147
1 University of Ljubljana, Biotechnical Faculty, 
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
2 Jožef Stefan Institute, Jamova cesta 39,  
SI-1000 Ljubljana, Slovenia
* Corresponding author:  
E-mail address: paula.pongrac@bf.uni-lj.si 
Citation: Bočaj, V., Regvar, M., Pongrac, 
P., (2025). Linking microbiome and 
hyperaccumulation in plants. Acta Biologica 
Slovenica 68 (2)
Received: 10.10.2024 / Accepted: 10.03.2025 /  
Published: 14.03.2025
https://doi.org/10.14720/abs.68.2.19957
This article is an open access article distributed 
under the terms and conditions of the Creative 
Commons Attribution (CC BY SA) license
Review
Linking microbiome  
and hyperaccumulation  
in plants
Valentina Bočaj 1, Marjana Regvar 2, Paula Pongrac 1,2*
Abstract
Hyperaccumulating plants can take up extraordinarily large concentrations of 
one or more metal(loid)s from the soil and accumulate it/them in the aboveground 
tissues without exhibiting any visible toxicity symptoms. Among more than 700 
plant taxa reported to have evolved this unique phenotype, the most common is 
the hyperaccumulation of nickel (Ni), and less common is the hyperaccumulation 
of arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese 
(Mn), lead (Pb), antimony (Sb), selenium (Se), thallium (Tl) or zinc (Zn). Metal(loid) 
hyperaccumulation is a result of several independent evolutionary events 
and despite considerable efforts, none of the proposed hypotheses on the 
environmental constraints driving these events has been supported fully to date. 
Among several tolerance strategies enabling hyperaccumulation is the allocation 
of metal(loid)s to competent cell types, typically away from photosynthetic 
apparatus, to limit damage to plant metabolism. Recently, the involvement 
of microorganisms colonizing roots in hyperaccumulation phenomenon has 
achieved increased attention due to the role of microorganisms in the mobilization 
of metal(loid)s in the soil. The complex interactions between hyperaccumulation 
and belowground microbiome are of primary interest for phytoremediation, a 
promising green technology for removing or immobilisation of metal(loid)s in the 
soil with the help of plants. In this review, we discuss and complement current 
reports on the contribution of microorganisms to metal(loid) hyperaccumulation.
Keywords
hyperaccumulating plants; arbuscular mycorrhizal fungi; dark septate 
endophytes
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Povezava med rastlinskim mikrobiomom in hiperakumulacijo
Izvleček
Hiperakumulacijske vrste lahko iz tal v svoja nadzemna tkiva privzamejo zelo velike koncentracije ene ali več 
(pol)kovin brez opaznih škodljivih učinkov pri rastlini. Pri več kot 700 taksonih, za katere poročajo, da so razvili ta 
edinstven fenotip, je najpogostejša hiperakumulacija niklja (Ni), manj pogosta pa je hiperakumulacija arzena (As), 
kadmija (Cd), kobalta (Co), kroma (Cr), bakra (Cu), mangana (Mn), svinca (Pb), antimona (Sb), selena (Se), talija (Tl) 
in cinka (Zn). Hiperakumulacija (pol)kovin je posledica več neodvisnih evolucijskih dogodkov. Kljub precejšnjim 
prizadevanjem pa nobena od predlaganih hipotez, ki bi razložila evolucijsko prednost hiperakumulacije, do danes 
ni bila v celoti podprta. Med številnimi tolerančnimi mehanizmi, ki omogočajo hiperakumulacijo, je tudi kopičenje 
presežnih koncentracij (pol)kovin v določenih tipih rastlinskih celic, običajno stran od fotosinteznega aparata. V 
zadnjem desetletju v ospredje raziskav hiperakumulacije stopa vloga mikroorganizmov, ki kolonizirajo korenine, pri 
mobilizaciji (pol)kovin v tleh. Zapletene interakcije med hiperakumulacijo in podzemnim rasltinskim mikrobiomom so 
zanimive predvsem z vidika fitoremediacije, t.j. zelene tehnologije, ki s pomočjo rastlin na naraven način odstrani 
(pol)kovine iz tal ali pa jih stabilizira. V tem preglednem članku razpravljamo in dopolnjujemo dosedanje študije o 
prispevku talnih in/ ali simbionstkih mikroorganizmov k hiperakumulaciji.
Ključne besede 
hiperakumulacijske vrste; arbuskularne mikorizne glive; temni septirani endofiti
Introduction
The term hyperaccumulator was introduced by Brooks et 
al. (1977) to describe plants with exceptional concentrations 
of one or more metal(loid)s in the aboveground biomass 
without showing any visible toxicity symptoms (Brooks et 
al., 1977; Rascio, 1977). Initially, these unique plants were 
studied in relation to geobotany, while today, they have 
been at the centre of discussions concerning environmental 
pollution and phytoremediation. Hyperaccumulation is more 
than tolerance because a typical metal(loid)-tolerant plant 
aims to limit translocation of metal(loid) from roots to shoots, 
whereas in aboveground tissue of a hyperaccumulating 
plant, up to 1000-fold larger concentrations of metal(loid)s 
can be found compared to non-hyperaccumulating plants 
(Rascio, 1977; Reeves, 2006). Among hyperaccumulators, 
the largest number of species hyperaccumulate Ni, followed 
by other metal(loid)s like As, Cd, Co, Cr, Cu, Mn, Pb, Sb, Se, 
Tl and Zn (Table 1) (Baker et al., 2000; Baker & Brooks, 1989; 
Reeves et al., 2018; White & Pongrac, 2017). To warrant the 
designation of a plant as a hyperaccumulating species, the 
following conditions must be met: (i) the plant has to accu-
mulate metal(loid) when grown in native soil, (ii) the shoot-
to-root concentration ratio for a metal(loid) must be higher 
than unity, and (iii) foliar concentration (in mg g-1 dry weight) 
of a metal(loid) must exceed the thresholds 0.1 for Cd, 1 for 
As, Co, Cr, Cu, Ni, Pb, Sb, Se, Tl, and 10 for Mn and Zn (Baker 
et al., 2000; Baker & Brooks, 1989; Krämer, 2010; McGrath & 
Zhao, 2003; Rascio & Navari-Izzo, 2011). To date, more than 
700 plant taxa are listed as hyperaccumulating, with several 
belonging to the Brassicaceae family (Baker et al., 2000; 
Krämer, 2010; Reeves et al., 2018; White & Pongrac, 2017).
Many authors argue that hyperaccumulation has inde-
pendently evolved multiple times (Krämer, 2010; Reeves, 
2006) as a response against herbivory and pathogens to 
which plants are constantly exposed (Boyd, 2007; Plaza et 
al., 2015). It is an extreme evolutionary trait, and although 
several hypotheses have been proposed, none of them is 
fully scientifically supported. However, the most accepted 
hypothesis among researchers remains the “defence 
against natural enemies” hypothesis, which predicts that 
plants (hyper)accumulate metal(loid)s in their aerial tissues 
to protect themselves from pathogens and herbivores. 
Furthermore, it also predicts the variety of defence organic 
compounds (Pollard, 2022; Rascio & Navari-Izzo, 2011) to 
complement large metal(loid) concentrations.
Although there are many studies on hyperaccumulating 
plants, many knowledge gaps still need to be filled to fully 
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understand the physiological mechanisms behind hyperac-
cumulation and furthermore, its complex interactions with 
the surrounding environment. One of the most prominent 
knowledge gaps is the understanding of the role microor-
ganisms have in hyperaccumulation, tolerance, and/or the 
overall fitness of hyperaccumulators. Therefore, the main 
aim of this review is to appraise existing knowledge and 
complement current reports on the contribution of microor-
ganisms to metal(loid) hyperaccumulation.
Plants are not alone
Seemingly, plants function as individuals. However, 
recent investigations revealed that they host numerous 
microorganisms (MO), referred to as phytomicrobiome. 
Phytomicrobiome consists of archaea, bacteria, fungi, 
nematodes, protists, and viruses (Bordenstein and Theis, 
2015; Trivedi et al., 2020). Together with their plant host, 
they form the-so-called holobiont (Fig. 1). The interactions 
of MO with plant hosts can be (anthropocentrically) labelled 
either beneficial, harmful or neutral (Arnold et al., 2000; 
Brundrett, 2006; Inácio et al., 2002; Lindow & Brandl, 
2003; Rosenblueth & Martínez-Romero, 2006), although 
gradients can be observed, namely a neutral interaction 
can become either beneficial or harmful, depending on 
different intrinsic and/or external factors.
Phytomicrobiome colonizes different parts of the plant, 
both vertically (Fig. 1) and horizontally. The MO colonizing 
the aboveground parts of the plant (e.g., leaves) inhabit 
the phyllosphere (Schlaeppi & Bulgarelli, 2015; Whipps 
et al., 2008), and those colonizing the underground (e.g., 
plant roots) inhabit the rhizosphere (Schlaeppi & Bulgarelli, 
2015). Microorganisms can be found either inside their 
host's tissues or on their surface and are therefore referred 
to as endophytic or epiphytic, respectively. Endophytic MO 
is associated with the host for the whole or only a part of 
their life cycle. The beneficial effects of phytomicrobiome 
on their host have been well documented and include (i) 
increased tolerance to abiotic stresses (Rolli et al., 2014), 
(ii) enhanced nutrient uptake (Van Der Heijden et al., 2015), 
(iii) priming plant immune system (van der Ent et al., 2009) 
and (iv) protection against pathogens (Ritpitakphong et 
al., 2016). Some authors hypothesise that the phytomicro-
Metal(loid) Number of species Selected plant species (family) Reference(s)
As 5 Pteris vittata (Pteridaceae) Xiao et al., 2021
Cd 7 Arabidopsis halleri, Noccaea caerulescens,  
N. praecox (Brassicaceae)
Bert et al., 2003; Vogel-Mikuš et al.  
2005; Wójcik et al., 2005
Co 42 Haumaniastrum robertii (Lamiaceae) Van Der Ent et al., 2019
Cr 1 Spartina argentinensis (Poaceae) Redondo-Gómez et al., 2011
Cu 53 Aeolanthus biformifolius (Lamiaceae) Van Der Ent et al., 2019
Mn 42 Grevillea meisneri (Proteaceae) Bihanic et al., 2021
Ni 532 Alyssum heldreichii, N. pindicum (Brassicaceae) Psaras et al., 2000
Sb At least 1 P. vittata (Pteridaceae) Wan et al., 2016
Se 41 Stanleya pinnata (Brassicaceae),  
Symphyotrichum ericoides (Asteraceae)
Cochran et al., 2018
Tl 2 Biscutella laevigata (Brassicaceae) Pošćić et al., 2013
Zn 20 A. halleri, N. caerulescens, N. praecox  
(Brassicaceae)
Kozhevnikova et al., 2017; Mijovilovich  
et al., 2020; Vogel-Mikuš et al., 2008
Table 1. List of metal(loid)s, number of species that hyperaccumulate corresponding metal(loid) and selected hyperaccumulating species with cor-
responding references. Note that one species can hyperaccumulate more than one metal(loid) and that not all combinations are presented in the 
current table. The number of species was summarized by Reeves et al. (2018).
Tabela 1. Seznam kovin(loidov), število vrst, ki hiperakumulirajo ustrezne kovine(loide), in izbrane hiperakumulirajoče vrste z ustreznimi referencami. 
Upoštevajte, da lahko ena vrsta hiperakumulira več kot eno kovino(loid) in da v tej tabeli niso predstavljene vse kombinacije. Število vrst je bilo 
povzeto po Reeves et al. (2018).
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biome is an extension of the plant genome (Rosenberg & 
Zilber-Rosenberg, 2016; Schlaeppi & Bulgarelli, 2015). 
The root system is a habitat for several MO, which 
inhabit both the surface of the roots and internal parts 
of this branched underground plant organ. Henceforth, 
we focus on three groups of rhizosphere-associated MO 
associated with hyperaccumulating plants: (i) mycorrhizal 
fungi, (ii) dark septate endophytes, and (iii) rhizoplane MO.
Do hyperaccumulators form mycorrhizal 
associations?
Mycorrhiza is one of the oldest and best-studied, presum-
ably beneficial endophytic interactions between plants, and 
there is robust evidence that this interaction has  played an 
important role in the evolution of land plants (Buscot, 2015; 
Martin & van der Heijden, 2024; Strullu-Derrien et al., 2018). 
Among different types of mycorrhiza, arbuscular mycor-
rhiza (AM) and arbuscular mycorrhizal fungi (AMF) are also 
associated with hyperaccumulating plants. Nevertheless, 
although they colonize most land plants, some plant fam-
ilies are believed not to form AM associations (Sharma et 
al., 2023). These include Brassicaceae, which hosts many 
known hyperaccumulators (Johnson et al., 1997; Harley 
& Harley, 1987; Veiga et al., 2013). However, some recent 
studies show that the non-mycorrhizal status of Brassica-
ceae might not be universal, at least in the sense of the 
plant family as a whole (Trautwig et al., 2023). For example, 
the presence of AMF was observed in Biscutella laevigata 
(Brassicaceae), a Tl hyperaccumulating plant (Pošćić et al., 
2013), both at metalliferous and non-metalliferous sites 
with all typical AMF structures, i.e., vesicles, coils, and 
arbuscules (Orłowska et al., 2002). A year later, Regvar 
et al. (2003) discovered that pennycresses (Noccaea 
spp.; Brassicaceae) form AMF associations too. The AMF 
colonization of a Cd and Zn hyperaccumulating N. praecox 
Figure 1. A representative plant with associated microorganisms.
Slika 1. Reprezentativna rastlina s pripadajočimi mikroorganizmi.
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was found at metal-polluted and non-polluted sites but was 
significantly lower or absent on the most polluted sites with 
the highest concentrations of Cd, Pb, and Zn (Vogel-Mikuš 
et al., 2005). This observation could mean there is a thresh-
old of metal(loid) concentration that AMF can tolerate and 
above which mycorrhizal interactions cannot be formed. 
A greenhouse inoculation experiment on N. praecox with 
an indigenous AMF mixture (Vogel-Mikuš et al., 2006) 
indicated that AMF association is favoured during the 
reproductive period of the plant when the requirements for 
nutrients are elevated. Moreover, inoculated plants showed 
significant improvement in nutrient uptake and decreased 
uptake of Cd and Zn compared to non-inoculated plants 
(Vogel-Mikuš et al., 2006). Pongrac et al. (2007) later 
confirmed similar results under field conditions. The higher 
AMF colonization levels during the reproductive phase 
could protect the plant from potentially toxic metal(loid)s 
when plants invest the majority of photosynthates to seed 
production. The protective role of AMF was revealed by 
their direct effect on Cd accumulation in N. praecox, where 
the accumulation of Cd in the shoots was decreased, pre-
venting potential over-accumulation in the seeds (Pongrac 
et al., 2007; Vogel-Mikuš et al., 2006). In addition, N. prae-
cox (population from Žerjav, Slovenia) and a Cd, Zn, and 
Ni hyperaccumulating N. caerulescens (Ganges ecotype) 
were successfully inoculated with AMF monospore inocula 
in a controlled pot experiment. The plants were grown for 
six months in the commercial substrate and in heavy-metal 
contaminated, field-collected soil from Žerjav as described 
previously but without metal amendments (Pongrac et al., 
2009). In addition, inoculated treatments in which 100 g 
of monospore AMF inoculum of Funneliformis mosseae 
or F. caledonium (both obtained as Glomus mosseae and 
G. caledonium from International Collection of Vesicular 
Arbuscular Mycorrhizal Fungi; reference numbers UK115 
and UK301, respectively) was mixed into the commercial 
substrate and 100 g of N. praecox indigenous fungal mixture 
to the heavy-metal contaminated, field-collected soil from 
Žerjav. After the first three months of growth, plants were 
exposed to lower temperatures to induce flowering, as 
this developmental stage has been shown to enhance the 
formation of the AM symbiosis in N. praecox (Pongrac et al., 
2007; Vogel-Mikuš et al., 2006) and plants were allowed to 
grow for three more months. At harvest, roots and shoots 
were separated, and carefully washed with deionized 
water, and a subsample of roots from all treatments was 
retained for AMF staining (Phillips & Hayman, 1970) and 
AMF colonization parameters (Vogel-Mikuš et al., 2006), 
whereas the rest of the shoots and roots were dried and 
weighed (dry weight). The concentrations of phosphorus 
(P), Zn, and Cd were determined after wet digestion using 
atomic absorption spectrometry (for Zn and Cd; (Pongrac, 
Zhao, et al., 2009)) and following the vanado-molybdate 
method according to Olsen & Sommers (1982) for P). 
In roots of inoculated Noccaea species AMF structures 
(hyphae, arbuscules, vesicles and coils) were observed 
(Fig. 2A). There was a stronger statistical difference 
between the species than between treatments observed 
in the dry biomass (Fig. 2B,). Only for N. caerulescens there 
was a significant decrease in root dry weight for inoculated 
treatments of commercial soil (Fig. 2B). 
Interestingly, inoculation with F. caledonium resulted 
in the highest shoot P, Zn and Cd concentrations for both 
species, compared to the control commercial substrate 
and the F. mosseae-inoculated commercial substrate (Fig. 
3). In field-collected soil, the inoculation with N. praecox 
indigenous inoculum increased Cd concentration in shoots 
only. In root Cd concentration, there was a decrease in 
the commercial soil-inoculated treatments, indicating an 
increase in the Cd translocation factors (Fig. 3). Again, in 
roots from the polluted site in Žerjav, AMF structures with 
distinct arbuscules have been observed (Fig. 4A).
Other hyperaccumulating species have also been 
reported to form AM. For example, in As hyperaccumu-
lating fern Pteris vitatta (Pteridaceae), an increase in the 
translocation of As has been observed in AMF-inoculated 
plants compared to non-AMF-inoculated plants (Trotta 
et al., 2006). In an effort to bridge the gap in our under-
standing of the distribution and the ecology of AMF, the 
GlobalAMFungi database (https://globalamfungi.com) has 
been recently developed (Větrovský et al., 2023). It will 
enable us to understand better the fungal taxa that form 
this remarkable interaction with plants.
Dark septate endophytes associated with 
hyperaccumulators
Another group of endophytic fungi has been discovered 
and studied extensively, especially with plants growing in 
extreme environmental conditions, namely dark septate 
endophytes (DSEs). DSEs comprise a miscellaneous group 
of ascomycetous anamorphic groups colonizing the plants 
inter- and/or intracellularly (Jumpponen, 2001). They have 
been found to colonize about 600 plant species, classified 
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Figure 1. Arbuscular mycorrhizal (AM) structures (hyphae, H; arbuscules, A; coils, C and vesicles, V) in roots of Noccaea praecox (Np) and 
N. caerulescens (Nc) inoculated by AM fungi Funneliformis mosseae (Fm) or F. caledonium (Fc) in a pot experiment (A) and root and shoot 
biomass of six months old plants (B) grown in commercial soil (CS) and inoculated with F. mosseae (CS+Fm) or F. caledonium (CS+Fc) or 
grown in heavy-metal polluted, field-collected soil (PS) and inoculated with Np-indigenous AM inoculum (PS+MI). Different letters above 
columns present statistically significant differences between treatments (at p<0.05) for CS and PS separately, and asterisks indicate statisti-
cally significant differences between species (*, p<0.05; **, p<0.01;) as determined by two-way ANOVA and Holm-Sidak post-hoc test. Shown 
are means (n=3) + standard errors.
Slika 1. Arbuskularne mikorizne (AM) strukture (hife, H; arbuskule, A; tuljave, C in vezikule, V) v koreninah Noccaea praecox (Np) in N. 
caerulescens (Nc), inokuliranih z AM glivami Funneliformis mosseae (Fm) ali F. caledonium (Fc) v lončnem poskusu (A ) ter biomasa korenin 
in poganjkov šest mesecev starih rastlin (B ), gojenih v komercialni zemlji (CS) in cepljenih z glivami F. mosseae (CS+Fm) ali F. caledonium 
(CS+Fc) ali gojenih v s težkimi kovinami onesnaženi, na polju zbrani zemlji (PS) in cepljenih z inokulumom Np-indigenous AM (PS+MI). 
Različne črke nad stolpci predstavljajo statistično pomembne razlike med obravnavami (pri p<0,05) ločeno za CS in PS, zvezdice pa označu-
jejo statistično pomembne razlike med vrstami (*, p<0,05; **, p<0,01;), kot je bilo določeno z dvosmerno ANOVA in Holm-Sidakovim post-hoc 
testom. Prikazana so povprečja (n=3) + standardne napake.
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Figure 3. Concentration of phosphorus (P), zinc (Zn) and cadmium (Cd) in roots and shoots of Noccaea praecox (Np) and N. caerulescens (Nc) 
grown in commercial soil (CS) and inoculated with monospore culture of arbuscular myorrhizal fungi Funneliformis mosseae (CS+Fm) and 
F. caledonium (CS+Fm) or grown in heavy-metal polluted, field-collected soil (PS) and inoculated with Np-indigenous mycorrhizal inoculum 
(PS+MI). Different letters above columns present statistically significant differences between treatments (at p<0.05) for CS and PS separately, 
and asterisks indicate statistically significant differences between species (*, p<0.05; **, p<0.01; ***, p<0.001) as determined by two-way 
ANOVA and Holm-Sidak posthoc test. Shown are means (n=3) + standard errors. DW, dry weight.
Slika 3. Koncentracija fosforja (P), cinka (Zn) in kadmija (Cd) v koreninah in poganjkih Noccaea praecox (Np) in N. caerulescens (Nc), gojenih 
v komercialni zemlji (CS) in inokuliranih z monosporno kulturo arbuskularnih mioriznih gliv Funneliformis mosseae (CS+Fm) in F. caledonium 
(CS+Fm) ali gojene v s težkimi kovinami onesnaženi, na polju zbrani zemlji (PS) in inokulirane z mikoriznim inokulumom Np-avtohtonih mikoriz 
(PS+MI). Različne črke nad stolpci predstavljajo statistično značilne razlike med tretiranji (pri p<0,05) ločeno za CS in PS, zvezdice pa označu-
jejo statistično značilne razlike med vrstami (*, p<0,05; **, p<0,01; ***, p<0,001), določene z dvosmerno ANOVA in Holm-Sidakovim posthoc 
testom. Prikazana so povprečja (n=3) + standardne napake. DW, suha masa.
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into 320 genera and 114 families (Jumpponen & Trappe, 
1998). DSEs benefit their host by facilitating carbon, nitro-
gen, and phosphorus uptake (Vergara et al., 2018; Yakti 
et al., 2018). Furthermore, they can induce the release 
of phytohormones like auxins (Berthelot et al., 2016) and 
protect plants against abiotic stress (Wang et al., 2016). It 
is known that DSEs colonize extreme habitats, like high 
salinity sites (Sonjak et al., 2009), dry habitats (Barrow, 
2003), and metal-enriched soils (Ban et al., 2012; Likar & 
Regvar, 2009, 2013). Although most studies have debated 
the beneficial role of DSEs (Ban et al., 2012; Likar & Regvar, 
2009, 2013; Newsham, 2011), some studies proposed that 
DSE taxa can form parasitic associations with their host 
(Wilcox & Wang, 1987).
In metal-enriched soils, DSEs have been shown to pos-
itively impact plant fitness. A particular plant species where 
DSEs frequently colonize the roots, especially in highly met-
al-contaminated soil, is goat willow (Salix caprea L.; Salica-
ceae). The study by Likar & Regvar (2013) revealed smaller 
Cd and Zn leaf concentrations, larger chlorophyll concentra-
tions and transpiration in DSE-inoculated S. caprea grown 
in metal-enriched soils compared to non-DSE-inoculated 
plants. It has been proposed that the binding of Cd and Zn 
to melanin in DSE may, through limiting the translocation of 
heavy metals, contribute to these observations (Potisek et 
al., 2021) in accordance with reports by Ban et al. (2012). 
These observations suggested that DSE associations are 
favorable for S. caprea under metal-enriched conditions. 
Furthermore, a larger diversity of DSE was found at sites 
with higher metal concentrations compared to low soil-
metal concentrations (Likar & Regvar, 2009).
Although several studies of DSEs were performed on 
non-hyperaccumulators (Ban et al., 2012; Barrow, 2003; He 
et al., 2019; Likar & Regvar, 2013; Stoyke & Currah, 1991), 
little is known about the associations between DSEs and 
hyperaccumulators. In a recent study DSE strains isolated 
from the roots of poplar growing at different trace element 
contaminated sites were used to successfully inoculate N. 
caerulescens (Yung et al. 2021). Moreover, in the highly 
contaminated soil, a specific strain of DSEs significantly 
increased the root biomass of N. caerulescens compared 
to the non-inoculated plant without affecting the nutrient 
status of the plant (Yung et al., 2021). The positive effect 
of DSE strains was also observed in the accumulation of 
Zn and Cd, which was larger in the roots in highly contam-
inated soil with inoculation of DSE (30% and 90% more, 
respectively) compared to non-DSE-inoculated plants 
(Yung et al., 2021). The DSEs were also observed in roots 
of N. praecox, a Cd and Zn hyperaccumulating plant, when 
grown on a highly contaminated site in Žerjav (Slovenia) 
(Fig. 4B) – an area still affected by the past mining and 
smelting activities (Bočaj et al., unpublished; Pongrac et 
al., 2009). DSEs may be important partners for the host, 
but more focused research is required, ideally by using 
DSE inoculum and performing controlled experiments to 
evaluate effects in different plants in different soils. 
Are, therefore, DSEs mycorrhizal or not? Jumpponen 
(2001) claimed that at least under some conditions, DSE 
should be treated as a mutualistic partner, in agreement 
with Treu et al. (1996). By contrast, Brundrett (2006) dis-
putes this by pointing out that in contrast to mycorrhizal 
symbiosis, the relationship between host plant and endo-
phyte (e.g., DSE) lacks three features typical for arbuscular 
mycorrhiza: a cellular interface where specialized struc-
tures (e.g., arbuscules in AM) occur, but the same is true for 
ectomycorrhizal fungi. The synchronization of the develop-
ment between the plant host and fungi and the benefits 
for both partners in the interaction are those that count. 
This demonstrates that there is a thin and often unclear line 
between beneficial or harmful, mycorrhizal or not.
Rhizosphere epiphytic microorganisms 
may also play an important role in 
hyperaccumulation
Sometimes overlooked in comparison to AMF and DSEs 
is the rhizosphere MO, one of the most complex micro-
bial communities on the earth with a large number and 
rich diversity (Mendes et al., 2013) because they play 
a crucial role in plant growth and development, nutrient 
acquisition, pest prevention, and yield improvement 
(Mendes et al., 2011). The diversity and complexity of the 
soil as a substrate and a habitat are reflected in different 
microbial communities between bulk soil and rhizosphere 
MO, as well as between rhizosphere and rhizoplane (i.e., 
the external surface of the root, including closely adher-
ing soil particles). The involvement of rhizosphere and 
rhizoplane MO in the ability of plants to hyperaccumulate 
metal(loid)s have been only poorly investigated so far. In 
a Cd and Zn hyperaccumulating Sedum alfredii (Crassu-
laceae), the diversity of four spatial compartments: bulk 
soil, rhizosphere, rhizoplane, and endosphere revealed 
that regardless of the soil type or genotype of S. alfredii, 
diversity diminished from rhizosphere to rhizoplane and 
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Figure 4. Representative structures of 
arbuscular mycorrhizal fungi (A) and 
dark septate endophytes (B) in roots of 
Noccaea praecox collected in Žerjav 
(Slovenia) in 2023. Roots were stained 
following the standard Tripan blue stain-
ing protocol (Philips & Haymann, 1970).
Slika 4. Reprezentativne strukture 
arbuskularnih mikoriznih gliv (A) in tem-
nosemenskih endofitov (B) v koreninah 
Noccaea praecox, nabranih v Žerjavu 
(Slovenija) leta 2023. Korenine so 
bile obarvane po standardnem pro-
tokolu barvanja s Tripan blue (Philips & 
Haymann, 1970).
endosphere (Luo et al., 2017). By contrast, the diversity 
increased from the bulk soil to the rhizosphere, which was 
confirmed by Xiao et al. (2021) and later by Kushwaha et 
al. (2022), claiming that the diversity is larger close to the 
roots than in nearby soil. This may be attributed to plant 
exudates, microbial chemotaxis towards exudates and 
nutrients (García-Salamanca et al., 2012), and changes in 
pH caused by plant exudates (Fan et al., 2017).
Interestingly, the taxa richness of MO was reported 
to differ between hyperaccumulators and non-hyperac-
cumulators. For example, comparing several Se-hyperac-
cumulators and non-hyperaccumulators from the same 
family sharing the same growing site revealed substantial 
differences in rhizosphere communities, with hyperac-
cumulators harboring a larger number of rhizobacterial 
species (Cochran et al., 2018). Similarly, a study by Martos 
et al. (2021) showed that in hyperaccumulators from the 
Brassicaceae an enrichment in previously described 
metal-tolerant bacteria and bacteria involved in nitrogen 
cycling was found compared to non-hyperaccumulating 
species (Martos et al., 2021). Unfortunately, these studies 
have so far been conducted on confamilial species and 
therefore, further in-depth species-specific investigations 
are required before meaningful conclusions can be made.
The importance of soil MO communities for hyper-
accumulation was studied by Muehe et al. (2015), who 
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Acta Biologica Slovenica, 2025, 68 (2)
compared the microbial community of A. halleri growing on 
metal-contaminated and gamma-irradiated soil, the latter 
having decreased diversity and species richness. Although 
no differences in aboveground biomass were observed 
during the experiment between treatments, A. halleri 
grown in untreated soil accumulated significantly more Cd 
and Zn (100% and 15%, respectively) than when grown in 
gamma-irradiated soil (Muehe et al., 2015). Mechanisms by 
which MO improve accumulation may lie in their ability to 
improve the bioavailability of metal(loid)s (Audet & Charest, 
2007). Whiting et al. (2001) used sterile soil where Zn was 
initially unavailable in water-soluble forms. Inoculation of N. 
caerulescens seeds with bacteria increased the solubility 
of Zn in the rhizosphere, resulting in a higher concentration 
of water-extractable Zn compared to axenic conditions 
(Whiting et al., 2001). In highly contaminated sites where 
hyperaccumulation could cause toxicity to plants, MO can 
also play an important role in reducing the bioavailability of 
metal(loid)s. Such an effect was shown for the Ni accumula-
tion in N. caerulescens whose inoculation with Ni-resistant 
bacteria did not result in the increase in Ni uptake, but it 
reduced the bioavailability of Ni in the serpentine soil and 
promoted plant growth compared to the axenic control 
(Aboudrar et al., 2013). 
All the above findings indicate the significant impor-
tance of soil MO on hyperaccumulation by altering 
metal(loid) bioavailability either positively or negatively.
Biological cleaners of 
metal(loid)s pollution  
and predictions
The use of hyperaccumulators to sustainably and in an 
environmentally friendly way remove toxic metal(loid) 
concentrations from the soil with commercial viability has 
been demonstrated for Ni only because plants with suffi-
cient biomass and Ni hyperaccumulation capacity have 
been identified. For other metal(loid)s, the main constraint 
remains the small aboveground biomass of hyperaccu-
mulators and/or limited accumulation capacity. Therefore, 
efforts into the discovery of new hyperaccumulators or 
enhancing biomass and hyperaccumulation capacity of 
known hyperaccumulators are essential if we are to remove 
other metal(loid)s using plants. According to Ernst (2005), 
an ideal plant for phytoextraction would have deep and 
well-branched root systems with AMF associations to take 
up as much metal(loid)s as possible, have efficient root-
to-shoot translocation with binding capacity in the roots 
smaller than in the shoots, without any biomass penalties. 
In addition, herbivores should not be attracted to these 
plants to prevent transmission through food chains, and 
harvesting should be easy and possible with conventional 
agricultural methods (Ernst, 2005). There is preliminary 
evidence showing that MO can affect several of these 
traits, therefore it may be viable to tailor MO associated 
with hyperaccumulators. However, our understanding of 
MO and their role in hyperaccumulation remains scarce. In 
this review, we underlined the indispensable role of MO 
for their hyperaccumulating hosts. Current knowledge of 
the link between functional profiles and ecological func-
tions in the MO communities is still scarce, and several 
questions, including the AM status of hyperaccumulators 
from the Brassicaceae, remain open. With the advances in 
analytical methods, such as cheaper and high-throughput 
next-generation sequencing and metabolomics, we can 
expect ground-breaking advancements in this novel field 
of research. Furthermore, with a better understanding of 
the hyperaccumulating plants and their interactions with 
MO, phytoremediation efforts may be improved and devel-
oped to the point that it becomes applicable to a variety of 
environmental remediation strategies.
Author Contributions
Conceptualization, M.R. and P.P.; investigation, V.B.; writ-
ing—original draft preparation, V.B.; writing—review and 
editing, M.R. and P.P.; supervision, P.P.
Acknowledgement
We thank Fang-Jie Zhao for Noccaea caerulescens Ganges 
ecotype seeds and Joseph Morton for AMF monospore 
inocula, both of which were used in the experiment we 
described in the text.
Funding
This research was founded by the Slovenian Research 
Agency (research core funding No. P1-0212, project fund-
ing numbers J4-3091, N4-0346 and J1-3014, and Young 
Researcher Scholarship to V.B.).
Conflicts of Interest
The authors declare no conflict of interest.
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Acta Biologica Slovenica, 2025, 68 (2)
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