Mycorrhizal status of Argania spinosa (L.) Skeels in northeastern of Morocco

The argan tree, an endemic species in Morocco, has been exhaustively studied in its southwestern range but neglected in its northeastern territory. Thus, the present study sets as objectives the identification of arbuscular mycorrhizal fungi and the evaluation of the argan tree’s roots’ mycorrhization level in two stations: one in the region of Béni Snassène (Berkane): Jebel Takermine with 7 prospected sites and the other at Jebel Aklim Alkabir with 2 sites. At the first station (Jebel Takermine), the mycorrhizal frequency of Argania spinosa roots varied between 64% and 100% with the root mycorrhizal intensity in the range of 30.77% and 66%. The arbuscular contents are higher at sites 2 (50.46%), 4 (50.33%), 7 (39.44%) and 1 (30.5%) against 18% and 20% at sites 6, 5 and 3. Argan trees from Jbel Aklim Alkbir, exhibited a high mycorrhization frequency and intensity ranging from 88% to 100% and between 39.4% and 73.4% respectively. Regarding arbuscular and vesicular rates, the highest values were associated to the roots of site 1 with 59.3% and 29.4% respectively compared to the lowest rates of 20% and 14% in those of site 2. Spore densities in the rhizosphere of the studied argan trees in the two stations were in the range of 78 and 697 spores/100 g of soil. The identification of isolated mycorrhizal spores revealed the presence of 28 species encompassing 7 genera: Acaulospora, Dentiscutata Claroideoglomus, Funneliformis, Glomus, Rhizophagus, Pacispora, and 5 Families: Glomeraceae (7 species), Acaulosporaceae (10 species), Pacisporaceae (2 species), Claroideoglomeraceae (2 species), Gigasporaceae (1 species). Maazouzi S et al. (2023). Not Sci Biol 15(2):11405 2


Introduction Introduction Introduction Introduction
The argan tree (Argania spinosa L. Skeels), exclusively endemic to the dry lowlands of Southwest Morocco, is of particular interest due to its ecological role and its many nutritional and cosmetic uses (Charrouf et al., 2009). It occupies an area of nearly 830,000 ha in southwest Morocco (M'Hirt et al., 1998). Thus, Morocco is one of a few countries in North Africa to have a range of remarkable endemic biodiversity ecosystems Boudy (1950) andEl Fasskaoui (2009).
The distribution area of argan trees in the northeastern sector of Morocco is much more complex and remains less studied (Charrouf et al., 2009). Previous works of Emberger (1925), El Alaoui (1999 and Quézel et al. (1992) with those of Benabid (1985), Haloui (1991) and Reda Tazi et al. (2003) that addressed the study of this stand in the east, claimed that it has not yet been precisely determined. The prospecting investigations carried out by Faouzi et al. (2014) in the northeastern argan tree area of Morocco made it possible to highlight a synthetic map of the argan formations' geographical area indicating its current state at the level of the western Beni-Snassen piedmonts and for the first time, its presence at the level of the eastern Rif on the Bou-Areg plain (Bled Arimane and Ouelad Mohand in Kari and Arekmane).
The argan tree grows well in the Souss plain, on the southern slopes of the Western High Atlas and on the northern and southern slopes of the Western Anti-Atlas up to altitudes of 1300 to 1500 m (Msanda, 2005). Beyond this geographical location, two small areas of the argan tree are registered in the upper Grou valley southeast of Rabat and in the northwestern piedmont of the Beni-Snassen, close Oujda (El Mousadik and Petit, 1996). There are only some argan trees scattered in the oriental sector where its plantations do not currently exceed 150 ha (Dommergues and Mangenot, 1970). Therefore, this area's expansion is recommended. The expansion will be necessary to mobilise the local forestry sector, but also to encourage the planting of argan trees on private land. Much work has been undertaken to optimise the plants growth in harsh environments and to control certain soil components likely to contribute to the rehabilitation of these degraded ecosystems (Pearson, 1982;Strutlu, 1991). Among the telluric components in particular involved in biological processes are mycorrhizal fungi. They establish a symbiotic relationship with argan plants to improve the efficiency of water drawn by the roots and succeed in improving the efficiency of water uptake by their roots and subsequently improve their nutrition and growth (Dommergues and Mangenot, 1970;Gianinazzi Pearson, 1982;Strutlu, 1991).
The objective of the present paper is to determine the biodiversity of the argan tree's endomycorrhizal fungi in the region of Beni Snassène (Berkane), where a few of this endemic species' saplings are encountered sparsely. This mycorrhizal biodiversity is seen for the first time in this area.

Sampling sites
Soil samples were collected from the rhizosphere of the argan trees located in the Beni Snassène region (Berkane) at two stations spaced 8.7 km apart: Jebel Takermine and Jebel Aklim Alkbir (Figure 1). In station 1, where the argan tree is generally well preserved, sampling was carried out at 7 sites of which four sites (1, 2, 3 and 4) are located on the mountain crest, two on the banks of the river (sites 5 and 6) and the last one (site 7) is located 14 m from up of the river (Oued); the argan trees of site 7, unlike those of the other sites, are stunted by grazing and present a dwarfed appearance. In station 2 where the argan tree is very degraded, sampling was done at 2 sites. The first one was done from the rhizosphere of argan trees deprived of thorns while the second site is featured by rocky substrate.
Samples are collected at a rate of 1 kg of soil per tree, at a depth of 0 to 20 cm, and a composite soil sample is taken per site. Very fine roots, more likely to be mycorrhized and more easily observable under the microscope are collected at the same time as the soil (

Root staining
The root samples taken from the argan trees' rhizosphere (at a depth of 20 cm), were removed from the substrate by washing them thoroughly with running water in a sieve, and only the finest roots were selected.
According to the thinning and staining technique of Philips and Haymann (1970), the roots were cut into segments of 1 cm length, then immersed in a solution of 10% KOH (potassium hydroxide) and placed in a water bath at 90 °C for 15 min. Then, the root segments were bleached by adding a few drops of H2O2 to the KOH solution. After 15 min, root fragments were rinsed then stained with cresyl blue, at 90 °C for 15 min. After the final rinse, thirty fragments were randomly selected and mounted, in groups of 10 to 15 segments, in glycerine between slides and coverslips.

Evaluation of mycorrhization rate
The evaluation of mycorrhization parameters was performed by observing thirty fragments of dyed roots of 1 cm length randomly selected (Trouvelot et al., 1986;Amir and Renard, 2003) and mounted, in groups of 10 to 15 segments, in glycerine between slide and coverslip.
The slides were examined under a microscope with each fragment being thoroughly checked over its entire length, at magnifications settings of x 100 and x 400 to observe and to note mycorrhizal frequency and the mycorrhizal structures (arbuscules, hyphae, vesicles, external hyphae, intra and intercellular hyphae and 4 even the endophytes structures). Vesicular and arbuscular frequencies and the content of endomycorrhizal fungi inside the roots were measured assigning a mycorrhization index ranging from 0 to 5 (Derkowska et al., 2008), 0: absence; 1: traces; 2: less than 10%; 3: 11-50%; 4: 51-90%; 5: more than 91%.

Spores' extraction
The spores were extracted by the wet sieving method described by Gerdemann and Nicolson (1963). In a beaker of 1L, 100 g of each soil sample was submerged in 0.5 L of tap water and it was stirred with a spatula for 1 minute. After 10 to 30 seconds of decantation, the supernatant was passed through four superposed sieves with a decreasing mesh size (500, 200, 80, and 50 microns). This operation was repeated twice. The content picked up by each mesh screen (200, 80 and 50 microns respectively) was transferred to centrifuge tubes and centrifuged at 2000 RPM for 5 min. The supernatant was discarded and a viscosity gradient was created by adding 4 ml of a solution of 40% sucrose in each centrifuge tube. The mixture was quickly stirred and the tube was handed back into the centrifuge for 1 min at 2000 RPM for 1 min, then a third centrifugation at 3000 RPM for 1 min was made. Unlike the first centrifugation process, the supernatant was poured into the sieve mesh of 50 microns; the substrate was rinsed with distilled water to remove the sucrose, and then disinfected with an antibiotic solution (streptomycin). The spores were then recovered with distilled water in an Erlenmeyer flask.
After extraction, the endomycorrhizal spores are counted and identified based on morphological characteristics. An estimation of the number of spores in the soil is made by counting the spores in 100 g of soil and extrapolating to the total volume (100 ml).

Richness and appearance frequency
Species richness is the total number of the observed species per site and the occurrence frequency of species corresponds to the percentage of sites where each species is detected.

Statistical analysis
The statistical treatment of results focused on the analysis of variance to a single criterion of classification (ANOVA).

Results Results
Results and Discussion and Discussion and Discussion and Discussion The results of the present study showed that the argan trees examined in the different forests were colonised by AMF. However, the AMF colonisation status varied significantly depending on sampling point. Roots collected at the level of the rhizospheric soil of argan trees in two studied stations revealed the presence of different mycorrhizal structures (hyphae, vesicles and arbuscules) and endophytes (Figure 2). The interaction extent of roots was marked by mycorrhizal frequencies and intensity in the range of 64% -100% and 31%-66% respectively in the prospected sites of the first station (Figure 3). As for arbuscular contents, they were higher at sites 2, 4, 7 and 1 with 50.46%, 50.33%, 39.44% and 30.5% respectively, while they were between 18% and 20% at sites 6, 5 and 3. Similarly, the vesicular contents varied from one site to another, with values ranging from 9.33% to 50.33% (Figure 4). In the second station, the infection degree expressed as mycorrhization frequency (or the roots colonisation level) matching to mycorrhizal intensity were high, varying between 100%-88% and from 73.4% to 39.4% respectively ( Figure 5).
The arbuscular and vesicular contents varied with the highest values of 59.3% and 29.4% were recorded respectively in site 1 while the lowest contents in the order of 20% (arbuscules) et 14% (vesicles) were registered in site 2 ( Figure 5). Likewise, Sellal et al. (2016), have detected a community of arbuscular mycorrhizal fungi in relation to argan trees sampled in two sites located in southern area where the frequency and intensity of mycorrhization of Argan trees reached 100% in the Toufalazt and Taroudant sites, nearly 97% in the sites of Elkhssass and Essaouira. Hence, the presence of diverse endomycorrhizal structures indicated that AMF had established effective symbiotic relationships with this tree species allowing us to classify the argan tree as a mycotrophic species.
Regardless of argan tree variety (thorny or spineless), a seasonal variation of arbuscular and vesicular rates during winter and spring seasons was reported by El Adib (2015). Indeed, many studies have shown that mycorrhizal symbioses with vesicles and arbuscules improve the growth of young plants (Nouaim and Chaussod, 1996;Giri et al., 2003;Citernesi et al., 1998;Atkinson et al., 2002) and induce morphological and physiological transformations that allow them to tolerate their environment's conditions (Porcel et al., 2006). Nicolson (1960) found that the intensity of colonisation is related to variation in soil organic matter content. According to Wang et al. (2019), AMF colonisation intensity in forest ecosystems was significantly and negatively correlated with organic matter content. AMF hyphae improve the water and mineral nutrition of the host plant by increasing the volume of soil prospected, and by mobilising phosphorus from complex soil compounds (Munns et al., 1981;Malaisse, 1979). The species richness of the argan trees' AMF differed widely among stations. The mean number of AMF spores ranged between 78 to 697 spores/100 g of soil.
At the same time, our assessment of the species' richness revealed some differences amongst the studied sites and stations. This variation has also marked the investigated sites of southern Morocco (Sellal et al., 2017;2021). A similar trend was observed for the number of spores in a 100 g soil; thereby, the highest spore density (585 spores/100 g soil) was related to site 4 of the first station followed by 518 spores/100 g soil, 204 spores/100 g soil, 201 spores/100 g soil, 154 spores/100 g soil, 101 spores/100 g soil, and 78 spores/100 g soil, detected respectively in sites 2, 5,7, 1,6 and 3. Dentiscutata nigra and Endogone versiformis were the most common species with the respective frequency of occurrence of 32.13% and 15.77%. In comparison, spores' number/ 100 g of soil was of 697 spores/100 g soil recorded in site 1 of the second station greater than that of site 2 in the order of 169 spores/100 g soil with a dominance of the genus Dentiscutata nigra (32.13%) and Acaulospora denticulata (14.77%) (Figure 7). These two species were more prevalent than Glomus etunicatum (16.26%), Acaulospora gedanensis (10.52%) or Glomus macrocarpum and much more represented in the AMF community structure in southern area (Sellal et al., 2017). Regarding our findings about the number of spores extracted from the soil of argan trees, they seem to be lower than those cited by El Maati et al. (2015) who found 1127.66 spores/100 g soil and Nouaim et al. (1991) work in the subsoils of Argania spinosa in south-west Morocco reporting a number of 900-2080 spores / 100 g soil. Weak densities in the range of 84 and 160 spores /100 g of soil related to the carob rhizosphere, were also reported in research results of El Asri et al. (2014) in five provinces covering east to south-west Morocco (Taroudant, Khenifra, Azilal, Beni Mellal and Nador).
Moreover, it is known that the same fungus can colonise many plant species. Conversely, a plant can be colonised by several species of AM fungi, sometimes at the same time (Steinkellner et al., 2007).
The relative low diversity and species richness is probably linked to the state of argan tree stands. According to Faouzi et al. (2014). The distribution of Argania spinosa at the piedmont of the western Beni-Snassen in the eastern Rif is presented as a matorral degraded by zone.
In fact, mycorrhizal associations may result in profound modifications of root structure and functioning for efficient acquisition and use of water by plants (Harley and Smith, 1983). Multiple factors influence the dynamics and structure of the AM fungal community such as soil texture, particle distribution, size, porosity, water retention capacity, cation exchange capacity, organic matter content, pH, macronutrients and micronutrients (Mohammad et al., 2003;Chaudhary et al., 2008). Additionally, the number of AMF propagules estimates is linked to experimental conditions as temperature and harvesting time, which may affect root and propagule growth (Wilson and Trinick, 1982). In this context, Mohammad et al. (2003) claimed that spore numbers are negatively correlated with soil phosphorus levels. Indeed, the good functioning of mycorrhization is noted under conditions of P-limitation especially. It was also demonstrated that the host plant presents greater mycorrhizal potential and soil infectivity where there is a deficiency of soil nutrients (Meddich et al., 2017).
Many plant species associated with endomycorrhizae exhibited a high tolerance to drought stress such as acacias (Maazouzi et al., 2020;Diem et al., 1981), Berberian thuja (Díaz and Honrubia 1993;El Khaddari et al., 2020), Argan (Nouaïme et al., 1991), Oleaster, Carob, Date Palm. This is due to the relevant role of vesicular and arbuscular endomycorrhizal allocation the plant to acquire mineral elements, especially those that are poorly mobile in the soil, such as phosphorus, copper and zinc (Strullu, 1991;Harrison, 1999). It is worth noting that the availability of water is the main environmental factor that limits forest production. Moreover, soil phosphorus availability is a limiting factor in the establishment of mycorrhizal symbiosis (Chen et al., 2007). The first mechanism by which symbiosis promotes the water regulation of trees is through its effect on mineral nutrition. If a fungus is particularly effective in phosphorus supply or potassium, it will indirectly contribute to the plant to better manage water (Bondonga et al., 2011). Nevertheless, (Wang et al., 1993) have also demonstrated the effect of pH level on the development of endomycorrhizae (Wang et al., 1993). An acidic pH level can limit the endomycorrhizal colonisation in the roots and may even inhibit it completely if it becomes too acidic (˂5) (Abbott and Robson, 1985).

Conclusions Conclusions Conclusions Conclusions
The argan tree's rhizosphere in northeastern Morocco has exhibited a significant specific richness of mycorrhizal fungi representing 28 taxa. The mycorrhizal community develops variable spores' densities across the site considered which remain relatively lesser in respect to those of argan trees in the southwest of the Kingdom.
We suspect that this difference is due to the irrational exploitation of unevenly distributed Argania spinosa in these two regions and abiotic factors such topography, soil properties which may influence the AMF community structure.

Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions
SM methodology and writing-original draft; JA, KS, MC, NM interpretation and validation; SM review and editing; S E, MA, MO, MK investigation and sampling; AOT supervision and AD Conceptualization and design of experiments. All authors read and approved the final manuscript Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.