Introduction

Since before the first millennium BC, southern Tuscany (Italy) was a region of concerted mining and metallurgical development: the Colline Metallifere, the Tolfa region and Monte Amiata were characterized by a large availability of a variety of metal ores (Harrison et al. 2010). During the Etruscan times (10th–1st century BC), the region showed highly sophisticated production of bronze and, increasingly, iron artefacts of all types. This activity would tend to put significant amounts of undesirable elements, especially arsenic, into the environment.

Also, due to natural deposits (alluvia from the Colline Metallifere) dating back to the Pleistocene and Holocene epochs, nowadays Tuscany exhibits extensive arsenic (As) anomalies in several areas of the region so that arsenic concentrations in soil may range from 5 to 2000 mg kg soil−1, both in topsoil and in deeper soils (Mascaro et al. 2001).

The dangerousness of this element for human and animal populations has been demonstrated by several studies (Valberg et al. 1997; Hughes 2002; Mandal and Suzuki 2002; Markley and Herbert 2009). Any risk assessment for soil contamination by trace elements needs an estimation of the bioavailability (concentration of exposition) to living organisms, which mainly depends on the soil chemistry and on the chemical behaviour of the element itself (Adriano 2001): pH, redox potential, organic matter content, texture, clay mineralogy, microbial activity and especially the presence of Fe- and Al-oxide/hydroxides (Ultra et al. 2007) and the competition between As(V) and phosphate for sorption sites strongly affect As availability (Jackson and Miller 2000). The main form (about 90 %) of arsenic in aerobic soil is pentavalent arsenate [As(V)O4 −3], which is adsorbed more strongly but with a faster sorption/desorption rate than phosphate (Lambkin and Alloway 2003). Trivalent arsenite [As(III)O3 −3] is the more labile but also the more toxic species in soil (Chatain et al. 2005). In terrestrial plants, as in soils, As occurs mostly in inorganic forms (Chen et al. 2004). In plants, As(V) acts as a phosphate analogue and is transported across the plasma membrane via a phosphate co-transport system (Meharg and Hartley-Whitaker 2002). Arsenate reductase (AR), detected in different terrestrial plants, plays an important role in reduction and detoxification of As(V) to As(III) and in its accumulation in plants (Yu et al. 2009). Concentrations of As commonly found in plants vary from 0.009 to 1.5 mg kg−1 dry weight (Kabata-Pendias and Pendias 2001).

Meharg et al. (1994), Abedin et al. (2002) and Esteban et al. (2003), in very-short-duration uptake studies, demonstrated that phosphate can effectively reduce plant uptake of As(V), depending on the different plant resistance characteristics to As and on the amounts of soluble P and As in the rhizosphere. In long-term studies with different plant species, on the contrary, Ultra et al. (2007), Pigna et al. (2009) and Puckett et al. (2012) observed increasing or decreasing uptake and translocation of As with P addition, depending on the experiment setup and mainly on the plants chosen.

As reported by Yu et al. (2009), arbuscular mycorrhizal (AM) fungi, which are ubiquitous in the rhizosphere, form symbiotic association with roots of the majority of plant species and are able to enhance the tolerance of host plants to soil contamination, generally decreasing metal concentration in the plant tissues, also in the case of arsenic (Smith and Read 1997; Gonzalez-Chavez et al. 2002; Bai et al. 2008). Nonetheless, the effect of AM on As depends on many factors and may vary between studies. For example, Chen et al. (2006) reported no effect of different arbuscular mycorrhizal fungi (Glomus mosseae, Glomus caledonium and Glomus intraradices) on As concentrations in tissues of Pteris vittata L. grown in a glasshouse experiment on soil polluted by both As and uranium. Xia et al. (2007), on the other hand, examined the influence of G. mosseae inoculation and P addition on maize after cultivation in a glasshouse on As-contaminated soil, and they found that AM decreased shoot As concentrations when no P was added and increased shoot and root As concentrations with P addition.

Orlowska et al. (2012), after having inoculated Plantago lanceolata L. grown on As-rich soil with AM, found higher root and shoot biomass and lower As concentration in roots than the non-inoculated plants, with significant differences in As concentration and accumulation between AM isolates. Yu et al. (2009), moreover, observed that AM inoculation increased P concentration in both roots and shoots. Maize is able to create particularly good environmental conditions for soil microorganisms and microfauna (Cattani et al. 2006). It is also the main crop cultivated in the sampling area to be used as livestock animal feed or for human consumption. Therefore, it was chosen for this study, which was designed to assess the amount of As(III) in the rhizosphere, the accumulation of As(III) and As(V) in the plant and their possible changes in relation to mycorrhizal inoculation and phosphorus application in a naturally contaminated soil. The obtained results represent a step in understanding the plant biology, with the ultimate purpose of understanding the best agronomic practices.

Materials and methods

Soil characterization and growth container

The soil (topsoil to a depth of approximately 0.3 m) used was collected from an agricultural field at Scarlino (Southern Tuscany, 42°55′45″ N, 10°48′31″ E), in a locality where As concentration is very high and much greater than the mean concentration of the earth crust (1.5 mg kg−1). In the work of Cattani et al. (2009), soil samples from the same location were used in observing the soil texture (typically clay loam), characterized by low permeability, which gave rise to significant stagnation of water, affecting plant growth and root development. For this reason, the soil was not directly used as a cultivation substrate, but it was mixed with coarse river sand (<2 mm) in the ratio 2:1 soil to sand. The analyzed properties of the modified soil were pH (1:2.5 soil to water) 8.22, organic matter 15.4 g kg−1, total CaCO3 35.1 g kg−1, total P 412 mg kg−1, extractable P (Olsen) 4.74 mg kg−1 and total As 194 mg kg−1. After having been previously sieved to 2 mm, homogenized, pasteurized and left to stabilize as reported by Orlowska et al. (2012), the modified soil was used to fill the rhizobox system created by Wenzel et al. (2001), adjusted to a bulk density of 1.3 g cm−3. This system is provided with an upper soil-plant compartment (416 cm3) and a lower compartment (409.5 cm3) where the root only compartment is physically separated from the rhizosphere-soil compartment with a nylon membrane inhibiting root penetration. After termination of the experiments, the soil compartment can be divided in layers parallel to the root plane. All construction materials are selected to minimize interaction with any element or organic compound.

Plants and AM fungus

Seeds of maize (Zea mays L.) were found in the market, washed and sterilized. The granular propagules of the endomychorrhizal fungus Rhizophagus irregularis DAOM197198 (Italpollina, Rivoli Veronese, Italy) used comprised a mixture of spores, mycelium, sandy soil and root fragments, containing 100 spore/cm3. According to Mathimaran et al. (2005), this fungus has been found to colonize rapidly a variety of cultivated plants, including maize, and also to be predominant in soils with high clay and As content, similar to that under investigation. Moreover, R. irregularis is the only fungus able to control nutrient uptake by individual hyphae, depending on differing phosphorus levels in the surrounding soil (Cavagnaro et al. 2005).

Experimental conditions

Plant cultivation was conducted from June to August 2013 in a phytotron, with a 14-h photoperiod, relative humidity constantly at 80 % and 28 °C/20 °C day/night temperatures for both germination and growth. During the light period, the light gradually increased to the maximum and decreased to the minimum to mimic sunrise and sunset and minimize the potential disturbance of plant processes by changing the light suddenly. The experiment comprised the following trials: (1) AM−/P− (not inoculated and not P-fertilized), (2) AM+/P− (inoculated and not P-fertilized), (3) AM−/P+ (not inoculated and P-fertilized) and (4) AM+/P+ (inoculated and P-fertilized), and each one was repeated in three replicates. The bulk density of the modified soil after the rhizobox filling was 1.5 g cm−3. Eight maize seeds were placed directly in the upper compartment of the rhizobox to test the growth efficiency in the different examined conditions. Where it was planned, a propagule was sown together with a seed, and phosphorus was added—as KH2PO4—at the rate of 25 mg kg−1, which corresponds to 100 kg ha−1. The P− treatments did not receive any supplemental potassium. After a period of about 2 weeks, the upper compartments were fixed on the lower compartment. The irrigation water was provided by the irrigation wicks, and root elongation was checked daily through the transparent acrylic window of the lower compartment. Plant harvest and soil sampling were realized after 4 weeks after rhizobox assembling, when P+ trials exhibited the maximum root elongation allowed by the compartment length and P− trials began to show growth inhibition and stress symptoms. Harvested plants were divided into roots and aerial parts, counted, weighed, air-dried and immediately analyzed.

After the plant harvest, the slicing device developed by Fitz et al. (2003) was used to divide the rhizosphere-soil compartment into different slices: 0–2 mm (rhizosphere, strictly speaking), 2–10 mm (‘extended rhizosphere’) and >10 mm (bulk soil). This distinction was realized because the membrane, characterized by a mesh size of 30 μm, can be penetrated by fungal hyphae and root hairs (with consequent rhizosphere widening), but not by roots (Luster et al. 2009), and the root-induced changes in the soil may be observed up to a distance of more than 7 mm from the root, depending on the soil texture and structure, the plant species, the monitored property and the presence of fungal hyphae (Norvell and Cary 1992). The mixed growth substrate allowed for easier management of the slicing device during sampling. The obtained soil samples were immediately submitted for analysis.

Mycorrhizal colonization was detected according to the modified method of Philips and Hayman (1970).

P and As measurements

Total arsenic

Ground soil samples (approximately 0.3 g dried weight) were analysed by ICP-MS (Agilent, 7500 ce) following nitric acid-hydrochloric acid digestion (Digiprep, Jr Model, SCP Science) and under vacuum filtration with a 0.45-μm Teflon filter membrane. ICP-MS operated in helium gas mode to remove any possible interference of 40Ar35Cl on m/z. The plant sample was analysed with the same procedure, following nitric acid-hydrogen peroxide-assisted digestion. Spike additions, certified reference materials at different arsenic concentrations (BCR 141 calcareous loam soil, BCR 143 sewage sludge amended soil for soil and BCR 402 white clover and BCR 482 lichen for plants) and analytical blanks were included for quality assurance. The recoveries ranged between 93 and 106 % with repeatability better than 8 %.

Total phosphorus

After digestion, soil and plant samples were analysed for P concentration by ICP-OES (Perkin Elmer, Optima™ 2100 DV) after having verified two wavelengths: 213.617 and 178.221 nm.

Arsenic species

Immediately after slicing, drying, grinding and sieving at 2 mm, 0.5 g of the soil samples was added with 10 mL of 1 M orthophosphoric acid and placed in PTFE vials at 60 W for 10 min. When the solution reached room temperature, it was transferred to a volumetric flask, diluted to 50 mL with mQ water and filtered through a 0.45-μm Millipore membrane as reported by Rahman et al. (2009). Spike additions of 1:1 As(III)/As(V) solutions were realized to verify any possible interconversion between the inorganic As forms during the extraction. Then, 0.1 g of the ground plant samples was added with 10 mL of water/methanol (1:1 v/v) mixture containing 1 % nitric acid in 15-mL polyethylene vials, which were shaken for 16 h at room temperature and centrifuged at 4000 rpm for 20 min for arsenic compound extraction. The supernatant was collected, and the residue was washed ultrasonically assisted with 10 water/methanol twice. The obtained combined supernatants were evaporated at 37 °C to dryness by Rotavapor, and the dry residue was redissolved in 20 mL of water and centrifuged. The final supernatant was filtered through a 0.22-μm nylon membrane. Efficiency of extraction was checked by analysing NIST 1568a-certified rice flour as standard reference material. The recovery was 92 % of As(III) and 94 % of As(V) (103 % of total As). Paik et al. (2010) used a very similar procedure to perform extraction and quantification of arsenic species in polished rice, obtaining very good validation results.

Analysis of arsenic species in soil, root and shoot plant extracts was carried out by HPLC (Agilent, LC 1100 series) interfaced with the described ICP–MS, according to the method described by Van den Broeck et al. (1998). Arsenic species were separated using an anion-exchange As speciation column (Agilent G3154-65001), fitted with a guard column (Agilent G3154-65002). The mobile phase was a solution of 3 mM NaH2PO4 and 0.2 mM Na2-EDTA (pH 6.0), which was pumped through the column isocratically at 1 mL min−1. The outlet of the separation column was connected to a MicroMist nebulizer and a Peltier-cooled spray chamber of the ICP–MS. Signals at m/z 75 (As) and 35 (Cl) were collected with a dwell time of 500 and 10 ms, respectively. Any possible polyatomic interference of 40Ar35Cl on m/z 75 was removed as for total arsenic. The calibration standards used for As speciation were previously controlled for their total As concentrations. Analysis of the samples at the beginning of the run was repeated at the end, and no changes in As speciation were observed during this period.

Statistics

Analysis of variance (ANOVA) and post hoc analysis with Tukey HSD test were performed using SAS (1996).

Results and discussion

Arsenic species in rhizosphere

The mycorrhizal colonization was satisfying (more than 60 % of mycorrhizal frequency estimated on the basis of aniline blue staining) for all the AM+ trials. No AM colonization was detected in the non-inoculated plants. The concentrations of As(III) in the soil layers are shown in Fig. 1. The As(III) concentration was very low, less than 1 % of total As (about 200 mg kg−1); thus, total As was mainly represented by As(V) in the studied soil. In the control (AM−/P−) rhizoboxes, no statistically significant differences were found in As(III) concentrations among the layers. A significant difference (p < 0.05) in As(III) concentration between the 0–2-mm layer and the other two layers was found for all treatments with respect to the control. Differences among treatments for the same layer were not significant. The effect of P on arsenic speciation in soil was less perceptible than that of AM. In any case, the effect observed for such nutritive element could be either direct, on As chemistry in soil, or indirect, through its interaction with development of both the roots and the rhizosphere microorganisms in the polluted soil. Regarding As speciation in the soil, Carbonell-Barachina et al. (1998) highlighted that the decreases of pH and redox potential induced in the rhizosphere by Spartina spp. may produce reduction of As(V) to As(III). Ultra et al. (2007) demonstrated that As(III) and As(V) showed gradients toward the root surface of Helianthus annuus L. and that the increase was more noticeable for As(III) in the P+ trials, and for As(V) in the AM+ trials, with results conflicting with those ones about our plants. To this end, Gonzalez-Chavez et al. (2002) reminded that the endomycorrhizal fungus Hymenoscyphus ericae, responsible for the colonization of arsenic-contaminated mine spoils by its host Calluna vulgaris, achieves the resistance to As(V) of its symbiont by reducing cellular As(V) to As(III) and then effluxing the As(III), which is also the mechanism used to achieve As(V) resistance in bacteria and yeasts.

Fig. 1
figure 1

Concentrations of As(III) in the soil layers collected at the end of the rhizobox experiments from the different trials. Black bars are for 0–2 mm, grey bars are for 2–10 mm, white bars are for bulk soil, AM − /P − not inoculated and not P-fertilized, AM+/P − inoculated and not P-fertilized, AM − /P+ not inoculated and P-fertilized and AM+/P+ inoculated and P-fertilized. Values are mean of three replicates. Significant differences (Tukey HSD test, at least p < 0.05) appear with different letters, uppercase for inter-treatment and lowercase for intra-treatment comparisons

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Most probably, the effect of P and AM treatments could be species and soil specific, peculiarly and simultaneously affecting not only the root activity but also the rhizosphere microbial activity, thus resulting in specific effects on As(V) reduction. In this context, and relatively to the microbiological aspect, Fitz and Wenzel (2002) already showed that the number of microorganisms in the rhizosphere is typically one order of magnitude greater than in the bulk soil, and the reduction of As(V) to As(III) may be also a widely distributed process of soil microbes.

Rahman et al. (2009), after having demonstrated the effectiveness of the extractant chosen (orthophosphoric acid) for both organic and inorganic arsenic extraction by soil, analysed 20 different soil samples from an As-contaminated area of Bangladesh and found that DMA was barely detectable as the only organic species in just two samples. It is normally expected in aerobic conditions, like in our case: as reminded by Fitz and Wenzel (2002), methylated arsenicals are generally produced under anaerobic conditions, and in very few cases trimethylarsine oxide (TMAO) and arsenobetaine (AB) have been detected as minor compounds in soil extracts. Regarding the organic forms of As, DMA and MMA were not detectable in any of our trials.

Plant growth and arsenic and phosphorus concentrations

For the P+ trials, germination rate and root and shoot dry biomass were significantly higher than for the P− trials (Table 1). Such effect might be ascribed to improved phosphorus nutrition, even if at the same time P-mediated alleviation of soil As toxicity could not be excluded. Gulz et al. (2005) reported similar results after arsenic application on two different soils, cultivated with maize (Z. mays), English ryegrass (Lolium perenne), rape (Brassica napus) and sunflower (H. annuus). Our findings, on the other hand, partially conflict with the results of Ultra et al. (2007), who observed that the As toxicity symptoms of sunflower (H. annuus L.), cultured in rhizoboxes, were most clearly alleviated in the AM+/P− treatment, whereas plant growth was promoted most strongly in the AM+/P+ treatment.

Table 1 Root and shoot dry biomass, germination rate and germination time of maize exposed to the different treatments (means of three replicates). Data with the same letters were not significantly different at Tukey test (p = 0.05)
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Figure 2 reports the concentrations of arsenic (total As, As(III) and As(V)) and P detected in maize after the different treatments. For what concerns total As, in all the treatments its shoot concentration was below the toxicity limits estimated by Kabata-Pendias and Pendias (2001). This result could be the effect of a very low bioavailability of the soil arsenic combined with a very strong retention of arsenic in the roots, the latter suggested by the huge difference in its root and shoot concentrations. Recent findings (Zhao et al. 2009) show that rice (Oryza sativa) is particularly efficient in As uptake from paddy soil, leading to accumulation in grain at concentrations hazardous for health. The same authors remind that As is generally only partially translocated from roots to shoots in plants, except in hyperaccumulators: for example, in wild-type Arabidopsis thaliana, only 2.6 % of As taken up by roots was found in shoots. The explanation is that arsenate is reduced to arsenite rapidly in roots, probably followed by complexation with thiols and possibly sequestration in the root vacuoles. The arsenic uptake and accumulation characteristics of maize, together with Chinese cabbage and tomato, were investigated by Yan et al. (2013) by using culture solutions with different arsenic concentrations. The transfer coefficient of maize exhibited a rising tendency with increasing arsenic concentrations, whereas in Chinese cabbage and tomato it increased and then declined.

Fig. 2
figure 2

Concentration of As and P in shoots and roots at the end of the rhizobox experiments. a Total As concentration. Black bars are for roots and white bars are for shoots. b As(III) and As(V) concentration. Light grey bars are for As(III) and dark grey bars are for As(V) in roots and shoots. c P concentration. Black bars are for roots and white bars are for shoots. AM−/P− not inoculated and not P-fertilized, AM+/P− inoculated and not P-fertilized, AM−/ P + not inoculated and P-fertilized and AM+/P+ inoculated and P-fertilized. Mean of three replicates. Values with different letters are significantly different at Tukey HSD test (p = 0.05)

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The low shoot arsenic levels observed by us (<1 mg kg−1 dry weight) were extremely encouraging for cultivating maize on a marginal land like the one studied here, even if further analyses are needed to assess the effective arsenic concentration in the maize edible parts. Many studies on arsenic around the world (Bhattacharya et al. 2010) have found that arsenic concentration in plants from arsenic-contaminated agricultural lands varied from less than 0.01 to about 5.0 mg kg−1, as in our study. The same authors reported that, in arsenic-affected areas of Murshidabad, West Bengal, India, the accumulation of arsenic in various food composites (potato skin, leaves of vegetables, rice, wheat, cumin, turmeric powder and cereals) ranged between <0.0004 and 0.693 mg kg−1. Rice grain can reach arsenic accumulation much above the WHO-recommended permissible level of 1.0 mg kg−1. Studies on the agricultural plants of China and Nepal have found that arsenic accumulated in the range 0.003–0.116 and <0.01–0.55 mg kg−1 dry weight, respectively. Similarly, a study of different plant parts in soils with varying arsenic levels found that the amount of arsenic in root crops, such as potatoes and onions, was correlated with the amount of arsenic in the soils in which they were grown (Dahal, et al. 2008) but did not exceed the FDA standard. Huang et al. (2006) demonstrated that radishes and onions accumulated arsenic when grown in soils with approximately 1 to 25 mg kg−1 of arsenic, although not in concentrations of concern. Finally, another study by Gaw et al. (2008) found that radishes and lettuce grown in soils that had formerly been treated with arsenical pesticides also accumulated arsenic, although not in concentrations that exceeded the FDA standard.

In all our treatments, arsenic concentration did not change in shoots, whereas in roots it significantly decreased (P < 0.05) with P application, in the absence of AM.

To evaluate if the differences in element concentrations could be related to any difference in the uptake, the apparent element uptake was calculated as in Arnetoli et al. (2008) and expressed as the total amount of element per plant per g root d.w. (Table 2). Arsenic uptake was significantly lower only in AM−/P+ treatment in respect to the other ones, and this effect, together with the data on element concentrations, intriguingly suggests a probable apparent nullification of the P effect in the presence of AM, either on plant As concentration or on apparent uptake. These results are partially in conflict with those of Ultra et al. (2007), who cultured sunflower (H. annuus L.) in rhizoboxes for 6 weeks and found that P application increased water-soluble As in the rhizosphere and also that shoot As concentration was slightly reduced by AM inoculation but enhanced by P application. In a long-term (2 months) study like the one presented in this work, Pigna et al. (2009), on the other hand, observed that P fertilization minimizes the translocation of As to the shoots and grain of wheat (Triticum durum L.), grown in uncontaminated soil irrigated with solutions containing As at three different concentrations. Also, the studies of Puckett et al. (2012) on arsenate uptake by an As-tolerant willow (Salix viminalis × Salix miyabeana) and an As-sensitive willow (Salix eriocephala) in hydroponic systems indicate that the presence of phosphate generally decreased plant uptake and translocation of arsenate.

Table 2 Apparent uptake for P and As, expressed as the total amount of element per plant per g root d.w. (means of three replicates). Data with the same letters are not significantly different at Tukey test (p = 0.05)
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As for the plant concentrations of the two arsenic forms (Fig. 2), the different treatments did not affect As(III) concentration in the roots and significantly increased it in the shoots. As(V) concentration was decreased in the case of AM−/P+ treatment in roots and in all the treatments in shoots. Therefore, the decrease in total As observed in roots after the P treatment could be ascribed to the well-known competition of P with As(V), which was able to produce a lowering in its accumulation in the roots. Most probably, the unaffected shoot concentration was due to the very low shoot As level that maize showed. Such concentrations were so low to probably obscure any possible effect of reduced As(V) root accumulation on shoot As concentration itself, at least for the experimental conditions used. Anyway, the decreased root concentration was enough to determine a lower apparent uptake at the level of the whole plant (Table 2).

Intriguingly, data on the concentrations of the different As forms showed that the apparently unaffected shoot total As concentration after the treatments was the result of an increased As(III) concentration and of a lowered As(V) concentration. As the uptake of As(III) was unchanged after the treatments, and that one of As(V) changed only in the AM+/P− case for the abovementioned reason, the changes in the As(III) and As(V) concentration in the shoots might be ascribed to an increased reduction of As(V) in this organ after the treatments. Probably, such higher efficiency of arsenic reduction could be a direct effect of the ameliorated plant growth in the case of P treatment, with or without AM. The fact that also the AM treatment alone produced such result, without any apparent effect on plant growth, remains hard to explain. Furthermore, our data did not depose in favour of any possible effect of mycorrhizal inoculation on the selective uptake and transport of different As species, leading to different accumulation of As species in maize, unaccountably in contrast with what was reported by Yu et al. (2009), who found that G. mosseae generally decreased concentrations of As(III) in maize roots and concentrations of As(III) and As(V) in the shoots. They also measured the arsenate reductase activity that was detected in maize roots and was reduced with mycorrhizal inoculation.

With respect to control and to P trials, phosphorus concentrations were significantly higher in roots and shoots only for AM+/P− trials (Fig. 2). As the P apparent uptake was higher in the AM+/P− treatment (Table 2), the difference in P concentration might rely on the improvement in P nutrition by AM. Interestingly, AM treatment increased the plant P concentration without any effect on plant growth. This lack of a higher plant biomass production in consequence of a higher P concentration was not due to a possible increase also in As concentration in the plant tissue. The higher P concentration resulted only in an increase in As(III) concentration and decrease in As(V) concentration in the shoots, without any apparent effect on plant growth. Our results were only partially in agreement with other reports. For example, Wang et al. (2008) verified that AM-treated maize had higher shoot biomass and root P and As concentration than the untreated.

In the P+ trials, the opposite situation occurred and higher plant growth was not coupled with higher P concentration in plants. Such effect could probably be a consequence of the higher plant biomass production stimulated by the higher P availability itself. For this reason, in the treatment AM+/P+, an increase in P concentration and uptake was not detected as it occurred only in the case of AM presence, the P-induced higher biomass production, obscuring the effect of AM on P accumulation and uptake themselves. In our plants, probably when P was supplemented directly, the plant readily absorbed it, and such increased its growth, thus rendering not detectable the higher amount of P taken up. On the contrary, when it was AM treatment to improve P nutrition, the process could have been slower and P could have had the time to reveal its higher accumulation inside the root and the shoot before having its effect on growth amelioration. This aspect could be very important to bear in mind in the use of AM.

Conclusions

The presence of arsenic in the examined soil was considerable, but not of concern, as speciation suggested that availability of this trace element was low and its concentration in the plant shoots was below the toxicity limits. Treatments with AM and P, separately or in combination, increased only moderately As(III) concentration close to the maize root. In plant shoots, all the treatments were able to increase the concentration of As(III) and decrease the concentration of As(V), but the shoot total concentration of this element did not change. In roots, only the treatment with P was able to decrease As(V) concentration. Moreover, the results of our study showed that phosphorus application at agronomic rate was much more effective than AM to improve the plant biomass production. This fertilization could be successfully used for cultivating maize on marginal lands like the one under investigation, whereas the effect of arbuscular mycorrhiza inoculation seemed to be of secondary relevance, at least for the experimental conditions applied. Further studies would be required to assess if different levels of fungal infection could be more helpful for plant resistance and growth.