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Article

Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity

by
Maria Androudi
1,2,
Vasiliki Liava
1,
Eleni Tsaliki
2,
Ioannis Ipsilantis
1 and
Evangelia E. Golia
1,*
1
Laboratory of Soil Science, School of Agriculture, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
2
Hellenic Agricultural Organization DIMITRA (ELGO Dimitra), Institute of Plant Breeding and Genetic Resources, 570 01 Thessaloniki, Greece
*
Author to whom correspondence should be addressed.
Soil Syst. 2024, 8(3), 100; https://doi.org/10.3390/soilsystems8030100
Submission received: 22 July 2024 / Revised: 10 September 2024 / Accepted: 14 September 2024 / Published: 16 September 2024
(This article belongs to the Special Issue Research on Heavy Metals in Soils and Sediments)
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Abstract

:
Although the phytoremediation strategy has been studied worldwide, little research data are available regarding the influence of mycorrhizae on the phytoremediation capacity of various plants grown in Cd-contaminated soils in Mediterranean environments. Therefore, a pot experiment was carried out to study the possible effectiveness of hemp plant (Cannabis sativa L.) in the remediation of moderately and heavily Cd-contaminated soils and additionally to quantify the effect of Cd on Arbuscular Mycorrhizal Fungi (AMFs). For this purpose, an alkaline clay soil collected from the Farm of Institute of Plant Breeding and Genetic Resources (North Greece) was contaminated with two levels of Cd (3 and 30 mg Cd kg−1, corresponding to Levels A and B, respectively—first factor) at two incubation times (10 and 30 days—second factor) and six treatments (Control_30d, Control_10d, CdA_30d, CdB_30d, CdA_10d, CdB_10d) were created. Soil Cd concentrations, both pseudo-total and available to plants, were determined after extraction with Aqua Regia mixture and DTPA solution, respectively, before and after the cultivation of hemp plants and after the harvesting. Cd concentrations in the aboveground and underground plant parts were also estimated after digestion with Aqua Regia, while root colonization by AMFs was determined with a microscope. The highest plant’s Cd concentration, more than 50%, was observed in its underground part, at all Cd-contaminated treatments, indicating a strong capacity for cadmium to gather up in the roots. Among different Cd levels and incubation days, significant differences were recorded in the rates of root colonization by AMFs. Among different Cd levels and incubation days, 3 mg Cd Kg−1 soil promoted AMF root colonization, particularly at 10-day incubation, while 30 mg Cd Kg−1 soil diminished it. Colonization was lower with longer incubation times at both levels of Cd. Hemp appears to be a viable option for phytostabilization in Cd-contaminated soils, enabling further utilization of AMFs to assist the phytoremediation process.

1. Introduction

Consistent with the rapid advancements of today’s society, the volume of waste being generated worldwide is steadily growing [1], leading to the incremental contamination of natural habitats with a wide array of both organic compounds and inorganic elements that typically do not occur in nature [2]. Heavy metal (HM) presence in soil is becoming more and more prevalent at elevated levels, creating considerable environmental and public health hazards [3]. This is mainly attributed to the fact that HMs are not subjected to biodegradation; thus, they can persist in the environment for extended periods, and they tend to build up in various segments of the food chain [4,5]. Khan et al. [6] reported that HMs might cause long-lasting detrimental effects on the vitality of soil ecosystems and negatively affect soil biological functions. At high levels, HMs can negatively impact microorganisms’ growth, structure, and metabolic functions, reducing the functional diversity within soil ecosystems [7].
Cadmium (Cd) stands out as a significant hazardous heavy metal in the environment and has detrimental impacts on all kinds of organisms [8,9], arising from diverse industrial and agricultural activities such as mining, metallurgy, pigment production, plastic stabilization, sewage sludge, manure, straw, fertilizer application, and nickel–cadmium battery manufacturing [9,10,11,12,13]. Its prolonged biological persistence renders it a cumulative toxin, causing adverse effects on soil microbial communities and susceptible plants (something that mainly depends on the factors affecting their bioavailability), thus ultimately affecting human health [14,15]. Cd, when discovered in soils, due to metal aging and soil characteristics may cause a change in the geochemical dispersion pattern [16]. In terms of weathering, it has been established that metals deposited slowly in soils over a long time tend to be least bioavailable [17]; the main reason behind this is that the metals move over time to inaccessible residual soil reservoirs (e.g., enclosed in Fe and Al oxides or in poorly exchangeable interstitial nodules of phyllosilicate clays) [18]. Regarding the properties of soils, the most important ones that affect metal uptake in plants are soil pH, percentage of CaCO3, redox potential, clay content, cation exchange capacity, organic matter, and mineral composition [19,20,21].
Arbuscular Mycorrhizal Fungi (AMFs) are some of the most widely known beneficial soil microorganisms, the key component of the rhizosphere. They form symbiotic relationships with most higher plants [22]. The mycorrhizal association has a crucial part in natural ecosystems by intracellular colonization of plant roots [23,24,25]. This interaction improves land assets, contributing to the effective management of environmental constraints [26,27,28,29]. Fungi strengthen the host plant’s ability to assimilate both nutritional elements and water, and in exchange the host plants supply fungal carbohydrates for their own expansion [30,31]. AMFs also provide essential minerals to plants [32] and enhance soil chemical and physical characteristics, serving as barriers to prevent the entry of harmful xenobiotics through their fungal networks [33]. Consequently, mycorrhizae function as a protective layer, forming a shield around the plant roots they inhabit [34]. Furthermore, it has been demonstrated that AMFs significantly increase plant resilience against both biotic and abiotic stresses, including the presence of heavy metals [35]. Given their crucial role as an intermediary between the earth and plant roots, they play an essential part in managing the presence and impact of heavy metals on plants [36]. AMFs have been reported to minimize the hazardous impacts of HMs, improving the plants’ resistance through different regulatory systems [37], like changing the chemical synthesis of the rhizosphere heavy metal-contaminated soil, enhancing plant nutritional status, and having a direct effect on the absorption and buildup of HMs [32,38,39].
In the last decades, the main and most suitable control technique used to eliminate HMs from the soil environment is phytoremediation [40]. This is an eco-friendly, economically efficient method, producing few side pollutants [41]. Phytoextraction and phytostabilization methods of phytoremediation seem to be well studied worldwide [42]. During phytoextraction, the plants used can build up the toxic elements in their overground parts by extracting metals from the soil [43,44]. Conversely, the second method involves immobilizing the HMs within the plant roots [42]. These mechanisms are used alone or in association between plants, as appropriate to the pollutant nature [38].
Industrial hemp (Cannabis sativa L.) is primarily cultivated for its fiber [45], seed production [46], medicinal purposes [47], and biomass for energy generation [48]; it presents highly favorable prospects as a phytoremediator. It exhibits rapid biomass growth and outperforms typical hyperaccumulators in heavy metal absorption, making it a valuable resource for environmental restoration [49]. C. sativa L. has a long radical system, keeps out of the trophic chain, provides easy cultivation and management, and has numerous other exceptional features [50]. It has been shown that mycorrhizal hemp may accumulate more Cd in the roots and may tolerate higher Cd concentrations [51], particularly with AMF inoculum with origins from a Cd contaminated area [52]; research has found that mycorrhization enhanced translocation of Cd from hemp root to shoot [53]. Inoculation with AMF combined with protein hydrolysate increased shoot Cd uptake [54]. Additionally, unlike most bioenergy plants, hemp provides the opportunity for contributing to a circular economy pattern [2]. These findings are therefore encouraging, yet little research data are currently available concerning the impacts of mycorrhiza on the phytoremediation capacity of various plants grown in Cd-contaminated soils in a Mediterranean environment with distinct climatic characteristics.
Considering these issues, this study aimed to investigate the cultivation of C. sativa, a plant species that could be utilized after phytoremediation, in Cd-contaminated soil.
Moreover, the aim was to study the impact of Cd stress on hemp’s root colonization by AMFs and additionally the influence of AMFs on the plant’s ability to tolerate and accumulate Cd. Thus, the objectives of this work were to evaluate (a) the effect of different Cd levels and incubation periods on the colonization of hemp (C. sativa L.) roots by AMFs, (b) the correlation between AMF colonization and the growth and resistance of hemp plants, and (c) the survey of the potential relations or interactions between Cd distributed in plant parts and Cd impact on AMF colonization using the proper statistical tools.

2. Materials and Methods

2.1. Sampling and Experimental Design

The soil sample (depth 0–20 cm) used for the experiment was collected from the Farm of the Institute of Plant Breeding and Genetic Resources (ELGO-DIMITRA) located at Thermi, Thessaloniki, in North Greece. Specifically, for the examined sample, six subsamples with the help of a wooden spade were taken from a roughly 150 cm radius area [55]. Composite surface soils were specifically developed to reduce the amount of analyses meant to provide a representative sample, which is necessary for replicates to be conducted [56]. The sample was then transported to the Soil Laboratory at the Aristotle University of Thessaloniki, where it was air-dried and put through a sieve with <2 mm pores in order to be subjected to various physicochemical analyses (pH, EC, CaCO3, particle size distribution, organic carbon content, available and pseudo-total Cd concentrations) [57,58].
The experimental design included one control (0 mg Cd kg−1) and two levels of Cd—first factor (3 and 30 mg Cd kg−1) at two incubation times—second factor (10 and 30 days). Six treatments with ten replicates per treatment were created. The treatment coding was outlined as shown in Table 1. The European Union’s Directive 86/278/EEC was the base on which the Cd concentrations were chosen. As a result, Level A represents moderately contaminated soils, while Level B is analogous to heavily contaminated soils [59].

2.2. Artificial (Laboratory) Contamination of Soil Sample and Pot Experiment

The soil sample was chosen to have an undetectable Cd concentration so that contamination could be carried out in a laboratory. To achieve this objective, appropriate calculated volumes of solutions of cadmium nitrate were evaluated and incorporated into the soil samples of each treatment at two levels (A and B), respectively, contaminated with 3 and 30 mg Cd kg−1. The Cd solutions were applied to the soils by spraying, and afterwards the mixture was manually stirred for 10 min. After mixing, the bags remained closed throughout the two periods of incubation time (for 10 and 30 days), during which the moisture content was kept around 70% with occasional aeration and dehumidification. Following the proper incubation period, the soil samples were placed into the pots, and seeding was carried out with the Felina 32 variety. Felina 32 is a monoecious variety of French origin well adapted to the Mediterranean climate, usually cultivated for seeds or fiber production. Furthermore, Fellina 32 is registered in the European Catalogue with Δ-9 tetrahydrocannabinol content <0.3% as required by the European legislation. The main factor for the choice of this variety was its height (around one meter) that can be considered ideal for an experiment in pots [60]. In total, 30 pots with a capacity of 7 L each were used for the experiment, in each of which 10 seeds of the variety were placed around the perimeter, early in May 2023 (on 2 May 2023). One month after sowing (on 1 June 2023), thinning was carried out to allow 3–4 female plants of similar size in each pot to grow without competition. The harvest was carried out during Stage 2305 of plant growth according to the UPOV protocol [61], early in August 2023 (on 8 August 2023) while flowering was completed and seed ripening began. From May to August, the pots were kept outdoors and automatically watered. The experiment was conducted without fertilization. Meteorological data from the growing season (April to August) are presented in Table 2 and are typical for Mediterranean climates [3,56].

2.3. Soil Chemical and Physical Analysis

After harvesting, soil samples from the pot experiments were subjected to physical and chemical analysis, as previously mentioned, according to Dimirkou et al. [58] and Golia [57]. Both electrical conductivity (EC) and soil reaction (pH) were calculated with the use of a 1:1 mixture of soil and water. For particle size distribution, the Bouyoukos method was used, while for the soil organic matter (OM) content appraisal, the modified Walkley–Black method was utilized, as described in Page [62]. With the use of a Bernard–Scheibler calcimeter, the appraisal of CaCO3% was performed. Soil cadmium concentrations, pseudo-total and available to plants, were determined both before the cultivation of hemp plants and after harvesting. Available concentration of Cd was determined with DTPA solution (1:2 diethylene-triamine-pentaacetic acid pH 7.3) [57], while the pseudo-total concentration of Cd was determined with Aqua Regia (HCl:HNO3 3:1 digested at 180 °C for 2 h [63]. All reagents used did not contain impurities (Merck, Darmstadt, Germany).
Atomic absorption spectrophotometry (AAS) was used to measure the Cd concentrations, utilizing either the flame (F-AAS) or graphite furnace (GF) technique. The analytical recovery was validated using Standard Reference soil material 2710 (National Institute of Standards and Technology, Gaithersburg, MD). The recovery ratio of standards ranged from 89.3% to 104.4% and 88.1% to 103.7% for Cd in soil and plant, respectively.

2.4. Plant Analysis

After harvesting, both the belowground and the stem and leaf parts of C. sativa were gathered to carry out the plant’s analyses. For this purpose, first, the aboveground parts were deposited in numbered bags made of paper, and the weight with water content of each plant part was measured. The roots were previously cleaned 2 to 3 times with the help of deionized water to remove the soil attached to them. A small weighted portion of the root (~0.5) was taken for the determination of AMF root length colonization. The specimens were subsequently heated at 60 °C for 48 h and weighed again when they reached the water concentration that could not be extracted with this method. Then, they were pulverized with the use of an electric grinder and/or porcelain mortar, and the pulverized samples (aboveground and roots) were stored for chemical analyses. A 3:1 mixture of concentrated acids of HCl and HNO3 was used for the Aqua Regia solution extraction method [63], which was conducted for Cd evaluation in the plant aboveground and underground parts.

2.5. Quantification of AMF Colonization in Roots: Clearing and Staining Roots

The following procedure was followed to quantify the root colonization by AMF [64]. Washed clean roots were placed in 10% KOH and remained there for 40 min at 80 °C. Then, after rinsing the KOH about 5 times with deionized water and acidifying the solution, the roots were stained with a 0.05% w/v trypan blue solution (1:1:1 v/v/v H2O: glycerin: lactic acid). Then, in order to observe and measure the colonization, slides were prepared by placing roots in PVLG (Polyvinyl-Lacto-Glycerol) on the slide. The percentage of AMF root length colonization was appraised under a microscope following the method described by McGonigle et al. [65] at ×100 magnification (400× for details).

2.6. Statistical Processing of Data

The data were recorded and subsequently analyzed statistically using appropriate statistical packages (Sigmaplot12 and Microsoft Office Excel, SigmaPlot 12 (Systat Software, San Jose, CA, USA) and Excel (Microsoft 365 Version 2408)). Prior to analysis, the normality test (Shapiro–Wilk) and the equal variance test were conducted to ensure normal distribution and homogeneity of variance. Then, a two-way analysis of variance (ANOVA) was conducted to evaluate the effect of Cd levels on contaminated soil (Factor A), the incubation times (Factor B) and their interaction effect (A × B) on Cd accumulation in plant parts, the dry weight of aboveground and underground parts, and the AMF colonization in roots. The differences between means were distinguished by Fisher’s least significant difference (LSD) test at p = 0.05.

3. Results and Discussion

3.1. Soil Characterization

Table 3 shows the physicochemical properties of the soil sample used in this study. These values are the averages of ten replicates. The studied soil was alkaline (pH > 7) and calcareous (CaCO3 = 13.24%) with low organic matter (2.9%) which is typical of Mediterranean climates [66]. Soil had high clay content (56% clay) and high electrical conductivity (1082 μS/cm). Thus, the soil sample had a clay–clay texture based on the categorization of soils after the determination of the percentages of sand, silt, and clay by the Bouyoukos method [62].
Alloway et al. [20] noted and illustrated in their experiments that soil reaction and soil texture are determinant factors in the physicochemical properties of the soil environment. Indeed, reports indicate that some soil types can significantly diminish metal availability to plants; these soils usually have a higher pH, which makes metal cations less soluble [18], causes higher content of CaCO3 [67] and clay minerals [60,68]. In this study, the CaCO3 rates are completely linked to soil pH values.
Plant uptake and bioavailability of Cd is negatively correlated with pH [67,69]. In addition, the presence of divalent cations in the soil solution, such as Ca2+ and Zn2+, may increase Cd bioavailability due to competition for absorption sites and competing ion activities [70,71]. Napoletano et al. [72] and Papadimou et al. [56] confirmed their observations and experimental results on soils with elevated electrical conductivity, primarily due to human activities. In this study, the soil samples exhibited high electrical conductivity values, indicating a significant presence of salts in the soil solution [20,73]. The percentage of organic matter suggests that the soil samples are representative of typical agricultural soils from the area of Thessaloniki. Golia and Diakoloukas [74] examined a large number of soils from agricultural regions spanning four soil orders and found that organic matter values typically fall below 3%, a pattern also observed in the current study.

3.2. Pseudo-Total and Available Concentration of Cd in Soil Samples

In Figure 1 and Figure 2, the pseudo-total and available Cd concentrations in soil samples are presented for 10 and 30 days of incubation, respectively, before (Initial) and after (Final) hemp cultivation in each treatment. It is evident from both figures that the control treatment, where no Cd was applied, exhibited undetectable levels of Cd concentrations. Conversely, in Cd treatments, measurable concentrations of Cd were observed across all treatments (CdA_30d, CdB_30d, CdA_10d, and CdB_10d). Comparing treatments with Cd at Level A (3 mg kg−1) to those at Level B (30 mg kg−1), considerably higher values were noted in the latter, indicating the pronounced impact of escalating Cd concentrations on extracted metal levels. Additionally, the influence of different incubation durations (10 and 30 days) on the Cd pseudo-total concentrations is noteworthy. Specifically, a shorter 10-day incubation period in the treatments with Cd at Level B resulted in lower pseudo-total Cd concentrations compared to the 30-day incubation period.
As expected, the minimum concentration of Cd extracted from the soil was found in the control where no Cd was applied, while in the other treatments, higher values were recorded in the following order: Level B > Level A > Control. Moreover, as presented in Figure 3, there was a significant positive correlation between the pseudo-total and available Cd concentrations, as well as between the initial and the final Cd concentrations in the soil. This finding is in agreement with the experimental results of Marković et al. [75], who reported that with increasing soil Cd concentration, the concentrations of Cd in all soil fractions increased. After harvesting the plants, however, it was observed that both pseudo-total and available Cd concentrations extracted from the soil of each treatment were lower. This result indicates that a part of the added Cd concentration was probably taken up by the hemp plants so that the concentrations that remained in the soil after harvesting the plants were lower than those extracted before plant cultivation [58]. From the Cd available concentrations, however, it was indicated that the concentration of Cd, which became available to the plants, was not influenced significantly by the incubation time. In contrast to our results, Marković et al. [75] reported in their study that prolonged incubation time (2 months) was an important factor in Cd stabilization in the investigated soils. Our different results may be attributed to the fact that in our study, the longer incubation period was only 1 month. Lu et al. [76] similarly reported that as incubation time increased (>30 days), Cd was barely transformed from its exchangeable condition to other types. Based on the present work, it is possible to conclude that even with a short-day exposure to Cd pollution, the available concentration, i.e., that which can be taken up by plants and cause potential risks to the health and functionality of organisms, appears to be the same as that corresponding to a threefold action time. Therefore, the aging or senescence of contamination is a way of limiting the possible risks, leading to appropriate decisions for the proper management of contaminated soil systems [17].

3.3. Cd Levels in C. sativa Cultivated in the Contaminated Soil Samples

When the experiment was completed, Cd concentrations were measured across cannabis plant parts to assess Cd uptake. Figure 4 presents the Cd concentration absorbed by cannabis roots and the aboveground plant part. As expected, in the control treatment, Cd concentration was undetectable in both plant parts. Conversely, a clear dose-dependent response was evident in the soils contaminated with Cd. Comparing treatments with Cd Levels A (3 mg Cd kg−1) and B (30 mg Cd kg−1), it is evident that the latter exhibited markedly higher Cd concentrations in each plant part as there is a positive correlation between the Cd concentrations in soil and plant parts (Figure 3). This observation aligns with similar studies conducted on various industrial hemp varieties, suggesting that as Cd concentrations in the growth medium increase, so does Cd accumulation in plant tissues [77,78]. Interestingly, varying incubation durations between treatments did not appear to significantly affect this parameter, as Cd uptake levels remained relatively consistent, with a slight upward trend noted for a 10-day incubation period. Considering the fact that at the shorter incubation time, an increasing trend was observed in the available Cd concentration in the soil, i.e., in the concentration extracted with the DTPA solution, it is understandable that the direct availability of water-soluble Cd artificially added to the soil samples of the experiment is directly taken up by the hemp plant. The low but increased concentration noted in both the roots and overground plant parts indicates the tendency for the metal to move towards soil fractions that make it less available as time progresses [56].
Among the different plant tissues, the roots accumulated the highest concentration of Cd, while significantly lower values were recorded in the aboveground parts of the plant.
In the following Table 4, the values of the Bio Concentration Factor (BFC), the ratio of Cd concentrations in hemp parts to Cd soil levels, are presented. The values of the calculated BFCs are significantly higher in the underground than in the aboveground part of hemp. Moreover, the greatest values were recorded in the lower level of Cd, especially after 10-day incubation.
This outcome is consistent with prior research on Cd bioaccumulation in cannabis plants, which reported that the highest metal concentrations were typically found in the underground part [53,60,79]. However, Shi et al. [80], in their investigation of the cumulative capacity of 18 different industrial hemp varieties in soil contaminated with 25 mg kg−1 of Cd, found that metal bioaccumulation varied among the different varieties. These findings are particularly encouraging, as they indicate that hemp, specifically the Felina 32 variety, tends to accumulate Cd in its roots. This characteristic allows for the utilization of its aboveground biomass, which holds significant economic importance for industrial purposes. Vasilou and colleagues [60] established that cultivating industrial hemp in Mediterranean soils suffering from anthropogenic cadmium pollution is an outstanding way of highlighting the important properties of the plant, as it can retain the pollutant in the soil, immobilizing it in the root, without allowing it to move to the upper parts of the plant from which cannabinoids (CBDs) are produced. Thus, hemp, it turns out, is an excellent plant that can successfully scavenge the soil, can produce the precious CBD, and can also provide its almost cadmium-free stems in order to produce ropes or other kinds of industrial products. Similarly, in Silybum marianum L., researchers Papadimou et al. [56,59] observed that there is potential for the exploitation of thistle weed for two important reasons: first, it can be directly used to recover soils contaminated not only with Cd, but also with Zn and Cu, yet simultaneously it can produce, unhindered, the human health valuable substance silymarin which is found in the seeds of the plant, even though it is grown in extremely contaminated soils, without deteriorating its quality.

3.4. Roots and Aboveground Plant Biomass

In Figure 5 and Figure 6, the dry biomass of the roots and aboveground parts, respectively, of C. sativa cultivated in soils with two different Cd levels and incubation periods are presented. A notable increase in plant biomass when the incubation period extended to 30 days was observed compared to the control at both Cd levels (A: 3 mg kg−1 and B: 30 mg kg−1). Conversely, for treatments with a 10-day incubation period, a slight downward trend in plant dry weight was determined after comparison to the control, but it was not statistically significant.
These findings suggest that Cd incorporation in soil at both levels did not adversely affect the produced plant biomass. Moreover, as presented in Figure 3, there are no correlations between the Cd concentration in soil and the dry weight of aboveground or underground parts. Thus, hemp can thrive under these Cd concentrations without experiencing degradation in biomass. This outcome aligns with the findings of Linger et al. [81], who noted no significant differences in hemp root dry weight when plants were exposed to Cd concentrations, even at notably high levels (up to 100 mg Cd kg−1). However, they did observe a significantly negative impact on leaf and shoot biomass when Cd concentrations exceeded 50 mg kg−1 in the soil. Similarly, Shi and Cai [82] conducted pot experiments investigating hemp growth at Cd concentrations ranging from 50 to 200 mg kg−1. They found that root growth was only suppressed in the treatment with 200 mg Cd kg−1. It is worth noting that previous studies have demonstrated a reduction in biomass for other plant species as Cd concentrations increased [83]. Different plants react variably to heavy metal stress, with their resilience and tolerance contingent upon the species’ ability to activate molecular mechanisms [84]. Plant development parameters are being used as appropriate biomarkers of trace element toxicity. In particular, within the context of phytoremediation, when high levels of biomass are needed, substantial decreases in plant growth determine the plant tolerance limits and thereby the perspectives of the potential candidate crop species. Thus, several plants have displayed a unique ability to develop defense mechanisms for withstanding the stress of heavy metals. These mechanisms include metal removal, scavenging, refining, sequestration, and accumulation to reduce the effects of metal toxicity. Proteomics has become quite a strong tool in biotechnological research to determine how plants respond or react to heavy metal pollution. A deeper investigation of the molecular mechanisms in plants involved in metal removal and pressure reaction could be a subject of future research [85,86].

3.5. Effect of Different Levels of Cd Concentration on AMF Colonization Rates (%) at Different Incubation Times

Figure 7 presents the colonization rates (%) of cannabis roots by AMF across the six treatments of the experiment. The addition of 3 mg kg−1 of Cd (CdA_30d and CdA_10d treatments) exhibited significantly higher colonization rates, within the 60–70% range, compared to the control treatments. Conversely, in the treatments where a higher concentration of Cd (30 mg kg−1) was added (CdB_30d and CdB_10d treatments), lower colonization rates were observed than with 3 mg kg−1 Cd, ranging from 35 to 55%. According to Figure 3, there was a negative relationship between the AMF colonization and the Cd concentration in the soil, owing to the values recorded at the higher level of Cd, although the correlation was not statistically significant. Also, notably, a comparison between the 30-day incubation treatments and the 10-day ones revealed that in the former case, the colonization of the roots was significantly lower.
To better describe the colonization of cannabis roots by AMFs, in Figure 8, observations from the microscope of cannabis blue-stained roots are presented. Specifically, in Figure 8a, the hyphae are illustrated, extending within the root and showing branching, while in Figure 8b,c, the intraradical vesicles and the arbuscules within the roots produced by AMF are presented, respectively.
These results indicate that the rate of colonization depends on the level of Cd added to the soil. This observation is consistent with the findings of Liu et al. [32], who demonstrated that the colonization rate of corn roots (Zea mays L.) varied between 30.3% and 72.3% across different Cd levels in the soil. Specifically, in our study, the addition of Cd at Level B (30 mg kg−1) led to a decrease in root colonization rates, contrasting with the significant positive effect observed when Cd was added at Level A (3 mg kg−1). According to Sun et al. [51], the increased colonization of cannabis roots by AMFs could be attributed to Cd presence in the rhizosphere, which may stimulate endoderm and root epidermis growth and promote secretion release, enhancing symbiosis between plants and fungi. Their study indicated that Cd presence in the soil enhanced the colonization of cannabis roots by fungus Rhizophagus irregularis. Therefore, an increase in the mycorrhizal population induced an increase in the ability of industrial hemp to accumulate soil cadmium, contributing to the remediation of mainly moderate soils. In the case of heavily contaminated soil, Molina et al. [87] found that applying 0.7–1.2 mg of available Cd kg−1 to soil did not adversely affect the colonization of soybean roots by Arbuscular Mycorrhizal Fungi. However, the findings of Kanwal et al. [88] suggested a negative impact of Cd addition on the colonization of alfalfa mycorrhizal roots (Medicago sativa L.), with a downward trend observed as Cd concentration in the soil increased. Citterio et al. [53], working with cannabis and high Cd soil levels, showed a decrease in mycorrhizal colonization. Sun et al. [51], who worked with cannabis, AMFs, and a range of Cd soil concentrations, did not report colonization but did show positive effects of AMFs on hemp growth and Cd stress alleviation.
In addition to the varying cadmium levels, different incubation periods were also found to have a notable effect on root colonization, with significantly higher values observed for the 10-day duration, indicating lower Cd toxicity relative to the respective 30-day incubation at both Cd levels. This result is in line with the findings of Hassan et al. [7], who examined the impact of cadmium (ranging from 0 to 200 mg kg−1) on microbial biomass over 10-, 20-, and 30-day incubation following soil contamination with Cd. They noted a direct correlation between Cd sensitivity and hatching time, indicating that Cd toxicity increased with prolonged incubation periods. Furthermore, Vig [89] estimated that Cd toxicity to soil biota varies depending on factors such as time, soil type, speciation, ageing, Cd source, organisms, and environmental conditions. Additionally, Sardar et al. [90] explored the breadth of the revocation, which significantly expanded with higher levels of heavy metals and varied across different incubation periods. Therefore, it was revealed in the present study that the growth and colonization of Arbuscular Mycorrhizal Fungi is significantly affected by the presence of toxic cadmium in the soil, but also by the residence time of this aqueous solution in the soil. Soil AMF microorganisms growing in the root appeared to proliferate normally when the Cd concentration was equal to the maximum permissible concentration as defined by the European Union. When the concentration is increased tenfold, then the microorganisms decrease, but still provide protection to the plant to cope with the toxic conditions of its immediate soil environment. The fact that the biomass of the plant both underground and aboveground increases when the Cd concentration is at the A level is therefore probably linked to the fact that there is an increase in the microroot population which helps the plant to cope and increase its mass and shoot, leaf, and root mass. Furthermore, at the A level of soil contamination, the highest rate of Cd transfer from soil to plant was observed, both above and underground, i.e., at the root. The respective Cd uptake rates for the aboveground and underground parts of the plant are 5% and 47.99% for 30-day incubation and 6.38% and 65.43% for 10-day incubation. At the B level of cadmium contamination of soils, a lower percentage movement of Cd from soil to plant was observed. The corresponding percentages of Cd movement were 7.39% and 21.54% for the above- and belowground part of the plant when the incubation lasted 30 days and 7.98% and 25.18% for the corresponding parts of the plant and 10-day incubation of Cd in the soil. Therefore, an increase in the mycorrhizal population induced an increase in the ability of industrial hemp to accumulate soil cadmium, contributing to the remediation of mainly moderate soils. In the case of heavily contaminated soils, which are not observed except in abandoned mine sites, the contribution of mycorrhizal AMFs to the remediation capacity of hemp appears to be significant but low.

4. Conclusions

In the current study, an approach was taken to find the possible relationship between the ability of industrial hemp to remediate cadmium-contaminated soils and the colonization of Arbuscular Mycorrhizal Fungi (AMFs). To this end, a pot experiment was conducted in which laboratory contamination of soils with two levels of cadmium corresponding to moderately and heavily contaminated soils was carried out, and in addition, two different incubation periods of the soils with cadmium-containing aqueous solutions added to the soil samples were studied. The ability of hemp to grow without problems in both levels of contaminated soils was observed, as no toxicity or inhibition of hemp growth was observed. The plant tended to accumulate most of the cadmium underground rather than aboveground. Therefore, the mechanism of phytoremediation that seems to prevail is phytostabilization. Cd accumulation from the soil to the cannabis root also appeared to be influenced by the aging of the pollution, as the plant accumulated greater amounts of metal when the pollution was fresh. A greater increase in plant mass was observed when the soil had the maximum concentration of cadmium allowed by the European Union, while at higher Cd concentrations, the mass increase was less significant. Under the same level of cadmium concentration, the highest rate of phytosuspension, i.e., a higher rate of uptake of Cd from the soil to the plant, as well as a higher rate of AMF colonization was observed. Therefore, the ability of industrial hemp to remediate Cd-impacted soils seems to have been significantly enhanced by the presence of AMFs, which contributed to the emergence of a valuable plant for Mediterranean soils while yielding quality aboveground plant tissue suitable for industrial use.

Author Contributions

Conceptualization, M.A., E.T. and E.E.G.; methodology, M.A., V.L., E.T., I.I. and E.E.G.; software, M.A. and V.L.; validation, M.A., V.L., I.I. and E.E.G.; formal analysis, M.A. and V.L.; investigation, M.A., V.L., E.T., I.I. and E.E.G.; resources, E.T. and E.E.G.; data curation, M.A., V.L. and E.E.G.; writing—original draft preparation, M.A. and E.E.G.; writing—review and editing, M.A, V.L., I.I. and E.E.G.; visualization, M.A., V.L. and E.E.G.; supervision, E.E.G.; project administration, E.E.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Pseudo-total and available Cd concentrations in soil (10 and 30 days of incubation time) before (Initial) hemp cultivation in each treatment (lowercase letters show the significant differences between the treatments for available Cd concentrations, while capital letters show the significant differences for pseudo-total Cd concentrations. Error bars indicate standard deviation. LSD5% values for pseudo-total and available Cd concentrations: 0.8102 and 0.3845, respectively).
Figure 1. Pseudo-total and available Cd concentrations in soil (10 and 30 days of incubation time) before (Initial) hemp cultivation in each treatment (lowercase letters show the significant differences between the treatments for available Cd concentrations, while capital letters show the significant differences for pseudo-total Cd concentrations. Error bars indicate standard deviation. LSD5% values for pseudo-total and available Cd concentrations: 0.8102 and 0.3845, respectively).
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Soilsystems 08 00100 g002
Figure 2. Pseudo-total and available Cd concentrations in soil (10 and 30 days of incubation time) after (Final) hemp cultivation in each treatment (lowercase letters show the significant differences between the treatments for available Cd concentrations, while capital letters show the significant differences for pseudo-total Cd concentrations. Error bars indicate standard deviation. LSD5% values for pseudo-total and available Cd concentrations: 0.6117 and 0.2326, respectively).
Figure 2. Pseudo-total and available Cd concentrations in soil (10 and 30 days of incubation time) after (Final) hemp cultivation in each treatment (lowercase letters show the significant differences between the treatments for available Cd concentrations, while capital letters show the significant differences for pseudo-total Cd concentrations. Error bars indicate standard deviation. LSD5% values for pseudo-total and available Cd concentrations: 0.6117 and 0.2326, respectively).
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Soilsystems 08 00100 g003
Figure 3. Heatmap of Pearson correlation coefficient.
Figure 3. Heatmap of Pearson correlation coefficient.
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Figure 4. Cd concentrations in the aboveground part and the roots of hemp in the six treatments of the experiment (lowercase letters show the significant differences between the treatments of roots, while capital letters show the significant differences for aboveground parts. Error bars indicate standard deviation. LSD5% values for aboveground and roots: 0.6772 and 0.6908, respectively).
Figure 4. Cd concentrations in the aboveground part and the roots of hemp in the six treatments of the experiment (lowercase letters show the significant differences between the treatments of roots, while capital letters show the significant differences for aboveground parts. Error bars indicate standard deviation. LSD5% values for aboveground and roots: 0.6772 and 0.6908, respectively).
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Figure 5. Dry biomass (g) of the roots of hemp in the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 0.2343).
Figure 5. Dry biomass (g) of the roots of hemp in the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 0.2343).
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Figure 6. Dry biomass (g) of the aboveground part of the hemp in the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 1.421).
Figure 6. Dry biomass (g) of the aboveground part of the hemp in the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 1.421).
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Figure 7. Colonization rates (%) of cannabis roots by AMFs across the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 11.602).
Figure 7. Colonization rates (%) of cannabis roots by AMFs across the six treatments of the experiment (The different letters show the significant differences between the treatments. Error bars indicate standard deviation. LSD5% = 11.602).
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Figure 8. Observations from the microscope of cannabis blue-stained roots: (a) hyphae, extending within the root, (b) intraradical vesicles, and (c) arbuscules within the roots produced by AMFs.
Figure 8. Observations from the microscope of cannabis blue-stained roots: (a) hyphae, extending within the root, (b) intraradical vesicles, and (c) arbuscules within the roots produced by AMFs.
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Table 1. The coding of the five treatments of the experiment.
Table 1. The coding of the five treatments of the experiment.
Codingmg Cd kg−1Incubation DaysReplicates
Control_10d01010
CdA_10d31010
CdB_10d301010
Control_30d03010
CdA_30d33010
CdB_30d303010
Table 2. Monthly and mean air temperature and precipitation during the growing period, C. sativa.
Table 2. Monthly and mean air temperature and precipitation during the growing period, C. sativa.
2023
AprilMayJuneJulyAugustTotal
Mean
Temperature (°C)		14.01823.328.827.8-
Precipitation (mm)75.670.085.89.040.0280.4
Table 3. Values of soil physicochemical properties (average of replicates, n = 10).
Table 3. Values of soil physicochemical properties (average of replicates, n = 10).
Physicochemical PropertiesMinMaxAverageSD
pH (1:1)7.69.18.3±0.6
EC (μS/cm)1082.010901086±3.3
OM (%)2.33.52.9±0.5
CaCO3 (%)12.114.413.2±1.0
Clay (%)535956±2.4
Sand (%)414644±2.0
Table 4. Values of Bio Concentration Factor (BFC) in hemp parts.
Table 4. Values of Bio Concentration Factor (BFC) in hemp parts.
UndergroundAboveground
TreatementsCplant/
Cpseudo-total		Cplant/
Cavailable		Cplant/
Cpseudo-total		Cplant/
Cavailable
Control_10d0.000.000.000.00
Control_30d 0.000.000.000.00
CdA_10d 0.655.060.060.49
CdA_30d 0.454.510.050.49
CdB_10d0.252.360.080.75
CdB_30d0.222.210.071.01
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Androudi, M.; Liava, V.; Tsaliki, E.; Ipsilantis, I.; Golia, E.E. Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity. Soil Syst. 2024, 8, 100. https://doi.org/10.3390/soilsystems8030100

AMA Style

Androudi M, Liava V, Tsaliki E, Ipsilantis I, Golia EE. Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity. Soil Systems. 2024; 8(3):100. https://doi.org/10.3390/soilsystems8030100

Chicago/Turabian Style

Androudi, Maria, Vasiliki Liava, Eleni Tsaliki, Ioannis Ipsilantis, and Evangelia E. Golia. 2024. "Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity" Soil Systems 8, no. 3: 100. https://doi.org/10.3390/soilsystems8030100

APA Style

Androudi, M., Liava, V., Tsaliki, E., Ipsilantis, I., & Golia, E. E. (2024). Use of Cannabis sativa L. for Improving Cadmium-Contaminated Mediterranean Soils—Effect of Mycorrhizal Colonization on Phytoremediation Capacity. Soil Systems, 8(3), 100. https://doi.org/10.3390/soilsystems8030100

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