Acta Bot. Croat. 79 (1), 3–14, 2020 CODEN: ABCRA 25 DOI: 10.37427/botcro-2020-001 ISSN 0365-0588 eISSN 1847-8476 The effect of salinity gradient and heavy metal pollution on arbuscular mycorrhizal fungal community structure in some Algerian wetlands Warda Sidhoum1,2*, Kheira Bahi2,3, Zohra Fortas1 1 Laboratory of Microorganisms Biology and Biotechnology, University of Oran 1 Ahmed Ben Bella, Oran, Algeria 2 Abdelhamid Ibn Badis University, Mostaganem, Algeria 3 Department of Biology, Faculty of Natural Science and Life, University of Oran 1 Ahmed Ben Bella, Oran, Algeria Abstract – Algerian natural wetlands suffer from anthropogenic disturbances due to industrial development and urbanization. This study was designed to draw attention to arbuscular mycorrhizal fungi (AMF) distribu- tion and community assemblages following heavy metal and salinity concentrations in two wetlands subjected to domestic and industrial effluents. Rhizospheric soil and roots of 18 plant species were collected in two wet- lands along a decreasing salinity gradient. The results showed that 72.72% of plant species exhibit an association within arbuscular mycorrhizas (AM), and 36.36% a dual association between AM and dark septate endophytes (DSE). A total of 33 AMF morphospecies were distinguished on the basis of morphological criteria dominated by taxa belonging to Glomeraceae and Acaulosporaceae. Soil contamination was investigated by determining metallic trace elements (MTE) (Cd, Cu, Ni, Pb, Cr and Zn) using an atomic absorption spectrophotometer. Val- ues of the pollution index revealed wetlands that were particularly polluted by lead. Two-way ANOVA showed significant variations in metal content among sampling locations and transects. Principal component analysis showed that species richness, and mycorrhizal frequency were slightly affected by MTE. This opens possibilities for their utilization in polluted soil remediation. Keywords: Dark septate endophytes, metallic trace elements, mycorrhizal association, saline wetlands, soil pollu- tion Introduction Oran is located in the north-west of Algeria, and con- presence of flooded or water saturated soils for at least part stitutes a wetland complex of eight zones, four of which of the growing season. These natural hydrosystems have are classified as of International Importance. Great Sebkha halophylic plants, such as Amaranthaceae (Ghodbani and and Macta (since 2001), Telamine Lake (LT) and les Salines Amokrane 2013, Megharbi et al. 2016). However, these eco- d’Arzew (since 2004), while others, though not be classi- systems can be modified by various factors, among them fied as Ramsar, have nonetheless received attention from aridity causing changes in soil properties and trace element the Ramsar Convention (Chenchouni and Si Bachir 2010). pollution. This is due to human activities, including, ur- These wetlands are ecologically important ecosystems, pro- banization processes, domestic sewage discharges, livestock viding important winter grounds for several world popu- wastewater and industrial effluent (Bouldjedri et al. 2011, lations of endangered bird species. In particular, species Domínguez-Beisiegel et al. 2016). belonging to the Anas and Tadorna orders, overwinter in Several studies have shown that arbuscular mycorrhizal significant numbers in these areas (Boucheker et al. 2011, fungi (AMF) exist in the roots of wetland plants and woody Samraoui et al. 2015). species grown on flooded soils (D’Souza and Rodrigues The wetland soils are mostly Solonchak types, contain- 2013), in aquatic macrophytes, freshwater wetland plant ing large amounts of exchangeable sodium and soluble salts communities and salt marshes (Xu et al. 2016). It has also (Benziane 2013). These habitats are characterized by the been shown that AM fungal diversity in wetlands is com- * Corresponding author e-mail: [email protected] ACTA BOT. CROAT. 79 (1), 2020 3 SIDHOUM W, BAHI K, FORTAS Z parable to that of most terrestrial ecosystems and for the sect was divided into three plots according to the salinity growth and development of wetland plant species, and thus gradient and plant distribution: from 0 m (where no vegeta- AM fungi are functionally essential (Tuheteru et al. 2015). tion was present) to 30 m, and electrical conductivity (EC) The primary abiotic factors: soil flooding, nutrient, oxy- varying between 6 to 9.5 dSm–1, and 30 m to 60 m (2 dSm–1 gen availability, salinity, and high levels of heavy metals in <EC<6 dSm–1), and more than 60 m (EC<2 dSm–1). soil strongly affect the abundance and distribution of AM A total of 24 soil samples representing 12 plots in each fungi in aquatic ecosystems (Millar and Bennett 2016). As wetland were selected. From each plot, one soil sample was previously reported, the intraradical and extraradical my- used for chemical analysis. Also, five replicates per plant celia of metallic stress adapted AMF isolates are capable of species of rhizospheric soil and roots of dominant plant sequestering heavy metals and alleviating metal toxicity to species were sampled as well. In order to establish AM fun- plants (Cabral et al. 2015). This indicates that these fungi gal diversity, about 500 g of rhizospheric soil was collect- have evolved a tolerance to metallic trace elements (MTE), ed from topsoil (10 to 30 cm depth), put into plastic bags, and play a role in the phytoremediation of metal polluted air-dried and stored at room temperature at the labora- sites, even in polluted aquatic and semi-aquatic habitats tory until use. (Wężowicz et al. 2015). Plant species encountered were recorded, with their rel- A number of surveys on AMF associated with wetland ative abundances estimated visually and rated on a scale of plants have been performed in order to investigate their di- I (very rare) to V (very abundant) (Bradai et al. 2015). The versity and colonization potential (D’Souza and Rodrigues determination of herbarium specimens for floristic inven- 2013, Kumar and Muthukumar 2014) along a soil hydrologi- tory was carried out using Flore de l’Afrique du Nord (Maire cal gradient (de Marins et al. 2009, Miller and Bever 1999, 1958–1976), and a previous study used as a reference on Turner et al. 2000), salinity (Roda et al. 2008, Saint-Etienne saline wetlands vegetation of Oran region (Quézel and Si- et al. 2006, Yang et al. 2010), or nutrient content (Cornwell monneau 1960). Plant nomenclature was brought up to date et al. 2001, Jayachandran and Shetty 2003). There has been according to the Synonymic Index proposed by Dobignard a small amount of research focused on AMF communities and Chatelain (2010-2013). in MTE polluted wetlands (Carrasco et al. 2006, Ban et al. 2017), but there has been no report of the simultaneous ef- Chemical analyses of soil fect of salinity and heavy metals on AMF distribution in Soil samples were dried at room temperature and sieved wetland habitats. The present study, therefore, was aimed in a 2 mm mesh size sieve. Dried soil samples were analyzed at evaluating the AM fungal diversity in heavy metals pol- for pH and electrical conductivity on 1:2.5 (soil: distilled wa- luted saline wetlands and at adding to knowledge on these ter suspension ratio) (Mathieu and Pieltain 2003). populated areas with heterogeneous plant species that have rarely been considered mycorrhizal, as well as at exploring Trace elements were extracted according to the aqua re- the impact of soil salinity gradient and trace element pollu- gia method ISO 11466: 1995. About 0.5 g of dried soil at tion on AMF community structures. 105 °C was digested in 10 mL of freshly prepared aqua re- gia solution (1/3 of HNO3 and HCl, v/v) on a hotplate for 2 h at 100 °C. After evaporation to near dryness, the extracts Materials and methods were cooled to room temperature before being filtered. The Study area filtrates were further transferred to 25 mL volumetric flasks and brought to volume with distilled water, then stored at This survey was carried out in two wetlands located in 4 °C until the spectrophotometric measurement using the Oran city (western region of Algeria), namely Telamine Lake AAS Shimadzu AA–7000 atomic absorption flame emission (LT) (35°42’50”N 0°23’30’’W) located in the district of Gdyel spectrophotometer. (eastern Oran) at 7 km from Hassi Amer industrial Zone II. Dayet Morsli (DM) (35°39’58”N 0°36’27”W) located in Es- Soil pollution index Sénia district in the south of Oran at a distance of 2 km north of the industrial Zone I of Es-Sénia. The altitude ranged be- Multiple contaminations by metallic trace elements in- tween 50 and 87 m a.s.l. The regional climate is of the semi- crease soil toxicity. Soil pollution index is the criterion that arid Mediterranean type characterized by a cold and rainy allows us to evaluate soil toxicity, to classify the soil contami- winter followed by a hot dry summer spread over 4 to 6 con- nation and to assess potential ecological risk (Müller 1979, secutive months where the average temperature varies be- Belabed et al. 2014). This index is calculated by the ratio of tween 14.1 °C and 22.5 °C and precipitation varies between heavy metal content in the soil (ppm) based on the corre- 250 and 400 mm per year. sponding values, according to the following formula pro- posed by Kloke (1979): Sampling Cd Cu Pb Zn Cr Ni +++++ The investigations were conducted at each site (LT and PI = 2 100 100 300 150 50 DM) along four transects 200 m long and 10 m wide. Start- 6 ing from the wetland water edge to the periphery. Each tran- where PI>1indicates that the soil is polluted. 4 ACTA BOT. CROAT. 79 (1), 2020 AMF DIVERSITY IN WETLAND STRESSES Assessment of AMF and DSE colonization sporen umbero fa speciesg ()enus RA = ×10 Young roots (with root tips) were washed in tap water to totaln umbero fi dentifiedds pore samples remove soil particles, and then fixed in FAA formalin, glacial acetic acid, and ethanol (1:1:18, v/v/v).