Phosphorus (P) is the second significant element required for plants growth and development (Razaq et al., 2017). With nitrogen (N) and potassium (K), P is considered as one of the three macronutrients necessary for plant growth and development, and is generally applied to soils through chemical fertilizers (Liu et al., 2014). P is an essential element of the energy metabolism, living cells since it is the main component of DNA, RNA, ATP, and phospholipids (the major component of cell membranes). It is, therefore, involved in cell division, the transmission of genetic information, the transfer and storage of energy (ATP), and also in the photosynthetic system. Plants take up P from the soil as orthophosphate form (Pi); however, the concentration of this latter in the soil rarely exceeds 10 mM (Daniel et al., 1998). Although the total P content (inorganic and organic fractions) of soils generally does not exceed 0.12%, only 0.1% of the total P exists in the soluble inorganic form, easily assimilated by plants (Goldstein, 1994). On the other hand, a very important amount of insoluble phosphate exists in different forms, such as dicalcium phosphate, tricalcium phosphate, rock phosphate, and hydroxyapatite.

Morocco, China, and the United States are the three main producers of rock phosphate in the world. They hold more than 80% of the exploitable deposits. This rock phosphate presents a good opportunity to be applied directly to the soils and considered as a cheaper fertilizer, affordable for low-income countries. However, there is a need for a smart way to convert insoluble rock phosphate to soluble form, easily available for the plants. The use of microorganisms presents one of the innovative and challenging approaches to RP solubilization. Microorganisms produce phosphatase enzymes (Stephen et al., 2015) and organic acids (Reyes et al., 2006). The latter chelate the cations attached to insoluble phosphates through their carboxylic groups, allowing them to be converted into soluble forms (Mackey and Paytan, 2009). These microorganisms directly benefit from the bioavailable P necessary for their growth (Azaroual et al., 2020). Likewise, other organisms are able to take advantage of solubilized P, such as symbiotic association between fungi and plants, largely named by mycorrhization, which is considered as one of the oldest associations between plants and microorganisms on earth. This association allows the both partners to grow, explore water and nutritional resources, and adapt to several kinds of environment and harsh conditions (Hazzoumi et al., 2015; Damin et al., 2020). Up to 80% of terrestrial plants species, including wild plants, trees, common and economically valuable crops, are estimated to be colonized by AMF (Tarkka and Frey-Klett, 2008).

One of the first benefits of AMF to the plants is the exploitation of water and nutrients, thanks to a very developed root system constituted principally by a huge hyphal network that connects plant interior to surrounding soil. Thus, hyphae are more extensive and more efficient than root hairs in taking up nutrients (Hazzoumi et al., 2017). However, plants’ nutritional profiles are mainly depending on macronutrients, mainly nitrogen and phosphorus (P). When soil is poor, AMF provide P and other micronutrients poorly available in the medium to the plants (Kamrun et al., 2020). Mutual beneficial association consists of supplying carbon (C) to the fungus from the plants and the P, microelements, and water by the fungus to the plants (Smith et al., 2003; Hazzoumi et al., 2017; Kamrun et al., 2020).

This association highly depends on the plant species, soil microorganisms (mycorrhiza and helping bacteria), disease, and environmental conditions. Under harsh conditions, Mycorrhizal associations help plants to ensure a better balance of nutrition uptake and tolerance to biotic and abiotic stresses (Damin et al., 2020; Ranjbar Sistani et al., 2020). According to some authors, the rates of root colonization and spores production by Glomus fasciculatum and Acaulosporalaevis in Sesamum indicum plants were highest in soil with increased concentration of Rock Phosphate (up to 22.5 mg/kg) (Sabannavar and Lakshman, 2009). On other hand, Kamrun et al. (2020) showed that the colonization may only be a suitable trait for improved P efficiency in farming systems where available P is consistently low. According to this study, P fertilizers reduced AMF colonization in wheat variety Carazinho and RAC875 that has a low-yield response to P fertilization.

The kind of fertilizer used and the concentration of free P in soil significantly impact mycorrhization efficiency. Kuramshina et al. (2020) investigated the effect of Gumi-Omi-Phosphorus organic fertilizer and the superphosphate mineral fertilizer on the biomass of roots and the formation of arbuscular mycorrhiza on Pisum sativum L., Avena sativa L., and Triticum aestivum L. Their results showed that, with the use of fertilizers, root biomass increased in all studied plants. At the opposite, the colonization intensity decreased when superphosphate was introduced into the soil. In unsuitable soil, the Gumi-Omi-Phosphorus could stimulate both partners to form an arbuscular mycorrhizal symbiosis. The unlikable impact of superphosphate is probably due to the presence of readily available phosphorus in greater quantities in the soil. Hence, better and efficient mycorrhization on the plants has been related to the rate of nutrients mobilization from soil to the plants (Bolan, 1991; Kobae, 2019) and helps this later to explore, accumulate, and transport the free phosphate (Pi). This fast movement of nutrients, mainly (P) into mycorrhizal hyphae, is achieved by increasing the affinity of this latter and by decreasing the threshold concentration required for the P absorption. However, in the plant colonized by many AMF, the ability of this latter regarding phosphate uptake differs (Kobae, 2019). Mycorrhization efficiency is also related to the dynamism of AMF, and, until now, this process is still poorly investigated and remained unknown. The different responses of the plants to AMF were mainly due to the high intraspecific variability in the fungal gene repertoires, which presents a major factor that explains this affinity between plants and fungi (Chen et al., 2018). In addition, the process by which AMF transform the organic phosphate (Po) to inorganic one (Pi) is named fine-tuning of phosphate homeostasis (PHO), which implicates the expression of PHO genes, including those encoding Pi transporters and the extracellular A. According to Saito and Ezawa (2016) and Tatsuhiro and Katsuharu (2018), uptake processes of Pi from soil to fungi implicate H⁺/Pi symporters and Na⁺/Pi symporters, driven by H⁺ATPases and Na⁺ATPases, respectively. Through these processes, the fungi are capable to accumulate a massive amount of polyP (up to 64% of total cellular phosphorus) (Tatsuhiro and Katsuharu, 2018); such a process is strictly regulated in the fungal cells. Another mechanism is consisting the mineralization of Po by its extracellular acid phosphatases (ACPs) (Koide and Kabir, 2000). Bulgarelli et al. (2020) explained that roots acquire phosphorus (P) as orthophosphate (Pi) through phosphate transporters of the PHT1 family with different affinities to Pi; this process is significantly influenced by arbuscular mycorrhizal (AM) symbiosis.

Generally, these microorganisms (AMF in association with bacteria) release three groups of enzymes able to mineralize P from organic amalgams in the soil environment (Azaroual et al., 2020; Etesami et al., 2021). The first group consisted on phytases that precisely cause phosphorus discharge from phytic acid; the second is related to non-specific phosphatases that accomplish dephosphorylation of phosphoester and phosphoric anhydride bonds in organic stuff, and, finally, phosphonatases and C–P lyases, enzymes that achieve carbon–phosphorus cleavage in organophosphonates (Lusk et al., 2017; Gong et al., 2018; Kour et al., 2020; Patel and Goswami, 2020; Rastegari et al., 2020).

In this work, we aimed to study and explore the diversity and the application of AMF isolated from two different environments: Moroccan phosphate mines and agricultural soil. Also, the ability of isolated AMF to improve the growth and nutrition by using different sources of phosphate (RP & TSP), their effects of the growth performance, and the physiological parameters of wheat plants were also investigated.

The physicochemical characteristics of the phosphate rock soils used along this work reflected an almost neutral pH (7, 83) and conductivity of 0.415 mS. Furthermore, for its mineral composition, the P content was around 51 mg/g, the N between 0.42 and 0.46 mg.g^–1, and K with average content of 12 mg.g^–1 (Table 2). On other hand, the agricultural Zone 1 is characterized by a slightly basic pH (8, 68) and high conductivity (35, 54); also, the two agricultural soils showed a considerable concentration of N, P, and K higher than in rock phosphate.

The AMF present one of the preferential microorganisms for supplying the phosphate to the plants (Damin et al., 2020; Ranjbar Sistani et al., 2020). In this work, we reported, for the first time, the isolation of AMF from phosphate mine in the vicinity of wild plants that grow in this particular environment. According to many scientists, the high amount of phosphate inhibits the multiplication and the association of mycorrhizal spores with the plant (Sabannavar and Lakshman, 2009). In addition, the studied phosphate mine is located in a harsh environment characterized by water scarcity, high temperatures fluctuation, lack of precipitations, and minimal content of organic compounds. This environment is particualrly inadequate to the plant growth. AMF spores isolated from rock phosphate showed a high ability to improve plant growth in the presence of RP, more efficient than spores isolated from normal agricultural soil.

The deleterious impact of phosphate mine on plant growth and distribution was investigated along a decade (Sylvia and Neal, 1990). According to this author, phosphate mining operations can reduce population densities of VAM fungi in soil by destroying plant communities and by mixing top soil with both sub-soil and mine-soil materials. To overcome this delicate situation, the author tried to inoculate the native plants with vesicular-arbuscular mycorrhizal fungi (e.g., Glomus etunicatum and Glomus intraradices) for phosphate mine land reclamation. The native plants used for the revegetation of phosphate mine lands in Florida were Aronia arbutifolia (L.) Ell. (red chokeberry), Clethra alnifolia L. (sweet pepperbush), Cornusfoemina Mill. (swamp dogwood), Ilex glabra (L.) Gray (gallberry), and Viburnum nudum L.

The results obtained in this work showed less plant diversity in the Moroccan phosphate mine samples. Three plant species; Digitariasanguinalis, Alternanthera caracasana, and Conyza canadensis that are native in this environment presented mycorrhizal richness. However, this richeness was significantly lower than that reported in agricultural soil. These species could be good candidates for indigenous spore multiplication. In fact, the phosphate mine is a very challenging environment for plant mycorrhization, not just because of the presence of inorganic phosphate but also the existence of the high rate of metals, which could explain the less abundance of wild-plant species (Mabrouk et al., 2020) and poor vegetation. Similar results were found in the Metlaoui phosphate mining basin (Gafsa, Tunisia), where only two native plants, Moricandia arvensis and Diplotaxis harra (Brassicaceae), were able to tolerate high concentrations of Cd, Ni, Zn, and Cr (Mabrouk et al., 2020).

The second factor that could explain the limited plant diversity and AMF in this environment arises from the allelopathic response of native plants against other weeds. For example, Digitariasanguinalis has a great ability to inhibit the growth of other plant species via allelopathic response. This plant exhibits its harmful allelopathic effects on crop species through different mechanisms, like phenolic acid compounds (Rashidi et al., 2020). This ability enhanced significantly with the presence of AMF. According to Rashidi et al. (2020), the inoculation of Digitariasanguinalis and S. nigrum plants with AMF showed greater competitive ability and allelopathic potential of this weed when associated with AMF, which makes it a good competitor against other plant species in the environment. According to Rashidi et al. (2020), this native plant seems to be more adapted to made association with mycorrhizae, especially with Funneliformismosseae, Rhizoglomusfasciculatum, and Rhizoglomusintraradices.

The poor abundance of host plants in the phosphate mine area impacted negatively the AMF diversity, which explains the abundance and diversity of AMF recorded in our study (Jan et al., 2006; Rosendahl and Matzen, 2008). Moreover, the chemical fertilizer (especially phosphorus) can decrease AMF root colonization and could have negative impact on AMF communities (Entry et al., 2002; Tang et al., 2005).

According to Xiao C. Q. et al. (2009), the plant species grown in the phosphate mine were more adapted to high phosphate concentration. In China, 38 plant species belonging to 7 families were collected from the phosphate mining areas. However, only 12 plant species were shown to have the ability to accumulate phosphorus. The two species, which have been more adapted, Pilea sinofasciata and Polygonum, could accumulate, respectively, 3.4 and 7 times phosphorus higher than in the non-mining areas.

One of the native plants investigated in this work (Digitariasanguinalis) has a great ability to establish association with mycorrhizae, especially Funneliformismosseae, Rhizoglomusfasciculatum, and Rhizoglomusintraradices (Xiao G. et al., 2009). According to Rashidi et al. (2020), this association improved the ability of Digitariasanguinalis to adapt to the harsh conditions by increasing the photosynthetic rate and biomass (from 26 to 49%). However, sometimes, the association could be ineffective or non-functional, which explains the non-adaptation of other species in the phosphate mine area.

In terms of spores diversity, according to Maazouzi et al. (2020), the Rhamna region holds great diversity of AMF spores, divided into three orders (Glomerales, Diversisporales, and Archaeosporales), and many Genus, Glomus (10 species), Rhizophagus (2 species), Funneliformis (4 species), Claroideoglomus (1 species), Dentiscutata (2 species), Gigaspora (3 species), Scutellospora (2 species), Racocetra (1 species), Acaulospora (6 species), Entrophospora (1 species), Diversispora (1 species), and Ambispora (1 species). These genera seem to be highly adapted to the host plants of the region but, of course, differ from one site to another one according to the above-cited parameters.

The native plants of the Rhamna region presented different rates of mycorrhization, which vary between 83.33 and 76.67% in Acacia gummifera and Acacia sp., respectively (Maazouzi et al., 2020), However, according to the same study, 50–60% of Acacia aneura and Prosopis juliflora roots were mycorrhized. This difference on mycorrhization frequency is probably due to soil composition, environmental conditions, and plants hots (Vivekanandan and Fixen, 1991; Hazzoumi et al., 2017; Maazouzi et al., 2020).

The main conclusion about the plants in phosphate mine and their associated AMF spores could be summarized as follows: the native plants growing in phosphate mine could be adapted to high concentrations of phosphate and heavy metals. In order to tolerate this environment, plants growing in this area develop great ability to make an association with AMF. This later helps the plants to persist and resist. The interaction plant AMF leads to an allelopathic response, which limits the growth of other plants. Simultaneously, the less abundance of plant hosts limits AMF spores multiplication and limits the AMF genera existing in this particular environment (Hazzoumi et al., 2017; Maazouzi et al., 2020).

Regarding inoculation assays in wheat plants, we can confirm that the presence of RP and TSP in a medium impacted significantly the plant growth; this source of phosphate nutrition was exploited differently according to the presence of the AMF inoculum or not, and also according to the type of inoculum. We previously reported that the mycorrhized plants presented a high rate of growth in both parts (the areal part and roots) (Hazzoumi et al., 2015), which is confirmed in other plants (ocimumbasilicum) (Ferahani et al., 2008; Zolfaghari et al., 2013). These studies showed that the inoculation of basil plants by G. intraradices and some indigenous MF enhanced the growth of both parts of the plants (82% for the areal part and more than 52% for the roots). Similar results were reported in other species, such as Lactuca sativa L., Lonicera confusa, and Vicia faba L. Omar (1998) recorded an increase in wheat plant growth after inoculation with G. constrictum. According to the same study, the greatest positive effect on growth was recorded after using a mixture of G. constrictum and RP, which increased phosphorus nutrition. Wang et al. (2020) showed that phosphorus nutrition increased the wheat plant growth, especially in the presence of AMF, which increased the nutrient absorption and, also, the resistance against insects and pathogens. Some studies reported the positive effects of AMF and RP association on plant growth, and explained how the fungi can lead to better exploitation of this phosphate source. Osorio and Habte (2013) showed that the AMF Glomus fistulosum had a positive effect on the growth and the nutritional profile of Leucaena plants in the presence of RP; however, the best efficient results were recorded in experiments using combination of G. fistulosum and phosphate-solubilizing fungi (Mortierella sp.). These results were confirmed by Omar (1998), who found that the better exploitation of RP was recorded with a mixture of inoculum of AMF and phosphate-solubilizing fungi (Aspergillus niger and Penicillium citrinum).

The results of the positive enhancement in morphologic parameters are supported by the physiological essays, especially the photosynthetic activity, which was impacted by the presence of RP and TSP in the medium and, also, the presence of AMF. The results of this study showed that the presence of RP and TSP in the medium increased the chlorophyll content of wheat leaves, especially with the inoculation of AMF spores. Similar effects were previously reported by Hazzoumi et al. (2015), which recorded a high chlorophyll content in basil plants inoculated by G. intraradices (more than 12%). This increase was higher under stress conditions (e.g., water stress) where the mycorrhization increased chlorophyll content of plants with more than 64%. According to the same authors, the nutritional profile (especially the phosphorus nutrition) was increased. The same results were confirmed by Rashidi et al. (2020) in Digitariasanguinalis, showing that inoculation with Funneliformismosseae, Rhizoglomusfasciculatum, and Rhizoglomusintraradices resulted in about 50% more biomass than uninoculated plants.

The mycorrhization leads to better exploitation and absorption of the nutrients, which explained the increase of chlorophyll content. At the same time, it provides to the plant good water exploitation (the main parameters that induce an alteration of the chlorophyll synthesis) (Hazzoumi et al., 2015). The combination of mycorrhizae and a phosphate source leads to a better chlorophyll synthesis, which is demonstrated by enhancements in the morphologic parameters.

The difference in chlorophyll content reported in plants inoculated by different spores could be explained by the rate of mycorrhization, as shown in Table 4, because a high rate of mycorrhization and arbuscule (A% = 42%) (an interface of nutrition change between plants and AMF). This implied better exploitation of nutrients and water and, also, plant protection. Moreover, there is an affinity between the inoculum and plants, which depend on medium composition. Indeed, plants inoculated with the spores from the phosphate mine environment seem more adapted when the RP is used as fertilizer. In the same sense, a better nutrition profile (TSP) with the presence of AMF led to a greater rate of chlorophyll content (the maximum value recorded in MN + TSP, Glomus sp2 + TSP), with content varies in the average of 3 mg/g FM (Figure 8).

Our results are in accordance with Aliferis et al. (2015) and Sunitha et al. (2018), who confirmed that AMF increased the chlorophyll synthesis in Salix purpurea L., Salicales: (Salicaceae) plants and positively impacted other physiological parameters. Levy and Krikun (1980), Allen et al. (1981), Kucey and Paul (1982), and Snellgrove et al. (1986) also confirmed these findings and demonstrated that mycorrhizae stimulated photosynthetic activities in many species. They reported that this photosynthesis improvement was due to the increase of the P nutrition, which is necessary for the photosynthetic reaction (the assimilation of CO₂ by the stomata). This stimulation could be linked also to an increase in the leaf area and the hydric status on the leaves (Snellgrove et al., 1982; Fredeen and Terry, 1988; Hazzoumi et al., 2015).

Total phenolic compounds content changed significantly according to the presence or the absence of AMF and, also, according to the presence of RP and TSP in the medium. Generally, the mycorrhization pushed the plant to synthesize phenolic compounds as natural response to the association plant/AMF. However, after the first phase of the association, we can record diminution of the TPC level if the plant is in an optimum medium of growth, the presence of RP in the medium pushed the plant to keep TPC synthesis, which explained this increase in TPC contents even in the absence of AMF inoculation (Hazzoumi et al., 2015). According to Khan et al. (2010), the P-solubilizing fungi produce more organic acids than bacteria and consequently exhibited greater P-solubilizing activity. These results are supported by the work of Wang et al. (2020) on wheat plants, which concluded that AMF increased the phenolic compounds level and contributed to plant defense against pathogens and, also, to P solubilization (Sunitha et al., 2018).

Generally, secondary metabolites are more influenced by a nutritional profile and soil microflora, and phenolic compounds are important secondary metabolites. The increased level of TPC in the roots is mainly explained by the direct contact of this part with RP and AMF. However, differences recorded on TPC content according to inoculation of different spores are mainly due to the mycorrhization level of each one and, also, to organic acid produced by the fungi and the plant for phosphate solubilization (Iker et al., 2004; Hazzoumi et al., 2015).

Wheat plants inoculated with the native spores of phosphate mine presented a high level of TPC synthesis, which allows the plant better exploitation of P existing in rock phosphate, as confirmed by the growth parameters and chlorophyll contents (Figure 8 and Table 5).

The soil pH in the non-mycorrhized plant is almost neutral, between 7 and 7.3, and we should mention that the RP slightly reduced the pH level (Table 6). In the mycorrhized soil without RP and TSP, we recorded a pH drop; however, the addition of TSP and RP on the medium leads to a significant reduction of the pH level, with the most reduction recorded in the plant inoculated with the MN spores with the RP (pH = 6.3) (Table 6).

This result supported our explanation about the impact of mycorrhizal fungi on the soil pH, as the first response to subtilize the phosphate rock. This diminution of the pH presented a mechanism that consists on the acidification of the medium, and many reactions of chelation and exchange in the rhizosphere environment (Cunningham and Kuiack, 1992). However, some fungi did not tolerate an acid medium, and, in the same time, they solubilized rock phosphate by some organic acid synthetized by the fungi in a tolerable amount (Omar, 1998).

In the study of Van Aarle et al. (2002), authors tested the response of arbuscular mycorrhizal fungi (AMF) Scutellosporacalospora and Glomus intraradices at different soil pHs (5 and 6), in association with Plantago lanceolata. Results showed that both fungi formed more extraradical mycelium at the higher pH, and both fungi formed more arbuscules and vesicles in low pH, with a high rate of colonization of plants by fungi.

The principal components analysis confirmed the previous results of growth and chlorophyll content, in relation with P sources and the AMF inoculation (Figure 10). The mycorrhization pulls the curve in the other sense than non-mycorrhized plants; in the same way, the presence of TSP and RP in the medium with the absence of P source. This means that the presence of AMF improved the chlorophyll content and the growth parameters; this positive impact was also influenced by the P sources (Figure 10A). However, the RP nutrition presented a very positive impact on all the parameters studied (chlorophyll content, growth of roots and stems), but with the inoculation of morphotypes able to explore this source of phosphate.

The AMF morphotypes affected positively the growth parameters and chlorophyll content. The inoculum isolated from the phosphate mine showed a positive correlation with all the parameters represented in the PCA contrariwise to the absence of the inoculum; however, Glomus sp1 and Acaulosporalongula almost act in the same way, and the positive control Glomusintraradices and Glomus sp2 almost reflected a similar effect (Figure 10B).

Boosting and improving plant growth and assimilation of nutriments present a crucial challenge in agriculture; in this vision, we put the light on the AMF isolated from agriculture soil and phosphate mine soil to improve thus parameters. The results showed high diversity of spores in the agricultural medium, in comparison to the phosphate mines area, with Glomus, Gigaspora, and Acaulospora present the main spores isolated.

The inoculation of wheat plants with AMF spores improved the growth of the plants via the increase of the root surface, which makes the plant able to explore a large surface of the soil and increases their nutritional profile, especially the spores isolated from phosphate mines, which present the results the more significant in the morphological and biochemical parameters (growth, chlorophyll phenolic compounds).

The results showed less diversity of AMF in the natural phosphate samples compared to soil samples.

Moreover, it is considered that there is an impact on the phosphate solubilization, which explained the positive results obtained in the mycorrhizal plant with rock phosphate (M + RP). Native plants and AMF from phosphate mines could present a reservoir of unexploited microbial diversity that could help in the agronomic efficiency of rock phosphate used as fertilizer.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ZH, SA, NE, YZ, RD, BB, and IMK participated in designing the study, data processing, spores identification, performed the statistical analysis, and the draft of the manuscript. All authors read and approved the final manuscript.

YZ was employed by OCP Group. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.