concentrations, which was not consistent with Hu et al. (2021). The reason might be that the wetland plants adsorbed more HMs due to the low concentrations and toxicity of combined HMs, thus resulting in the reduction of combined HMs in the substrates. Besides, AMF accumulated HMs in their intra-radical and extra-radical mycelium, thus resulting in lower HMs contents in the water. Wu et al. (2016) showed that the extraradical mycelium of AMF could absorb and transport Cr to mycorrhizal roots from a distance, which could contribute to promoting the ability of the wetland system to purify HMs-polluted wastewater. In addition, the effect of AA on the mass balance of combined HMs in aquatic habits was mainly related to the enhanced microorganisms activities and improved HMs uptake of wetland plants. Therefore, we found that AMF inoculation and AA showed significant synergistic effect on the removal of combined HMs from VFCWs.