Effect of Endomycorrhizal Inoculation on the Young Cork Oak Plants ( Quercus suber) Growth

Inoculation with endomycorrhizae had a significant effect on the cork oak ( Quercus suber ) seedlings growth. The average length (55.37 cm) and weight (12.6 g) of the aerial part, the length (41 cm) and weight (26.41 g) of the root system, the rod diameter (0.56 cm) and the leaves number (120) of the plants inoculated with mycorrhizae AM were higher than those of control, respectively, 41.75 cm; 10.1 g; 28.5 cm; 16.75 g; 0.53 cm; 112.75. Frequency (90%) and intensity (71%) of mycorrhization were higher at the root levels of plants inoculated with endomycorrhizae than those of control plants that did not show any mycorrhization. However, the study of morphological spores criteria of AM fungi isolated from the rhizosphere of plants inoculated with endomycorrhizae made it possible to identify 18 different species (9 species belong to Glomus genus, 4 belong to Acaulospora genus, 2 belong to Scutellospora genus and one to Gigaspora genus, Diversispora genus and Redeckera genus).


INTORDUCTION
Mycorrhizal associations play a key role in the functioning and stability of terrestrial ecosystems that are heavily involved in the mechanisms governing the spatiotemporal evolution of ecosystems [1]. Some plant species have the property of contracting symbiotic relationships with the two types of fungal symbiosis; Mycorrhizal arbuscular fungi (endomycorrhizae) and ectomycorrhizae [2,3,4]. These include the Acacia genus from Australia, Casuarina, Eucalyptus, and Quercus [5,6,7,8], Salicaceae [9,10], Populus [11,12].
In the establishment of the mycorrhizal procession, the arbuscular mycorrhizal fungi settle early and are followed by the ectomycorrhizal symbionts [13]. Ducousso et al. [14] showed that the double inoculation of Acacia holosericea reduced by 39% the rate of ectomycorhization by Pisolithus sp. in the presence of Glomus mosseae (endomycorrhizae arbuscular). On the contrary, Chen et al. [6] observed an increase in the rate of ectomycorhization at Eucalyptus globulus and E. urophylla inoculated with ectomycorhizal fungi (Laccaria genus) in the presence of arbuscular mycorrhizal fungi (Glomus invermaium, Acaulospora laevis or Scutellospora calospora). On the other hand, Founoune et al. [7] showed that the dual inoculation improved the development of Acacia holosericea, compared to the growth of the plants inoculated with one or more of the symbiotes.
Similarly, other studies have shown that specific ectomycorrhizal fungi are able to improve the juvenile growth of certain forest species and mitigate the effects of the transplant crisis [15][16][17][18][19][20]. In addition, the cork oak (Quercus suber) is a mycotropic tree, frequently associated with endomycorrhizae and/or more rarely with ectoendomycorrhizae. The arbuscular mycorrhizae are dominant in young seedlings while Ectomycorrhizal are mainly detected in adult trees [21,22].
In this context, the objective of this work was to study the effect of endomycorrhizae on the mycorrhizal parameters and growth of cork oak seedlings (Quercus suber).

Plant Material
The study was carried out on six-month-old cork oak seedlings, which were brought from a nursery located in the region of Kenitra in the north-west of Morocco.

Production and Multiplication of the Endomycorrhizal Inoculum
An endomycorrhizal inoculum was collected from the rhizosphere of carob tree (soil and roots) from different Moroccan regions (Taroudant, Khénifra, Afourar, Nador and Ksiba). This inoculum, made up by 30 endomycorrhizal species [23,24], was multiplied on two mycotrophic species: maize and sorghum. The seeds of these two species were disinfected with sodium hypochlorite (5%) for two minutes, then rinsed with tap water and sown in pots containing mycorrhizal soil and carob tree root fragments. These pots were placed under greenhouse and watered regularly with distilled water. After three months of cultivation, mycorrhizal roots of maize and sorghum were used to inoculate cork oak plants.

Enodmycorrhizal Inoculation
Inoculation consists of cork oak transplanted seedlings in pots filled with the disinfected Mamora's sand forest containing a layer of mycorrhizal roots. The control plants are transplanted into pots containing only the soil of the disinfected Mamora forest. All plants are watered with distilled water to facilitate the installation of mycorrhizae.

Experimental Apparatus
The experiment was carried out between January and June 2015. The experimental design was designed in random blocks with one plant per pot and eight replicates for each treatment. The pots were placed in a greenhouse whose temperature varies between 18 and 25 C°.

Evaluation of cork oak plants agronomical parameters
After 5 months of cultivation, the pots were brought back from the greenhouse and the cork oak plants were cut at the collar. The growth parameters evaluated were: the number of leaves, number of branches, length and fresh weight of the aerial part, diameter of the main stem, fresh weight and length of the root part and the percentage of leaves that show symptoms of chlorosis or necrosis, calculated according to the formula: %Fsym: Percentage of leaves with symptoms.
: Number of leaves showing symptoms.
: Total number of leaves.

Mycorrhizal roots
After five months of inoculation, the colonization of the cork oak plants roots by AM fungi was carried out using the root staining technique of [25]. The roots were recuperated from the substrate and washed with water. The finest roots were cut into pieces of 1 cm length, soaked in a solution containing 10% KOH and a few drops of hydrogen peroxide (H 2 O 2 ), and placed in an oven at 90 C° for 45 min. These fragments were then rinsed with distilled water and heated to 90 C° for 15 min in the Cresyl blue.
Thirty randomly selected fragments were used for microscopic observation and for the calculation of mycorrhizal parameters, namely mycorrhizal frequency (MF%), mycorrhizal intensity (MI%), arbuscular and vesiclar contents, according to index Mycorhizal of Trouvelot et al. [26].
The mycorrhizal frequency (M.F. %) reflects the importance of the colonization of the root system and was calculated using the following formula: M.F. % = 100 × (N -n0) / N N: Number of observed fragments, n0: Number of non-mycorrhizal fragments.

Spore's extraction
Spores are extracted according to the wet sieving method described by Gerdemann and Nicolson [27]. Indeed, in a 1 L beaker, 100 g of each composite sample of the soil is immersed with 0.5 L of running water and stirred for 1 minute with a spatula. After 10 to 30 seconds of decanting, the supernatant is passed through two superposed sieves with decreasing meshes (315 and 50 μm). This operation is repeated twice. The contents retained by the sieves are distributed in tubes and centrifuged for 5 min at 2000 towers/min. The supernatant is discarded and a viscosity gradient is created by adding 20 ml of a 40% sucrose solution to each centrifuge tube [28]. The mixture was rapidly stirred and the tube returned to the centrifuge for 1 min at 3000 turns/min.
Unlike the first centrifuging operation, the supernatant is poured onto the 50 μm mesh sieve. The substrate obtained is rinsed with distilled water to remove the sucrose and then disinfected with an antibiotic solution of Streptomycin at 10 mg / L. The spores were then recovered in an Erlenmeyer flask with a little distilled water. The AM fungi are then identified based on their morphological characters.

Statistical Analysis
The statistical treatment of the results obtained involved the analysis of the variance with a single classification criterion (ANOVA 1) at the 5% threshold using the STATISTICA software.

Evaluation of Cork Oak Plants Agronomical Parameters
The lengths and average fresh weights of aerial and root parts of the cork oak plants, after 5 months of inoculation, vary according to the type of treatment (Fig. 1

Roots mycorhization
The microscopic observation of the root fragments after 5 months of inoculation (Fig. 3) revealed the presence of different mycorrhizal structures, namely: arbuscules (A, E), vesicles (D), Hyphae (B) and endophytes (C).
The frequency and intensity of the cork oak roots mycorrhization (Fig. 4) were elevated in roots inoculated with mycorrhiza (90%, 71%, respectively). Whereas the roots of the control plants were not mycorrhizal. On the other hand, the contents of arbuscules and vesicles in the inoculated roots were respectively in the order of (60%; 25%) compared to the roots of the control plants which had neither arbuscules nor vesicles (Fig. 4).
Moreover, the number of spores (114 spores / 100 g of soil) was high in roots inoculated with mycorrhizae while their density in the control plants rhizosphere was nulle.

B-extraction of spores
The study of the morphological criteria of the AM fungi spores isolated from the inoculated plants rhizosphere, allowed identifying 18 different species. Nine species belong to the Glomus genus, four Acaulospora, both to Scutellospora and one to Gigaspora, Diversispora and Redeckera. Glomus etunicatum was the most abundant species, with a frequency of occurrence which reached 20% (Fig. 5).

DISCUSSION
In this work, the beneficial effect of endomycorrhizal fungi on the growth of the cork oak plants has been proved. This effect was mainly due to an increase in biomass, axial, and root growth. This is consistent with the work done on tomato plants [29,30], the carob tree [31] and Eucalyptus [32] which also demonstrated that plant inoculation with AM fungi stimulated the weight and length of the aerial and root parts of these plants. Indeed, the development of the root system is due to the formation of a greater number of rootlets, confirming that the AM fungi increase the rooting zone [33].
The exploration of the soil volume by extrametrical mycelium and its ability to mobilize nutrients from primary minerals promote phosphate plant nutrition [34,35,36]. This improvement in plant mineral nutrition also concerns other macro elements and trace elements [37,38]. On the other hand, mycorrhizal associations play a significant role in decomposition and mineralization of organic matter from land-based nutrients and mobilize the benefit on the host plant [39] and an improving water nutrition of the host plants in exchange of photosynthates [39,40].
In addition, our results showed that inoculation with AM fungi reduced the percentage of leaves showing symptoms relative to control, which would be partly responsible for growth stimulation in these plants. Many results attribute to mycorrhizal symbiosis a bioprotective effect via a pathogen reduction effect of certain plantagents [41,42,43].
The seedlings roots of Quercus suber showed the presence of different structures of arbuscular mycorrhizae (arbuscules, vesicles, hyphae), with a high frequency and intensity of mycorrhization, while the control plants roots were not mycorrhizae. Ian et al. [44], working on the seedlings roots of Quercus rubra, noted the abundance of hyphae and vesicles, and the absence of arbuscules. The same result was recorded on Q. imbricaria [45].
However, other authors have reported the presence of arbuscules on Quercus rubra and Q. falcata [46], on Q. rubra and Q. palustris [47]. Moreover, other studies on Eucalyptus revealed the presence of different structures of arbuscular mycorrhiza [32,48,49,50,51]. On the other hand, microscopic observation revealed the presence of 18 species, whose Glomus etunicatum is the most dominant. This is consistent with the results of El Asri et al. [23], who noted the dominance of Glomus etunicatum in the carob tree's rhizosphere in five regions of Morocco.
The same result was recorded at the maize rhizosphere [31]. Chliyeh et al. [30] also noted the dominance of Glomus etunicatum at the root zone of the olive tree. This specie also dominates in the rhizosphere of the plant species in the Atlantic forest of Brazil [52].

Fig. 1. Effect of AM (Myc) fungi on average length (cm) (LPA, LPR) and weight (g) (PPA, PPR) of the aerial part and root of the cork oak plants after 5 months of Inoculation compared to control (T)
Two affected results of the same letter of the same parameter do not differ significantly with the threshold from 5%, according to ANOVA test  Among the vascular plant species that simultaneously or successively harbor endomycorrhizae and ectomycorrhizae, Eucalyptus [53,32], Populus [11,12] and Quercus [46]. This double colonization increases the ecological amplitude of the plant host by improving its mineral supply: Endomycorrhizal and ectomycorrhizal symbionts facilitate respectively phosphate and nitrogen nutritions [54,55]. Indeed, although Quercus and Eucalyptus are typically ectomycorrhizal, some authors [56,57] reported that endomycorrhizal fungi are predominantly present in the root system of young plants and when these plants develop, endomycorrhizae are reinforced by ectomycorrhizae.

CONCLUSION
The obtained results show the potential effect of inoculation with mycorrhizae on the growth and development of cork oak plants. Thus, the use of a functional inoculums based on AM fungi as a biotechnological technique allows better exploitation of nutrients from culture substrates in order to obtain a better improvement in the growth of horticultural crops.
This inoculum can also be used in reforestation, restoration of degraded ecosystems and enhancement of cork oak plants strength in nurseries.